Conference Abstracts 2005 – 2009

2009

J. Castro, M. Bosch, Y. Hayashi, M.Sur
Ultrastructural reorganization after long term potentiation of a single dendritic spine
Soc. Neurosci., 2009.

It is commonly accepted that structure and molecular composition of the spine change with the input received but the exact dynamics of those changes at a single spine in a small time frame have not been shown until recent experiments combining two-photon imaging and glutamate uncaging stimulation. These experiments show an absence of changes in postsynaptic density (PSD) protein trafficking and concentration in the early stages after long term potentiation (LTP). However a validation of those observations with a high spatial resolution is absolutely necessary. We have tried to confirm these findings with the combination of a precise (temporal and spatially) paradigm of stimulation and a method to track the structural changes with electron microscopy (EM) resolution at a single spine level. We have imaged CA1 pyramidal neurons in organotypic slices and analyzed stimulated spines 1, 7 and 30 minutes after LTP induction, using two-photon microscopy to visualize, elicit LTP with glutamate-uncaging and photo-generate landmarks to pinpoint the stimulated spines for further study with EM serial reconstruction. We have developed a reliable method to locate a specific spine which enables us to reconstruct and analyze volume and surface areas of the head and neck as well as the PSD and other form factors like the sphericity from stimulated spines. Compared to non-stimulated spines, the overall volume of the LTP-induced spine increased, with the highest increment in the 1 minute potentiated group while the PSD apparently did not show any significant change of its area. The increase in volume seemed to be due to an overall build up and not an abnormal enlargement of a specific part or increase in length of the spine. The surface complexity measured as a ratio between the surface area and volume of a given structure did not change as the surface area increased accordingly with the volume. These results suggest that the structural plasticity in the whole spine and the PSD are independently regulated :

Society for Neuroscience Abstract, 2009.

M. Bosch, J. Castro, M.Sur, Y. Hayashi
Remodelación estructural y molecular de una única espina dendrítica durante su potenciación a largo plazo
XIII Cong. SENC, Tarragona, Spain, Sept. 18, 2009.

Synapses undergo long-lasting modifications of their structure, function and composition to store information, but the precise evolution of these changes has never been observed in real time.

Here we present the chronological sequence of the protein reorganization that takes place during the early phase of long-term potentiation (LTP), induced in a single dendritic spine by two-photon glutamate uncaging. Using fluorescent and photoactivatable protein fusions we identified three different patterns of protein dynamics: (1) A set of proteins that moves rapidly to the spine in a transient initial phase (1-2 min), including actin and, specially cofilin 1, which gets massively concentrated in the spine. (2) During the stabilization of the spine growth (2-7 min), a second group of proteins, including CaMKII a and ß, the GluR1 subunit of AMPAR, a-actinin 2, Drebrin A and Profilin IIa, gradually translocate to the spine, at different rates, to consolidate the new structure. (3) Surprisingly, the main scaffolding components of the postsynaptic density (PSD), like PSD-95, Homer 1b, Shank 1b and SAP-97, do not change their total amount, spine sub-localization or turn-over during the subsequent 60 min. This suggests a totally independent structural plasticity between the whole spine and its PSD. To confirm it, we visualized the fine ultrastructure of a single potentiated spine at different moments of LTP induction, by combining two-photon uncaging, imaging and photo-localization with electron microscopy.

These results provide a broad view with high spatial and temporal resolution of the structural and molecular changes that underlie synaptic plasticity in individual spines.

Society for Neuroscience Abstract, 2009.

M. Bosch, J. Castro, R. Narayanan, M. Sur, Y. Hayashi
Structural and molecular reorganization of a single dendritic spine during long-term potentiation
Soc. Neurosci., 2009.

Synapses undergo long-lasting modifications of their structure, function and composition to store information, but the precise evolution of these changes has never been observed in real time.

Here we present the chronological sequence of the protein reorganization that takes place during the early phase of long-term potentiation (LTP), induced in a single dendritic spine by two-photon glutamate uncaging. Using fluorescent and photoactivatable protein fusions we identified three different patterns of protein dynamics: (1) A set of proteins that moves rapidly to the spine in a transient initial phase (1-2 min), including actin and, specially cofilin 1, which gets massively concentrated in the spine. (2) During the stabilization of the spine growth (2-7 min), a second group of proteins, including CaMKII a and ß, the GluR1 subunit of AMPAR, a-actinin 2, Drebrin A and Profilin IIa, gradually translocate to the spine, at different rates, to consolidate the new structure. (3) Surprisingly, the main scaffolding components of the postsynaptic density (PSD), like PSD-95, Homer 1b, Shank 1b and SAP-97, do not change their total amount, spine sub-localization or turn-over during the subsequent 60 min. This suggests a totally independent structural plasticity between the whole spine and its PSD. To confirm it, we visualized the fine ultrastructure of a single potentiated spine at different moments of LTP induction, by combining two-photon uncaging, imaging and photo-localization with electron microscopy.

These results provide a broad view with high spatial and temporal resolution of the structural and molecular changes that underlie synaptic plasticity in individual spines.

Society for Neuroscience Abstract, 2009.

N. Chen, N. Wilson, G. Tan, M. Sur
High-throughput connectivity mapping in mouse visual cortex: probing the spatial extent of interlaminar and intralaminar connections
Soc. Neurosci., 2009.

The diversity of functions performed by the neocortex is believed to be supported at least in part by a single “canonical” circuit that is replicated for various functional contexts. To understand how cognitive processes such as perception, emotions and decision-making emerge from neuronal architecture, it is therefore necessary to map the functional properties and spatial extent of communication between units of this circuit. In a novel approach to probing cortical function, we combine a high-throughput 60 electrode microstimulation system that we have recently engineered to stimulate multiple points across the mouse visual cortex with population-level calcium imaging to measure in vitro neural response in coronal sections of mouse cortex. We find that neuronal activity spreads vertically throughout the width of the cortex and horizontally to differing extents from the locus of stimulation at each cortical electrode. This is well accounted for by the organization of local cortical circuits into vertical bands (analogous to cortical columns in higher mammals) with horizontal intralaminar connections.

Such spread is thought to be stabilized by the balance of glutamatergic excitation and GABA-mediated inhibition. We investigated the functional spread of synaptic transmission by fitting a Gaussian basis function to their horizontal response profiles comparing parameters of amplitude (reflecting response magnitude), and full-wave half maximum (FWHM), reflecting extent of spread invariant to magnitude. We observed decreased amplitude of response around layer IV compared to its adjacent layers, but no consistent interlaminar differences in FWHM. We then investigated the effect of modulation by pharmacological receptor blockade of glutamate and GABA. Both vertical and horizontal spread is inhibited by blocking glutamatergic transmission. Removal of GABAergic inhibition by the antagonist bicuculline caused a uniform increase in activity, with additional activation in the deeper cortical layers which supported consistent interlaminar differences in FWHM. We thus characterize disparate extents of interlaminar and intralaminar responses, and pharmacologically dissect the functional components of excitation and inhibition that shape these responses.

Society for Neuroscience Abstract, 2009.

Y. Hayashi, A. F. Mower, S. Kwok, H. Yu, A. Majewska, K. Okamoto, M. Sur
Eye domain-specific synaptic CaMKII activation during ocular dominance plasticity in vivo
Soc. Neurosci., 2009.

Persistent modification of cortical neuronal responses upon alternations in sensory experience is believed to be the neural correlate of memory and learning. Ocular dominance (OD) plasticity, a process in which changes in visual input cause a shift in cortical responsiveness, is a paradigmatic model for studying experience-dependent plasticity. To understand the rapid processes of OD plasticity, we combined chronic in vivo two-photon microscopy and intrinsic signal optical imaging to monitor the activity of CaMKII-alpha, a protein kinase critically involved in the induction of long-term potentiation (LTP), in the visual cortex of ferrets. We found that in layer II/III pyramidal neurons located in the deprived eye domain, CaMKII-alpha activity in the spines and the adjacent dendritic regions increased significantly after 4 hr of monocular deprivation (MD). This increase was also seen in the binocular eye domain. However, the overall increase in CaMKII-alpha activity was not observed in the open eye domain. These observations were specific to MD as control experiments did not show such changes. Furthermore, in spines that were eliminated after 4 hr MD, the basal level of CaMKII-alpha activity was low. These results lend support to the model that both Hebbian, as well as homeostatic compensatory mechanism subserve OD plasticity.

Society for Neuroscience Abstract, 2009.

B. Jarosiewicz, J. Schummers, W. Q. Malik, E. N. Brown, M. Sur
Differences in the tuning properties of ferret V1 neurons that project to the dorsal vs. ventral visual streams
Soc. Neurosci., 2009.

Optical tools present new opportunities for studying the relationship between neural circuitry and function. Using fluorescent neuronal tracing together with in vivo two-photon calcium imaging, we tested whether neurons in ferret primary visual cortex that project to dorsal vs. ventral visual stream areas differ accordingly in their physiological responses to visual stimuli. We injected two different colored tracers (Cholera Toxin B conjugated to Alexa Fluor 555 and 594), one into Posterior Suprasylvian Sulcus, or ‘PSS’, the probable analog of primate dorsal stream area MT, and one into Area 21, the probable analog of primate ventral stream area V4. After neurons in V1 were retrogradely filled (4-10 days after the injection), we implanted a cranial window over V1, loaded a region containing both sets of retrogradely labeled cells with the calcium indicator dye Oregon Green-BAPTA, and characterized their activity in response to visual stimuli using 2-photon imaging. Using improved signal extraction methods to obtain tuning curves from the raw fluorescence time series (see Malik et al., this session), we found that V1 neurons projecting to PSS are more direction-selective than neurons projecting to Area 21, but that the two cell groups do not differ in their orientation selectivity. This difference in direction selectivity also exists within individual imaging sessions, when both sets of projection neurons are imaged simultaneously from the same ~250×250 um window. Additionally, we found that neurons projecting to PSS prefer shorter bars than neurons projecting to Area 21, i.e., PSS-projecting cells show behavior consistent with end-stopping (end-inhibition), whereas Area 21-projecting cells show behavior consistent with length summation. This difference also holds within individual imaging sessions. We also confirmed our previous finding that V1 neurons projecting to PSS prefer higher temporal frequencies (TF) and lower spatial frequencies (SF) than neurons projecting to Area 21, though this was true only within individual imaging sessions, not when all cells across all sessions were treated as independent samples. The existence of such ‘sorting’ of outputs from V1 into the dorsal and ventral streams might help constrain possible explanations for how functional modularity arises in higher-order visual areas.

Society for Neuroscience Abstract, 2009.

W. Q. Malik, J. Schummers, B. Jarosiewicz, M. Sur, E. N. Brown
A Statistical Modeling Framework for Two-Photon Calcium Imaging
Soc. Neurosci., 2009.

Two-photon laser-scanning microscopy has emerged as a powerful tool for in vivo functional imaging of the brain at the cellular and subcellular resolution. One prominent application is the estimation of neural coding properties from changes in somatic calcium, taken from time series imaging of tissue loaded with fluorescent calcium indicators. The true potential of this emerging modality for physiological imaging has not yet been achieved, primarily because the analysis methods developed so far are rather rudimentary. We present a new signal processing framework for two-photon imaging data that facilitates the statistical modeling of neuronal responses to the stimuli presented. We use a signal-plus-noise model for the measured calcium fluorescence, noting that the latter consists of the neuronal response corrupted by background activity and various sources of noise. A multiple harmonic regression approach is used to model the stimulus-evoked response (the signal component), while an autoregressive (AR) process is used to capture the stimulus-free response (the colored noise component). The joint estimation of signal and noise is achieved by a cyclic descent approach, making use of the Burg and Durbin-Levinson algorithms for the estimation of the AR parameters and the iterative covariance estimation. This approach helps us decompose an involved nonlinear estimation problem into two multivariate linear regression problems, yielding near-optimal estimates with high computational efficiency. The optimal model orders for the harmonic regression and AR models are determined by the Akaike information criterion and other related measures. A complete statistical description, including the confidence intervals and significance test results, are obtained for the model parameters and the signal and noise components. The decomposition of the data into these two distinct components not only offers insight into the underlying physiology but also leads us to obtain a principled estimate of the neuronal signal-to-noise ratio. We use this method to model the responses of neurons in the ferret primary visual cortex to visual stimuli varying periodically or episodically in the parameter of interest, such as orientation, direction of motion, spatial frequency or temporal frequency. The images reconstructed from our model applied to each pixel’s time series data have substantial contrast enhancement and noise rejection compared to conventional processing. The response tuning curve estimates are also significantly improved, enabling reliable inference of the functional characteristics of the imaged neurons.

Society for Neuroscience Abstract, 2009.

C. McCurry, H. Sugihara, M. Sur
Disparate plasticity in putative excitatory and inhibitory cells of Arc KO mice
Soc. Neurosci., 2009.

A major question in neuroscience is how excitatory glutamatergic and inhibitory GABA-ergic cells differ in their response to altered sensory experience. The immediate early gene Arc is specific to excitatory cells and we have shown that it is involved in ocular dominance plasticity. We use functional two-photon calcium imaging in critical period heterozygote and homozygote Arc-GFP mice, in which GFP is under the control of the Arc promoter, to assay how genetic deletion of the Arc gene in GFP expressing putative excitatory cells influences response properties of these cells and their nearby neighbors not expressing GFP (putative inhibitory cells). In response to 5-6 days of monocular deprivation both GFP-positive and –negative cells in Arc heterozygotes show a significant shift in ocular dominance. However, a reduced shift occurs in GFP-positive cells within Arc homozygotes. Surprisingly, GFP-negative cells in Arc homozygotes show a normal shift in ocular dominance suggesting that plasticity in putative inhibitory neurons can operate independently of excitatory cell plasticity. To our knowledge, we provide the first in vivo calcium imaging data from the visual cortex of genetically identified knockout cells at single–cell resolution.

Society for Neuroscience Abstract, 2009.

S. Merlin, A. Sawatari, L. R. Marotte, S. Horng, M. Sur, C. A. Leamey
Impact of a subcortical interocular mismatch, induced by deletion of Ten-m3, on the organisation and function of V1
Soc. Neurosci., 2009.

The mechanisms which coordinate the generation of visuotopically aligned eye-specific inputs, such that a cohesive map of visual space is generated in the primary visual cortex (V1), have been the subject of intense scrutiny over recent decades. Recent work by our group has identified a developmentally expressed protein, Ten-m3, that plays a unique and important role in the generation of visuotopically aligned eye-specific projections to subcortical visual centres in mice. In the absence of Ten-m3, ipsilateral, but not contralateral inputs are dramatically mistargeted in the dorsal lateral geniculate nucleus (dLGN). Behavioural testing revealed a marked visual deficit which, remarkably, was reversed by acute monocular inactivation. This led us to hypothesise that altered interocular interactions may act to suppress vision in Ten-m3 knockouts (KOs). The aim of this study is to determine how the subcortical axon guidance errors impact the anatomical and functional organisation of primary visual cortex (V1), and thus whether they may underlie the remarkable behavioural phenotype. Transneuronal tracing with 3H-proline revealed that ipsilateral inputs map to medial, normally monocular V1 in Ten-m3 KOs, rather than remaining confined to the lateral region as in wildtype (WT) mice. Immunoreactivity for c-fos, a marker of activity, shows that the ipsilateral inputs are able to drive activity in medial V1 under monocular conditions in KOs. This contrasts with WTs where activity is confined to lateral V1. Interestingly, the ipsilaterally-driven cortical neurons are clustered in patches across V1 in KOs suggesting that there may be some functional segregation of eye-specific inputs. Optical imaging of intrinsic signals confirms the presence of ipsilaterally driven patches in medial V1 and further suggests that contralateral topography is unaltered in Ten_m3 KOs. In vivo single-unit electrophysiological recording supports this and further shows that individual cortical neurons receive topographically appropriate inputs from the contralateral eye and topographically inappropriate inputs through the ipsilateral eye. The demonstration that spatially divergent inputs from the two eyes converge on single cortical neurons suggests that the altered subcortical mapping has the capacity to cause profound changes in interocular interactions which may thus underlie the reversible visual deficit in Ten-m3 KOs.

Society for Neuroscience Abstract, 2009.

D. T. Page, O. J. Kuti, B. Karki, M. Sur
Maternal immune activation modifies autism-relevant brain and behavioral effects of Pten haploinsufficiency
Soc. Neurosci., 2009.

Immune activation during brain development in utero has been identified as a risk factor for the neurodevelopmental disorders autism and schizophrenia. We hypothesize that the effects maternal immune stimulation on brain and behavior may be exacerbated in genetic backgrounds that: 1) are sensitized to the consequences of immune stimulation, and 2) carry mutations or chromosomal rearrangements that confer risk for neurodevelopmental disorders. PTEN encodes a repressor of the PI3K signaling pathway and is involved in a variety of immune responses. Haploinsufficiency for PTEN is linked with both autoimmunity and impairments in cognition, including autism. Here we test whether a gene-environment interaction may exist between Pten and maternal immune activation. We show that stimulation of the maternal immune system using the viral ssRNA mimic and Tlr3 ligand Poly I:C leads to elevated rates of lethality in Pten haploinsufficient offspring as compared to wild type littermates. Using a relatively low dose of Poly I:C, we find that maternal immune activation acts as a modifier and exacerbates abnormal social behavior, anxiety, and sensorimotor gating phenotypes in Pten haploinsufficient offspring. In addition, brain size is modified in Pten haploinsufficient offspring of Poly I:C-treated dams. To investigate the molecular basis of the putative gene-environment interaction, we probed activation of the PI3K pathway, and find that AKT activation is elevated in the brains of embryos treated with Poly I:C in utero, a phenotype shared with Pten haploinsufficiency. This work shows how an environmental risk factor can lead to differential outcomes on autism-relevant phenotypes based on genetic background and points to dysregulation of the Pten-PI3K-Akt pathway as a mechanism contributing to the neurodevelopmental consequences of maternal immune activation.

Society for Neuroscience Abstract, 2009.

C. A. Runyan, A. Van Wart, S. Kuhlman, J. Schummers, R. Mao, Z. J. Huang, M. Sur
Characterization of visual response properties of parvalbumin-expressing inhibitory interneurons using in vivo two-photon calcium imaging
Soc. Neurosci., 2009.

In primary visual cortex (V1), inhibition shapes the tuning properties, plasticity, and integrative responses of excitatory cells. The genetic, morphological, and physiological heterogeneity of cortical interneurons suggests different subclasses may uniquely influence the firing properties of target cells. Of particular interest is the parvalbumin-expressing (Pv) class, which is composed of predominantly fast-spiking basket and chandelier cells, whose broad axonal arbors target the soma and proximal dendrites of pyramidal cells. Pv cells mediate local and long-range horizontal inhibition, and their perisomatic synapses are strategically placed to modulate the firing threshold and spike output of hundreds of target cells. Because of the difficulty in specifically identifying and manipulating interneuron subtypes in vivo, little is known about the visually-evoked responses of such cell classes, or their role in cortical information processing. In this study, we have fluorescently labeled Pv-expressing cells in vivo by introducing an adeno-associated virus carrying a floxed-stop-RFP construct into mice expressing Cre-recombinase under the Pv promoter [Kuhlman & Huang 2008]. Our data indicate that all RFP-labeled cells are indeed Pv-positive, and that Pv-negative cells do not express detectable RFP. We subsequently performed in vivo two-photon calcium imaging at the viral injection site, to analyze the visual receptive field properties, including orientation tuning, spatial frequency tuning, and receptive field size, of the fluorescently labeled Pv cells in layer 2/3. We find that the orientation tuning properties of Pv cells are generally broad, though some non-labeled cells have even flatter tuning. Pv cells also have large receptive fields when compared to the entire population of responsive cells. Spatial frequency preferences of the Pv cells, however, do not differ from the population mean. Such large, broadly selective receptive fields are consistent with a class of inhibitory cells that sparsely tiles the cortex, and acts as a general modulator of firing threshold in excitatory cells. Our findings are the first to describe the stimulus-specificity of perisomatic inhibition, and should further our understanding of how local excitatory/inhibitory balance modulates information processing.

Society for Neuroscience Abstract, 2009.

J. Schummers, H. Yu, M. Sur
Responses of astrocytes and neurons to varying contrast in ferret visual cortex
Soc. Neurosci., 2009.

Brain tissue comprises a complex milieu of cell types, including a multitude of neuronal, as well as non-neuronal, cell types. Astrocytes are one of the most prominent non-neuronal cell classes and can constitute close to half of all cells in some brain regions and species. Astrocytes are intimately linked with neurons, anatomically, metabolically and physiologically. They are positioned and equipped to both respond to neural activity, and provide feed-back to neurons. An extensive body of work in vitro has demonstrated the ability of astrocytes to modulate neural activity on multiple time scales, raising the possibility that astrocytes have substantial interactions with neural networks in vivo. Previous work has demonstrated that cortical astrocytes respond to sensory stimulation and have receptive field properties similar to those of nearby neurons. Furthermore, blocking astrocyte activity leads increased visual responses in neighboring neurons, suggesting the possibility that astrocytes regulate neural responses. In order to understand the role of astrocytes in cortical network activity, it will be necessary to carefully describe the relationship between evoked neuronal activity and astrocyte activity. Several lines of evidence suggest that this relationship is not linear; astrocytes are more sharply tuned, and more sensitive to depth of anesthesia. To examine this issue in detail, we have measured the contrast-response functions, contrast invariance of orientation tuning, and contrast adaptation in layer 2/3 visual cortical neurons and astrocytes in the ferret using two-photon imaging of bulk-loaded calcium indicators in vivo. We find that the contrast-response functions of neighboring neurons are highly heterogeneous, and those of astrocytes are less so. In general, astrocytes have higher contrast thresholds and steeper contrast-response functions than neurons. As with neurons, orientation tuning in astrocytes is fairly contrast-invariant. Interestingly contrast adaptation is more pronounced in astrocytes than in neurons. Together, these results suggest a complex interaction between the levels of neural activity and astrocyte activity.

Society for Neuroscience Abstract, 2009.

J. Sharma H. Sugihara M. Sur
Temporal expectation differentially modulates neuronal responses in macaque V1 and V4
Soc. Neurosci., 2009.

Attention in the temporal domain impacts behavioral responses leading to faster response times. Human studies have shown that expectation of an impending behavioral response is reflected in progressively shorter reaction times with increasing trial duration. In this ongoing series of experiments conducted in awake, behaving monkeys we are investigating whether neurons in early visual pathway signal impending events in time that require a behavioral response. The behavioral task required monkeys to maintain fixation on a central spot while attentively tracking a peripherally presented spot for a variable period at the end of which the attention spot disappeared. The monkey had to release a lever within a fixed time window to earn reward. The timing of attention spot disappearance was varied between 900ms to 2300ms, with equal probability over entire time window. However, the conditional probability increased with trial duration. The visual stimulus consisted of two identical patches of sinusoidal gratings of rapidly changing orientations, one of which was centered at the RF of the neuron, and other on the contralateral side. We have previously shown that V1 neuron responses in monkeys performing a temporal attention task exhibit late modulation that starts to ramp up between 150-200ms before the monkeys’ behavioral response and its magnitude increases with the trial duration. Furthermore, the time course of neuronal modulation showed significant negative correlation with their response time. Here we extend our investigations by simultaneously recording neuronal spike response and local field potentials in areas V1 and V4 in two macaque monkeys. Neurons in V4 are known to be robustly modulated by spatial attention that appears early, possibly under fronto-parietal influence, whereas V1 responses only show modest attentional modulation that usually follow attentional changes in V4. We wanted to know if temporal attention followed similar temporal dynamics such that the change in responses with expectation of a behavioral response would arrive earlier in V4 and V1 would follow suit. The analysis of time course of attentional modulation did show a late modulation in V1 and V4 that ramped up before the monkey’s impending behavioral response. However, contrary to our expectation, the modulatory influences arrived earlier in V1, roughly 50-80 ms before those seen in V4. Our results indicate that neurons in V1 and V4 signal behavioral expectations that are expressed through gating of top down attentional processes, but unlike spatial attention, these influences exhibit distinct temporal dynamics that suggests involvement of distinct neuronal networks.

Society for Neuroscience Abstract, 2009.

D. Tropea, N. R. Wilson, E. Giacometti, C. McCurry, R. Jaenisch, M. Sur
Amelioration of RTT-like symptoms in MeCP2 mutant mice by the active peptide and recombinant human forms of IGF1
Soc. Neurosci., 2009.

Rett Syndrome (RTT) is an X-linked neurological disorder and the leading known genetic cause of autism in girls. The pathogenesis of Rett Syndrome is, in the majority of cases, linked to mutations in a single gene coding for methyl CpG-binding protein 2 (MeCP2), and mutant mice that lack MeCP2 or express a truncated MeCP2 protein recapitulate many of the features of RTT. We have used one of these mouse models to explore the hypothesis that deficits of RTT arise from a recoverable failure of synaptic and circuit development. By carrying out molecular and physiological analyses of cortical maturation and plasticity in RTT mice, we identified a novel therapeutic strategy and potential drug target for the disorder, where a host of RTT symptoms can be ameliorated through the activation of the signaling pathway for insulin-like growth factor 1 (IGF1).

While our previous work examined the deficits of RTT and their amelioration via the active tri-peptide (1-3)IGF1, we have now performed similar rescue experiments using the recombinant human form of IGF1 (rhIGF1), to further explore the potential for clinical applications. Systemic treatment with both the peptide (1-3)IGF1 and the recombinant rhIGF1 significantly activated IGF1 signaling and increased the expression of PSD95, a critical synaptic scaffolding protein that is down-regulated in RTT mice and required for synapse stabilization and excitatory transmission. Acute administration of both (1-3)IGF1 and rhIGF1 to MeCP2 knockout brain slices of visual cortex in turn induced a multi-fold increase in both excitatory transmission and spontaneous firing rate, measures that are typically reduced in RTT mice. Similarly, measurements of ocular dominance plasticity, a widely used protocol to assess the stability and maturation of cortical synapses and circuits, indicated that RTT mice retain an immature susceptibility to cortical reorganization, whereas mice treated with both (1-3)IGF1 and rhIGF1 exhibited dramatically stabilized circuitry in the face of aberrant sensory experience. These results suggest that (1-3)IGF1 and rhIGF1 exhibit comparable efficacy in activating key signaling pathways and ameliorating critical synaptic and circuit phenotypes that are characteristic to mouse models of Rett Syndrome.

Society for Neuroscience Abstract, 2009.

N. R. Wilson, N. Chen, F. Wang, A. Daitch, S. Yan, B. Shi, M. Sur
Patterned spatiotemporal stimulation via computer-controlled electrode arrays evokes network plasticity in acute visual cortex slices
Soc. Neurosci., 2009.

The response properties of cortical neurons are sculpted and entrained by the pattern of synaptic activity that they receive. While much is known about plasticity mechanisms across single synapses (LTP, STDP, etc), little is known about plasticity involving the temporal interplay of multiple synapses, with potential relevance to synaptic competition, sequence detection, input comparison, and other forms of computation through which a postsynaptic neuron engages more than one partner. A system was thus sought by which to track the relative strengths of multiple synaptic inputs and precisely control their temporal interplay. We developed a 64-channel analog stimulator capable of controlling commercial multi-electrode arrays with sub-millisecond precision across all channels, and connected it to a versatile “pattern engine” in MATLAB via a USB connection. By interfacing the multi-electrode arrays to acute coronal slices of mouse visual cortex, and sending patterned stimulation through the array, it is possible to micro-stimulate small areas simultaneously, asynchronously, or in rapidly changing patterns. The neurons that are activated in turn project presynaptic drive to a neuron of interest, which is recorded via intracellular patch clamp or calcium imaging to reliably quantify transmission and detect changes in synaptic drive and neuronal output. Finally, patch clamp software that is also implemented in MATLAB provides a feedback loop to the pattern engine for real-time acquisition, analysis, and adjustment to the stimulus parameters. Using this semi-automated search to map stimulus patterns to response, we have been able to establish reliable control over many synaptic pathways, influence their weights independently via LTP protocols, and drive them in temporal patterns to instigate forms of network plasticity that may approximate sequence detection. The ability to supply more complex and rich input patterns to an in vitro system, which avails direct optical, pharmacological, and mechanical access to the tissue of interest, may enable the detailed study of new classes of network plasticity together with associated structural and molecular mechanisms.

Society for Neuroscience Abstract, 2009.

2008

L. C. CARMODY, R. L. NEVE, M. SUR
The role of Rac1 in structural changes in primary visual cortex (V1)
Soc. Neurosci., 2008.

The primary visual cortex (V1) has often been used as a model system to understand synaptic plasticity within intact cortical circuits. During the “critical period” of early development, V1 is extremely sensitive to changes in activity, such that brief manipulations in visual input cause functional changes in visually driven responses together with alterations in the structure of synapses on individual neurons and of connections between neurons. Structural changes at the level of dendritic spines – specialized F-actin rich protrusions on dendrites that receive the majority of excitatory input – are thought to underlie synaptic development and plasticity. In part, the Rho-family GTPase, Rac1, regulates the formation of spines and changes in their morphology. In this study we examined the correlation between structural changes in spines and Rac1 expression and activity. First, we monitored the expression level of proteins thought to regulate spine formation, such as spine-associated proteins (PSD95, GluR1) and Rac-signaling proteins (Rac, PAK, Actin). Critical period mice, aged ca P28, that were monocularly deprived (MD) for two days showed an increase in Rac1 expression in V1 contralateral to the lid suture. However, no difference in PSD95, GluR1, PAK, or beta actin expression was observed in immunoblotting or immunohistochemical analysis. Next, we examined the role of Rac in regulating the structure of spines and dendrites on V1 neurons before and after MD. A herpes simplex virus expressing two separate gene products, dsRed2 red fluorescent protein (RFP) and mutant Rac1 (constitutively active CA-Rac, or dominant negative DN-Rac), were expressed in critical period mice. The number and spine type (mushroom, stubby, or filopodia) was determined by measuring RFP-filled dendrites. After 4 days of expression, mutant Rac1 constructs altered number and class of spine type, thereby suggesting a role for Rac in structural changes at synapses during visual cortex plasticity.

S. GORLIN, J. SHARMA, M. MENG, H. SUGIHARA, M. SUR, P. SINHA
Splitting the difference between stimulus-driven and prior information
Soc. Neurosci., 2008.

Prior information and experience with visual stimuli enhance our ability to recognize images, but where and how does this facilitation occur in the brain? Using degraded stimuli we can tease apart the effects of bottom-up visual processes, and top-down, experience-dependent processes, as prior knowledge of the fully coherent images makes them easier to recognize. Using machine learning algorithms like Support Vector Machines (SVM’s), we can then quantify the amount of information a given brain region contains about the stimulus as the subject learns the coherent image. Here we show how distinct brain regions from prefrontal cortex to V1 contain more information about degraded stimuli with prior knowledge, and that regional information in the brain persists in line with behavior. Interestingly, this effect depends critically on the complexity of the stimuli, so that prior information seems to be encoded over complex, real-world features, but not simple stimuli such as oriented gratings.

S. HORNG, M. BLANK, K. MILLEN, M. SUR
A role for Zic4 in visual pathway patterning and function
Soc. Neurosci., 2008.

Forebrain pathways of the mammalian visual system are organized into topographic maps during development. Early patterning cues likely translate regional gradients of gene expression into precise diagrams of retinogeniculate and geniculocortical wiring, though the molecular mechanisms of these processes are not well understood. Here we examine the role of Zic4 and Zic1, two transcription factors currently known to control aspects of cerebellar differentiation, in specifying retinotopic organization within the dorsal lateral geniculate nucleus (dLG) and primary visual cortex (V1). Zic4 and Zic1 are both highly expressed in gradients of the embryonic and perinatal retina, dLG and posterior cortex. Mice with null mutations in Zic4 and Zic1/4 were used to investigate whether these genes are necessary for the proper formation of retinotopic maps. Retinal cholera toxin-B (CTB) injections at postnatal day 28 (P28) were used to assay retinogeniculate targeting in its mature state. We found that ipsilateral retinal projections to the dLG are disordered in Zic4 mutants, with abnormal increases in clustering, compression in the medial-lateral axis and ectopic projections to the lateral edge of the dLG. Eye specific segregation is normal, suggesting no disruptions in activity-dependent refinement of retinogeniculate terminals. Intrinsic signal optical imaging of V1 in Zic4 mutant and control mice at P28 was performed to examine functional maps of retinotopy in the cortex. Zic4 mutants exhibit a compression in size of the binocular zone, while the total size of V1 remains unchanged. Mice with Zic4 mutations demonstrate deficits in visually mediated behavior using a visual cliff test, including the number of crossing over a presumptive cliff. These data indicate that Zic4 is necessary for the proper patterning of ipsilateral axons in the dLG, the formation of normal binocular maps in V1, and the detection of behaviorally relevant landmarks in visual space.

B. JAROSIEWICZ, J. SCHUMMERS, M. SUR
V1 origins of distinct visual processing streams: Illuminating the link between connectivity and function using 2-photon calcium imaging
Soc. Neurosci., 2008.

How does anatomical connectivity relate to the function of neurons within a cortical region? As a first step towards addressing this question, we have combined retrograde fluorescent labeling with in vivo 2-photon calcium imaging to characterize the activity of subpopulations of ferret V1 neurons that have different downstream projection targets. We injected two fluorescent tracers (Cholera Toxin B conjugated to Alexa Fluor 555 and 594), one into a “dorsal stream” target of V1 (Posterior Suprasylvian Sulcus, or ‘PSS’, the probable analog of primate MT) and one into a “ventral stream” target of V1 (Area 21, the probable analog of primate V4). After neurons in V1 were retrogradely filled (4-10 days after the injection), we implanted a cranial window over V1, loaded a region containing both sets of retrogradely labeled cells with the calcium indicator dye Oregon Green-BAPTA, and characterized their activity in response to visual stimuli using 2-photon imaging. We found that V1 neurons projecting to PSS are more direction-selective and prefer higher temporal frequencies than neurons projecting to Area 21, and that neurons projecting to Area 21 have higher spatial frequency preferences than neurons projecting to PSS. Some aspects of these distinct functional profiles might arise from biased retinal inputs into the two populations of cells, which are further enhanced by the local circuitry within V1; e.g., PSS-projecting V1 cells might obtain their preference for high temporal frequencies from Y-cell-biased inputs, and Area 21-projecting V1 cells might obtain their preference for high spatial frequencies from X-cell-biased inputs. Other aspects of their functional properties, e.g. the direction selectivity of PSS-projecting V1 cells, might arise within the local circuitry of V1. We propose that the functional specificity of dorsal and ventral stream areas arises in a similar manner: biased inputs from V1 and elsewhere are enhanced within the local circuitry to give rise to the functional specificity of that area. Combining these methodologies across multiple stages of processing, and adding molecular and/or pharmacological manipulations, promises to help illuminate how information is transformed at each stage of cortical processing and what factors contribute to the self-organization of distinct functional modules.

R. MAO, J. SCHUMMERS, A. VAN WART, B. CRONIN, G. PRUSKY, N. ALAM, C. KIM, J. L. R. RUBENSTEIN, M. SUR
Effects of subtype-specific loss of inhibitory interneurons on receptive field properties in visual cortex
Soc. Neurosci., 2008.

Inhibitory interneurons are vital to cortical development and function. These cells comprise 20-30% of neocortical neurons, and exhibit great diversity in morphological, physiological, molecular and synaptic characteristics. Given the diversity in their properties, it is probable that different classes of interneurons have distinct roles in modulating integrative responses and receptive field properties in the visual cortex. The Dlx homeobox transcription factor family regulates the development of inhibitory interneurons. To probe the function of these interneuron classes, we utilized a Dlx1-/- mouse line (Cobos et al., Nat Neurosci 2005) that shows a specific loss of cortical and hippocampal calretinin- and somatostatin-expressing subclasses in early adulthood but no discernible effect on the parvalbumin-expressing subclass. Calretinin and somatostatin are expressed in double bouquet, bipolar, bitufted and Martinotti cells (which target dendrites and apical tufts), whereas Parvalbumin is expressed in basket cells (which target somata and proximal dendrites) and chandelier cells (which target axon initial segments). Immunohistochemistry on Dlx1-/- mice showed a partial reduction (33.6 ± 1.3%) of calretinin-positive neurons in visual cortex at postnatal day 60+ compared to wild types. Parvalbumin immunohistochemistry was unchanged in the knockout mice. Given previous evidence for a critical role of inhibition in sharpening orientation selectivity, we postulated that the interneuron deficit would result in a disruption of tuned responses in the primary visual cortex (V1). Consistent with this hypothesis, there appeared to be fewer tuned cells and significantly broadened orientation tuning in Dlx1-/- mice (n>100 cells) than in wild types (n>50 cells) based on single-unit recording experiments in adult V1. This effect is specific, as intrinsic signal optical imaging maps of retinotopy and ocular dominance were unchanged in adult Dlx1-/- mice. Visual cliff tests, visual water maze tests and threshold tests of optokinetic tracking on Dlx1-/- mice did not detect overt deficits in vision. Our findings reveal a key role for this dendritic-targeting class of inhibitory interneurons in the fine tuning of stimulus-specific visual responses.

C. MCCURRY, J. SHEPHERD, D. TROPEA, K. WANG, M. BEAR, M. SUR
Reduced ocular dominance plasticity in the visual cortex of juvenile arc null mice
Soc. Neurosci., 2008.

During a critical period in development the mouse primary visual cortex (V1) is extremely sensitive to changes in activity. In wildtype (WT) animals brief monocular deprivation (MD) results in a decrease in cortical responsiveness to the deprived eye within the binocular zone of V1. It has been suggested that endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is the mechanism mediating loss of the deprived eye response. The immediate early gene encoding activity regulated cytoskeleton associated protein (Arc) has recently been shown to regulate AMPAR internalization. Using Arc null animals we investigated whether loss of a putative mechanism for AMPAR endocytosis might prevent the depression seen after MD. Arc null animals display normal localization and size of V1. In addition, binocular visual response, acuity, and retinotopy are not significantly different from WT animals. To assess plasticity, critical period WT and Arc null animals were monocularly deprived for 4 days via lid suture and the response in cortex assessed using both intrinsic signal imaging and visually evoked potentials (VEPs). In keeping with the literature, WT animals show a significant shift in ocular dominance mediated by a depression in the contralateral eye response. Interestingly, Arc null animals display a reduced shift in OD, with no change in the contralateral eye response magnitude. These results suggest that Arc protein is required for juvenile ocular dominance plasticity. In addition, our data implicate normal trafficking of AMPARs as a prerequisite for plasticity during the critical period.

A. F. MOWER, S. KWOK, H. YU, A. MAJEWSKA, K. OKAMOTO, W. WANG, A. MEDINA, Y. HAYASHI, M. SUR
Dynamics of CaMKIIa activation induced by changes in physiological activity in visual cortex
Soc. Neurosci., 2008.

Calcium/calmodulin-dependent protein kinase type II alpha (CaMKIIa) is known to play a role in both structural and functional correlates of plasticity such as learning, memory, and changes in dendritic spine size. Most studies investigating the role of CaMKIIa have been performed in vitro using tetanic stimulation protocols which are well outside the range of physiological activity occurring in the intact brain, while studies showing a requirement for CaMKIIa in cortical plasticity have been performed in knockout mice. For this reason we used an engineered FRET probe (Camuia), packaged into a herpes simplex virus (HSV), to look at changes in CaMKIIa activation that occur in vivo with physiological changes in activity. HSV-Camuia was intracortically injected into the visual cortex of ferrets during the critical period, a cranial window was implanted and chronic in vivo 2-photon imaging combined with optical imaging for determination of ocular dominance was performed. Our results revealed that changing visual cortical activity via short periods (4 hours) of monocular deprivation (MD) leads to an increase in CaMKIIa activation in spines and dendrites of cells in deprived eye dominated domains, a decrease in open eye dominated domains and no significant change relative to pre-MD levels in binocular regions of the cortex. Control experiments using a T305D/T306D mutant which renders CaMKIIa unable to bind calmodulin and therefore inactive, or sham MD experiments, showed no significant change in CaMKIIa activation. These results were further supported using western blot analysis of visual cortex contralateral or ipsilateral to the deprived eye. To date, literature investigating activity dependent changes in CaMKIIa activation have shown that large increases in neuronal activity, and calcium influx, via tetanic stimulation result in an increase in CaMKIIa activation. In contrast, our results show an increase in CaMKIIa activation in areas of the visual cortex where calcium influx is likely to be decreased due to a decrease in visual cortical drive resulting from MD. Our findings suggest that the activation/deactivation dynamics of CaMKIIa within physiological ranges of activity differ markedly from those seen at artificially induced ranges.

D. T. PAGE, O. J. KUTI, C. PRESTIA, M. SUR
Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior: a model for polygenic autism
Soc. Neurosci., 2008.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders that share deficits in sociability, communication, and restrictive and repetitive interests. ASD is polygenic in origin in most cases, but we presently lack an understanding of the relationships between ASD susceptibility genes and the neurobiological and behavioral phenotypes of ASD. Two genes that have been implicated as conferring susceptibility to ASD are PTEN and Serotonin transporter (SLC6A4). The PI3K and serotonin pathways, in which these genes respectively act, are both potential biomarkers for ASD diagnosis and treatment. Biochemical evidence exists for an interaction between these pathways; however, the relevance of this for the pathogenesis of ASD is unclear. We find that Pten haploinsufficient (Pten+/-) mice are macrocephalic and have a corresponding increase in neuronal soma size, and this phenotype is exacerbated in Pten+/-; Slc6a4+/- mice. Furthermore, Pten+/- mice are impaired in social approach behavior, a phenotype that is exacerbated in Pten+/-; Slc6a4+/- mice. While increased brain size correlates with decreased sociability across these genotypes, within each genotype, increased brain size correlates with increased sociability, suggesting that environmental influences during development interact with genetic factors in influencing the phenotype. These findings provide insight into a potential mechanism of multifactorial ASD and point toward the use of compound mutant mice to validate biomarkers for ASD against biological and behavioral phenotypes. We are making use of this model to investigate gene-environment interactions potentially relevant to ASD by examining the anatomical and behavioral effects of Poly I:C-mediated maternal immune stimulation in a Pten haploinsufficient background.

C. A. RUNYAN, A. VAN WART, J. SCHUMMERS, B. CRONIN, H. YU, M. SUR
Single-cell mapping of multiple stimulus response features in rodent primary visual cortex using in vivo two photon calcium imaging
Soc. Neurosci., 2008.

Unlike the primary visual cortex of higher mammals, such as ferrets, cats, and primates, mouse V1 is generally considered to be lacking in the ordering of receptive field properties such as ocular dominance, orientation preference, and spatial frequency preference. It is possible, however, that such ordering does exist on a smaller scale in the rodent and that it has been overlooked with electrophysiological and optical imaging techniques, which lack the spatial resolution that may be required in order to observe such small-scale mapping. Indeed, some clustering of orientation selectivity has been noted in sequentially recorded cells in single electrode penetrations in mouse V1. In vivo two-photon calcium imaging fills this requirement, enabling precise cell-by-cell mapping of visual receptive field properties across patches of the cortex. Using this method, we were able to measure the receptive field location and ocular dominance index as well as describe the spatial frequency and orientation tuning curves for each visually responsive cell within a given focal plane. Consistent with previous reports, preliminary data indicated that only a fraction of visually responsive neurons in mouse V1 were tuned for orientation, and that within this group, tuning width was broad when compared to higher mammals. The distribution of spatial frequency preferences encompassed a wide range that was skewed in favor of low spatial frequencies, and spatial frequency tuning curves were best described by either a Gaussian function with a low spatial frequency roll-off, or more often by a sigmoid function demonstrating little sideband inhibition. We then examined the spatial distribution of each receptive field property across a small (ca 400µm2 ) patch of cortex to determine whether cells in mouse V1 may follow rules of mapping similar to those of the visual cortices of higher mammals. Pixel-based analyses indicated a highly ordered and smooth mapping of retinotopy along the elevation and azimuth axes, which held true at the single cell level. In contrast, there was an apparent lack of ordered orientation and spatial frequency preference mapping on the cellular level. Such an organization does not preclude an inverse relationship between gradients of orientation and spatial frequency, as predicted by rules of map continuity and coverage.

J. M. SCHUMMERS, H. YU, M. SUR
Contrast response functions of neurons and astrocytes in ferret visual cortex
Soc. Neurosci., 2008.

Astrocytes respond to nearby neuronal activity via intracellular calcium activity, and astrocytes in visual cortex exhibit visually-evoked calcium responses. We have recently described the orientation and spatial frequency tuning of astrocyte calcium responses in ferret primary visual cortex (V1). One intriguing finding is that astrocyte tuning is sharper than that of neurons, suggesting that astrocyte responses are only triggered by strong local neural activity. This notion of thresholded astrocyte responses is supported by a sharp dependence of astrocyte responses on anesthetic concentration. As astrocytes have the ability to influence neuronal activity both negatively and positively, and on several time scales, it is important to develop a quantitative understanding of the dependence of astrocyte responses on nearby neuronal activity levels. To further explore the relationship between neural and astrocyte responses, we now examine the contrast-dependence of neighboring neuronal and astrocyte responses. It is well known that the firing rates of neurons in visual cortex are sharply dependent on stimulus contrast. Neuronal contrast-response functions are typically characterized by a sigmoid function, with a steep rising phase, and response saturation (or even super-saturation) at high contrasts. We have begun to examine the contrast-response characteristics of neurons and astrocytes by two-photon imaging of bulk-loaded calcium indicators in visual cortex (V1) of the ferret. We find that the contrast response functions of neurons are diverse, with a wide range of half-maximal contrast values (C50) as low as 4%, possibly reflecting the diversity of neuronal cell types. Preliminary data demonstrate that in astrocytes, the contrast response functions are generally characterized by more sharply rising curves and moderate C50 values (9.8 +/- 0.6 %). This suggests that astrocyte contrast response functions have subtle differences from those of nearby neurons, indicative of a non-linear coupling between neurons and astrocytes. Further characterization of these differences will help elucidate the precise nature of neuronal-astroctye interactions.

J. SHARMA, H. SUGIHARA, J. SCHUMMERS, M. SUR
Spatial and temporal expectations modulate neuronal responses in macaque primary visual cortex
Soc. Neurosci., 2008.

Attention is a flexible conduit that biases perceptual processing and prioritizes behavioral responses according to internal states such as anticipation or expectation. While spatial attention improves detection or processing of stimulus attributes, attention in the temporal domain has direct implications on behavior, such as faster response times. Investigations of the neural underpinnings of temporal orienting point to a distributed processing network, mainly involving fronto-parietal areas. To investigate whether neurons in early visual cortex also dynamically signal impending events in time that require a behavioral response, two rhesus monkeys were trained to maintain fixation on a central spot while attentively tracking a peripherally presented spot that appeared in one of 4 locations. We used 3 different time schedules, short (900ms +/-300ms), medium (1500ms +/-300ms) or long (2100ms +/-300ms), randomly interleaved, at the end of which the attended spot disappeared and required the monkey to release a lever to earn reward. While the probability of attention spot disappearance was equally distributed over three time schedules, its conditional probability or the hazard function increased with time. That the monkey attentively tracked time was clearly seen in its response time, which significantly (p<0.01) and systematically reduced as a function of trial length. We simultaneously recorded from single neurons in V1 while the monkeys performed the task. Of the 4 locations at which the attention spot could appear in a random sequence, two locations were close to the receptive field (RF) of the recorded neuron, and two away from it, in the contralateral hemifield. Two stimulus movies, one centered on the RF and other on the contralateral side, consisted of patches of sinusoidal gratings of rapidly varying orientations, that monkeys essentially ignored. Analysis of single neuron responses in V1 revealed modest early modulation in responses when the monkey’s attention was directed towards the RF. This was followed by a late modulation that ramped up around 200ms before the monkeys’ impending behavioral response and increased with the duration of the trial. There was a close correlation between monkey’s response time and neuronal responses. In line with human psychophysical studies, our data suggest that a monkey’s expectation of an impending behavioral response is reflected in faster reaction times with increasing trial duration. Furthermore, a concomitant change in neuronal responses indicates that V1 neurons reflect behavioral expectations that are expressed through gating of top down attentional processes.

D. TROPEA, N. R. WILSON, E. GIACOMETTI, C. BEARD, C. MCCURRY, E. SOUGANIDIS, D. FU, R. FLANNERY, R. JAENISCH, M. SUR
Partial reversal of Rett-like symptoms by insulin growth factor 1 in MeCP2 mutant mice
Soc. Neurosci., 2008.

Rett Syndrome (RTT) is a severe form of X-linked mental retardation caused by mutations in the gene coding for methyl CpG-binding protein 2 (MECP2). Here we show that adult Mecp2 mutant mice exhibit physiological signatures indicative of immature cortical circuitry, including weaker synaptic function in vitro and persistent cortical plasticity in vivo. Systemic treatment with (1-3)IGF-1, a 3 amino acid active fragment of insulin-like growth factor 1 (IGF-1), restored the circuitry of the adult RTT mouse to more mature levels, by stimulating synaptic function and stabilizing the plasticity of the circuit. Additionally, treatment with the tri-peptide ameliorated bradycardia, improved locomotor function, and extended the life span of the knockout mice, which we observed to be significantly impaired in these areas. Our results suggest (1-3)IGF-1 as a strong candidate for pharmacological treatment of Rett Syndrome and potentially of other CNS disorders caused by delayed synapse maturation.

A. M. VAN WART, N. WILSON, M. SUR
TNF-alpha is a molecular substrate for activity-dependent scaling of visual responses in vivo
Soc. Neurosci., 2008.

During the critical period, cortical networks employ a number of homeostatic feedback mechanisms in order to balance feedforward modifications to synaptic strength. Reducing visual drive during the critical period with intraocular injection of TTX or brief dark-rearing (DR) leads to a scaling up of AMPA mEPSC amplitudes [Desai et al, Nat Neurosci, 8:783; Goel & Lee, J Neurosci, 27(25):6692], which may help compensate for deprivation-induced response attenuation within the cortical network. Recent in vitro work has shown that glial release of the cytokine tumor necrosis factor-alpha (TNFa) is necessary for scaling up mEPSC amplitudes at hippocampal synapses in response to activity blockade [Stellwagen & Malenka, Nature, 440:1054]. We asked whether this glial-derived factor is important for activity-dependent scaling of visual responses in vivo, measured by shifts in the balance of feedback and feedforward mechanisms induced by visual deprivation. We find that TNFa immunoreactivity is indeed present in both glia and a population of neurons throughout V1. In vivo intrinsic signal optical imaging was performed on wildtype (WT) and TNFa-/- mice at the peak of the critical period (age P28 – P29) to measure visual response strengths after 4 – 5 days of DR or binocular lid suture (BLS). We detect a significant enhancement (p < 0.05) of binocular responses after 4 – 5 days DR, consistent with an early feedback response that offsets decreases in visual drive. In contrast, we observe a significant response decrease (p < 0.05) in mice lacking TNFa, consistent with feedforward depression that is not countered by feedback enhancement. Further, we also observe a modest response attenuation in TNFa-/- mice after BLS that is not seen in WT mice. Thus TNFa is critical for activity-dependent increases in visual responses, which are necessary to prevent or counter response depression during sensory deprivation. Other aspects of visual circuitry are likely unchanged in these mice, as maps of retinotopy and ocular dominance were indistinguishable from WT. Interestingly, baseline visual responses were on average 38% greater in nondeprived TNFa-/- mice than their WT counterparts, suggesting TNFa may also be important developmentally for establishing the set point for firing rates. These data indicate a central role for TNFa in maintaining a balanced level of excitation in vivo.

K. WIMMER, R. MARTIN, M. STIMBERG, J. SCHUMMERS, M. SUR, K. OBERMAYER
Dependence of orientation tuning dynamics on map location in a network model of V1
Soc. Neurosci., 2008.

The primary visual cortex (V1) in highly visual mammals is organized into two-dimensional maps of stimulus features including orientation, spatial frequency and visual space. Intrinsic signal optical imaging, which defines maps at a scale of ca 100 um resolution, has revealed an inverse-gradient relationship between certain sets of maps: high gradient regions of orientation, ocular dominance and spatial frequency maps avoid each other in a two dimensional cortical space. This finding is consistent with a model of map self-organization that maximizes continuity and coverage. We have now probed map organization on a cell-by-cell basis, and further examined whether similar relationships exist in visual space, at a scale below the resolution of intrinsic signal measurements. We used bulk loading of fluorescent calcium indicators and two-photon imaging in ferret V1 in vivo to measure multiple tuning properties of individual, spatially identified, adjacent neurons in the same patch of cortex. By combining calcium imaging with intrinsic signal optical imaging, we targeted injections of calcium indicator specifically to orientation pinwheel centers, as well as to specific locations within spatial frequency maps. We find that neurons with the same preferred spatial frequency cluster in a columnar way, and different spatial frequency columns separated by less than 100 um can be clearly distinguished. Retinotopic mapping and receptive field progression is evident across cells spread over 100 um of cortical distance. Receptive field centers have small but significant jumps at one side of the orientation pinwheel centers, yet at locations more than 50 um away from them. Such sites are seen consistently at multiple depths of superficial visual cortex. At orientation pinwheels, where the preferred orientation of neurons changes rapidly, preferred spatial frequencies of the same group of neurons are very similar and show very little scatter. Away from pinwheel centers and on the opposite side of the retinotopic jump, where both preferred orientations and retinotopy change very little, preferred spatial frequencies change rapidly at some regions. Thus, the retinotopic, orientation and spatial frequency maps appear to have a highly precise, fine-scale inter-relationship, so that the rapidly changing high-gradient areas of multiple feature maps are exquisitely positioned to avoid each other in space.

H. YU, J. SCHUMMERS, M. SUR
The relationship between multiple feature maps in ferret visual cortex at single cell resolution
Soc. Neurosci., 2008.

The primary visual cortex (V1) in highly visual mammals is organized into two-dimensional maps of stimulus features including orientation, spatial frequency and visual space. Intrinsic signal optical imaging, which defines maps at a scale of ca 100 um resolution, has revealed an inverse-gradient relationship between certain sets of maps: high gradient regions of orientation, ocular dominance and spatial frequency maps avoid each other in a two dimensional cortical space. This finding is consistent with a model of map self-organization that maximizes continuity and coverage. We have now probed map organization on a cell-by-cell basis, and further examined whether similar relationships exist in visual space, at a scale below the resolution of intrinsic signal measurements. We used bulk loading of fluorescent calcium indicators and two-photon imaging in ferret V1 in vivo to measure multiple tuning properties of individual, spatially identified, adjacent neurons in the same patch of cortex. By combining calcium imaging with intrinsic signal optical imaging, we targeted injections of calcium indicator specifically to orientation pinwheel centers, as well as to specific locations within spatial frequency maps. We find that neurons with the same preferred spatial frequency cluster in a columnar way, and different spatial frequency columns separated by less than 100 um can be clearly distinguished. Retinotopic mapping and receptive field progression is evident across cells spread over 100 um of cortical distance. Receptive field centers have small but significant jumps at one side of the orientation pinwheel centers, yet at locations more than 50 um away from them. Such sites are seen consistently at multiple depths of superficial visual cortex. At orientation pinwheels, where the preferred orientation of neurons changes rapidly, preferred spatial frequencies of the same group of neurons are very similar and show very little scatter. Away from pinwheel centers and on the opposite side of the retinotopic jump, where both preferred orientations and retinotopy change very little, preferred spatial frequencies change rapidly at some regions. Thus, the retinotopic, orientation and spatial frequency maps appear to have a highly precise, fine-scale inter-relationship, so that the rapidly changing high-gradient areas of multiple feature maps are exquisitely positioned to avoid each other in space.

2007

Cronin, B., Sur, M. and Körding, K.
A Bayesian framework for explaining multiple timescales of pattern adaptation in sensory physiology and perception
Soc. Neurosci., 2007.

Sensory systems in the brain must compute reliably in the presence of significant internal and external fluctuations that unfold over many time scales. The nervous system thus needs to compute reliably with an ever changing substrate. In order to achieve stable computational outcomes in this environment, the nervous system must compensate for ongoing changes in the properties of neurons. This leads to a statistical estimation problem: in order to minimize changes in its computation, a neuron must implicitly estimate the excitability level of those neurons which comprise its inputs.
We present a simple yet general model of sensory adaptation, using orientation selectivity in primary visual cortex as an example. The two key elements of the model are Bayes-optimal temporal credit assignment (Kording et al., Nature Neuroscience, 2007), and an assumption of sparse firing in sensory cortex. We show that this model reproduces several key results from physiological experiments (Dragoi et al., Neuron, 2000; Saul and Cynader, Visual Neuroscience, 1989), including the repulsive adaptation of the orientation tuning curve which is observed in individual neurons, as well as a separation of the time scales for different components of this adaptation. In addition, the model is consistent with perceptual effects such as the tilt after-effect. It therefore explains why a simple computational principle leads to observations in both physiology and perception. The model is able to explain these effects using only a simple linear architecture. Indeed, the model is consistent with a broad range of circuit-level architectures, including those that rely on feed-forward mechanisms for orientation tuning (Troyer et al., J Neuroscience, 1998), and others that invoke recurrent connectivity to produce sharp orientation tuning (Somers et al., J Neuroscience, 1995; Ben-Yishai et al., PNAS, 1995).
In spite of its simplicity, this model provides significant explanatory power beyond that provided by previous models which have incorporated adaptation mechanisms into existing mechanistic models of orientation tuning (Teich and Qian, J Neurophysiology, 2003; Zin, Dragoi et al., J Neurophysiology, 2005). Furthermore, this approach supposes a fundamentally different justification for sensory adaptation than some previous work (e.g., Stocker and Simoncelli, NIPS, 2005), which posits that sensory adaptation represents an attempt by the sensory system to adjust to changes in the statistical composition of incoming stimuli. Rather, our model views adaptation across time scales as the best solution to maintaining stable computation in the face of a fluctuating nervous system.

Gorlin, S., Sharma, J., Sugihara, H., Sur, M. and Sinha, P.
Hysteresis and object recognition: imaging prior information in the visual system
Soc. Neurosci., 2007.

One of the least understood aspects of mammalian vision is the ability to recognize scenes through significant degradations in image quality. Neural receptive fields have traditionally been described with coherent structures – for example, oriented gratings in V1. However, this does not address how neurons respond to noisy, less coherent visual input, which is arguably more prevalent in the natural world. Previous studies with natural images show that recognition is highly non-linear with respect to noise, and more importantly, that recognition in noise is facilitated by prior experience with the stimuli (Sadr and Sinha, 2004). These studies entail using RISE sequences (Random Image Structure Evolution) to present subjects with structured images evolving from noise. Specifically, the direction of RISE evolution – ascending or descending in information content – allows us to control for low-level image features, such as luminance, while trending towards or away from a neuron’s experimentally defined preferred stimulus. Any difference in response to ascending and descending stimuli thus reflects prior knowledge facilitating visual recognition in noise. Expanding on our previous work, subjects were presented RISE stimuli in an MRI scanner. Recent machine-learning techniques allowed us to classify the BOLD images (predicting the current visual stimulus) while subjects performed the same recognition task in the scanner. Classification was better for degraded images when subjects had prior knowledge of the coherent stimuli. We show that this hysteresis in machine classification matches the subject’s behavior, indicating more information in the brain with prior knowledge of the stimulus. Furthermore, we can localize this information by showing a graded hysteresis response from V1 through prefrontal cortex, suggesting that prior knowledge affects lower and higher visual areas in different ways. We also compare the location of hysteresis with respect to the kinds of stimuli used, to elucidate what types of image features are facilitated with prior knowledge and which are strictly processed in a feed-forward manner.

Horng, S., Blank, M., Kreiman, G., Millen, K. and Sur, M.
Characterization of Zic1 and Zic4 expression and potential downstream targets in the developing murine visual system
Soc. Neurosci., 2007.

During development of the vertebrate forebrain, regional patterns of gene expression contribute to the functional differentiation of parallel sensory pathways. In a microarray screen comparing visual and auditory regions of the perinatal murine thalamus, two genes in the Zic family of transcription factors, Zic1 and Zic4, were identified as strongly enriched in the lateral geniculate nucleus (LGN), a relay and processing target for incoming retinal afferents. These results were confirmed with RT-PCR and in situ hybridization. In the mouse, Zic1 and Zic4 have been shown to be required for proper cerebellar and dorsal spinal cord morphology, while other members of the Zic family, Zic2 and Zic3, have been implicated in the axonal pathfinding of retinal ganglion cells. In order to correlate Zic1 and Zic4 to various time points in visual pathway development, we characterized Zic1 and Zic4 expression in the developing retina and thalamus at different ages. Both Zic1 and Zic4 are expressed in a nasal/low to temporal/high gradient in the P0 retina and in a ventral-lateral/low to dorsal-medial/high gradient in the LGN from E15.5 through P5. Zic4 expression in the LGN is absent by P16, while Zic1 expression decreases by P28 and persists at a low level into adulthood. To elucidate a possible role in functional differentiation of the LGN, we examined cell-type specificity of Zic1 and Zic4 expressing cells using glial, differentiated neuronal, and excitatory and inhibitory cell markers. In order to examine potential downstream pathways of Zic1 and Zic4 regulation, we performed a transcription factor binding site analysis probing for consensus Zic1 DNA binding sites among promoter regions of putative target genes. This analysis identified several Eph receptor genes as potential targets of Zic regulation. In situ hybridizations for these genes will be performed on thalamic sections of control and Zic1, Zic4 and Zic1/4 +/- and -/- mice to test for potential regulation by Zic1 or Zic4. Preliminary experiments using intraocular CTB injections to test for retinogeniculate patterning defects in control and Zic4 knock-out mice showed no gross differences. Further experiments to test the role of Zic1 and Zic4 in the patterning of geniculocortical projections will be pursued.

Leamey, C.A., Merlin, S., Sawatari, A., Dharmaratne, N., Lattouf, P., Nguyen, M., Faessler, R. and M. Sur, M
Effects of Ten_m3 deletion on the visual system: aberrant wiring of ipsilateral retinal projections, an interocular mismatch and visual deficits.
Soc. Neurosci., 2007.

The visual system is characterised by the precise topographic representation of the visual world. This is considered vital for the normal processing of sensory information. We have investigated a role for the transmembrane protein, Ten_m3, in the development of connectivity in the visual pathway. We found that Ten_m3 is expressed in gradients at all levels of the visual pathway, which are consistently highest in regions which represent dorsal visual field. Anterograde tracing from the eye showed that the targeting of ipsilateral retinal axons is aberrant in Ten_m3 knockout (KO) mice. Unlike in wildtype (WT) mice, where ipsilateral terminals are confined to the dorsomedial region of the dorsal lateral geniculate nucleus (dLGN), in Ten_m3 KOs ipsilateral projections are elongated along the dorsomedial to ventrolateral axis of the nucleus. Ipsilateral projections to the superior colliculus (SC) are also more widely distributed than in WTs. Retrograde labeling revealed no change in the number or distribution of ipsilaterally projecting retinal ganglion cells (RGCs). Focal retinal injections showed no change in the topography of contralateral retinal projections, but a major change was observed for the ipsilateral projections. Most notably, 2 distinct terminal zones were present in the ipsilateral dLGN of all KOs examined. Consequently, the projections from the two eyes are not aligned in Ten_m3 KOs. The topography of geniculocortical projections was unchanged, however, suggesting that the interocular mismatch is conveyed to the visual cortex. The impact of this on vision was investigated using behavioural tests. We found that Ten_m3 KOs have pronounced deficits in the performance of visually-mediated behavioural tasks such as the visual–cliff and placement tests. Interestingly, these were reversed by acute monocular inactivation. This suggests that the deficits are a direct consequence of the interocular mismatch, and raises the possibility that the mismatch may mediate a functional suppression. This is being further investigated using c-fos staining and electrophysiology.

Liu J.V., Sharma, J., Moore, C.I. and Sur, M.
Measuring visual field maps and orientation-selective responses in ferret visual cortex with high-resolution functional MRI
Soc. Neurosci., 2007.

Primary visual cortex in ferrets is a model system for studying the plasticity and development of neuronal feature maps, such as maps of retinotopy, orientation and spatial frequency. Previous studies examining the nature of these maps and their relationships have primarily utilized optical imaging of intrinsic signals, and have focused on the superficial layers of V1 (Sharma et al., 2000; Yu et al., 2005). To extend our understanding to the deep layers of V1 and to other cortical areas, we have developed chronic, non-invasive procedures to measure visual feature maps in anesthetized, paralyzed ferrets using high-resolution functional magnetic resonance imaging (fMRI). Central to our measurements is a skull implant that has allowed us to precisely place a small coil (1.0-1.5 cm diameter) to ensure consistent coil position and coverage across imaging sessions. We have made three types of measurements in normal ferrets. First, we measured fMRI responses to gratings (~0.1 cpd, 1 Hz) compared to blank, using a 1-shot spin-echo EPI sequence with 0.5 mm iso-voxel resolution (TE: 30 ms, TR: 2.4 s, 14 slices). Robust responses (>1% amplitude, p <0.01) could be readily observed across the visual cortex within just 3 minutes of imaging time. Next, we measured fMRI responses to a single bar presented at a series of visual locations using the same EPI sequence, and obtained maps of visual elevation and azimuth that were found to be comparable to those from optical imaging. Third, fMRI responses to gratings in a series of orientations (0, 45, 90, 135 degrees) were measured using spin-echo EPI (TE: 30 ms) in 1-shot (TR: 2.4 s, 0.3X0.3X0.6 mm) or 2-shot (TR: 2 s, 0.2X0.25X0.6 mm) with 6 slices tangential to cortical surface. Data averaged from 1 hour of imaging time showed clusters of voxels with weak but reliable orientation-selective responses (0.5% amplitude, p < 0.05). Systematic and detailed investigation of these maps of retinotopy and orientation across the different visual areas in ferret brain are underway. Similar procedures were applied to image visual responses in auditory cortex of “rewired” ferrets (Sharma et al., 2000). Robust visual responses were found in some areas of rewired auditory cortex in response to high contrast grating stimuli.

Mao, R., Schummers, J., Page, D.T., Lee, C.M., Kim, C., Rubenstein, J.L.R. and Sur, M.
Effects of subtype-specific loss of inhibitory interneurons on stimulus-specific responses in visual cortex
Soc. Neurosci., 2007.

Primary visual cortex in ferrets is a model system for studying the plasticity and development of neuronal feature maps, such as maps of retinotopy, orientation and spatial frequency. Previous studies examining the nature of these maps and their relationships have primarily utilized optical imaging of intrinsic signals, and have focused on the superficial layers of V1 (Sharma et al., 2000; Yu et al., 2005). To extend our understanding to the deep layers of V1 and to other cortical areas, we have developed chronic, non-invasive procedures to measure visual feature maps in anesthetized, paralyzed ferrets using high-resolution functional magnetic resonance imaging (fMRI). Central to our measurements is a skull implant that has allowed us to precisely place a small coil (1.0-1.5 cm diameter) to ensure consistent coil position and coverage across imaging sessions. We have made three types of measurements in normal ferrets. First, we measured fMRI responses to gratings (~0.1 cpd, 1 Hz) compared to blank, using a 1-shot spin-echo EPI sequence with 0.5 mm iso-voxel resolution (TE: 30 ms, TR: 2.4 s, 14 slices). Robust responses (>1% amplitude, p <0.01) could be readily observed across the visual cortex within just 3 minutes of imaging time. Next, we measured fMRI responses to a single bar presented at a series of visual locations using the same EPI sequence, and obtained maps of visual elevation and azimuth that were found to be comparable to those from optical imaging. Third, fMRI responses to gratings in a series of orientations (0, 45, 90, 135 degrees) were measured using spin-echo EPI (TE: 30 ms) in 1-shot (TR: 2.4 s, 0.3X0.3X0.6 mm) or 2-shot (TR: 2 s, 0.2X0.25X0.6 mm) with 6 slices tangential to cortical surface. Data averaged from 1 hour of imaging time showed clusters of voxels with weak but reliable orientation-selective responses (0.5% amplitude, p < 0.05). Systematic and detailed investigation of these maps of retinotopy and orientation across the different visual areas in ferret brain are underway. Similar procedures were applied to image visual responses in auditory cortex of “rewired” ferrets (Sharma et al., 2000). Robust visual responses were found in some areas of rewired auditory cortex in response to high contrast grating stimuli.

McCurry, C., Tropea, D., Wang, K.H. and Sur, M.
A role for Arc in constraining ocular dominance plasticity in adult visual cortex
Soc. Neurosci., 2007.

 

Page, D.T., Kuti, O.J., Prestia, C. and Sur, M.
Characterization of heterozygote PTEN knockout mice as a model for gene and environment interactions in autism
Soc. Neurosci., 2007.

Autism spectrum disorders (ASD) are characterized by pervasive social, cognitive, language, motor and sensory processing abnormalities. How genetic and environmental factors interact on the backdrop of normal brain development to bring about these phenotypes is unclear. Cowden syndrome and macrocephaly/autism syndrome are two disorders on the autism spectrum in which affected individuals are heterozygous carriers of mutant alleles for PTEN, a repressor of PI3-kinase signaling. To develop a mouse model to investigate gene interactions underlying ASD, we are characterizing heterozygous Pten knockout mice (Pten+/-) for phenotypes relevant to ASD. At the level of behavior, we have tested these mice in a social approach assay and find that Pten+/- mice spend significantly less time interacting with a stranger mouse than do wild type mice. Furthermore, we find that Pten+/- mice have deficits in prepulse inhibition of the acoustic startle response, a measure of sensorimotor gating that is abnormal in at least some individuals with ASD. We are further characterizing these mice for neuroanatomical and molecular phenotypes relevant to ASD, including cell packing density and morphology in frontal cortex, amygdala, hippocampus and cerebellum, as well as profiling expression of a panel of putative ASD markers at the levels of mRNA and protein. Pten+/- mice provide a sensitized background in which PI3-kinase signaling is perturbed; this pathway is also implicated in the pathology of tuberous sclerosis with autism, thus making it an attractive target for modifier screening. Our results provide the basis for experiments that we are pursuing, examining modification of ASD-related phenotypes in these mice using double heterozygosity with other ASD candidate genes, as well as pharmacological and environmental manipulations.

Schummers J., Yu, H. and Sur, M.
Role of astrocytes in translating neuronal activity to hemodynamic responses in visual cortex.
Soc. Neurosci., 2007.

Astrocytes are a major class of non-neuronal cells in the brain that were long thought to act as a support network for neurons. Recently, evidence has accumulated for a more active role for astrocytes in brain function. Astrocytes have processes that are closely apposed to synapses, and they respond to a number of neurotransmitters, in large part via pathways that lead to the release of internal calcium. In addition, astrocytes contact vascular networks and can influence cerebral microcirculation. We have simultaneously imaged the activity of astrocytes and neurons with two-photon imaging of bulk loaded calcium indicators in primary visual cortex (V1) of the ferret. We have found that astrocytes have robust calcium responses to visual stimuli, which are delayed 2-4 seconds relative to neurons. The responses are sharply tuned for stimulus orientation and spatial frequency. Furthermore, their preferred orientation closely matches nearby neurons, and the organization of preferred orientation at pinwheel centers is equally precise for astroyctes. Astrocyte tuning is narrower than neuronal tuning, suggesting that astrocyte calcium responses have a high threshold for activation. Consistent with this suggestion, we discovered that astrocyte responses are remarkably sensitive to anesthetic (isoflurane) concentration, with a dramatic decrease in amplitude over a narrow range of anesthetic concentration, over which neuronal responses are only minimally reduced. We took advantage of this sensitivity to investigate the role of astrocyte calcium responses in hemodynamic responses. We obtained orientation preference maps with intrinsic signal optical imaging at anesthetic conditions that enabled us to selectively turn astrocyte responses on and off. By comparing responses at multiple wavelengths, we find that the delayed, visually-evoked, increase in blood flow is extremely sensitive to astrocyte activity levels. The blood flow response is reduced threefold when astrocytes calcium responses are reduced by high isoflurane. The early portion of hemodynamic signals is unaffected, suggesting a mechanistic link between the astrocyte calcium responses and blood flow increases. The mapping portion of the intrinsic signal is also dramatically reduced, suggesting that astrocytes are critical for translating local neuronal activity into localized increases in blood flow. The combination of highly organized response selectivity of astrocytes, and their role in regulating blood flow, likely contributes to the spatial localization of intrinsic signal responses.

Tropea, D., Majewska, A. and Sur, M.
The structural basis of functional plasticity: correlation analysis using in vivo imaging in mouse V1.
Soc. Neurosci., 2007.

The visual cortex has been used extensively as a model for the study of activity-dependent plasticity. Changes in the visual input are stimulus-dependent and result in functional reorganization at the cortical level. Alongside functional changes, the structure of cortical neurons is altered in animals subjected to different levels of visual drive. This structural rearrangement can be observed at the synaptic level in the structure of dendritic spines, which are subcellular structures that constitute the postsynaptic sites of most excitatory synapses in the CNS. Changes in spine density, morphology and motility have been described in visual cortex during normal development and with sensory deprivation.
In this study we examined the correlation between structural and functional changes in visual cortex in animals that have been deprived of sensory input from birth (dark rearing-DR) and that have been re-exposed to a normal light environment for different periods.
We addressed this issue in mice 28 days after birth (P28); we used time-lapse two-photon laser scanning microscopy to assay motility of spines on V1 neurons in vivo, in mice expressing GFP in a subset of cortical layer 5 neurons; for the measurement of cortical activity, we used optical imaging of intrinsic signals.
We found that dark rearing caused a significant increase in spine motility (about 20%) with respect to age matched controls; moreover the dark reared animals showed a significant increase (p-value <0.05 t-test) in thin spines (from 11% to 21%) and a decrease in stubby spines with respect to control (from 26% to 15%). The evoked cortical activity was four times lower in dark reared animals compared to controls and the organization of the retinotopic map was poor in absence of visual drive. Re-exposure to light for 2 days did not show any change in the motility of the whole population or in the magnitude of the visual evoked response, although the number of stubby spines returned to control levels and the retinotopic map was substantially improved. Only after 7 days of light re-exposure the structural as well as the functional measurements were comparable to those in control animals.
These results suggest that in visual cortex structural and functional plasticity are dependent on sensory experience and changes in spine dynamics and cortical activity have a consistent time course.

Van Wart, A., Tropea, D. and Sur, M
Enhanced JAK/STAT1 signaling restricts ocular dominance plasticity in visual cortex.
Soc. Neurosci., 2007.

The primary visual cortex (V1) is exquisitely sensitive to changes in activity during a critical period of early development. In mice, suturing the lids of one eye (monocular deprivation, MD) during this time shifts the strength of cortical responses in the binocular zone in favor of the open eye. In an effort to uncover the molecular mechanisms underlying this ocular dominance plasticity, previous DNA microarray studies were performed examining gene expression changes after MD (Tropea et al., Nature Neurosci., 2006). Interestingly, these studies identified the Janus kinase/Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway as being significantly upregulated in V1 in response to MD. While studied mainly for its role in cell-mediated immunity, JAK/STAT1 signaling also functions in the nervous system, modifying gene expression in response to cytokines and growth factors during development, nerve injury, and disease. STAT1 is also a prime locus for modulating plastic changes during MD, as it is a point of convergence for multiple signaling pathways involved in neuronal plasticity.
In order to confirm JAK/STAT upregulation at the protein level, we examined cortical STAT1 expression using slice immunohistochemistry and western blot analysis. We found that MD increases immunostaining for activated STAT1 in diverse cortical cell types contralateral to the deprived eye. We also observed increased STAT1 levels in cortical protein extracts after 7 days of MD, an effect that could be mimicked by 7 days systemic administration of the STAT1-specific agonist interferon-gamma (IFN). In order to understand the effects of JAK/STAT1 modulation during MD, STAT1 signaling was enhanced via daily administration of IFN, in mice with or without concurrent critical period MD. Changes in ocular dominance were measured using intrinsic signal optical imaging. Strikingly, IFN treatment during MD prevented the shift in cortical responses toward the nondeprived eye, and had no significant effect on ocular dominance in nondeprived mice. Furthermore, in addition to reducing functional plasticity, IFN treatment also minimized deprivation-induced changes in spine morphology, measured from layer 5 pyramidal cell apical dendrites located in layers 2/3. Contrary to untreated mice receiving 7 days of MD, IFN-treated mice exhibited no significant decrease in the proportion of stubby spines, nor an increase in filopodia after 7 days of MD. Altogether, these data point to a homeostatic role for JAK/STAT signaling during MD, counterbalancing the functional and structural changes that take place during sensory deprivation.

Supported by: EY015068 and EY017098 and 1F32EYO17240 (DT)

Yu, H., Schummers, J., and Sur, M.
The relationship of multiple stimulus features in ferret visual cortex measured by two-photon calcium imaging.
Soc. Neurosci., 2007.

The primary visual cortex (V1) in highly visual mammals is organized into two-dimensional maps of stimulus features including orientation, spatial frequency and visual space. Intrinsic signal optical imaging, which defines maps at a scale of ca 100 um resolution, has revealed an inverse-gradient relationship between certain sets of maps: at regions where the orientation map changes rapidly, spatial frequency and ocular dominance maps change relatively slowly. We have now probed a crucial hypothesis underlying map relationships, that individual cells that are each tuned to multiple features show subtle variations in response tuning on a cell-by-cell basis, consistent with the inverse-gradient principle of multiple maps. Furthermore, we have examined whether additional relationships, such as between the maps of orientation and visual space, exist at a scale below the resolution of intrinsic signal measurements. We used bulk loading of fluorescent calcium indicators and two-photon imaging in ferret V1 in vivo to measure multiple tuning properties of individual, spatially identified, adjacent neurons in the same patch of cortex. By combining calcium imaging with intrinsic signal optical imaging, we targeted injections of calcium indicator specifically to orientation pinwheel centers, as well as to specific locations within spatial frequency maps. We find that neurons with the same preferred spatial frequency cluster in a columnar way, and different spatial frequency columns separated by less than 100 um can be clearly distinguished. Furthermore, the orientation and spatial frequency maps measured at single cell resolution by calcium imaging match closely the maps obtained by intrinsic signal imaging. At orientation pinwheels, where the preferred orientation of neurons changes rapidly, preferred spatial frequencies of the same group of neurons are very similar and show very little scatter. At spatial frequency column borders, where preferred spatial frequencies change rapidly, preferred orientations change very little. Thus, the inverse-gradient relationship between orientation and spatial frequency representations indeed exists at the level of single cell responses. Retinotopic mapping and receptive field progression is evident across cells spread over 100 um of cortical distance. Receptive field centers have small but significant jumps near pinwheel centers, yet at locations distinct from them. Such sites are seen consistently at multiple depths of superficial visual cortex. Thus, the retinotopic map also appears to have a fine-scale relationship with the orientation map.

2006

Cronin, B., Schummers, J., Kording, K. and Sur, M.
Bayesian sampling methods for the analysis of reverse correlation data.
Soc. Neurosci., 2006.

Reverse correlation paradigm can be used to examine the fine temporal structure of the stimulus-evoked response of neurons in visual cortex. In primary visual cortex, key scientific questions include whether tuning properties such as preferred orientation and tuning width change over the course of the response, whether the response is better characterized by a traditional Gaussian tuning curve or as a difference of two Gaussians (Mexican hat), and whether any of these properties covary with the neuron’s location in the orientation map (i.e., pinwheel vs. domain). It has proven challenging to draw definitive conclusions from such data, however, because of the difficulty inherent in evaluating the confidence associated with the relevant statistical summaries. In order to address such difficulties, we have employed the framework of Bayesian statistics, including the specification of a generative model which provides a formal, stochastic description of the process by which the data is generated. We then used Markov Chain Monte Carlo sampling methods to infer the joint posterior distribution of the model parameters. Finally, we applied the results of these sampling procedures to directly address all of the questions outlined above. Here we discuss both the application of the Bayesian statistical framework, and the results we obtain with it on data obtained using the reverse correlation paradigm in the primary visual cortex of the cat. We describe results both from individual neurons, and aggregate results from the population as a whole.

Farley, B.J.,Yu, H., Jin, D.Z. and Sur, M.
Experimental effects of removing the ocular dominance map on the organization of the remaining feature maps in visual cortex.
Soc. Neurosci., 2006.

In visual cortex, receptive field properties change in an orderly way across the tangential dimension, forming multiple overlapping maps of retinotopy, orientation, ocular dominance, and other features. The mechanisms underlying the development of these feature maps are poorly understood. Some computational models can successfully reproduce the structures of multiple feature maps and the characteristic spatial relationships between them; these models suggest that the individual maps develop inter-dependently, in a way that balances smooth mapping of each feature and equal representation of each feature combination. The models predict that changing the structure of (or removing) one of the feature maps early in development should alter the structures of the remaining maps. To test this prediction, we monocularly enucleated ferrets on the day of birth. Anatomically, this manipulation had previously been shown to cause the remaining eye to project uniformly throughout the LGN, rather than to eye-specific patches. Functionally, we find that monocular stimulation in monocularly enucleated animals activates the cortex more uniformly than monocular stimulation in normal animals, and as uniformly as binocular stimulation in normal animals. This indicates that the cortical ocular dominance map is functionally removed following neonatal monocular enucleation. Despite the complete loss of input from one eye, cortical maps of orientation and spatial frequency develop both contralaterally and ipsilaterally to the remaining eye, and their structures are largely unperturbed. Further, their interrelations remain: regions where preferred orientation changes rapidly across the cortex coincide with regions where spatial frequency changes slowly, and vice versa. The results indicate that characteristics of the orientation and spatial frequency maps develop independently of the pattern of ocular dominance, and thus inform theories of feature map development.

Gorlin, S., Sharma, J., Sugihara, H., Jarosiewicz, B., Sur, M. and Sinha, P.
Finding Signals in Noise: The Neural Advantage of Prior Information.
Soc. Neurosci., 2006.

One of the less understood aspects of mammalian vision is the ability to recognize scenes through significant degradations in image quality. Neuronal receptive fields have traditionally been described with coherent structures – for example, oriented gratings in V1. However, this does not address how neurons respond to more noisy, less coherent visual input, which is arguably more prevalent in the natural world. Previous studies with natural images have shown that recognition is highly non-linear with respect to noise, and more importantly, that recognition in noise is facilitated by prior knowledge or experience with the stimuli (Sadr and Sinha, 2004). We extend these studies by using RISE sequences (Random Image Structure Evolution) to present oriented gratings evolving from noise, both in behavioral assays in humans, and in simultaneous multi-electrode recordings in Macaque V1 and V4. Specifically, the direction of RISE evolution ascending or descending in information content allows us to control for low-level image properties, such as luminance, while trending toward or away from a neurons classically defined preferred stimulus. By recording simultaneously in discrete regions, we show the differential response to noise throughout the visual processing hierarchy. In line with previous behavioral studies using high-order stimuli, we find that prior knowledge of a stimulus, as in the descending paradigm, facilitates recognition in noise. We also present evidence for hysteretic neuronal facilitation in low-level visual areas, which can account for this improvement in performance.

Supported by EY07023 to MS and the John Merck Scholars Award to PS

Horng, S., Kreiman, G., Ellsworth, C. and Sur, M.
Analysis of Functional Gene Sets in the Neonatal Murine Lateral Geniculate Nucleus and Medial Geniculate Nucleus.
Soc. Neurosci., 2006.

Regions of the developing murine thalamus exhibit characteristic patterns of gene expression. These patterns are believed to regulate the differentiation of thalamic cells into functional nuclei processing channels of modality specific information. From late embryogenesis (E15) to P5, the lateral geniculate nucleus (LGN) and medial geniculate nucleus (MGN) acquire sensory afferents from the retina and inferior colliculus (IC), respectively. The specificity of this targeting, as well as the acquisition of sensory specific processing features, likely depend on unique patterns of gene expression. To discover gene pathways involved in these processes, we performed a differential gene expression screen on the LGN and MGN of P0 mice using Affymetrix u74v2 microarrays (4 replicates/group). Enriched sets of single genes (n=13 for MGN; n=21 for LGN) were isolated using Significance Analysis of Microarrays (SAM) software (FPR<0.01%). Differential gene expression of 13/13 genes from the MGN set and 20/21 genes from the LGN set were confirmed using relative quantitative real-time PCR. In situ hybridization further confirmed that transcript expression of selected genes from both the MGN and LGN enriched sets was nuclei specific. To analyze the coordinated regulation of genes in each thalamic nucleus, enriched gene clusters involved in functional pathways were assembled using Gene Set Enrichment Analysis (GSEA). 20 gene sets of 199/487 enriched LGN gene sets were significant (p<0.05) and included Wnt signaling and cell adhesion pathways. 17 gene sets of 288/487 enriched MGN gene sets were significant (p<0.05, FDR< 25%) and included rho, integrin and cell adhesion pathways. Analysis of co-regulatory transcription factor elements with both single gene and gene cluster sets is in progress. The analysis of LGN and MGN specific gene sets from the neonatal mouse will allow us to generate functional hypotheses on the processes by which these two regions acquire modality specific inputs and visual or auditory processing features.

Liu, J.V., Sharma, J., Moore, C.I. and Sur, M.
Visually driven responses in ferret measured with high-resolution functional MRI at 9.4T.
Soc. Neurosci., 2006.

We developed non-invasive procedures to repetitively image individual ferrets using high-field (9.4 T) functional magnetic resonance imaging (fMRI). Ferrets were initially anesthetized with ketamine and xylazine (IM), and later maintained with isoflurane (0.8-1.5% in 1:1 N2O:O2) delivered through a cuffed endotracheal tube. They were artificially respired to maintain end-tidal CO2 at around 4%, and immobilized in a custom stereotaxic frame constructed to fit the scanner bore internal diameter (12 cm). FMRI responses were acquired using a single-shot spin-echo EPI sequence (TE: 30 ms, TR: 1.2 or 2.4 s, matrix: 56×40 or smaller) in 14 coronal slices with 0.6 mm iso-voxel resolution. Anatomical images were acquired with matched TE, TR and geometry. Visual stimuli were presented binocularly on a screen (9 cm diameter) placed at a distance of 12 cm. Stimuli in each run comprised 6 cycles; each cycle included an “On” period of drifting gratings (0.05 or 0.15 cpd, 1 Hz), followed by an “Off” period of a black field. At 50% duty cycle (“On”=”Off”=12 or 18 s), robust responses to “On” period (> 1% amplitude, p < 0.05) could be readily observed within a short data averaging period (< 20 min). Visually responsive regions were identified based on anatomical images with strong responses obtained in primary visual cortex. At low duty cycle (“On”=2 s, “Off”=34 s), time courses of fMRI responses in these regions were equivalent to hemodynamic response functions. These time courses showed no significant “initial dip”, reached quickly to maximum responses (at ~4 s), then reached a significant post-stimulus undershoot (at ~9 s) before returning to baseline, in the 2 ferrets studied so far. Such rapid bi-phasic hemodynamic components were reflected in the 50% duty cycle data as well. In summary, visually driven responses in ferrets can be readily measured with spin-echo fMRI at 0.6 mm resolution. The non-invasive data acquisition and three-dimensional spatial coverage of fMRI facilitate studies of neural plasticity and development using ferret as model system. Specifically, we plan to extend our studies to adult and developing “rewired” ferrets whose retinal inputs are surgically re-routed into auditory thalamo-cortical pathway at birth, such that auditory cortices of these ferrets are driven by visual rather than auditory inputs (Sharma et al., 2000; Sur et al., 2005).

Page, D. and Sur, M
A screen for autism candidate gene homologues capable of influencing morphogenesis in the developing mouse neocortex.
Soc. Neurosci., 2006.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with a strong genetic component, characterized by cognitive, social, language and motor impairments. Neuropathology studies have implicated diverse brain regions as structurally abnormal in individuals with ASD, and several susceptibility genes have been identified. However, the link between genotype and phenotype in this disorder is obscure. We hypothesize that we can uncover, from the phenotype-up, common genetic pathways relevant to ASD by identifying candidate genes that are capable of influencing specific cellular processes potentially related to the neuropathology of ASD, such as growth, cell migration and neurite morphogenesis. To this end, we have carried out a dominant gain-of-function misexpression screen using the technique of in utero electroporation in embryonic mouse. Our aim is to identify genes capable of influencing these processes when misexpressed in precursors of layer II/III pyramidal cells in anterior neocortex. We have screened through over 20 positional and functional candidate genes for ASD, focusing on homologues of genes located at or near an ASD susceptibility locus on human chromosome 17, and report here results for two genes previously uncharacterized regarding function in neocortical development. These include genes encode a transcription factor capable of altering the number and migration of layer II/III cells, and a serine/threonine kinase that can alter the dendritic orientation of these cells. A general trend amongst the ASD candidate genes screened is that misexpression alters parameters of cortical growth. We are currently characterizing the endogenous expression patterns of these genes in detail and further investigating mechanisms of action by knocking down expression levels in the developing brain using siRNA, as well as probing interactions with other ASD candidate genes. The genes isolated in this screen represent focused candidates for further studies of brain development, and for polymorphism screening in human ASD gene-finding studies. Furthermore, this screening approach can be applied to investigating the functions of candidate genes at other loci for ASD and neurodevelopmental disorders such as Williams Syndrome and schizophrenia.

Schummers J., Yu, H. and Sur, M.
Single-cell resolution mapping of multiple stimulus features in ferret visual cortex with two-photon calcium imaging.
Soc. Neurosci., 2006.

The primary visual cortex in highly visual mammals is organized into two-dimensional maps of stimulus features including orientation, visual space, spatial frequency and direction of motion. Recent advances in cell labeling and imaging methodologies have enabled single-cell resolution imaging of the activity of large populations of superficial cortical neurons in response to sensory stimulation. We have applied bulk loading of fluorescent calcium indicators and two-photon imaging in the ferret visual cortex in vivo to measure the tuning properties of populations of neurons in small patches of cortex. We have succeeded in mapping the tuning of responses to orientation, direction, retinotopic space and spatial frequency. By combining calcium imaging with intrinsic signal optical imaging, we have been able to target injections of calcium indicator to orientation domains, pinwheel centers, orientation fractures, and direction fractures. We find that the boundaries of columns representing different stimulus features determined with calcium imaging match those obtained with intrinsic signal imaging very closely. Furthermore, the boundaries of columns representing stimulus orientation, direction and retinotopy are fairly precise, with relatively little scatter on a cell by cell basis. However, there is considerable scatter in the representation of preferred spatial frequency.
The neuropil surrounding cell bodies is also strongly labeled, and shows robust visual responses. The tuning of the neuropil is generally similar to that of nearby neurons; however, near pinwheel centers, the neuropil is much more broadly tuned for orientation than neuronal tuning. Given that the neuropil response is likely dominated by the pooled pre-synaptic activity, this result is consistent with the broad tuning of synaptic inputs to pinwheel centers. We have also examined the spatial arrangement of the mapping of retinotopy relative to the orientation preference map. We do not find large jumps in the mapping of retinotopic space near pinwheel centers; the scatter of receptive field position is much smaller than the average receptive field size, suggesting no positive correlation between the gradient of receptive field and orientation near pinwheels.

Sharma, J., Sugihara, H., Schummers, J. and Sur, M.
Distinct modes of visuo-spatial attention in macaque V1 and V4.
Soc. Neurosci., 2006.

Psychophysical studies suggest that spatially directed attention mediated by both top-down and bottom-up processes can improve perceptual sensitivity to an attended location. While the top-down control is goal-directed and deployed voluntarily, the bottom-up component is stimulus driven. The stimulus driven component should therefore engage neurons in primary visual cortex (V1) at the onset of attentional process whereas the voluntary, top-down component should initially engage V4 and higher areas. However, most physiological studies find robust modulation with attention in V4 neurons and only a weak, delayed modulation of V1 neurons, that is taken as the signature of top-down mediation of attention. In an ongoing series of experiments, we have examined spatial and temporal dynamics of attentional modulation in V1 and V4 by using a reverse-correlation technique. This approach, employed in simultaneous recordings of V1 and V4 neurons, has helped us study attentional dynamics on a millisecond timescale. Two monkeys were trained to covertly attend to a small attention spot that could appear near the receptive field (RF) of a neuron or away from it in the contralateral hemifield. The spot could extinguish after a random interval following which the monkey had to release a bar within 350-400 ms to earn a juice reward. The stimuli, consisting of patches of sinusoidal gratings at 8 different orientations, two different contrasts and two sizes, were flashed at 75 Hz. Identical stimuli were presented overlapping the RF and in the contralateral location, while we recorded from neurons in V1 and V4. We find that attention towards the receptive field shows distinct modes of response modulation in V1 and V4. Whereas V4 neurons show strong modulation early on when the animal allocates covert attention towards the RF, no such modulation was seen in V1. However, V1 neurons show clear modulation when attention is required to achieve a behavioral goal that culminates in a motor response. Neurons in V4 also show similar modulation, but this modulation follows V1 responses in time. Furthermore, attention delays contextual influences in V4 but does not influence their magnitude. In V1, attention accentuates contextual influences: a suppressive surround becomes more suppressive whereas surround facilitation gets more facilitated with attention.

Tropea, D., Kreiman, G. and Sur, M.
Role of Insulin-like Growth Factor 1 in activity-dependent plasticity in visual cortex.
Soc. Neurosci., 2006.

Alteration of visual activity during specific periods of postnatal development causes adaptive changes in the organization of visual cortical circuitry; in particular, suturing the eyelids of one eye (monocular deprivation, MD) increases the proportion of neurons that respond to the open eye in the binocular portion of primary visual cortex (V1). DNA microarray studies in mouse V1 examining changes in gene expression after MD, combined with computational analyses, have pointed at Insulin-like growth factor 1 (IGF1) as an important modulator of activity-dependent plasticity (Tropea et al., Nature Neurosci., 2006). IGF1 is a growth factor involved in growth and maintenance of tissues and has recently been shown to have a role in brain development and repair. Its action is modulated by IGF binding proteins (IGFBPs) that prevent activation of the IGF1 signaling pathway. We have shown that the expression of IGFBP5 is increased after MD, both at the RNA and protein level. Furthermore, signals downstream of IGF1, including PI3Kinase and p-Akt, are downregulated after MD. By using optical imaging of intrinsic signals, we have shown that exogenous application of IGF1 prevents the shift in ocular dominance following MD. In order to understand the mechanisms of action of IGF1, we have asked how RNA and protein levels of signaling pathways are altered by addition of IGF1 concurrent with MD. We did a microarray analysis comparing mRNA from the visual cortex of three groups of mice at P28: control (non-deprived); monocularly deprived (MD, P24-P28); and, monocularly deprived treated with IGF1 (MD-IGF1, P24-P28). RNA samples were run on the 430.2 microarray chip set (Affymetrix) covering 45000 cDNA target sequences. The effects of IGF1 were determined by comparing gene expression levels in animals treated with IGF1 with the expression levels in MD animals: 439 genes were down-regulated in MD versus MD-IGF1 animals, whereas 355 genes were up-regulated in MD versus MD-IGF1 animals (criterion level p<0.05, student’s t-test). PI3Kinase and p-Akt expression were both restored after IGF1 application, whereas levels of IGFBP5 were downregulated. Interestingly, IGF1 treatment led to a several-fold increase in the RNA expression of Brain Derived Neurotrophic Factor (BDNF), suggesting that IGF1 may exert its role by activating the expression of this neurotrophin.

van der Vlies, M., Konya, D., Mower, A.F., Saigal, R., Yu, D., George, P.M., Li, J., Sidman, R.L., Sur, M. and Teng, Y.D.
Extracellular matrix remodeling in the injured spinal cord: a novel mechanism partially repairing mature CNS.
Soc. Neurosci., 2006.

Recent research on neurotrauma and neuroregeneration points to up-regulation of developmental molecular components in the post-insult adult CNS, and suggests that rejuvenation of a microenvironment permissive for neuroplasticity may be an effective strategy for functional repair. We previously reported that critical period plasticity in visual cortical synaptic organization can be modified via remodeling of extracellular matrix (ECM) constituents by the tPA/plasmin proteolytic cascade (Oray, Majewska & Sur: Neuron 2004, 44:1021). Here we propose that a similar mechanism may be critical in the recovery process after spinal cord injury. To test our hypothesis, ECM remodeling in the post-lesion spinal cord was activated by local delivery of tPA into the cord parenchyma adjacent to the injury epicenter 3 d after contusion. This treatment elevated immunoreactivity levels of neurofilament-M and GAP-43 markers associated with neurites and their outgrowth/sprouting in the remaining cord. We also found that compared to control vehicle microinfusion, tPA treatment significantly increased the presence scale of synapsin, a protein which binds synaptic vesicles to regulate neurotransmitter release. Importantly, the number of descending serotonin-immunoreactive axons, mediating primary motofunction, was markedly higher in the tPA-treated spinal cord compared with controls. Also, tPA-increased corticospinal tract sprouting/regeneration was prominent. Thus, neuroregeneration and tissue repair in the post-developmental CNS can be facilitated by elevating levels of ECM remodeling factors that promote neuroplasticity.

Wimmer, K., Stimberg, M., Martin, R., Schummers, J., Sur, M. and Obermayer, K.
Temporal response dynamics of orientation tuned neurons in a network model of V1.
Soc. Neurosci., 2006.

The time course of orientation tuned responses in primary visual cortex (V1) can provide insight into the circuitry underlying tuning. While several models of orientation tuning in V1 have been proposed, most of the research focused on the steady-state behavior of the simulated networks. Here, we characterize the temporal response dynamics of a large-scale network using forward- and reverse-correlation analysis of the responses to time-varying stimuli. The network is composed of more than 20000 excitatory and inhibitory Hodgkin-Huxley neurons whose orientation preferences depend on the location of the neurons in an artificial orientation map. Varying both the strength of the excitatory and inhibitory recurrent connections, we assess implications for the response dynamics. Our findings confirm previous results regarding the likely operating regime of V1 [1]: The model operates in a balanced regime in which recurrent excitation and inhibition together with feedforward input lead to sharp spike tuning irrespective of the position of a neuron in the orientation preference map. Interestingly, this map-location invariance also holds for the mean temporal response. The variance of responses may, however, correlate with differences in properties between orientation domains and pin-wheel centers [1], as observed in recent experimental findings regarding the temporal response dynamics of V1 neurons. [1] Mariño, J. et al., Nat Neurosci 8, 194ff (2005).

Wilson, N.R., Shi, B., Daitch, A.L. and Sur, M.
A Windows-programmable multi-channel stimulator for generating patterned input and inducing plasticity in cortical networks in vitro.
Soc. Neurosci., 2006.

The response properties of synapses, neurons and maps in cortical networks are believed to be specified at least in part by patterned activity from their inputs. To understand the mechanisms by which activity patterns are transduced into connectivity changes, we sought the ability to deliver programmable stimulation patterns across multiple sites of a network of cortical neurons cultured on a multi-electrode array (MEA). Such tools have recently been developed commercially and by research groups in the form of multi-channel stimulators, and we aimed to develop a version that made use of standard, readily available parts, and that was compatible with the most ubiquitous interface (USB), operating system (Windows) and programming environment (Matlab) to facilitate customization by researchers. We present a design that allows waveforms to be arbitrarily programmed in Matlab and then delivered to 64 channels in user-defined patterns, either in disparate sequences or in tandem. The timing of delivery is controlled by hardware and is not subject to operating system delays, and the “plug and play” nature of the system makes the interface apparent to the user only for the customization of stimuli. Most importantly, this system allows patterned stimulation to be arbitrarily integrated with other computer-controlled modules, such as patch clamp recording, to enable ‘feedback loops” between stimulation and recording. We present one application of this system in which patterned stimulation is used to drive multiple inputs converging on a single neuron, and the concurrent measurement of the strength of these inputs enables a computer-assisted search for patterns of activity that result in heterosynaptic changes of the cell’s response properties.

Yu, H., Schummers, J. and Sur, M.
Orientation tuning of visually-evoked increases in calcium concentration in astrocytes in visual cortex of the ferret.
Soc. Neurosci., 2006.

Two-photon imaging allows cellular level imaging in the intact brain in vivo. In combination with cellular loading with fluorescent calcium indicators, neural activity can be simultaneously monitored in large populations of neurons as a consequence of the tight correlation between firing rate and somatic calcium influx in neurons. We have employed bulk loading of Oregon Green Bapta (OGB) to image the spiking activity of superficial neurons in ferret visual cortex in response to visual stimuli. We have targeted injections to pinwheel centers and orientation domains based on orientation maps obtained with intrinsic signal optical imaging. This method labels both neurons and astrocytes, which were identified by loading with sulfarhodamine101 (SR101). As has been previously reported, we find strong stimulus evoked fluorescence changes in neurons. Surprisingly, we also find that astrocytes labeled with OGB show visually evoked changes in calcium concentration. The responses in astrocytes are delayed approximately 3 seconds relative to the stimulus onset (and the nearby neural response). Although the exact mechanism responsible for the calcium response is unknown, it is likely that astrocytes are responding to glutamate release from local axon boutons. Furthermore, the responses are sharply tuned for orientation, and have confined spatial receptive fields. The preferred orientation and receptive field location are well matched to those of the nearby neurons. This is particularly striking near pinwheel centers, where astrocytes separated by only 50 um can have sharp tuning for orthogonal orientations. This suggests that astrocytes sample neural activity over a well defined region, confined by the orientation map structure. Furthermore, their activation may serve to link neuronal responses and population map obtained with intrinsic signal imaging.

2005

Cronin, B., Wimmer, K., Sharma, J., Schummers, J., Obermayer, K. and Sur, M.
Attention modulates the discriminability and information content of orientation response in macaque primary visual cortex.
Soc. Neurosci., 2005.

 

Horng, S., Ellsworth, C., Kreiman, G. and Sur, M.
Differential gene expression in the developing lateral geniculate nucleus and medial geniculate nucleus.
Soc. Neurosci., 2005.

The thalamus serves as the principal relay, as well as processing, station for nearly all incoming sensory information to the cortex. During development, the diencephalon gives rise to dorsal and ventral subdivisions of the thalamus, which further parcellate into functional nuclei within sensory-specific pathways. These nuclei include the lateral geniculate nucleus (LGN) and medial geniculate nucleus (MGN), well-studied targets of visual and auditory afferents, respectively. As in the cortex, the molecular mechanisms of thalamic arealization are not well understood. Experiments in which visual afferents are rerouted to a structurally intact MGN after auditory deafferentation suggest that intrinsic factors maintain the structural integrity of thalamic nuclei, while extrinsic factors potentially modulate functional connectivity. In order to discover candidate molecules for intrinsic thalamic patterning, we used Affymetrix u74v2 microarrays to catalog differential gene expression between the LGN and MGN of mouse neonatal (P0) thalamus. From a screen of 34,600 probe sets, only sixty-four probes showed differential expression levels (p<0.05) and a fold-change >2, corresponding to 22 genes more highly expressed in the LGN, and 19 genes in the MGN. SQ-PCR confirmed the expression data of selected genes and in situ hybridization characterized expression anatomically. We report a group of novel genes, including 4 members of the Zic family of transcription factors, that are significantly enriched in the neonatal LGN. For the first time, we identify a group of MGN-enriched molecules, including the strong histologic markers, Foxp2, Crabp2, and Ssbp2. Finally, using a computational analysis that searches for common cis-regulatory elements among co-expressed genes, we propose a regulatory pathway for functional development within the LGN. This pathway involves two parallel streams regulated by Pax6 and Lhx1 and provides a potential conceptual framework for understanding the differentiation of the dorsal thalamus.

Majewska, A., Newton, J.R. and Sur, M.
Stability of dendritic spines and axon terminals in diverse sensory regions of adult mouse cortex.
Soc. Neurosci., 2005.

Cortical synapses consist of postsynaptic dendritic spines and presynaptic axon terminals. Although plastic changes are thought to occur in adult cortex, it remains unclear whether these changes require remodeling of cortical circuitry whereby synapses are formed and eliminated, or whether they rely on changes in synaptic strength of existing synapses. In order to determine the structural stability of synapses in vivo, several groups have used chronic in vivo imaging to monitor synaptic structure over time. These experiments have yielded contradictory results, and it remains unclear to what extent dendritic spines turn over in the adult cortex. Additionally, the stability of the presynaptic partner has not been explored. Here, we report that dendritic spines on the apical tuft of layer 5 cortical neurons are remarkably stable (>80%; n=2644; 26 animals) even in young adult animals (P40) over a period of 1 to 3 weeks. At these ages spine loss (13%) is more prominent than spine gain (5%). Over a one week period, spines in different sensory cortical areas (primary visual, somatosensory, and auditory cortex) had similar rates of turnover. After 3 weeks of observation, most spines (70-80%) could still be identified in all areas studied. Spines in auditory cortex, however, appeared more stable than those in visual or somatosensory cortices (p<0.05). Interestingly, rewiring visual input into auditory cortex did not alter this increased stability, suggesting that intrinsic cues may determine the level of spine turnover in different cortical areas. Axon terminals were equally stable (>90%; n=2137; 26 animals) in all cortical areas studied and showed less turnover than dendritic spines (p<0.01), with matched levels of terminal loss (~5%) and gain (~5%). These data suggest that limited remodeling of connectivity occurs in the adult cortex, and that this remodeling is importantly mediated by turnover of the postsynaptic partner.

Mower, A.F., Kwok, S.M., Majewska, A., Yu, H., Okamoto, K., Hayashi, Y. and Sur, M.
In Vivo imaging of CaMKII activity in the primary visual cortex of ferrets using FRET.
Soc. Neurosci., 2005.

Most excitatory synapses in the brain are made on small dendritic protrusions called spines. Spines are motile structures that undergo activity-dependent morphological plasticity involving Ca +2 influx through glutamate receptors and voltage-gated Ca +2 channels. Rapid structural changes at the level of spines have been shown to occur following brief periods of monocular deprivation (MD). At the molecular level, this activity-dependent plasticity requires the transformation of synaptic depolarization into changes in synaptic weight. CaMKIIa and ß are known to play a role in this transformation through their interaction with the actin cytoskeleton. Autophosphorylation of CaMKIIa is also known to be required for ocular dominance (OD) plasticity. To examine activity dependent changes in CaMKIIa and ß activation in the primary visual cortex of ferrets in vivo, we performed intracortical injections of engineered FRET probes, Camuia and ß, packaged into herpes simplex virus. An optical window was then implanted to allow chronic 2-photon microscopic imaging and optical imaging of intrinsic signals for the derivation of OD maps. HSV-Camuia and ß specifically allow for the detection of calmodulin binding and autophosphorylation at threonine 286(a)/287(ß), which renders the enzyme constitutively active. Chronic imaging in normal animals, and following MD, showed robust expression of HSV-Camuia and ß in both dendrites and spines. This expression lasted for several days and allowed for high resolution imaging as indicated by the clear visibility of individual spine structure. Chronic in vivo imaging of HSV-Camuia and ß showed little fluctuation in FRET signal over time in normal animals. These experiments demonstrate the feasibility of utilizing FRET for studying CaMKII activition during OD plasticity in vivo .

Sharma, J., Schummers, J., Hosseini, P. and Sur, M.
Attention spans multiple stimulus dimensions in macaque visual cortex.
Soc. Neurosci., 2005.

Visuo-spatial attention involves a two way process of enhancing perception of the attended stimulus while inhibiting non-attended stimuli. Attentional modulations have been likened to contrast gain mechanisms that serve to bias competitive interactions between multiple stimuli towards the attended stimulus. However, the locus and mechanism underlying such biased processing remains unresolved. We have investigated the effect of attention on responses of V1 neurons in monkeys trained to attend to a cue, by asking whether the same visual stimulus, placed on the receptive field of the neuron, leads to different responses when attention is directed towards the receptive field (attend-to) compared to when attention is directed away from the receptive field (attend-away). In 128 orientation tuned V1 neurons recorded from 2 monkeys, we find that in 65% of neurons there was significant increase in response modulation (p<0.01) in attend-to condition. We also find that attention differentially affects superficial and deep layer neurons. In superficial layers, significantly more neurons were modulated in attend-to condition whereas deeper layers, neurons showed preferential modulation in the attend-away condition. Next we studied temporal dynamics of attentional modulation in various stimulus dimensions in striate (V1) and extra-striate (V4) cortex. Analysis of the time course of attentional modulation shows significant differences in individual neurons for stimulus size, orientation and contrast. Our results suggest that visual spatial attention directed towards the receptive field of V1 and V4 neurons modulate responses in multiple stimulus dimensions. This argues against the notion of a “hard wired” substrate that enhances contrast of the attended stimulus, as a mechanism for attentional modulation. Further, in many neurons, this modulation is a non-linear enhancement in response to the preferred stimulus feature. These data implicate attention as a dynamic interaction between top-down and bottom-up processes.

Tropea, D., Majewska, A. and Sur, M.
Influence of dark-rearing and subsequent light exposure on spine dynamics of primary visual cortical neurons in vivo.
Soc. Neurosci., 2005.

Dendritic spines are subcellular structures that constitute the postsynaptic sites of most excitatory synapses in the CNS. Changes in spine density, morphology and motility have been described both in vivo during normal development and with sensory deprivation, and in vitro following protocols that elicit changes in synaptic strength. Dark rearing has been shown to perturb the normal physiological properties of neurons in primary visual cortex, V1 and in particular to delay the maturation of visual cortex circuitry. Re-exposure to light rapidly restores orientation selectivity and responsiveness and the expression of activity-dependent molecules. In this study, we measured spine dynamics in V1 of mice dark reared from birth. Experiments were performed at postnatal day 28 (P28), which corresponds to the peak of the critical period. Animals were anesthetized in the dark prior to imaging. We used time-lapse two-photon laser scanning microscopy to assay motility of spines on V1 neurons in vivo, in mice expressing GFP in a subset of cortical layer 5 neurons. Compared to controls, dark-rearing caused a small but significant (p<0.001) increase in the motility of different types of spines: mushroom 20%, stubby 31%, thin 37%, filopodia 13%. The total increase in motility, considering all spines, was ~20%. Interestingly, the types of spines present in our sample was affected by dark rearing: there was a decrease in the percentage of stubby spines (from 26% to 15%) and an increase in the percentage of thin spines (from 11% to 21%) and filopodia (from 2% to 4%). Since thin spines and filopodia are generally the most motile protrusions and are also thought to form transient synapses, it is likely that dark rearing destabilizes existing spines and increases the number of transient connections in order to counteract decreased activity in visual cortex. Surprisingly, re-exposure to light for two days was not sufficient to restore either spine class composition or spine motility levels to those observed in control animals.

Wiesing, P., Beck, O., Schwabe, L., Marino, J., Schummers, J., Lyon, D.C., Sur, M.
Balanced computation of orientation selectivity in network models of the primary visual cortex.
Soc. Neurosci., 2005.

 

Yu, H., Majewska, A. and Sur, M.
Rearrangement of dendritic spines during short term monocular deprivation in the primary visual cortex of the ferret in vivo.
Soc. Neurosci., 2005.

Spines are small protrusions on dendrites that receive excitatory synapses. Previous studies have revealed that dendritic spines are dynamic, indicating that spines have the potential to consolidate functional cortical plasticity at the micro-structural level. In this study, we examined whether changes in visually-driven activity elicit rapid modifications of dendritic spine morphology in ferret primary visual cortex (V1). In order to compare changes in subcellular structure to changes in network activity, structural two-photon imaging of GFP-labeled neurons and functional intrinsic signal optical imaging were performed in the same cortex in vivo. Monocular deprivation (MD) for 3-6 hours can induce a significant shift of the ocular dominance map in V1 of a critical period ferret. The deprived eye response decreases, and this shift can be reversed by 3-6 hours of binocular visual experience. Functional changes after MD are accompanied by a significant loss of dendritic spines (control 5%; MD 20%), suggesting that the decrease in deprived eye responses may be mediated by a rapid loss of cortical connections subserving the deprived eye. This hypothesis is supported by the observation that in regions dominated by the deprived eye, spine loss is more pronounced than in the binocular zone. Importantly, the spine number recovers after 1 day of binocular vision, partially due to the reappearance of some spines that were lost following MD, and also due to the appearance of spines at new sites. Spines that are likely to turn over during MD and recovery include small mushroom spines, stubby spines and filopodia; large mushroom spines rarely turn over. The correlation between dendritic spine turnover and the magnitudes and timescales of ocular dominance shifts at the same loci suggests that rapid structural changes at synapses are closely tied to functional remodeling elicited by visual activity.