Cortical circuit mechanisms underlying sensorimotor behavior
Gerald Pho*, Rafiq Huda*, Liadan Gunter, Ian Wickersham, Wasim Malik, Emery Brown, Mriganka Sur
2016 BRAIN Initiative Investigators Meeting
A fundamental building block of voluntary behavior is the ability to respond to information from the environment with appropriate motor actions. While seemingly simple, this process of transforming sensory information into motor commands consists of at least two distinct steps – detecting sensory stimuli relevant to behavioral goals in a noisy environment, and selecting appropriate motor actions based on those stimuli. While neural substrates of sensorimotor mapping and attention have been widely studied in non-human primates, many questions remain due to lack of tools that can selectively probe and manipulate neural activity, especially in a projection-specific manner.
In this project, we developed visual sensorimotor tasks for head-fixed mice to probe the contribution of specific cortical regions and projections in attention and sensorimotor mapping. Using optogenetics and large-scale calcium imaging during a go/no-go lick-based task, we previously identified the posterior parietal cortex (PPC) as an important locus for sensorimotor transformation (Goard et al 2016). By varying task parameters such as attentional engagement, stimulus contrast, and reward contingency, we discovered that PPC encodes multiple task variables across heterogeneous cell types in an engagement-specific manner. We are now developing statistical models to describe how sensory and motor variables are combined with network activity to generate PPC responses.
We have additionally developed a two-alternative sensorimotor task that requires sustained attention. Using retrograde tracing, we identified a prefrontal region, the anterior cingulate cortex (ACC), that is anatomically poised to contribute to both attentional processing, owing to its top-down projections to visual cortex, and sensorimotor transformation, due to its feed-forward projections to superior colliculus. Optogenetic inactivation of ACC compromised task performance, and projection-specific inactivation revealed dissociable contributions of corticocortical versus corticotectal neurons to attention and sensorimotor transformation, respectively. We are now combining retrograde tracers with calcium imaging to investigate the physiological signatures of these projection neurons.
Massive-scale multi-area single neuron recordings to reveal circuits underlying short-term memory
Murat Yildirim, Michael J Goard, Christopher J. Rowlands, Gerald Pho, Rafiq Huda, Peter So, Mriganka Sur
2016 BRAIN Initiative Investigators Meeting
Short-term memory is a fundamental cognitive process underlying an array of complex abilities, but its neural mechanism is not fully understood. Many brain regions are implicated in memory-guided decisions, including visual, association, and motor cortices as well as subcortical structures. However, it is not mechanistically understood what regions are involved when, what neuronal subsets are recruited within these regions, or how they interact to represent information relevant to behavior. Conventional two-photon imaging systems offer high resolution imaging of the required brain regions, but the field of view is typically smaller than a cortical area in a mouse (e.g., primary visual cortex), which in turn inhibits the simultaneous measurement of neural activity in different brain regions. In this project, we aim to elucidate the role of specific cortical regions and neuronal subsets in a visually-cued memory-guided discrimination task, in order to understand the neural mechanism of short-term memory. For this purpose, we will perform very large scale (>10 mm2) calcium imaging through two-photon microscopy in behaving mice to measure activity of thousands of neurons simultaneously across multiple brain regions. To obtain larger scale (>10 mm2) calcium imaging through two-photon microscopy, we have developed a novel system which includes non-descanned multifocal microscopy with a single photon counting camera, custom-made intermediate optics, and a custom made objective and tube lens (5x/0.7NA) to preserve the signal to noise ratio (SNR) and high resolution across the entire field of view. In parallel, we have designed and implemented three-photon microscopy to perform structural and functional imaging for discovering how task-relevant information is coded in deeper cortical layers and brain structures. We acquired an intrinsic third harmonic generation (THG) signal and three photon fluorescence (3PEF) images. In these three-photon images, we can excite either green fluorescent protein (GFP) or green genetically encoded calcium indicator (GCamp6s) in the whole visual cortex, external capsule, and hippocampal region. In addition, we have performed THG imaging in healthy intact human cerebral organoids cleared with SWITCH. In these THG images, nuclei are clearly delineated and cross sections demonstrate the depth penetration capacity (> 1mm) that extends throughout the organoid. Imaging control and MeCP2-deficient human cerebral organoids in 2D sections reveals structural and protein expression-based alterations that we expect will be clearly elucidated via both THG and three-photon fluorescence microscopy.
MeCP2 deficient astrocytes have altered signaling pathway activation and reduced visually-evoked microdomain sizes
R. GARCIA, R. V. RIKHYE, J. PETRAVICZ, C. DELÉPINE, M. SUR
Society for Neuroscience, 2016
Loss of function mutations in the X-linked gene encoding for MeCP2 are the underlying genetic cause for Rett Syndrome (RTT), a devastating neurodevelopmental disorder that primarily affects girls. Loss of function mutations in this ubiquitously expressed transcriptional regulator leads to imbalances in excitation and inhibition and disruption to neuronal circuit function. While the function of this transcriptional regulator remains elusive and complex, recent focus has turned to downstream signaling pathways as putative targets for novel therapeutics. The complexity of MeCP2 function is compounded by the heterogeneity of cell types in the brain, with recent evidence implicating glia cells in RTT pathophysiology. MeCP2 expression has been detected in astrocytes, and selective deletion or re-introduction of MeCP2 in astrocytes alone has been sufficient to induce or ameliorate pathological symptoms, respectively. Previously, we identified signaling pathways upstream of synaptic function that are impaired in MeCP2 mouse models, yet the downstream molecular and signaling effects resulting from a loss of MeCP2 function in astrocytes remains unknown. Here we measure signaling and astrocyte-specific proteins in a heterogeneous MeCP2-expressing population. We find that activated mTOR and AKT are reduced in astrocytes lacking MeCP2, while levels of cortical glutamate transporter 1 (GLT-1) are upregulated. We have recently shown that astrocytes in layer 2/3 of rodent visual cortex can respond to visual stimuli with robust and reliable microdomain Ca2+ elevation and that this effect is influenced by the availability and function of GLT-1. In MeCP2-/+ astrocytes, we find that the microdomain areas evoked during visual stimulation are reduced, in line with reduced circuit function. These data identify novel, cell-specific effects in astrocytes lacking MeCP2 and offer insight on their signaling and circuit interactions.
Circuit mechanisms of prefrontal contribution to visual behavior
R. HUDA, G. PHO, L. GUNTER, I. WICKERSHAM, M. SUR
Society for Neuroscience, 2016
A fundamental building block of voluntary behavior is the ability to respond to information from the environment with appropriate motor actions. While seemingly simple, this process of transforming sensory information into motor commands consists of at least two distinct steps – detecting sensory information relevant to current behavioral goals from a noisy environment and selecting appropriate motor actions based on those stimuli. While neural substrates of sensorimotor mapping and attention have been widely studied in non-human primates, many questions remain due to lack of tools that can selectively probe and manipulate neural activity, especially in a projection-specific manner. The availability of optogenetic and viral-based gene expression tools in mice enables the interrogation of cell type-specific contributions to circuit-level mechanisms underlying these cognitive phenomenon. Here, we devised a visual sustained-attention, sensorimotor task for head-fixed mice to probe the selective contribution of specific neuronal subsets. First, we used rabies viruses to identify a caudal midline prefrontal region (anterior cingulate cortex) that is anatomically poised to contribute to both attentional processing of visual stimuli, owing to its top-down projections to visual cortex, and sensorimotor transformation, due to its feed-forward projections to superior colliculus. Mice were trained to report the spatial location of a visual stimulus by rotating a ball. We made the task attention-demanding by including a variable foreperiod that introduces uncertainty in temporal expectancy for visual stimulus onset. In the first set of experiments, we found that optogenetic inactivation of this prefrontal area compromises performance on the task. Subsequently, two-photon microscopy and projection-specific optogenetic inactivation has allowed us to probe the functional contribution of visual cortex- and superior colliculus-projecting prefrontal neurons to sustained attention and sensorimotor transformation, respectively.
Third harmonic generation imaging of intact human cerebral organoids to assess key components of early neurogenesis in Rett Syndrome
M. YILDIRIM, D. FELDMAN, T. WANG, D. OUZOUNOV, S. CHOU, J. M. SWANEY, K. CHUNG, C. XU, P. SO, M. SUR;
Society for Neuroscience, 2016
Rett Syndrome (RTT) is a pervasive, X-linked neurodevelopmental disorder that predominantly affects girls. It is mostly caused by a sporadic mutation in the gene encoding methyl CpG-binding protein 2 (MeCP2).The clinical features of RTT are most commonly reported to emerge between the ages of 6-18 months and implicating RTT as a disorder of postnatal development. However, a variety of recent evidence from our lab and others demonstrates that RTT phenotypes are present at the earliest stages of brain development including neurogenesis, migration, and patterning in addition to stages of synaptic and circuit development and plasticity. We have used RTT patient-derived induced pluripotent stem cells to generate 3D human cerebral organoids that can serve as a model for human neurogenesis in vitro. We aim to expand on our existing findings in order to determine aberrancies at individual stages of neurogenesis by performing structural and immunocytochemical staining in isogenic control and MeCP2-deficient organoids. In addition, we aim to use Third Harmonic Generation (THG) microscopy as a label-free, nondestructive 3D tissue visualization method in order to gain a complete understanding of the structural complexity that underlies human neurogenesis. As a proof of concept, we have performed THG imaging in healthy intact human cerebral organoids cleared with SWITCH. We acquired an intrinsic THG signal with the following laser configurations: 400 kHz repetition rate, 65 fs pulse width laser at 1350 nm wavelength. In these THG images, nuclei are clearly delineated and cross sections demonstrate the depth penetration capacity (> 1mm) that extends throughout the organoid. Imaging control and MeCP2-deficient human cerebral organoids in 2D sections reveals structural and protein expression-based alterations that we expect will be clearly elucidated via both THG and three-photon fluorescence microscopy.
Growth, differentiation and connectivity of implanted human neuronal precursor cells in the mouse visual cortex
J. BENOIT, H. WU, V. BRETON-PROVENCHER, J. P. K. IP, D. FELDMAN, S. CHOU, R. JAENISCH, M. SUR
Society for Neuroscience, 2016
The use of induced pluripotent stem cells (iPSCs) to recapitulate the effect of human genetic diseases in an experimentally tractable system requires both a rich genomic as well as cellular context. Current models for neurological diseases generally consist of human induced-neuronal (iN) cells engineered with the specific mutations of interest, or derived from patient cells with those same mutations, and grown in vitro in a 2D culture. In order to create a more natural and 3D environment in which to grow and assess human iNs, we differentiated “wild-type” AAVS1-CAG-tdTomato human neuronal precursor cells (NPCs) which were then transplanted into the mouse cortex to form a “humanized” functional network in a model system. We injected approximately 10,000 NPCs into the primary visual cortex (V1) of SCID immunodeficient mice at P21 and then using a craniotomy, examined their morphological development in vivo from 6 weeks post-injection onwards. Of the injected NPCs, several hundred survived and were localized mainly to the injection tract although some cells in deeper layers (~300 um from pia) were well-intercalated between endogenous mouse cells within several hundred um of the injection. NPCs sent wide-ranging projections, some of which reached to adjacent cortical areas and extended beyond the craniotomy (1.5 mm lateral distance) in some cases. We found evidence of filopodia and potential immature spines on human dendrites which suggests that the NPCs have differentiated into a neuronal phenotype and may be forming synapses with endogenous mouse neurons. We are currently examining calcium responses in these cells to determine if they possess functional contacts with the mouse cortical circuit and are therefore responsive to visual input. We posit that this system represents a more realistic environment with superior experimental validity in which the development of normal and patient-derived human neurons can be studied.
A computational model of astrocyte induced modulation of synaptic plasticity and normalization
V. Sreerag 1, Ryan Phillips1, Srinivasa Chakravarthy1, Mriganka Sur2
Society for Neuroscience, 2016
1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, IIT Madras, India; 2Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
It has been suggested that perisynaptic astrocytic processes have a role in synaptic plasticity. Here we propose a simple model for astrocytic modulation of synaptic plasticity. The model consists of presynaptic, postsynaptic and astrocyte compartments of the tripartite synapse. Presynaptic activation initiates neurotransmitter release into the synaptic cleft. This neurotransmitter binds with NMDA receptors leading to calcium influx into the postsynaptic compartment. However, NMDAR opening is also contingent upon postsynaptic potential-dependent magnesium blockage. The postsynaptic compartment is influenced by both the presynaptic compartment and astrocytic processes. Astrocytes release gliotransmitters including D-serine and glutamate, which in turn regulate the synaptic neurotransmitter concentration and influence the postsynaptic calcium concentration. Following the ‘calcium control hypothesis,’ we assume that the postsynaptic calcium controls the “synaptic strength”, w, which corresponds to the strength of response of AMPA receptors to synaptic glutamate. This synaptic strength is modelled by a cubic nonlinearity exhibiting bistable dynamics.
The proposed model is comprised of a presynaptic variable (firing rate) and postsynaptic voltage (controlled by an external current), which are independently varied to display LTD and LTP. Interestingly, the model exhibits BCM-like dynamics wherein the plasticity switches from LTD to LTP at a threshold value of postsynaptic voltage. A key feature of the model is that this threshold voltage is modulated by gliotransmitter released by the astrocyte. We next simulate weight dynamics at two synapses supplied by the same astrocyte such that that the total gliotransmitter released to the two synapses is conserved. Simulations showed a mechanism of normalization of the two synaptic weights in that growth in one synapses is accompanied by attenuation in the other.
In summary, the model demonstrates: (1) astrocyte induced modulation in LTD to LTP threshold, and (2) weight normalization of multiple synapses controlled via astrocytic gliotransmitter release.
Development of an Image-Based High-Content Screening Assay for Tau Clearing Drugs in a Human iPSC-Derived Neuronal Cell Model of Frontotemporal Dementia
Chialin Cheng, Surya A. Reis, Emily T. Adams, M. Catarina Silva, Krista M. Hennig, Daniel M. Fass, Danielle A. Feldman, Mriganka Sur, Bradford C. Dickerson, Kenneth S. Kosik, Tau Consortium & Stephen J. Haggarty
Autosomal dominant mutations in the microtubule-associated protein gene (MAPT) encoding the protein tau cause frontotemporal dementia spectrum disorders (FTD-s). These MAPT mutations are associated with pathologically abnormal tau phosphorylation levels and intracellular accumulation of aggregated protein predominantly in neurons (“tauopathy”). Recently, a rare variant of tau, Tau-A152T, located N-terminal of the microtubule-binding domain has been described. This variant of tau has decreased affinity for binding microtubules in vitro, and has been shown to increase the risk for FTD-s, Alzheimer’s disease, and synucleinopathies. Here we used human induced pluripotent stem cells (iPSC) from a FTD-s subject diagnosed with progressive supranuclear palsy carrying this Tau-A152T variant as a genetically accurate cell model of tauopathy. To utilize these cells to create a rapid and robust biological cellular assay system capable of supporting the discovery of novel therapeutics for tauopathies, we have adapted strategies for the inducible expression of the pro-neural transcription factor Neurogenin 2 in stably transduced iPSC-derived neural progenitor cells (iNgn2-NPCs). We demonstrate the ability to efficiently and reproducibly generate nearly limitless numbers of excitatory, glutamatergic-like neurons from these iNgn2-NPCs in a 96-well plate format with abundant expression of tau with enhanced polarized distribution to axonal processes. In order to monitor potential mutation-induced aberrant subcellular tau distribution, as well as drug-induced tau clearance, we developed a high-content image-based screen utilizing automated confocal microscopy and an advanced image-processing pipeline optimized for analysis of morphologically complex neuronal cultures. We summarize the results of a pilot screen for tau clearing compounds targeting autophagy and protein homeostasis pathways with an emphasis on clinically used FDA-approved drugs with potential for repurposing. This strategy will aid in expediting the translational research in elucidating novel targets for therapeutic intervention for neurological diseases involving tauopathy such as FTD-s.
Major vault protein, a candidate gene in 16p11.2 microdeletion syndrome, is required for homeostatic regulation of cortical plasticity
Jacque Pak Kan Ip, Ikue Nagakura, Jeremy Petravicz, Jamie Benoit, Erik A.C. Wiemer, Mriganka Sur
Society for Neuroscience, 2016
Microdeletion of a region in the chromosome 16p11.2 increases susceptibility to autism and accounts for up to 1% of this population. Although this region contains 29 genes, disrupting only a small piece of this region, which spans 5 genes, is sufficient to cause autistic traits. One candidate gene in this region is the major vault protein (MVP), which has been implicated in the regulation of several cellular processes including transport mechanisms and multidrug resistance. We found that MVP expression levels in MVP Het mice closely phenocopy those of 16p11.2 mice, suggesting MVP Het mice may serve as a model of MVP function in 16p11.2 microdeletion. However, the function of MVP in the central nervous system, in particular its role in brain function and plasticity, has not been investigated. To determine the role of MVP in experience-dependent synaptic and circuit plasticity, we first measured ocular dominance plasticity (ODP) in primary visual cortex (V1). We found that MVP Het mice show impairment in strengthening of open eye responses in V1 after 7 days monocular deprivation (MD), resulting in reduced overall plasticity. Furthermore, electrophysiology experiments showed that the frequency of mEPSCs was decreased in MVP Het mice after 7 days MD, suggesting a decrease in the number of functional synapses, which may underlie the reduced plasticity in MVP Het mice. By a biotin-labeling assay, we found impaired homeostatic upregulation of surface GluA1 in MVP Het mice after longer term MD. To investigate the underlying molecular mechanism, we measured intracellular signaling in MVP WT and MVP Het mice and found ERK activation was significantly increased in MVP MVP Het mice, while activation of other signals such as Akt and JAK were normal. STAT1 is a downstream molecule of JAK signaling and reported to be inhibited by MVP. We found a tendency towards increased expression of STAT1 in MVP Het mice, suggesting the possibility of MVP inhibition on STAT1. We have previously examined ODP in STAT1 KO mice and shown that they have an accelerated increase in open eye responses and enhanced plasticity after 4 days of MD. These results suggest that MVP may interact with STAT1 to regulate plasticity, and one function of MVP may be to negatively regulate STAT1. Collectively, we find a highly specific role for MVP as a critical molecule in the homeostatic or response-restoring component of activity-dependent synaptic plasticity. Thus, this study helps reveal a new mechanism for an autism-related gene in brain function, and suggests a broader role for neuro-immune interactions in circuit level plasticity.
Keji Li, Rajeev V. Rikhye, Abhishek Banerjee, and Mriganka Sur
Depolarizing GABA receptor causes cortical network deficits in Rett Syndrome
Society for Neuroscience, 2016
Rett Syndrome (RTT), a form of autism spectrum disorder, is mainly caused by mutations of a single gene, Methyl CpG binding protein 2 (MECP2). Symptoms of RTT include developmental regression of acquired motor and language skills, and severe cognitive impairment. The mechanism behind these symptoms are not yet clear, but in mouse RTT models the disruption of excitation/inhibition (E/I) balance in the brain has been observed. The E/I balance is crucial for the normal functioning of cortical networks. Even subtle E/I imbalance is reflected in changes in spike rate and timing, measured by signal-to-noise-ratio (SNR), and coding reliability and sparseness. Indeed, all these properties are decreased in RTT model mice. This E/I imbalance is moved in the direction of hyper-excitation in RTT, yet RTT also seems to feature hyper-connection of inhibitory neurons and reduced excitation.
Reduced efficacy of inhibitory GABAergic transmission following Mecp2 mutation is a likely mechanism for the above observations in RTT. At the synaptic level, Tang et al. (2015) found that K⁺/Cl⁻ cotransporter 2 (KCC2) was a critical downstream target of Mecp2, and KCC2 reduction following MECP2 mutation lowered intracellular Cl⁻. Low intracellular Cl⁻ concentration caused depolarizing postsynatpic responses of GABAAR, and weakened inhibitory transmission. However, it is not clear whether this synaptic mechanism can cause the observed E/I balance shift in cortical circuits in vivo.
To specifically rescue the abnormal intracellular Cl⁻ levels and observe its effect on cortical network behavior, we used bumetanide, a Na⁺K⁺Cl⁻ cotransporter 1 (NKCC1) antagonist that raises intracellular Cl⁻ concentration, and measured the network responses of V1 superficial layer neurons to a range of visual stimuli. In particular, we evaluated SNR, sparseness and reliability, in response to natural movies. After daily injections of bumetanide for a week, RTT model mice showed increased SNR and sparseness compared to sham-injected RTT model control, showing clear rescue of both properties. These results indicate that the altered intracellular Cl⁻ level, which leads to depolarizing GABAergic transmission, underlies a major portion of deficits observed in cortical network responses in RTT.
M. HU, R. V. RIKHYE, M. J. GOARD, M. SUR
Dissecting functional organization of mouse visual cortex
Society for Neuroscience, 2016
Although it is known that mice have roughly ten distinct visual areas, the specific function of each area and interactions between areas remains poorly understood. In this study, we used both wide-field and single-cell calcium imaging from awake, head-fixed mice, which transgenically expressed GCaMP6f, to functionally segment the entire visual cortex. First, to identify the gross organization of the visual areas, we performed retinotopic mapping using a custom-built wide-field epi-fluorescence microscope. Next, we characterized the responses of neurons within each segmented area to drifting gratings with various directions and spatial temporal frequencies. Within V1, we found a gradient of bias of averaged peak orientation (from vertical to horizontal orientations) along binocular to monocular axis. We also found systematic variations in averaged peak spatial and temporal frequencies along the same axis. The averaged peak temporal frequency in area LM exhibit a similar distribution (a mirror of V1) in visual space, indicating its role as the second visual area along the feedforward visual pathway. These results suggest that the different visual areas are sensitive to different visual features. To further test this hypothesis, we perturbed either the phase or the amplitude content of natural movies. The phase spectrum of natural movies contains information about salient image features, such as edges; whereas the amplitude spectrum contains low-level information, such as luminance. Interestingly, we found that medial regions (such as medial posterior part of V1 and PM) was more selective to phase, while lateral regions (such as lateral anterior part of V1 and AL) was more selective to the amplitude spectrum. Together, our results reveal that mouse visual cortex has organized representation of different visual features, which is globally extended through an entire set of areas. This distribution may already present in V1 and further elaborated in different higher visual areas.
V. BRETON-PROVENCHER, M. SUR
Interaction between parasympathetic and sympathetic pathways on prediction of noradrenergic activity by pupil size.
Society for Neuroscience, 2016
Pupil diameter has been used as a predictor of brain arousal. However, little is known about the neuromodulators responsible for pupil-mediated brain state, and to what extent pupil size can predict these neuromodulatory tones. Here we recorded the activity of noradrenergic (NE) neurons by using both functional imaging of NE axons in the cortex, and by single unit recordings in the locus coeruleus (LC) of awake mice. We show that pupil dilation predicts an increase of correlated NE activity both at a single cell and population level. The increase in LC-NE firing rate is linearly correlated with the amplitude of dilation events. This coherence between NE and pupil signals peaks in the low frequency range (10-2 to 10-1 Hz). Direct activation of LC-NE neurons by optogenetics further demonstrates the causal relationship between NE and pupil dilation. We are currently investigating the interaction between the pathways governing light mediated pupil constriction and internal state driven dilation. Altogether, our results show that pupil diameter can be used as a tool to track noradrenergic tone in the brain.
J. SHARMA , R. LANDMAN , J. HYMAN, L. BRATTAIN , K. JOHNSON , T. QUATIERI , K. SRINIVASAN , A. WISLER , G. FENG , M. SUR , R. DESIMONE
Asymmetry in vocal communication in marmosets – influence of social context and gender differences
Society for Neuroscience, 2016
Common marmosets are highly gregarious animals and use a rich vocal repertoire while communicating with conspecifics during social interactions. Recent studies indicate that they take turns in uttering calls (antiphonal calling). There is also evidence that these exchanges are modulated by social context and specific calls are used to locate a group member when out of visual contact, when under threat or to show anxiety. To study these vocal/social interactions in a naturalistic home-cage environment, we developed a wireless lightweight flexible neck collar, equipped with a microelectromechanical system (MEMS) acoustic microphone, a non-acoustic contact microphone for detecting caller vocal fold vibration, and a Bluetooth module for wireless data transmission. The initial testing was done on marmoset dyads and spectral analysis of their calls was performed to identify individual caller. Approximately 80% of calls could be attributed to an individual based on relative sound pressure alone. The remaining 20% were attributed with addition of data from the contact microphone. Cross-correlation between audio channel and vibrational signal allows identification of most likely caller. We examined vocal interactions between dyads within the home-cage environment. We find that antiphonal calling occurs not only for ‘phee’calls, but also for other calls such as ‘trill’, and even while in visual contact. We selectively removed one member of a dyadic pair (male or a female) from the home cage for short periods, while within or without visual contact. Analysis shows that there is directionality in these interactions when the female is out of the home cage but within view, there is an increase in temporal coherence, where one animal calls at a lag of about 0.5 sec after his/her partner. An asymmetry between dyads is also found when one animal is taken out of the room, but within audible range. When the female is taken out, dyads ‘phee’call back and forth, but when the male is taken out, there is no ‘phee’calling. The first ‘phee’call is typically uttered by the animal that is outside. Directionality in vocal interactions may be thus associated with the sex of the animal, social context, dominance and relatedness. Further research is underway on multiple dyads to confirm and to explore neural underpinnings and behavioral consequences of this asymmetry.
G. N. PHO, M. J. GOARD, B. CRAWFORD, M. SUR
Distinct roles of mouse visual and parietal cortex during perceptual decisions
Soc. Neurosci., 2015.
The posterior parietal cortex (PPC) has been implicated in perceptual decisions, but its specific role at the interface between sensation and action remains unresolved. Here, we provide evidence that mouse PPC, in functional analogy to primates, is neither a pure sensory area, nor directly involved in control of motor output, but rather is important for the mapping of sensory inputs to motor commands. Mice were trained on a visual discrimination task with distinct stimulus and motor epochs. We first tested the necessity of both PPC and the primary visual cortex (V1) during the different task epochs using VGAT-ChR2 transgenic mice, which express ChR2 in inhibitory neurons. Optogenetic inactivation revealed that both V1 and PPC were necessary during the stimulus period, but not for execution of the motor response. We then used two-photon calcium imaging to measure population activity in V1 and PPC, both during engagement in the task and during passive viewing of the same stimuli. Whereas V1 responses were driven by visual stimuli alone and only mildly modulated by task engagement, PPC responses were strongly gated by engagement and signaled the impending response. PPC responses exhibited both signatures of classical decision neurons: they reflected both the animal’s choice on error trials, as well as the degree of sensory evidence, which was manipulated using stimuli of varying contrasts. Lastly, to test whether PPC primarily encoded information about the stimulus or the choice, we re-trained mice with a reversed stimulus-reward contingency, and imaged the same neurons before and after the switch. We found that stimulus selectivity in PPC, but not V1, was dramatically reversed after retraining on the new contingency. Our results are consistent with a role of the mouse posterior parietal cortex in transforming sensory information to motor commands during perceptual decisions.
P. IP, N. MELLIOS, D. FELDMAN, S. D. SHERIDAN, S. KWOK, B. ROSEN, B. CRAWFORD, Y. LI, R. JAENISCH, S. J. HAGGARTY, M. SUR
Human and Mouse Models of Rett Syndrome exhibit altered prenatal cortical development due to alterations in neurogenesis
Soc. Neurosci., 2015.
Rett Syndrome (RTT) is a neurodevelopmental disorder that, in the vast majority of cases, arises from mutations in the X-linked gene MECP2. MeCP2 is an epigenetic modulator of gene expression that has recently been shown to interact with miRNA machinery. In addition, MeCP2 itself has been implicated in several neurodevelopmental disorders. Multiple lines of evidence point to the importance of miRNA-mediated pathways downstream of MeCP2 in different stages of brain development and plasticity. We hypothesized that the pleiotropic effects of MeCP2 in prenatal development are mediated via a set of early regulated miRNAs. Towards that end, we used induced pluripotent stem cell (iPSC) RTT lines generated from patients, virally-mediated knockdown of MeCP2 in human embryonic stem cells (ESCs), TALEN-derived isogenic ESC RTT lines, and an Mecp2 mutant mouse model as complementary approaches to identify novel MeCP2-regulated miRNAs and examine their respective influence on neurogenesis and neuronal differentiation.. Via BrdU pulse labelling, we found that the proliferation rate of patient-derived and MeCP2-deficient neuronal progenitor cells was significantly altered relative to control cells; this was accompanied by reductions in the expression of early neuronal markers and immature dendritic morphology. Our findings to date implicate aberrant regulation of prenatal neurogenesis as a result of MeCP2 deficiency. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 capable of influencing neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders. Ongoing experiments are focused on elucidating the mechanisms of disease-related impairments in neurogenesis in both mouse and human organoid models of RTT, and translating these findings to scalable assays for novel therapeutic discovery.
H. SUGIHARA, N. CHEN, M. SUR
Acetylcholine drives cortical microcircuit and modulates temporal dynamics in V1
Soc. Neurosci., 2015.
Acetylcholine (ACh) modulates cortical functions including information processing and plasticity. To understand the physiological basis of these functions, it is critical to identify the cortical circuit elements involved. We have previously shown that cholinergic activation of astrocytes and their facilitatory influences on pyramidal neurons (PYR) are crucial to induce plasticity (Chen N, Sugihara H et al., PNAS 2012). In this work, we aim to dissect the neural circuit involved in cholinergic modulation of sensory processing. Specifically, we focus on the temporal dynamics of cortical activity: decorrelation of neuronal responses and desynchronization of local field potential (LFP) using L2/3 mouse primary visual cortex (V1) as a model. Recent studies suggest that inhibitory neurons are important for mediating temporal changes in neural activity. Candidate neurons include regular-spiking inhibitory neurons: somatostatin-expressing (SOM), vasoactive intestinal peptide-expressing (VIP) and layer 1 (L1) neurons. We recorded the cholinergic responses of these inhibitory neuronal subtypes in slice preparations. ACh induced concentration-specific responses in these neurons: SOM neurons were activated by a range of ACh concentrations while VIP/L1 neurons were activated only at high concentration. We further show that this is likely due to the active shaping of inhibitory neuronal responses through defined inhibitory connections between them: SOM neurons inhibit VIP/L1 neurons and this counters the ACh-induced facilitatory responses in the VIP/L1 neurons. In addition, we show that ACh-activated SOM (but not VIP/L1) induced inhibitory currents in parvalbumin-expressing (PV) and PYR neurons. This suggests the presence of an ACh-activated neural circuit comprising direct SOM-PYR and indirect SOM-PV-PYR connections. We next tested the causal relationship between this SOM-driven circuit and decorrelation/desynchronization through hyperpolarizing Arch-expressing SOM neurons in vivo. Indeed, hyperpolarization of SOM neurons blocked the cholinergic-mediated desynchronization/decorrelation. Hyperpolarization of VIP neurons did not affect the LFP desynchronization. Finally, we stimulated SOM neurons directly by expressing ChR2 in these neurons. Photostimulation of SOM neurons, in the absence of cholinergic stimulation, induced LFP desynchronization. This suggests that direct activation of SOM-driven circuit is sufficient to change temporal dynamics of V1. Collectively, these findings reveal the powerful role of SOM neurons in dynamically shaping the temporal pattern of cholinergic-mediated responses.
R. GARCIA, R. RIKHYE, M. SUR
Robust and reliable Ca2+ response in microdomains of astrocytes
Soc. Neurosci., 2015.
Astrocytic intracellular Ca2+ signaling has come to light as a prominent feature of neuronal-glial interactions. The majority of astrocyte Ca2+ signaling studies are performed in either culture or in situ brain slices, both of which rely on electrical stimulation or pharmacological methods to examine the spatial and temporal coding of astrocyte Ca2+ signals. We have investigated visually evoked Ca2+ responses in astrocytes of the visual cortex of awake, head-fixed mice using two-photon microscopy. Initially, our studies involved the use of viral-mediated delivery of genetically encoded calcium indicators. However, in order maintain an intact and unperturbed cortex, we have chosen to use a recently developed line of conditional transgenic animals that express GCaMP5G in astrocytes throughout the mouse brain. We have found that Ca2+ transients in distal processes of cortical astrocytes are more frequent than those observed in anesthetized preparations, exhibiting a variable relationship to somatic responses. Furthermore, we found discrete structural regions of distal processes of single astrocytes that responded to sinusoidal drifting gratings and were tuned to specific orientations. Natural movies (NM) are known to evoke sparse, but reliable, responses from V1 pyramidal neurons. Surprisingly, we found discrete processes of astrocytes also respond reliably to natural movies. Responses to sinusoidal gratings were also less reliable than to natural movies. We hypothesize that these reliable astrocytic microdomain Ca2+ transients are due to the synchronous activation of neighboring ensembles of synapses. Together our results suggest that astrocytes could play an important role in modulating information processing in V1, potentially by modulating response reliability at pyramidal cell synapses.
J. SHARMA, R. LANDMAN, F. YOSHIDA, H. SUGIHARA, M. SUR
Two photon imaging with genetically -encoded calcium indicators in new world primates
Soc. Neurosci., 2015.
Two photon imaging using calcium sensors has provided important insights into neural circuit mechanisms with unprecedented detail, particularly in mice. With the advent of highly sensitive and targettable genetically encoded-calcium indicators (such as variants of GCaMP) riding on viral backbones of various vectors and aided by cell -specific promoters, the same population of neurons can be repeatedly imaged over several weeks or months to investigate neural circuit function (and dysfunction), and study neural mechanisms and plasticity underlying perception, cognition, learning and memory. However, achieving a similar level of sophistication in imaging higher mammals, particularly primates with more complex and dense cerebral architecture, poses new challenges. From the published literature, it is already clear that a new set of tools needs to be developed for this purpose. These include hardware for animal stabilization, movement correction of the imaging data, the development of chronic optical windows for accessing population of neurons expressing florescent proteins over several weeks, and the optimization of viral vectors and promoters while keeping brain tissue physiologically viable and in good health. Here we present our experience in developing these tools for 2 photon calcium imaging with GCaMP6 variants for chronic imaging in anesthetized new world monkeys, from several striate and extrastriate areas of the visual cortex. We modified a 2 photon imaging system (Sutter Instruments) to couple with a custom-designed movable objective that affords maximal rotational degrees of freedom along X and Y axes. We also developed a flexible imaging platform that provides precise alignment with the imaging plane of the objective, and can be tailored to suit primates of various sizes. For head stabilization we designed a dual, low-profile head post system that provides excellent rigidity while allowing flexibility to orient the head in any position to target multiple areas on the cortical surface. To minimize body movement due to respiration, we designed a simple trampoline suspension. We have also developed and tested several versions of chronic imaging windows, including sealed, removable and replaceable optical windows that are easy to maintain, while minimizing risk of infection and with flexibility to re-inject viral vectors or remove interfering pial-tissue growth. Large field ex-vivo confocal imaging was used to confirm GCaMP expression in several layers of the cortex with hSynapsin and CaMKII promoters. Two photon imaging in new world primates provides exciting possibilities as the next generation of transgenic primates come online.
R. HUDA, G. PHO, I. R. WICKERSHAM, M. SUR
Circuit mechanisms underlying visual responses of the anterior cingulate cortex
Soc. Neurosci., 2015.
Neural dynamics in sensory cortices are shaped by bottom-up inputs relaying the physical features of sensory stimuli and by top-down projections that modulate their encoding. The anterior cingulate division of the prefrontal cortex is known to provide top-down input to the visual cortex. Here, we use multiple approaches to delineate the functional role of visual inputs to the anterior cingulate cortex (ACC) and of the feedback from ACC to V1. Using rabies virus-mediated anatomical tracing to identify sources of inputs to the ACC, we found that V1 as well as other cortical and subcortical brain regions project to the ACC. Using rabies viruses that express the genetically encodable calcium indicator GCaMP6f and two-photon microscopy, we characterized the functional properties of ACC-projecting visual cortex neurons in passively viewing, awake head-fixed mice. We found that many of these neurons are tuned to the orientation and direction of drifting gratings. Next, we expressed GCaMP6s in the ACC and imaged the calcium activity of ACC axons found in layer 1 of V1. A subset of ACC axons were visually driven and displayed sharply tuned responses to the orientation and direction of drifting gratings. To assess the contribution of V1 to this property, we used a chemogenetic approach. We expressed the inhibitory hM4Di DREADD (designer receptors exclusively activated by designer drugs) in V1, GCaMP6s in the ACC, and monitored the calcium responses of ACC axons to oriented drifting gratings before and after systemic application of the DREADD agonist clozapine-N-oxide (CNO). While CNO application in control animals had no effect on the visual responses of ACC axons, it reduced responses in DREADD expressing animals. Together, these findings show that a projection from the visual cortex contributes to the visual responsiveness of the ACC. Since the ACC has been proposed to play a crucial role in cognition, and in particular reward processing, we propose that the ACC processes visual information in the context of its behavioral significance and relays a saliency signal back to the visual cortex to modulate the encoding of relevant visual stimuli.
R. V. RIKHYE, M. SUR
Dissecting the inhibitory mechanisms of reliable coding in mouse primary visual cortex
Soc. Neurosci., 2015.
Neurons in the primary visual cortex (V1) respond to full-field natural scenes with spike trains that are highly reliable between trials. While it has been argued that local inhibitory interneurons are responsible for modulating reliable coding, no study has yet systematically detailed the role of different interneuron subtypes. Our goal was to show how Parvalbumin (PV), Somatostatin (SST) and Vasoactive Intestinal Peptide (VIP) expressing interneurons modulate reliable coding in mouse V1. Specifically, we aimed to: (1) show how subnetworks of these interneurons process natural scenes and (2) determine how they contribute to reliable coding. To address these questions, we performed in vivo two-photon calcium imaging in awake, head-fixed mice by conditionally expressing GCaMP6f in PV, SST or VIP neurons. This allowed us to minimize the effect of contamination from nearby excitatory neurons and permitted us to study population coding within these interneuron subnetworks. SST neurons also preferred lower spatial frequencies than PV neurons, consistent with their role in integrating information from a larger visual area. Not surprisingly, VIP neurons responded poorly to gratings. PV neurons responded strongly, but unreliably, to full-field natural scenes. In contrast, SST neurons were more selective and were highly reliable between trials. SST cell reliability was comparable to excitatory neurons. This suggests that SST neurons are selectively driven by specific features in natural scenes and, consequently, provide reliable dendritic inhibition on their target cells. We also found that VIP neurons responded more strongly to natural scenes than gratings, suggesting that these interneurons are driven more by “salient” stimuli. Next, we investigated how these interneurons modulated pyramidal cell reliability. To do so, we conditionally expressed ChR2 in both PV or SST neurons and GCaMP6f in pyramidal neurons. We reasoned that reliability arose due precisely timed excitatory (E) and inhibitory (I) synaptic currents. Thus, we used a stimulation protocol to decorrelate these E- and I-currents in pyramidal cells. Specifically, we pulsed a blue LED for 100ms at random times during a natural movie. This allowed us to activate PV/SST neurons during periods when pyramidal cells were most reliable. We discovered that activating SST neurons during epochs of reliability increased reliability. In contrast, stimulating PV neurons reduced reliability. Taken together, our work demonstrates that SST neurons play an important role in shaping the reliability of pyramidal cell responses to natural scenes in mouse visual cortex.
N. MELLIOS, D. FELDMAN, S. D. SHERIDAN, P. K. IP, S. KWOK, B. ROSEN, B. CRAWFORD, Y. LI, R. JAENISCH, S. J. HAGGARTY, M. SUR
Robustly dysregulated miRNAs downstream of MeCP2 control human prenatal brain development through differential effects on autism-related signaling pathways
Soc. Neurosci., 2015.
Rett Syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in methyl-CpG-binding protein 2 (MECP2), a potent epigenetic regulator whose role in prenatal brain development is poorly understood. Given the known effects of MeCP2 on miRNA biogenesis, we hypothesized that neurogenesis may be impacted in RTT via MeCP2-regulated miRNAs that are enriched in early brain development; and hence modulate critical molecular components of neuronal progenitor proliferation and differentiation. Focusing on the most dysregulated miRNAs we found two prenatal brain-enriched miRNAs – miR-199 and miR-214 – to be robustly increased in human patient-derived culture, cerebral organoid, and mouse models of MeCP2 deficiency. Increases in miR-199 and miR-214 in MeCP2 mutant or deficient neuronal progenitors were a consequence of altered miRNA biogenesis and were associated with reduced expression of their targets PAK4 and PTEN, which in turn resulted in differential changes in Erk and Akt phosphorylation. Inhibiting miR-199 or miR-214 expression in induced pluripotent stem cell-derived neuronal progenitors deficient in MeCP2 restored Akt and Erk activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Collectively, our data suggest that MeCP2-mediated dysregulation of miR-199 and miR-214 expression influences early neurogenesis through the differential regulation of molecular pathways with known links to autism spectrum disorders.