Functional and Anatomical Investigation of Sensory Processing in the Rodent Somatosensory Cortex

Of all sensory cortical areas, barrel cortex is among the best understood in terms of circuitry, yet least understood in terms of sensory function. Because sensory cortical areas have stereotyped anatomies, understanding computations in one sensory area may inform us of computations being performed by other sensory areas or sensory microcircuits all over the brain. Functional studies of barrel cortex are therefore important for marrying our immense and increasing knowledge of the cortical circuitry with the computations being performed in a cortical microcircuit. This thesis is an investigation of barrel cortex function as it pertains to 1) site specific sensory evoked plasticity in cortical microcircuit and 2) sensory receptive fields of the different cortical lamina in S1. The brain's capacity to rewire is thought to diminish with age. It is widely believed that development stabilizes the synapses from thalamus to cortex and that adult experience alters only synaptic connections between cortical neurons. We combined whole-cell recording from individual thalamocortical neurons in adult rats with a newly developed automatic tracing technique to reconstruct individual axonal trees. Whisker trimming substantially reduced thalamocortical axon length in barrel cortex but not the density of TC synapses along a fiber. Thus, sensory experience alters the total number of TC synapses. After trimming, sensory stimulation evoked more tightly time-locked responses among thalamorecipient layer 4 cortical neurons. Axonal plasticity was topographically specific, with robust changes in L4 and modest changes in the septal and infragranular layers. These results indicate that plasticity is mediated by interactions with the local cortical subcircuit and may be suggestive of laminar specific roles in sensory learning/coding. Next we sought to examine spatiotemporal coding properties of neurons in the different layers of the cortical microcircuit in S1. We combined intracellular recording and a novel multi-directional multi-whisker stimulator system to estimate receptive fields by reverse correlation of stimuli to synaptic inputs. Spatiotemporal receptive fields were identified orders of magnitude faster than by conventional spike-based approaches, even for neurons with little or no spiking activity. Given a suitable stimulus representation, a simple linear model captured the stimulus-response relationship for all neurons with unprecedented accuracy. In contrast to conventional single-whisker stimuli, complex stimuli revealed dramatically sharpened receptive fields, largely due to the effects of adaptation. Surprisingly, this phenomenon allows the surround to facilitate rather than suppress responses to the principal whisker. Optimized stimuli enhanced firing in layers 4-6, but not 2/3, which remained sparsely active. Surround facilitation through adaptation may be required for discriminating complex shapes and textures during natural sensing

All-optical interrogation of neural circuits during behaviour

This thesis explores the fundamental question of how patterns of neural activity encode information and guide behaviour. To address this, one needs three things: a way to record neural activity so that one can correlate neuronal responses with environmental variables; a flexible and specific way to influence neural activity so that one can modulate the variables that may underlie how information is encoded; a robust behavioural paradigm that allows one to assess how modulation of both environmental and neural variables modify behaviour. Techniques combining all three would be transformative for investigating which features of neural activity, and which neurons, most influence behavioural output. Previous electrical and optogenetic microstimulation studies have told us much about the impact of spatially or genetically defined groups of neurons, however they lack the flexibility to probe the contribution of specific, functionally defined subsets. In this thesis I leverage a combination of existing technologies to approach this goal. I combine two-photon calcium imaging with two-photon optogenetics and digital holography to generate an “all-optical” method for simultaneous reading and writing of neural activity in vivo with high spatio-temporal resolution. Calcium imaging allows for cellular resolution recordings from neural populations. Two-photon optogenetics allows for targeted activation of individual cells. Digital holography, using spatial light modulators (SLMs), allows for simultaneous photostimulation of tens to hundreds of neurons in arbitrary spatial locations. Taken together, I demonstrate that this method allows one to map the functional signature of neurons in superficial mouse barrel cortex and to target photostimulation to functionally-defined subsets of cells. I develop a suite of software that allows for quick, intuitive execution of such experiments and I combine this with a behavioural paradigm testing the effect of targeted perturbations on behaviour. In doing so, I demonstrate that animals are able to reliably detect the targeted activation of tens of neurons, with some sensitive to as few as five cortical cells. I demonstrate that such learning can be specific to targeted cells, and that the lower bound of perception shifts with training. The temporal structure of such perturbations had little impact on behaviour, however different groups of neurons drive behaviour to different extents. In order to probe which characteristics underly such variation, I tested whether the sensory response strength or correlation structure of targeted ensembles influenced their behavioural salience. Whilst these final experiments were inconclusive, they demonstrate their feasibility and provide us with some key actionable improvements that could further strengthen the all-optical approach. This thesis therefore represents a significant step forward towards the goal of combining high resolution readout and perturbation of neural activity with behaviour in order to investigate which features of the neural code are behaviourally relevant

Corticothalamic Spike Transfer via the L5B-POm Pathway in vivo

The cortex connects to the thalamus via extensive corticothalamic (CT) pathways, but their function in vivo is not well understood. We investigated "top-down" signaling from cortex to thalamus via the cortical layer 5B (L5B) to posterior medial nucleus (POm) pathway in the whisker system of the anesthetized mouse. While L5B CT inputs to POm are extremely strong in vitro, ongoing activity of L5 neurons in vivo might tonically depress these inputs and thereby block CT spike transfer. We find robust transfer of spikes from the cortex to the thalamus, mediated by few L5B-POm synapses. However, the gain of this pathway is not constant but instead is controlled by global cortical Up and Down states. We characterized in vivo CT spike transfer by analyzing unitary PSPs and found that a minority of PSPs drove POm spikes when CT gain peaked at the beginning of Up states. CT gain declined sharply during Up states due to frequency-dependent adaptation, resulting in periodic high gain-low gain oscillations. We estimate that POm neurons receive few (2-3) active L5B inputs. Thus, the L5B-POm pathway strongly amplifies the output of a few L5B neurons and locks thalamic POm sub-and suprathreshold activity to cortical L5B spiking

The Timescales of Transformation Across Brain Structures in the Thalamocortical Circuit

Sensory processing requires reliable transmission of sensory information across multiple brain regions, from peripheral sensors, through sub-cortical structures, to sensory cortex, ultimately producing the sensory representations that drive perception and behavior. Despite decades of research, we do not yet have a mechanistic understanding of how neural representations are transformed across these critical brain structures. This is primarily due to the fact that what we know at the circuit level has been mainly derived from electrophysiological recordings targeted at single regions and upon gross anatomical connection patterns across brain regions without specific, precise knowledge of synaptic connectivity. To fill this gap in knowledge and to uncover how signaling changes across brain regions in response to changes in the sensory environment, this thesis work has two primary contributions. First, we developed a work-flow of topographic mapping and histological validation for extracellular multi-electrode recordings of neurons in the thalamocortical circuit in rodents, followed by a novel statistical approach for inferring synaptic connectivity across the brain regions. Specifically, we developed a signal-detection based classification of synaptic connectivity in the thalamus and S1 cortex, with an assessment of classification confidence that is scalable to the large-scale recording approaches that are emerging in the field. Utilizing this experimental and computational framework, we next investigated the neural mechanisms that underlie an important sensory phenomenon that emerges in this early sensory circuit: rapid sensory adaptation. While this phenomenon has been well-studied over very rapid timescales of hundreds of milliseconds, other studies suggest that longer time scales of 10’s of seconds may also be relevant. Here, we demonstrated that the thalamus and the thalamorecipient layer 4 excitatory and inhibitory neurons in S1 exhibit differential adaptation dynamics, and that the neuronal dynamics across these different regions and cell types show common signatures of multiple timescales in response to sensory adaptation. We characterized the adaptation profiles at the TC junction and further identified several mechanisms that potentially underlie the adaptation effects on the circuit dynamics, including synaptic depression of the TC synapse in identified monosynaptically connected thalamic and cortical neurons, and changes in spike timing and synchronization in the thalamic population. These mechanisms together mediate a dynamic trade-off in the theoretical detectability and discriminability of stimulus inputs. These results suggest that adaptation of the thalamocortical circuit across timescales results from a complex interaction between distinct mechanisms, and notably the engagement of different mechanisms can shift depending on the timescale of environmental changes.Ph.D

Structure and dynamics of the corticothalamic driver pathway in the mouse whisker system

To generate a sensory percept of the environment, the brain needs to analyze and integrate spatially and temporally distributed sensory signals. Consequently, sensation on a neuronal basis is a distributed, non-linear and dynamic process. Following sensory receptor activation the signal travels through many brain regions wherein the pathway is split, loops back onto itself and joins together with others. At each step, neurons dynamically transform and filter the signal. To understand how the brain arrives at a sensory percept, it is therefore essential to determine the neuronal connectivity along the processing chain, the stimulus specificity of responses as well as the input-output transformations at each station. An interesting model system for investigating these dynamical processes is the rodent whisker system. Rodents can solve highly complicated tasks with their whiskers alone, distributed receptors at the follicles require spatial integration and rhythmic movements suggest temporal processing components. The posterior group nucleus of the thalamus (PO) is in a key position of the whisker sensory system. In addition to being part of the ascending paralemniscal pathway it is mainly driven by somatosensory barrel cortex (BC) and projects to many cortical and subcortical areas. Due to its poor excitability by whisker deflections, its function is unclear. The origin of the corticothalamic drive onto PO neurons are ‘thick-tufted’ layer 5B cortical neurons, which have large synaptic terminals in thalamus. One of those synapses alone has a strong influence on postsynaptic target neurons – a very unusual property for cortical synapses. Here, using quantitative anatomy, in vivo electrophysiology and optogenetics I characterize the organization and input-output computations along the BC L5B to PO pathway. Using a dual anterograde tract tracing approach and large scale anatomical reconstructions we demonstrate that BC L5B synaptic boutons divide PO in 4 subregions with different projection parameters. The lateral area (POm lateral) receives most boutons with the highest density. Additionally, L5B neurons innervate two inhibitory nuclei in thalamus and midbrain that both inhibit PO. In all 6 regions we report map specific projections, with different map orientations, showing that somatotopic projections are the rule in these cortico-subcortical projections. Next we investigated the L5B to POm action potential transfer efficacy during spontaneous slow oscillations in anesthesia. Using pharmacology and cell-type specific optogenetics we show that cortical activity is necessary and L5B activation is sufficient to evoke large excitatory postsynaptic potentials (EPSPs) in POm, typical for L5B inputs. Simultaneous cortical local field potential and L5B as well as POm juxtasomal recordings demonstrate that the gain of action potential transmission is high following periods of relative cortical silence, but dynamically decreases during periods of higher cortical activity. Isolation of individual EPSPs allowed us to determine the frequency dependent adaptation of the L5B to POm synapse in vivo. We determined that approximately half of the recorded POm neurons follow a simple rule of EPSP adaptation, suggesting that the subthreshold activity in these neurons originates from a single active L5B input. Using two independent modeling approaches, we determined that on average POm neurons receive 2-3 functional inputs from BC L5B. Finally we investigated how whisker deflection signals reach POm. We found that POm neurons fall into two groups. Approximately one third of the recorded neurons were activated at a relatively short latency by large EPSPs and fired action potentials following whisker stimulation. All neurons had long latency sub- and suprathreshold responses, due to Up-state initiation by the whisker stimulation. POm whisker responses were entirely dependent on cortex and were blocked by optogenetic cortical inactivation. Taken together we quantified the anatomical and physiological properties of the L5B to POm projection. The connection is sparse, parallel, strong and the dominant input for POm spontaneous activity as well as whisker evoked responses. Its gain is dynamically regulated and depends on cortical activity states

Two-photon calcium imaging of neocortical projection neurons in whisker somatosensory cortex during goal-directed sensorimotor learning

Abstract Excitatory projection neurons of the neocortex are thought to play important roles in per-ceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical and functional organization of these inter-areal connections are unknown. The mouse primary somatosensory whisk-er barrel cortex (S1) serves as an important model for investigating the mammalian neo-cortex, and, here, I firstly investigate the structure and secondly the function of a specific subset of S1 cortico-cortical long-range projection neurons. In the first part of my thesis, I studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in S1. As a population, these neurons densely projected to secondary whisker somatosensory cortex (S2) and primary/secondary whisker motor cortex (M1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. The execution of a goal-directed behavior requires the brain to process incoming sensory information from the environment in a context-, learning- and motivation-dependent manner in order to perform specific motor actions. Cortico-cortical communica-tion in the context of goal-directed sensorimotor transformation has begun to be studied, but little is known about how signaling between interconnected cortical areas is modified by sensorimotor learning, as well as in response to changes in reward contingencies. Hence, in the second part of my thesis, I studied cortico-cortical dynamics in primary whisker somatosensory barrel cortex (S1) of mice during a combined whisker and audito-ry task. Using transgenic mice expressing GCaMP6f combined with two-photon micros-copy and retrograde labeling techniques, I chronically monitored the activity of excitatory layer 2/3 neurons in S1 projecting to M1 or S2, while mice learned the behavioral switch task. The results demonstrated that both classes of neurons responded after whisker and auditory stimulation. However, the whisker stimulus evoked response was stronger than the auditory stimulus evoked response. Neurons projecting to S2 exhibited stronger re-sponses compared to neurons projecting to M1 neurons. Those responses remained rela-tively stable across training sessions and under different reward conditions. Furthermore, both classes of neurons responded during spontaneous licking, but neurons projecting to S2 had larger licking-related responses compared to neurons projecting to M1. This work therefore furthers our knowledge of the structure and function of specific types of cortical projection neurons, which is a necessary step towards detailed under-standing of how sensory information might be signaled from primary sensory areas to downstream brain regions for further processing

Properties and function of somatostatin-containing inhibitory interneurons in the somatosensory cortex of the mouse

GABAergic inhibitory interneurons play a pivotal role in balancing neuronal activity in the neocortex. They can be classified into different classes according to their variable morphological, electrophysiological, and neurochemical properties, including two major groups: parvalbumin-containing (PV+), fast-spiking (FS) cells and somatostatin-containing (SOM+) cells. Using transgenic mice, we identified two subgroups, distinct by all criteria, of SOM+ cells in the somatosensory (barrel) cortex of the mouse, one (called X94) in layer 4 and 5B, and the other one (X98) in deep layers (Ma et al., 2006). We found that X98 cells were calbindin-expressing (CB+), infragranular, layer 1--targeting Martinotti cells, and had a propensity to fire low-threshold calcium spikes, whereas X94 cells did not express CB, targeted mostly layer 4, discharged in stuttering pattern and with quasi fast-spiking properties. In the barrel cortex, it was previously shown that SOM+ cells mediate disynaptic inhibition in supragranular and infragranular layers. However, the roles of layer 4 SOM+ cells remain largely unknown. We used dual whole-cell recording to elucidate the synaptic circuits in layer 4 and the function of layer 4 SOM+ cells during cortical network activities. We found that layer 4 X94 SOM+ cells received strongly facilitating excitatory input and generated relatively slow rising inhibitory postsynaptic currents (IPSCs) compared to those evoked by FS cells. Strikingly, our data showed that SOM+ cells mediated strong synaptic inhibition of FS cells with connection probability greater than 90% in layer 4, but received very little reciprocal inhibition from FS cells, and no reciprocal inhibition from other SOM+ cells. Moreover, 100% of recorded SOM+-SOM+ cell pairs were electrically coupled with higher coupling ratio compared to that of electrically coupled FS cell pairs. In order to examine the functions of SOM+ cells, we applied 0 Mg2+ artificial cerebrospinal fluid (ACSF) to induce episodes of cortical network activity and observed that, during episodes of network activity, SOM+ cells fired robustly and synchronously, and produced strong inhibition of regular-spiking (RS) excitatory cells and inhibitory FS cells, especially the latter. Taken together, our data reveal that SOM+ cells in the barrel cortex can be sub-divided into different subtypes, and that layer 4 SOM+ cells exert a powerful inhibitory effect during high frequency network activity