The balance between excitatory and inhibitory synaptic transmission within and across neurons in active networks is crucial for cortical function and may allow for rapid transitions between stable network states. GABAergic interneurons mediate the majority of inhibitory transmission in the cortex, and therefore contribute to the global balance of activity in neuronal networks. Disruption in the network balance due to impaired inhibition has been implicated in several neuropsychiatric diseases (Marin 2012). Both schizophrenia and autism are two highly heritable cognitive disorders with complex genetic aetiologies but overlapping behavioural phenotypes that share common imbalances in neuronal network activity (Gao & Penzes 2015). An increasing body of evidence suggests that functional abnormalities in a particular group of cortical GABAergic interneurons expressing the calcium-binding protein parvalbumin (PV) are involved in the pathology of these disorders (Marin 2012). As deficits in this neuronal population have been linked to these disorders it could be useful to target them and increase their activity. A conserved feature in PV cells is their unusually low input resistance compared to other neuronal populations. This feature is regulated by the expression of leak K+ channels, believed to be mediated in part by TASK and TREK subfamily two-pore K+ channels (Goldberg et al. 2011). The selective blockade of specific leak K+ channels could therefore be applied to increase the activity of PV cells. In this thesis, specific TASK-1/3 and TREK-1 channel blockers were applied in cortical mouse slices in an attempt to increase the output of PV cells. The blockade of either channel did not successfully increase the amplitude of PV cell-evoked inhibitory postsynaptic currents (IPSCs) onto principal cells. However, while the blockade of TASK-1/3 channels failed to depolarise the membrane or alter the input resistance, the blockade of TREK-1 channels resulted in a small but significant depolarisation of the membrane potential in PV cells. Interestingly, TREK-1 channel blockade also increased action potential firing of PV cells in response to given current stimuli, suggesting that TREK-1 could be a useful target for PV cell modulation. These results demonstrate for the first time the functional effects of using specific two-pore K+ channel blockers in PV cells. Furthermore, these data provide electrophysiological evidence against the functional expression of TASK-1/3 in PV cells. It could therefore be interesting to further characterise the precise subtypes of leak K+ channels responsible for their low resistivity. This would help to classify the key contributors of the background K+ conductances present in PV cells in addition to finding suitable targets to increase their activity.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains vii, 218 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Neurons are constantly integrating information from external and internal sources, causing them to spike at particular times. The exact timing of spikes is determined by a neuron's intrinsic properties, as well as the interplay between local excitatory and inhibitory inputs. Although inhibitory interneurons have been extensively studied, their contribution to neuronal integration and spike timing remains poorly understood. To elucidate the functional role of GABAergic interneurons during cortical activity, we combined molecular identification of interneurons, two photon imaging and electrophysiological recordings in mouse thalamocortical slices. In this preparation, cortical UP states, a network state characterized by prolonged periods of depolarization and synchronized spiking, can be evoked by thalamic stimulation and can also occur spontaneously. To assay the role of inhibition, we first characterized the firing properties of Parvalbumin (PV) and Somatostatin (SOM) interneurons during UP states activity, and found a higher probability and rate of spiking in these two subtypes compared to excitatory cells. These subtypes did not display differential timing of activation during the evoked response. Furthermore, calcium imaging showed low correlations among PV and SOM interneurons, indicating that neurons sharing these neurochemical markers do not coordinate their firing. Intracellular recordings confirmed that nearby interneurons, known to be electrically coupled, do not display more synchronous spiking than excitatory cells, suggesting that this coupling may not function to synchronize the activity of interneurons on fast time scales¬¬¬. After characterizing inhibitory interneuron outputs, we next studied the timing and correlation of inhibitory inputs, which we isolated from excitatory inputs by voltage clamping at the reversal for excitation (0mV) or inhibition (-70mV). In both thalamically triggered and spontaneous activations, IPSCs between cell pairs were remarkably well correlated, with correlation coefficients reaching over .9 in some cases. This high degree of correlation has previously been assumed to be due to interneuron synchrony, but our population imaging and paired recordings did not support this view. In addition, we found that the connection rate between interneurons is very high (~80%), and quantal analysis revealed that each IPSC recorded in neighboring cells during an UP state could be due to a single presynaptic interneuron. Therefore, we explain the high IPSCs correlations in nearby pyramidal cells are emerging from the common input from individual interneurons, rather than from synchronization of interneuron activity across the population. In a final set of experiments, we found that a partial pharmacological block of inhibitory signaling increased EPSC correlations. Our data support a model in which inhibitory neurons do not fire in a correlated fashion but have strong, dense connections to pyramidal neurons that serve to prevent local excitatory synchrony during UP states. This would mean that inhibition may not, as previously thought, serve to synchronize the firing of excitatory cells, but have precisely the opposite effect, decorrelating their activity by breaking down their coordinated firing. This is consistent with the hypothesis that pyramidal cells are carrying out an essentially integrative function in the circuit and that interneurons expand the temporal dynamic range of this integration.

In this thesis, I describe the use of two different techniques for the targeting and functional inactivation of individual populations of GABAergic interneurons located within the cerebellum and dentate gyrus of the hippocampus. Through functional inactivation of these interneuron types, I demonstrate their behavioural relevance for the processes of learning and memory. In chapter 2, I describe a genetic approach for the removal of GABAA-mediated signalling from molecular layer interneurons (MLIs) onto Purkinje cells within the cerebellum. Using the Cre lox P system to delete post-synaptic GABAA receptors on Purkinje cells, I have shown a previously unappreciated role for MLIs in fear memory. Deficits were specific to the acquisition and long-term retention of fear memories suggesting that feed-forward inhibition from MLIs onto Purkinje cells is critical for these processes. In chapter 3, I describe a further development to this project through the creation of a novel dual recombinase mouse line. This intersectional approach of combining Cre and Flpo recombinase systems together would allow direct targeting of MLIs for the first time and circumvent drawbacks associated with using a static genetic knockout approach. In chapter 4, I describe an adeno-associated viral (AAV) approach to target a specific population of GABAergic interneurons located within the hilar region of the dentate gyrus. These hilar perforant path-associated (HIPP) cells are characterised by their expression of the neuropeptide somatostatin (SST) and regulate granule cell activity through feedback inhibition. However, up until now their behavioural relevance has been unknown. Through Cre-mediated viral expression of tetanus toxin light chain (TeLC), neurotransmission was prevented in SST interneurons revealing an involvement in spatial working memory and spatial reference memory precision. In addition, preliminary immediate early gene data suggests that SST interneurons increase memory precision through maintaining the sparse activity of the granule cell population through feedback inhibition.

Thesis (doctoral)--Umeå Universitet, Sweden, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Umeå Universitet, Sweden, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Local inhibitory microcircuit of amygdala is an active component in processing emotional information. Despite prominent evidence of its importance, our understanding of GABAergic cell types, their connectivity and role in amygdala network is limited. The aim of this thesis is to understand connectivity and physiology of two specific components of GABAergic microcircuit of amygdala: so-called intercalated cells and neuropeptide Y (NPY) expressing interneurons. Intercalated cells (ITCs) of the amygdala are clusters of GABAergic neurons that surround the basolateral complex of amygdala (BLA). There is growing evidence suggesting that ITCs are required for the expression of fear extinction. The main intercalated nucleus (Im) is the largest of the ITC clusters and could be also important for emotional processing. Using patch-clamp whole-cell recordings paired with subsequent anatomical analysis I described basic physiology and anatomy of neurons within the Im. I found that these neurons share common characteristics to earlier described neurons within the medial ITC cluster, yet they can be divided into three distinct groups. Next, I provided anatomical and functional evidence that Im neurons project to central and basal nucleus of amygdala and that they are reciprocally connected with medial and lateral ITCs clusters. I found that Im neurons receive excitatory inputs from BLA as well as cortex; next I verified that heterogeneous inputs do not interact with each other. I have shown that the Im neurons express both AMPA and NMDA receptors, suggesting that they may undergo NMDA-dependent plasticity. I have reported that dopamine hyperpolarizes Im neurons via dopamine receptor 1, therefore providing a cellular substrate for disinhibition of the amygdala at the systemic level. Thus, the Im is likely to be an additional site of integration of the distributed network underlying acquisition, expression and extinction of conditioned fear. In another project, I report novel interneuron type of the BLA and call it neurogliaform cell (NGFC) of amygdala. I used a mouse line expressing green fluorescent protein (GFP) under NPY promoter and patch clamp technique combined with pharmacology and electron microscope analysis. I performed paired recordings between presynaptic NPY-GFP positive (+) cells and postsynaptic principal neurons (PNs). Presynaptic NPY-GFP+ neurons display small soma and short dendrites embedded in a cloud of highly arborized axon. I showed that NPY-GFP+ cells are source of GABAA receptor-mediated slow inhibitory postsynaptic currents (IPSCs, decay time constant > 30 ms) evoked in PNs and in themselves (autapses). These slow IPSCs are known in literature as GABAA,slow. My results indicate that the slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. Physiologically-relevant in vivo firing re-played in NPY+-NGFCs in vitro evoked a transient depression of the IPSCs. Presynaptic GABAB receptors controlled the strength of this short-term plasticity. Interestingly, synaptic contacts made by NGFCs showed close appositions, without identifiable classical synaptic structures, between presynaptic boutons of the recorded cells and postsynaptic profiles. Thus, volume transmission of GABA is likely to be generated by this interneuron of the amygdala. NPY+-NGFC is a novel interneuron type of the BLA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Hippocampal GABAergic interneuron deficits are implicated in the pathophysiology of schizophrenia. Postmortem histological analyses show alteration in number and/or function of parvalbumin-expressing (PV+) GABAergic interneurons in the cerebral cortex of these patients. A parallel line of research using functional imaging of cerebral blood flow or volume has shown that hyperactivity of the hippocampus may contribute to psychotic symptoms as well as cognitive deficits in schizophrenia. It is not known if changes in GABA transmission, particularly in the number and function of PV+ interneurons, are causally related to hippocampal hyperactivity and expression of behavioral and cognitive abnormalities in schizophrenia. To help answer this question, we used genetic mouse models with deficits in cortical GABAergic interneuron development to test the hypothesis that a selective deficit in PV+ interneurons in the hippocampus can lead to schizophrenia relevant phenotypes such as hippocampal hyperactivity, dysregulation of the mesolimbic dopamine system, enhanced psychomotor responsiveness to amphetamine, and disruption of hippocampal dependent cognition. Here I describe my studies primarily on a mouse model with a deletion of the cell-cycle gene cyclin D2 (cD2 null). This mutation disrupts interneuron development in the medial ganglionic eminence (MGE), leading to a partial and selective deficit in PV+ interneurons in the neocortex and the hippocampus. I show that the cD2 null mouse shows regionally heterogeneous, persistent structural and functional deficit in PV+ interneurons, with a relatively larger and more functional deficit in the hippocampus. The GABAergic deficit in the hippocampus is associated with signs of disinhibition, such as increased cerebral blood volume as found by functional magnetic resonance imaging (fMRI).Upon establishing the evidence for hippocampal disinhibition in the cyclin D2 null mouse, I examined the relationship between this disinhibition and two areas of neural function know to be altered in psychosis and schizophrenia: Mesostriatal DA system function and hippocampus-mediated cognition. I found that the cD2 null mice showed increased dopamine population activity in the ventral tegmental area and enhanced psychomotor response to amphetamine. The latter was eliminated by a partial lesion of the ventral hippocampus, indicating hippocampal disinhibition as the driver of DA neuron dysregulation. In addition, cD2 null mice showed deficits in cognitive functions that recruit and depend on the hippocampus, such as the contextual and cued fear conditioning. Lastly, to test for a causal relationship between the PV+ interneuron deficit in the hippocampus, and the abnormalities in hippocampal metabolism, imaging phenotype, the mesolimbic dopamine dysfunction and contextual learning and memory, I examined the effects of replacing GABAergic interneurons to the hippocampus. I used transplantation of GABAergic interneuron precursors derived from the medial ganglionic eminence (MGE) into the adult hippocampus of cyclin D2 null mutants. MGE-derived progenitor cells developed into structurally and functionally mature PV+ and other GABAergic cells, and normalized hippocampal hypermetabolism. In addition, the MGE transplants normalized VTA dopamine cell activity, normalized amphetamine sensitivity and improved hippocampus-dependent learning and memory. Taken together, these studies establish the plausibility of a causal relationship between hippocampal PV+ interneuron pathology and psychosis-relevant pathophysiological and cognitive phenotypes. Moreover, they provide a rationale for limbic cortical GABAergic-interneuron-targeted treatment strategies in psychotic disorders.

Locomotion, including running, walking, and swimming, is a complex behavior enabling animals to interact with the environment. Vertebrate locomotion depends upon sets of interneurons in the spinal cord, known as the central pattern generator (CPG). The CPG performs multiple roles: pattern formation (left-right alternation and flexor-extensor alternation) and rhythm generation (the onset and frequency of locomotion). Many studies have begun to unravel the organization of the neuronal circuits underlying left-right and flexor-extensor alternation. However, despite pharmacologic, lesion, and optogenetic studies suggesting that the rhythm generating neurons are ispilaterally-projecting glutamatergic neurons, the precise cellular identification of rhythm generating neurons remains largely unknown. Traditionally, CPG networks (both pattern formation and rhythm generation) are thought to reside upstream of motor neurons, which serve as the output of the spinal cord. Recently however, it has been discovered that direct stimulation of lumbar motor neurons using the intact ex vivo neonate mouse spinal cord preparation can activate CPG networks to produce locomotor-like behavior. Furthermore, depressing motor neuron discharge decreases locomotor frequency, whereas increasing motor neuron discharge accelerates locomotor frequency, suggesting that motor neurons provide ongoing feedback to the CPG. However, the circuit mechanisms through which motor neurons can influence activity in the CPG in mammals remain unknown. Here, I used motor neurons as a means of accessing CPG interneurons by asking how motor neuron activation might induce locomotor-like activity. Through intracellular recording and morphological assays, I discovered that ventral spinocerebellar tract (VSCT) neurons are activated monosynaptically following motor neuron axon stimulation through chemical and electrical synapses. A subset of VSCT neurons were located close to or within the motor neuron nucleus. VSCT neurons were found to be excitatory, have descending spinal axon collaterals, and influence motor neuron output, suggesting that VSCT neurons are positioned advantageously to initiate and maintain locomotor-like rhythmogenesis. Intracellular recording from VSCT neurons revealed that they exhibit rhythmic activity during locomotor-like activity. VSCT neurons were found to contain the rhythmogenic pacemaker Ih current and to be connected to other VSCT neurons, at least through gap junctions. Optogenetic and chemogenetic manipulation of VSCT neuron activity provided evidence that VSCT neurons are both necessary and sufficient for the production of locomotor-like activity. Silencing VSCT neurons prevented the induction of such activity, whereas activation of VSCT neurons was capable of inducing locomotor-like activity. The production of locomotor-like activity by VSCT neuron photoactivation was dependent upon both electrical communication through gap junctions as well as the pacemaker Ih current. The evidence presented in this thesis suggests that VSCT neurons are critical components for rhythm generation in the mammalian CPG and are key mediators of locomotor activity.

During nervous system development, thousands of distinct neuronal cell types are generated and assembled into highly precise circuits. The proper wiring of these circuits requires that developing neurons recognize their appropriate synaptic partners. Analysis of a vertebrate spinal circuit that controls motor behavior reveals distinct synaptic connections of two types of inhibitory interneurons, a ventral V1 class that synapses with motor neurons and a dorsal dI4 class that selectively synapses with proprioceptive sensory neuron terminals that are located on or in close proximity to motor neurons. What are the molecular and cellular programs that instruct this remarkable synaptic specificity? Are only subsets of these interneurons capable of integrating into this circuit, or do all neurons within the same class behave similarly? The ability to answer such questions, however, is hampered both by the complexity of the spinal cord, where many different neuronal cell types can be found synapsing in the same area; as well as by the challenge of obtaining enough neurons of a particular subtype for analysis. Meanwhile, pluripotent stem cells have emerged as powerful tools for studying neural development, particularly because they can be differentiated to produce large amounts of diverse neuronal populations. Mouse embryonic stem cell-derived neurons can thus be used in a simplified in vitro system to study the development of specific neuronal cell types as well the interactions between defined cell types in a controlled environment. Using stem cell-derived neurons, I investigated how the V1 and dI4 cardinal spinal classes differentiate into molecularly distinct subtypes and acquire cell type-specific functional properties, including synaptic connectivity. In Chapter Two, I describe the production of lineage-based reporter stem cell lines and optimized differentiation protocols for generating V1 and dI4 INs from mouse embryonic stem cells, including confirming that they have molecular and functional characteristics of their in vivo counterparts. In Chapter Three, I show that a well-known V1 interneuron subtype, the Renshaw cell, which mediates recurrent inhibition of motor neurons, can be efficiently generated from stem cell differentiation. Importantly, manipulation of the Notch signaling pathway in V1 progenitors impinges on V1 subtype differentiation and greatly enhances the generation of Renshaw cells. I further show that sustained retinoic acid signaling is critical for the specific development of the Renshaw cell subtype, suggesting that interneuron progenitor domain diversification may also be regulated by spatially-restricted cues during embryonic development. In Chapter Four, using a series of transplantation, rabies virus-based transsynaptic tracing, and optogenetics combined with whole-cell patch-clamp recording approaches, I demonstrate that stem cell-derived Renshaw cells exhibit significant differences in physiology and connectivity compared to other V1 subpopulations, suggesting that synaptic specificity of the Renshaw cell-motor neuron circuit can be modeled and studied in a simplified in vitro co-culture preparation. Finally, in Chapter Five, I describe ongoing investigations into molecular mechanisms of dI4 interneuron subtype diversification, as well as approaches to studying their synaptic specificity with proprioceptive sensory neurons. Overall, my results suggest that our stem cell-cell based system is well-positioned to probe the functional diversity of molecularly-defined cell types. This work represents a novel use of embryonic stem cell-derived neurons for studying inhibitory spinal circuit assembly and will contribute to further understanding of neural circuit formation and function during normal development and potentially in diseased states.