The Reelin signaling pathway is critical for neuronal migration during development and the function of excitatory synapses in the adult forebrain. Despite a growing body of evidence implicating impaired Reelin signaling in the pathogenesis of numerous neuropsychiatric and neurodegenerative disorders, including Schizophrenia and Alzheimer's disease, little is known still regarding the specific molecular and cellular mechanisms whereby Reelin signaling modulates the function of synapses to enable normal learning and memory. In this dissertation, we addressed these knowledge gaps by identifying mechanisms of Reelin proteolysis following synaptic potentiation (Chapter 2) and dissociated the synaptic function of Reelin signaling at excitatory (Chapter 3) and inhibitory synapses (Chapter 4). In the adult brain, Reelin is secreted by GABAergic interneurons into the extracellular space, after which it is cleaved by unknown proteases to generate active fragments that signal downstream. In Chapter 1, we demonstrate that tissue plasminogen activator (tPA) and its major in vivo substrate, plasminogen, cleave Reelin under cell-free conditions to generate major Reelin fragments found in vivo. Since manipulation of tPA levels under basal conditions had no effect on Reelin processing, we hypothesized that synaptic activity may be required to render Reelin susceptible to proteolysis by tPA. Indeed, the modulation of Reelin processing by synaptic potentiation of ex vivo hippocampal slices required the presence of tPA. These data are the first to demonstrate a specific context in which Reelin signaling may be initiated in the intact brain and further emphasize that extracellular proteolysis of Reelin by tPA and other yet-to-be identified proteases is important to consider when trying to understand how altered Reelin processing and/or expression contribute to cognitive impairments associated with disease states. In Chapters 2 and 3 of this dissertation, we describe recent attempts by our lab to elucidate cellular mechanisms of Reelin signaling in the adult brain. To do this, we generated two conditional knockout mutants that lack the obligate downstream adaptor protein, Disabled-1 (Dab1), specifically in postnatal excitatory neurons (eKO) or GABAergic interneurons (iKO). Despite some overlap of Reelin and Dab1 in a subset of GABAergic interneurons, we found that their expression was generally juxtaposed, with Dab1 being primarily expressed by principle neurons and a more widespread population of Reelin-negative GABAergic interneurons. While eKO mice exhibited normal forebrain lamination, dendritic architecture, and dendritic spine density, they did have reductions in spine volume and a loss of basal and activity-dependent Akt and MAPK activation. These changes culminated in impairments in short-term and long-term synaptic plasticity, as well as impairments in associative learning and spatial memory. Taken together, our observations in the eKO mice are the first to definitively establish a synaptic function of Reelin signaling in the adult hippocampus. While characterizing the eKO mice, we also observed that GABAergic interneurons expressed Dab1, which motivated us to explore the inhibitory synapse as a novel locus of Reelin signaling (Chapter 4). Although loss of Dab1 in GABAergic interneurons did not affect forebrain development or the overall patterning of inhibitory synapses, iKO mice presented with an ataxic gait, resting tremor and cerebellar hypoplasia. Interestingly, loss of Dab1 in interneurons led to altered expression of some major glutamatergic synapse proteins (i.e. NMDA receptor subunits NR1 and NR2B), while other excitatory and inhibitory synapse proteins were normal (e.g. NR2A, GAD67/65, and gephyrin). Hippocampal field recordings further demonstrated that even partial loss of Dab1 expression in iHET mice, led to enhanced presynaptic activation and impaired theta-burst induced LTP. These data establish the inhibitory synapse as a novel locus of Reelin signaling in the developing and adult brain. Taken together, data discussed herein should prove useful for understanding and treating disorders associated with Reelin signaling impairments (e.g. AD and Schizophrenia).

No description available.

GABAergic interneurons in the basolateral complex (BLC) of the amygdala are a part of the emotional-learning circuitry of the brain and receive excitatory inputs from all sensory modalities via cortex and thalamus. Although the BLC, which is made up of the lateral amygdala (LA), basal amygdala (BA) and accessory basal nucleus, is under the influence of a strong inhibition brought about by local interneurons, little is known about the diversity, characteristics and functioning of these interneurons. In this study, I have characterised the BLC interneuron population using a transgenic mouse model in which enhanced green fluorescent protein has been tagged to the GAD67 promoter. This promoter is specifically expressed in all GABAergic interneurons, enabling us to visualise interneurons under UV light. Whole-cell recordings were made from GAD67 interneurons in the BLA to study their membrane and synaptic properties. On the basis of their firing properties, interneurons in the BLC were classified into six distinct groups. The calcium-binding proteins calbindin, calretinin and parvalbumin were found to be expressed differently in the LA and BA interneurons, with the majority of the interneurons in the LA expressing calretinin, whereas those in the BA mostly expressed parvalbumin. We also found diversity in the expression of postsynaptic glutamate receptors in the BLC. Long-term potentiation induced at the interneurons was specific to the cortical inputs in the LA. LTP was expressed only in interneurons that either lacked NMDA receptors or had NMDA receptors with fast decay kinetics. This form of LTP was mediated by calcium-permeable AMPA receptors and required a postsynaptic calcium rise for its induction This study shows that the interneurons in the BLC are a heterogenous population with respect to the expression of calcium-binding proteins, axonal morphology, synaptic and membrane properties. This heterogeneity in interneuron population may be essential for the specialised roles various types of interneurons play in the functioning of the amygdala and in emotional learning.

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 47-50).

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Schizophrenia is a relatively poorly understood, debilitating psychiatric disorder affecting around 0.5% of the population worldwide. The main characteristics of the disease are hallucinations, delusions and cognitive impairment such as difficulty in learning. It has been recently suggested that Disrupted-in-Schizophrenia-1 (DISC1) might be one of the main genetic risk factors for this disease. Mouse Disc1 has been implicated in brain development, mainly in neurite outgrowth, integration of newborn neurons, neuronal precursor proliferation/differentiation and neuronal migration. Disc1 function in the cortical excitatory cells was studied in fair detail but there is little data on Disc1 role in cortical interneuron development. In this study I have investigated development of the cortical interneurons in 21 days old mice with ENU-induced point mutations in the mouse Disc1 sequence - L100P and Q31L; previously characterized as ‘schizophrenic-like’ and ‘depressive-like’ respectively. Bin analysis was performed on five brain regions: frontal and central primary somatosensory (fSSp and SSp respectively) cortices, ventral auditory (vAud) cortex, visual (Vis) cortex and medial prefrontal cortex (MPFC); for four major interneuronal markers: parvalbumin (PV), somatostatin (STT), calretitnin (CLR) and glutamate decarboxylase 67 (GAD67). A significant decrease in PV (protein and mRNA) expression was observed in a subclass of the cortical interneurons in the fSSp, SSp, vAud and Vis cortices of L100P homozyogous (L100P) and heterozygous (L100P +/-) mouse brains when compared to their wild-type (WT) littermates. No such difference in the PV positive cells was found in the MPFC in the L100P mouse brain. Other interneuronal markers expression was not different in the L100P and L100P +/- brain from that in the WT littermate controls. Furthermore, there was no significant difference in any of the interneuronal markers expression in the Q31L mouse brain cortex. A minor change in the relative distribution of the interneurons (GAD67 positive cells) was found in the L100P but not Q31L brain. With no difference in the number of the interneurons and the nature of PV expression regulation, the cell non-autonomous effect of L100P Disc1 on this subpopulation of intereneurons was investigated. Overexpression of the mouse Disc1-100P in utero in the radial glia cells born at E14.5 (future layer II/III and IV excitatory cells) resulted in a significant decrease in the PV positive cells in all of the electroporated regions (fSSp, SSp, vAud and Vis cortices) when compared to mouse WT Disc1 overexpression. Furthermore, a decrease in the PV cells on the contralateral side was observed in the SSp and Vis cortices. This study demonstrates that mouse Disc1 is involved in the generation of parvalbumin expressing interneurons within the cortex in a cell non-autonomous way. The L100P point mutation in Disc1 led to downregulation of parvalbumin, which in turn would result in abnormal inhibitory properties of this interneuron subtype.

The inhibitory circuitry of the prefrontal cortex (PFC) is involved in working memory and modulation of brain oscillations. Alterations in this network and especially GABAergic cells that express cholecystokinin (CCK), parvalbumin (PV), or somatostatin (SST) may underlie some of the cognitive deficits observed in schizophrenia. To assess the involvement of CCK+, PV+, and SST+ interneurons in PFC-dependent behaviours, we selectively inactivated these in prelimbic and infralimbic PFC via virus-mediated expression of tetanus toxin light chain (TeLC). We found that functional removal of CCK+ or PV+, but not SST+ neurons leads to specific impairments in working memory, and these represent the main cognitive domains affected in schizophrenia. PV-TeLC and CCK-TeLC mice displayed significant Y-maze alternation index reduction (p < 0.05). Targeting of PV+ prefrontal cells causes anxiety-like phenotype. Moreover, PV+ and SST+, but not CCK+ interneurons, appear to play a role in latent inhibition. Functional removal of CCK+, PV+ and CCK+ cells from PFC does not affect circadian activity and does not cause anhedonia. The involvement of PV-network in generation of neuronal activity and acetylcholine homeostasis was assessed. For neurophysiological recordings, each arm of the Y-maze divided into two equal-sized zones – proximal (close to the central decision point) and distal (far end). Zone entry was event-mapped onto continues local field potential recordings from medial PFC and CA1 region of the hippocampus. PV-TeLC animals displayed significantly lower prefrontal power in the decision zone. This suggests that the PV-TeLC animals are unable to modulate neuronal activity depending on the cognitive demand. Functional removal of prefrontal PV+ interneurons also leads to disturbed acetylcholine homeostasis. These results show that prefrontal GABAergic cells drive different behaviours and control task-relevant neuronal activity in different brain regions engaged with working memory such as hippocampus. Similar signalling anomalies may thus underlie cognitive deficits found in schizophrenia.

Autism spectrum disorder (ASD) is a complex disorder with large individual variability, where every case has differences in the type and severity of symptoms. Despite the recent increase in diagnoses, scientists have advanced considerably less in their understanding of the mechanisms of ASD because few individual genes that are implicated in ASD are mutated in much more than 1% of patients. One proposed mechanism is that the dysfunction of GABAergic interneurons may play a role in the development and progression of the disorder by interrupting the excitatory and inhibitory balance of neural networks. In our research, we elucidate the role of one class of interneurons in ASD by knocking out a high-risk gene (phosphatase and tensin homologue on chromosome ten, or PTEN) selectively in somatostatinexpressing (SOM+) interneurons. Since many symptoms of autism spectrum disorder present themselves as social anxieties, we test our mouse model in a variety of settings to observe social interaction and social preference, anxiety-like behavior, and repetitive stereotyped behavior. We found that in the SOM+ conditional knockout of PTEN, mice had elevated levels of anxiety and fear recall, suggesting a potential disruption of amygdala function. We then investigated potential dysfunction at the cellular and circuit levels using confocal microscopy, electrophysiology, and 2P local circuit mapping. We found that SOM+ cells lacking PTEN were overgrown morphologically, with larger cell bodies and larger, more complex dendritic arbors. Additionally, SOM+ cells in the central amygdala (CeA) lacking PTEN had elevated levels of excitatory drive from the basolateral amygdala (BLA) as well as a drastic disruption of lateral inhibition within the CeA, seen by decreased connection probability and reduced inhibitory post synaptic currents. Given what is known about central amygdala circuitry, these deficits in CeA SOM+ neuron activity conceivably underlie the fear and anxiety-related phenotype observed in mice with a conditional SOM+ PTEN knockout. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

The medial ganglionic eminence (MGE) is a progenitor domain in the subpallium that produces both locally-projecting interneurons which undergo tangential migration in structures such as the cortex as well as long-range projection neurons that occupy subcortical nuclei. Very little is known about the transcriptional mechanisms specifying the migratory behavior and axonal projection patterns of these two broad classes of MGE-derived neurons. In this study, I identify St18 as a novel transcriptional determinant specifying projection neuron fate in the MGE lineage. St18 is transiently expressed in the MGE subventricular zone (SVZ) and mantle, and I assessed its function using an ES cell-based model of MGE development. Induction of St18 is sufficient to direct ES-derived MGE neurons to adopt a projection neuron-like identity as defined by migration and morphology. Through gene expression analysis I identified a downstream effector of St18, Cbx7, which is a component of Polycomb repressor complex 1. I find that Cbx7 is essential for projection neuron-like migration and is not involved in St18-mediated projection neuron-like morphology. Using genetic loss-of-function in mice, I find that St18 is required for the production of globus pallidus pars externa (GPe) prototypic projection neurons. Single cell RNA sequencing revealed that St18 regulates MGE output of specific neuronal populations: in the absence of St18, I observe a large expansion of cortical interneurons at the expense of putative GPe neurons. I also find that, following St18 genetic loss of function, mouse walk cycles are disrupted downstream of a loss of a critical neuronal projection from the GPe to the sub thalamic nucleus (STN). These results characterize a novel transcriptional determinant that directs GPe prototypic projection neuron identity within the MGE lineage. Further, I have identified a downstream target of St18, Cbx7, which regulates only the migratory behavior of long-range projection neurons, suggesting that specific features of MGE projection neuron identity may be governed in a compartmentalized fashion by distinct transcriptional modules downstream of St18. I’ve also demonstrated the role of the GPe PV+ prototypic neurons in the production and maintenance of mouse locomotor gait.

GABAergic inhibitory neurons are known to play a critical regulatory role in memory formation and learning. During motor learning, pyramidal neurons (PNs) of the primary motor cortex (M1) undergo spine reorganization and firing pattern refinement. Cortical PNs are directly inhibited and regulated by two inhibitory neuronal subtypes: somatostatin-expressing interneurons (SOM-INs) and parvalbumin-expressing interneurons (PV-INs). Interestingly, SOM-mediated inhibition has been shown to regulate the observed dynamics of PNs during motor learning. Despite our expanded understanding, the molecular mechanisms that underlie these processes remain unclear. Here, I identified that the immediate-early gene transcription factor, NPAS4, is selectively expressed in a subset of SOM-INs, but not in PV-INs or PNs, during the head-fixed pellet reaching motor learning task. Furthermore, I characterized its expression pattern within the SOM-INs of M1 and found that there was no change at early phases; but as training progressed, there was a gradual increase and plateau in the number of NPAS4-expressing SOM-INs. In collaboration with other lab members, we showed that Npas4 region- and cell-type specific deletion within SOM-INs of M1, impaired motor skill acquisition and disrupted the motor learning-induced spine reorganization. In addition, I validated and employed the novel NRAM system to examine if NPAS4 is continually expressed within the same subset of SOM-INs and found that an ensemble of SOM-INs repetitively express NPAS4 at various phases of learning. Lastly, chronic in vivo two-photon Ca²⁺ imaging during training showed that the ensemble of NPAS4-expressing SOM-INs had reduced activity during task-related movements compared to other SOM-INs. Together, our results reveal an important instructive role of NPAS4 within the microcircuits of M1, in which it modulates the inhibition of a distinct subset of SOM-INs during motor learning to promote spine stabilization of downstream task-related PNs that are important for motor skill acquisition.