Cholinergic interneurons are relatively rare neurons in the rat striatum. These sparsely distributed neurons display a synchronous pause in their tonic firing pattern during reward-related learning. It has been hypothesised that a specialised fast-conducting crossed-corticostriatal pathway is involved in synchronising the pause in tonic firing of these interneurons. This study aimed to detail the innervation of cholinergic interneurons by mapping their proximal and distal inputs and to describe the innervation of the crossed-corticostriatal pathway in male Wistar rats. In vivo electrophysiological recording methods were used to label single crossed-corticostriatal neurons but inadequately labeled their axons. Thus, an anterograde neuronal tracing study was conducted. Biotinylated dextran amine (BDA; 1.2 [mu]l) was pressure-injected into the left cerebral hemisphere. Six days later, the rat was perfused-fixed and the brain sectioned. BDA-labelled axons were traced to both the ipsilateral and contralateral striata. Cholinergic interneurons in the right striatum were double-immunolabelled using an optimised protocol including a polyclonal rabbit anti-m2-muscarinic receptor antibody and a monoclonal goat anti-choline acetyltransferase antibody. All sections were processed for transmission electron microscopy. Serial ultrathin sections were montaged and distal (from non BDA-labelled tissue) and proximal synapses were each mapped separately. A reconstructed distal dendrite from a cholinergic interneuron, located 225 [mu]m from the soma, was analysed. It had an average width of 1 .25[mu]m and 0.726 synapses per [mu]m. This was compared to dendrites in the same tissue and from BDA-labelled tissue. Two dendrites were presumed to be distal profiles of either cholinergic or somatostatin interneurons, while the third was thought to belong to another interneuronal cell type. In terms of surface area, there were less somal synapses compared to those made onto the distal dendrite of the cholinergic interneuron. Somal synapse counts were similar to those reported previously from our laboratory, where symmetric synapses were most common. Crossed-corticostriatal BDA-labelled axons were found to course across proximal dendrites and somas of immunolabelled cholinergic interneurons. Varicosities from these axons were found in close proximity to proximal dendrites and somas of cholinergic interneurons. Of all cholinergic interneurons in an adjacent section, 77% showed closely associated proximal varicosities. Of these, 76% of varicosities were associated with the soma, 11% to proximal dendrites and 13% to both locations. Twenty-nine BDA-labeled axons were analysed using transmission electron microscopy. Most were observed making asymmetric synaptic contact with unlabelled spines. In two cases spines were traced to medium spiny projection neurons. Two axon segments were seen touching the proximal regions of separate cholinergic interneurons. At these contact sites interrupted membrane thickenings were observed. It is proposed here that synapses may form at these sites during reward-related learning. However labelling of the contact sites with a postsynaptic marker would be necessary to confirm their synaptic nature. The current study has gathered information about the distal and proximal innervation patterns of these neurons and described the termination pattern of the crossed-corticostriatal pathway in relation to these neurons for the first time. These findings support the crossed-corticostriatal pathway as one possible anatomical substrate for synchronising the pause response on both sides of the brain.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Chez les rongeurs, les interneurones (INs) corticaux sont issus de l’éminence ganglionnaire (EG) médiale (MGE) et caudale (CGE), expriment une combinaison de facteurs définis et migrent tangentiellement puis radialement pour atteindre leur position laminaire définitive. La diversité et la spécification des sous-types d’INs provenant de la MGE ont suscité de nombreuses études, en revanche les mécanismes moléculaires contrôlant la migration et la spécification des INs issus de la CGE demeurent toujours obscurs. Dans cette étude, les voies de migration de ces INs ont été examinées grâce à une lignée de souris rapportrices des interneurones issus de la CGE avant d’analyser le rôle de deux facteurs de transcription, COUP-TFI et COUP-TFII, hautement exprimés dans la CGE. Deux voies de migration non précédemment caractérisées ont alors été identifiées : une voie dorsale (CLMS) où les INs migrent vers l’EG latérale (LGE) et une voie ventrale (CMMS) où les INs migrent vers la MGE. Le CLMS et le CMMS ont donc été analysés, ainsi que la voie de migration caudale (CMS), à différents stades de développement et l’expression spécifique de certains gènes a pu être identifiée. En inactivant conditionnellement COUP-TFI et/ou COUP-TFII dans les INs, les voies de migration sont altérées ainsi que l’expression des marqueurs moléculaires. Comme probable conséquence, les souris mutantes adultes montrent une distribution altérée des sous-populations d’INs en particulier de celles issues de la CGE. Mon étude a donc permis d’identifier et de caractériser deux nouvelles voies de migration pour les INs provenant de la CGE et a montré que COUP-TFs contribuent à leur modulation. / In rodents, cortical interneurons (INs) originate from the medial (MGE) and caudal ganglionic eminence (CGE) according to precise temporal schedules, express a defined combination of factors, and reach their final laminar position through tangential and radial cell migration. The diversity and fate-specification of MGE-derived interneuron subtypes are well characterized however the molecular mechanisms controlling the migration and specification of CGE-derived INs are still vague. In this study, I have first investigated the migratory paths of cortical INs using a reporter line specific to the CGE, and then I have assessed the involvement of COUP-TFI and COUP-TFII, which are highly expressed in the embryonic CGE during development, in these paths. My data unravelled two major previously non-characterized migratory streams from the subpallium to the pallium: a dorsal stream (CLMS) in which CGE-derived cells migrate to the lateral GE (LGE), and a ventral one (CMMS) in which CGE-derived cells migrate to the MGE. I have characterized both streams and the already well-described caudal stream (CMS) during different stages of development and identified a series of genes expressed in the migrating cells. By inactivating COUP-TFI and/or COUP-TFII in the developing INs, these streams together with their molecular marker expression are perturbed. As a consequence, adult mutant mice have an altered distribution of interneuron subpopulations, particularly the ones derived from the CGE. Taken together, my study identified and characterized two novel CGE-derived interneuron migratory routes to the cortex and showed that COUP-TFs contribute in modulating these paths.

The striatum is the largest nucleus of the basal ganglia, and acts as a point of convergence for thalamic, cortical and midbrain inputs. It is involved in both motor and associative forms of learning, and is composed of spiny projection neurons (SPNs) whose output along the so-called "direct pathway" and "indirect pathway" is modified by the activity of diverse sets of interneurons. Four "classical" or major classes of striatal interneuron can be identified according to the selective expression of the molecular markers parvalbumin (PV), calretinin (CR), nitric oxide synthase (NOS) or choline acetyltransferase (ChAT). Although the interneurons within a class are generally considered to be homogeneous in form and function, there is emerging evidence that some classes encompass multiple types of neuron, and that the heterogeneity in striatal interneurons extends beyond these four classes. Defining the extent of interneuron heterogeneity is important for understanding how the striatum processes distinct, topographically-organized inputs from the cortex and thalamus in order to govern a wide range of behaviors. To address these issues, a combination of immunofluorescence microscopy and stereological cell counting approaches was used in striatal tissue from rat, mouse and non-human primate. This was supplemented by in vivo recording and juxtacellular labelling of single neurons in rat. A first set of experiments showed that secretagogin (Scgn), a calcium-binding protein, is expressed by a large number of interneurons in the dorsal striatum of rat and primate, but not in the mouse. In all species tested, secretagogin was expressed by a subset of PV+ interneurons and a subset of CR+ interneurons in the dorsal striatum, but also labelled a group of interneurons that did not express any of the classical markers of striatal interneurons. A second set of experiments in the rat demonstrated that the selective co-expression of Scgn by PV+ interneurons delineates two topographically-, physiologically- and morphologically-distinct cell populations. These topographical differences in distribution were largely conserved in the primate caudate/putamen. In rats, PV+/Scgn+ and PV+/Scgn- interneurons differed significantly in their firing rates, firing patterns and phase-locking to cortical oscillations. The axons of PV+/Scgn+ interneurons were more likely to form appositions with the somata of direct pathway SPNs than indirect pathway SPNs, whereas the opposite was true for the axons of PV+/Scgn- interneurons. These two populations of GABAergic interneurons provide a potential substrate through which either of the striatal output pathways can be rapidly and selectively inhibited, and in turn mediate the expression of behavioral routines. A third set of experiments showed that CR+ interneurons of the dorsal striatum can be separated into three populations based on their molecular, topographical and morphological properties. Small-sized ("Type 3") CR+ interneurons co-expressed Scgn and were restricted in their distribution towards the rostro-medial poles of the striatum in both rats and primates. In rats, these neurons also expressed the transcription factor SP8, suggesting that they may be newly generated throughout adulthood. Large-sized, ("Type 1") CR+ interneurons did not express Scgn, but could be further distinguished by their expression of the transcription factor Lhx7. Medium-sized ("Type 2") CR+ interneurons did not express Scgn or Lhx7, and had heterogeneous electrophysiological properties in vivo. The expression of Scgn, but not other classical interneuron markers, identified a group of interneurons that were restricted in their distribution towards the ventro-medial aspects of the dorsal striatum. A fourth set of experiments showed that these neurons are also present in the core and the shell of the nucleus accumbens. Unlike the case of dorsal striatum, however, PV+ interneurons and CR+ interneurons of the nucleus accumbens did not co-express Scgn. Moreover, many of the interneuron populations studied had greater densities in the ventral striatum compared to the dorsal striatum, and had quantifiably strong biases in their distribution towards a variety of axes within both the core and the shell of the nucleus accumbens. These data thus highlight some major differences in the constituent elements of the microcircuits of dorsal striatum and nucleus accumbens. In conclusion, these studies have revealed a great deal of molecular, topographical, electrophysiological and structural heterogeneity within the interneuron populations of the striatum. As several of these interneuron populations were not evenly distributed throughout the striatum, this ultimately suggests that the microcircuit of the striatum is specialized according to regions that differ in their cortical, thalamic and dopaminergic inputs.

Earlier human and animal studies have indicated abnormal striatal GABAergic interneurons relating to autism spectrum disorder’s (ASD) core features such as stereotypic repetitive behaviors, impaired language and motor skills, and social interactions. Purkinje cells (PCs) in the cerebellum are of great interest in ASD; earlier research has reported a loss of PCs, irregularities within deep cerebellar nuclei, a lower level of GAD67 (glutamic acid decarboxylase) mRNA expressed on PCs, and reduced parvalbumin (PV)-positive interneurons in cortex and hippocampus. In this study, in-situ hybridization was used to quantify the levels of PV mRNA in PCs in post-mortem human autism and control cerebellum sections. Two-tailed t-test analysis of the data showed a significant decrease (p<0.05) in PV mRNA level on PCs in autism compared to control sections. In addition, when comparing two groups (seizure and no seizure) in autism sections, no statistical significance was observed. Post-mortem interval (PMI) and age was compared between the PV mRNA levels in autism and control. Only weak negative correlation was found among age and PV mRNA levels in both groups. This report of decreased PV mRNA level in autism cases further supported previous research findings related to PCs and also confirmed interference with the inhibitory function of PCs to deep cerebellar nuclei and the cortex resulting in behavioral and motor impairments in ASD.

No description available.

Striatal oscillatory activity associated with movement, reward, and decision-making is observed in several interacting frequency bands. Rodent striatal local field potential recordings show dopamine- and reward-dependent transitions between a 'spontaneous' state involving beta (15-30 Hz) and low gamma (40-60 Hz) and a 'dopaminergic' state involving theta (4-8 Hz) and high gamma (60-100 Hz) activity. The mechanisms underlying these rhythmic dynamics and their functional consequences are not well understood. In this thesis, I construct a biophysical model of striatal microcircuits that comprehensively describes the generation and interaction of these rhythms as well as their modulation by dopamine and rhythmic inputs, and test its predictions using human electroencephalography (EEG) data. Chapter 1 describes the striatal model and its dopaminergic modulation. Building on previous work suggesting striatal projection neuron (SPN) networks can generate beta oscillations, I construct a model network of striatal fast-spiking interneurons (FSIs) capable of generating delta/theta (2-6 Hz) and gamma rhythms. This FSI network produces low gamma oscillations under low (simulated) dopaminergic tone, and high gamma activity nested within a delta/theta oscillation under high dopaminergic tone. In a combined model under high dopaminergic tone SPN network beta oscillations are interrupted by delta/theta-periodic bursts of gamma-frequency FSI inhibition. This high dopamine-induced periodic inhibition may enable switching between beta-rhythmic SPN cell assemblies representing motor programs, suggesting that dopamine facilitates movement in part by allowing for rapid, periodic changes in motor program execution. Chapter 2 describes the model's response to square-wave periodic cortical inputs. Comparing models with and without FSIs reveals that the FSI network: (i) prevents the SPN network's generation of phase-locked beta oscillations in response to beta's harmonic frequencies, ensuring fidelity of transmission of cortical beta rhythms; and (ii) limits or entrains SPN activity in response to certain gamma frequency inputs. Chapter 3 describes an analysis of phase-amplitude coupling at cortical electrodes in human EEG data during a reward task. The alternating rhythms predicted by the model appear in response to positive feedback. While the origins of these rhythms remain unclear, if they represent striatal signals, they provide a direct link between human behavior and striatal cellular function.

The spatial patterning of neurons is a fundamental problem in neuroscience. The functions of the brain are rooted in the cellular architecture that underlies the structure of the brain. In the cerebral cortex, the functions of the cortex depend on the proper assembly of circuits made up of long-range excitatory neurons and locally-projecting inhibitory interneurons. Interneurons are incredibly diverse from a morphological and functional perspective and are found in every cortical area. Unlike excitatory cortical neurons, interneurons are born outside of the cortex and migrate long distances into the cortex and distribute across the cortex broadly. How do these diverse cells that essentially invade the cortex properly distribute? How do different developmental stages contribute to the final patterning of interneuron subtypes, and what are the molecules that influence this process? In this dissertation, I will present my original research which has advanced our knowledge of the answers to these fundamental questions in the field of developmental neuroscience. I addressed these questions by applying a range of techniques including mouse genetics, immunohistochemistry, confocal microscopy, and point pattern analysis. My research has shown that cortical interneuron subtypes are spatially independent. Spatial patterns of cortical interneuron subtypes are non-random within subtypes, but are randomly positioned with respect to other subtypes. I also explored the effects of loss of diversity within the clustered protocadherin family of adhesion molecules. Though these molecules do not appear to play a role in subtype specific spatial independence, I found that loss of clustered protocadherin diversity alters the density and laminar distribution of cortical interneuron subtypes. I also contributed to the development of genetic tools which could help us further understand how developmental stages contribute to final interneuron distribution. My original research has collectively advanced our knowledge of how cortical interneurons achieve their final distributions during development and has opened up new avenues of scientific inquiry for future research in developmental neuroscience.

Calretinin (CR) and parvalbumin (PV) neurons are inhibitory interneurons (INs) that play important roles in the modulation of excitatory pyramidal neurons. They are found in many species are and throughout the neocortex. However, their characteristics vary between species and brain region. The aim of this study was to compare the density, distribution, and inhibitory signaling of CR and PV neurons in monkey primary visual cortex (V1), monkey lateral prefrontal cortex (LPFC), mouse V1 and mouse frontal cortex (FC). Coronal brain slices from each of the species and brain regions were stained using immunohistochemistry and then the slices were scanned using high-resolution confocal imaging. High resolution image stacks were used to count the somata of CR and PV. The vesicular gamma aminobutyric acid (GABA) transporter (VGAT), CR and PV particles were analyzed to quantify these inhibitory markers in monkey V1, LPFC, and mouse V1 and FC. There were significant differences in the laminar distribution of CR and PV neurons in that CR neurons were concentrated in L2/3 and PV neurons were concentrated in L2-5. In L2/3, Monkey V1 had more CR neurons than did monkey LPFC. Furthermore, there were a greater number of PV neurons in monkey and mouse V1 compared to monkey LPFC and mouse FC. In L2/3, monkey V1 had the highest number of PV neurons. In L5, there significantly greater PV neurons in mouse V1 compared to monkey V1. There was significantly higher density of CR neurons in the upper middle layers of Monkey V1 compared to mouse V1 and monkey LPFC compared to mouse FC. The upper middle layers of monkey V1 had significantly higher density of PV neurons compared to monkey LPFC and mouse V1. There was significantly higher density of VGAT particles in monkey V1 and LPFC compared to mouse V1 and FC, which indicates more inhibitory synapses. There were significantly more VGAT+ boutons colocalized with PV+ boutons than CR+ boutons. Finally, discriminant analysis and hierarchical cluster analysis show that species is the largest separating factor between monkey V1, LPFC and mouse V1 and FC. Mouse V1 and FC are very similar, and monkey V1 and LPFC are dissimilar from one another. This data, united with comparative data on pyramidal neurons, demonstrates that neurons have differences between species, and monkeys have more regional specialization than mice.

Neural circuits within the hippocampus, a mammalian brain structure critical for both the encoding and consolidation of episodic memories, are composed of intimately connected excitatory pyramidal cells and inhibitory interneurons. While decades of research have focused on how the in vivo physiological properties of pyramidal cells may support these cognitive processes, and the anatomical and physiological properties of interneurons have been extensively studied in vitro, relatively little is known about how the in vivo activity patterns of interneurons support memory encoding and consolidation. Here, I have utilized Acousto-Optic Deflection (AOD)-based two-photon calcium imaging and post-hoc immunohistochemistry to perform large-scale recordings of molecularly-defined interneuron subtypes, within both CA1 and CA3, during various behavioral tasks and states. I conclude that the subtype-specific dynamics of inhibitory circuits within the hippocampus are critical in supporting its role in memory encoding and consolidation.