The claustrum of the sheep and its connections to the visual cortex.

The present study analyses the organization and selected neurochemical features of the claustrum and visual cortex of the sheep, based on the patterns of calcium-binding proteins expression. Connections of the claustrum with the visual cortex have been studied by tractography. Parvalbumin-immunoreactive (PV-ir) and Calbindin-immunoreactive (CB-ir) cell bodies increased along the rostro-caudal axis of the nucleus. Calretinin (CR)-labeled somata were few and evenly distributed along the rostro-caudal axis. PV and CB distribution in the visual cortex was characterized by larger round and multipolar cells for PV, and more bitufted neurons for CB. The staining pattern for PV was the opposite of that of CR, which showed densely stained but rare cell bodies. Tractography shows the existence of connections with the caudal visual cortex. However, we detected no contralateral projection in the visuo-claustral interconnections. Since sheep and goats have laterally placed eyes and a limited binocular vision, the absence of contralateral projections could be of prime importance if confirmed by other studies, to rule out the role of the claustrum in stereopsis.

On the G Protein-Coupled Receptor Neuromodulation of the Claustrum.

G protein-coupled receptors modulate the synaptic glutamate and GABA transmission of the claustrum. The work focused on the transmitter-receptor relationships in the claustral catecholamine system and receptor-receptor interactions between kappa opioid receptors (KOR) and SomatostatinR2 (SSTR2) in claustrum. Methods used involved immunohistochemistry and in situ proximity ligation assay (PLA) using confocal microscopy. Double immunolabeling studies on dopamine (DA) D1 receptor (D1R) and tyrosine hydroxylase (TH) immunoreactivities (IR) demonstrated that D1R IR existed in almost all claustral and dorsal endopiriform nucleus (DEn) nerve cell bodies, known as glutamate projection neurons, and D4R IR in large numbers of nerve cell bodies of the claustrum and DEn. However, only a low to moderate density of TH IR nerve terminals was observed in the DEn versus de few scattered TH IR terminals found in the claustrum. These results indicated that DA D1R and D4R transmission in the rat operated via long distance DA volume transmission in the rat claustrum and DEn to modulate claustral-sensory cortical glutamate transmission. Large numbers of these glutamate projection neurons also expressed KOR and SSTR2 which formed KOR-SSTR2 heteroreceptor complexes using PLA. Such receptor-receptor interactions can finetune the activity of the glutamate claustral-sensory cortex projections from inhibition to enhancement of their sensory cortex signaling. This can give the sensory cortical regions significant help in deciding on the salience to be given to various incoming sensory stimuli.

Mice Lacking Connective Tissue Growth Factor in the Forebrain Exhibit Delayed Seizure Response, Reduced C-Fos Expression and Different Microglial Phenotype Following Acute PTZ Injection.

Connective tissue growth factor (CTGF) plays important roles in the development and regeneration of the connective tissue, yet its function in the nervous system is still not clear. CTGF is expressed in some distinct regions of the brain, including the dorsal endopiriform nucleus (DEPN) which has been recognized as an epileptogenic zone. We generated a forebrain-specific Ctgf knockout (FbCtgf KO) mouse line in which the expression of Ctgf in the DEPN is eliminated. In this study, we adopted a pentylenetetrazole (PTZ)-induced seizure model and found similar severity and latencies to death between FbCtgf KO and WT mice. Interestingly, there was a delay in the seizure reactions in the mutant mice. We further observed reduced c-fos expression subsequent to PTZ treatment in the KO mice, especially in the hippocampus. While the densities of astrocytes and microglia in the hippocampus were kept constant after acute PTZ treatment, microglial morphology was different between genotypes. Our present study demonstrated that in the FbCtgf KO mice, PTZ failed to increase neuronal activity and microglial response in the hippocampus. Our results suggested that inhibition of Ctgf function may have a therapeutic potential in preventing the pathophysiology of epilepsy.

Spexin and nesfatin-1-expressing neurons in the male human claustrum.

Neuropeptides are involved in numerous brain activities being responsible for a wide spectrum of higher mental functions. The purpose of this concise, structural and qualitative investigation was to map the possible immunoreactivity of the novel regulatory peptides: spexin (SPX) and nesfatin-1 within the human claustrum. SPX is a newly identified peptide, a natural ligand for the galanin receptors (GALR) 2/3, with no molecular structure similarities to currently known regulatory factors. SPX seems to have multiple physiological functions, with an involvement in reproduction and food-intake regulation recently revealed in animal studies. Nesfatin-1, a second pleiotropic neuropeptide, which is a derivative of the nucleobindin-2 (NUCB-2) protein, is characterized by a wide distribution in the brain. Nesfatin-1 is a substance with a strong anorexigenic effect, playing an important role in the neuronal circuits of the hypothalamus that regulate food intake and energy homeostasis. On the other hand, nesfatin-1 may be involved in several important brain functions such as sleep, reproductive behaviour, cognitive processes, stress responses and anxiety. For the first time we detected and described a population of nesfatin-1 and SPX expressing neurons in the human claustrum using immunohistochemical and fluorescent methods. The study presents the novel identification of SPX and nesfatin-1 immunopositive neurons in the human claustrum and their assemblies show similar patterns of distribution in the whole structure.

Claustrum and dorsal endopiriform cortex complex cell-identity is determined by Nurr1 and regulates hallucinogenic-like states in mice.

The Claustrum/dorsal endopiriform cortex complex (CLA) is an enigmatic brain region with extensive glutamatergic projections to multiple cortical areas. The transcription factor Nurr1 is highly expressed in the CLA, but its role in this region is not understood. By using conditional gene-targeted mice, we show that Nurr1 is a crucial regulator of CLA neuron identity. Although CLA neurons remain intact in the absence of Nurr1, the distinctive gene expression pattern in the CLA is abolished. CLA has been hypothesized to control hallucinations, but little is known of how the CLA responds to hallucinogens. After the deletion of Nurr1 in the CLA, both hallucinogen receptor expression and signaling are lost. Furthermore, functional ultrasound and Neuropixel electrophysiological recordings revealed that the hallucinogenic-receptor agonists' effects on functional connectivity between prefrontal and sensorimotor cortices are altered in Nurr1-ablated mice. Our findings suggest that Nurr1-targeted strategies provide additional avenues for functional studies of the CLA.

Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum.

The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

The claustrum-prelimbic cortex circuit through dynorphin/κ-opioid receptor signaling underlies depression-like behaviors associated with social stress etiology.

Ample evidence has suggested the stress etiology of depression, but the underlying mechanism is not fully understood yet. Here, we report that chronic social defeat stress (CSDS) attenuates the excitatory output of the claustrum (CLA) to the prelimbic cortex (PL) through the dynorphin/κ-opioid receptor (KOR) signaling, being critical for depression-related behaviors in male mice. The CSDS preferentially impairs the excitatory output from the CLA onto the parvalbumin (PV) of the PL, leading to PL micronetwork dysfunction by disinhibiting pyramidal neurons (PNs). Optogenetic activation or inhibition of this circuit suppresses or promotes depressive-like behaviors, which is reversed by chemogenetic inhibition or activation of the PV neurons. Notably, manipulating the dynorphin/KOR signaling in the CLA-PL projecting terminals controls depressive-like behaviors that is suppressed or promoted by optogenetic activation or inhibition of CLA-PL circuit. Thus, this study reveals both mechanism of the stress etiology of depression and possibly therapeutic interventions by targeting CLA-PL circuit.

A multifaceted architectural framework of the mouse claustrum complex.

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CLCX), this has yet to be fully accomplished. The CLCX is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are debated. To address this, we conducted a multifaceted analysis of fiber- and cytoarchitecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to CLCX, including an online reference atlas. Our data indicated four distinct subregions within CLCX, subdividing both CL and DEn into two. Additionally, we conducted brain-wide tracing of inputs to CLCX using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of CLCX and its subregions. Myelinated fibers were abundant dorsally in CL but absent ventrally, whereas PV expressing fibers occupied the entire CL. CB staining revealed a central gap within CL, also visible anterior to the striatum. The Nr2f2, Npsr1, and Cplx3 genes expressed specifically within different subregions of the CLCX, and Rprm helped delineate the CL-insular border. Furthermore, cells in CL projecting to the retrosplenial cortex were located within the myelin sparse area. By combining own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework.

Mice are a highly tractable model for studying the claustrum complex (CLCX). However, without a consensus on how to delineate the CLCX in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CLCX, to match the level of detail needed to study its functional properties. To improve and expand upon preexisting delineation schemes, we used the combinatorial expression of several markers to create a comprehensive guide to delineate the CLCX and its subregions, including an online reference atlas. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CLCX.

Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum.

The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum-core region and dorsal and ventral shell regions-the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.

Dopamine receptor 1 on CaMKII-positive neurons within claustrum mediates adolescent cocaine exposure-induced anxiety-like behaviors and electro-acupuncture therapy.

Adolescent cocaine exposure (ACE) increases risk of developing psychiatric problems such as anxiety, which may drive relapse in later life, however, its underlying molecular mechanism remains poorly understood. Methods: ACE male mice model were established by exposing to cocaine during adolescent period. Elevated plus maze (EPM) were used to assess anxiety-like behaviors in mice. Within claustrum, local injection of SCH-23390, a specific antagonist for dopamine receptor 1 (D1R), or D1R knocking-down virus were used to regulate D1R function or expression on CaMKII-positive neurons (D1RCaMKII) in vivo. Electro-acupuncture (EA) treatment was performed at acupoints of Baihui and Yintang during withdrawal period. Results: We found that ACE mice exhibited anxiety-like behaviors, along with more activated CaMKII-positive neurons and increased D1RCaMKII levels in claustrum during adulthood. Inhibiting D1R function or knocking-down D1RCaMKII levels in claustrum efficiently reduced claustrum activation, and ultimately suppressed anxiety-like behaviors in ACE mice during adulthood. EA treatment alleviated ACE-evoked claustrum activation and anxiety-like behaviors by suppressing claustrum D1RCaMKII. Conclusion: Our findings identified a novel role of claustrum in ACE-induced anxiety-like behaviors, and put new insight into the D1RCaMKII in the claustrum. The claustrum D1RCaMKII might be a promising pharmacological target, such as EA treatment, to treat drug-induced anxiety-like behaviors.

The Claustrum in the Squirrel Monkey.

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD65/67 and GABAA receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439-1454, 2020. © 2019 American Association for Anatomy.

Immunolocalization of cannabinoid receptor 1 (CB1), monoglyceride lipase (MGL) and fatty-acid amide hydrolase 1 (FAAH) in the pig claustrum.

The claustrum (Cl) is a subcortical nucleus present in all mammalian species examined so far, whose function is still a puzzling problem. There is a wealth of data on its general anatomy, cytoarchitecture, and chemoarchitecture including the connectivity with both cortical and subcortical structures; instead, much less is known about the presence of the endocannabinoid system (ECs) an important neuromodulatory complex in this brain area. In an attempt to better understand the role of the ECs within the Cl circuitry, we undertook an immunohistochemical analysis to describe the distribution of the CB1 and of the endogenous cannabinoids degrading enzymes MGL and FAAH in the pig Cl as well as their relationship with both the catecholaminergic system and with parvalbumin (PV) expressing neurons. CB1, FAAH and MGL were expressed throughout the entire Cl. CB1 was expressed by fibers and puncta, while FAAH and MGL were mainly localized in the neuropil. FAAH also showed a faint cell body localization that colocalized with PV. Tyrosine hydroxylase positive fibers (catecholaminergic system), did not demonstrate the presence of CB1. Taken together, the results described herein provide evidence for an anatomical distribution of a CB1/PV signaling system in the pig Cl suggesting that PV cells may play a role within the ECs.

The relationship between the claustrum and endopiriform nucleus: A perspective towards consensus on cross-species homology.

With the emergence of interest in studying the claustrum, a recent special issue of the Journal of Comparative Neurology dedicated to the claustrum (Volume 525, Issue 6, pp. 1313-1513) brought to light questions concerning the relationship between the claustrum (CLA) and a region immediately ventral known as the endopiriform nucleus (En). These structures have been identified as separate entities in rodents but appear as a single continuous structure in primates. During the recent Society for Claustrum Research meeting, a panel of experts presented data pertaining to the relationship of these regions and held a discussion on whether the CLA and En should be considered (a) separate unrelated structures, (b) separate nuclei within the same formation, or (c) subregions of a continuous structure. This review article summarizes that discussion, presenting comparisons of the cytoarchitecture, neurochemical profiles, genetic markers, and anatomical connectivity of the CLA and En across several mammalian species. In rodents, we conclude that the CLA and the dorsal endopiriform nucleus (DEn) are subregions of a larger complex, which likely performs analogous computations and exert similar effects on their respective cortical targets (e.g., sensorimotor versus limbic). Moving forward, we recommend that the field retain the nomenclature currently employed for this region but should continue to examine the delineation of these structures across different species. Using thorough descriptions of a variety of anatomical features, this review offers a clear definition of the CLA and En in rodents, which provides a framework for identifying homologous structures in primates.

The Claustrum of the Pig: An Immunohistochemical and a Quantitative Golgi Study.

Despite increasing interest in the claustrum (Cl) over the last decades, its function is still a puzzling problem. Among the experimental species of potential use in Cl research, the pig is considered an interesting model, because of the similarities of its brain with the corresponding cortical and subcortical human structures. The swine Cl presents a peculiar morphology, characterized by a wide posterior enlargement, ideal for physiological investigations. There is a wealth of data on general anatomy, cytoarchitecture, and chemo architecture of the Cl, but much less is known about the dendritic morphometry of its neurons. Dendritic length and branching pattern are key features to understand the organization of the microcircuitry, and thus the delineation of the structure-function relationships of the Cl. To this effect, we undertook (a) a quantitative study of the dendrites of the spiny neurons of the swine Cl, employing the Golgi staining; and (b) an immunohistochemical analysis to describe the distribution of the parvalbumin (PV)-immunoreactive interneurons throughout the same nucleus. Taken together, the results that we report here show that the dendritic architecture and the distribution of the PV expressing interneurons change when the Cl of this species changes its shape along the rostro-caudal axis, thus suggesting a potentially specific function for the large posterior puddle. Anat Rec, 302:1638-1646, 2019. © 2019 American Association for Anatomy.

Gad1-promotor-driven GFP expression in non-GABAergic neurons of the nucleus endopiriformis in a transgenic mouse line.

Transgenic animals have become a widely used model to identify and study specific cell types in whole organs. Promotor-driven reporter gene labeling of the cells under investigation has promoted experimental efficacy to a large degree. However, rigorous assessment of transgene expression specificity in these animal models is highly recommended to validate cellular identity and to isolate potentially mislabeled cell populations. Here, we report on one such mislabeled neuron population in a widely used transgenic mouse line in which GABAergic somatostatin-expressing interneurons (SOMpos INs) are labeled by eGFP (so-called GIN mouse, FVB-Tg(GadGFP)45704Swn/J). These neurons represent a subpopulation of all SOMpos INs. However, we report here on GFP labeling of non-GABAergic neurons in the nucleus endopiriformis of this mouse line.