Rat Claustrum Research Articles

Neuropeptides in the rat claustrum – An immunohistochemical detection

Neuropeptides are involved in numerous brain activities and are responsible for a wide spectrum of higher mental functions. The main purpose of this outline structural qualitative study was to identify the possible immunoreactivity of classical neuropeptides, as well as novel ones such as nesfatin-1, phoenixin (PNX), spexin (SPX), neuromedin U (NMU) and respective receptors within the rat claustrum for the first time. The study shows the novel identification of peptidergic neurotransmission in the rat claustrum which potentially implicates a contribution of this neuropeptide to numerous central neurosecretory mechanisms.

Place cells in the claustrum remap under NMDA receptor control.

Place cells are cells that exhibit location‐dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto‐cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment, and that this remapping is NMDA‐receptor‐dependent. Eliminating visual input decreases claustral delta‐band oscillatory activity, increases theta‐band oscillatory activity, and increases simultaneously recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs.

Developmental Patterning and Neurogenetic Gradients of Nurr1 Positive Neurons in the Rat Claustrum and Lateral Cortex.

The claustrum is an enigmatic brain structure thought to be important for conscious sensations. Recent studies have focused on gene expression patterns, connectivity, and function of the claustrum, but relatively little is known about its development. Interestingly, claustrum-enriched genes, including the previously identified marker Nurr1, are not only expressed in the classical claustrum complex, but also embedded within lateral neocortical regions in rodents. Recent studies suggest that Nurr1 positive neurons in the lateral cortex share a highly conserved genetic expression pattern with claustrum neurons. Thus, we focus on the developmental progression and birth dating pattern of the claustrum and Nurr1 positive neurons in the lateral cortex. We comprehensively investigate the expression of Nurr1 at various stages of development in the rat and find that Nurr1 expression first appears as an elongated line along the anterior-posterior axis on embryonic day 13.5 (E13.5) and then gradually differentiates into multiple sub-regions during prenatal development. Previous birth dating studies of the claustrum have led to conflicting results, therefore, we combine 5-ethynyl-2′-deoxyuridine (EdU) labeling with in situ hybridization for Nurr1 to study birth dating patterns. We find that most dorsal endopiriform (DEn) neurons are born on E13.5 to E14.5. Ventral claustrum (vCL) and dorsal claustrum (dCL) are mainly born on E14.5 to E15.5. Nurr1 positive cortical deep layer neurons (dLn) and superficial layer neurons (sLn) are mainly born on E14.5 to E15.5 and E15.5 to E17.5, respectively. Finally, we identify ventral to dorsal and posterior to anterior neurogenetic gradients within vCL and DEn. Thus, our findings suggest that claustrum and Nurr1 positive neurons in the lateral cortex are born sequentially over several days of embryonic development and contribute toward charting the complex developmental pattern of the claustrum in rodents.

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.

Resting State Functional Connectivity of the Rat Claustrum.

The claustrum is structurally connected with many cortical areas.A major hurdle standing in the way of understanding claustrum function is the difficulty in assessing the global functional connectivity (FC) of this structure. The primary issues lie in the inability to isolate claustrum signal from the adjacent insular cortex (Ins), caudate/putamen (CPu), and endopiriform nucleus (Endo). To address this issue, we used (7T) fMRI in the rat and describe a novel analytic method to study claustrum without signal contamination from the surrounding structures. Using this approach, we acquired claustrum signal distinct from Ins, CPu, and Endo, and used this claustrum signal to determine whole brain resting state functional connectivity (RSFC). Claustrum RSFC was distinct from the adjacent structures and displayed extensive connections with sensory cortices and the cingulate cortex, consistent with known structural connectivity of the claustrum. These results suggest fMRI and improved analysis can be combined to accurately assay claustrum function.

Comparative investigation of neuronal nitric oxide synthase immunoreactivity in rat and human claustrum

We compared the distribution, density and morphological characteristics of nitric oxide synthase-immunoreactive (NOS-ir) neurons in the rat and human claustrum. These neurons were categorized by diameter into three main types: large, medium and small. In the human claustrum, large neurons ranged from 26 to 40μm in diameter, medium neurons from 20 to 25μm and small neurons from 13 to 19μm. In the rat claustrum, large neurons ranged from 19 to 23μm in diameter, medium neurons from 15 to 18μm and small neurons from 10 to 14μm. The cell bodies of large and medium neurons varied broadly in shape – multipolar, elliptical, bipolar and irregular, consistent with a projection neuron phenotype. The small neurons were most seen as being oval or elliptical in shape, resembling an interneuron phenotype. Based on a quantitative comparison of their dendritic characteristics, the NOS-ir neurons of humans and rats displayed a statistically significant difference.

Claustrum of the short-tailed fruit bat, Carollia perspicillata: Alignment of cellular orientation and functional connectivity.

The claustrum is a gray-matter structure that underlies neocortex and reciprocates connections with cortical and subcortical targets. In lower mammals, the claustrum is directly adjacent to neocortex, making the definition of claustral boundaries challenging. Latexin, an endogenous inhibitor of metallocarboxypeptidases, localizes to claustral cells, enabling a clear delineation of claustrum. Given its proportionately large claustrum, we hypothesized that the short-tailed fruit bat, Carollia perspicillata, can be a useful model for claustral structure-function relations. We used latexin immunohistochemistry to identify claustral boundaries and intrinsic structure and multielectrode recordings from brain slices to explore intrinsic excitatory connectivity of the claustrum. Carollia's claustrum contains cells whose intrinsic connectivity and alignment permit the generation of spontaneous, synchronous population events and mirror their pattern of spread in disinhibited brain slices over millimeters. Carollia shows cellular alignment and spontaneous population-activity spread along both horizontal and dorsoventral axes. Carollia claustrum possesses intrinsic excitatory connectivity sufficient to: 1) generate single, spontaneous, synchronized burst discharges, 2) support activity spread along axes where claustral cells are aligned, and 3), because of multiple axes for cell alignment, support activity spread along both rostrocaudal and dorsoventral axes. The smaller event sizes in bat claustrum compared with rat claustrum are consistent with events occurring in population subsets rather than the full claustral cell population. The overall size of claustrum, its pronounced vascularity, and its more complex intrinsic connectivity than rat suggest that the bat is an animal model for claustral structure and function that will permit unique access to claustrum's processing capabilities. J. Comp. Neurol. 525:1459-1474, 2017. © 2016 Wiley Periodicals, Inc.

Rat Claustrum Coordinates But Does Not Integrate Somatosensory and Motor Cortical Information

The function of the claustrum is a fundamental issue in neuroscience. Anatomical data indicate that the rat claustrum is part of an interhemispheric circuit that could be involved in the bilateral coordination of whisker movements. Given that whisking is a somesthetic-guided motor behavior, the goal of the current study was to elucidate the connections of the claustrum with respect to the whisker representations in the primary somatosensory (wSI) and motor (wMI) cortical areas. Anterograde tracer injections showed that wMI projects most densely to the claustrum in the contralateral hemisphere, whereas wSI does not project to the claustrum in either hemisphere. Injections of different retrograde tracers into wMI and wSI of the same animal revealed intermingled populations of labeled neurons in the claustrum, as well as many double-labeled neurons. This indicates that the same part of the claustrum projects to the whisker representations in both SI and MI. Finally, injections of different anterograde tracers in the wMI regions of both hemispheres were combined with a retrograde tracer injection in wSI, and this produced dense terminal labeling around retrogradely labeled neurons in the claustrum of both hemispheres. Although the rodent claustrum is probably involved in the interhemispheric coordination of the MI and SI whisker representations, it does not receive inputs from both of these cortical regions. Hence, the claustrum should not be universally regarded as an integrator of somesthetic and motor information.

Functional specificity of claustrum connections in the rat: interhemispheric communication between specific parts of motor cortex.

Recent evidence indicates that the rat claustrum interconnects the motor cortical areas in both hemispheres. To elucidate the functional specificity of the interhemispheric connections between the claustrum and primary motor (MI) cortex, anterograde tracer injections in specific parts of MI were paired with retrograde tracer injections in homotopic sites of the opposite hemisphere. In addition to injecting the MI forepaw (Fp) region in both hemispheres, we injected the region associated with whisker retractions (Re) and the more caudal rhythmic whisking (RW) region. While the MI-Fp region has few connections with the claustrum of either hemisphere, both whisker regions project to the contralateral claustrum, with those from the MI-RW region being denser and more extensive than those originating from the MI-Re region. Retrograde tracer injections in the MI-RW region produced more labeled neurons in the ipsilateral claustrum than retrograde tracer injections in the MI-Re. Consistent with these patterns, the overlap of labeled terminals and soma in the claustrum was greatest when both tracers were injected into the MI-RW region. When retrograde tracers were injected into the claustrum, the highest density of labeled neurons in MI appeared in the contralateral RW region. Tracer injections in the claustrum also revealed hundreds of labeled neurons throughout its rostrocaudal extent, thereby establishing the presence of long-range intraclaustral connections. These results indicate that the intrinsic and extrinsic connections of the rat claustrum are structured for rapid, interhemispheric transmission of information needed for bilateral coordination of the MI regions that regulate whisker movements.

Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat

The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin. We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0–P120; P: postnatal day). Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons. In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period.

Proteomic Analysis Illuminates a Novel Structural Definition of the Claustrum and Insula

The claustrum is a prominent but ill-defined forebrain structure that has been suggested to integrate multisensory information and perhaps transform percepts into consciousness. The claustrum's shape and vague borders have hampered experimental assessment of its functions. We used matrix-assisted laser desorption ionization-imaging mass spectrometry to reveal a novel protein marker, G-protein gamma2 subunit (Gng2), which is enriched in the claustrum but not adjacent structures of the rat forebrain. The spatial pattern of Gng2 expression suggests key differences from commonly held views of the claustrum's structure. Using anatomical methods, we found that the rat claustrum is present only at striatal levels of the telencephalon and does not extend to frontal cortical territories. Moreover, the claustrum is surrounded on all sides by layer VI insular cortex cells in both the rat and primate. Using these defining characteristics of the claustrum, we found that the claustrum projects to cortical but not to subcortical sites. The definition of the claustrum as a cortical site is considered. The identification of a claustrum-specific protein opens the door to selective molecular lesions and the subsequent evaluation of the role of the claustrum in cognition.

The anatomical relationships between the serotonergic afferents and the neurons containing calciumbinding proteins in the rat claustrum

Claustrum is a telencephalic structure integrating information of various modalities. Proper functioning of this structure depends on the presence of a network of intrinsic connections. This includes GABA-ergic neuronal populations that also contain calcium-binding proteins (CaBPs). The goal of this study was to analyze qualitative and quantitative the 5-HTcontaining fibers in the rat claustrum and to assess the relationships between these fibers and the populations of claustral neurons expressing CaBPs. We used the methods of immunocytochemistry and morphometry. The serotonergic fibers in the claustrum are heterogeneous, both with respect to their morphology and spatial distribution. Thin varicose fibers are more numerous and are homogeneously distributed within the claustrum. Remaining fibers were thicker and possessed larger varicosities. They were present mainly in the ventral part of the claustrum. Although the serotonergic fibers are found in the vicinity of claustral cells containing CaBPs, direct contacts between these fibers and cells are rare. Other mechanisms, including volume transmission, may possibly mediate serotonergic influences.

Differentiation in the immunocytochemical features of intrinsic and cortically projecting neurons in the rat claustrum — combined immunocytochemical and axonal transport study

Retrograde axonal transport method of the fluorescent tracer FluoroGold (FG) was combined with immunocytochemistry to investigate the occurrence of nitric oxide synthase (NOS), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in both intrinsic and cortically projecting neurons of the rat claustrum. Only NOS was detected in both the scattered projecting neurons and internal neurons of the claustrum. Approximately 20% of NOS-immunoreactive neurons in the claustrum were also retrogradely labeled with FG after tracer injections into the frontal cortex. The other substances were exclusively confined to the population of interneurons, which mainly displayed an oval, round or fusiform shape and a medium size. Apart from the neuronal somata, the proximal parts of the dendritic arborization were clearly visible. The immunoreactive neurons were randomly distributed in the claustrum and their neuronal size and shape did not differ in the various parts of the studied structure. Co-localization of NOS and SOM or NOS and NPY was reported. In conclusion, SOM, VIP and NPY do not appear to play a significant role in the claustro-cortical projection but are most probably involved in modulation and information transfer in the claustrum. The appearance of NOS in both cortically projecting and intrinsic neurons of the claustrum may be indicative of a fundamentally different role in the functioning of the claustro-cortical loop.

The limbic zone of the rabbit and rat claustrum: a study of the claustrocingulate connections based on the retrograde axonal transport of fluorescent tracers.

The claustrum is a subcortical structure lying under the insular and piriform cortices, whose function is still not clear. Although data exist on connections of the claustrum and the limbic cortex, the topography of the limbic zone in the rabbit and rat claustrum has not been studied extensively. The study was performed on 17 adult Wistar rats and 12 New Zealand rabbits. Two percent water solutions of fluorescent retrograde tracers fast blue and nuclear yellow were injected into the various regions of the limbic cortex. The limbic zone is localized throughout the whole rostrocaudal extent of the claustrum, mainly in its ventromedial portion lying close to the external capsule. Although this zone of the claustrum is localized similarly in both rat and rabbit, some differences between these two species exist. In the rat, neurons projecting to all limbic areas are localized mainly in the anterior and central parts of the claustrum, whereas in the rabbit, the majority of the neurons projecting to the cingulate cortex are present in the anterior and central parts of this structure, while neurons sending axons to the retrosplenial cortex are localized in the central and posterior parts. In both species, double-labeling study showed that neurons projecting to various limbic regions are intermingled and that neurons sending axons into two different limbic regions are seen only occasionally. Our findings give support to the role of the claustrum in integrating information between different areas of the cerebral cortex and the limbic system.

3H]MK-801 binding and the mRNA for the NMDAR1 subunit of the NMDA receptor are differentially distributed in human and rat forebrain

The distributions of [ 3H]MK-801 binding and the NMDA NR1 subunit mRNA were studied using receptor autoradiography and in-situ hybridization in rat and human brain whole-hemisphere coronal sections. Receptor protein detected by radioligand autoradiography and the mRNA for the key subunit of the receptor presented similar distributions in the forebrain, with a few areas showing an imbalance between the levels of mRNA and receptor protein. Human frontal cortex showed a relative abundance of NMDAR1 mRNA as compared to [ 3H]MK-801 binding. The same area in rat brain did not show any difference in the two distributions. In comparison, the rat claustrum presented a relative excess of NMDAR1 mRNA which was not detected in human sections. Human caudate nucleus exhibited relatively high levels of [ 3H]MK-801 binding that were unmatched in rat caudate. The hippocampi of either species presented similar levels of [ 3H]MK-801 binding and NMDAR1 mRNA, but when the two signals were measured in specific subfields of the hippocampal formation, the differential distribution of the two signals reflected the anatomy of hippocampal connections assuming a preferential dendritic distribution for MK-801 binding. Interestingly, rat and human hippocampi also showed some important species-dependent difference in the relative distribution of the receptor protein and mRNA. The data presented show an overall good correlation between the mRNA for the key subunit of the NMDA receptor and the functional receptor detected with radioligand binding and highlight the presence of local differences in their ratio. This may reflect different splicing of the mRNA for the NMDAR1 subunit in specific brain areas of rat and human. The species-dependent differences in the relative distribution of the mRNA for the key subunit of the NMDA receptor and that of a marker of functional receptors also highlights important differences in the NMDA function in rat and human brain.

Rat's claustrum shows two main cortico-related zones

Methods of retrograde axonal transport were employed to evaluate the topography and overlap of claustroneocortical connections in the rat. Fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) were injected simultaneously in various combinations into the motor, somatosensory, auditory and visual cortical areas. Experiments showed that claustroneocortical projections are organized in two main cortico-related zones: sensorimotor and visuoauditory. The sensorimotor zone occupies the anterodorsal part whereas the visuoauditory occupies the posteroventral part of the claustrum. Between these two main zones only a scanty overlap was observed. In the sensorimotor zone a large overlap between neurons projecting to the motor and somatosensory cortical areas exists. The visuoauditory zone is characterized by a full overlap of neuronal populations projecting to the visual and auditory areas.

Parvalbumin and calbindin in the rat claustrum: An immunocytochemical study combined with retrograde tracing from frontoparietal cortex

The distribution of the calcium binding proteins parvalbumin and calbindin D-28k was examined in the claustrum of the rat by means of immunohistochemistry. The two proteins displayed a different and largely complementary pattern of distribution. Parvalbumin-immunostaining was intense in the neuropil of the dorsal claustrum and virtually absent in the neuropil of the ventral claustrum; parvalbumin-immunoreactive neuronal cell bodies were relatively numerous in the dorsal claustrum and were detected only occasionally in the ventral region. On the other hand, calbindin-immunostaining was prevalent in the ventral claustrum; very few calbindin-positive neurons were seen in the dorsal sector of the nucleus, whereas they were relatively more numerous in the ventral claustrum. The cell bodies of the majority of the claustral parvalbumin- or calbindin-immunoreactive neurons were oval or round, but immunostained polymorphous neurons were also observed. The surface of the immunopositive dendritic branches was smooth, with no evidence of spines. Fluorescent retrograde tracing was combined with immunohistofluorescence to determine whether the parvalbumin-containing claustral cells project to the frontoparietal cortex. Neurons labelled after large fluorogold injections in frontoparietal cortical fields were highly intermingled in the dorsal claustrum with parvalbumin-immunoreactive cells but the two neuronal populations were separate. These data show that parvalbumin-immunoreactive claustral neurons do not project to the frontoparietal cortex. In addition, although these cells may project to other cortical or subcortical targets, the present findings suggest that they may represent, at least in part, local circuit claustral neurons, corresponding to the aspiny intrinsic neurons described in the rat claustrum in studies based on Golgi impregnation.

Neuropeptide content and connectivity of the rat claustrum

The rat claustrum has a homogeneous distribution of the neuropeptides somatostatin (SOM), cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) along its rostrocaudal axis. In general, neuropeptide levels are comparable to those of overlying pyriform cortex. Visualization of mRNA encoding SOM, CCK and VIP in cell bodies of the claustrum by in situ hybridization histochemistry demonstrates that all 3 neuropeptides are contained in intrinsic claustral neurons. Mid-coronal section of the claustrum itself, or interruption of potential rostral, caudal or medial connections between the claustrum and the rest of the brain did not significantly alter levels of VIP, SOM or CCK in claustrum, cerebral cortex, or basal ganglia. Isolation of the claustrum from the cerebral cortex immediately dorsal to it along its rostrocaudal aspect caused no change in peptide levels in claustrum indicating that VIP, SOM and CCK projections to claustrum do not arrive from dorsal cortical areas. Transection of the external capsule above the claustrum caused a 50–100% elevation of all 3 peptides on the contralateral side of the lesion, suggesting that VIP, SOM and CCK synthesis and/or release within the claustrum may be regulated by projections from the contralateral side. VIP, SOM and CCK are candidates for neurotransmitters contained in neurons whose cell bodies are within the claustrum and possibly also immediately overlying lateral neocortex, and have their terminals mainly within the claustrum itself.

Diencephalic and mesencephalic afferents of the rat claustrum.

The retrograde fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) have been used to study subcortical afferents of the claustrum. DY or FB was injected into the claustrum. The greatest amount of labeled cell bodies were observed in the posterior thalamic nuclear complex. They were especially abundant in its caudal part, lying between the medial geniculate body and the pretectal area. In comparison to the numerous labeled cells near the diencephalic-mesencephalic junction, the number of fluorescing neurons in the brain stem was considerably lower. These neurons were mostly concentrated in the monoaminergic cell groups. The results indicate the presence of a substantial projection from the posterior thalamic and anterior pretectal region to the claustrum.

The rat claustrum: Afferent and efferent connections with visual cortex

We have examined the afferent and efferent projections between the claustrum and visual cortex in the Long-Evans rat using anterograde and retrogade axonal transport techniques. Injections of either wheat germ agglutinin/horseradish peroxidase (WGA/HRP) or Fast Blue were made into each of the main visual regions (17, 18a or 18b) as well as directly into the claustrum. The cortical injections were placed in either the upper, middle or deep layers so as to assist in determining the laminar organization of these connections. Of the 3 visual areas, only area 18b appears to have extensive and reciprocal connections with the claustrum. After a WGA/HRP injection of this area, dense labeled terminals and numerous labeled cells were found intermixed throughout the full extent of the claustrum. The density of this labeled activity was found to vary directly with the amount of the infragranular layers involved by the injections. Injections in the other visual areas did produce labeled cells in the claustrum, but their number was always small or even negligible. There was never any evidence of anterograde labeled terminals in the claustrum from any injection of areas 17 or 18a. Tracer injections directly in the claustrum confirmed and extended these findings by showing that the labeled terminals and/or labeled cells were localized predominantly in layer VI of area 18b of visual cortex. On the basis of these injections, two major conclusions are reached. First, the pattern of connections between the claustrum and visual cortex in the rat differs fundamentally with that found in other species. Secondly, given the extensive labeling in the claustrum observed after the injections of a single cortical locus (area 18b), these results argue against a simple topographical relationship between cortex and the claustrum.