Parvalbumin expression in the claustrum of the adult dog. An immunohistochemical and topographical study with comparative notes on the structure of the nucleus

Although the detailed structure and function of the claustrum remain enigmatic, its extensive reciprocal connection with the cortex suggests a role in the integration of multisensory information.Claustrum samples, obtained from necropsy of four dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for parvalbumin (PV). We focused on PV because in cortical and hippocampal areas it is a marker of the fast-spiking interneurons which have an important role in the information transmission and processing. Soma area, perimeter and circularity were considered as morphological parameters to quantitatively group the PV positive somata by k-means clustering.The histological investigation revealed a superior pyramidoid puddle and a posterior puddle characterized by a “cloud” of neurons in its dorso-lateral part. Immunostaining showed positive somata and fibers throughout the rostro-caudal extent of the dog claustrum, localized principally in the dorsal region. k-Means clustering analysis enabled neuron classification according to size, identifying respectively big (radius = 11.42 ± 1.99 μm) and small (radius = 6.33 ± 1.08 μm) cells. No statistical differences in soma shape were observed. The topographical distribution of PV immunoreactivity suggests that the dog dorsal claustrum might be functionally related to the processing of visual inputs.Taken together our findings may help in the understanding the physiology of claustrum when compared with anatomical and functional data obtained in other species.     

Parvalbumin immunoreactive neurons and fibres in the teleost cerebellum

Summary The distribution of parvalbumin-(PV) immunopositive cell bodies and fibres in the cerebellum of two species of freshwater teleosts (Salmo gairdneri and Barbus meridonalis) was studied using a monoclonal antibody and the avidin-biotin immunoperoxidase technique. A clear laminated pattern of PV immunoreactivity was observed. After PV-immunostaining, Purkinje cells were strongly labelled in their cell bodies, the initial segments of the axons and the dendritic trees. In the molecular layer, only the dendritic branches of the Purkinje cells were PV-positive. In the granule cell layer, extensive axonal plexuses and scattered cell bodies were observed. Most of the immunopositive perikarya were unequivocally identified as displaced Purkinje cells, whereas a reduced number of smaller neurons with unstained dendrites was also found. Eurydendroid cells, the efferent neurons of the teleost cerebellum, were negative; however, they were impinged upon by numerous PV-positive boutons, corresponding to terminals of Purkinje cell axons. Parallel fibres and climbing fibres, as well as stellate cells and granule cells were negative. Basket cells (or deep stellate cells) whose existence in the teleost cerebellum is discussed, were also not observed. The immunoreactivity distribution pattern for PV in the teleost cerebellum differs from previous observations on the localization of this protein in the cerebellum of amniotes.     

Auditory response properties of neurons in the claustrum and putamen of the cat

Summary The auditory response properties of single neurons in claustrum and putamen were studied in response to simple dichotic stimuli (viz. noise- and tone-bursts) in chloralose-anaesthetized cats. Neurons in claustrum were commonly weakly driven with long latency, were broadly tuned and were excited by stimulation of either ear (EE). Putamen neurons, in contrast, were securely driven with short latency, showed irregular tuning with a preference for low frequencies and were either EE or excited only by the contralateral ear (EO). The differences between claustrum and putamen responses can be related to differences in connections with the auditory cortical fields and with auditory thalamus. Some neurons were also tested for visual responsiveness: auditory and visual cells were intermingled in both nuclei and only a small percentage of cells were bimodal. In contrast to the visual and somatosensory input to claustrum, which are derived from primary cortical fields, the auditory input to claustrum is apparently derived from non-primary cortical regions, suggesting a fundamentally different role for processing of auditory information in claustrum.     

Neuropeptide Y immunoreactivity in the cat claustrum: A light- and electron-microscopic investigation

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular, primary sensory areas. However, despite renewed and growing interest amongst investigators, there remains a paucity of data concerning its peptidergic profile. The aim of the present study was to examine the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir) neurons and fibers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All animals received human and ethical treatment in accordance with the Principles of Laboratory Animal Care. Subjects were irreversibly anesthetized and transcardially perfused with fixative solution containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postfixed and sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin–biotin–peroxidase complex method adopted by our Department of Anatomy, Histology and Embryology. NPY-ir neurons and fibers were found to be diffusely distributed throughout the claustrum, with no obvious topographic or functional patterning other than larger numbers in its central/broadest part (stereotaxic planes A12–A16). Neurons were generally classified by diameter into three sizes: small (under 17 μm), medium (17–25 μm) and large (over 25 μm). Staining density is varied with some neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that gender-specific differences were not observed.     

Identification of the projection from the visual cortex to the claustrum by anterograde axonal transport in the cat

Summary Visual cortex, including areas 17, 18, and sometimes 19, was injected with tritiated leucine. Terminal labelling could be detected by autoradiography in the dorsocaudal part of the ipsilateral claustrum in all cases.     

Ultrastructure of the dorsal claustrum in cat. II. Synaptic organization

The claustrum is a bilateral subcortical nucleus situated between the insular cortex and the striatum in the brain of all mammals. It consists of two embryologically distinct subdivisions - dorsal and ventral claustrum. The claustrum has high connectivity with various areas of the cortex, subcortical and allocortical structures. It has long been suggested that the various claustral connections have different types of synaptic contacts at the claustral neurons. However, to the best of our knowledge, the literature data on the ultrastructural organization of the different types of synaptic contacts in the dorsal claustrum are very few. Therefore, the aim of our study was to observe and describe the synaptic organization of the dorsal claustrum in the cat. We used a total of 10 adult male cats and conducted an ultrastructural study under a transmission electron microscope as per established protocol. We described a multitude of dendritic spines, which were subdivided into two types - with and without foot processes. Based on the size and shape of the terminal boutons, the quantity and distribution of vesicles and the characteristic features of the active synaptic zone, we described six types of synaptic boutons, most of which formed asymmetrical synaptic contacts. Furthermore, we reported the presence of axo-dendritic, axo-somatic, dendro-dendritic and axo-axonal synapses. The former two likely represent the morphological substrate of the corticoclaustral pathway, while the remaining two types have the ultrastructural features of inhibitory synapses, likely forming a local inhibitory circuit in the claustrum. In conclusion, the present study shares new information about the neuropil of the claustrum and proposes a systematic classification of the types of synaptic boutons and contacts observed in the dorsal claustrum of the cat, thus supporting its key and complex role as a structure integrating various information within the brain.     

Parvalbumin immunoreactive Cajal-Retzius and non-Cajal-Retzius neurons in layer I of different cortical regions of human newborn

Neurons of layer I play an important role in the development of the basic structural and functional organization of the mammalian cerebral cortex. Basic data, however, concerning the spatial and temporal distribution of the neuron populations in layer I are still limited, especially for human material. The present study investigates the distribution of Cajal-Retzius (CR) and non Cajal-Retzius (NCR) neurons in thirteen cortical areas in the newborn human in terms of their relative density and possible subtypes. Neuronal populations were identified by immunohistochemistry for parvalbumin. Three main results are reported. First, parvalbumin-immunoreactive (Parv-ir) CR cells were observed in all of the neocortical areas examined. These areas also had a Parv-ir horizontal fiber plexus in deep layer I, confirming to the horizontal plexus classically associated with CR neurons. Second, many Parv-ir CR cells showed clear signs of degeneration. Third, in addition to the large CR cells, smaller Parv-ir NCR neurons occurred in many of the neocortical areas examined. These were morphologically heterogeneous and may represent several subtypes. By sampling across several areas, we were able to establish that these NCR cells occurred at higher density in primary sensory areas 3, 1, 17, and 41. Because of this variability in density of Parv-ir NCR cells, the ratio of Parv-ir CR to Parv-ir NCR cells is selectively lower in primary sensory areas. Recent investigations in somatosensory cortex of early postnatal rat report complex spatiotemporal patterns of correlated spontaneous activity among neurons in layer I (Schwartz et al. 1998). An interesting possibility is that regional variability in this activity may play a major role in the organization of cortical circuitry in different areas. Accepted: 20 October 1999     

The role of the claustrum in the bilateral control of frontal oculomotor neurons in the cat

Summary The effect of claustrum (CL) stimulation on the spontaneous unitary activity of ipsi and contralateral frontal oculomotor neurons, was studied in chloralose-anaesthetized cats. A total of 205 units was bilaterally recorded in the medial oculomotor area, homologous of the primate frontal eye fields 127 neurons were identified as projecting to the superior colliculus; for 33 of these last units stimulation of the ipsilateral CL provoked an excitatory effect lasting 10–25 ms and appearing with a latency of 5–15 ms; on 8 units the excitatory effect was followed by an inhibition lasting 100–250 ms. Ninety-eight of the 127 neurons were also tested through activation of the contralateral CL: 13 cells showed an excitatory effect lasting 10–35 ms and appearing with a latency of 20–50 ms. In three of the thirteen units the excitatory effect was followed by an inhibition lasting 100–150 ms. Complete section of the corpus callosum abolished the contralateral CL effect, suggesting the existence of a direct claustro-contralateral oculomotor cortex pathway running through the corpus callosum. The results could support the hypothesis that the CL may play a role in the bilateral control of the visuomotor performance.     

Diencephalic and mesencephalic afferents of the rat claustrum

Summary 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 calustrum.P.S. was a fellow of the Alexander von Humboldt Foundation, on leave from the Department of Anatomy, Medical Academy, Debinki 1, 80-211 Gdansk, Poland     

Immunohistochemical distribution of the cannabinoid receptor 1 and fatty acid amide hydrolase in the dog claustrum

Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum.     

大鼠屏状核的传入联系

采用WGA－HRP和CB－HRP法，追踪了16只大鼠屏状核的传入纤维联系，结果表明大脑皮质的躯体感觉区，视皮质及扣带皮质有细胞发出纤维投射到屏状核，后脑腹侧核，未定带，中缝背核及脑脚周核投射到屏状核，下后脑外侧核，视前大细胞核，斜角带核水平支和蓝斑青少量纤维投射到屏状核。     

Neuronal types in the claustrum of man

Summary Neuronal types of the human clastrum have been investigated by means of a transparent Golgi technique which enables one to study the characteristics of not only the cellular processes but also the marking features of the nuclei, the cellular organelles, and the paraplasmic substances of various types of nerve cells.Five varieties of neurons have been distinguished:Type I represents a class of spiny nerve cells varying to a certain extent in size and shape. These cells contain fine and widely dispersed lipofuscin granules which can only faintly be tinged by aldehydefuchsin.Type II cells are large aspiny neurons. Their cell bodies contain a great number of deeply stained coarse pigment granules.Type III cells are large aspiny neurons devoid of pigment deposits.Type IV is a small pigment-laden aspiny neuron.Type V is a small aspiny neuron devoid of lipofuscin granules.The pattern of pigmentation revealed by the different types of nerve cells turns out to be highly characteristic. It can well be used for classification of the various types of nerve cells which occur within the reaches of the claustrum.     

Parvalbumin and calretinin in the avian thymus

The avian thymic hormone, known to support the maturation of T-lymphocytes, is biochemically similar to parvalbumin. However, its exact cellular distribution in the thymus is unknown. We have therefore studied the occurrence of parvalbumin and other related calcium-binding proteins in this organ by immunohistochemistry during development and after hatching. Parvalbumin immunoreactivity appears in the epithelial cells on embryonic day 9, and is expressed in the cortical reticular cells in the adult. Calretinin is found in the clusters of medullary epithelial cells from embryonic day 11, whereas calbindin D-28k is absent from this organ. Thus, calcium binding-proteins are excellent markers for different compartments of the avian thymus in embryonic life and after hatching, and their expression seems to coincide with their functional maturation.     

Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method,the disector

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-m-thick sections, 3 m distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 × 10³/mm³; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 × 10³/mm³, 272.4 × 10³/mm³, and 1.9 × 10³/mm³, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 × 10³/mm³ and 1.1 × 10³/mm³, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV + neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA + and/or PV + neurons was 2.5 × 10³/mm³ and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 × 10³/mm³, 6.2 × 10³/mm³, and 5.4 × 10³/mm³, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.     

Calcium-binding protein parvalbumin-immunoreactive neurons in the rat olfactory bulb

The laminar distribution and morphological features of parvalbumin-immunoreactive [PV(+l)] neurons, one of the subpopulations of GABAergic neurons, were studied in the rat olfactory bulb at a light microscopic level. In the main olfactory bulb of adult rats, PV(+) neurons were mainly located in the external plexiform layer (EPL), and a few were scattered in the glomerular layer (GL), mitral cell layer (ML), and granule cell layer (GRL); whereas PV(+) neurons were rarely seen in the accessory olfactory bulb. The inner and outer sublayers of the EPL (ISL and OSL) appeared to be somewhat different in the distribution of PV(+) somata and features of PV(+) processes. PV(+) somata were located throughout the OSL, and PV(+) processes intermingled with one another, making a dense meshwork in the OSL; whereas, in the ISL, PV(+) somata were mainly located near the inner border of the EPL, and PV(+) processes made a sparser meshwork than that in the OSL. PV(+) neurons in the EPL were apparently heterogeneous in their structural features and appeared to be classifiable into several groups. Among them there appeared five distinctive types of PV(+) neurons. The most prominent group of PV(+) neurons in the OSL were superficial short-axon cells, located in the superficial portion of this sublayer and giving rise to relatively thick processes, in horizontal or oblique directions, which usually bore spines and varicosities. Another prominent group of PV(+) neurons extended several short, branched dendrites with spines and varicosities, which appeared to intermingle with one another, making a relatively small, spherical or ovoid dendritic field around the cell bodies; most of them resembled Van Gehuchten cells reported in previous Golgi studies. A third distinctive and most numerous group of PV(+) neurons were of the multipolar type; their somata and processes were located throughout the EPL. Their relatively smooth processes with frequent varicosities and a few spines were extended horizontally or diagonally throughout the EPL. A fourth group, which could be a subtype of the multipolar type, were located in or just above th ML and extended several thin, smooth dendrites in the EPL, some of which appeared to reach the border between the GL and EPL. Occasionally, axonlike processes arose from their cell bodies and extended into the ML. This fourth type of PV(+) neuron was named inner short-axon cells. A fifth group of neuron was located in the ML; processes of these neurons were extended horizontally, so they were named inner horizontal cells. PV(+) processes from the fourth and the fifth group of cells appeared to make contacts on mitral cell somata. In the GL some presumably periglomerular cells were also PV(+). In the GRL, PV(+) neurons were small in number, but they were also heterogeneous in their structural features; Some were identified as Golgi cells. This study shows a tremendous heterogeneity in morphological features of a chemically defined subpopulation of GABAergic interneurons in the olfactory bulb.     

Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus

Summary The structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic cells.     

Gap junctions on GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus (CA1 region)

Summary Gap junctions were identified for the first time on chemically defined neurons in the central nervous system. Gap junctions were thus demonstrated on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 m thick) sections were cut alternately and consecutively from osmium-fixed tissue which was embedded in epoxy resin and usable for conventional electron microscopic studies. The semithin sections were processed for postembedding immunocytochemistry using an anti-PV serum. Structures corresponding to the PV-immunoreactive (PV-I) profiles on the semithin sections were easily identified on electron micrographs from the adjacent thin sections. Using this technique gap junctions were found (1) between PV-I dendrites, (2) between PV-I dendrites and PV-I somata and (3) between PV-I dendrites and small processes whose origin could not be identified. Despite a systematic search, we did not find gap junction between PV-negative processes.     

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

 The claustrum is topographically and reciprocally connected with many different cortical areas, and anatomical and physiological data suggest it is composed of functionally distinct subdivisions. We asked if the distribution of cells immunoreactive for three calcium-binding proteins, parvalbumin, calbindin D-28k and calretinin would delineate functional subdivisions in the claustrum. We also asked if, as in cortex, different cell types were immunoreactive for the different proteins. We found that cells with parvalbumin-ir were large, multipolar cells. Cells immunoreactive for calretinin were bipolar cells with elongated cell bodies and beaded dendrites. There were three different types of cells immunoreactive for calbindin. The most numerous were small cells with round or oval cell bodies and numerous fine, winding processes. A second type were large multipolar, cells that resembled the parvalbumin-ir cells. The third class were bipolar cells with large, elongated cell bodies. Each type of cell resembles a cell type described in earlier Golgi studies, and each has a morphological cortical counterpart. While the different cell types varied in density, each was seen over the anterior-posterior and dorsal-ventral extent of the claustrum. Accepted: 21 July 1998