Parvalbumin in the cat claustrum: ultrastructure, distribution and functional implications

The presence of the calcium-binding protein (CaBP) parvalbumin (PV) in the neuronal elements of the cat's dorsal claustrum was studied by immunohistochemistry at the light- and electron-microscopic level. PV-immunoreactive neurons and fibers were detected in all parts of the claustrum. The PV-immunoreactive neurons were divided into several subtypes according to their size and shape. Approximately 7% of all PV-immunoreactive neurons were classified as large, while approximately half of the labeled neurons were medium-sized. The small PV-immunoreactive neurons were 45% of the total PV-immunoreactive neuronal population. Ultrastructurally, many spiny and aspiny dendrites were heavily immunolabeled, and the reaction product was present in dendritic spines as well. Several types of synaptic boutons containing reaction product were also found. These boutons terminated on both labeled and unlabeled postsynaptic targets (soma, dendrites, etc.), forming asymmetric or symmetric synapses. Approximately 70% of all PV-immunoreactive terminals contained round synaptic vesicles and formed asymmetric synapses. The majority of these boutons were of the 'large round' type. A lesser percentage were of the 'small round' type. This paper represents the first study demonstrating the existence of PV, a CaBP, in the cat claustrum, and its distribution at the light and electron microscope level. Beyond the relevance of this research from the standpoint of adding to the paucity of literature on PV immunoreactivity in the claustrum of various other mammals (e.g. monkey, rabbit, rat, mouse), it is of particular significance that the cat claustrum is more similar to the rabbit claustrum than to any other mammalian species studied thus far, noted by the existence of four distinct morphologic subtypes. We also demonstrate a lack of intrinsic, and possibly functional, heterogeneity as evidenced by the uniform distribution of PV throughout the cat claustrum, across the four cell subtypes (i.e. inhibitory interneurons as well as projection neurons). Indeed, the association with, and influence of, the cat claustrum on diverse multisensory mechanisms may have more to do with its afferent than efferent relationships, which speaks strongly for its importance in the sensory hierarchy. Exactly what role PV plays in the claustrum is subject to discussion, but it can be postulated that, since CaBP is associated with GABAergic interneurons, synaptogenesis and neuronal maturation, it may also serve as a neuroprotectant, particularly with regard to pathologies associated with the aging process, such as in Alzheimer's disease.     

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.     

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.     

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.     

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.     

Sustained expression of parvalbumin immunoreactivity in the hippocampal CA1 region and dentate gyrus during aging in dogs

The hippocampus contains a heterogeneous population of interneurons. Parvalbumin (PV) positive neurons constitute an abundant subpopulation of cells that express GABA. The authors observed PV immunoreactivity in the hippocampal CA1 region and dentate gyrus of variously aged dogs. In 1-year-old dogs, PV immunoreactive neurons were detected in the stratum oriens of the CA1 region, and in the polymorphic layer of the dentate gyrus. In addition, weak PV immunoreactive fibers were observed in all layers in the CA1 region and dentate gyrus. In 3-year-old dogs, PV immunoreactivity was significantly higher in the CA1 region and dentate gyrus, and this was maintained in 10-year-old dogs. This finding suggests that PV immunoreactive interneurons may show high resistance to age-dependent neurodegenerative processes.     

Differential expression of calcium-binding proteins in the red nucleus of the developing and adult human brain

The adult human red nucleus consists of two parts: (1) the parvocellular part, which is clearly separated from (2) the magnocellular part. The latter and its rubrospinal projection is known to be rudimentary in the adult human brain. Information concerning the fetal or neonatal features of the red nucleus is sparse. This study is aimed at providing a detailed account of the distribution of three calcium-binding proteins: calretinin (CR), calbindin (CB), and parvalbumin (PV), which are known to be expressed in distinct neuronal populations. Special attention has been paid to transient phenomena. CB was the most abundant protein in the magnocellular part in fetal and perinatal brains; immunoreactive (ir) neurons appeared numerous and densely packed. In the adult only few and widely spaced ir nerve cells were present. CR-expression largely corresponds to that of CB, except that fewer neurons were immunolabelled. In double- labellings the majority of neurons expressed both CB and CR; a moderate number of nerve cells solely expressing CR was present in the magnocellular part. PV-ir fibers and a moderate number of small cells were observed in the fetal, perinatal as well as the adult parvocellular part. A few PV-ir neurons were seen in the magnocellular part of the fetal and perinatal brains. Our results indicated that: (1) the magnocellular and parvocellular parts of the red nucleus were well-demarcated portions from fetal life onwards, thus a dominance of the parvocellular part over the magnocellular occurred during development; (2) the magnocellular part was more prominent in the fetal period than in adulthood; (3) neurons in the red nucleus were heterogeneous with respect to the immunoreactivities towards the three calcium-binding proteins examined; (4) the transient prominence of the magnocellular part might be a substrate for a specific transitory pattern of motor behaviour. Accepted: 7 September 2000     

An electron microscopic study of the pars distalis of the dog adenohypophysis

Summary The parenchymal cells of the pars distalis of the dog adenohypophysis were studied with the electron microscope. Criteria for the differentiation of four cell types included the size and shape of the cells and the size, shape and distribution of organelles and secretory granules. Three secretory granule-containing cells designated as I, II and III were observed. Cell type I was further divided into subtypes (Ia, Ib, Ic) on the basis of the granule size of maximal frequency. A fourth cell type (IV) contained no or only a few secretory granules. It was unique in having a highly irregular shape with cytoplasmic processes extending between the other cell types and also occasionally enclosing follicular cavities.These studies were supported by USPHS training grant, GM-155 and by Public Health Service, GRS grant 5394. The author wishes to thank Dr. J. M. Wolfe and Dr. E. V. Crabill for their valuable assistance during the course of this investigation.     

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. Accepted: 11 June 1999     

Correlation in Lewy pathology between the claustrum and visual areas in brains of dementia with Lewy bodies

We investigated Lewy pathologies in the claustrum and the related cerebral cortices and subcortical nuclei of dementia with Lewy bodies (DLB) brains using alpha-synuclein-immunohistochemistry to clarify the relationship between Lewy pathology in the claustrum and visual misidentification of DLB patients. The claustrum is known to have strong reciprocal connections with the visual areas. Consequently, the claustrum demonstrated many Lewy bodies (LB) and LB-related neurites. The insular and inferior temporal cortices, amygdala, BA 18, 19, transentohrinal and cingulate cortices showed stronger or similar Lewy pathology as compared with the claustrum, while BA 17, precentral, postcentral and transverse temporal cortices showed weaker Lewy pathology. Comparing the correlation coefficient of Lewy pathology between the clausturm and other regions, BA 18 and 19 as well as the insular and transentorhinal cortices demonstrated a higher correlation coefficient. These findings suggest that Lewy pathology in the claustrum is more closely associated with that in visual areas than in auditory, somatosensory or motor areas, and that dysfunction of the visuo-claustral pathway participates in visual misidentification in addition to the visuo-amygdaloid pathway. The paralimbic cortices including the insular and transentorhinal cortices may connect visual areas with limbic areas by relay of the visuo-claustral or visuo-amygdaloid pathway.     

An evoked potential study of spinal pathways projecting to the cerebral somatosensory areas in the dog

Summary Evoked potentials from stimulation of the hindlimbs were recorded in the cerebral somatosensory areas of dogs using Nembutal anaesthesia. Various transections of the low thoracic spinal cord were made in order to determine the pathways projecting to these areas. The shortest latency cortical activation was obtained via two pathways: the dorsal column pathway and the spinocervical tract which is located in the dorsomedial part of the lateral funiculus (DLF). Cortical activation after longer latency was obtained via ventral pathways in both the contralateral and ipsilateral spinal halves. The cortical potentials elicited by adequate (hair) stimuli depended mainly on transmission via the dorsal column and DLF pathways.     

犬眶下动脉终末支逆行插管导药入眼实验研究

目的 为探讨眼底病新的给药治疗方法，进行动物实验研究。方法 用犬做了眼动脉解剖，并做了眼动脉逆行插管注药实验。结果 在犬的解剖中发现眼动脉发自上颌动脉，起点处与眶下动脉成夹角。眶下动脉逆行插管给药成功，注药人体后，动脉眼部及全身均无不良反应。结论眼动脉终末支逆行插管给药的方法及安全可行的。     

Development of the endopiriform nucleus and the claustrum in the rat brain.

Long-survival [3H]thymidine autoradiography was used to quantitatively determine the time of origin of neurons in the endopiriform nucleus and the claustrum in rats killed on postnatal day 60 after their dams received two consecutive daily injections of [3H]thymidine on embryonic day E13 and E14, E14 and E15, ... E21 and E22. The claustrum originates late, on E15 and E16, and has a strong gradient in the longitudinal direction, posterior (older) to anterior (younger). In contrast, the endopiriform nucleus originates early, on E14 and E15, and lacks a longitudinal gradient but has a strong one in the vertical direction, ventral (older) to dorsal (younger). Sequential-survival [3H]thymidine autoradiography was used to qualitatively determine the germinal sources and settling sites of endopiriform and claustral neurons in embryonic rats. The dams received a single injection of [3H]thymidine on either E14 (to heavily label older endopiriform neurons) or E16 (to heavily label younger claustral neurons) and were killed in sequential 24-h intervals. Neurons in the presumptive endopiriform nucleus settle within two to three days after their peak time of neurogenesis while those in the presumptive claustrum take approximately five days to settle after their peak. It is postulated that endopiriform neurons are generated in the palliostriatal ventricular angle, the neuroepithelium that forms a wedge between the primordia of the neocortex and the basal ganglia, and that claustral neurons are generated in the neocortical neuroepithelium. Divergent developmental patterns between the endopiriform nucleus and the claustrum support the anatomical evidence that these nuclei have different connections. Furthermore, neurogenetic gradients in the claustrum correlate with the pattern of anatomical connections between the claustrum and the neocortex.     

An immunohistochemical study of the expression of p53 protein in colon cancer.

A total of 471 cases of colonic adenocarcinomas and 28 cases of colonic adenomas were examined immunohistochemically to evaluate the expression of p53 protein in the light of their relationship with various prognostic factors. A monoclonal antibody, p53 DO-7, was used in the study. Two hundred and fourteen adenocarcinomas (45.5%) showed positive staining for p53, however only three of the adenomas (10.3%) were positive (P < 0.05). p53 was stained to neoplastic nuclei. Adjacent normal mucosal cells were negative. There were no significant correlations between p53 expression and prognostic parameters such as age, sex, gross configuration, modified Astler-Coller stages, microscopic tumor growth patterns, tumor depth, tumor size and lymph node involvements. However, left sided adenocarcinomas (49.3%) expressed p53 more often than right sided adenocarcinomas (35.6%) (P = 0.01). The positive rates were different according to the histologic differentiation; 45.2% in well differentiated, 51.3% in moderately well differentiated, 23.8% in poorly differentiated, and 26.5% in mucinous carcinomas (P = 0.011). The mean survival periods of the p53 positive and negative groups were 29 months and 32 months, respectively (P = 0.385). However, overall survival for patients with grade one and two positive p53 was better than those of grade three and four positive cases (P = 0.028). In conclusion, the result of this multivariate analysis suggests that immunohistochemically strong p53 protein expression (more than 30% of tumor cells) has value in estimating a prognosis for patients with colorectal adenocarcinomas.     

Up-regulation of parvalbumin expression in newborn and adult rat heart

Parvalbumin (PV), a cytoplasmic calcium-binding protein, functions as a relaxing factor and has recently been detected in rat heart. Developmental changes in PV localization and expression were investigated in the heart of Wistar rats at different ages. Ten hearts from newborn, 3-month-old (young), 6-month-old (young adult), and 12-month-old (adult) rats were processed for immunohistochemistry and Western blot assay. PV was detected in hearts of all the age groups of the rats from newborn to 12-month-old by both immunohistochemistry and Western blotting. A variable distribution of PV immunoreactivity was present in newborn cardiac myocytes. In the 3-, 6-, and 12-month-old rat hearts, identical PV immunoreactivity was found in all cardiac myocytes and the intensity of PV immunoreactivity increased with increasing age. By using Western blotting, it was found that the expression of PV was low in the newborn rat heart and increased with increasing age. The presence of PV may correlate with the physiological age, and possibly serves to maintain proper relaxation of the cardiac myocytes to cope with an increasing workload of the heart during body growth.     

Sialosyl-Tn expression in normal and pathological conditions of human endometrium. An immunohistochemical study

To assess the clinico-prognostic relevance of the cell surface carbohydrate glycoprotein in normal and pathological conditions of human endometrium, Sialosyl-Tn (STn) antigen was immunohistochemically studied in normal (n = 10), hyperplastic (n = 18), and neoplastic (n = 60) endometrial lesions. There was no STn antigen reactivity in the proliferative endometrial slides, while weak staining was observed in all secretory endometria. STn expression was noted in 8/18 (44%) hyperplastic endometrial cases and in 40/60 (67%) endometrial carcinomas. Positive staining was observed throughout the cytoplasm of the glandular cancer cells, at the cell membranes, and in an intraluminar mucus. This antigen was mostly expressed heterogeneously as far as the distribution of positive cells is concerned. There was a statistically significant association between STn expression and the histological grading of cancer (p = 0.019). Advanced clinical stage (III-IV; p = 0.014) and infiltration of the myometrial wall (more than 1/2 of the myometrial wall; p = 0.004), but no STn immunoreactivity, were reported to be independent prognostic variables during follow-up. Our study shows that a) STn is not constantly expressed during the menstrual cycle, and is increased at the secretory phase of the cycle; b) Sialosyl-Tn reactivity decreases with the degree of tumor differentiation, but there was no relationship with other clinicopathological variables of cancer; c) this cell surface carbohydrate glycoprotein does not appear to predict the outcome of endometrial cancer patients.     

Interspecies differences in expression of cytokeratin polypeptides within thymic epithelium: a comparative immunohistochemical study.

Cytokeratin (CK) polypeptide expression within the thymic epithelium of several mammalian species (mouse, rat, calf, pig, rabbit, and human) has been analyzed by the streptavidin-biotin immunoperoxidase method. Comparative analysis by a large panel of 17 monoclonal antibodies (mAbs) specific for individual CK polypeptides, pairs, or groups showed considerable heterogeneity of thymic epithelial cells (TEC) in each species. In addition, extreme interspecies difference in CK contents was observed. Four main phenotypic zones: the subcapsule/perivascular area, cortex, medulla, and Hassall's corpuscles (HC) were clearly identified, each characterized by different CK expression. Medullary TEC were more heterogenous and shared common CK polypeptides either with subcapsular/perivascular TEC, cortical TEC, or HC, in most species.     

The rarity of rhabdomyosarcomas in the adult. A morphologic and immunohistochemical study

An analysis was made of the rhabdomyosarcomas diagnosed in the Dept. of Pathology of the University of Groningen between 1971 and 1983. Ten cases diagnosed in patients over 30 years of age were studied in detail. After review the diagnosis was discarded on morphologic criteria in all cases. In 9 cases it was changed into malignant fibrous histiocytoma (MFH) and in one case this diagnosis was favoured, but inconclusive. In 5 cases immunohistochemical studies could be performed. In all cases staining for the muscle specific intermediate filaments desmin appeared negative and for the mesenchymal intermediate filaments vimentin positive. These cases were also positive for one or more of the histiocytic markers alpha-1-antichymotrypsin, alpha-1-antitrypsin and lysozyme. It is concluded that rhabdomyosarcoma in older patients is extremely rare and the possible relationship between MFH in the adult and rhabdomyosarcoma in childhood is discussed.     

Topographical localization of substance P in the human post-mortem brainstem. An immunohistochemical study in the newborn and adult tissue

The localization of substance P-immunoreactive nervous structures in the newborn and adult human brainstem is studied by the indirect immunofluorescence technique of Coons and collaborators. Substance P-containing perikarya, nerve fibres and terminals are widely and unevenly distributed in several areas of the human brainstem, with a location similar to that described in other mammals; however, some remarkable differences are detectable, which speak in favour of the existence of species differences. For example, in the human brain there is a dense plexus of substance P-immunoreactive fibres in the cuneate fasciculus. The use of the newborn tissue is very helpful in the detection of the immunoreactive neuronal cell bodies. The comparative study of the newborn and the adult tissue reveals that the distribution of the substance P-like immunoreactivity changes in man during ontogeny and results in a more complete map of the substance P-containing neuron system.