Naturally occurring parvovirus-associated feline hypogranular cerebellar hypoplasia-- A comparison to experimentally-induced lesions using immunohistology.

Three cases of feline cerebellar hypoplasia are presented. At the time of examination, the ages of the cats ranged from 2 months to 1 year. Necropsy revealed cerebellar and pons hypoplasia. Polymerase chain reaction for parvoviral deoxyribonucleic acid was positive in cerebellar tissue. Cell-specific immunolabeling was used to characterize the lesions, which were characterized into 2 types. In type 1 lesions, the cortex was nearly agranular, with an extremely thin molecular layer; the Purkinje cells were randomly placed and oriented, and their stunted main dendrite produced a thorn-covered atrophic dendritic tree; the basket cell axons ran randomly and had dysmorphic endings; and myelinated fibers were severely reduced in folia axes. In type 2 lesions, the cortex was hypogranular; the Purkinje cells were linearly organized, but their main dendrite extended too far in the molecular layer before giving up smooth, bent secondary dendrites; many basket cells were located along the cerebellar surface, and their axons ran at right angle to the surface; myelinated fibers were moderately reduced. Defects in climbing fiber synapse translocation and elimination were evident in both types of lesion. This immunohistologic study allowed a comparison between lesions in these spontaneous cerebellar hypoplasia cases with those documented when using silver impregnation studies after perinatal experimental cerebellar damage. Such a comparison is consistent with viral infection that occurs before birth in all 3 cases. Progress in parvovirus biology knowledge suggests that viral NS1 protein cytotoxicity might explain degenerative changes in the Purkinje cells that were present, in addition to the development defect.

Olivopontocerebellar atrophy in two adult cats, sporadic cases or new genetic entity.

Two otherwise healthy adult cats were presented with progressive cerebellar signs of different severity. Owners requested euthanasia. Necropsy disclosed whole cerebellum and pontine atrophy, with a severity paralleling the neurologic dysfunction. We used cell type-specific immunolabelings to characterize the lesions. The severity of the cerebellar cortex atrophy followed a general gradient from the midvermis toward the hemispheres and a local gradient from the depth of the folia toward their tip. Along these gradients, Purkinje cells were the first to disappear, followed by basket, Golgi, and stellate cells, and eventually by granule cells. Bergmann glia cells and unipolar brush cells were preserved. Pontine nuclei and the olivary complex were also severely depopulated. Neurons in the cerebellar nuclei, vestibular nuclei, and other cerebellar system-associated structures were preserved, as well as substantia nigra. Olivopontocerebellar atrophy (OPCA) in a domestic animal species was rarely reported. Some features allow tentative linking to either familial or sporadic OPCA of humans. However, the ordered disappearance of all cortical neuronal types has never been described before. Either this entity is cat specific or it might pinpoint the need for increased knowledge about differential gene expression depending on genetic background, i.e., among different species. It also would open prospects about gene product interactions within neurons.

Laminin alpha2 deficiency-associated muscular dystrophy in a Maine coon cat.

A European case of laminin alpha2 deficiency-associated muscular dystrophy in a 12-month-old, female Maine coon pedigree cat is reported. The history and eventual clinical presentation of this cat differed from those of two cats reported in the USA. In this case, the myopathy was characterised by progressively worsening weakness, muscle atrophy and joint contracture. Tendon reflexes were diminished, and motor nerve conduction velocities were slowed. Muscle biopsy demonstrated a dystrophic phenotype with endomysial fibrosis. Occasional thinly myelinated nerve fibres were present within a peripheral nerve specimen. Poorly myelinated fibres were also found at the root level on necropsy specimens. Immunohistochemical staining revealed the absence of laminin alpha2. The cat's family history did not indicate genetic transmission of the disease.

Eriochrome Black T inhibits endothelial cell growth through S-phase blockade.

We used human umbilical vein endothelial cells (HUVEC) cultures to investigate in vitro the antiproliferative effects of suramin and of its analogue, Eriochrome Black T. The cell cycle phases of interest were characterised with specific immune sera raised against cyclin D(1), cyclin E and proliferating nuclear cell antigen (PCNA). Simultaneous detection of two cell cycle markers was ensured by double colour immunofluorescence. Both compounds inhibited the endothelial cell growth while Eriochrome Black T was more potent than suramin. Suramin induced HUVEC to accumulate in G1-phase as an increase of the number of cells expressing both cyclin D(1) and PCNA was observed. Eriochrome Black T preferentially blocked them in the early S-phase, as it increased the proportion of cyclin E positive cells. These results suggest that in addition of its more potent antiproliferative effect on endothelial cell growth, Eriochrome Black T acts at another molecular level than suramin.

Immunolocalization of calbindin D28k and calretinin in the dog cochlea during postnatal development.

The calbindin (CB) and the calretinin (CR) immunoreactivities were studied in the dog cochlea during its postnatal maturation from birth to the 33rd postnatal day. At birth, CB was expressed in the Kölliker's organ, in the immature inner (IHC) and outer hair cells (OHC), in neurons of the spiral ganglion, and in nerve fibers running in the basilar membrane of the apical turn. During the cochlear maturation, non-sensorineuronal structures, such as the Kölliker's organ, the rods of Corti, and the inner sulcus cells, displayed a transient CB-staining. In the adult-like dog cochlea, CB was found in the cytoplasm, the cuticular plate, and the stereocilia of the IHC and OHC. All the neurons of the spiral ganglion and some nerves fibers in the modulius were CB-positive. At birth, CR exhibited a neuronal distribution: about 75% of the spiral ganglion neurons, some nerve fibers in the modulius and nerve fibers running in the basilar membrane were CR-labeled. During the postnatal maturation, a CR-immunostaining appeared around the IHC body and CR was expressed transiently in the OHC. In the adult-like dog cochlea, a CR-positive network surrounded the unlabeled IHC. The neuronal CR-labeling remained unchanged from birth.

Bilateral deafness in a maltese terrier and a great pyrenean puppy: inner ear morphology.

Two puppies, a 4-month-old female Maltese terrier and a 6-week-old male Great Pyrenean, were presented for confirmation of bilateral deafness by electrophysiological testing. In both puppies, brainstem auditory potentials were not evoked by 90 dB NHL click stimulation of each ear. Examination of the inner ear revealed a bilateral cochleo-saccular degeneration in both animals. The lesions were characterized by generalized atrophy of the stria vascularis, collapse of the cochlear duct, degeneration of the organ of Corti, an abnormal tectorial membrane, and saccular collapse, with a normal spiral ganglion. The cochlear duct was entirely obliterated throughout the cochleae in the Maltese terrier puppy, but was locally and asymmetrically affected in the Great Pyrenean. The abnormalities observed in the Maltese terrier puppy were identical with those previously described in deaf Dalmatian puppies; the lesions observed in the Great Pyrenean, however, were less typical. This is the first histopathological description of cochleo-saccular degeneration in the Maltese terrier and Great Pyrenean breeds. In both puppies the defect was probably congenital.

Pterin-4alpha-carbinolamine dehydratase in rat brain. I. Patterns of co-localization with tyrosine hydroxylase.

The bifunctional protein, PCD/DCoH, is both a pterin-4alpha-carbinolamine dehydratase (PCD) and a dimerization cofactor of the hepatic nuclear factor 1alpha (DCoH). In association with brain tyrosine hydroxylase (TH), which is required for dopamine synthesis, PCD catalyses dehydration and thus recycling of the cofactor tetrahydrobiopterin (BH(4)). PCD immunoreactivity in the catecholaminergic system of the rat brain was studied using a rabbit polyclonal antibody. Double immunofluorescence was performed to establish intracellular co-localization with TH. PCD immunoreactivity was found to be high and consistently present in all the neuron groups expressing TH. More than 90% of the TH+ cells were also expressing PCD. The highest co-expression (99-100% of TH+ cells) was observed in pontine catecholaminergic cell groups including locus coeruleus. Lower co-expression was observed in substantia nigra (17% of TH+ cells without PCD) and particularly in arcuate nucleus (41% of TH+ cells without PCD). Our results argue in favor of a generalized recycling of BH(4) in catecholaminergic neurons except when the neuron terminal field is located outside the blood-brain barrier. The respective roles of synthesis and recycling of BH(4) in the control of TH activity are discussed.

Overexpression of pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 in human colon cancer.

Pterin-4a-carbinolamine dehydratase (PCD) is a bifunctional protein also known as DCoH (dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)). PCD/DCoH modulates the DNA binding specificity of HNF1, thus acting on its transcriptional activity. In addition, it participates in the recycling of tetrahydrobiopterin (BH(4)), an essential cofactor of several metabolic reactions. We investigated colorectal tumors and colorectal tumor cell lines as compared to normal colon samples in search of a potential differential expression of PCD/DCoH. Immunohistochemistry was conducted on 20 human colorectal tumors and 20 normal samples using a specific polyclonal antibody. Immunoblotting and RT-PCR analysis for PCD/DCoH and HNF1 were also performed on both human tissues and CACO-2 and HT-29 cell lines. All of the 20 tumors and both colon cancer cell lines presented a strong and widespread immunoreactivity for PCD/DCoH, contrasting with the absence of expression in the normal epithelia. We thus report the massive overexpression of PCD/DCoH in colon tumors, which is in striking contrast with the absence of staining in normal counterparts. The sharp contrast in the expression of a modulator of transcriptional activity between tumoral and normal cells may have a physiopathological role. PCD/DCoH could potentially be a new marker of malignant colon cells in vivo.

Immunohistochemical localisation of pterin-4alpha-carbinolamine dehydratase in rat peripheral organs.

The bifunctional protein PCD/DCoH is both a pterin-4alpha-carbinolamine dehydratase (PCD) involved in the recycling of tetrahydrobiopterin (BH4) and a dimerisation cofactor (DCoH) of the hepatic nuclear factor 1alpha (HNF-1alpha). An antiserum raised against rat PCD/DCoH was used to localise the protein in peripheral organs. In liver, all the hepatocytes but not the other cell types are immunoreactive. In kidney, the protein is prevalent in the proximal and distal convoluted tubules. In adrenals, all the cells of the medulla are labelled. Positive nerve cells occur in myenteric ganglia of the whole gastrointestinal tract and in the intestinal submucous ganglia. Many positive endocrine cells are present in the epithelium. The immunoreactivity is either cytoplasmic (hepatocytes, convoluted tubules of the kidney and part of the gastrointestinal endocrine cells) or prominently nuclear (kidney collecting tubules, adrenals, intestinal neural plexuses and part of the gastrointestinal endocrine cells). Our results show that PCD/DCoH is present in cells expressing enzymes that use BH4 as a cofactor and/or HNF-1alpha. In addition, PCD/DCoH is present in other cells, for example, neurons in the submucosal plexus. This fact and the prominent nuclear immunoreactivity found in all the positive cells derived from the neural crests argue in favour of a new, still unknown function for the protein.

Expression of transforming growth factor beta receptors in normal human colon and sporadic adenocarcinomas.

BACKGROUND & AIMS: An absence or a presence of mutated transforming growth factor (TGF)-beta receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-beta. Mutations involving microsatellite-like regions of the type II TGF-beta receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-beta receptors in sporadic colorectal cancers and normal tissues. METHODS: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-beta receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. RESULTS: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. CONCLUSIONS: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-beta receptors that could explain a resistance to TGF-beta-mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.

Abnormalities in the cerebellum and brainstem in homozygous lurcher mice.

The lurcher mutation induces Purkinje cell degeneration in heterozygous mice, and neonatal death in homozygous animals. Using the D6Mit16 Simple Sequence Length Polymorphic marker in F2 hybrids between AKR +/+ mice and B6+/Lc mice, homozygous lurcher fetuses and newborns as well as heterozygous and normal littermates were identified, and their brain morphology was analysed. In homozygous lurcher embryos at embryonic day 18 and neonates the cerebellum was hypotrophic, particularly in the posterior half. Purkinje cells were smaller in the whole cerebellum and showed a maturational delay. Calretinin-positive cells were less frequently observed in the depth of the vermis than in normal mice. Both Purkinje cells and the vermal calretinin-positive cells were more abnormal in fetuses at day 19 and newborn mutants than one day earlier. An abnormal number of pycnotic cells were observed in the cerebellum, especially in newborn mutants. Brainstem abnormalities were characterized by abnormal curvature, caudal displacement of the pontine gray nuclei which were located caudally along the ventral border of the superior olivary complex, a drastic decrease in Purkinje cell axons in all the vestibular nuclei and the presence of dystrophic processes in at least two calbindin-positive cell groups of the dorsal pontine region. These results show that the mutation, which is semidominant in Purkinje cells, is recessive in other cell groups of the cerebellum and brainstem. They reveal that the sequence leading to Purkinje cell death appears to be similar in homozygous and heterozygous mice, although occurring earlier and worsening more quickly in the former. Lastly, they confirm the absence of effect of the mutation on the neurons of the inferior olivary complex.

Different expression of transforming growth factor beta 1 in pancreatic ductal adenocarcinoma and cystic neoplasms.

Pancreatic neoplasms harbor different prognoses according to their histological type: a benign course for serous cystadenoma, a low malignant potential for intraductal papillary mucinous neoplasms (IPMN), and high aggressiveness for ductal adenocarcinoma (ADC). Transforming growth factor beta 1 (TGF beta 1) may regulate tumor growth. The present study analyzes and compares the expression of its precursor beta 1-latency-associated peptide (beta 1-LAP), its latent binding protein (LTBP), and its mRNA in ductal adenocarcinoma (n = 10), in IPMN (n = 8), in serous cystadenoma (n = 2), and in normal tissues (n = 5). LTBP is thought to play a strategic role in the processing and active secretion of latent TGF beta 1 and its stockage in the extracellular matrix. Localization of beta 1-LAP and LTBP was assessed by immunohistochemistry using specific antibodies and expression of TGF beta 1 mRNA by reverse-transcriptase polymerase chain reaction analysis. beta 1-LAP was only slightly expressed in normal specimens, while LTBP was not detected. beta 1-LAP was detected in the cytoplasm of neoplastic cells in 9 of 10 patients with ADC. An intense staining was present in stromal cells surrounding the neoplastic glands in all cases except in one carcinoma in situ. LTBP was detected only in stromal cells and in the surrounding extracellular matrix. In IPMN with mild-grade dysplasia and in cystadenoma, beta 1-LAP was strongly expressed in the epithelial cells, while it was poorly detected in invasive IPMN; stromal cells were poorly or not all stained by beta 1-LAP, except in invasive IPMN (n = 2). LTBP was detected in neoplastic cells of three cases with benign IPMN and two of two cases with cystadenoma, while stroma was not immunostained. TGF beta 1 mRNA was strongly expressed in most of the tumors and no difference in expression was observed between the different types of neoplasms. There is no quantitative difference in expression of TGF beta 1 in ADC and in IPMN or cystadenoma. However, the latter are able to secrete TGF beta 1 efficiently, in contrast to ductal ADC as shown by the ability of the neoplastic cells to express both beta 1-LAP and LTBP. Invasive stroma reaction was associated with enhanced beta 1-LAP and LTBP expression in stromal cells and could be mediated by TGF beta 1 via LTBP

GTP-cyclohydrolase-I like immunoreactivity in rat brain.

GTPCH-I immunoreactive structures in the rat brain were studied using a polyclonal antibody raised in the chick. General mapping was made using the avidin-biotin-peroxidase technique and compared with the distribution of tyrosine hydroxylase and serotonin immunoreactivities. Double immunofluorescence was performed in order to establish real intracellular colocalization. GTPCH-I immunoreactivity was generally found to be low. Immunostained neurons were present in all the serotonin cell groups. In catecholaminergic neurons, although tyrosine hydroxylase immunoreactivity was always very high, GTPCH-I immunoreactivity was extremely variable, from relatively strong (substantia nigra, ventral tegmental area) to low (locus coeruleus, caudal part of the hypothalamus), extremely low (rostral hypothalamus, ventral brainstem) or almost absent (dorsal brainstem, some hypothalamic nuclei). When feasible, double immunolabeling revealed that all the serotonin cells and most of the tyrosine hydroxylase cells were also expressing GTPCH-I. Our results argue in favor of a regulation of tyrosine hydroxylase activity by the intracellular synthesis of BH4.

Localization of transforming growth factor-beta 1 precursor and latent TGF-beta 1 binding protein in colorectal adenomas.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine and is thought to be involved in colorectal tumorigenesis as a regulator of cell growth and differentiation. This role is mainly supported by in vitro studies while its role in vivo remains unclear. The aim of the present study was to investigate whether the TGF-beta 1 precursor (beta 1-LAP) and the latent TGF-beta 1 binding protein (LTBP) are expressed in colorectal adenomas, the presumed precursors of most of colorectal adenocarcinomas. TGF-beta 1 precursor and LTBP were examined in 35 adenomas and 10 normal colonic mucosa specimens by immunohistochemistry, using specific polyclonal antibodies. In normal colonic mucosa, beta 1-LAP was moderately expressed in epithelial crypt cells and in the stromal cells in the lamina propria. In adenomas, beta 1-LAP was localized in epithelial cells with an heterogeneous pattern and was also present in stromal cells around the adenomatous glands. LTBP was not detected in epithelial cells but was observed in stromal cells and in the extracellular matrix (ECM). beta 1-LAP expression in epithelial cells did not correlate with the grade of dysplasia, while LTBP localized in stromal cells and ECM appeared to be closely associated with areas of higher grade of dysplasia. This study is the first demonstration of both beta 1-LAP and LTBP in colorectal adenomas with different dysplasia grades. Our results suggest that TGF-beta 1 might be involved in the mechanisms controlling in vivo colorectal tumorigenesis and support a role for the stromal-associated TGF-beta 1.

Transforming growth factor beta promotes development of fibrosis after repeated courses of acute pancreatitis in mice.

BACKGROUND & AIMS: Transforming growth factor beta (TGF-beta) is a putative mediator of fibrosis in several chronic diseases. Recently, chronic pancreatitis was suggested to be related to acute pancreatitis in the so-called necrosis-fibrosis sequence hypothesis. The present study investigated whether TGF-beta is able to promote chronic fibrosis after repeated courses of necrotizing acute pancreatitis induced by cerulein in mice. METHODS: Six episodes of acute pancreatitis were repeatedly induced at weekly intervals in mice receiving either recombinant TGF-beta (4 micrograms in 4 days) or excipient alone at each induction. One week after the last induction, pancreatic lesions and collagen deposition were histologically assessed. Expression of pancreatic fibronectin messenger RNA was also examined in both groups. RESULTS: TGF-beta had no influence on a single course of acute pancreatitis. After six courses of acute pancreatitis, only mild inflammatory changes were observed in the control group. In contrast, important areas of perilobular and intralobular fibrosis were observed adjacent to inflammatory and necrotic foci in the TGF-beta group. Fibronectin messenger RNA expression was significantly higher in this group. CONCLUSIONS: TGF-beta promotes development of pancreatic fibrosis after recurrent episodes of acute pancreatitis. This model of pancreatic fibrosis could be used as a model of chronic pancreatitis consistent with the necrosis-fibrosis sequence hypothesis.

Localization of transforming growth factor beta 1 and its latent binding protein in human chronic pancreatitis.

BACKGROUND/AIMS: Transforming growth factor beta 1 (TGF-beta 1) is thought to be the mediator of fibrosis in liver, glomerular, and pulmonary fibrosis. This study investigated the expression of TGF-beta 1 precursor (beta 1 latency-associated peptide), latent TGF-beta 1-binding protein (LTBP), and TGF-beta 1 messenger RNA (mRNA) in chronic pancreatitis. METHODS: Beta 1 latency-associated peptide and LTBP expression were studied by immunohistochemistry, and TGF-beta 1 mRNA expression was studied by reverse-transcription polymerase chain reaction analysis in normal pancreatic parenchyma and in tissues from patients with chronic pancreatitis of different etiologies. RESULTS: In normal specimens, TGF-beta 1 precursor was present in islet cells and in a few ductal and acinar cells but not in periductal connective tissue. No immunoreactivity for LTBP was detected. In chronic pancreatitis, TGF-beta 1 precursor was detected mainly in mononuclear cells located in the fibrotic areas and also in ducts damaged by fibrosis, more frequently in calcifying chronic pancreatitis. LTBP was present predominantly in mononuclear cells and in the extracellular matrix around them. TGF-beta 1 mRNA was either not expressed or was faintly expressed in normal tissue, whereas intense signals were detected in chronic pancreatitis. CONCLUSIONS: The findings suggest the involvement of TGF-beta 1 in the development of fibrosis in chronic pancreatitis and the important role of inflammatory cells.

A subset of calretinin-positive neurons are abnormal in Alzheimer's disease.

The distribution of the calcium-binding protein calretinin was investigated by immunohistochemistry in the hippocampus, the subicular areas, and the entorhinal cortex in patients with Alzheimer's disease and in control subjects. By double immunolabelling, the calretinin immunoreactivity was compared to the immunoreactivity for beta/A4 amyloid or for tau proteins. Calretinin-positive neurons were mainly observed in the molecular layer of the gyrus dentatus, the stratum radiatum of the Ammon's horn, and in layers II and III of the entorhinal cortex. The general pattern of calretinin immunoreactivity was conserved in Alzheimer's disease. Calretinin-positive neurons appeared normal in the hippocampus but had a reduced dendritic tree in the entorhinal cortex. Dystrophic calretinin immunoreactive fibres were often observed in the outer molecular layer of the gyrus dentatus and in the CA4 sector in Alzheimer's disease. Most neurons containing neurofibrillary tangles were not calretinin immunoreactive and most senile plaques were not associated with calretinin positive fibres. These results show that entorhinal calretinin-positive neurons are affected in Alzheimer's disease in spite of an absence of systematic association with neurofibrillary tangles and senile plaques.

Calretinin and calbindin-D28k in rat brain: patterns of partial co-localization.

Calretinin and calbindin-D28k are homologous calcium-binding proteins, each present in a variety of neurons in the brain. Their distributions in the rat brain have been compared at the cellular level to determine whether they tend to occur in the same or in different cells, and to determine whether calbindin-positive cells show any common features once crossreaction with calretinin has been eliminated. The results show great heterogeneity. Most cells which contain one of the proteins do not contain the other, but many cells do contain both; even in the ventral cochlear nucleus, where there is abundant calretinin and most calbindin-like immunoreactivity is due to crossreaction, a few cells contain both proteins. In the substantia nigra and ventral tegmental area, many cells are double-positive but some only contain one or the other protein. Only the triangular septal nucleus is uniformly positive for both proteins. Cells which look like local-circuit neurons in many forebrain areas (cortex, hippocampus, olfactory bulb, anterior olfactory nucleus) are exclusively positive for either calretinin or calbindin, in spite of their similar morphology. In the more heterogeneous parts of the brain (including hypothalamus central gray and substantia gelatinosa), there are mixtures of calretinin-positive, calbindin-positive, and double-positive cells. In comparison with previous data on the chick, some aspects of the distributions are conserved, but double-positive cells are more frequent in the rat. The degree of heterogeneity observed, even within comparatively well-defined neuronal populations, makes it difficult to infer in what neuronal properties these proteins could be involved.

Calretinin in rat brain: an immunohistochemical study.

Calretinin is a calcium-binding protein related to calbindin-D28k; both are present in different though overlapping sets of neurons in brains of birds and mammals. We describe in detail the pattern of calretinin immunoreactivity in the rat brain. As in chick brain, calretinin immunoreactivity is abundant in various sensory pathways (particularly certain cells and fibres of the cochlear nuclei and olfactory bulb), in the heterogeneous parts of the brainstem and in parts of the hypothalamus. Many primary sensory fibres are strongly positive. Major groups of calretinin-positive neurons also include the thalamic reticular nucleus, triangular septal nucleus, lateral mammillary nucleus and substantia nigra pars compacta. Many other calretinin-positive cells are recognizable as local inhibitory neurons. Calretinin is absent from all but a few cells in the cerebral cortex, and is never found in motor neurons. There are also some distinctive positive structures whose identity is uncertain, notably irregular "shells" of cells and fibres around the thalamus and in the amygdala and an unnamed cell type in the vestibulocerebellum.