Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum

An antiserum prepared in sheep against rat brain glutamic acid decar?ylase, the biosynthetic enzyme for γ-aminobutyric acid, has been employed to localize, at the light and electronmicroscopic levels, neurons containing glutamate decar?ylase in the cerebellum of adult rats using the unlabeled antibody enzyme method of Sternberger (1979).With high dilutions of the primary antiserum (1:4000 and higher) glutamate decar?ylase-immunoreactivity was localized in synaptic terminals of basket, stellate and Golgi cells and Purkinje cell axon recurrent collaterals in the cerebellar cortex, as well as in axosomatic and axodendritic synaptic terminals in the deep cerebellar nuclei, all of which contain pleomorphic synaptic vesicles. The reaction product was present in the axoplasm and at the cytoplasmic aspect of the plasmamembrane, the membrane of synaptic vesicles and axoplasmic reticulum, the outer mitochondrial membrane and also the microtubules. Lower dilutions of antiserum (1:1500) revealed light, specific immunoreactivity in somata of Purkinje, basket, stellate and Golgi-cells of the cerebellar cortex. We obtained specimens in which the vast majority of the members of these cells were stained. Staining of stellate cells was the least intense of all positive neurons in the cerebellar cortex. Very light labeling of some small cell bodies of the deep cerebellar nuclei was also observed. Granule cells, parallel fibers, mossy fibers and climbing fibers in the cerebellar cortex, the large neurons of the cerebellar nuclei, and glial cells were negative.When the antiserum was absorbed against acetone-dried rat liver powder, the same staining pattern was obtained, however, dilutions of 1:500 were necessary to obtain equally strong staining of neuronal cell bodies.These data indicate the specificity of the new anti-serum to rat brain glutamate decar?ylase. They confirm and complement earlier immunocytochemical work with an antiserum to mouse brain glutamate decar?ylase. The new antiserum not only shows that nearly all inhibitory axons in the cerebellum are glutamate decar?ylase positive but also detects immunoreactivity in neuronal cell bodies in the cerebellar cortex without administration of blockers of axoplasmic transport.     

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

γ-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I–III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I–III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons.

At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals.

These results confirm data obtained withl-glutamate decar☐ylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.     

Purkinje cell axon collaterals terminate on Cat-301+ neurons in Macaca monkey cerebellum

The monoclonal antibody Cat-301 identifies perineuronal nets around specific neuronal types, including those in the cerebellum. This report finds in adult Macaca monkey that basket cells in the deep molecular layer; granule cell layer (GCL) interneurons including Lugaro cells; large neurons in the foliar white matter (WM); and deep cerebellar nuclei (DCN) neurons contain subsets of Cat-301 positive (+) cells. Most Cat-301+ GCL interneurons are glycine+ and all are densely innervated by a meshwork of calbindin+/glutamic acid decarboxylase+ Purkinje cell collaterals and their synapses. DCN and WM Cat-301+ neurons also receive a similar but less dense innervation. Due to the heavy labeling of adjacent Purkinje cell dendrites, the innervation of Cat-301+ basket cells was less certain. These findings suggest that several complex feedback circuits from Purkinje cell to cerebellar interneurons exist in primate cerebellum whose function needs to be investigated. Cat-301 labeling begins postnatally in WM and DCN, but remains sparse until at least 3 months of age. Because the appearance of perineuronal nets is associated with maturation of synaptic circuits, this suggests that the Purkinje cell feedback circuits develop for some time after birth.     

An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture

Mouse-derived cerebellar explants were exposed for 5 days to cytosine arabinoside, an inhibitor of deoxyribonucleic acid synthesis. They were then maintained in normal nutrient medium until fixation for electron microscopy at 15–20 days in vitro. The cerebellar cortex lacked granule cells, but Purkinje cells, Golgi neurons and a few basket and stellate cells survived. Astrocytes and oligodendrocytes were diminished in number and myelination was absent. Purkinje cell recurrent axon collaterals increased in number and formed synapses with the surviving cortical neurons and their processes.The ultrastructural alterations that occurred in the cytosine arabinoside-treated cultures were consistent with an interpretation of cortical remodeling in which Purkinje cell axon collaterals were the dominant inhibitory elements.     

Calbindin‐D28K Immunoreactivity in the Human Cerebellar Cortex

Calbindin‐D28k (CB) is a calcium‐binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer‐specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed. Anat Rec, 297:1306–1315, 2014. © 2014 Wiley Periodicals, Inc.     

Noradrenergic innervation of the cerebellar cortex in normal and in Purkinje cell degeneration mutant mice: evidence for long term survival following loss of the two major cerebellar cortical neuronal populations

Purkinje cell degeneration mutant mice were examined during the course of Purkinje cell death (26 and 35 days old) and at 3, 5, 9 and 12 months of age. Glyoxylic acid fluorescence histochemistry for catecholamines was used to investigate possible alterations or reorganization of the noradrenergic fibers from the coeruleo-cerebellar system in response to the degeneration of two major cell types in the cerebellar cortex, of which one, the Purkinje cell, is reported to be the major target neuron. In control mice, noradrenergic fibers traveled in linear and tortuous profiles through the granule cell layer, formed pericellular arrays alongside Purkinje cell somata, and branched profusely into both radially oriented and longitudinally oriented chains. The density of noradrenergic varicosities diminished in the molecular layer, there was with age. In the mutants, concomitant with the progressive shrinkage of the molecular layer, there was a progressive increase in the density of noradrenergic varicosities. This was most conspicuous at 9 and 12 months of age, at which time the molecular layer has been depleted not only of Purkinje cell dendrites, but also of parallel fibers. Noradrenergic fibers in these zones formed dense parallel bundles of varicose profiles whose density reached 621.3 +/- 122.8% (mean +/- SD, n = 4) at 9-12 months of age, compared with age-matched controls. Neurochemical measurement of norepinephrine content in whole cerebellum of the Purkinje cell degeneration mutants revealed no change compared with age-matched controls. We conclude that noradrenergic innervation persists in the cerebellar cortex despite the death of Purkinje cells and most of the granule cells. Although we found an increased density of varicosities in the molecular layer of mutant mice, progressing with age, we believe that this can be explained on the basis of the resultant geometry of the altered cerebellar cortex. It appears that the health of the environment surrounding the noradrenergic fibers in cerebellar cortex has little influence on their anatomical integrity.     

Differential Ca2+ binding properties in the human cerebellar cortex: distribution of parvalbumin and calbindin D-28k immunoreactivity

Summary The distribution of the Ca²⁺-binding proteins parvalbumin (PV) and calbindin D-28k (CaBP) was investigated in the human cerebellar cortex. Purkinje cells contain both PV and CaBP. PV but not CaBP stains stellate and basket cells in the molecular layer. In the granular layer Golgi neurons can be subdivided into a majority, devoid of both Ca²⁺-binding proteins, and a scanty population which appears to be PV- and CaBP-immunoreactive. Thus GABAergic neurons in the human cerebellar cortex show selective differences in their Ca²⁺-binding properties, and these differences might reflect a heterogeneity in the processing of Ca²-mediated events.Abbreviations CaBP calbindin D-28k - CNS central nervous system - GABA -aminobutyric acid - -IR immunoreactivity - LTD long-term depression - PV parvalbumin     

Enzyme histochemistry of glutamate dehydrogenase in ageing rat cerebellar cortex

The influence of ageing on glutamate dehydrogenase activity was studied in the cerebellar cortex of 3-month-old (young), 12-month-old (adult) and 26-month-old (aged) male Sprague-Dawley rats by using an enzyme histochemical technique. In young rats the enzyme reactivity was observed in the neuropil of the molecular layer as well as in the perikarya of basket cells and of stellate cells; within the cytoplasm of Purkinje neurons and in synaptic glomeruli of the granular layer. Glutamate dehydrogenase activity was significantly increased in the cerebellar cortex of adult rats and decreased in old animals. The synaptic glomeruli of the granular layer were the structures of the cerebellar cortex more remarkably affected by age-related changes. The possibility that decreased glutamate catabolism occurring in the ageing cerebellar cortex may result in an excess of the amino acid and may contribute to the nerve cell loss occurring in the cerebellum of old rats is discussed.     

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the “late” time intervals investigated (30–90 days after kainic acid).

These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at “late” time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide.

It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.     

Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cells

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT 1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.     

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals,but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum

Summary The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.     

NMDAR1和GABA_A受体的α_1及α_3亚单位在成年猫小脑的定位分布

用免疫组织化学反应及 Nissl染色探讨了 NMDAR1及 GABAA受体α1 和α3亚单位在成年猫小脑皮质及小脑核的定位分布。结果表明 ,NMDAR1免疫反应产物主要分布在 Purkinje细胞胞质和分子层的树突 ,分子层的星形细胞和篮细胞以及颗粒细胞层的颗粒细胞和胶质细胞呈中等强度的阳性反应 ,小脑核的神经元胞质和部分突起着色明显。 GABAA受体的α1 亚单位免疫反应产物主要分布在 Purkinje细胞胞质和树突 ,分子层的星形细胞和胶质细胞呈弱阳性 ,小脑核的神经元阳性反应明显。GABAA 受体的α3亚单位免疫反应产物主要分布在 Purkinje细胞胞质和树突 ,分子层的星形细胞和篮细胞着色明显 ,胶质细胞呈免疫反应弱阳性 ,小脑核神经元及纤维着色明显。在 Purkinje细胞层 NMDAR1、GABAA受体α1 及α3亚单位免疫阳性神经元分别占 Purkinje细胞总数的 80 % ,61% ,88%。结论 :NMDAR1、GABAA受体的α1 及α3亚单位在成年猫小脑具有广泛的分布。这些受体在介导小脑的复杂功能中可能发挥重要的作用。     

Stimulus-dependent activation of c-Fos in neurons and glia in the rat cerebellum

The intent of the present study was to use chemical or electrical stimulation of cerebellar afferents to determine how different stimulation paradigms affect the pattern of activation of different populations of neurons in the cerebellar cortex. Specifically, we analyzed immediate changes in neuronal activity, identified neurons affected by different stimulation paradigms, and determined the time course over which neuronal activity is altered. In the present study, we used either systemic (harmaline) or electrical stimulation of the inferior cerebellar peduncle (10 and 40 Hz) to alter the firing rate of climbing and mossy fiber afferents to the rat cerebellum and an antibody made against the proto-oncogene, c-fos, as a marker to identify activated neurons and glia. In control animals, only a few scattered granule cells express nuclear Fos-like immunoreactivity. Although no other cells show Fos-like immunoreactivity in their nuclei, Purkinje cells express Fos-like immunoreactivity within their somatic and dendritic cytoplasm in control animals. Within 15 min of chemical or electrical stimulation, numerous granule and glial cells express Fos-like immunoreactivity in their nuclei. Cells in the molecular layer express Fos-like immunoreactivity following harmaline stimulation in a time and lobule specific manner; they do not appear to be activated in the electrical stimulation paradigm. Following harmaline injections, there is an initial loss of Fos-like immunoreactivity in the cytoplasm of Purkinje cells; 90 min later, nuclear staining is observed in a few scattered Purkinje cells. Following electrical stimulation, the cytoplasmic staining in Purkinje cells is enhanced; it is never present in the nucleus. Data derived from this study reveal cell-specific temporal and spatial patterns of c-Fos activation that is unique to each paradigm. Further, it reveals the presence of an activity dependent protein in the cytoplasm of Purkinje cell somata and dendrites.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.     

Brain-derived neurotrophic factor modulates dendritic morphology of cerebellar basket and stellate cells: an in vitro study

The dendrites of cerebellar basket/stellate cells show a highly stereotyped orientation relative to granule cell axons (parallel fibers) and Purkinje cell dendrites. This specific morphology is acquired during the early postnatal phase of cerebellar development, when basket/stellate cells become synaptically integrated with Purkinje neurons and granule cells. In the present study, we used primary cerebellar cultures to test how the spatial arrangement of granule cell axons affects basket/stellate cell dendritic morphology. In addition, we sought to determine whether active signals as might be provided by granule cells, i.e. synaptic input and the neurotrophin, brain-derived neurotrophic factor, affect basket/stellate cell development. Our results confirm the critical role of parallel fiber orientation for basket/stellate dendritic morphogenesis. Moreover, we found that both electrical activity and brain-derived neurotrophic factor increased basket and stellate cell dendritic arborization.Together with previously published findings, our data led to the conclusion that both structural cues and active interneuronal signaling collaborate to bring about the precise morphogenesis of cerebellar basket/stellate cells. The distinct responses of various cerebellar phenotypes towards the morphogenetic effects of brain-derived neurotrophic factor suggest that this neurotrophin, within the developing cerebellum, enhances synaptic connectivity by concerting the formation of appropriate pre- and postsynaptic structures.     

Cytology and organization of rat cerebellar organ cultures

Roller tube cultures of parasagittal cerebellar slices were taken from young rats aged 9-11 days, and maintained in vitro for 1-2 weeks. Morphological aspects of cell types and synaptic relationships in such organ cultures were examined at light and electron microscopic levels. Some neurons were marked by intracellular injections of horseradish peroxidase for subsequent identification of their connection patterns. Cytoarchitecture of the cerebellar cortex was largely preserved in the organ cultures. Dendritic trees of Purkinje cells exhibited isoplanar organizations that often resembled their orientation at the time of explanation. Other cerebellar neurons, namely granule cells, Golgi cells, basket cells, stellate cells, all differentiated within the organ cultures. In addition, some neurons of the deep cerebellar nuclei remained viable during the period of culture. Mossy fibers most probably of cerebellar nuclear origin were found terminating on the dendrites of granule cells and Golgi cells. Quite unexpected were certain types of direct synapses of afferent fibers on short necked spines arising from Purkinje cell smooth dendrites and somata. Such terminals resembled climbing fibers. They were most likely modified mossy fiber afferents, since the organ cultures did not include neurons of the inferior olive which are well spearated from the cerebellar mass at postnatal stages. These "ascending" mossy fibers presumably occupied postsynaptic surfaces that were either vacated by deafferentation or induced by the afferent fibers themselves. Intracellularly labeled Purkinje cells had widely distributed axonal collateral branches. Labeled axons were distributed within the Purkinje cell layer. Several recurrent Purkinje cell axon collaterals stained with reaction products of horseradish peroxidase tracer were followed at the ultrastructural level. In one case, labeled terminals were examined in an area of approximately 2 mm2. Terminals of Purkinje cell collaterals formed symmetric synapses with somata of basket cells and dendrites of Golgi cells, but not Purkinje cell somata. Some large boutons of serially traced Purkinje cell axon collaterals formed asymmetric contacts with profiles interpreted as Golgi cell dendrites. In contrast to the apparent axonal sprouting in cerebellar organ cultures, maturation of dendritic processes remained static. Astroglia cells of diverse shapes were observed following immunocytochemical staining with antisera to glia filament proteins. The distribution patterns of immunoreactive astrocytes changed dramatically in cerebellar slice cultures maintained for 3-6 weeks in vitro.     

Immunohistochemical study on the distribution of the voltage-gated potassium channels in the gerbil cerebellum

Although there have been many studies on the regional distribution of Kv channels in the rat and mouse cerebellum, there are no reports about Kv channel distribution in the gerbil, which is used as an ischemia animal model. Therefore, we aimed to investigate differences in the spatial patterning of Kv channel alpha-subunit isoforms in the gerbil cerebellum. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Kv1.1 immunoreactivity was also concentrated in this area although the staining intensity was relatively lower. Both Purkinje cell layer and granular layer were intensely stained with anti-Kv1.3 and Kv1.6 antibodies, whereas immunoreactivities for Kv1.4 and Kv1.5 were detected in the Purkinje cell bodies with much lower intensity in the molecular and granular layers. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong immunoreactivities for Kv1.2, Kv1.4, and Kv1.6 with moderate staining for Kv1.3 and Kv1.5 in the cell bodies. This study on the differential localization patterns of Kv1 channel subunits in the gerbil cerebellum may provide helpful guidelines for correlating current types with particular channels and useful data for the future investigations on the pathological conditions such as ischemia and epilepsy.     

Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat

Immunohistochemical and biochemical techniques were used to look for serotonin in the adrenal medulla of the rat. Using antibodies to serotonin, noradrenaline and adrenaline, it could be shown that the adrenaline-storing cells are highly immunoreactive for serotonin. Noradrenaline-storing cells were not stained even after administration of the precursors l-tryptophan and 5-hydroxytryptophan, or of serotonin itself. Specificity of the immune reaction was studied by both absorption and inhibition experiments. Chemical assays showed that rat adrenals contain significant amounts of serotonin (1.4 ± 0.11 μg/g wet weight) which is about 0.4% of the adrenaline levels. Serotonin could be reduced to about 10% of control by a high dose of reserpine. From differential and sucrose gradient centrifugation experiments it was concluded that serotonin is probably stored in granules also containing adrenaline. Administration of 5-hydroxytryptophan led to a marked increase of the serotonin level, preferentially in the granular fraction. This increase could be blocked almost completely by a decar☐ylase inhibitor. Serotonin administration did not result in a statistically significant increase of the serotonin concentration. Serotonin levels were not changed either after administration of l-tryptophan or the tryptophan hydroxylase inhibitor H22/54. These results indicate that there is no significant synthesis of serotonin from l-tryptophan.

It is suggested that the serotonin present in the adrenaline-storing cells is derived from circulating serotonin and/or 5-hydroxytryptophan. Serotonin taken up directly from the circulation or formed by decar☐ylation from 5-hydroxytryptophan is subsequently incorporated in the chromaffan granules.     

Developmental changes of chick cerebellar cortex revealed by monoclonal antibodies

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramón y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.