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To truly understand cerebellar function, it is essential to address the effect of the numerous peptides present within cerebellar circuits and the role they play in modulating neuronal activity in the cerebellum. To date, at least 22 neuropeptides have been identified in the cerebellum. However, relatively little is conclusively known about the modulatory role of the vast majority of these peptides. The potential role of three peptides is reviewed. Future research should focus on defining transduction pathways activated following binding of these peptides to their G-protein-coupled receptors, defining the function of peptides produced by cerebellar neurons such as Purkinje cells or Golgi cells, and describing how neuropeptides modulate cerebellar nuclear neurons, which represent the output of the cerebellum.

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet–Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.

Thermoregulatory responses are partially controlled by the preoptic area and anterior hypothalamus (PO/AH), which contains a mixed population of temperature-sensitive and insensitive neurons. Immunohistochemical procedures identified the extent of various ionic channels in rat PO/AH neurons. These included pacemaker current channels [i.e., hyperpolarization-activated cyclic nucleotide-gated channels (HCN)], background potassium leak channels (TASK-1 and TRAAK), and transient receptor potential channel (TRP) TRPV4. PO/AH neurons showed dense TASK-1 and HCN-2 immunoreactivity and moderate TRAAK and HCN-4 immunoreactivity. In contrast, the neuronal cell bodies did not label for TRPV4, but instead, punctate labeling was observed in traversing axons or their terminal endings. On the basis of these results and previous electrophysiological studies, Hodgkin–Huxley-like models were constructed. These models suggest that most PO/AH neurons have the same types of ionic channels, but different levels of channel expression can explain the inherent properties of the various types of temperature-sensitive and insensitive neurons.

Corticotropin releasing factor (CRF), localized in extrinsic afferents in the mammalian cerebellum, is defined as a neuromodulator within cerebellar circuits, and appears to be an essential element in the generation of long term depression, a proposed mechanism for motor learning. These physiological studies are based on exogenous application of CRF and do not address potential mechanisms that may influence endogenous release of the peptide. In the present study, immunohistochemistry was used to analyze changes in the lobular distribution of CRF-like immunoreactivity (LIR). In addition radioimmunoassay (RIA) was used to quantify changes in levels of the peptide in the cerebellum following stimulation of the inferior cerebellar peduncle (ICP) at 10 or 40 Hz or the inferior olivary nucleus (ION) at 1, 5, 10, or 20 Hz. Results indicate that there is a greater distribution of CRF-like-immunolabeled climbing fibers, mossy fibers, and astrocytes in all lobules of the cerebellum that is directly related to stimulation frequency. Maximal effects were elicited with 40 Hz ICP and 5–10 Hz ION stimulation. Quantitatively, the RIA studies indicate that there is a significant increase in CRF levels in the vermis, hemispheres and flocculus that correlates closely with stimulation frequency. In conclusion, stimulation of cerebellar afferents induces a significant change in the distribution and levels of CRF-LIR in climbing fibers, mossy fibers and glial cells. This suggests that the modulatory effects ascribed to CRF may influence a greater number of target neurons when levels of activity in afferent systems is increased.

Corticotropin releasing factor (CRF) is present in the adult, as well as in the embryonic and postnatal rodent cerebellum. Further, the distribution of the type 1 CRF receptor has been described in adult and postnatal animals. The focus of the present study is to determine the distribution and cellular relationships of the type 1 CRF receptor (CRF-R1) during embryonic development of the cerebellum. Between embryonic day (E)11 and E12, CRF-R1 immunoreactive puncta are uniformly distributed in the ventricular zone, the site of origin of Purkinje cells, nuclear neurons, and GABAergic interneurons, as well as the germinal trigone, the birthplace of the precursors of granule cells. Between E13 and 18, the distribution of immunolabeled puncta decreases in both the ventricular zone and the germinal trigone and increases in the intermediate zone, as well as in the dorsal aspect of the cerebellar plate. Between E14 and 18, antibodies that label specific populations of cerebellar neurons were combined with the antibody for the receptor to determine the cellular elements that expressed CRF-R1. At E14, CRF-R1 immunoreactivity is co-localized in neurons immunolabeled with PAX-2, an antibody that is specific for GABAergic interneurons. These neurons continue to express CRF-R1 as they migrate dorsally toward the cerebellar surface. Between E16 and 18, Purkinje cells, immunolabeled with calbindin, near the dorsal surface of the cerebellum express CRF-R1 in their cell bodies and apical processes. CRF has been shown to have a depolarizing effect on adult and postnatal Purkinje cells. Further, CRF has been shown to contribute to excitability of hippocampal neurons during embryonic development by binding to CRF-R1; depolarization induced excitability appears to be critical for cell survival. The location of the type one CRF receptor and the presence of its primary ligand, CRF, in the germinal zones of the cerebellum and in migrating neurons suggest that this receptor/ligand interaction could be important in the regulation of neuronal survival through cellular mechanisms that lead to depolarization of embryonic cerebellar neurons.

BACKGROUND Although donor leukocytes are only thought to prolong survival when administered before transplantation, recent evidence shows that they are effective at transplantation. This study aims to identify the leukocyte subset that is most active in prolonging kidney allograft survival and examine the cytokine expression in long-term acceptance. METHODS PVG rat kidneys were transplanted to completely MHC class I and class II-mismatched DA recipients. Donor B cells or T cells, purified by negative selection, were injected i.v. at the time of transplantation. Expression of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma, and transforming growth factor-beta mRNA was measured by quantitative real-time polymerase chain reaction (PCR). Immunohistochemical analysis and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining was used to identify infiltrating cells and apoptotic cells, respectively, in sections of kidney allografts. RESULTS Median kidney graft survival time (MST) of B cell-treated animals (n=5) was >300 days, compared with 7 days in untreated animals (n=7) (P=0.003), whereas animals treated with the same number of T cells (n=6) had a MST of 17 days (P=0.1 vs. untreated, P=0.03 vs. B cell-treated). Examination of the long-term (>300 days) accepted grafts from B cell-treated recipients showed little evidence of kidney damage but a moderate perivascular infiltrate consisting of T and B cells. This infiltrate seemed to be quiescent because there was no detectable expression of IL-2 receptors or of apoptotic cells. It produced little or no cytokine mRNA, because expression in the long-term accepted grafts was similar to levels in normal kidneys or syngeneic transplants. There was a marked increase of cytokine mRNA early after transplantation in both leukocyte-treated and untreated grafts, with more rapid appearance of IFN-gamma and IL-10 in leukocyte-treated grafts. CONCLUSIONS Donor B cells efficiently induce long-term acceptance of transplanted kidneys in a fully MHC-mismatched rat model when administered at transplantation, by a mechanism that seems to be independent of Th2 cytokine expression within the long-term accepted graft.

Cocaine's main pharmacological actions are the inhibition of the dopamine, serotonin, and norepinephrine transporters. Its main behavioral effects are reward and locomotor stimulation, potentially leading to addiction. Using knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) we have shown previously that inhibition of the dopamine transporter (DAT) is necessary for both of these behaviors. In this study, we sought to determine brain regions in which DAT inhibition by cocaine stimulates locomotor activity and/or produces reward. We used adeno-associated viral vectors to re-introduce the cocaine-sensitive wild-type DAT in specific brain regions of DAT-CI mice, which otherwise only express a cocaine-insensitive DAT globally. Viral-mediated expression of wild-type DAT in the rostrolateral striatum restored cocaine-induced locomotor stimulation and sensitization in DAT-CI mice. In contrast, the expression of wild-type DAT in the dorsal striatum, or in the medial nucleus accumbens, did not restore cocaine-induced locomotor stimulation. These data help to determine cocaine's molecular actions and anatomical loci that cause hyperlocomotion. Interestingly, cocaine did not produce significant reward - as measured by conditioned place-preference - in any of the three cohorts of DAT-CI mice with the virus injections. Therefore, the locus or loci underlying cocaine-induced reward remain underdetermined. It is possible that multiple dopamine-related brain regions are involved in producing the robust rewarding effect of cocaine.

Background The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. Methods. We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. Results. A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST > 100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. Conclusions. These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.

Background Increased numbers of macrophages and myofibroblasts are observed to occur in chronic renal allograft rejection (CR). The aim of this study was to examine the expression of cellular markers for the macrophage and myofibroblast in early renal transplant biopsy specimens and correlate these findings with allograft outcome. Methods The first postengraftment biopsy specimens from 53 patients who underwent renal transplantation between January 1993 and December 1995 were studied using immunohistochemistry with antibodies to alpha-smooth muscle actin, which identifies myofibroblasts and CD68, a marker for monocytes and macrophages. Patients were followed until December 1998 (mean follow-up 4.7+/-1.2 years). Results Nine patients had progressed to CR by the time of the study, whereas 44 patients continued to have stable renal function. A marked increase in both macrophages (P=0.02) and myofibroblasts (P=0.04) was noted in the first biopsy specimen obtained after engraftment in the patients who developed CR compared with those with stable allograft function. There was a positive correlation between alpha-smooth muscle actin and collagen expression (P=0.0001). Conclusion Significant increases in macrophages and myofibroblasts occur in the first renal biopsy specimen in those patients who later develop CR.

In the adult cerebellum, corticotropin releasing factor (CRF), that is localized in climbing fibers, mossy fibers, and a fine varicose plexus along the Purkinje cell layer, modulates the responsiveness of Purkinje cells to excitatory amino acids. During development, CRF has been detected in the primitive cerebellar anlage as early as embryonic day (E)10, and is continuously expressed throughout embryonic and postnatal cerebellar ontogeny. To investigate a possible trophic role for CRF during cerebellar development, cerebellar culture studies using E18 mouse embryos were carried out. In our culture paradigm, that used serum-free defined medium to suppress cell proliferation, CRF induced proliferation of cells in a dose-dependent manner in a range of concentrations between 0.1-10 microM. The proliferating cells were identified as astrocytes based on their expression of vimentin and GFAP. BrdU incorporation studies supported the proposed mitogenic effect of CRF on developing astrocytes. The mitogenic effects of CRF seemed to be primarily on immature astrocytes determined by their differential expression of vimentin and GFAP. Astrocytes at more advanced stages of development, as determined by the extent of process outgrowth and GFAP expression, incorporated less BrdU compared to immature astrocytes. CRF receptors were localized in astrocytes, and the proliferation of astrocytes induced by CRF was inhibited by astressin, a competitive CRF receptor antagonist. In conclusion, CRF induces proliferation of astrocytes derived from the developing cerebellum, that suggests a gliotrophic role for CRF during cerebellar development.

Background Fully allogeneic liver grafts from piebald virol glaxo to dark agouti rats are spontaneously tolerated, whereas kidney transplants between these strains are rejected. Liver tolerance is broken by donor irradiation or peritransplant corticosteroid treatment of recipient rats, both of which interfere with the activation of recipient cells. Methods In this study we used a combination of immunohistochemical staining, reverse transcription-polymerase chain reaction, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling and Annexin-V apoptosis assays to compare donor cell migration, cytokine profiles, and leukocyte apoptosis in grafts and lymphoid organs from tolerant liver and rejecting kidney recipients. We then examined the effect on apoptosis of treatments which abrogate liver tolerance. Results Liver transplantation in this tolerant strain combination is accompanied by rapid migration of many passenger leukocytes to the recipient spleen and lymph node, concurrent with a marked but transient increase in the amount of mRNA for the cytokines interleukin-2 and interferon-gamma. Apoptotic cells appear promptly in the spleen, their numbers reaching a peak 2 days earlier than has been previously shown for the graft infiltrate. Both CD4+ and CD8+ T cells undergo apoptosis and apoptotic cells are most concentrated among CD25+ T cells. In contrast, renal transplant rejection is associated with limited donor cell migration to lymphoid tissues and significantly less up-regulation of interleukin-2 and interferon-gamma in the spleen. Few apoptotic cells are detected in spleen or graft infiltrate during rejection, whereas apoptotic renal tubular and glomerular cells are found from day 5. Either recipient steroid treatment or donor irradiation significantly reduced the number of apoptotic cells in liver graft infiltrates and recipient spleen. Conclusions Taken together, these findings suggest that a mechanism akin to activation-induced cell death, with apoptosis of alloreactive recipient cells may be responsible for the induction of spontaneous liver transplant tolerance.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide that has been localized throughout the mouse cerebellum on postnatal day (P0). The wide-spread distribution of CRF within this brain region at birth suggests that it likely is present during embryonic stages of development. Thus, the intent of this study was to use immunohistochemical techniques to determine when CRF is first present in the cerebellar anlage, to analyze its distribution within the developing cerebellum, and to correlate these findings with early events in cerebellar ontogeny. CRF can first be detected in the cerebellum on embryonic day (E) 10 in scattered puncta that appear to approximate cell bodies throughout the cerebellar plate. Between E11 and E14 the number of puncta increase in the intermediate zone and more dorsal aspect of the cerebellum and decrease in the ventricular zone. At E14, in addition to the puncta, lightly immunolabeled cell bodies are observed in the ventricular zone. Just prior to birth at E17, CRF-immunoreactive varicosities distribute along the multitiered Purkinje cell layer and the intermediate zone. The CRF-positive cell bodies increase in number and intensity of staining. The majority remain within the ventricular zone, although a few also are present in the intermediate zone; it is postulated that these may be glial cells or neurons that are transiently expressing CRF. In conclusion, CRF-positive punctate elements derived from an as yet unknown source are present in the embryonic cerebellum just prior to and during the birth of Purkinje cells and nuclear neurons. The presence of this peptide at this critical stage of cerebellar development and its continued expression throughout the postnatal period of ontogeny suggests that CRF may play an important developmental role.

The intent of the present study is to define the brainstem nuclei which give rise to CGRP-immunolabeled afferents to the inferior olivary complex of the mouse. A technique which combines retrograde transport of fluorescent microspheres with immunohistochemistry was used to address this question. In the present study, intensely labeled CGRP neurons were localized within several cranial nerve nuclei including the hypoglossal, facial, oculomotor, motor nucleus of the trigeminal nerve and nucleus ambiguus, as well as in the parabrachial nucleus, locus coeruleus and medullary and pontine reticular formation. In addition, lightly labeled CGRP neurons were identified within the deep cerebellar nuclei, the inferior olivary complex, lateral reticular nucleus, medial and lateral vestibular nuclei, nucleus Darkschewitsch, interstitial nucleus of Cajal, the central gray area adjacent to the third ventricle, and the zona incerta. The origin of the projection to the inferior olivary complex primarily arises from the deep cerebellar nuclei, the locus coeruleus, and the central gray matter of the mesodiencephalic area. In addition, a small CGRP input is derived from the superior and lateral vestibular nuclei as well as the zona incerta. In conclusion, we have identified several extrinsic sources of CGRP to the inferior olivary complex and have localized it within afferents that have been shown to have either excitatory (mesodiencephalic nuclei) or inhibitory (cerebellar nuclei) effects on olivary circuits. The presence of CGRP in these functionally diverse brainstem and cerebellar afferents suggests that the peptide may act as a co-transmitter to modulate the activity of olivary neurons.

Background Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy. Methods. Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease. Results. In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.

Background.Liver transplants in the rat strain combination PVG-to-Dark Agouti are spontaneously tolerated, whereas kidney transplants in the same strain combination are rejected in 7-9 days.Methods.To identify organ-specific differences that might yield further information about the mechanism of tol

The indirect antibody peroxidase-antiperoxidase technique was used to determine the laminar and lobular distribution of catecholaminergic afferents in the adult mouse, opossum, and cat cerebellum. A monoclonal antibody to tyrosine hydroxylase (TH) revealed a plexus of thin varicose fibers that exhibited a different density and distribution pattern for each species. In the cat, TH-immunoreactive fibers were sparsely distributed to all laminae, lobules, and nuclei of the cat cerebellum except for an area of elevated density in the ventral folia of lobules V and VI. In the opossum, TH-positive fibers were uniformly and densely distributed in the granule and Purkinje cell layers; they were more abundant in vermal lobules V–VI than in more anterior and posterior lobules, particularly I and X. Numerous TH-immunoreactive fibers were found in all four cerebellar nuclei of the opossum. In the mouse, TH-positive fibers formed a dense plexus within all cerebellar lobules, laminae, and nuclei. The mouse also exhibited numerous TH-immunoreactive Purkinje cells that were localized predominantly within vermal lobules VI–X, the paraflocculus, and flocculus. In addition to the interspecies differences in the distribution of catecholaminergic fibers within the cerebellum, comparison of this plexus to that previously described for serotonin in these species reveals that the relative densities and distribution patterns of catecholaminergic and serotoninergic fibers also vary between species. It is thus hypothesized that in each species a given monoamine has a unique net effect on cerebellar output that is determined by its effects on different neuronal populations within the cerebellum. J. Comp. Neurol. 379:443–454, 1997. © 1997 Wiley-Liss, Inc.

Cholecystokinin-B (CCK-8) is an octapeptide that was initially described in the gastrointestinal tract. Recent studies have shown that this peptide also has an extensive distribution in the central nervous system, including the cerebellum of the opossum. In addition to the protein, binding sites for CCK-8 also have been described in the granule cell and molecular layer of this species. These anatomical data suggest that CCK-8 has a functional role in cerebellar circuitry. In the present study we have determined the physiological effects of CCK-8 on spontaneous and amino acid-induced activity. The results indicate that this peptide has both excitatory and inhibitory effects on spontaneous activity as well as the excitatory responses elicited by application of the excitatory amino acids aspartate, glutamate and quisqualate. The data suggest that CCK-8 may influence more than one population of cerebellar neurons. The findings support a neuromodulatory role for this peptide in cerebellar circuitry.

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.

Calcitonin gene related peptide (CGRP) has been localized within specific populations of mossy fibers in the cat's cerebellar cortex. The intent of the present study was to determine the physiological role of this peptide in cerebellar circuitry. CGRP was iontophoretically applied and its effects on spontaneous, amino acid-induced, and synaptically-mediated activity were recorded. In addition, interactions between CGRP and serotonin (5HT), another neuromodulator in cerebellar circuitry, also were analyzed. The findings of this study reveal that the primary effect of CGRP is to suppress spontaneous and excitatory amino acid-induced activity. However, CGRP has a more potent effect in suppressing aspartate- and quisqualate-induce activity as compared to that elicited by glutamate. CGRP slowed or completely blocked synaptic activity mediated by stimulation of the inferior cerebellar peduncle. Finally, the individual suppressive effects of 5HT and CGRP were potentiated when both were applied simultaneously. However, the potentiation was greater when the neuron was exposed to 5HT before CGRP was applied. In summary, the presence of CGRP in selected populations of mossy fibers, together with serotoninergic afferents, decreases the responsiveness of Purkinje cells to excitatory amino acids as well as synaptically-driven activity. Thus, activation of an afferent system to the cerebellum can elicit distinct effects on different populations of neurons that are dependent on the microenvironment of the cell at a particular point in time.

Liver allografts in the fully allogenic combination of LEW donor liver to DA recipient (LEW-->DA) are spontaneously tolerated (TOL) with no requirement for immunosuppression, while DA-->LEW allografts are rejected in 12-15 days (REJ). We investigated the mechanism of tolerance induction by identifying differences between TOL and REJ grafts from day 1 to day 9 after transplantation and in normal livers and syngeneic liver graft controls. Infiltrating cell populations were counted after immunohistochemical staining of liver graft sections. There were occasional minor differences between TOL and REJ grafts in the T cell or CD11b/c+ (monocyte/macrophage/granulocyte) infiltrate. In contrast, there was a major difference in B cell infiltrate between TOL and REJ liver grafts. Membrane IgD+ cells were significantly greater in TOL (1796 +/- 225) versus REJ (569 +/- 281) (P = 0.004) portal tracts, as were B220+ cells (1086 +/- 100 vs. 181 +/- 105, P = 0.0004) and CD45RC+ cells (2317 +/- 456 vs. 597 +/- 194, P = 0.004). IgG1, IgG2a, IgM, and IgD deposition in liver allografts, identified by immunohistochemical staining of tissue sections, revealed no IgG or IgD in normal rat liver and low levels of IgM. Deposition of IgG1 was observed in REJ but not in TOL liver on days 7 and 9. IgM was increased in both TOL and REJ liver and appeared to be associated mainly with hepatocytes in REJ and with infiltrate in TOL liver. There was a parallel increase in IgG1-expressing plasma cells in the spleen and lymph nodes of REJ but not TOL animals. Cytokine mRNA was analyzed by reverse transcription and semiquantitative polymerase chain reaction amplification of liver RNA. Increased levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, transforming growth factor-beta, and IFN-gamma were observed, with similar levels of expression in TOL compared with REJ liver. Cytokine mRNA in syngeneic grafts was not different from normal except for IL-6 and transforming growth factor-beta, which were increased. There is no major difference in the T cell component of the infiltrate or in the extent of upregulation of cytokine mRNA between TOL and REJ grafts. There is a major difference in the B cell compartment, with more B cells in TOL livers and deposition of IgG1 in REJ grafts.

In liver allograft rejection, interlobular bile ducts are thought to be the main target of rejection. In contrast, in other organ allografts the capillary bed of the graft appears to be the primary target. To determine whether portal tract microvasculature is destroyed in liver allografts during rejection, we have identified portal tract microvasculature in 11 normal livers and 38 liver allograft biopsy specimens using monoclonal antibodies to capillary endothelium in immunohistochemical staining. E1.5, CD31 and EL-4 antibodies identified portal microvascular endothelium in normal liver that had the morphology of capillaries. In allograft biopsies the number of microvascular structures per portal tract was reduced markedly in acute cellular rejection to 1.1 +/- 0.6 (n = 25) and to 0.65 +/- 0.9 (n = 15) in chronic ductopenic rejection compared with nonrejecting allografts (2.8 +/- 0.6) (n = 4) or normal liver (3.8 +/- 0.7) (n = 11). To determine whether loss of microvascular structures preceded bile duct destruction in rejection, sections were double-stained to identify both microvasculature and bile ducts. The number of microvascular structures per bile duct was significantly lower in acute cellular rejection (0.5 +/- 0.4) (n = 18) or chronic rejection (0.3 +/- 0.4) (n = 8) compared with normal liver (2.3 +/- 0.6) (n = 7) (P < 0.0001), demonstrating that components of the portal vasculature are destroyed prior to bile ducts. There was a correlation between the severity of rejection and the loss of microvascular structures per bile duct (P < 0.001). In conclusion, in common with other allografted organs, the microvasculature of liver allografts appears to be an early target of rejection.

Serotonin is present in a fine beaded plexus in the cerebellar cortex of several mammalian species. In the cat, serotoninergic afferents arise from neurons located within the lateral, paramedian and peri-olivary reticular nuclei (Kerr and Bishop, J. Comp. Neurol., 304 (1991) 502-515). In addition to serotoninergic afferents, these same nuclei also contain a separate population of neurons that give rise to mossy fibers to the cerebellar cortex. Physiological studies have shown that mossy fibers are excitatory to their target neurons. The intent of the present study was to determine the physiological effects of serotonin in the cat's cerebellum in an in vivo preparation and to identify the receptor(s) that mediate the observed responses. Iontophoretic application of serotonin (5HT) onto Purkinje cells reduces the spontaneous firing rate of all cells tested (n = 12). Serotonin also blocks the excitatory effects elicited by the application of aspartate in 17 of 19 units tested and of glutamate (n = 62) in all cases. In addition, 5HT potentiated the inhibitory action of GABA (n = 12). Iontophoretic application of the 5HT1A agonists, 8-OH-DPAT and ipsapirone, mimic the suppressive action of serotonin in a dose-dependent manner. This response, as well as the 5HT mediated suppression are blocked by the application of spiperone, a 5HT1A antagonist. Compounds selective for the 5HT1C,2 and 3 receptors are physiologically ineffective. The present data are in partial agreement with previous studies in the rat's cerebellar cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

The present account provides a developmental timetable for the maturation of cholecystokinin (CCK)-positive fibers in the cerebellar cortex and cerebellar nuclei of the opossum. CCK-positive fibers are in the cerebellar peduncle by postnatal day (PD) 1, however they wait until PD 7 to penetrate the cerebellar anlage. Between PD 7 and PD 20 the fibers wait again in the medullary core of the cerebellum. After PD 20, there are 2 distinct patterns of CCK localization within the overlying cortical layers. The first pattern develops between PD 20-26 when CCK puncta are present in restricted foci within the Purkinje cell layer of the anterior lobe vermis. They distribute in 4 parasagittal bands, 2 on either side of the midline, that extend from the primary fissure rostrally into the anterior lobe of the cerebellum. By PD 33 two additional parasagittal bands are present in the posterior lobe vermis. The vast majority of these CCK puncta are transient in nature as all but a few disappear by PD 84. Those that remain progress through a series of developmental stages characteristic of climbing fiber ontogeny. These climbing fibers persist in lobules V, VII and VIII of the adult cerebellum. Further, there is a transient expression of CCK-immunoreactivity within inferior olivary neurons. These observations support the interpretation that the transient population of CCK-IR puncta are immature climbing fiber axons derived from the inferior olive. The second pattern of CCK localization is evident between PD 30-33, the time when granule cells first can be recognized in a histologically distinct internal granule cell layer (IGL). Between PD 30 and PD 68 there is a differential pattern of distribution of CCK-IR profiles within the lobules of the cerebellum. Initially, CCK-IR axons are only present in the anterior vermis where they are aligned in register with the bands of CCK puncta in the Purkinje cell layer. CCK-IR puncta are not present in the posterior lobe vermis or hemispheres until later stages of development. Further, a sagittal organization is not evident in either of these latter 2 areas. Initially, CCK-IR profiles in the IGL cannot be identified as mossy fibers based on their terminal morphology. When they first enter the IGL they appear as punctate elements. Over time they become increasingly more complex in shape and between PD 68-84 develop morphological characteristics of adult mossy fiber rosettes. The cerebellar nuclei can be distinguished histologically by PD 18, but CCK-IR fibers are not evident among these neurons until PD 36 which corresponds to about the time they can be visualized in the IGL. In addition, CCK-IR cell bodies first appear in the cerebellar nuclei between PD 26-30; these are present in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of serotonin immunoreactivity in the cat cerebellum was studied by using the indirect antibody peroxidase-antiperoxidase (PAP) technique. Furthermore, the origin of these chemically defined afferents was determined by combining the retrograde transport of horseradish peroxidase (HRP) with the PAP technique. In the cerebellar cortex, serotonin immunoreactivity is present in a plexus of beaded fibers that is confined almost exclusively to the granule and Purkinje cell layers; a few fibers are present in the molecular layer. Serotoninergic axons and varicosities have a dense and uniform distribution throughout all lobules of the cerebellum with the exception of lobule X where the fiber density is sparse. Serotonin cell bodies were not found within the cerebellar cortex. However, following pretreatment with pargyline and L-tryptophan, serotonin positive cell bodies were found in all deep cerebellar nuclei as well as the raphe and reticular nuclei in the brainstem. The present study demonstrates that the serotoninergic projection to the cat's cerebellum has some degree of topographical organization. Serotoninergic fibers in the anterior vermis (lobules I-V) were shown to arise from neurons located within the paramedian reticular nucleus, the lateral reticular nucleus, and the lateral tegmental field. Injections of HRP into either the posterior vermis (lobule VI-IX) or the paramedian lobule, labeled serotoninergic neurons exclusively in the lateral reticular nucleus. Lobus simplex, crus I and crus II (the hemisphere) receive a serotoninergic input from cells located in the lateral tegmental field, the peri-olivary reticular formation and the paramedian reticular nucleus. In no cases were neurons in the raphe double-labeled, although there were cells positive for HRP or serotonin alone. The data indicate that there is a topographical organization in the serotoninergic projection from the caudal brainstem to specific regions of the cat's cerebellar cortex. In addition to climbing and mossy fibers, this projection represents a third major source of cerebellar afferents based on its dense and widespread distribution as well as its morphological and chemical characteristics.

The rostral hypothalamus, especially the preoptic-anterior hypothalamus (POAH), contains temperature-sensitive and -insensitive neurons that form synaptic networks to control thermoregulatory responses. Previous studies suggest that the cyclic nucleotide cGMP is an important mediator in this neuronal network, since hypothalamic microinjections of cGMP analogs produce hypothermia in several species. In the present study, immunohistochemisty showed that rostral hypothalamic neurons contain cGMP, guanylate cyclase (necessary for cGMP synthesis), and CNG A2 (an important cyclic nucleotide-gated channel). Extracellular electrophysiological activity was recorded from different types of neurons in rat hypothalamic tissue slices. Each recorded neuron was classified according to its thermosensitivity as well as its firing rate response to 2–100 μM 8-bromo-cGMP (a membrane-permeable cGMP analog). cGMP has specific effects on different neurons in the rostral hypothalamus. In the POAH, the cGMP analog decreased the spontaneous firing rate in 45% of temperature-sensitive and -insensitive neurons, an effect that is likely due to cGMP-enhanced hyperpolarizing K+ currents. This decreased POAH activity could attenuate thermoregulatory responses and produce hypothermia during exposures to cool or neutral ambient temperatures. Although 8-bromo-cGMP did not affect the thermosensitivity of most POAH neurons, it did increase the warm sensitivity of neurons in other hypothalamic regions located dorsal, lateral, and posterior to the POAH. This increased thermosensitivity may be due to pacemaker currents that are facilitated by cyclic nucleotides. If some of these non-POAH thermosensitive neurons promote heat loss or inhibit heat production, then their increased thermosensitivity could contribute to cGMP-induced decreases in body temperature.

In order to determine the distribution of the peptide cholecystokinin (CCK) within the cerebellum and medullary precerebellar nuclei of the adult opossum, sections of these brain regions were processed for peroxidase-antiperoxidase immunohistochemistry. Within the inferior and superior cerebellar peduncles, fine-beaded fibers are evident and a beaded plexus of fibers is present in all the cerebellar nuclei. In the overlying cerebellar cortex, CCK-positive mossy fiber rosettes are present in all lobules, where their morphology varies from simple enlargements to more complex rosettes. However, their distribution varies particularly in vermal lobules II, III, VII, and IX where they are organized in parasagittal bands. Climbing fibers that are positive for CCK are present in very restricted areas of vermal lobules IV, VII, and VIII. After colchicine pretreatment, CCK-positive cell bodies are seen in restricted regions of the posterior interposed and fastigial nuclei as well as within several precerebellar nuclei known to give rise to mossy fibers. Such nuclei include the lateral cuneate nucleus, the nucleus prepositis hypoglossi, the nucleus reticularis lateralis, the nucleus raphe obscurus, the paramedian reticular nucleus, the nucleus reticularis gigantocellularis, and the medial vestibular nucleus. To localize the brainstem origin(s) of the CCK fibers in the cerebellum, a double-label paradigm employing a retrograde tracer and CCK immunohistochemistry was used. These experiments indicate that CCK mossy fibers originate primarily within the lateral cuneate nucleus, the perihypoglossal complex, and the lateral reticular nucleus. Some also originate within the medial vestibular nucleus and the nucleus reticularis gigantocellularis. In addition, double-labeled cell bodies are present within the caudal medial accessory inferior olive, the likely source of the CCK-positive climbing fibers. These data indicate that specific populations of climbing fibers and mossy fibers may utilize CCK to alter the firing rate of their target neurons.

Solitary primary cilia project from nearly every cell type in the human body. These organelles are considered to have important sensory and signaling functions. Although primary cilia have been detected throughout the mammalian brain, their functions are unknown. The study of primary cilia in the brain is constrained by the scarcity of specific markers for these organelles. We previously demonstrated that type III adenylyl cyclase (ACIII) is a marker for primary cilia on neonatal hippocampal neurons in vivo and in vitro. We further showed that ACIII localizes to cilia on cultured glial cells. Here, we report that ACIII is a marker for primary cilia throughout many regions of the adult mouse brain. Furthermore, we report that ACIII localizes to primary cilia on choroid plexus cells and some astrocytes in the brain, which to our knowledge is the first report of a marker for visualizing cilia on glia in vivo. Overall, our data indicate that ACIII is a prominent marker of primary cilia in the brain and will provide an important tool to facilitate further investigations into the functions of these organelles.

In a previous study, it was shown that populations of climbing fibers, derived from the inferior olivary complex (IOC) contain the peptide corticotropin releasing factor (CRF) and that the expression of this peptide in climbing fibers could be modulated by the level of activity in olivary afferents. The intent of this study was to determine if there was comparable plasticity in the distribution of the type 1 CRF receptor (CRF-R1) in the cerebellum of the rat. Our results indicate that CRF-R1 was localized primarily to Purkinje cell somata and their primary dendrites and granule cells. In addition, scattered immunolabeling was present over the somata of Golgi cells, basket cells and stellate cells, as well as Bergmann glial cells and their processes. IOC stimulation for 30 min at 1 Hz increased CRF-R1 expression in molecular layer interneurons and processes of Bergmann glial cells. Little to no effect on CRF receptor distribution was observed in Purkinje cells, granule cells, or Golgi cells. IOC stimulation at 5 Hz however, increased CRF-R1 expression in the processes of Bergmann glial cells while decreasing its expression in basket, stellate and, to some extent, in Purkinje cells. The present results suggest that there is activity-dependent plasticity in CRF-R1 expression that must be considered in defining the mechanism by which the CRF family of peptides modulates activity in cerebellar circuits. The present results also suggest that the primary targets of CRF released from climbing fibers are Bergmann glial cells and interneurons in the molecular layer. Further, interneurons responded with a decrease in receptor expression following more intense levels of stimulation suggesting the possibility of internalization of the receptor. In contrast, Bergmann glial cells showed an increased expression in receptor expression. These data suggest that CRF released from climbing fibers may modulate the physiological properties of basket and stellate cells as well as having a heretofore unidentified and potentially unique effect on Bergmann glia.

Corticotropin releasing factor (CRF) and its cognate receptors, defined as Type 1 and Type 2 have been localized within the cerebellum. The Type 2 CRF receptor (CRF-R2) is known to have both a full length (CRF-R2alpha) and a truncated (CRF-R2alpha-tr) isoform. A recent study documented CRF-R2alpha primarily in Bergann glia and astrocytes, as well as in populations of Purkinje cells in the adult cerebellum. The goal of the present study is to determine if CRF-R2alpha is present in the postnatal cerebellum, and if so to describe its cellular distribution. RT-PCR data showed that CRF-R2alpha is expressed in the mouse cerebellum from birth through postnatal day 21. Between birth and P14, CRF-R2alpha-immunoreactivity was localized within the somata of Purkinje cells, and migrating GABAergic interneurons. GFAP-immunoreactive astrocytes, including Bergmann glia, also expressed CRF-R2alpha-immunoreactivity from P3-P14. There is a change, however, in CRF-R2alpha immunolabeling within neurons as the cerebellum matures. Compared to its expression in the adult cerebellum, Purkinje cells, and GABAergic interneurons showed more extensive CRF-R2alpha immunolabeling during early postnatal development. We postulate that CRF-R2alpha could be involved in developmental events related to the survival and differentiation of Purkinje cells and GABAergic neurons, whereas in the adult, this isoform of the CRF receptor family is likely involved in modulating Bergmann glia that have been shown to play a role in regulating the synaptic environment around Purkinje neurons.

Primary cilia are cellular appendages that provide important sensory functions and defects in primary ciliary signaling have been implicated in the pathophysiology of human diseases and developmental abnormalities. Almost all human cell types possess a primary cilium. Neurons throughout the brain possess primary cilia on which certain receptors localize, suggesting that neurons possess cilia-mediated signaling. However, the functional significance of neuronal cilia is unknown. Although there is a great deal of interest in understanding the functions of neuronal cilia, their study is hampered by the lack of an in vitro model system. We report that the majority of hippocampal neurons cultured from postnatal mice possess primary cilia in vitro. Further, we describe cilia proteins that can be labeled to readily visualize neuronal primary cilia in culture. These findings are the first characterization of neuronal primary cilia in vitro and should greatly facilitate further investigations into the function of these organelles.

Corticotropin releasing factor (CRF), localized in afferent inputs to the cerebellum, binds to two receptors defined as the Type 1 (CRF-R1) and the Type 2 (CRF-R2alpha). CRF-R1 has been localized to the cerebellum, as has a truncated isoform of CRF-R2alpha. Evidence for the presence of the full length isoform of CRF-R2alpha in the cerebellum is conflicting. We used RT-PCR, immunohistochemical, and physiologic techniques to resolve this conflict. RT-PCR data show low levels of CRF-R2alpha in the vermis and hemisphere of the cerebellum. These observations were confirmed by the Gene Expression Nervous System Atlas (GENSAT) database. A CRF-R2alpha antibody was used to determine the cellular distribution of the receptor in the cerebellum. The vast majority of the receptors are localized to Bergmann glial cells located throughout the cerebellum, as well as astrocytes in the granule cell layer. Neuronal labeling is present in sub-populations of Purkinje cells, Golgi cells, basket cells, and cerebellar nuclear neurons. Physiologic data show that urocortin II, which binds selectively to CRF-R2alpha, increases the firing rate of both Purkinje cells and nuclear neurons; this response can be blocked by the CRF-R2alpha-specific antagonist, antisauvagine-30. The present results confirm that CRF-R2alpha is present in the cerebellum and functions in circuits that modulate the firing rate of Purkinje cells and cerebellar nuclear neurons. A comparative analysis showed that the patterns of distribution of CRF-R1, CRF-R2alpha and CRF-R2alpha-tr are distinct. These data indicate that the CRF family of peptides modulates cerebellar output by binding to multiple CRF receptors.

Previous studies have described the embryonic and postnatal development of CRF, as well as the type 1 CRF receptor in the mouse cerebellum. The present immunohistochemical study localizes the cellular distribution of the type 2 CRF receptor (CRF-R2) during postnatal development of the mouse cerebellum. Western blot analysis indicates that the antibody used in this analysis recognizes both a full-length and a truncated isoform of the type 2 receptor. We propose that each isoform has a unique cellular distribution. In the present study, the postnatal (P) development (P0-P14) and cellular localization of CRF-R2 in different cell types was analyzed using PAP and double-label fluorescent immunohistochemistry; cell-specific antibodies were used to identify cells expressing CRF-R2 at different stages of postnatal development. At P0, CRF-R2 immunoreactivity was localized within the somata of Purkinje cells and migrating GABAergic interneurons. CRF-R2 was first observed in the initial axonal segments of some Purkinje cells at P5, and was evident in many Purkinje cell axon hillocks at P8. Punctate immunoreactivity is present in the molecular layer by P5 and is interpreted to be immunolabeled parallel fibers. Between P8 and P14, CRF-R2 immunostaining is present in the initial axonal segments of Golgi cells, within the internal granule cell layer. Finally, CRF-R2 is present in both radial glia in the molecular layer as well as in astrocytes in the white matter and internal granule cell layer from P5 to P14. The present results suggest that CRF-R2, both the truncated and the full-length isoforms, are present in the developing cerebellum, each with a unique cellular distribution. The immunohistochemical evidence indicates that the truncated isoform of the type 2 CRF receptor is in the axons of several different types of cerebellar cortical neurons, and suggests that CRF could play a role in cerebellar development by modulating the release of transmitters from excitatory and/or inhibitory interneurons, which in turn could directly alter the maturation of cerebellar circuits. In contrast, the binding of a ligand to the full-length isoform of CRF-R2 or to CRF-R1, both in a postsynaptic location, may have a more direct effect on regulating the responsiveness of these cells to growth factors or neurotransmitters released from afferent axons by regulating permeability of ion channels or altering second messenger systems.

The distribution of corticotropin-releasing factor (CRF), the development of CRF-binding sites, and the age at which application of CRF elicits a physiological response have been described previously in the postnatal mouse cerebellum. The intent of the present study was to determine the cellular and subcellular distribution of the CRF type 1 receptor (CRF-R1) in the vermis of the postnatal mouse cerebellum and to correlate these data with those presented in previous studies. On P0, CRF-R1 is present in the apical processes of migrating Purkinje cells. Between P0 and P8, CRF-R1 immunostaining is confined to a supranuclear position in Purkinje cell bodies. Between P9 and P14, the receptor immunolabeling circumscribes Purkinje cell nuclei and extends into their primary dendrites. An adult-like distribution is achieved between P16 and P21. Between P0 and P14, the CRF-R1 antibody also labels processes of migrating GABAergic interneurons that are directed toward the pial surface. By P12, labeling begins to circumscribe the nucleus of GABAergic cells in the internal granule cell layer. Finally, astrocytic processes in the white matter, as well as radial glial processes, show focal labeling with the CRF-R1 antibody beginning at P3 and throughout postnatal development. A previous study demonstrated that CRF does not elicit a physiological response in Purkinje cells until P9. This observation, together with the data presented in this study, suggests that the binding of CRF to the type 1 receptor may be involved in regulating the development of cerebellar neurons and glia immediately after birth, before CRF assumes its function as a neuromodulator later in postnatal development and in the adult.

Corticotropin-releasing factor (CRF), present in climbing and mossy fiber afferents to the adult mouse cerebellum, acts as a neuromodulator to enhance the spontaneous and amino-acid-induced firing rate of Purkinje cells. CRF also is present during development of the mouse cerebellum, at ages that precede synaptogenesis, which suggests that it may have a different function during development compared to its modulatory role in the adult. The intent of this study was to determine when CRF begins to affect the firing rate of Purkinje cells as well as the time course over which this effect matures. The earliest effect of CRF was elicited at postnatal day (P) 9 at which time a weak enhancement in the amplitude of the firing rate was recorded. However, the amplitude, time to peak, sustainability, and duration of the response were significantly different from that recorded in the older animals or adults. The excitatory effect of CRF became stronger and the duration of the response increased progressively from P9 until it was adult-like by P20. Purkinje cells in the posterior lobe vermis developed a mature response before those in the anterior lobe or hemispheres. Data from previous studies have shown that CRF and its type 1 receptor are present in the cerebellum before birth and that both undergo major reorganization around P10. Taken together, these immunohistochemical observations and the present physiologic data indicate that CRF does not modulate the activity of Purkinje cells until the peptide begins to assume an adult-like distribution in cerebellar afferents.

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.

Microsatellite polymorphisms provide highly informative readily detectable markers for human linkage studies. This paper reports apparent non-Mendelian inheritance of a dinucleotide repeat polymorphism due to allele non-amplification. The previously hidden allele was revealed and Mendelian inheritance restored when a new CA strand primer was used for amplification. Sequencing of the hidden allele identified a single base substitution at the 3'-most position of the binding site for the original CA strand primer. Allele non-amplification is a potential source of confusion in linkage studies employing polymorphisms detected by the polymerase chain reaction. It should be considered whenever apparent non-Mendelian inheritance or non-paternity are encountered.

Corticotropin-releasing factor (CRF) has been described within varicosities that have a uniform distribution throughout the cerebellar nuclei of the cat. To date, however, no data are available as to the source of these nuclear afferents. Thus, a double-label technique was used to identify brainstem neurons which give rise to the CRF-containing afferents in the nucleus interpositus anterior (NIA) of the cat's cerebellum. Injections of fluorescent-tagged microspheres, which are retrogradely transported by cells with axons in the injection site, were made into lateral and medial aspects of the nucleus. The same sections were also processed for CRF immunohistochemistry. The primary source of CRF afferents to the NIA are the medial and dorsal accessory olivary nuclei. In addition to the inferior olive, several other brainstem nuclei also provide CRF afferents to the cerebellar nuclei. The medial aspect of the NIA receives afferents from the lateral reticular nucleus, external cuneate nucleus, perihypoglossal nucleus, medial vestibular nucleus and inferior central raphe nucleus. Additional afferents to more lateral aspects of the NIA are derived from the lateral reticular nucleus, external cuneate nucleus, and the magnocellular, lateral and gigantocellular tegmental areas. The brainstem nuclei that give rise to the CRF projection to the NIA receive input primarily from the spinal cord and likely relay information related to the status of an ongoing movement. A previous physiological study by Bishop has shown that CRF enhances the excitatory activity of nuclear neurons. CRF released from these afferents likely would enhance nuclear cell activity and thus provide a stronger or more prolonged effect on their respective target neurons in the brainstem.

The inferior olivary complex is the sole source of climbing fibers to the cerebellar cortex. Physiologically these afferents have been shown to have a powerful excitatory effect on their target neurons, namely Purkinje cells. Thus, any modulation of olivary firing rate or responsiveness will alter Purkinje cell firing and ultimately cerebellar function. Neuropeptides have been shown to modulate neuronal activity in several systems. The intent of the present study is to determine the olivary distribution of five peptides previously shown to be present and functional in cerebellar circuitry including cholecystokinin, calcitonin gene-related peptide, corticotropin releasing factor, enkephalin and substance P. These studies were carried out in the adult C57BL/6J mouse using the peroxidase anti-peroxidase immunohistochemical technique. All five peptides labeled varicosities of varying sizes. Varicosities labeled for cholecystokinin, calcitonin gene-related peptide and corticotropin releasing factor were densely distributed throughout the inferior olive. In contrast, varicosities immunostained for substance P and enkephalin, were more restricted in their distribution. The overlap in the distribution of these peptides suggests that they may be colocalized with each other as well as with excitatory or inhibitory amino acids known to be present in afferents to the inferior olive. Because of the extensive distribution of the peptides, it is likely that they are derived from multiple brainstem sources. These findings serve as baseline data for future physiological studies designed to address the functional role of peptides in olivary circuitry.

It is well established that cerebellar efferents originate from neurons located within the cerebellar nuclei. Neurons within these nuclei receive excitatory inputs derived from the axons that arise from cells in several different regions of the brainstem and spinal cord, some of which continue on to terminate as mossy fibers and climbing fibers in the cerebellar cortex. GABA-induced inhibition in the nuclei is derived primarily from Purkinje cells located in the overlying cortex and possibly from axonal collaterals of a population of small, GABAergic nuclear neurons. In addition, a third chemically defined system of afferents that contain the monoamine serotonin forms a dense plexus of fibers throughout the cat's cerebellar nuclei. The intent of this study is to determine the physiological effects of serotonin on the spontaneous activity of cerebellar nuclear cells as well as that induced by application of the excitatory amino acids glutamate and aspartate in an adult in vivo preparation. Iontophoretic application of serotonin in anesthetized preparations suppresses both spontaneous and excitatory amino acid induced activity. In addition, interactions between serotonin and the amino acid analogs quisqualate and NMDA were analyzed; 5HT suppresses the excitatory responses of neurons to both analogs. However, there is a stronger suppressive effect on quisqualate-induced excitation as compared to that elicited by NMDA. In addition to modulating the effects of the excitatory amino acids, serotonin also potentiates the inhibitory effects of GABA. However, the effect was greatest if the neuron was initially preconditioned with GABA. In summary, serotonin acts to suppress amino acid induced activity in cerebellar nuclear neurons and to enhance gABA-mediated inhibition. The net effect is a decrease in nuclear cell activity and consequently in cerebellar output.

The purpose of the present study is to determine the distribution of CRF containing afferents, and correlate these findings with the distribution of CRF binding sites and the neuronal localization of mRNA for the CRF1 receptor in the cerebellum of a single species, the mouse. Corticotropin releasing factor (CRF) has been localized within climbing fibers and mossy fibers throughout the cerebellar cortex of the mouse using immunohistochemistry. CRF immunoreactive, axonal varicosities also are present within all four of the cerebellar nuclei. 125I-labeled CRF binding sites are evident throughout all three layers of the cerebellar cortex (molecular, Purkinje and granule cell layers), but are not seen within the cerebellar nuclei. In situ hybridization histochemistry was employed using an antisense riboprobe corresponding to the full length sequence of the rat mRNA for the CRF1 receptor. Positive signal is present throughout the cerebellum in Purkinje cells and the granule cell layer. CRF1 receptor mRNA also is expressed within all four of the cerebellar nuclei. Further experiments are required to reconcile the lack of CRF binding sites in the cerebellar nuclei with the positive mRNA receptor expression and the presence of immunoreactive axonal varicosities. In previous physiological experiments, iontophoretic application of CRF enhances spontaneous as well as quisqualate-induced activity of Purkinje cells in slice preparations of the mouse cerebellum. When the results of the anatomical techniques are compared to the physiological data, there is convergent evidence to suggest that CRF influences the firing rate or responsiveness of Purkinje cells directly via release of the peptide from the climbing fiber system and indirectly via the mossy fiber-granule cell-parallel fiber circuit. Taken together, these anatomical and physiological data provide strong evidence to suggest that, in the adult cerebellum, CRF functions as a neuromodulator.