[The patient in the psychiatric emergency ambulance: diagnoses, reasons and comparison of layperson vs. physician viewpoints]. / Der Patient in der psychiatrischen Notambulanz: Erstdiagnosen, Vorstellungsgründe und Vergleich der Laien- vs. Arztsicht.

A survey in specialties other than psychiatry showed that "emergency room"-patients have factors other than the presenting disease that determine the usage of urgent medical evaluation. In the following prospective study 104 outpatients presenting at daytime in a university psychiatric emergency care unit were included over 6 months. Apart from social and epidemiological data, illnesses according to ICD-10, reason for presentation from the patient's point of view and in this regard the physician's evaluation were included. The most prevalent diagnoses were depression, adjustment disorders and anxiety disorders, comprising together 75 %. Organic disorders or addictive disorders were less frequent; psychoses were found in 8 %. Concerning the presentation as an emergency, 70 % of patients reported a subjective clinical deterioration but only 44 % were regarded as an urgent need in the responsible physician's point of view (Cohen's kappa 0.39). Our findings show that patients presenting as "psychiatric emergency cases" without appointment mainly suffer from depression, adjustment disorders and panic disorders. Furthermore, the layperson's point of view of clinical deterioration justifying an emergency presentation differs from physician's evaluation. The most likely cause for this disagreement between physicians and patients in the assessment to utilise a medical emergency care service in psychiatry might be dysfunctional or, respectively, negative-biased cognitions accompanying depressive syndromes.

Cost of a 5-year lung cancer survivor: symptomatic tumour identification vs proactive computed tomography screening.

BACKGROUND: Our objective was to analyse the cost effectiveness of computed tomography (CT) screening for lung cancer in terms of the cost per long-term survivor, which has not been evaluated to date. METHODS: Estimations were computed based on data from the Surveillance, Epidemiology, and End Results registries covering years 1999-2003. The design framework of our model allowed for the incorporation of multiple values taken from the epidemiological and clinical literature to be utilised for cost inputs, scope of patients screened, diagnostic staging, and survival percentages applied separately to two cohorts: age 40-79 and 60-79 years. This enabled the analysis of over 1400 scenarios, each containing a unique set of input values, for which the estimated cost per 5-year survivor (CP5YS) was compared between the symptom-detected and proactive screening approaches. RESULTS: Estimated CP5YS were higher for the symptom-detected approach in all 729 scenarios analysed for the cohort ages 60-79 years, ranging from approximately $5800 to $116,700 increased cost per 5-year survivor (CP5YS). For the cohort ages 40-79 years, 75% of the 729 scenarios analysed showed increased CP5YS for the symptom-detected approach ranging from $5700 to $110,000 increased CP5YS. Total costs and total 5-year survivors were higher for the proactive screening method for all scenarios analysed across both cohorts with increases ranging from 50-256% and 98-309%, respectively. CONCLUSION: The predicted increase in long-term survival with CT screening and the potential for better utilisation of health-care dollars in terms of CP5YS, particularly when screening patients over the age of 60 years, are critically important considerations in directing effective future lung cancer management strategy.

Modulation of synapse formation by cyclic adenosine monophosphate.

Synapses between neuroblastoma-hybrid cells and myotubes exhibit a high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) levels of the hybrid cells for several days results in the appearance of functional voltage-sensitive Ca2+ channels, which are required for evoked secretion of acetylcholine. The results show that cyclic AMP regulates synaptogenesis by regulating the expression of voltage-sensitive Ca2+ channels, and suggest that cyclic AMP affects posttranslational modifications of some glycoproteins and cellular levels of certain proteins.

Genotoxic effects of the cyanobacterial pentapeptide nodularin in HepG2 cells.

The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1 µg/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine.

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

Genomic organization and alternative splicing of the human and mouse RPTPrho genes.

BACKGROUND: Receptor protein tyrosine phosphatase rho (RPTPrho, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPrho gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPrho expression is restricted to the central nervous system. RESULTS: The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPrho genes are described. The two genes are comprised of at least 33 exons. Both RPTPrho genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning approximately 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment. CONCLUSIONS: We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPrho isoforms. Our findings suggest that RPTPrho extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period.

Topographical distribution of muscarinic cholinergic receptors in the cerebellar cortex of the mouse, rat, guinea pig, and rabbit: a species comparison.

Light microscopic autoradiography of [3H]quinuclidinyl benzilate (QNB) binding sites was used to study the distribution of muscarinic acetylcholine receptors in the mouse, rat, guinea pig, and rabbit cerebellar cortex. In the mouse, the laminar distribution of grain density was similar throughout the cortex, with slightly higher levels over lobules IX and X. The highest [3H]QNB labeling was present over the granule cell layer, and low levels were observed over the molecular layer. In the rat, the general distribution was similar to that of the mouse in that the granule cell layer was most densely labeled and the highest concentration of [3H]QNB binding sites was present in lobules IX and X of the archicerebellum. In these lobules, however, the laminar distribution of grain density was reversed so that the molecular layer was more densely labeled than the granule cell layer. In addition, several discrete columns of elevated grain density traversed the granule cell layer in caudal regions of lobule IX. The distribution of [3H]QNB binding sites in the guinea pig cerebellum was similar to that of the rat in that the molecular layer of lobules IX and X was again more intensely labeled than other cerebellar regions. In the remaining lobules, grain density was equal over the granule cell and molecular layers. In the rabbit cerebellar cortex, slightly higher grain density was observed in the granule cell layer than in the molecular layer. In lobules IX and X and in the hemisphere of X, the Purkinje cell layer was most densely labeled; parasagittal columns of very high grain density were present over the molecular layer of several cortical regions, including lobules, I, II, III, IV, V, IX, X, and the hemispheres of IX and X. Since muscarinic receptors have previously been found on blood vessels, there is a possibility that some proportion of receptor labeling may be localized to these structures. Microvessels and capillaries in each of the species examined were more numerous in the granule cell layer than in the molecular layer and white matter. The distribution of blood vessels in many cerebellar lobules of mice, rats, and guinea pigs corresponded quite closely to the general distribution of [3H]QNB binding sites. Unique patterns of labeling in lobules IX and X were not accompanied by corresponding patterns of blood vessel distribution, however. In the mouse, there was a slight increase in muscarinic receptor density observed in the archicerebellum, with no corresponding increase in the density of blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Embryonic and postnatal expression of four gamma-aminobutyric acid transporter mRNAs in the mouse brain and leptomeninges.

The distribution of gamma-aminobutyric acid (GABA) transporter mRNAs (mGATs) was studied in mouse brain during embryonic and postnatal development using in situ hybridization with radiolabeled oligonucleotide probes. Mouse GATs 1 and 4 were present in the ventricular and subventricular zones of the lateral ventricle from gestational day 13. During postnatal development, mGAT1 mRNA was distributed diffusely throughout the brain and spinal cord, with the highest expression present in the olfactory bulbs, hippocampus, and cerebellar cortex. The mGAT4 message was densely distributed throughout the central nervous system during postnatal week 1; however, the hybridization signal in the cerebral cortex and hippocampus decreased during postnatal weeks 2 and 3, and in adults, mGAT4 labeling was restricted largely to the olfactory bulbs, midbrain, deep cerebellar nuclei, medulla, and spinal cord. Mouse GAT2 mRNA was expressed only in proliferating and migrating cerebellar granule cells, whereas mGAT3 mRNA was absent from the brain and spinal cord throughout development. Each of the four mGATs was present to some degree in the leptomeninges. The expression of mGATs 2 and 3 was almost entirely restricted to the pia-arachnoid, whereas mGATs 1 and 4 were present only in specific regions of the membrane. Although mGATs 1 and 4 may subserve the classical purpose of terminating inhibitory GABAergic transmission through neuronal and glial uptake mechanisms, GABA transporters in the pia-arachnoid may help to regulate the amount of GABA available to proliferating and migrating neurons at the sub-pial surface during perinatal development.

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.

GABAA/benzodiazepine receptor alpha 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice.

The gamma aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor is a multisubunit (alpha, beta, gamma, delta, and rho) ligand-gated chloride channel; there are several variants of the alpha, beta, and gamma subunits, each of which has been localized throughout the central nervous system. A large number of GABAA/BZ subunit variants are expressed within the cerebellar cortex. In previous studies from other laboratories, alpha 6 subunit mRNA has been reported to be present exclusively in cerebellar granule cells. The developmental expression of alpha 6 mRNA in cerebellar and cochlear granule cells is of interest because it has been suggested that each of these cell types is derived from a common precursor pool. The polymerase chain reaction was used to generate a cDNA fragment encoding a portion of the M3-M4 intracellular loop of the alpha 6 subunit of the GABAA/BZ receptor. A [35S] riboprobe, transcribed from this cDNA fragment, was used to examine the expression of the alpha 6 subunit mRNA by in situ hybridization in developing normal mice and in adult mutant mice with known deficits in synaptic circuitry. A strong hybridization signal was observed over the granule cell layers of both the cerebellum and cochlear nuclei in adult mice. The signal over the cochlear nuclei appeared after birth toward the end of postnatal week 1, coinciding with the appearance of labeling in the cerebellar cortex. The intensity of the hybridization signal in both regions increased rapidly until postnatal day 14, after which it increased more gradually, reaching adult levels during postnatal week 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the expression of gamma-aminobutyric acidA/benzodiazepine receptor subunit mRNAs in the murine inferior olivary complex.

The pharmacological and physiological properties of ligand-gated ion channels are dependent on their subunit composition; spontaneously occurring changes in subunit composition during neuronal development may result in dramatic functional differences between embryonic and adult forms of the receptor complex. In the present study, in situ hybridization with antisense cRNA probes was used to examine the subunit composition of the gamma-aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor in the developing inferior olivary complex. This receptor is thought to be a pentameric chloride channel comprised of selected alpha, beta, gamma, delta, and rho subunits, the majority of which have several isoforms: alpha 1-6, beta 1-4, gamma 1-4, and rho 1,2. Among the 13 subunit variants present in the mammalian central nervous system, alpha 2-5, beta 3, and gamma 1,2 mRNAs are expressed at significant levels in the inferior olivary complex. Two clearly different temporal patterns of GABAA/BZ receptor subunit mRNA expression were observed: The expression of alpha 3, alpha 5, beta 3, and gamma 2 mRNAs was at a peak during embryonic and early postnatal development followed by rapid down-regulation thereafter. Conversely, alpha 2, alpha 4, and gamma 1 mRNA expression was very low or absent during early development, and a pronounced increase was observed at the end of postnatal week 1. These studies suggest that there are developmental changes in the subunit composition of the GABAA/BZ receptor in inferior olivary neurons. These changes in subunit expression, which occur during a period of major alterations in afferent and efferent synaptic connections, may subserve a change in the role of GABA from its function as a neurotrophic factor to that of an inhibitory neurotransmitter.

Novel receptor protein tyrosine phosphatase (RPTPrho) and acidic fibroblast growth factor (FGF-1) transcripts delineate a rostrocaudal boundary in the granule cell layer of the murine cerebellar cortex.

We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.

Harmaline-induced changes in gamma aminobutyric acid(A) receptor subunit mRNA expression in murine olivocerebellar nuclei.

Increased CNS activity in the form of electrically or chemically induced seizures is known to alter the properties of GABA(A) receptors. The tremorgen, harmaline, causes a bursting pattern of activity in inferior olivary neurons, the effects of which are transmitted throughout the olivocerebellar circuit to other regions of the CNS. In situ hybridization was used to determine the effect of this increased activity on gamma aminobutyric acid(A) (GABA(A)) receptor subunit gene expression in the cerebellar Purkinje cell layer, deep cerebellar nuclei and inferior olivary complex of adult mice. In Purkinje cells, the expression of alpha(1), beta(2), and gamma(2) mRNAs was increased only slightly (<5%) by harmaline administration, while in deep cerebellar neurons, beta(2) transcript levels were initially elevated (26%), but dropped to control levels immediately thereafter. The expression of alpha(2), alpha(4), beta(3) and gamma(1) mRNAs in olivary neurons was affected differentially by harmaline administration. The alpha(4) transcript was increased, reaching >60% above control levels at 6 h post-injection. A smaller increase was observed for alpha(2) mRNA, while beta(3) and gamma(1) transcripts dropped below control levels during the same period. The expression of corticotropin-releasing factor mRNA was also elevated in the olivary complex. These data indicate that while Purkinje cells and deep cerebellar neurons are only minimally affected, harmaline induced changes in cellular properties may result in increased numbers of alpha(4)-containing, diazepam-insensitive, GABA(A) receptors in olivary neurons.

Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of beta 3 subunit mRNA and [3H]muscimol binding sites.

The GABAA/benzodiazepine receptor consists of at least four subunits, alpha, beta, gamma and delta, each comprised of several variants. The developmental expression of the alpha 1, beta 1-3, gamma 2 and delta subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [3H]flunitrazepam and [3H]muscimol binding sites, alpha and beta subunit-specific ligands respectively, were also studied autoradiographically. The beta 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of alpha 1 subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of beta 1, beta 2, and gamma 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while delta mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABAA/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.

GABAA/benzodiazepine receptor gamma 2 subunit gene expression in developing normal and mutant mouse cerebellum.

Recent studies have identified several subunits (alpha, beta, gamma and delta) of the gamma-aminobutyric acidA/benzodiazepine receptor; each consists of several variants. The gamma 2 subunit appears to mediate the interaction of the alpha and beta subunits making the receptor capable of modulation by benzodiazepines. In the present studies, the expression of mRNA encoding the gamma 2 subunit was examined in the cerebellum during development and in adult Purkinje cell degeneration, lurcher and reeler mutant mice. In the normal adult cerebellum, in situ hybridization with [35S]cRNA probes revealed a strong signal over the Purkinje cell layer and deep cerebellar nuclei, and a weaker signal over basket, stellate and granule cells. Labeling over Purkinje cells was detectable at birth, gradually becoming stronger and more punctate during postnatal weeks 1 and 2, as Purkinje cells formed a monolayer between the molecular and granule cell layers. Adult levels of grain density were reached by P20. The external germinal layer, which contained proliferating granule cells, was unlabeled throughout development; however, weak labeling was detected over the internal granular layer at the end of postnatal week 1, as granule cells began their migration across the molecular layer. During the second postnatal week, punctate labeling became visible over the molecular layer in a distribution indicative of basket and stellate cells. In adult Purkinje cell degeneration and lurcher mutants, in which Purkinje cells have degenerated, no punctate labeling characteristic of mature Purkinje cells was detected. In adult and developing reeler mutants, where all classes of cells are malpositioned throughout the cerebellum, the punctate hybridization signal was present and clearly associated with Purkinje cells in all cortical regions. Our results suggest that developing Purkinje cells express the gamma 2 gene at a time prior to receiving GABAergic inhibitory input, and that the continued expression in the adult is not affected by the absence of afferents.

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11.

We have established that the GABAA receptor alpha 6 (Gabra6) and alpha 1 (Gabra 1) subunit genes are tightly linked on mouse chromosome 11 by analysing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant inbred strains. These results further demonstrate clustering of the GABAA receptor subunit genes on mouse chromosomes and the synteny for these clusters between the mouse and human genomes.

Identification and characterization of RPTP rho, a novel RPTP mu/kappa-like receptor protein tyrosine phosphatase whose expression is restricted to the central nervous system.

We describe the cloning, chromosomal localization and characterization of RPTPrho, a new member of the RPTPmu/kappa phosphatase subfamily. Receptor tyrosine phosphatases in this subfamily are comprised of a MAM domain near the N-terminal, an immunoglobulin-like domain, four fibronectin type III repeats, a single transmembrane domain, and a large juxtamembrane segment followed by two intracellular phosphatase domains. An alternatively spliced mini-exon was identified in the extracellular segment of RPTPrho, between the fourth fibronectin type III repeat and the transmembrane domain. The RPTPrho gene was mapped to human chromosome 20 and mouse chromosome 2. Northern blot analysis demonstrated that RPTPrho expression was restricted to the central nervous system, and in situ hybridization studies showed that the RPTPrho transcript was distributed throughout the murine brain and spinal cord. Exceptionally high levels of the transcript were present in the cortex and olfactory bulbs during perinatal development, but were down-regulated during postnatal week two. The motifs found in the extracellular segment of type II receptor protein tyrosine phosphatases are commonly found in neural cell adhesion molecules, suggesting that RPTPrho may be involved in both signal transduction and cellular adhesion in the central nervous system.

Light-microscopic distribution and parasagittal organisation of muscarinic receptors in rabbit cerebellar cortex.

Recent studies on the effects of intrafloccular injections of muscarinic agonists and antagonists on compensatory eye movements in rabbit, indicate that muscarinic receptors may play a modulatory role in the rabbit cerebellar circuitry. It was previously demonstrated by Neustadt et al. (1988), that muscarinic receptors in rabbit cerebellar cortex are distributed into alternating longitudinal zones of very high and very low receptor density. In the present study, the zonal and cellular distribution of muscarinic receptors in the rabbit cerebellar cortex is investigated in detail using in vitro ligand autoradiography with the non-selective high-affinity antagonist [3H]quinuclidinyl benzilate (QNB), and the M2-specific antagonist [3H]AF-DX384, and immunocytochemistry with a monoclonal antibody specific for the cloned m2 muscarinic receptor protein. [3H]QNB and [3H]AF-DX384 binding sites and m2-immunoreactivity had similar overall distributions: dense labeling occurred in the dendritic arbors of a subset of Purkinje cells that are organized into parasagittal bands. A high level of muscarinic receptor labeling was also observed in a thin substratum of the molecular layer immediately above the Purkinje cell layer of the vestibulo-cerebellar lobules, i.e. the nodulus, the ventral uvula and the flocculus. Labeling in this stratum was associated with densely packed fibres, which were putatively identified as parallel fibres. Also Golgi cells, which were localized in part in the molecular layer, and a subset of mossy fibre rosettes, primarily concentrated in lobule VI, were immunoreactive for the m2 receptor. The parasagittal band of labeled Purkinje cell dendrites were most prominent in the anterior lobe (lobules I-V), in crus 1 and 2, in the flocculus, the ventral paraflocculus and the rostral folium of the nodulus. In other lobules, only infrequent Purkinje cells contained muscarinic receptors. The parasagittal organisation of muscarinic receptors differed from that of zebrin I, a Purkinje cell-specific protein which is often used as a marker of parasagittal parcelation of the cerebellar cortex. In the anterior lobe, however, there was a partial correspondence between muscarinic receptor and zebrin I bands. In the flocculus the distribution of muscarinic-receptor-positive Purkinje cells was related to the distinct white matter compartments as revealed with acetylcholinesterase (AChE) histochemistry. Muscarinic receptor-containing Purkinje cells were located primarily in the floccular zone 1, which is implicated in the control of eye movements about a horizontal axis. In order to relate the distribution of muscarinic receptor labeling to that of cholinergic nerve terminals, [3H]QNB binding sites and sodium-dependent [3H]hemicholinium-3 binding were compared. Sodium-dependent [3H]hemicholinium-3 binding sites mainly occurred in the granule cell layer of the vestibulo-cerebellum, which corresponds well with the distribution of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). However, sodium-dependent [3H]hemicholinium binding complemented, rather than co-localized with, muscarinic receptors which were primarily distributed in the molecular layer of the lobules of the vestibulo-cerebellar lobules. Their functional significance is puzzling, since their distribution does not correspond to that of markers of cholinergic innervation.

The ontogeny of [3H]muscimol binding sites in the C57BL/6J mouse cerebellum.

The characteristics of [3H]muscimol binding were investigated in cerebellar sections from 7-day-old mice. The binding sites were found to possess the kinetic properties and pharmacological specificity characteristic of high-affinity GABAA receptors. [3H]Muscimol binding sites in the developing C57BL/6J mouse cerebellum were visualized by light microscopic autoradiography. A distinct band of labeling situated over the molecular layer was apparent from day 1 to day 7. The external granule cell layer remained unlabeled throughout development. Labeling over the internal granule cell layer gradually increased from birth; it became more dense and well defined until adult levels of grain density were reached at 35-42 days of age. The deep cerebellar nuclei were moderately labeled at birth and gradually decreased in density thereafter. The observed ontogeny of granule cell [3H]muscimol binding sites suggests that the synthesis of receptors is initiated at a time immediately after cessation of cell division, coinciding with the beginning of granule cell translocation across the molecular layer. Since, at this time, granule cells have not yet formed synapses with the GABAergic Golgi II cells, nor have they, in turn, formed the vast majority of synaptic contacts with Purkinje cells, it follows that receptor appearance precedes the formation of afferent connections, and may also precede efferent synaptic contacts. The timing of the appearance of [3H]muscimol binding sites raises the possibility that their initial acquisition may be related to developmental events other than the interaction of the granule cell with its pre- or postsynaptic neuronal partners.

Cerebellar benzodiazepine receptors: cellular localization and consequences of neurological mutations in mice.

The distribution of cerebellar [3H]flunitrazepam binding sites was studied autoradiographically in Purkinje cell degeneration (pcd/pcd), weaver (wv/wv), staggerer (sg/sg) and reeler (rl/rl) mutant mice. In the normal 78-day-old C57BL/6J mouse cerebellum, the highest concentration of [3H]flunitrazepam binding sites was observed over the molecular layer. Intermediate grain density was present over the Purkinje cell layer and intermediate to high density over the deep cerebellar nuclei. Low labeling was observed over the granule cell layer. Negligible concentrations of binding sites were seen in the white matter. In 45-49-day-old Purkinje cell degeneration mutants, where essentially all Purkinje cells have disappeared by day 45, there was a small decrease in grain density over the cerebellar cortex. Concomitantly, a substantial increase in grain density was observed over the deep cerebellar nuclei of the pcd/pcd mutants when compared to littermate controls. A significant increase in [3H]flunitrazepam labeling was observed over the cerebellar cortex of 81-86-day-old wv/wv mutants; this was most pronounced in the vermis where the granule cell loss was greatest. Over the hemispheres, where fewer granule cells degenerate, a lower density of binding sites was seen. Grain density over the wv/wv deep cerebellar nuclei was comparable to that of littermates. Substantially lower [3H]flunitrazepam labeling was detected over the cerebellar cortex of 25-27-day-old sg/sg mutants in which the number of granule, Purkinje and Golgi cells is greatly reduced; the labeling over the deep nuclei, however, was significantly increased. In 27-29-day-old rl/rl mutant cerebella, where all classes of cells are malpositioned, labeling density over all areas of the cerebellar cortex, including the Purkinje cell masses, was increased. Our autoradiographic data suggest that a proportion of cerebellar cortical benzodiazepine receptors are associated with Purkinje cells; we propose that the remainder of the receptors are localized on Golgi cells, while granule cells are devoid of receptors. In the deep cerebellar nuclei, the observed increase in benzodiazepine receptors in the pcd/pcd and sg/sg mutants may be a manifestation of denervation supersensitivity subsequent to the loss of innervation by Purkinje cell axon terminals. The finding of a high receptor density in the Purkinje cell masses of the rl/rl mutant, where Purkinje cells are devoid of afferent basket cell input, suggests that benzodiazepine receptors are expressed and maintained in the absence of a full complement of GABAergic afferents.