Parallin, a cerebellar granule cell protein the expression of which is developmentally regulated by Purkinje cells: evidence from mutant mice

In this paper we report on monoclonal antibody 3H6 with unique specificities for development of the cerebellum. Immunohistochemical studies on normal and mutant mice suggest that it is primarily located in or on granule cell parallel fibers in the cerebellum. The only other region showing immunoreactivity is a small region of the hippocampus. The antigen is detected immunohistochemically as early as postnatal day 11 in the molecular layer of the cerebellum. In adult wild-type mice parallin expression is seen in the molecular layer and to a lesser degree in the internal granular layer. In the cerebella of two neurological granule cell-deficient mutants, weaver (wv) and staggerer (sg), parallin is not detected. However, in two Purkinje cell-deficient mutants, Purkinje cell degeneration (pcd) and nervous (nr), a more complex and interesting pattern is observed. These two mutants do have granule cells and parallel fibers and 3H6 immunoreactivity is observed. However, in both of these Purkinje cell-deficient mutants the 3H6 immunoreactivity is drastically reduced in regions where Purkinje cells have degenerated. Furthermore, in nr mutants, the antigen appears to be concentrated in regions of the parallel fiber that are in close proximity to Purkinje cells, suggesting its possible association with synapses. Taken together these results suggest that parallin is a marker of granule cells and their parallel fibers, its onset correlates with the formation of granule cell synapses on developing Purkinje cells, and it requires Purkinje cells for the maintenance of expression.     

Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the beta-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1-3 days post-injection (1-3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells.     

Cerebellar granule cell differentiation in mutant and X-irradiated rodents revealed by the neural adhesion molecule TAG-1

In the external granular layer of the cerebellum, the granule cell precursors express the transient axonal glycoprotein TAG-1, a molecule involved in adhesion and neurite outgrowth. Granule cells express TAG-1 transiently, just as they extend neurites before migrating over the radial glia. The present study aims to investigate whether the expression pattern of TAG-1 is altered when granule cells develop abnormally. We studied in vivo models in which Purkinje and/or granule cell defects occur during postnatal development. These include the cerebellar mutant mice staggerer and lurcher as well as rats irradiated during postnatal development. Neither alterations in Purkinje cell differentiation nor the related granule cell loss in the mouse mutants impairs the ability of the surviving granule cell precursors to express TAG-1. Also, early granule cell loss in the X-irradiated rats do not disturb the TAG-1 expression phase in the patches of surviving granule cell precursors. Ectopic granule cells found in the adult cerebellum of X-irradiated rats do not bear the molecule, although they are located in the most superficial part of the molecular layer, occupied by the immunopositive cells a few days earlier. Thus, TAG-1 marks a very precise stage of granule cell differentiation, and the inward migration process itself is not required for the cessation of the expression. We postulate that TAG-1 may be involved in local differentiation steps restricted to the deep external granular layer such as parallel migratory routes or synchrony of axonal growth.     

Localization of neuronal and glial glutamate transporters

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.     

N-Acetylglucosaminyl transferase regulates the expression of the sulfoglucuronyl glycolipids in specific cell types in cerebellum during development

In the adult cerebellum, sulfoglucuronyl glycolipids (SGGLs) are specifically localized in Purkinje cells and their dendrites in the molecular layer. Other major cell types such as granule neurons and glial cells lack SGGLs. To explain the cell specific localization and the known biphasic expression of SGGLs, enzymic activities of four glycosyltransferases involved in the biosynthesis of SGGLs were studied in murine cerebellar mutants, in distinct cellular layers of rat cerebellum, and in isolated granule neurons during development. The enzymes studied were lactosylceramide: N-acetylglucosaminyl transferase (GlcNAc-Tr), lactotriaosylceramide:galactosyltransferase, neolactotetraosylceramide:glucuronyltransferase, and glucuronylglycolipid:sulfotransferase. In the cerebellum of Purkinje cell-deficient mutants, such as (pcd/pcd) and lurcher (Lc/+) where Purkinje cells are lost, GlcNAc-Tr was absent, but the other three glycosyltransferase were not severely affected. This indicated that the latter three enzymes were localized in other cell types, such as in mature granule neurons and glial cells, in addition to that in Purkinje cells, and the lack of SGGLs in these mutants was due to absence of GlcNAc-Tr. Analyses of the enzymes in the specific micro-dissected cellular layers also showed that Purkinje cell layer and molecular layer (where Purkinje cell dendrites are localized) contained all four enzymes. However, granule neurons and glial cells in the white matter lacked GlcNAc-Tr, but expressed the other three enzymes. It was concluded that the absence of SGGLs in adult granule neurons and glial cells was due to specific deficiency of the GlcNAc-Tr. Although adult granule neurons lacked GlcNAc-Tr and therefore SGGLs, isolated granule neurons from the neonatal cerebellum contained all four enzymes necessary for the synthesis of SGGLs. With development, the activity of GlcNAc-Tr in the isolated granule neurons declined but the other enzymes were not as affected, indicating that immature granule neurons were capable of synthesizing SGGLs and with maturation the synthesis was down-regulated. This also explains the biphasic expression of SGGLs in the developing cerebellum.     

Peak mitral inflow velocity predicts mitral regurgitation severity

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. Here we report the cloning of rat EAAT4 (rEAAT4) cDNA from rat cerebellum. The nucleotide sequence of rEAAT4 was 88% identical to the human sequence, and the predicted peptide was 89% identical to the human protein. The transport activity encoded by rEAAT4 has high affinity for L-glutamate. In Xenopus laevis oocytes expressing rEAAT4, L-glutamate and other transporter substrates elicited a current predominantly carried by chloride ions. Like human EAAT4, the rEAAT4 mRNA was largely restricted to cerebellar Purkinje cells; the rEAAT4 protein was localized to Purkinje cell somas and dendrites.     

Plastic changes in Ara C-treated and "transplanted" coeruleocerebellar cultures

Locus coeruleus axons project to cerebellar cortex in coeruleocerebellar cultures, where they make functional contacts, and also appear as fine fibers in the outgrowth zones. The predominant catecholamine of locus coeruleus neurons in culture is dopamine. When coeruleocerebellar cultures are exposed to cytosine arabinoside to destroy cerebellar granule cells and functionally compromise glia, there is a resultant increase of Purkinje cell survival and a sprouting of Purkinje cell recurrent axon collaterals, plus an increase of catecholaminergic axons accompanied by a doubling of tissue dopamine content. If such reorganized cultures are transplanted with granule cells and glia, a second round of plastic changes ensues in which the Purkinje cell population and the recurrent axon collaterals are reduced to control levels, but catecholaminergic axons and dopamine content remain increased. The maintenance of catecholaminergic axons does not appear to depend on the persistence of target neurons.     

Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion

Immunoreactivity to beta-amyloid precursor protein (APP) was present in the inner plexiform, ganglion cell and optic fibre layers, as well as in blood vessels, at birth in normally developing rat retinas. In the inner plexiform layer immunoreactivity disappeared by postnatal day (P) 14. A small population of ganglion cells was immunoreactive at birth, but none were visible at P7. From P14 onwards, however, there was weak immunoreactivity in ganglion cells again, and strong staining in Müller glia. Retinas affected by neonatal optic tract lesions contained more immunoreactive ganglion cells at P4 than did controls, but by P14 there was a severe loss of ganglion cells. These observations are consistent with APP being involved in retinal differentiation, including maturation of glia and neurones, synaptogenesis and possibly neuronal survival.     

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.     

Distinct modes of neuronal migration in different domains of developing cerebellar cortex

As postmitotic neurons migrate to their final destinations, they encounter different cellular microenvironments, but functional responses of migrating neurons to changes in local environmental cues have not been examined. In the present study, we used a confocal microscope on acute cerebellar slice preparations to examine real-time changes in the shape of granule cells, as well as the mode and rate of their migration as they transit different microenvironments. The rate of granule cell movement is fastest in the molecular layer, whereas their elongated somata and long leading processes remain in close contact with Bergmann glial fibers. Cell movement is slowest in the Purkinje cell layer after granule cells detach from the surface of Bergmann glia and the somata become transiently round, whereas the leading processes considerably shorten. Surprisingly, after entering the internal granular layer, granule cells re-extend both their somata and leading processes as they resume rapid movement independent of Bergmann glial fibers. In this last phase of migration, described here for the first time, most granule cells move radially for >100 micron (a distance comparable to that observed in the molecular layer) until they reach the deep strata of the internal granular layer, where they become rounded again and form synaptic contacts with mossy fiber terminals. These observations reveal that migrating neurons alter their shape, rate, and mode of movement in response to local environmental cues and open the possibility for testing the role of signaling molecules in cerebellar neurogenesis.     

Reactive astroglia-neuron relationships in the human cerebellar cortex: a quantitative, morphological and immunocytochemical study in Creutzfeldt-Jakob disease

In order to investigate the role of neuron-glia interactions in the response of astroglial to a non-invasive cerebellar cortex injury, we have used two cases of the ataxic form of Creutzfeldt-Jakob disease (CJD) with distinct neuronal loss and diffuse astrogliosis. The quantitative study showed no changes in cell density of either Purkinje or Bergmann glial cells in CJ-1, whereas in the more affected CJ-2 a loss of Purkinje cells and an increase of Bergmann glial cells was found. The granular layer in both CJD cases showed a similar loss of granule cells (about 60%) in parallel with the significant increase in GFAP+ reactive astrocytes. GFAP immunostaining revealed greater reactivity of Bergmann glia in CJ-2 than in CJ-1, as indicated by the thicker glial processes and the higher optical density. Granular layer reactive astrocytes were regularly spaced. In both CJD cases there was strict preservation of the spatial arrangement of all astroglial subtypes--Fa?anas cells, Bergmann glia and granular layer astrocytes. Reactive Fa?anas and Bergmann glial cells and microglia/macrophages expressed vimentin, while only a few vimentin+ reactive astrocytes were detected in the granular layer. Karyometric analysis showed that the increase in nuclear volume in reactive astroglia was directly related with the level of glial hypertrophy. The number of nucleoli per nuclear section was constant in astroglial cells of human controls and CJD, suggesting an absence of polyploidy in reactive astroglia. Ultrastructural analysis revealed junctional complexes formed by the association of macula adherens and gap junctions. In the molecular layer numerous vacant dendritic spines were ensheathed by lamellar processes of reactive Bergmann glia. Our results suggest that quantitative (neuron/astroglia ratio) and qualitative changes in the interaction of neurons with their region-specific astroglial partners play a central role in the astroglial response pattern to the pathogenic agent of CJD.     

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.     

Synaptically evoked glutamate transport currents may be used to detect the expression of long-term potentiation in cerebellar culture

Cerebellar long-term potentiation (LTP) is a use-dependent increase in the strength of the granule cell-Purkinje neuron synapse that occurs after brief stimulation of granule cell axons at 2-8 Hz. Previous work has shown that cerebellar LTP also may be seen when synaptic currents are evoked in granule cell-glial cell pairs in culture. This finding suggests a model in which cerebellar LTP is expressed presynaptically and therefore may be detected by either neuronal or glial postsynaptic cells. However, synaptic currents evoked in both granule cell-glial cell pairs and granule cell-Purkinje neuron pairs in culture are mediated primarily by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, raising the possibility that cerebellar LTP might be expressed postsynaptically in both glial cells and Purkinje neurons in a similar manner. To address this question, glutamate transport currents were recorded in granule cell-glial cell pairs in culture by pharmacological isolation. These currents were increased by substitution of internal Cl with NO3 and were blocked by -pyrrolidine-2,4-dicarboxylate, both characteristics of the major cloned Bergmann glial cell glutamate transporter, EAAT1. After acquisition of baseline responses, LTP of isolated transport current was evoked by stimulation at 4 Hz (100 pulses) and could be blocked by removal of external Ca during this stimulation. The expression of LTP was associated with a decrease in the rate of synaptic failures and a decrease in the degree of paired-pulse facilitation. These findings, when taken together with the previous observation that both Purkinje neuron and glial AMPA/kainate responses can be used to detect cerebellar LTP, strongly suggest that the expression of cerebellar LTP is, at least in part, presynaptic. This strategy should also be useful in illuminating the locus of expression of other model systems of information storage such as hippocampal LTP/long-term depression.     

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.     

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.     

Role of arachidonic acid and its metabolites in the priming of NADPH oxidase in human polymorphonuclear leukocytes by peritoneal dialysis effluent

Immunohistochemical techniques were used to examine the distribution of prostaglandin H synthase (PGHS)-2 and neuronal nitric oxide synthase (nNOS) in piglet brain. Samples from parietal cortex, hippocampus, and cerebellum were immersion fixed in 10% formalin, sectioned at 50 microm, and immunostained using specific antibodies against PGHS-2 and nNOS. Immunoreactivity for PGHS-2 was extensive throughout the areas examined. For example, PGHS-2 immunoreactive cells were present in all layers of the cortex, but were particularly dense among neurons in layers II/II, V, and VI. In addition, glial cells associated with microvessels in white matter showed PGHS-2 immunoreactivity. In contrast, nNOS immunoreactive neurons were limited in number and widely dispersed across all layers of the cortex and thus did not form a definable pattern. In the hippocampus, heavy PGHS-2 immunoreactivity was present in neurons and glial cells in the subgranular region, stratum radiatum, adjacent to the hippocampal sulcus, and in CA1 and CA3 pyramidal cells. Immunostaining for nNOS displayed a different pattern from PGHS-2 in the hippocampus, and was mainly localized to the granule cell layer of the dentate gyrus and the mossy fiber layer. In the cerebellum, PGHS-2 immunoreactivity was heavily represented in the Bergmann glia and to a lesser extent in cells of the granular layer, whereas nNOS was detected only in Basket cells. There are four conclusions from this study. First, PGHS-2 immunoreactivity is widely represented in the cerebral cortex, hippocampus, and cerebellum of neonatal pigs. Second, glia cells as well as neurons can show immunoreactivity for PGHS-2. And third, the distribution of nNOS is different from PGHS-2 immunoreactivity in the cerebral cortex, hippocampus, and cerebellum.     

Kaposiform hemangioendothelioma of infancy and childhood. An aggressive neoplasm associated with Kasabach-Merritt syndrome and lymphangiomatosis

After we identified several novel cDNAs by screening a neonatal (P1) heterozygous weaver (wv/+) cerebellar cDNA expression library with a rabbit anti-mouse granule cell antiserum, we characterized and sequenced one cDNA, GCAP-8 (standing for granule cell antiserum positive, clone number 8). In this study we examined its expression and cellular distribution in adult cerebellar mutant mice as evidenced by in situ hybridization histochemistry. In wild-type (+/+) brain, strong hybridization signal is seen in cerebellum, hippocampus, substantia nigra (SN), and cerebral cortex; in the cerebellum, hybridization signal is seen in granule cells, Purkinje cells, and in cells of the deep cerebellar nuclei. In the granuloprival weaver (wv/wv) cerebellum, hybridization signal is seen mainly in Purkinje cells. GCAP-8 expression is reduced in wv/wv SN pars compacta, which is known to lose dopamine (DA) neurons. In Purkinje cell degeneration (pcd/pcd) mutants, granule cells show hybridization signal, but overall expression is decreased owing to the absence of Purkinje cells. In reeler (rl/rl) cerebellum, the strongest hybridization signal is found in a thin granule cell layer without the typical foliation pattern, while grain clusters representing ectopic Purkinje cells are observed in the subcortical white matter and the area of the deep cerebellar nuclei. GCAP-8 expression in the reeler hippocampus and cerebral cortex shows a mixing of layers, which is known to be an aspect of the histological phenotype of this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N-terminal and central parts), GLAST (C-terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)-triphosphate (IP3)-receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na+-dependent "binding" of D-[3H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N-terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT.     

Expression of the human excitatory amino acid transporter 2 and metabotropic glutamate receptors 3 and 5 in the prefrontal cortex from normal individuals and patients with schizophrenia

A disturbance of glutamatergic transmission has been suggested to contribute to the development of schizophrenic pathophysiology based primarily on the ability of glutamate receptor antagonists to induce schizophrenic-like symptoms, and recent studies suggesting reduced glutamatergic function in the prefrontal cortex (PFC) of individuals with a diagnosis of schizophrenia. In order to investigate this hypothesis further, the expression of several 'glutamatergic' markers, the metabotropic glutamate receptors (mGluRs; mGluR3, 5) and the human excitatory amino acid transporter (EAAT2) were compared in the PFC of normal individuals and schizophrenics. The present results showed that glial cells in the pyramidal layers of the PFC from schizophrenics had decreased EAAT2 mRNA content relative to controls in Brodmann areas 9 and 10. The cellular levels of expression of the two mGluR signals investigated (mGluR3, and 5) were not significantly changed relative to controls except for an increase in the neuronal mGluR5 in the pyramidal cell layers of area 11. Comparing the ratio of cellular mGluR expression to that of EAAT2, the mGluR/EAAT2 ratio showed that schizophrenics had a significantly increased mGluR/EAAT2 ratios in the pyramidal cell layers of all three PFC regions examined. The glutamate content of consecutive sections analyzed by high pressure liquid chromatography (HPLC), although decreased in schizophrenics did not reach significance and did not correlate with either EAAT2 or mGluR mRNA content. These results are discussed in the light of current results on the neurochemistry and pharmacology of schizophrenia.     

Dynorphin and the kappa 1 ligand [3H]U69,593 binding in the human epileptogenic hippocampus

The distribution of dynorphin (DYN), one of its binding sites (kappa 1 receptor) and their relationship to neuronal loss and granule cell hyperexcitability was examined in hippocampi from patients with temporal lobe epilepsy (TLE). In hippocampi that were not the seizure focus (mass associated temporal lobe epilepsy, MaTLE; and paradoxical temporal lobe epilepsy, PTLE) DYN-like immunoreactivity was localized in the dentate granule cells and their mossy fiber terminals within the hilus and area CA3. In hippocampi that were the seizure focus (MTLE), 89% showed an additional band of immunoreactivity confined to the inner molecular layer (IML) of the dentate gyrus, representing recurrent mossy fiber collaterals. In 11% of MTLE patients no staining was found in the IML (MTLE/DYN-). The MTLE/DYN- hippocampi were also characterized by a significantly lower degree of cell loss than in MTLE hippocampi in the dentate granule cell layer, the hilus and CA3. Both MTLE and MTLE/DYN- hippocampi showed evoked epileptiform bursting in granule cells while MTLE showed greater polysynaptic EPSPs and spontaneous excitatory activity. Thus granule cell recurrent collateral sprouting may account for only some aspects of hyperexcitability. In 30% of the MTLE group, hilar neurons of a variety of morphological types expressed DYN immunoreactivity in their somata and dendrites. The density of [3H]U69,593 binding sites in MaTLE and PTLE patients was highest in areas CA1 and the subiculum-regions having little or no DYN-staining. In the dentate molecular layer, hilus and CA3--regions with the most DYN immunoreactivity--there was a low density of ligand binding. The significance of this transmitter/receptor mismatch is yet unknown.