Sexually dimorphic long-term effects of an early life experience on AMPA receptor subunit expression in rat brain.

Neonatal handling, an experimental model of early life experiences, is known to affect hypothalamic-pituitary-adrenal (HPA) axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. AMPA receptors (AMPARs) mediate fast synaptic transmission at excitatory glutamatergic synapses in the CNS and are crucial during neuronal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as AMPA glutamate receptor subunits (GluA1, GluA2, GluA3 and GluA4), which combine to form tetramers. The present study addressed the question of whether neonatal handling (15min daily maternal separation from postnatal day 1 (PND1) to PND21) might have an effect on GluA1-4 mRNA levels in adult rat male and female brain using in situ hybridization. We have identified selective sexually dimorphic effects of neonatal handling on the mRNA expression levels of AMPAR subunits in adult rat hippocampus and nuclei of the amygdaloid complex. In the dorsal hippocampus GluA1 mRNA levels were increased in handled males, while they were decreased in handled female animals. In the ventral hippocampus and the amygdaloid complex GluA2 mRNA was lower in handled females, while no effect was observed in handled males. Furthermore, we observed that neonatal handling induced in both sexes decreases of GluA2 mRNA in the dorsal hippocampus, as well as in the somatosensory and occipital cortex, of GluA3 mRNA in most hippocampal areas, amygdaloid complex and cortical regions studied, and of GluA4 mRNA in the ventral hippocampus. These results show that glutamatergic transmission is markedly affected by an early experience. The neonatal handling-induced alterations in AMPAR subunit composition are in line with the increased brain plasticity, the more effective HPA axis function, and in general the more adaptive behavioral phenotype known to characterize the handled animals.

Selective effects of neonatal handling on rat brain N-methyl-D-aspartate receptors.

Neonatal handling, an experimental model of early life experiences, is known to affect the hypothalamic-pituitary-adrenal axis function thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. A molecule that plays a most critical role in such processes is the N-methyl-D-aspartate (NMDA) receptor, a tetramer consisting of two obligatory, channel forming NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. Since the subunit composition of the NMDA receptor affects brain plasticity, in the present study we investigated the effect of neonatal handling on NR1, NR2A and NR2B mRNA levels using in situ hybridization, and on NR2B binding sites, using autoradiography of in vitro binding of [(3)H]-ifenprodil, in adult rat limbic brain areas. We found that neonatal handling specifically increased NR2B mRNA and binding sites, while it had no effect on the NR1 and NR2A subunits. More specifically, neonatally handled animals, both males and females, had higher NR2B mRNA and binding sites in the dorsal CA1 hippocampal area, as well as the prelimbic, the anterior cingulate and the somatosensory cortex, compared to the non-handled. Moreover NR2B binding sites were increased in the dorsal CA3 area of handled animals of both sexes. Furthermore, neonatal handling had a sexually dimorphic effect, increasing NR2B mRNA and binding sites in the central and medial amygdaloid nuclei only of the females. The neonatal handling-induced increase in the NR2B subunit of the NMDA receptor could underlie the higher brain plasticity, which neonatally handled animals exhibit.

Dopamine receptor and transporter levels are altered in the brain of Purkinje Cell Degeneration mutant mice.

The Purkinje Cell Degeneration (Nna1pcd, pcd) mutant mouse is mainly characterized by the complete, primary loss of the Purkinje cells and the secondary, partial, retrograde loss of the granule and inferior olive neurons and is considered a model of human degenerative ataxia. We determined, by in vitro quantitative autoradiography and in situ hybridization, the effects of the Purkinje cell deprivation on the dopaminergic system of the Nna1pcd mutant mouse. The dopamine transporters, as determined by [3H]WIN35428 binding, were increased compared with wild-type mice in the ventral mesencephalic dopaminergic nuclei and in the lateral striatum, motor cortex and septum. In the cerebellum of Nna1pcd mice, the dopamine transporters showed a significant increase in the deep cerebellar nuclei, but were significantly decreased in the molecular layer. The D1-like receptors, as determined by [3H]SCH23390 binding, increased significantly in the Nna1pcd substantia nigra. The D2/D3 receptors, as determined by [3H]raclopride binding, exhibited a significant decrease in lateral divisions of the striatum. Significant increases in D2-like receptors, as determined by [3H]nemonapride binding, were observed in most divisions of the striatum as well as in septum, hippocampus, and piriform cortex. This D2-like fraction most probably corresponds to the D4 receptor subtype. In the cerebellum of Nna1pcd mice, D2-like receptors were significantly decreased in the molecular layer. The results suggest an increased excitatory input on the dopaminergic mesencephalic neurons and an alteration of the dopaminergic neurotransmission in basal ganglia, cortical and limbic regions of the Nna1pcd mutant mouse. In the cerebellum, the significant downregulation of the dopamine transporters and D2-like receptors in the mutant cerebellar molecular layer is possibly due to the absence of the Purkinje cells.

Differential effect of dopamine deficiency on the expression of NMDA receptor subunits in the weaver mouse brain.

The weaver mutant mouse is characterized by degeneration of the dopaminergic mesencephalic neurons. The role of the dopaminergic system in the regulation of N-methyl-d-aspartate (NMDA) receptor subunit expression was addressed in the present study. In situ hybridization experiments were conducted to determine the expression levels of the NMDA receptor subunit mRNAs, z1, epsilon1 and epsilon2, in striatum, nucleus accumbens, olfactory tubercle and cerebral cortical regions of 26-day-, 3- and 6-month-old weaver mice. Data indicated statistically significant increases in z1 and epsilon2 mRNA levels in 6-month-old weaver striatum, whereas at the same age epsilon1 mRNA expression was decreased in all striatal regions, as well as in the cortex. In the 26-day-old weaver striatum and nucleus accumbens, statistically significant increases were observed in epsilon1 mRNA levels, whereas no changes were observed in the other two subunits. In the somatosensory cortex of 26-day-old weaver brain an increased expression of all three subunits was observed. The upregulation of NMDA receptor subunit expression observed in the somatosensory cortex can be attributed to a decreased activity of the glutamatergic thalamocortical pathway, following the degeneration of the nigrostriatal dopaminergic fibres. In the striatum, the present results demonstrate a differential control on the expression of z1 and epsilon2 subunits on the one hand, and epsilon1 subunit on the other. It is suggested that dopamine exerts a negative control on the expression of z1 and epsilon2 subunits, through a downregulation of transcription factors associated with the AP1 regulatory site, which is mediated by the activation of striatal dopamine D2 receptors.

Somatostatin, cholecystokinin and neuropeptide Y mRNAs in normal and weaver mouse brain.

The distribution of mRNAs encoding for somatostatin, cholecystokinin and neuropeptide Y was determined by in situ hybridization histochemistry in the weaver (wv/wv) mouse, a model of dopamine deficiency as well as in normal (+/+) controls. Weaver mutants did not show any appreciable departure from the normal pattern of expression for mRNA encoding for neuropeptide Y. In contrast, an 82% increase in mRNA encoding for somatostatin was observed in the reticular thalamic nucleus, whereas increases in the order of 20-87% were observed in different hypothalamic nuclei of the weaver brain. In addition, a 47-103% increase of the hybridization signal encoding for cholecystokinin was observed in the cerebral cortex, hippocampus and thalamus of the weaver brain. It can be assumed that the elevated and region-specific somatostatin and cholecystokinin levels observed in the weaver brain may be due to a secondary or compensatory response under conditions of altered neurotransmitter levels.

AMPA receptor subunit RNA transcripts and [(3)H]AMPA binding in the cerebellum of normal and pcd mutant mice: an in situ hybridization study combined with receptor autoradiography.

The expression of AMPA receptor subunit mRNAs and the binding of [(3)H]AMPA were studied in the cerebellum of normal and "Purkinje cell degeneration" ( pcd) mutant mouse. In the pcd cerebellum, [(3)H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA.

Partial restoration of striatal GABAA receptor balance by functional mesencephalic dopaminergic grafts in mice with hereditary parkinsonism.

Levels of inhibitory amino acid receptors were studied in the weaver (wv/wv) mouse model of dopamine (DA) deficiency after unilateral intrastriatal transplantation of fetal mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935. The average increase in [3H]GBR 12935 binding in grafted dorsal striatum compared to nongrafted wv/wv striatum was 60% 3 months after grafting. Autoradiography of 8 nM [3H]flunitrazepam and 12 nM [3H]muscimol binding was carried out to visualize the distribution of GABAA receptors in +/+ mice and in recipient weaver mutants. A 17% increase in [3H]flunitrazepam binding and a 20% increase in [3H]muscimol binding was found in the nongrafted dorsal striatum of weaver mutants compared to +/+. The functional mesencephalic grafts had a partial normalizing effect on both [3H]flunitrazepam and [3H]muscimol binding in the dorsal striatum of the weaver recipients. The normalization brought about by the grafts was around 20% for [3H]flunitrazepam binding and more than 40% for [3H]muscimol binding. The results are discussed in the context of the important interaction between the converging glutamatergic corticostriatal and DAergic nigrostriatal pathways in controlling the functional GABAergic output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

[3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts.

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Pharmacological characterization, anatomical distribution and sex differences of the non-NMDA excitatory amino acid receptors in the quail brain as identified by CNQX binding.

The distribution of non-N-methyl-D-aspartate binding sites was studied in coronal and sagittal sections through the brain of adult Japanese quail by quantitative autoradiography, using tritiated 6-cyano-7-nitroquinoxaline-2,3-dione as a radioligand. Saturation binding experiments were, in addition, carried out in areas showing high levels of binding (cerebellar molecular layer, nucleus anterior medialis and nucleus infundibularis) and demonstrated that the binding of tritiated ligand was specific and saturable. Competition studies with alpha-amino-3-hydroxy-methyl-4-isoxazole propionic acid and kainic acid indicated that kainic acid strongly inhibited ligand binding in all brain areas. alpha-Amino-3-hydroxy-methyl-4-isoxazole propionic acid was only a weak inhibitor in the hypothalamic nuclei whereas in the cerebellar molecular layer both high and low affinity inhibitions were detected. The highest binding levels of tritiated ligand were observed in the molecular layer of the cerebellum. Very high levels of binding were detected in various preoptic/hypothalamic sites including the nucleus suprachiasmaticus pars medialis, nucleus anterior medialis hypothalami, nucleus infundibularis, nucleus mammillaris medialis, nucleus posteromediale hypothalami and nucleus hypothalami ventromedialis. High levels of binding were also detected in the bulbus olfactorius, bed nucleus commissuralis anterior, bed nucleus commissuralis pallii, nucleus accumbens, bed nucleus striae terminalis and nucleus interpeduncularis. In the preoptic area/hypothalamus, high levels of binding were clearly present in all areas that contain gonadotropin releasing hormone cells or fibers. In the pons and mesencephalon, moderate levels of binding were associated with catecholaminergic areas such as the area ventralis tegmentalis (area ventralis of Tsai) and the locus coeruleus. Saturation analysis demonstrated the presence of a higher number of binding sites in females than in males in the cerebellar molecular layer, nucleus infundibularis and nucleus anterior medialis. This latter difference was confirmed in the one point assays that also identified higher levels of specific binding in the nucleus suprachiasmaticus pars medialis of males as compared with females. These anatomical data suggest a possible implication of non-N-methyl-D-aspartate receptors in the synthesis and/or release of both gonadotropin releasing hormone and catecholaminergic neurotransmitters that should now be tested by pharmacological experiments.

Cerebellar grafts partially reverse amino acid receptor changes observed in the cerebellum of mice with hereditary ataxia: quantitative autoradiographic studies.

We used quantitative autoradiography of [3H]CNQX (200 nM), [3H]muscimol (13 nM), and [3H]flunitrazepam (10 nM) binding to study the distribution of non-NMDA and GABA(A) receptors in the cerebellum of pcd mutant mice with unilateral cerebellar grafts. Nonspecific binding was determined by incubation with 1 mM Glu, 200 microM GABA, or 1 microM clonazepam, respectively. Saturation parameters were defined in wild-type and mutant cerebella. In mutants, non-NMDA receptors were reduced by 38% in the molecular layer and by 47% in the granule cell layer. The reduction of non-NMDA receptors in the pcd cerebellar cortex supports their localization on Purkinje cells. [3H]CNQX binding sites were visualized at higher density in grafts that had migrated to the cerebellar cortex of the hosts (4.1 and 11.0 pmol/mg protein, respectively, at 23 and 37 days after grafting) than in grafts arrested intraparenchymally (2.6 and 6.2 pmol/mg protein, respectively, at 23 and 37 days after grafting). The pattern of expression of non-NMDA receptors in cortical vs. parenchymal grafts suggests a possible regulation of their levels by transacting elements from host parallel fibers. GABA(A) binding levels in the grafts for both ligands used were similar to normal molecular layer. Binding was increased in the deep cerebellar nuclei of pcd mutants: the increase in [3H]muscimol binding over normal was 215% and the increase in [3H]flunitrazepam binding was 89%. Such increases in the pcd deep cerebellar nuclei may reflect a denervation-induced supersensitivity subsequent to the loss of Purkinje axon terminal innervation. In the deep nuclei of pcd mutants with unilateral cerebellar grafts, [3H]muscimol binding was 31% lower in the grafted side than in the contralateral nongrafted side at 37 days after transplantation; [3H]flunitrazepam binding was also lower in the grafted side by 15% compared to the nongrafted side. Such changes in GABA(A) receptors suggest a significant, albeit partial, normalizing trend of cerebellar grafts on the state of postsynaptic supersensitive receptors in the host cerebellar nuclei.

Changes in [3H]AMPA and [3H]kainate binding in rat caudate-putamen and nucleus accumbens after 6-hydroxydopamine lesions of the medial forebrain bundle: an autoradiographic study.

The binding parameters of [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and [3H]kainate binding were examined in caudate-putamen and nucleus accumbens of rat striatum after unilateral lesions of the right medial forebrain bundle (MFB) using in vitro receptor autoradiography. Lesioning of the dopaminergic fibres in the MFB with 6-hydroxydopamine (6-OHDA) resulted, after one or four weeks, in a significant decrease in the levels of [3H]GBR 12935 (1-[2-diphenylmethoxy)-ethyl]-4-(3-phenylpropyl) piperazine) in ipsilateral caudate-putamen and nucleus accumbens (62 and 43%, respectively). A comparison of the dissociation constants (Kd) of [3H]AMPA and [3H]kainate binding in caudate-putamen and nucleus accumbens between control and MFB-lesioned side did not indicate any significant change. However, the maximum number of [3H]AMPA and [3H]kainate binding sites (Bmax) were significantly decreased in caudate-putamen and nucleus accumbens of the MFB-lesioned side of the brain. This decrease was between 17 and 26%. Our results suggested that at least one-fourth to one-fifth of AMPA and kainate receptors in rat caudate-putamen and nucleus accumbens are localized on the presynaptic endings of dopamine fibres that follow the MFB. A role of non-NMDA glutamate receptors in the presynaptic regulation of dopamine release in rat striatum is therefore supported.

Autoradiographic characterization of [3H]6-cyano-7-nitroquinoxaline-2,3-dione binding sites in adult chick brain.

The non-N-methyl-D-aspartate binding sites have been characterized in chick brain using quantitative autoradiography, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) as a radioligand. [3H]CNQX binding sites were densely localized in the molecular layer of cerebellum. Other areas of prominent binding were the superficial layers of optic tectum, one of the isthmic nuclei, the hippocampus, the hyperstriatum accessorium and the archistriatum ventrale. Analysis of equilibrium binding data in the cerebellar molecular layer indicated that [3H]CNQX bound to a single class of sites (Kd = 78.9 +/- 11.8 nM and Bmax = 41.2 +/- 3.0 pmol/mg protein). Competition experiments in six different regions of chick brain gave the Ki and Bmax values for the inhibition of [3H]CNQX binding by various standard compounds and indicated that: (i) [3H]CNQX labelled non-N-methyl-D-aspartate binding sites with high affinity of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate, (ii) the displacement curves for AMPA and kainate were biphasic in all regions studied, and (iii) the potencies of AMPA and kainate in displacing [3H]CNQX binding were different in the regions studied. Our results indicate that [3H]CNQX labelled non-N-methyl-D-aspartate binding sites in chick brain. In the cerebellar molecular layer, these sites were more sensitive to kainate than AMPA, as were the binding sites in the superficial layers of optic tectum and nucleus isthmi magnocellularis. However, non-N-methyl-D-aspartate binding sites in forebrain regions such as hippocampus and hyperstriatum ventrale appeared to be different in being equally sensitive to AMPA and kainate.

Autoradiographic characterization of the non-N-methyl-D-aspartate binding sites in human cerebellum using the antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione.

Using quantitative autoradiography, we have characterized the binding properties of the non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in adult human cerebellum. Saturation experiments revealed [3H]CNQX binding to a single class of sites with similar affinity in the molecular and granule cell layer (Kd = 89.0 +/- 6.4 and 83.3 +/- 9.9 nM, respectively). The maximum number of [3H]CNQX binding sites was much higher in the molecular compared to the granule cell layer (Bmax = 16.2 +/- 1.1 and 2.8 +/- 0.5 pmol/mg protein, respectively). Inhibition experiments were performed in order to examine the pharmacological profile of [3H]CNQX binding in the molecular layer. [3H]CNQX labeled sites with high affinity for both non-NMDA agonists, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate. Dose-response curves for inhibition of [3H]CNQX by AMPA and kainate were biphasic. The potency of AMPA for displacement of [3H]CNQX binding (Ki: 2.8 +/- 0.8 nM and 12.5 +/- 0.8 microM) was 4- to 6-fold greater than the corresponding potency of kainate (Ki: 18.1 +/- 5.7 nM and 48.7 +/- 9.3 microM). In conclusion, the pharmacological analysis of [3H]CNQX binding in the human cerebellar molecular layer reflects the existence of multiple binding sites of the non-NMDA receptor that have different affinities for both AMPA and kainate.

Quantitative autoradiographic study of L-glutamate binding sites in normal and atrophic human cerebellum.

In the present work the distribution of L-glutamate binding sites in the different layers of human cerebellum of normal individuals and of seven patients who died with olivopontocerebellar atrophy (OPCA) was examined with the technique of quantitative autoradiography. Specific L-[3H]glutamate binding was higher in the molecular than in the granule cell layer of normal cerebellar tissue. A significant decrease of L-[3H]glutamate specific binding was observed in the molecular layer of all OPCA tissues. In the granule cell layer L-[3H]glutamate binding was decreased only in two patients who suffered from late-onset sporadic OPCA and in one patient who suffered from a form of OPCA inherited in a dominant manner. Quisqualate-sensitive binding sites were the most abundant binding sites in the molecular layer of normal cerebella, whereas N-methyl-D-aspartic acid (NMDA)-sensitive binding sites were the most abundant type in the granule cell layer. A significant decrease of quisqualate-sensitive and an increase in NMDA-sensitive binding sites were observed in the molecular layer of OPCA cerebellar tissues. No significant changes were observed in the granule cell layer of these tissues.

Localization of L-glutamate binding sites in chick brain by quantitative autoradiography.

Quantitative autoradiography was used for the localization of L-[3H]glutamate binding sites in chick brain. The highest concentration of these sites was observed in the molecular layer of cerebellum. High concentrations were also observed in the hippocampus, area parahippocampalis, neostriatum, neostriatum intermedium and hyperstriatum ventrale. In most areas binding of L-[3H]glutamate was increased when incubation was done in chloride-containing medium. This increase was statistically significant in only few of these areas.

L-glutamate binding sites of normal and atrophic human cerebellum.

The binding kinetics, pharmacologic properties, ontogeny and localization of L-glutamate binding sites were studied in membrane preparations and sections of normal and olivopontocerebellar atrophy (OPCA) human cerebellum. One binding component was found with a Kd value in the order of 150 x 10(-9) M. No significant changes of Kd values were observed with age, whereas the highest Bmax value was observed at the age of 1 year. L-Aspartate, ibotenate, quisqualate and L-homocysteic acid were potent inhibitors of L-[3H]glutamate binding. Quantitative densitometric measurements indicated the presence of L-glutamate sites in both the molecular and granule cell layer. In OPCA cerebella a very significant decrease of L-[3H]glutamate specific binding (Bmax) was observed, whereas Kd values were found unchanged. The pharmacologic properties of L-[3H]glutamate binding sites of OPCA cerebellar tissues were similar to those of normal cerebellum. [3H]quinuclidinyl benzylate binding, expressed in fmol/mg protein, did not show significant differences between normal and OPCA cerebella.

L-aspartate and L-glutamate binding sites in developing normal and 'nervous' mutant mouse cerebellum.

This study concerns the ontogeny and the cellular localization of L-aspartate and L-glutamate binding sites in normal and 'nervous' mutant mouse cerebellar membranes. The binding kinetics revealed for L-aspartate a single binding system (Kd = 750 nM) and for L-glutamate also a single binding component of higher affinity (Kd = 344 nM). The pharmacological study, using various amino acid analogues, revealed a differential specificity for the binding sites of the two amino acids. The developmental study showed that the binding sites of both amino acids appear mainly during the second and third week of life, a period when parallel and climbing fiber synaptogenesis occurs, but they follow a slightly different developmental pattern. The study using 'nervous', mutant mouse cerebellum showed an age-dependent decrease of L-aspartate and L-glutamate binding, which coincides in time with the Purkinje cell degeneration in this mutant, indicating a cellular localization of these binding sites on the Purkinje cell membranes. These results suggest that L-aspartate and L-glutamate binding sites may be respectively associated with the postsynaptic target of climbing and parallel fibers on the Purkinje cell dendrites. However, the decrease of specific binding in 'nervous' mutant mouse cerebellum was about 50% for L-aspartate and 60% for L-glutamate, implying that a significant number of L-aspartate and L-glutamate binding sites are located on cerebellar elements other than the Purkinje cell membranes.

The superior vestibular nucleus: an intracellular HRP study in the cat. I. Vestibulo-ocular neurons.

Superior vestibular neurons were penetrated with horseradish peroxidase (HRP)-loaded glass microelectrodes in anesthetized cats. Responses to electrical stimulation of the oculomotor complex and the vestibular nerves were characterized and selected neurons were injected with HRP. Neurons antidromically activated by oculomotor complex stimulation were generally monosynaptically excited by the ipsilateral vestibular nerve. Notable was the absence of strong commissural inhibition by stimulation of the contralateral vestibular nerve. Light microscopy of antidromically identified injected cells demonstrated that these cells are predominantly located at the central levels of the superior vestibular nucleus along the incoming vestibular nerve fibers but a few are found at more caudal levels. Cell bodies, elongated or pyramidal, are mainly medium-sized to large (30-50 micrometers). Dendritic trees extend in a plane at an acute to the collaterals of the vestibular nerve fibers. Dendrites remain within the nuclear territory and generally display an isodendritic branching pattern. Dendritic spines and appendages are mainly distributed on secondary and distal dendrites. A few terminal enlargements similar to growth cones are observed in these neurons. Axons of these neurons project rostrally via the medial longitudinal fasciculus, while a minor projection via the brachium conjunctivum is also found. Axon collaterals, when present, originate in the nucleus itself and in the pontine reticular formation.

The superior vestibular nucleus: an intracellular HRP study in the cat. II. Non-vestibulo-ocular neurons.

Superior vestibular neurons were penetrated with horseradish peroxidase (HRP)-loaded glass microelectrodes in anesthetized cats and identified electrophysiologically following electrical stimulation of the vestibular nerves and oculomotor complex. Neurons that were not antidromically activated from the oculomotor complex were stained by intracellular injection of horseradish peroxidase. Three types of neurons are identified according to their initial axonal trajectories into the cerebellum, the dorsal pontine reticular formation, or the brachium conjunctivum. Ipsilateral vestibular nerve input to all neurons is primarily monosynaptic and excitatory, whereas the contralateral is inhibitory. The neurons are located in the periphery of the superior vestibular nucleus. Soma diameters range from 20.5 micrometers to 44 micrometers. Most neurons exhibit globular and ovoid cell bodies. The dendritic arbors are intermediate between iso- and allodendritic branching patterns. The few spines and dendritic appendages present are distributed mainly distally on the dendrites. Soma size does not correlate with axon diameter, number of dendrites, or dendritic territories.