Cytochrome P450 17α-hydroxylase/C(17,20)-lyase immunoreactivity and molecular expression in the cerebellar nuclei of adult male rats.

Several probes have been developed to identify steroidogenic activity in the brain of vertebrates. However, the presence of the cytochrome P450 17α-hydroxylase/C(17,20)-lyase (P450C(17)), an enzyme that converts pregnenolone and progesterone into dehydroepiandrosterone and androstenedione, in specific areas of the cerebellum such as the deep cerebellar nuclei, remains virtually unexplored. Using Western blot analysis and immunohistochemistry, we found molecular expression of P450C(17) in the lateral, interposed and medial deep cerebellar nuclei. Moreover, double immunofluorescence procedures enabled localization of P450C(17) mainly in neurons, axons and glutamatergic synapses. Taken together, these data demonstrate the occurrence of P450C(17) in the deep cerebellar nuclei, and enable the chemical characterization of the cells that express the cytochrome.

Compartmentalization of the deep cerebellar nuclei based on afferent projections and aldolase C expression.

The distribution of aldolase C (zebrin II)-positive and -negative Purkinje cells (PCs) can be used to define about 20 longitudinally extended compartments in the cerebellar cortex of the rat, which may correspond to certain aspects of cerebellar functional localization. An equivalent compartmental organization may exist in the deep cerebellar nuclei (DCN). This DCN compartmentalization is primarily represented by the afferent projection pattern in the DCN. PC projections and collateral nuclear projections of olivocerebellar climbing fiber axons have a relatively localized terminal arbor in the DCN. Projections of these axons make a closed olivo-cortico-nuclear circuit to connect a longitudinal stripe-shaped cortical compartment to a small subarea in the DCN, which can be defined as a DCN compartment. The actual DCN compartmentalization, which has been revealed by systematically mapping these projections, is quite different from the cortical compartmentalization. The stripe-shaped alternation of aldolase C-positive and -negative narrow longitudinal compartments in the cerebellar cortex is transformed to the separate clustering of positive and negative compartments in the caudoventral and rostrodorsal DCN, respectively. The distinctive projection of aldolase C-positive and -negative PCs to the caudoventral and rostrodorsal DCN underlies this transformation. Accordingly, the medial cerebellar nucleus is divided into the rostrodorsal aldolase C-negative and caudoventral aldolase C-positive parts. The anterior and posterior interposed nuclei generally correspond to the aldolase C-negative and -positive parts, respectively. DCN compartmentalization is important for understanding functional localization in the DCN since it is speculated that aldolase C-positive and -negative compartments are generally associated with somatosensory and other functions, respectively.

Molecular, topographic, and functional organization of the cerebellar nuclei: analysis by three-dimensional mapping of the olivonuclear projection and aldolase C labeling.

The olivocerebellar climbing fiber projection pattern is closely correlated with the pattern of aldolase C expression in cerebellar Purkinje cells. Based on this expression pattern, the olivocerebellar projection can be classified into five "groups" of functional compartments. Each group originates from a subarea within the inferior olive that projects to multiple cortical stripes of Purkinje cells, all of which are either aldolase C positive or aldolase C negative. However, no equivalent compartmental organization has been demonstrated in the cerebellar nuclei (CN). Thus, in the CN of the rat, we systematically mapped the location of olivonuclear projections belonging to the five groups and determined their relationship to the expression of aldolase C in Purkinje cell axonal terminals. The CN were divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts. The olivonuclear terminations from the five groups projected topographically to five separate compartments within the CN that partly crossed the traditional boundaries that define the fastigial, interposed, and dentate nuclei. Each compartment had mostly uniform cytoarchitecture and the same aldolase C expression (either positive or negative) that was found in the corresponding olivocortical projection. These results suggest a new view of the organization of the CN whereby the pattern of olivonuclear terminations links portions of different CN together. We propose that each compartment in the CN, along with its corresponding olivary subarea and cortical stripes, may be related to a different aspect of motor control.

Transglutaminase activity, protein, and mRNA expression are increased in progressive supranuclear palsy.

Transglutaminases catalyze the covalent cross-linking of substrate proteins to form insoluble protein complexes that are resistant to degradation. Our previous studies demonstrated that transglutaminase-induced cross-linking of tau proteins occurs in Alzheimer disease and progressive supranuclear palsy (PSP). The current study was designed to measure transglutaminase enzyme activity and the mRNA and protein levels of 3 transglutaminase isoforms that are expressed in human brain. Overall, transglutaminase activity was significantly increased in the globus pallidus (182% of control) and pons in PSP (171% of control) but not the occipital cortex (a region spared from pathology). Using a Spearman rank correlation test, we found that tissues with more transglutaminase-activity had more neurofibrillary tangles. Protein and mRNA levels of transglutaminase 1 were increased in globus pallidus of PSP as compared to controls. There were also significantly higher mRNA levels of the short form of transglutaminase 2 in globus pallidus of PSP (974% of control). Transglutaminase 1 mRNA and the long isoform of transglutaminase 2 mRNA (2212% of control) were significantly higher in PSP in the dentate of cerebellum. Together, these findings suggest that transglutaminase 1 and 2 enzymes may be involved in the formation and/or stabilization of neurofibrillary tangles in selectively vulnerable brain regions in PSP. These transglutaminases may be potential targets for therapeutic intervention.

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia.

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca(2+)-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca(2+)-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.

Immunohistochemical studies of the structural bases of inhibition in the central cerebellar nuclei in mice.

The distribution of glutamate decarboxylase-immunoreactive structures in the central nuclei of the cerebellum, its first afferent component, was studied at the light and electron microscope levels. Axosomatic, axodendritic, and axospinous synapses were detected, in which the presynaptic parts contained glutamate decarboxylase (GDC); this enzyme is involved in GABA synthesis. Additionally, these investigations revealed axoaxonal synapses in which both poles were GDC-reactive. The central nuclei of the cerebellum were found to have an intrinsic GABAergic system.

Learning induces a CDC2-related protein kinase, KKIAMRE.

To elucidate molecular mechanisms in learning and memory, we analyzed expression of mRNAs in brains of rabbits undergoing eyeblink conditioning. Infusion of the transcription inhibitor actinomycin D into the cerebellar interpositus nucleus reversibly blocked learning but not performance of the conditioned response. Differential display PCR analysis of cerebellar interpositus RNAs from trained and pseudotrained rabbits identified a 207 bp band that was induced with learning. The fragment was used to isolate a cDNA from a lambdagt11 rabbit brain library containing a 1698 bp open reading frame. The deduced amino acid sequence contains the KKIAMRE motif, which is conserved among cell division cycle 2 (cdc2)-related kinases. These results suggest that there is a new category of cdc2-related kinases in the brain whose function may be important in learning and memory.

A role of climbing fibers in regulation of flocculonodular lobe protein kinase C expression during vestibular compensation.

The behavioral recovery from unilateral labyrinthectomy (UL) in rats is accompanied by asymmetric expression of Protein kinase C (PKC) in parasagittal regions of the flocculonodular lobe within 6 h after UL, which resolves to the control, symmetric pattern within 24 h. These changes consist of a regionally selective increase in the number of PKC-immunopositive Purkinje cells contralateral to the lesion. This study tested the hypotheses (1) that climbing fiber innervation inhibits PKC expression and (2) that climbing fibers are essential for the observed changes in PKC expression within 6 h after UL. The patterns of flocculonodular lobe Purkinje cell PKCdelta expression were analyzed 6 h post-operatively in both UL and sham-operated that had been treated previously with 3-acetylpyridine to destroy the inferior olive. These data were compared with previous results from rats with an intact olive. The results suggest that at least two signals regulate the zonal distribution of Purkinje cell PKCdelta expression in the flocculonodular lobe during the early period of compensation from UL. Climbing fiber activation appears to reduce PKC expression, while extraolivary mechanisms appear to up-regulate PKC expression. It is suggested that the climbing fiber signals may act as a molecular 'filter' or 'automatic gain control' which adjusts the contributions of these kinases to synaptic plasticity within the context of the background activity of climbing fibers.

Differential expression of glutamate decarboxylase messenger RNA in cerebellar Purkinje cells and deep cerebellar nuclei of the genetically dystonic rat.

The genetically dystonic rat exhibits a motor syndrome that closely resembles the human disease, generalized idiopathic dystonia. Although in humans dystonia is often the result of pathology in the basal ganglia, previous studies have revealed electrophysiological abnormalities and alterations in glutamate decarboxylase, the synthetic enzyme for GABA, in the cerebellum of dystonic rats. In this study, we further characterized the alterations in cerebellar GABAergic transmission in these mutants by examining the expression of the messenger RNA encoding glutamate decarboxylase (67000 mol. wt) with in situ hybridization histochemistry at the single cell level in Purkinje cells and neurons of the deep cerebellar nuclei. Glutamate decarboxylase (67000 mol. wt) messenger RNA levels were increased in the Purkinje cells and decreased in the deep cerebellar nuclei of dystonic rats compared to control littermates, suggesting opposite changes in GABAergic transmission in Purkinje cells and in their target neurons in the deep cerebellar nuclei. In contrast, levels of glutamate decarboxylase (67000 mol. wt) messenger RNA in the pallidum, and of enkephalin messenger RNA in the striatum, were unaffected in dystonic rats. The data indicate that both the Purkinje cells and GABAergic neurons of the deep cerebellar nuclei are the site of significant functional abnormality in the dystonic rat.

[Immunohistochemical study of structural basis of inhibition in central cerebellar nuclei]. / Immunogistokhimicheskoe izuchenie strukturnykh osnov tormozheniia v tsenral'nykh iadrakh mozzhechka u myshei.

Glutamatic acid decarboxylase (GAD): a synthetic enzyme for inhibitory neurotransmitter GABA, was studied in the central cerebellar nuclei. Synapses were found: morphological structures providing of inhibition in the central cerebellar nuclei.

Protein kinase C in central vestibular, cerebellar, and precerebellar pathways of the rat.

The protein kinase C family of enzymes is composed of at least ten different isoforms that display a variety of distinct biochemical specificities. Many of these isoforms are highly expressed in brain, and some show regional specificity in their distribution, suggesting that they may serve specific functions. By using immunocytochemistry to localize the betaI, betaII, gamma, or delta isoforms of protein kinase C in the central vestibular system of the adult rat, we found the vestibular ganglion and its peripheral and central processes of the eighth nerve to be heavily labeled with protein kinase C betaI immunoreactivity. Labeled axons and terminals were also found in all four vestibular nuclei. Some neurons of the vestibular ganglion were weakly stained with the antibody to protein kinase C betaII, as were scattered axons in the eighth nerve, and scattered axons and terminals were found in all four vestibular nuclei among weakly labeled neurons. A few axons in the vestibular portion of the eighth nerve were labeled with protein kinase C gamma immunoreactivity, and neurons of the spinal, lateral, and superior vestibular nuclei were heavily decorated with synapses, presumably derived from Purkinje neurons, which were also strongly immunoreactive. Neurons of the medial vestibular nucleus were not as heavily innervated. With the antibody to protein kinase C delta, we found scattered, weakly immunoreactive neurons in the vestibular portion of the eighth nerve. Myelinated fiber bundles of the spinal vestibular nucleus contained moderate numbers of labeled axons, and the other vestibular nuclei were well innervated by protein kinase C delta axons and terminals. Most of these probably derive from Purkinje cells, which were labeled in longitudinal bands interspersed with bands of labeled basket cells. These data suggest that particular protein kinase C isoforms play specific roles in vestibular and cerebellar function.

Transient changes in flocculonodular lobe protein kinase C expression during vestibular compensation.

Protein kinase C (PKC) is a family of intracellular signal transduction enzymes, comprising isoforms that vary in sensitivity to calcium, arachidonic acid, and diacylglycerol. PKC isoforms alpha, gamma, and delta are expressed by cerebellar Purkinje cells and neurons in the cerebellar nuclei and vestibular nuclei of the Long-Evans rat. In control rats, these PKCs are distributed symmetrically in the flocculonodular-lobe Purkinje cells. Behavioral recovery from vestibular dysfunction produced by unilateral labyrinthectomy (UL) is accompanied by asymmetric expression of PKC isoforms in these regions within 6 hr after UL. These expression changes were localized within parasagittal regions of the flocculus and nodulus. The distribution of PKCalpha, -gamma, and -delta were identical, suggesting that they are coregulated in cerebellar Purkinje cells during this early compensatory period. The pattern of Purkinje cell PKC expression returned to the control, symmetric distribution within 24 hr after UL. It is hypothesized that these regional changes in Purkinje cell PKC expression are an early intracellular signal contributing to vestibular compensation. In particular, regulation of PKC expression may contribute to changes in the efficacy of cerebellar synaptic plasticity during the acute post-UL period.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Distribution of nitric oxide synthase in the central nervous system of Macaca fuscata: subcortical regions.

The distribution of nitric oxide synthase-immunoreactive neurons was studied in the Macaca fuscata by immunohistochemistry using antiserum against nitric oxide synthase. In the macaque lower brainstem, many nitric oxide synthase-containing cell bodies were found in the gigantocellular and parvocellular reticular nuclei, the nucleus of the spinal tract of trigeminal nerve, the cochlear nucleus, the prepositus hypoglossi and the nucleus of the solitary tract. Many nitric oxide synthase-immunoreactive perikarya were observed in the laterodorsal-pedunculopontine tegmental nucleus complex of the macaque pontine and midbrain tegmentum. In addition, there were many highly immunoreactive cell bodies in the superficial layers of the inferior and superior colliculi. In the forebrain, numerous cell bodies were observed in the caudate nucleus, putamen, nucleus accumbens, nucleus of the diagonal band, anterior perforated substance and amygdaloid complex. Whereas most of these neurons were labeled highly intense for nitric oxide synthase, there were also many lightly labeled nitric oxide synthase-immunoreactive neurons in the substantia innominata, globus pallidus, ansa peduncularis and lateral hypothalamic nucleus. The present observation indicated some species difference in the distribution of central nitric oxide synthase-containing neurons. Furthermore, the present neuroanatomical evidence that nitric oxide synthase is distributed in a variety of specific neuronal systems, with some differences in the patterns of cytoplasmic localization, further indicates the neural messenger role of nitric oxide in the central nervous system.

Cholinergic innervation of the primate hippocampal formation. I. Distribution of choline acetyltransferase immunoreactivity in the Macaca fascicularis and Macaca mulatta monkeys.

The cholinergic innervation of the hippocampal formation of Macaca fascicularis (cynomolgus) and Macaca mulatta (rhesus) monkeys was investigated by immunohistochemical procedures using a monoclonal antibody directed against choline acetyltransferase. The distribution of choline acetyltransferase in the monkey demonstrated both similarities and differences with the staining patterns observed in the rat or with acetylcholinesterase in the monkey. While both of these latter preparations demonstrated labeled cells, for example, no choline acetyltransferase labeled neurons were observed in the monkey hippocampal formation. Choline acetyltransferase activity was restricted to fibers which varied in thickness and number of varicosities and in their regional and laminar distribution. The highest densities of labeled fibers were observed in the uncal portion of the hippocampus, in the parasubiculum, and in the entorhinal cortex; the lowest densities of labeled fibers were observed in CA1 and in midrostrocaudal levels of the dentate gyrus. In the dentate gyrus, immunoreactive fibers were densely distributed in the molecular layer and in an infragranular plexus. One of the few striking noticeable interspecies differences was observed in the dentate gyrus. In the rhesus monkey, labeled fibers in the molecular layer were divided into a superficial denser and an inner lighter lamina, whereas in M. fascicularis, the cholinergic fibers were distributed more homogeneously throughout the molecular layer. In the hippocampus proper, there was a progressive decrease in the density of ChAT-immunoreactive fibers from CA3/CA2 into CA1. The subiculum also demonstrated modest labeling which was nonetheless higher than in CA1; the border of these fields demonstrated increased fiber labeling. The density of choline acetyltransferase staining was high in the presubiculum and parasubiculum. In the entorhinal cortex, a relatively clear boundary was observed between the more heavily stained superficial layers (I, II, and III) and the more weakly labeled deep layers (V and VI), especially in the intermediate and caudal fields. A transverse decreasing gradient was observed with the densest plexus of cholinergic fibers found in the medially situated olfactory field of the entorhinal cortex and the lowest density in the laterally located caudal and lateral fields.

Microanatomical and electrophysiological changes of the rat dentate gyrus caused by lesions of the nucleus basalis magnocellularis.

The effect of unilateral or bilateral lesions of the nucleus basalis magnocellularis (NBM) on the dentate gyrus of the hippocampus were assessed using microanatomical and electrophysiological techniques. NBM is the main cholinergic basal forebrain nucleus that supplies the fronto-parietal cortex. Lesions were induced using the neurotoxin ibotenic acid or a radio-frequency system and did not affect glutamic acid decarboxylase activity both in the frontal cortex and in the hippocampus. At 4 weeks after lesioning, a loss of choline acetyltransferase (ChAT) activity and of ChAT-immunoreactive fibres was observed in the frontal cortex but not in the hippocampus and no changes in the density of granule neurons of the dentate gyrus or in the hippocampal long-term potentiation (LTP) were noticeable. At 8 weeks after lesioning the loss of both ChAT activity and of ChAT-immunoreactive fibres persisted in the frontal cortex of NBM-lesioned rats. Moreover, at this time a significant decrease in the density of granule neurons in the dentate gyrus accompanied by a reduced probability of dentate LTP induction were observed in both ibotenic acid- and radio-frequency-lesioned rats. These findings have shown that although NBM does not send direct cholinergic projections to the hippocampus, lesions of this cholinergic nucleus are accompanied by delayed neurodegenerative changes involving the dentate gyrus. This suggests the occurrence of indirect connections between NBM and the hippocampus, the functional relevance of which should be explored.

The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.

Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed the distribution of glutamic acid decarboxylase (GAD), a synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), in the cerebellar nuclei of the mouse. Pre-embedding immunocytochemistry was carried out on brain sections fixed with high concentrations of glutaraldehyde and sodium metabisulfite. After incubations with a monoclonal antibody to GAD and a 1.4-nm NanoGold-labeled secondary antibody, sections were silver-enhanced with N-propyl gallate as a reducing agent and MES as a new buffer system. In the cerebellar nuclei, GAD label was specifically localized in axon terminals over clusters of synaptic vesicles. These terminals formed axosomatic and axodendritic contacts. The majority of GAD-labeled terminals had cytological characteristics indicating their origin from Purkinje cells, which are known to contain GAD.

Tyrosine hydroxylase- and dopamine-beta-hydroxylase-positive neurons and fibres in the developing human cerebellum--an immunohistochemical study.

Six human fetuses of gestational ages 16-28 weeks were employed. The immunocytochemical avidin-biotin-peroxidase complex method combined with the silver Bodian technique was used to evaluate the presence of tyrosine hydroxylase and dopamine-beta-hydroxylase neurons and afferent and efferent fibres in the cerebellum during development. Our results illustrated that by 16-18 weeks, immunoreactivity of the Purkinje cells and the granule cells was evident. By 23 weeks, the positive Purkinje cells were tightly packed together and the perinuclear granules began to extend into the processes. The positive cells next to Purkinje cells were the basket cells and stellate cells. By 26-28 weeks, all positive cells increased in number and size. Mossy and climbing fibres appeared early in development (16-18 weeks of gestation) and were seen synapsing with the positive granule cells. At the same time, some parallel fibres were observed. At later stages, the tyrosine hydroxylase- and dopamine-beta-hydroxylase-positive Purkinje cells were surrounded by abundant climbing fibres, while parallel fibres were also evident in the molecular layer. In the deep cerebellar nuclei, positive tyrosine hydroxylase and dopamine-beta-hydroxylase neurons were present by 16-18 weeks of development. Those in the dentate nucleus were more polymorphic but smaller in size. Some afferent fibres were also spotted around 16-18 weeks of gestation and their numbers increased later. Positive efferent fibres were present by 26 weeks. All these observations point to an early presence of tyrosine hydroxylase and dopamine-beta-hydroxylase components in cerebellar development.

Glutamate dehydrogenase in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

Applying quantitative microscopic histochemistry, the activity of the mitochondrial glutamate dehydrogenase which is localized in astrocytes was determined in the molecular layer of the dentate gyrus of the rat hippocampus. This hippocampal region contains the important terminations of the glutamatergic perforant path. For comparison, determinations of the mitochondrial succinate dehydrogenase were performed, which is localized preferentially in terminals and dendrites. Two age groups of animals were examined: young adults (three months old) and aged subjects (24 months old). Both age groups were divided into controls, and animals killed three, 21 and 90 days following unilateral electrolytic lesion of the entorhinal cortex. The post-lesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of enzymatic data. Statistic analysis revealed that ipsilateral to the lesion side there was a significant decrease of glutamate and succinate dehydrogenase activities in the terminal field of the perforant path three, 21 and 90 days following lesion. It is reasonable to assume that the decrease of succinate dehydrogenase activity (50-60%) was caused by the loss of mitochondria localized in degenerating terminals, whereas the decrease of glutamate dehydrogenase activity (20-30%) was related to the decrease of glutamatergic transmission following lesion. In the terminal field of the perforant path contralateral to the lesion side both significant increases and decreases of enzyme activities were measured following lesion. From these results it is concluded that the hippocampus contralateral to the lesion side cannot be considered as an appropriate intraindividual control. The comparison between young and aged animals showed no differences in the demonstration of glutamate dehydrogenase and only restricted differences in the activity level of succinate dehydrogenase post-lesion. Therefore, it is reasonable to assume that the post-lesional reactivity of the enzymes studied was very similar in both age groups.

Amygdala kindling alters protein kinase C activity in dentate gyrus.

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.