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Calcium sensing receptor (CaSR), isolated for the first time from bovine and human parathyroid, is a G-protein-coupled receptors that has been involved in diverse physiological functions. At present a complete in vivo work on the identification of CaSR mRNA-expressing cells in the adult brain lacks and this investigation was undertaken in order to acquire more information on cell type expressing CaSR mRNA in the rat brain and to analyse for the first time its expression in different experimental models of brain injury. The expression of CaSR mRNAs was found mainly in scattered cells throughout almost all the brain regions. A double labeling analysis showed a colocalization of CaSR mRNA expression in neurons and oligodendrocytes, whereas it was not found expressed both in the microglia and in astrocytes. One week after kainate-induced seizure CaSR was found in the injured CA3 region of the hippocampus and very interestingly it was found up-regulated in the neurons of CA1-CA2 and dentate gyrus. Similarly, 1 week following ibotenic acid injection in the hippocampus, CaSR mRNA expression was increased in oligodendrocytes both in the lesioned area and in the contralateral CA1-CA3 pyramidal cell layers and dentate gyrus. One week after needle-induced mechanical lesion an increase of labeled cells expressing CaSR mRNA was observed along the needle track. In conclusion, the present results contribute to extend available data on cell type-expressing CaSR in normal and injured brain and could spur to understand the role of CaSR in repairing processes of brain injury.

In order to understand the role of metabotropic glutamate receptors (mGluRs) in the brain, it is important to know how the mGluRs are differentially expressed among the different cell types. At present, the cellular expression of mGluR3 and mGluR5 has been mostly studied in terms of proteins with observations suggesting the expression of both mGluR3 and mGluR5 in neuronal and in glial cells. In order to verify the brain cell type-expressing mGluR3 and mGluR5 mRNAs, both in normal and injured brain, we performed a double labeling analysis, by in situ hybridization for mGluR3 or mGluR5 mRNA and immunohistochemistry for specific cellular markers. This approach allowed us to find mGluR3 mRNA expressed in neurons (NeuN-positive cells), and in glial cells, such as astrocytes (GFAP-positive cells) and oligodendrocytes (CNPase-positive cells). The same analysis showed that only NeuN-positive cells express mGluR5 mRNA. The time course of mGluR3 mRNA expression in two models of hippocampal formation lesion, kainate-induced seizures or ibotenic acid injection, showed an increased expression of mGluR3 in the area of lesion. This effect appears 1 week after the injury and was localized in GFAP- and CNPase-positive cells. In contrast, mGluR5 was not found expressed in the area of lesion. The present results contribute to extend available data on cell type-expressing mGluR3 and mGluR5 in normal and injured brain and could be relevant to understand the mechanisms that drive neuron-glial cells interaction both in normal and repairing processes.

The identification of connexins (Cxs) expressed in neuronal cells represents a crucial step for understanding the direct communication between neurons and between neuron and glia. In the present work, using a double-labelling method combining in situ hybridization for Cx mRNAs with immunohistochemical detection for neuronal markers, we provide evidence that, among cerebral connexins (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45 and Cx47), only Cx45 and Cx36 mRNAs are localized in neuronal cells in both developing and adult rat brain. In order to establish whether connexin expression is influenced in vivo by abnormal neuronal activity, we examined the short-term effects of kainate-induced seizures. The results revealed an unexpected expression of Cx26 and Cx45 mRNA in neuronal cells undergoing apoptotic cell death in the CA3-CA4, in the hilus of the hippocampus and in other brain regions involved in seizure-induced lesion. However, the expression of Cx26 and Cx45 mRNAs was not associated with detectable expression of corresponding proteins as evaluated by immunohistochemistry with specific antibodies. Moreover, in the same brain regions Cx32 and Cx43 were up-regulated in non-neruronal cells whereas the neuronal Cx36 was down-regulated. Taken together the present results provide novel information regarding the specific subpopulation of neurons expressing Cx45 and raise the question of the meaning of connexin mRNA expression in the neuronal apoptotic process.

Glial connexins (Cxs) make an extensively interconnected functional syncytium created by a network of gap junctions between astrocytes and oligodendrocytes. Among Cxs expressed in the brain, Cx30 is expressed in grey matter astrocytes, as shown at the protein level by immunoistochemistry. In the present study we aimed to perform a detailed study of the regional distribution of Cx30 mRNA in the adult and postnatal developing rat brain, analyzing its expression by in situ hybridization, and determining its cell type localization by double labeling. Recently, it has been suggested that neuronal activity may control the level of intercellular communication between astrocytes through gap junctions channels. Thus, a second aim of the present study was to investigate the short-term effects of kainate-induced seizures on Cx30 expression. The results showed that, in basal condition, Cx30 was expressed only in grey matter astrocytes with distinct regional patterns in developing and adult brain. Kainate treatment induced strong and region-specific changes of astroglial Cx30 mRNA levels and expression of Cx30 mRNA in neuronal cells undergoing cell death, suggesting a direct or indirect involvement of this connexin in the neuronal apoptotic process.

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the gene structure is that typical of the Cx35/36 subgroup observed in the other species (presence of a single intron located within the coding region, 71 bp after the translation initiation site). The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal among the cerebral areas examined by in situ hybridization was observed in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling was also observed in several myelencephalic nuclei, in specific cells of the the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus, and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells were revealed in all the lamina of the spinal cord gray matter. The chromosomal localization of the human Cx36 gene was determined by fluorescence in situ hybridization. The results allowed assignment of the gene to band 15q14, thus making it a possible candidate gene for a form of familial epilepsy previously linked to the same chromosomal band. The knowledge of the human Cx36 gene sequence, of its chromosomal localization, and of its pattern of expression opens new avenues for the analysis of its possible involvement in human genetic and acquired neuropathology., (Copyright 1999 Wiley-Liss, Inc.)

GFAPbeta mRNA is an alternative transcript of the glial fibrillary acidic protein (GFAP) gene, whose transcriptional start site is located 169 nucleotides upstream to the classical GFAPalpha mRNA. By an RT-PCR method with primers on separate exons, we were able to confirm the presence of GFAP transcripts with a longer 5' untranslated region in all the examined areas of rat brain and in primary cultures of astroglial cells. Northern blot analysis, using an oligoprobe specific for the 5' region of GFAPbeta, revealed a single hybridization band of 2.9 kb in all the brain regions examined and in primary cultures of astroglial cells. The availability of the quantitative Northern blot assay allowed further studies on the regulation of GFAPbeta expression in vivo. Since it is well-known that neuronal brain injury is one of the most powerful inducers of GFAP, we examined the expression of GFAPalpha and beta after a neurotoxic lesion in the rat hippocampus. Results obtained show a parallel increase in both GFAP transcripts with an identical time-course, suggesting that regulatory regions of the gene influence in similar way the rate of transcription at the two different start sites (alpha and beta) or that a similar post-transcriptional mechanism is involved in regulating both mRNA isoforms.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPalpha, beta, and gamma, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1-14; Zelenika et al. [1995] Mol. Brain Res. 30:251-258), we called this novel mRNA isoform GFAPdelta. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPdelta transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (alpha, beta, and gamma) produce hypothetical translation products differing in the amino-terminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain.

The levels of glial fibrillary acidic protein mRNA were analysed by in situ hybridization during the first 6 h in experimental models of status epilepticus in the rat. Two different models of status epilepticus were studied: one is produced by the administration of pilocarpine to lithium-treated rats and the other by the intracerebroventricular administration of kainate. Results obtained in the present study showed a very rapid (as early as 1.5 h in periventricular zones of hypothalamus, cerebral cortex, and hippocampal area) up-regulation of GFAP mRNA levels following the pharmacological induction of seizures. Several other areas showed a GFAP activation starting at 3 h such as septum, habenular nuclei, corpus callosum, and cingulum. The comparison of the results obtained in the two models of status epilepticus revealed interesting differences in some brain areas, such as cerebellum and striatum, which can be related to the specific neurotransmitter receptors and neurochemical pathways stimulated by the drugs. Interestingly, some brain areas whose neurons are strongly activated by pilocarpine and kainate (amygdala and CA3 hippocampal field) and that undergo neuronal degeneration did not show the early GFAP response. An interesting spatial feature was observed in several brain regions examined (striatum, septum, and hypothalamus): the response first appeared in the periventricular zones and then diffused to the rest of the brain area. In general GFAP responses in the periventricular zones were early and intense.

Description of constitutive and inducible transcription factors in brain cells is a prerequisite for understanding phenomena of long-term neuronal plasticity. In this report we report that basal levels of NF-kappa B-like transcription factor DNA binding activity (measured by electrophoretic mobility shift assay) are observed in two kinds of cultured rat brain cells, namely neurons of fetal cerebral hemispheres and astroglia derived from newborn brains. In the latter culture, phorbol ester induces additional forms of DNA binding activity, whereas L-glutamate fails to do so. In neurons, neither treatment is effective in inducing NF-kappa B-like DNA binding activity over the basal level. These results are in contrast to the fact that L-glutamate in both neurons and glia elevates DNA binding activity of AP-1, another transcription factor. These data, besides describing behavior of NF-kappa B-like DNA binding activities, also provide some evidence that L-glutamate exerts its modulatory functions on neurons and glia through specific transcription factors like AP-1.

In the present investigation we address the question of whether one of the responses to increased neuronal activity is a modification of the expression of the different subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, GluR-2, GluR-3). Thus, we used two different models of generalized status epilepticus, as widespread elevated neuronal activity, to study in vivo responses of the AMPA receptor mRNA expression in rat forebrain. By Northern blot analysis and in situ hybridization, we show that one of the delayed responses to LiCl/pilocarpine-induced status epilepticus is a dramatic change in the mRNA level of some subunits of AMPA-selective glutamate receptors. These effects, which appear between 6 and 12 h after the drug treatment, are subunit and brain region specific. The most striking example of differential expression of the three examined GluR mRNAs can be observed in the dentate gyrus of the hippocampus. In this specific brain subregion an increase of GluR-3 mRNA level is induced 12 h after LiCl/pilocarpine treatment, while a clear decrease in GluR-1 mRNA level and no significant change in GluR-2 mRNA level can be observed in the same area under these experimental conditions. Both the GluR-1 decrease and the GluR-3 increase are transient effects and a return to basal level can be observed after 48-72 h. In the CA1 layer of the hippocampus, a parallel decrease of both GluR-1 and GluR-3 expression is found 12-24 h after drug treatment, followed by a recovery of the expression to control values at 48 h. In kainate-induced epilepsy we could reproduce the late increase (12-24 h) in GluR-3 mRNA in the dentate gyrus; however, under this experimental condition, no clear decrease of GluR-1 expression can be observed in this area. A general decrease in mRNA level for the AMPA receptor subunits (GluR-1-3) in the hippocampal layers, in particular in CA3 and CA4 subfields, was also observed. In conclusion the results reported in the present paper reveal a specific regulation of GluR gene expression in the granule cells of the hippocampal dentate gyrus and stimulate further investigation on the functional role of the GluR-3 subunit in the receptor-channel complex.

By northern blot analysis and ribonuclease protection assay, we observed the presence of a high level of trkB mRNA in primary brain cultures devoid of neuronal cells and highly enriched in glial fibrillary acidic protein-positive astroglial cells prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum. In primary astroglial cultures, the more abundant trkB transcripts code for the truncated receptor without tyrosine kinase activity; probes specific for the full-length trkB mRNA did not detect any signal in northern blot analysis. By the sensitive ribonuclease protection assay, we could show the presence of trkC mRNA in cultured astrocytes, whereas no trkA mRNA was detected. We confirmed the presence of relatively high levels of nerve growth factor and neurotrophin-3 mRNA, and very low basal level of brain-derived neurotrophic factor mRNA. Moreover, we demonstrated that another member of the neurotrophin family, neurotrophin-4, is also expressed in cultured astroglial cells. In view of the fact that many functional receptors for conventional neurotransmitters or neuropeptides present on astroglial cells may act via the adenylate cyclase system, we studied also the effect of agents able to increase the intracellular cyclic AMP concentration. A sharp increase in the trkB mRNA level was observed after treatment of primary astroglial cultures with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. On the contrary, trkC mRNA levels were unaffected by treatment with cyclic AMP-elevating agents. All the neurotrophin mRNAs examined, except neutrophin-4, were increased by 3-isobutyl-1-methylxanthine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR-1 to -4) and at the protein level (GluR-1 and GluR-2/3/4c) in "primary astroglial cultures" (non-neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum and in "brain non-neuronal cell cultures" (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined AMPA subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (GluR-1,-2,-3,-4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti-GluR-1 and anti-GluR-2/3/4c polyclonal antibodies confirmed the presence of low level of immunoreactive proteins of the same size of those identified in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR-1, -2, and -3 were mainly expressed in cortical cultures, while GluR-4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR-4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutamate ionotropic receptors of the AMPA type in primary astroglial cultures and suggest that GluR-4 is the main AMPA receptor subunit expressed in non-neuronal cells of the central nervous system.

We have characterized the pharmacological profile of activation of metabotropic glutamate receptors negatively linked to adenylyl cyclase (mGluR decreases cAMP) in brain slices. Among the putative mGluR agonists, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), were the most potent inhibitors of forskolin-stimulated cAMP formation in hippocampal slices, followed by ibotenate, L-2-amino-3-phosphonopropionate (AP3), quisqualate, L-glutamate and beta-N-methylamino-L-alanine (BMAA). Inhibition of forskolin-stimulated cAMP formation by DL-2-amino-4-phosphonobutanoate (AP4) was biphasic, suggesting that the drug interacts with more than one mGluR decreases cAMP subtype. Both L-AP4 and L-serine-O-phosphate (a restricted analogue of AP4) were much more effective in inhibiting forskolin-stimulated cAMP formation than their D-isomers, indicating that interaction of these drugs with the mGluR decreases cAMP is stereoselective. Despite the fact that DCG-IV and ibotenate behave as NMDA receptor agonists, their effect was insensitive to MK-801. The regional pattern of expression of mGluR decreases cAMPS, as estimated by using 1S,3R-ACPD as an agonist, did not correlate with the steady-state levels of mGluR2 mRNA. Thus, 1S,3R-ACPD inhibited forskolin-stimulated cAMP in slices from hippocampus, cerebral cortex, corpus striatum, olfactory tubercle or hypothalamus, but not in slices from olfactory bulb or cerebellum; in contrast, mGluR2 mRNA levels were high in the olfactory bulb and very low in the corpus striatum. 1S,3R-ACPD also inhibited forskolin-stimulated cAMP formation in cortical membranes, excluding the involvement of trans-synaptic mechanisms in the activity of mGluR decreases cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the expression of metabotropic glutamate receptor (mGluR) mRNA by Northern blot analysis with a specific cDNA probe (the pmGR1 probe). In 1-day-old rats, the steady state levels of mRNA were higher in the hypothalamus and olfactory bulb, with intermediate levels in the cerebellum and low levels in the hippocampus and cerebral cortex. In the olfactory bulb, hypothalamus, and cerebral cortex, the expression of mGluR mRNA remained constant at 8 and 30 days of postnatal life. In contrast, in the cerebellum and hippocampus, mRNA levels increased progressively with age. There was no correlation between levels of mGluR mRNA and stimulation of polyphosphoinositide hydrolysis by 1-aminocyclopentane-1S,3R-dicarboxylic acid (trans-ACPD), which was much greater in brain slices from 8-day-old rats and was nearly absent in the adult cerebellum and olfactory bulb, where we have found the highest levels of mRNA. In addition, mGluR mRNA was detectable in cultured cerebellar granule cells but not in cultured neurons from cerebral hemispheres or in cultured astrocytes, which responded to trans-ACPD with an increased formation of [3H]inositol monophosphate. The discrepancies between levels of mGluR mRNA detected with the pmGR1 probe and trans-ACPD-stimulated polyphosphoinositide hydrolysis suggest either that different subtypes of mGluRs exist or that mRNA levels are not critical for the dynamic changes in the activity of mGluRs during development.

Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP-ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression. In the present study the process of ADP-ribosylation of chromosomal proteins (histone and non-histone proteins) in some rat brain regions during postnatal development was investigated; also the effect of epidermal growth factor (EGF) on this process in fetal brain slices was studied. It has been found that the process of ADP-ribosylation of total histones extracted from rat cerebral cortex and cerebellum at 1, 10 and 30 days of age, increases from 1 to 10 days of age (i.e. the period of maximal cell proliferation) and decreases thereafter, while the process of ADP-ribosylation of non-histone proteins (NHPs) sharply decreases during the same developmental period. The addition of EGF to fetal brain slices causes a significant increase of ADP-ribosylation of total histones (particularly of the histone H1 fraction) and also of NHPs and microsomal proteins. This result is in agreement with the effect of EGF as a mitogen factor, previously shown in astroglial cell cultures.

The AA. have investigated the effects of acute or chronic injection of typical and atypical antidepressants on the activity of the GABA--synthesizing enzyme glutamic acid decarboxylase (GAD, EC 4.1.1.15) in discrete brain regions. Very significant changes in GAD activity were only observed with sulpiride and nomifensine, two atypical antidepressants that selectively influence dopaminergic transmission and, in turn, prolactin secretion.

Addition of quisqualate (a heterocyclic analogue of glutamate) reduced [methyl-3H]thymidine incorporation and cell proliferation in primary cultures of rat cortical astrocytes. The inhibitory action of quisqualate was mimicked by glutamate and ibotenate, whereas kainate, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were inactive. These results suggest that activation of a specific class of excitatory amino acid receptors contributes to the regulation of growth and proliferation of glial cells in primary culture.

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

Glutamic acid decarboxylase activity was assayed in rat substantia nigra, corpus striatum, medial basal hypothalamus and cerebellum under conditions of hyperprolactinemia induced by transplantation of adenopituitary under the kidney capsule. The results indicate that hyperprolactinemia induces a slight increase in the glutamic acid decarboxylase activity of substantia nigra and medial basal hypothalamus and a more evident increase at striatal level. No change was observed in the cerebellum.

Previous data indicate that the injection of dopaminergic drugs induces changes in cerebellar 3',5'-guanosine monophosphate (cGMP) content. Accordingly, we have investigated the effects of haloperidol, sulpiride, or apomorphine on cerebellar prostaglandin (PG) concentration, a parameter related to cGMP content. Results obtained show that dopamine receptor blocking agents, such as haloperidol and sulpiride, significantly decrease cerebellar PGE2 and PGF2 alpha concentrations, while opposite changes are induced by apomorphine, a dopamine receptor agonist.

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-14C]thymidine incorporation into DNA of cerebral cortex and cerebellum during aging was observed. The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging. In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A) RNA and poly(A) RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.