Noradrenergic abnormalities in the genetically epilepsy-prone rat

The genetically epilepsy-prone rat (GEPR) has central nervous system noradrenergic deficits as compared to normal rats. It is possible that these deficits contribute to seizure predisposition because they are exhibited by seizure-naive as well as by seizure-experienced GEPRs. On the basis of pharmacological studies, it is hypothesized that there is an inverse relation between seizure predisposition and levels of noradrenergic activity in brain. Neurochemical studies indicate that deficits exist in areas innervated by both the locus ceruleus and the lateral tegmental noradrenergic systems. These deficits exist in GEPRs without seizure experience and are more pronounced in the severe seizure strain as compared to the moderate seizure strain. We review eight experimental steps undertaken to identify more precisely the anatomical location of noradrenergic determinants of seizure predisposition. These steps illustrate the theoretical bases for the studies and describe the specific experiments completed. Evidence supports the hypothesis that noradrenergic deficits in the superior colliculus and/or ventrally adjacent regions are determinants of seizure predisposition.     

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.     

Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function.     

The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.     

The acetylcholine, GABA, glutamate triangle in the rat forebrain

The present overview demonstrates that stress, fear, novelty, and learning processes are associated with arousal and increases of extracellular levels of cortical and hippocampal ACh, independently of increases of motor activity. Forebrain cholinergic systems appears to be regulated by GABAergic and glutamatergic inputs. However, several other neurotransmitter systems play a role.     

GABAergic neurotransmission in rat taste buds: immunocytochemical study for GABA and GABA transporter subtypes

Gamma-aminobutyric acid (GABA) is known to be a candidate for the neurotransmitter involved in the sense of taste. We hereby studied GABA and its termination system, GABA transporters, in rat taste buds by immunocytochemical approaches. Immunoblot analysis of three GABA transporter subtypes (GAT1, GAT2 and GAT3) revealed that the immunoreactive bands of GAT2 and GAT3, but not GAT1, were detected in the tongue. GAT3-immunoreactive band was recognized only in the circumvallate papilla containing a large number of taste buds while GAT2-immunoreactive bands were seen in all areas of the tongue. GAT2 immunoreactivity appeared to be specifically in the nerve fibers beneath the lingual epithelium. Both GAT3 and GABA immunoreactivities were detected only in taste buds. A few GAT3-immunoreactive cells were found in a cross-section of each taste bud but most GAT3-immunoreactive cells were localized in the margin of the taste bud. GAT3 was predominantly concentrated in the distal portion of the GAT3-immunoreactive cells. In contrast, GABA-immunoreactive cells were seen more frequently within each taste bud and the immunoreactivity was distributed throughout the perikarya of the cells. These results suggest that the GABA-uptake system is present in the taste buds and the GABAergic neurotransmission involved in the sensation of taste is terminated by the uptake of GABA into certain taste cells via GAT3.     

Cloning and expression of a neuronal rat brain glutamate transporter

Recent evidence suggests that nitric oxide (NO) may function as a second messenger in the intracellular signal transduction pathways. We explored the possibility that NO was involved in the signal for triggering apoptosis in smooth muscle cells (SMCs). Chemical NO donors induced SMCs apoptosis in a concentration- and time-dependent manner. The membrane-permeable cGMP analogue, dibutyryl-cGMP, did not induce SMCs apoptosis, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the induction of NO-induced SMCs apoptosis. Inhibitor of ADP-ribosyltransferase slightly attenuated the induction of SMCs apoptosis by S-nitroso-N-acetyl penicillamine (SNAP). The inhibitor of Na+-H+ antiporter, amiloride, completely inhibited the induction of SMCs apoptosis by SNAP. These results demonstrate for the first time that NO can induce apoptosis in SMCs, suggesting that NO acts as a mediator in the development of atherosclerosis lesion via alterations in the number of SMCs. In addition, the results suggest that NO exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.     

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system.     

Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4 h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of N-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs. Combined with previous data on the expression of neurotrophin messenger RNAs following similar injuries, our results support the hypothesis that increased levels of neurotrophins and their receptors could protect against neuronal damage following a brain insult.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.     

Status of somatostatin receptor messenger RNAs and binding sites in rat brain during kindling epileptogenesis

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.     

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

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

The stimulus-evoked release of glutamate and GABA from brain subregions following transient forebrain ischemia in the rat

The release of glutamate and GABA in response to K+ depolarization was determined for tissue prisms prepared from brain subregions removed from rats following 30 min of forebrain ischemia or recirculation periods up to 24 h. There were statistically significant effects of this treatment on release of both amino acids from samples of the dorsolateral striatum, an area developing selective neuronal degeneration. However, for at least the first 3 h of recirculation the calcium-dependent and calcium-independent release of both amino acids in this region were similar to pre-ischemic values. Differences were observed under some conditions at longer recirculation times. In particular there was a decrease in calcium-dependent GABA release at 24 h of recirculation and a trend towards increased release of glutamate at 6 h of recirculation and beyond. No statistically significant differences were seen in samples from the paramedian neocortex, a region resistant to post-ischemic damage. These results suggest that changes in the ability to release glutamate and GABA in response to stimulation are not necessary for the development of neurodegeneration in the striatum but rather that release of these amino acids may be modified as a result of the degenerative process.     

Reduction of vesicular acetylcholine transporter mRNA in the rat septum following lead exposure

We have previously observed that maternal exposure to lead (Pb) results in a reduction of levels of mRNA coding for cholineacetyltransferase (ChAT) in the septum of developing rat without affecting the dams. Here we report that Pb similarly affects the expression of vesicular acetylcholine transporter (VAChT) mRNA in the rat septum. In close agreement with the time course of ChAT mRNA expression, septal VAChT mRNA levels increased from 30% at postnatal day 7 to 78% and 100% of adult levels at days 14 and 21, respectively. Maternal exposure to 0.2% lead acetate in drinking water from gestational day 16 resulted in an approximately 30% reduction of VAChT in 7 and 21-day-old rat pups without affecting VAChT mRNA levels in the dams. These results indicate a developmental stage-dependent interference by Pb with ChAT/VAChT gene expression in the rat septum.     

A liver-specific isoform of the betaine/GABA transporter in the rat: cDNA sequence and organ distribution

This paper discusses the findings of a quantitative study of nurses' willingness to care for patients with AIDS. It identifies several factors that enable nurses to confront the fear of AIDS nursing and to turn fear into compassion, and negative attitudes into positive ones. These factors include professional values, organizational support, group identity, patients' responses, spirituality, family and friends with AIDS, knowledge about AIDS, and the importance of personal choice. The value of these factors are further supported through personal statements made by nurses on AIDS-dedicated units as they express their feelings, thoughts, and positive experiences and expectations regarding AIDS nursing.     

Differential expression of protein kinase C isozyme messenger RNAs in dunning R-3327 rat prostatic tumors

Adenocarcinoma of the prostate is the second leading cause of cancer deaths in men. The protein kinase C (PKC) family of signal transducing kinases has been implicated in neoplastic transformation and progression in other tissues, and some evidence suggests roles for PKC in prostate growth and neoplasia. We have detected expression of eight specific PKC isozyme mRNAs (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) in normal rat whole prostate and found some of these to be differentially expressed in certain Dunning R-3327 rat prostatic adenocarcinoma sublines. PKC zeta mRNA was detected in normal prostate and Dunning H tumor, whereas an alternatively spliced form of PKC zeta RNA was found in Dunning G tumor and normal brain. Both forms of PKC zeta RNA were markedly reduced in the androgen insensitive, highly metastatic Dunning AT-3, MAT-Lu, and MAT-LyLu tumors. We have cloned and report the sequence of the novel portion of the alternatively spliced form of PKC zeta RNA, which is polyadenylated and present in cytoplasm.     

Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of alpha- and beta-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific alpha- and beta-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of alpha-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in beta-globin mRNA sequences is not detected until 20-24 hr after culture. In cells exposed to hemin, both alpha- and beta-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of alpha/beta-mRNA is maintained during induction. In maximally induced cells, the alpha/beta-globin mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3-0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of alpha- and beta-like genes. In addition, the relative accumulation of alpha- and beta-globulin mRNAs in induced cells differs with various types of inducers.