A role for hippocampal opioids in long-term functional plasticity.

In the granule cells of the hippocampus, glutamate coexists with opioid peptides derived from the proenkephalin and prodynorphin genes. The functional significance of this coexistence has been unclear but recent evidence suggests that the dynorphins and enkephalins play a crucial role in regulating the efficiency of neurotransmission at granule-cell synapses. Together with evidence that the level of opioid activity in this pathway can change dramatically according to the physiological or pathological state of the tissue, this information focuses attention on granule-cell opioids as primary mediators of hippocampal plasticity.

Changes in mouse granulosa cell gene expression during early luteinization.

Changes in gene expression during granulosa cell luteinization have been measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with pregnant mare serum gonadotropin (PMSG) or PMSG followed, 48 h later, by human chorionic gonadotropin (hCG). Granulosa cells were collected from preovulatory follicles after PMSG injection or PMSG/hCG injection and SAGE libraries generated from the isolated mRNA. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by hCG and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in steroidogenesis as well as clusters of genes involved in modeling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signaling. The SAGE libraries described here provide a base for functional investigation of the regulation of granulosa cell luteinization.

Histidine mutants requiring adenine: selection of mutants with reduced hisG expression in Salmonella typhimurium.

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leady types), and they map throughout the hisG gene.

A refined map of the hisG gene of Salmonella typhimurium.

The hisG gene is the most operator-proximal structural gene of the histidine operon; it encodes the feedback-inhibitable first enzyme of the biosynthetic pathway. Previously, hisG mutants were mapped into seven intervals defined by the availble deletion mutations having endpoints in the hisG gene. The map has been refined using over 60 new deletion mutants. The new map divides the gene into 40 deletion intervals, which average approximately 30 base pairs in length. The map has been used to analyze the distribution of insertion sites for the transposable element Tn10 and has permitted conclusions on the diistribution of duplication endpoints. The map promises to be useful in analysis of his regulation and, more particularly, in the determination of the possible role of the hisG enzyme in this mechanism.

Coexistence of NADPH diaphorase with GABA, glycine, and acetylcholine in rat spinal cord.

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motoneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies.

Plasmapheresis in children with Guillain-Barré syndrome.

Between 1987 and 1989, we plasmapheresed six children with Guillain-Barré syndrome (GBS). None of the children could walk independently at the start of the treatment, and one was being ventilated. Five patients showed clinical improvement during pheresis, and no significant side effects occurred. The median time from onset of weakness to independent walking for these six children was 17 days. This compares with 43 days for 18 children with GBS in this institution who were given supportive measures only.

Cardiac assessment in childhood carriers of Duchenne and Becker muscular dystrophies.

Cardiac disease in adult female carriers of the X-linked dystrophinopathies, Duchenne and Becker muscular dystrophies, is a well-recognised entity. A single study has reported a 15% incidence of cardiac abnormalities in female carriers under 16 years. Our study aims, clinically and with electrocardiograph and echocardiograph, to determine the incidence of cardiac abnormality in young girls who are proven carriers of X-linked dystrophinopathies. Twenty-three girls aged 6.2-15.9 years were assessed. All had normal cardiac examination. None had electrocardiograph abnormalities consistent with dystrophic cardiomyopathy. Left ventricular fractional shortening ranged from 33 to 55% (normal>28%). Septal thickness, posterior wall thickness and wall thickness ratio were within normal limits. No cardiac abnormalities have been demonstrated in young girls who are proven carriers of X-linked dystrophinopathies in our study. This has important implications for planning timing of carrier determination and cardiac assessment.

Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release.

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.

Nitric oxide alters proenkephalin and prodynorphin gene expression in hippocampal granule cells.

Application of N-methyl-D-aspartate on to the dendrites of hippocampal granule cells dramatically decreased prodynorphin messenger RNA levels in the affected cells while increasing proenkephalin messenger RNA levels. Sin-1 molsidomine (an agent which releases nitric oxide) and 8-bromo-cyclic GMP were similarly effective, and the actions of sin-1 molsidomine were blocked by inhibition of cyclic GMP-dependent protein kinase. Since, in this region, dynorphins act to inhibit potentiation of synaptic transmission, while enkephalins have excitatory effects, this switch in opioid gene expression is likely to have a prolonged effect on the efficiency of the mossy fibre synapses. In addition, the results demonstrate a powerful role for nitric oxide in the long-term regulation of hippocampal excitability.

Induction of c-fos gene expression is not responsible for increased proenkephalin mRNA levels in the hippocampal dentate gyrus following NMDA stimulation.

The increased levels of proenkephalin mRNA in the dentate gyrus following hippocampal stimulation have been assumed to be a consequence of the transient induction of c-fos. Injection of 50 microM NMDA in vivo onto the dendrites of a small number of granule cells causes a pronounced but highly localised elevation in proenkephalin mRNA levels 24 h later, whereas vehicle has no effect. In contrast, there is a widespread induction of c-fos mRNA throughout the dentate gyrus, 45 min after injection of either vehicle or NMDA, suggesting that induction of c-fos expression is unrelated to the subsequent, anatomically discrete, increase in proenkephalin mRNA levels.

N-methyl-D-aspartate and nitric oxide regulate the expression of calcium/calmodulin-dependent kinase II in the hippocampal dentate gyrus.

Injection of small volumes of N-methyl-D-aspartate (NMDA) or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). We report here that at short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction [54]. However, we also report that 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected, and occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or NO release.

zif/268 does not mediate increases in proenkephalin mRNA levels after NMDA receptor stimulation.

THE immediate-early gene zif/268 is induced in the hippocampal dentate gyrus by stimuli leading to long-term potentiation (LTP), and is though then to alter the rate of transcription of other genes involved in hippocampal plasticity, such as the proenkephalin (Penk) gene. Injection of 50 microM NMDA in vivo on to the granule cells of the dentate gyrus results in a highly localized increase in Penk mRNA levels 24 h later, while vehicle has no effect. In contrast, there is a widespread induction of zif/268 mRNA throughout the dentate gyrus, after injection of either NMDA or vehicle. This suggests that zif/268 induction in the hippocampal dentate gyrus is not sufficient to increase Penk gene expression.

Further evidence supporting the existence of the celiac artery compression syndrome.

A 23-year-old man presented with prolonged postprandial epigastric pain and an epigastric bruit with systolic and diastolic components, the intensity of which decreased with inspiration as demonstrated by abdominal phonography. Arteriography demonstrated significant narrowing of the origin of the celiac artery. At operation, the origin of the celiac artery was found to be constricted by fibers of the median arcuate ligament of the diaphragm, and this ligament was divided. Intraoperative flow measurements demonstrated an increase in blood flow through the main branches of the celiac axis, after division of the ligament. Four years following successful surgery, the patient has continued to be in good health without symptoms, and the bruit has remained absent. Further abdominal arteriography has demonstrated the normality of the celiac artery. We believe this to be a well-proven case of the "celiac artery compression syndrome."

Immunohistochemical evidence that Met-enkephalin and GABA coexist in some neurones in rat dorsal horn.

A pre-embedding immunohistochemical method to detect Met-enkephalin was combined with postembedding immunohistochemistry with GABA and glycine antisera, in order to determine whether or not Met-enkephalin coexisted with either of these inhibitory transmitters in neuronal cell bodies within the superficial dorsal horn of the rat. The distribution of immunostaining with the three antisera was similar to that which has been described previously. Of 74 enkephalin-immunoreactive neurones in laminae II and III, 51 were immunoreactive with the GABA antiserum and 23 were not. All of the neurones which were not GABA-immunoreactive were located in lamina II. None of the enkephalin-immunoreactive cells showed glycine-like immunoreactivity. These results suggest that enkephalin is present both in GABAergic neurones and in neurones which do not contain GABA within the rat superficial dorsal horn. It is likely that the population of neurones immunoreactive with both enkephalin and GABA antisera includes lamina II islet cells and that the population which were enkephalin-immunoreactive but not GABA-immunoreactive includes stalked cells. In addition, this latter group may correspond to those cells which possess both enkephalin- and substance P-like immunoreactivity and which have been described previously in this area.

Selective regulation of dendritic MAP2 mRNA levels in hippocampal granule cells by nitric oxide.

Application of NMDA, or agents releasing nitric oxide (NO), onto the dendrites of hippocampal granule cells increased the levels of the mRNA encoding MAP2, a cytoskeletal component induced during periods of neurite outgrowth. Furthermore, local increases in the hybridisation signal in the molecular layer, representing dendritic MAP2 mRNA, occurred independently of changes in MAP2 mRNA levels in the cell body layer. The selective modulation of MAP2 mRNA in dendrites reveals a mechanism allowing a sustained stimulation of dendritic outgrowth to be confined to those regions of a neuron's dendritic arbour local to glutamate receptor stimulation.

Neuronal expression of protease-nexin 1 mRNA in rat brain.

Protease-nexin 1 (PN1), also known as glia-derived nexin, is a protease inhibitor secreted by cultured fibroblasts and glioma cells, with postulated roles in regeneration and the regulation of neurite outgrowth. In this study we have localised the sites of PN1 gene expression in rat brain using in situ hybridisation. As expected, cultured cortical astrocytes contained relatively high levels of PN1 mRNA. However, the mRNA localisation in rat brain suggested that the primary sites of synthesis in the CNS are neuronal. Relatively high levels of PN1 mRNA were found in the olfactory nerve layer of the olfactory bulb, in layer V of the cerebral cortex, in magnocellular neurones of the basal forebrain, and in scattered neurones of the striatum. The results show that PN1 gene expression occurs in discrete populations of neurones in the brain, and suggest that these neurones may therefore play a role in the local regulation of neurite outgrowth.

Lorazepam as a sedative-amnesic in an intensive care unit.

A clinical study was carried out to evaluate the usefulness of intravenous lorazepam, given for sedation instead of opiate narcotics or diazepam, in 25 seriously-ill patients being treated in a respiratory and intensive care unit. All but 3 patients were on assisted ventilation. Standard doses of 4 mg lorazepam were given at 4 or 6-hourly intervals for periods up to 25 days. ECG, haemodynamic stability and biological determinations were monitored constantly. Apart from some delay in onset of action, lorazepam proved to be a useful sedative with diminished recall on the part of the patients. No side-effects were reported, nor was there any local reaction to the injection. Cardiac output was measured in 9 patients following intravenous administration of a single-dose of either 4 mg or 8 mg lorazepam. No significant changes were recorded.

Severe muscular dystrophy in girls.

Twelve girls and 2 boys with severe but not congenital muscular dystrophy were found in a national survey. An autosomal recessive gene is likely to account for most if not all of these cases. The condition differs slightly from X-linked Duchenne muscular dystrophy in showing prominent early toe-walking, a milder course, relatively more weakness of the deltoid muscles, normal intelligence, a normal ECG and a more focal pattern of muscle pathology.

Perinatal exposure to morphine affects adult sexual behavior of the male golden hamster.

Duromorph, a long-acting form of morphine, was administered to pregnant golden hamsters and/or their pups over the last 4 days of pregnancy and/or the first 4 days after birth. As adults, offspring were gonadectomized, primed with estrogen and progesterone, and tested for their ability to display feminine sexual behavior when placed with a stud male. They were then given testosterone over a 4-week period and tested for their ability to display masculine sexual behavior in the presence of a receptive female. Perinatal morphine exposure had little effect on the females' ability to display either feminine or masculine sexual behavior. In contrast, feminine sexual behavior was significantly enhanced in males exposed to morphine over the perinatal period. This suggests that exposure to opiates during the critical period of sexual differentiation may prevent the defeminization process in this species.

Neonatal serotonin reduction alters the adult feminine sexual behaviour of golden hamsters.

HPLC analysis of hamster hypothalamic 5HT indicated higher levels in females than in males on day 12 after birth. Levels of 5HT and 5HIAA could be reduced in both sexes by pCPA administration. Male and female hamster pups were treated on days 1-7 or 7-14 after birth with either pCPA, 5HTP or buffer, and tested for feminine and masculine sexual behaviour in adulthood. 5HTP had no effect on behaviour in either sex. pCPA had no effect on masculine sexual behaviour nor did it affect feminine sexual behaviour when given between days 1-7. When administered on days 7-14, pCPA significantly decreased the time that females spent displaying feminine sexual behaviour, while significantly increasing it in males. We, therefore, suggest that serotonin may be modulating a neural substrate already differentiated by androgens.