Glutamate receptors and gene induction: signalling from receptor to nucleus.

Activation of glutamate receptors has been linked to a diversity of lasting physiologic and pathologic changes in the mammalian nervous system. The cellular and molecular mechanisms underlying permanent modifications of nervous system structure and function following brief episodes of neuronal activity are unknown. Immediate early genes (IEGs) have been implicated in the conversion of short-term stimuli to long-term changes in cellular phenotype by regulation of gene expression. Many of the long-term consequences of glutamate receptor activation correlate with increases in specific IEGs; the intracellular signalling pathways coupling activation of receptors at the cell surface with induction of IEGs in the nucleus are incompletely understood. Analysis of mechanisms of how extracellular factors control gene expression implicate activation of second messenger systems and protein kinases. Activation of glutamate receptors results in an initial increase in intracellular calcium; the route of calcium influx may differ depending on the specific receptor subtype activated. Intracellular calcium is often the first messenger in response to an extracellular stimulus and can be the trigger for activating numerous other signalling pathways. Results obtained over the past several years support a hypothesis where selective activation of distinct intracellular signalling pathways and IEG responses, following activation of different glutamate receptor subtypes, involve spatial restriction of key enzymes to sites of local calcium increases. The specificity in long-term neuronal responses following brief synaptic activation may depend on the specific intracellular signalling mechanisms triggered and the unique array of IEGs transcribed.

Prostaglandin F2alpha is required for NMDA receptor-mediated induction of c-fos mRNA in dentate gyrus neurons.

Activation of NMDA receptors has been linked to a diversity of lasting physiological and pathological changes in the mammalian nervous system. The cellular and molecular mechanisms underlying permanent modifications of nervous system structure and function after brief episodes of neuronal activity are unknown. Immediate-early genes (IEGs) have been implicated in the conversion of short-term stimuli to long-term changes in cellular phenotype by regulation of gene expression. The intracellular signaling pathways coupling activation of receptors at the cell surface with induction of IEGs in the nucleus are incompletely understood. NMDA produces a striking increase in the IEG c-fos in dentate gyrus (DG) neurons in vitro; this induction is dependent, in part, on the arachidonic acid cascade. Here we show that NMDA receptor activation triggers the synthesis of the prostaglandins PGF2alpha and PGE2, but not PGD2, in rat cerebral cortical neurons in vitro. We further demonstrate that PGF2alpha, but not PGE2 or PGD2, is necessary but not sufficient for NMDA induction of c-fos mRNA in DG neurons. These findings provide insight into the molecular events coupling activation of the NMDA receptor with regulation of the IEG c-fos and identify the diffusable messenger PGF2alpha as obligatory for NMDA receptor-mediated transcription of a nuclear IEG.

N-methyl-D-aspartate receptors activate transcription of c-fos and NGFI-A by distinct phospholipase A2-requiring intracellular signaling pathways.

Activation of N-methyl-D-aspartate (NMDA) receptors is required for induction of some lasting changes in nervous system structure and function. The cellular mechanisms involved in transducing receptor stimulation into long-lasting changes in cellular activity are unknown. Immediate-early genes (IEGs) have been implicated in the conversion of short term stimuli to long term changes in cellular phenotype, by regulation of gene expression. Activation of NMDA receptors on dentate gyrus neurons triggers the transcriptional activation of several IEGs. To determine whether the same intracellular pathways transduce the signal from this ligand-gated ion channel to the nucleus, we compared NMDA induction of two IEGs. NMDA was sufficient to produce a striking increase in both c-fos and NGFI-A mRNAs in dentate granule neurons, in a calcium-dependent manner. The induction of both IEGs was blocked by structurally distinct inhibitors of phospholipase A2, an enzyme that catalyzes phospholipid degradation and formation of arachidonic acid. Arachidonic acid itself is catalyzed to biologically active metabolites by multiple enzymes, including cyclooxygenase and lipoxygenase. Selective inhibitors of cyclooxygenase attenuated NMDA induction of c-fos but not NGFI-A. Conversely, structurally distinct inhibitors of lipoxygenase blocked NMDA induction of NGFI-A but not c-fos. The signaling pathways linking NMDA receptors to the transcriptional activation of c-fos and NGFI-A are related but distinct. We suggest that phospholipase A2 and the arachidonic acid cascade play a pivotal role in NMDA receptor regulation of gene expression.

Ionotropic glutamate receptor subtypes activate c-fos transcription by distinct calcium-requiring intracellular signaling pathways.

N-Methyl-D-aspartate (NMDA) or non-NMDA receptor activation is sufficient to induce transcription of the immediate early gene c-fos in a calcium-requiring manner. We sought to determine whether the calcium-dependent mechanisms inducing c-fos transcription are identical following activation of these two receptor subtypes. We used in situ hybridization and fura-2 imaging to detect c-fos mRNA and intracellular calcium in individual dentate gyrus neurons maintained in vitro. Structurally distinct inhibitors of phospholipase A2 and cyclooxygenase abolished NMDA--but not kainic acid-induced increases of c-fos mRNA. Conversely, the calmodulin antagonist calmidazolium markedly inhibited kainic acid--but not NMDA-mediated increases of c-fos mRNA. We propose that the dissociation in the mechanisms transducing the calcium influx signals to the nucleus following NMDA and non-NMDA receptor activation is due to spatially distinct sites of calcium entry, resulting in activation of different enzymes located at distinct sites in the cell.

NMDA and non-NMDA receptor-mediated increase of c-fos mRNA in dentate gyrus neurons involves calcium influx via different routes.

We examined the effects of selective agonists of ionotropic excitatory amino acid (EAA) receptor subtypes on induction of the immediate early gene c-fos. We used in situ hybridization to measure c-fos mRNA and fura-2 imaging to measure intracellular calcium (Ca2+i) in individual dentate gyrus neurons maintained in vitro. Activation of either NMDA or non-NMDA receptor subtypes is sufficient to induce the rapid and dramatic increase of c-fos mRNA. Activation of either NMDA or non-NMDA receptors also induces a rapid and dramatic increase of Ca2+i, effects blocked by the removal or chelation of extracellular calcium (Ca2+e). c-fos mRNA induction by either receptor subtype is Ca2+ dependent, since chelation of Ca2+e with EGTA prevents c-fos mRNA induction by both NMDA and non-NMDA receptor agonists. The increase in Ca2+i induced by activating non-NMDA receptors is inhibited either by removal of extracellular sodium (Na+e) or by the voltage-sensitive calcium channel (VSCC) blocker nifedipine. By contrast, the increase of Ca2+i induced by activating NMDA receptors is not inhibited by removal of Na+e or nifedipine. Consistent with these effects on Ca2+i, nifedipine inhibits induction of c-fos mRNA by non-NMDA, but not by NMDA, receptor agonists. These findings indicate that Ca2+ serves as a second messenger coupling ionotropic EAA receptors with transcriptional activation of c-fos mRNA. The route of Ca2+ entry into dentate neurons, however, depends on the EAA receptor subtype stimulated. Non-NMDA receptor activation results in Ca2+ influx indirectly via VSCCs, whereas NMDA receptor activation results in Ca2+ influx directly through the NMDA channel itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuron-associated astroglial cells express beta- and alpha 1-adrenergic receptors in vitro.

In vivo, astroglial cells are closely interrelated with neurons. The present studies were undertaken to determine if certain astroglial properties are influenced when maintained in a heterogeneous cellular environment. Astrocytes and neurons were co-cultured from hippocampal tissue and the parameters examined included astroglial morphology and expression of beta- and alpha 1-adrenergic receptors (AR). Astroglial cells exhibit an extremely elongated morphology when growing in direct contact with neurons. Astroglia growing under the same culture conditions, but not in direct contact with neurons, exhibited a polygonal morphology. One hundred percent of the elongated astroglia expressed the beta-AR with an average density of 1320 binding sites/1000 microns 2. In contrast, only 40-50% of these elongated astroglia expressed alpha 1-ARs. Our results indicate that astroglia, maintained in the presence of neurons, continue to express beta- and alpha 1-ARs. These results suggest that under in vivo conditions, where astroglia normally exist in close contact with neurons, astrocytes may express surface receptors which enable them to sense neuronal activity and to selectively respond to such activity. The elongated astroglia described here may exhibit morphological features more reminiscent to that which exists in vivo.

Astroglial cells in vitro are heterogeneous with respect to expression of the alpha 1-adrenergic receptor.

A wide variety of approaches have been used to examine the expression of neuroligand receptors by cultured astroglial cells. The results of such studies make it clear that these cells exhibit most, if not all, of the different receptors known to be associated with central neurons. However, it has been more difficult to determine if there are multiple populations of astroglia that can be distinguished on the basis of their complement of neuroligand receptors. To address this question, we established a radioligand binding assay that enabled us to visualize alpha 1-adrenergic receptors (alpha 1-ARs) on immunocytochemically defined neural cells. Saturation, time course, and competition binding experiments determined that 125I-HEAT could be used to identify alpha 1-AR binding sites on immunocytochemically defined astroglial cells. Our results indicate that approximately 66% of cortical polygonal astroglia express detectable numbers of alpha 1-ARs. 3H-thymidine labeling experiments indicate that both dividing and nondividing astroglia exhibit alpha 1-ARs. These results greatly contrast with studies on beta-adrenergic receptor expression (beta-AR), in which 100% of polygonal astroglia express beta-ARs. Process-bearing astroglia also exhibit alpha 1-ARs, which is in marked contrast to the lack of beta-AR expression by these cells. The results presented here suggest that astroglia, like neurons, differ in the receptors they express and therefore may be able to selectively respond to extracellular stimulation.

Postnatal methyl mercury exposure: effects on ontogeny of renal and hepatic ornithine decarboxylase responses to trophic stimuli.

The effects of postnatal methyl mercury exposure on the ontogeny of renal and hepatic responsiveness to trophic stimuli were examined. Increased ornithine decarboxylase (ODC) activity was used as an index of tissue stimulation. In the rat, renal ODC responsiveness to growth hormone, angiotensin, vasopressin, isoproterenol, and serotonin was absent at birth and matured 3 to 4 weeks later. However, pups exposed to methyl mercury showed marked, ODC responses to these same agents as early as 10 to 19 days of postnatal age, accompanied by a significant renal hypertrophy. In contrast to the kidney, the liver of normally developing rats was responsive to trophic factors even in the neonate. In this tissue, there was no consistent effect of neonatal methyl mercury treatment on ODC responses at any developmental stage tested; although absolute liver weights were reduced, liver/body weight ratio was not affected. These results demonstrate that postnatal methyl mercury exposure causes a precocious onset of ODC responses to trophic agents specifically in the kidney. Altered responsiveness may mediate some of the effects of this organomercurial on overall renal development and function.

Bupropion does not antagonize cardiovascular actions of clonidine in normal subjects and spontaneously hypertensive rats.

Tricyclic antidepressants (TADs) are known to antagonize the hypotensive and sedative actions of clonidine. We compared the effects of bupropion and imipramine pretreatment on the acute hypotensive and sedative actions of clonidine in eight normotensive male subjects in a randomized, double-blind crossover study. Pretreatment with bupropion, 100 mg by mouth three times a day for 9 days, had no effect on baseline supine blood pressure (BP) and heart rate (HR) and did not modify the hypotensive, bradycardic, and sedative actions of clonidine. Imipramine, 25 mg by mouth three times a day for 9 days, increased supine and standing HR and decreased standing systolic BP. In half the subjects the hypotensive action of clonidine was reduced 40% to 50% by imipramine. The specific binding of 3H-yohimbine to alpha 2-receptors of platelet membranes was not affected by pretreatment with either antidepressant. In spontaneously hypertensive rats, 16 days of bupropion, 25 mg/kg subcutaneously, had no effect on baseline BP and HR and did not antagonize the hypotensive and bradycardic effects of clonidine, 5 mg/kg iv. Pretreatment with desipramine, 5 mg/kg subcutaneously for 16 days, accelerated baseline HR and reduced cardiovascular actions of clonidine. These observations suggest that not all antidepressants antagonize the effects of clonidine. If the negative interaction between TADs and clonidine is a result of sensitivity of alpha 2-receptors, these receptor changes are not the common denominator of antidepressant activity and may only be seen with TADs.

Neonatal methylmercury poisoning in the rat: effects on development of peripheral sympathetic nervous system. Neuronal participation in methylmercury-induced cardiac and renal overgrowth.

The effects of neonatal CH3-Hg exposure on development and function of peripheral catecholaminergic synapses were examined by measuring tissue norepinephrine (NE) levels and turnover rates and cardiac biochemical responses to sympathetic reflex stimulation. In the rat, cardiac sympathetic neurotransmission normally develops towards the end of the first week postnatally; however, pups given CH3-Hg showed responses to sympathetic reflex stimulation as early as 2 days of age. The accelerated maturation of cardiac sympathetic effect was accompanied by initial enhancement of NE levels and turnover. This effect appeared to be specific to the heart, as kidney displayed subnormal NE levels in CH3-Hg-treated animals. Since neonatal CH3-Hg produces heart and kidney overgrowth, we examined the potential role of sympathetic input in altered tissue growth, utilizing chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Sympathectomy inhibited the early phase of renal overgrowth, suggesting that sympathetic nerves participate in the initial effect of CH3-Hg on this tissue; however, 6-OHDA did not influence later phases of renal enlargement nor did it alter the CH3-Hg-induced cardiac overgrowth. These results indicate that neonatal exposure to CH3-Hg alters the synaptic development of peripheral catecholamine neurons, which may play a role in some of the subsequent effects on tissue development.

Role of ornithine decarboxylase and the polyamines in nervous system development: Short-term postnatal administration of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase.

The role of ornithine decarboxylase (ODC) and the polyamines in development of central and peripheral catecholaminergic neurons was examined through the use of α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Short-term postnatal administration of DFMO (500 mg/kg daily on days 1-6) to neonatal rats resulted in effective inhibition of ODC and depletion of both putrescine and spermidine in brain, heart and kidney; after cessation of DFMO administration, polyamine levels returned to normal by 10-13 days of age. There were no signs of generalized toxicity of short-term DFMO treatment, as body weight gains were largely unaffected over the course of study (through weaning). However, development of peripheral sympathetic neurons was severely retarded by DFMO, with persistent and profound deficits of both cardiac and renal norepinephrine; the catecholamine deficiencies were unrelated to effects on end-organ growth, as cardiac weights were essentially normal whereas kidney weights were adversely affected by DFMO. Development of the adrenal medulla, a peripheral catecholaminergic tissue which displays approximately the same developmental profile as do sympathetic neurons but which does not develop axonal projections, was not slowed by DFMO treatment; similarly, central noradrenergic and dopaminergic neurons, which undergo the majority of axonal outgrowth and synaptogenesis during the second to third postnatal week (just after the period in which polyamines returned to control levels), developed normally as assessed by measurements of transmitter levels, tyrosine hydroxylase activity and synaptosomal uptake of [(3)H]norepinephrine or [(3)H]dopamine. Extension of the period of DFMO treatment and consequent depletion of polyamines into the period in which central synaptogenesis occurs does, however, produce slowing of development of brain catecholamine neuronal projections. Thus, the ODC/polyamine system appears to play a critical postnatal role in catecholamine systems specifically undergoing active synaptogenesis.

Critical periods for the role of ornithine decarboxylase and the polyamines in growth and development of the rat: Effects of exposure to α-difluoromethylornithine during discrete prenatal or postnatal intervals.

The roles of ornithine decarboxylase (ODC) and the polyamines in fetal and neonatal development were examined through the use of α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Administration to pregnant rats of 500 mg/kg of DFMO every 12 h for a 4-day period (8 DFMO injections) resulted in fetal and neonatal death; DFMO early in gestation produced fetal resorption whereas late gestational exposure did not compromise fetal viability but instead resulted in a delayed toxic effect, with high mortality in the first postnatal week. Generalized toxicity of DFMO was not apparent in later developmental periods, as 4 days of DFMO treatment begun postnatally did not produce any neonatal death. Shortening the course of gestational DFMO treatment to 2.5 days (5 DFMO injections) also did not adversely affect fetal or neonatal viability and thus permitted identification of critical periods in which various tissues are sensitive to DFMO. Examination of growth patterns of brain, heart and kidney and of neurochemical development of central and peripheral catecholaminergic neurons indicated that different critical periods exist for effects of DFMO on each tissue or even on the various cell types within a tissue. The separable sensitivities were apparent even though the effects of DFMO on ODC and the polyamines for any given treatment period were fairly uniform in all tissues studied. These results indicate that the ODC/polyamine system plays multiple roles in fetal survival and in tissue growth during discrete periods of development; because the time course of cellular maturation differs for each tissue or cell population, DFMO administered during any one brief period can produce organ-specific developmental deficits.

Grief among healthcare workers: a comparative study.

This investigation examined the prevalence and nature of grief in response to patient suffering, loss, or death among healthcare workers employed at a general hospital and a skilled nursing facility. A questionnaire was constructed for this purpose. Approximately two-thirds of the skilled nursing facility personnel remembered experiencing bereavement as a reaction to the crises of their geriatric patients. Mourning occurred among virtually all of the general hospital personnel who usually serve younger patients. Healthcare personnel mourned most often for those persons who suffered or died of cancer. A selected sample of respondents recalled that psychological symptoms of grief were more evident than physical. Both symptom types often persisted for more than 1 month. Since grief is common among nurses and aides employed at the two medical settings, bereavement counseling is suggested for those healthcare personnel who require this service.

Ornithine decarboxylase and polyamines in tissues of the neonatal rat: effects of alpha-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase.

To evaluate the role of ornithine decarboxylase (ODC) and the polyamines in tissue growth and development, neonatal rats were given daily injections of alpha-difluoromethylornithine, a specific, irreversible inhibitor of ODC. Enzyme activity in brain, heart and kidney was effectively inhibited, leading to prompt reductions in putrescine levels which were apparent throughout the 4-week period of drug treatment. Deficits in spermidine levels appeared within several days and remained significant in all three tissues, although some recovery toward control levels was apparent after 2 weeks postnatally. Spermine levels were not decreased and in some cases were actually increased during the course of alpha-difluoromethylornithine administration; assessment of total organ content of spermine or total polyamines per organ (putrescine + spermidine + spermine) indicated that the tissues were still actively increasing their net polyamine content despite continued inhibition of ODC. Growth of the kidney and brain were affected within several days of commencing alpha-difluoromethylornithine treatment, well before the onset of body weight or heart weight deficits. By 14 days of age and thereafter, animals displayed delayed eye-opening, deficient fur growth and shorter body length. These data suggest that the ODC/polyamine system does serve as a modulator of tissue growth during neonatal mammalian development and that differences exist among various tissues in the degree and time course of dependence of growth on polyamines, particularly putrescine and/or spermidine.

Impaired development of central and peripheral catecholamine neurotransmitter systems in preweanling rats treated with alpha-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase.

Daily administration of alpha-difluoromethylornithine (DFMO) to neonatal rats results in persistent inhibition of ornithine decarboxylase, depletion of polyamines and rapid onset of brain growth deficits. Animals treated with DFMO displayed marked retardation of synaptic development of catecholaminergic systems in the brain, evidenced by slowed development of synaptosomal uptake of [3H]norepinephrine and of tyrosine hydroxylase activity. Fundamental alterations in brain membrane metabolism also could be detected through measurements of phospholipid incorporation of 33Pi; DFMO suppressed the developmental increments in phospholipid synthesis normally accompanying synaptic outgrowth. Although the content of norepinephrine and dopamine in the brain was unchanged by DFMO, the drug did cause initial reductions, and subsequent elevations, in catecholamine turnover. Effects of DFMO on development of peripheral sympathetic neurons were even more profound, with substantial deficits in norepinephrine content throughout preweanling development, again accompanied by biphasic alterations of turnover. The adrenal medulla, a sympathetic tissue which does not undergo catecholaminergic axonal outgrowth and synaptogenesis, was spared the deleterious effects of DFMO on development. These results support the view that ornithine decarboxylase and the polyamines play an obligatory role in synaptic maturation, with the greatest sensitivity to DFMO-induced alterations occurring during periods of rapid development.