Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system.

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

Corticotropin-releasing factor (CRF) and related peptides confer neuroprotection via type 1 CRF receptors.

Corticotropin-releasing factor (CRF) receptors are members of the superfamily of G-protein coupled receptors that utilise adenylate cyclase and subsequent production of cAMP for signal transduction in many tissues. Activation of cAMP-dependent pathways, through elevation of intracellular cAMP levels is known to promote survival of a large variety of central and peripheral neuronal populations. Utilising cultured primary rat central nervous system neurons, we show that stimulation of endogenous cAMP signalling pathways by forskolin confers neuroprotection, whilst inhibition of this pathway triggers neuronal death. CRF and the related CRF family peptides urotensin I, urocortin, and sauvagine, which also induced cAMP production, prevented the apoptotic death of cerebellar granule neurons triggered by inhibition of phosphatidylinositol kinase-3 pathway activity with LY294002. These effects were negated by the highly selective CRF-R1 antagonist CP154,526. CRF even conferred neuroprotection when its application was delayed by up to 8 h following LY294002 addition. The CRF peptides also protected cortical and hippocampal neurons against death induced by beta-amyloid peptide (1-42), in a CRF-R1 dependent manner. In separate experiments, LY294002 reduced neuronal protein kinase B activity while increasing glycogen synthase kinase-3, whilst CRF (and related peptides) promoted phosphorylation of glycogen synthase kinase-3 without protein kinase B activation. Taken together, these results suggest that the neuroprotective activity of CRF may involve cAMP-dependent phosphorylation of glycogen synthase kinase-3.

Neuronal protein kinase signaling cascades and excitotoxic cell death.

Perturbation of normal survival mechanisms may play a role in a large number of disease processes. Glutamate neurotoxicity, particularly when mediated by the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, has been hypothesized to underlie several types of acute brain injury, including stroke. Several neurological insults linked to excessive release of glutamate and neuronal death result in tyrosine kinase activation, including p44/42 mitogen activated protein (MAP) kinase. To further explore a role for MAP kinase activation in excitotoxicity, we used a novel tissue culture model to induce neurotoxicity. Removal of the endogenous blockade by Mg2+ of the NMDA receptor in cultured hippocampal neurons triggers a self perpetuating cycle of excitotoxicity, which has relatively slow onset, and is critically dependent on NMDA receptors and activation of voltage gated Na+ channels. These injury conditions led to a rapid phosphorylation of p44/42 that was blocked by MAP kinase kinase (MEK) inhibitors. MEK inhibition was associated with protection against synaptically mediated excitotoxicity. Interestingly, hippocampal neurons preconditioned by a sublethal exposure to Mg(2+)-free conditions were rendered resistant to injury induced by a subsequently longer exposure to this insult; the preconditioning effect was MAP kinase dependent. The MAP kinase signaling pathway can also promote polypeptide growth factor mediated neuronal survival. MAP kinase regulated pathways may act to promote survival or death, depending upon the cellular context in which they are activated.

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition.

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.

Early responses to Salmonella typhimurium infection in mice occur at focal lesions in infected organs.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid, with key roles for cytokines and possibly also inducible nitric oxide synthase (iNOS), in mediating host responses to infection. In this paper we demonstrate that iNOS mRNA, protein and enzyme activity is induced within spleens and livers of infected mice as early as 5 h post-infection. Immunohistochemistry and in situ hybridization indicated that iNOS expression occurs predominantly in macrophages in localized, discrete foci in the infected organs. iNOS activity in spleen and liver was not detectable in uninfected control mice. The presence of mRNA encoding pro-inflammatory cytokines (TNFalpha, IL-1beta and IFNgamma) in infected organs was measured using RT-PCR, all three being present from 2 h post-infection onwards, but not before. These data show that there is a very early host response to S. typhimurium infection in mice, limited to foci within the infected organs.

Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: a possible role for mast cells.

Excessive glutamatergic neurotransmission, particularly when mediated by the N:-methyl-D-aspartate (NMDA) subtype of glutamate receptor, is thought to underlie neuronal death in a number of neurological disorders. Histamine has been reported to potentiate NMDA receptor-mediated events under a variety of conditions. In the present study we have utilized primary hippocampal neurone cultures to investigate the effect of mast cell-derived, as well as exogenously applied, histamine on neurotoxicity evoked by excessive synaptic activity. Exposure of mature cultures for 15 min to an Mg(2+)-free/glycine-containing buffer to trigger synaptic transmission through NMDA receptors, caused a 30-35% neuronal loss over 24 h. When co-cultured with hippocampal neurones, activated mast cells increased excitotoxic injury to 60%, an effect that was abolished in the presence of histaminase. Similarly, addition of histamine during magnesium deprivation produced a concentration-dependent potentiation (+ 60%; EC(50) : 5 microM) of neuronal death which was inhibited by sodium channel blockers and NMDA receptor antagonists, although this effect did not involve known histamine receptors. The histamine effect was further potentiated by acidification of the culture medium. Cultures 'preconditioned' by sublethal (5 min) Mg(2+) deprivation exhibited less neuronal death than controls when exposed to a more severe insult. NMDA receptor activation and the extracellular regulated kinase cascade were required for preconditioning neuroprotection. The finding that histamine potentiates NMDA receptor-mediated excitotoxicity may have important implications for our understanding of conditions where enhanced glutamatergic neurotransmission is observed in conjunction with tissue acidification, such as cerebral ischaemia and epilepsy.

Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage.

Fractalkine is a recently identified chemokine that exhibits cell adhesion and chemoattractive properties. It represents a unique member of the chemokine superfamily because it is located predominantly in the brain in which it is expressed constitutively on specific subsets of neurons. To elucidate the possible role of neuronally expressed fractalkine in the inflammatory response to neuronal injury, we have analyzed the regulation of fractalkine mRNA expression and protein cleavage under conditions of neurotoxicity. We observed that mRNA encoding fractalkine is unaffected by experimental ischemic stroke (permanent middle cerebral artery occlusion) in the rat. Similarly, in vitro, levels of fractalkine mRNA were unaffected by ensuing excitotoxicity. However, when analyzed at the protein level, we found that fractalkine is rapidly cleaved from cultured neurons in response to an excitotoxic stimulus. More specifically, fractalkine cleavage preceded actual neuronal death by 2-3 hr, and, when evaluated functionally, fractalkine represented the principal chemokine released from the neurons into the culture medium upon an excitotoxic stimulus to promote chemotaxis of primary microglial and monocytic cells. We further demonstrate that cleavage of neuron-derived, chemoattractive fractalkine can be prevented by inhibition of matrix metalloproteases. These data strongly suggest that dynamic proteolytic cleavage of fractalkine from neuronal membranes in response to a neurotoxic insult, and subsequent chemoattraction of reactive immune cells, may represent an early event in the inflammatory response to neuronal injury.

Molecular characterization and in situ localization of a full-length cyclic nucleotide-gated channel in rat brain.

Ion channels gated directly by cyclic nucleotides are required for the transduction of sensory signals in photoreceptor cells and olfactory cells. Cyclic nucleotide-gated (CNG) channels may also be expressed in the central nervous system because partial transcripts that share homology with CNG channels have been found therein. We have now isolated and cloned a full-length CNG channel cDNA from adult rat brain. The sequence is identical to that of the alpha-subunit originally found in the olfactory epithelium (CNCalpha3). In situ hybridization, using probes specific for the CNCalpha3 mRNA, suggest that this channel is expressed widely in the rat brain, albeit mostly at relatively low levels. Certain neuronal populations, however, such as deep cerebellar nuclei, olfactory bulb mitral cells and cerebellar Purkinje neurons, appeared specially enriched. The study demonstrates for the first time that a full-length CNG channel mRNA is expressed in the brain, supporting the possibility that CNG channels are involved in central neural communication and plasticity. The sequence reported in this paper has been deposited in the GenBank data base (accession no. AF126808).

Temporal expression of inducible nitric oxide synthase in mouse and human placenta.

The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12-20 although the level of calcium-independent NOS activity declined with advancing gestation. RT-PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16-18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.

Nitric oxide in cerebral ischemic neurodegeneration and excitotoxicity.

The observation that the free radical nitric oxide (NO) acts as a cell signaling molecule in key physiological processes such as regulation of blood pressure and immunological host-defense responses is probably one of the most important and exciting findings made in biology in the last decade. Likewise, in the brain NO has been implicated in a number of fundamental processes, including memory formation, sexual behavior and the control of cerebral blood flow. This has radically altered the accepted dogma of brain physiology and has placed NO at the center stage of neuroscience research. Evidence suggests that some of the actions of NO in the brain may be intimately linked to those of the classic excitatory neurotransmitter glutamate. The historical view that aberrations in glutamate signal transduction may underlie central neurodegeneration following, for example, cerebral ischemia, has implicated NO, by default, as a potential mediator of neuronal death. Indeed, with the advent of potent and specific compounds that interact with NO synthesizing (NOS) enzymes and with the NO signaling cascade, there is now ample evidence to suggest that NO can mediate neurodegeneration, although its involvement is paradoxical. Its cerebrovascular effects may act to limit ischemic damage by preserving tissue perfusion and preventing platelet aggregation, while NO produced in the parenchyma, either directly following the ischemic insult or at a later stage as part of a neuroinflammatory response, may be deleterious to the outcome of ischemia. Nonetheless, significant efforts are made into the potential therapeutic use of chemical NO donors and specific NOS inhibitors in the treatment of cerebral ischemia and other central neurodegenerative disorders. Here, the latest concepts and developments in our understanding of the role of NO in cerebral ischemic neurodegeneration are discussed.

Effect of annexin-1 on experimental autoimmune encephalomyelitis (EAE) in the rat.

Annexin-1, a calcium-dependent phospholipid binding protein, has been shown to act as an endogenous central neuroprotectant, notably against cerebral ischaemic damage. In the present study we extend these findings to an animal model of multiple sclerosis, EAE, and report that endogenous annexin-1 is expressed in ED1+ macrophages and resident astrocytes localized within the lesions in the central nervous system (CNS). Intracerebroventricular (i.c.v.) administration of an NH2-terminal fragment spanning amino acids 1-188 of annexin-1 after the onset of the clinical symptoms significantly reduced both the neurological severity as well as weight loss of mild EAE. Immunoneutralization of endogenous brain annexin-1 failed to exacerbate the clinical features of EAE. Thus, although the role of endogenous annexin-1 in the pathogenesis of EAE remains to be determined, our findings suggest that annexin-1 may be of therapeutic benefit to the treatment of multiple sclerosis.

The nitric oxide-cyclic GMP pathway is required for nociceptive signalling at specific loci within the somatosensory pathway.

The involvement of nitric oxide in nociceptive processing was examined at the main loci of synaptic transmission within the rat somatosensory pathway from the caudal sural cutaneous nerve. Intrathecal (lumbar 1-3) administration of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (30 micrograms), inhibited nitric oxide synthase in this region of the spinal cord by greater than 80% but had no significant effect on nitric oxide synthase in parietal cerebral cortex, thalamus or medulla/pons. In a rat model of peripheral neuropathy (one to two week ligation of the caudal sural cutaneous nerve), intrathecal administration of the same dose of N omega-nitro-L-arginine methyl ester prevented the hyperalgesic response to thermal stimuli. Administration of 30 micrograms N omega-nitro-L-arginine methyl ester into the lateral ventricle had no effect on nitric oxide synthase in the lumbar 1-3 region of the spinal cord but gave substantial inhibition in higher areas of the somatosensory pathway (parietal cerebral cortex, thalamus and medulla/pons). Nitric oxide synthase in the parietal cerebral cortex (but not thalamus) was inhibited to a greater extent in the hemisphere ipsilateral to the site of administration. Administration of 30 micrograms N omega-nitro-L-arginine methyl ester into the lateral ventricle decreased thermal hyperalgesia, but only when N omega-nitro-L-arginine methyl ester was administered contralateral to the ligated caudal sural cutaneous nerve and therefore ipsilateral to the cortical nociceptive processing from this nerve. Intrathecal and intracerebroventricular administration of the selective inhibitor of nitric oxide-sensitive guanylyl cyclase, 1-H-[1,2,4]oxadiazalo[4,3-a]quinoxalin-1-one, also decreased the hyperalgesic response to thermal stimuli. These data demonstrate that, in a model of neuropathic pain, nitric oxide is involved in nociceptive processing at spinal and cerebrocortical synaptic loci of the somatosensory pathway and that its actions appear to be mediated through guanylyl cyclase.

Vicious cycle involving Na+ channels, glutamate release, and NMDA receptors mediates delayed neurodegeneration through nitric oxide formation.

The mechanisms by which neurons die after cerebral ischemia and related conditions in vivo are unclear, but they are thought to involve voltage-dependent Na+ channels, glutamate receptors, and nitric oxide (NO) formation because selective inhibition of each provides neuroprotection. It is not known precisely what their roles are, nor whether they interact within a single cascade or in parallel pathways. These questions were investigated using an in vitro primary cell culture model in which striatal neurons undergo a gradual and delayed neurodegeneration after a brief (5 min) challenge with the glutamate receptor agonist NMDA. Unexpectedly, NO was generated continuously by the cultures for up to 16 hr after the NMDA exposure. Neuronal death followed the same general time course except that its start was delayed by approximately 4 hr. Application of the NO synthase inhibitor nitroarginine after, but not during, the NMDA exposure inhibited NO formation and protected against delayed neuronal death. Blockade of NMDA receptors or of voltage-sensitive Na+ channels [with tetrodotoxin (TTX)] during the postexposure period also inhibited both NO formation and cell death. The NMDA exposure resulted in a selective accumulation of glutamate in the culture medium during the period preceding cell death. This glutamate release could be inhibited by NMDA antagonism or by TTX, but not by nitroarginine. These data suggest that Na+ channels, glutamate receptors, and NO operate interdependently and sequentially to cause neurodegeneration. At the core of the mechanism is a vicious cycle in which NMDA receptor stimulation causes activation of TTX-sensitive Na+ channels, leading to glutamate release and further NMDA receptor stimulation. The output of the cycle is an enduring production of NO from neuronal sources, and this is responsible for delayed neuronal death. The same neurons, however, could be induced to undergo more rapid NMDA receptor-dependent death that required neither TTX-sensitive Na+ channels nor NO.

Ex vivo measurement of brain tissue nitrite and nitrate accurately reflects nitric oxide synthase activity in vivo.

The ex vivo tissue concentration of nitrite and nitrate (NOx) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N omega-nitro-L-arginine (L-NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NOx by up to 75% but had no effect on plasma NOx. Selective inhibition of central NOS by intracerebroventricular administration of L-NA methyl ester produced similar decreases in levels of whole-brain NOx. A residual concentration of NOx of 10-15 microM remained in all brain regions even after complete inhibition of brain NOS. Brain NOx content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NOx was found to reflect the in vivo efficacy of several different types of NOS inhibitor: L-NA, N omega-monomethyl-l-arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate L-arginine increased brain NOx concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NOx is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.

Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.

Neurotoxic mechanisms of transactivating protein Tat of Maedi-Visna virus.

Infection by lentiviruses such as human immunodeficiency virus (HIV) and Maedi-Visna virus (MVV) is associated with neurodegenerative disorders. We have investigated the neurotoxic mechanisms of a synthetic peptide of transactivating protein tat of MVV in striatal neuronal cultures. Tat peptide (but not control peptide) caused neuronal death, without affecting glial viability, in a time- and dose-dependent manner. Significant neuronal death was not observed until 6-8 h after tat peptide application (2.35-2350 nM), whereas half maximal and maximal cell death was observed after 12 and 24 h respectively. Tat peptide neurotoxicity could be partially inhibited by blockade of either N-methyl-D-aspartate (NMDA)- or non-NMDA receptors, suggesting that excessive neuroexcitation by glutamate or its analogues may contribute to tat-neurotoxicity. Furthermore, when both these glutamate receptor subtypes were blocked simultaneously, an increased degree of neuroprotection was observed. Finally, tat peptide toxicity was also reduced by blockade of L-type calcium channels. Calcium imaging revealed that intracellular calcium increases slowly upon tat application, predominantly due to entry of extracellular calcium. These results indicate that cellular calcium entry through voltage-gated calcium channels following activation of both NMDA and non-NMDA receptors, and subsequent accumulation of intracellular calcium may contribute to the neuronal death induced by tat protein.

Cytokines in neurodegeneration and repair.

Cytokines have diverse actions in the brain, some of which may facilitate either neurodegeneration or neuroprotection. The expression of cytokines, particularly interleukins-1 and -6 (IL-1, IL-6) and tumor necrosis factor alpha, is rapidly and markedly induced in response to experimentally induced or clinical neurodegeneration. We have demonstrated that central administration of the IL-1 receptor antagonist (IL-1ra) markedly inhibits neurodegeneration induced by focal cerebral ischaemia, local infusion of glutamate receptor agonists or traumatic brain injury in the rat. In contrast, IL-1ra offers no protection against degeneration of primary cortical neurones in culture caused by exposure to agonists of ionotrophic or metabotrophic receptors. In vivo, administration of IL-1 beta exacerbates ischaemic brain damage, whereas in cell culture, exogenous IL-1 is neuroprotective at concentrations in the nM range, an effect which appears to be mediated by release of endogenous nerve growth factor. Higher concentrations of IL-1 (microM range) are neurotoxic to neurones in culture and may mimic the involvement of IL-1 in neurodegeneration in vivo. Thus, excessive production of cytokines such as IL-1 appears to mediate experimentally induced neurodegeneration in vivo, while neuroprotective effects of low concentrations of the cytokine suggest a dual role for IL-1 in neuronal survival.

Substantial regional and hemispheric differences in brain nitric oxide synthase (NOS) inhibition following intracerebroventricular administration of N omega-nitro-L-arginine (L-NA) and its methyl ester (L-NAME).

Nitric oxide synthase (NOS) enzyme activity was determined in a comprehensive selection of regions of the rat brain. The effects of lateral ventricular administration of N omega-nitro-L-arginine (L-NA, 30 micrograms) and its methyl ester (L-NAME, 3-100 micrograms) on NOS activity were examined in the ipsilateral and contralateral areas of 4 of these brain regions and in the cerebellum. NOS activity was determined using a new and rapid ex vivo assay method which ensures minimal dissociation of the enzyme-inhibitor complex. Following infusion of L-NAME, NOS activity was rapidly and dose-dependently inhibited in all brain regions studied (cerebral cortex, striatum, hippocampus, cerebellum and thalamus). However, NOS activity of brain regions within the contralateral hemisphere was inhibited significantly less than in ipsilateral regions, with the exception of the thalamus. The degree of NOS inhibition varied markedly between brain regions within each hemisphere and correlated with their ventricular proximity to the site of NOS inhibitor administration. Therefore, NOS in the thalamus was inhibited most effectively and NOS in the cerebral cortex the least. Within the cerebral cortex further regional differences could be observed, with NOS in the frontal/parietal areas inhibited more effectively than NOS in the temporal/occipital areas. Maximal inhibition of NOS was sustained for approx 6 hr after administration of 30 and 100 micrograms L-NAME. No inhibition of NOS was observed 24 hr after administration. Lateral ventricular administration of the metabolite and active moiety of L-NAME, L-NA, resulted in a similar degree of inhibition and time of inhibitory onset. In contrast, when L-NAME was administered i.p., a significant delay in the onset of NOS inhibition was observed in the above brain regions compared to L-NA. However, no regional or hemispheric differences in NOS inhibition were detected following peripheral administration of these inhibitors. These results indicate that central administration of NOS inhibitors yields a complex pattern of NOS inhibition and that data obtained on brain physiology following the i.c.v. administration of NOS inhibitors, or for that matter any other CNS effector, should therefore be interpreted with extreme caution.

Interleukin-1 beta attenuates excitatory amino acid-induced neurodegeneration in vitro: involvement of nerve growth factor.

Certain cytokines have been reported to exert neurotrophic actions in vivo and in vitro. In the present study, we investigated the possible neuroprotective actions of the cytokine human recombinant interleukin-1 beta (hrIL-1 beta) against excitatory amino acid (EAA)-induced neurodegeneration in cultured primary cortical neurons. Brief (15 min) exposure of cultures to submaximal concentrations of glutamate, NMDA, AMPA, or kainate caused extensive neuronal death (approximately 70% of all neurons). Neuronal damage induced by the EAAs was significantly reduced (up to 70%) by pretreatment with 500 ng/ml (6.5 x 10(3) U/ml) hrIL-1 beta for 24 hr. The neuroprotective effect of hrIL-1 beta was reversed by coapplication of an IL-1 receptor antagonist (IL-1ra, 50 micrograms/ml). Neuroprotective actions of hrIL-1 beta were also reduced by administration of a neutralizing monoclonal antibody to NGF (65% inhibition). In concordance, the neurotoxic actions of EAAs were significantly reduced (by 40%) after pretreatment with NGF (100 ng/ml for 48 hr). Furthermore, an additive neuroprotective effect of approximately 75% was observed when cultures were exposed to a combination of hrIL-1 beta and NGF. In contrast, exposure of cultures to high concentrations hrIL-1 beta alone (100 micrograms/ml, 1.3 x 10(6) U/ml) for periods up to 72 hr resulted in neurotoxicity, which was reversed by IL-1ra (1 mg/ml). These findings suggest that hrIL-1 beta can limit EAA-induced neuronal damage. These effects appear to be may be mediated, at least in part, via NGF. These findings may be relevant to the understanding of neurodegenerative diseases.

Corticotrophin-releasing factor antagonist inhibits neuronal damage induced by focal cerebral ischaemia or activation of NMDA receptors in the rat brain.

This study investigated the involvement of corticotrophin-releasing factor (CRF) in acute neuronal damage induced by focal cerebral ischaemia or pharmacological activation of NMDA receptors in the rat brain. Intracerebroventricular injection of a CRF receptor antagonist (alpha-helical CRF9-41), markedly inhibited ischaemic (61%) and excitotoxic (41%) brain damage. Peripheral injection of a glucocorticoid antagonist (RU38486) did not affect ischaemic damage. Ischaemic and excitotoxic damage caused increased hypothalamic concentrations of CRF. These data indicate that CRF mediates ischaemic and excitotoxic neuronal damage in the rat, but that this effect is not dependent on glucocorticoids.