Heterogeneity of the Endocannabinoid System Between Cerebral Cortex and Spinal Cord Oligodendrocytes.

In the last years, regional differences have been reported between the brain and spinal cord oligodendrocytes, which should be considered when designing therapeutic strategies for myelin repair. Promising targets to achieve myelin restoration are the different components of the endocannabinoid system (ECS) that modulate oligodendrocyte biology, but almost all studies have been focused on brain-derived cells. Therefore, we compared the ECS between the spinal cord and cerebral cortex-derived oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (OLs). Cells from both regions express synthesizing and degrading enzymes for the endocannabinoid 2-arachidonoylglycerol, and degrading enzymes increase with maturation, more notably in the spinal cord (monoglyceride lipase-MGLL, alpha/beta hydrolase domain-containing 6-ABHD6, and alpha/beta hydrolase domain-containing 12-ABHD12). In addition, spinal cord OPCs express higher levels of the synthesizing enzymes diacylglycerol lipases alpha (DAGLA) and beta (DAGLB) than cortical ones, DAGLA reaching statistical significance. Cells from both the cortex and spinal cord express low levels of NAEs synthesizing enzymes, except for the glycerophosphodiester phosphodiesterase 1 (GDE-1) but high levels of the degrading enzyme fatty acid amidohydrolase (FAAH) that increases with maturation. Finally, cells from both regions show similar levels of CB1 receptor and GPR55, but spinal cord-derived cells show significantly higher levels of transient receptor potential cation channel V1 (TRPV1) and CB2. Overall, our results show that the majority of the ECS components could be targeted in OPCs and OLs from both the spinal cord and brain, but regional heterogeneity has to be considered for DAGLA, MGLL, ABHD6, ABHD12, GDE1, CB2, or TRPV1.

Spinal cord injury induces a long-lasting upregulation of interleukin-1ß in astrocytes around the central canal.

Under inflammatory conditions, interleukin-1ß (IL-1ß) modulates neural stem cells at neurogenic niches. Here we show that spinal cord injury in rats increases IL-1ß expression in astrocytes located around the spinal cord ependyma, a region that also holds a neurogenic potential. IL-1ß increases from day 1 after lesion, reaches maximal levels between days 3 and 7, and declines from 14 days to low levels after 28 days. At the time of maximal expression, periependymal upregulation of IL-1ß extends beyond 5 mm from the epicenter of the lesion both rostral and caudally. Since IL-1ß controls proliferation and cell fate of neural stem/precursor cells, its modulation in periependymal astrocytes might create an appropriate environment for cell replacement after injury.

CB1 receptor antagonism/inverse agonism increases motor system excitability in humans.

CB1 receptor is highly expressed in cerebral structures related to motor control, such as motor cortex, basal ganglia and cerebellum. In the spinal cord, the expression of CB1 receptors has also been observed in ventral motor neurons, interneurons and primary afferents, i.e., in the cells that may be part of the circuits involved in motor control. It is known that the antagonist/inverse agonist of CB1 receptors Rimonabant penetrates the blood-brain barrier and produces a broad range of central psychoactive effects in humans. Based on the occurrence of central effects in humans treated with Rimonabant and on the location of CB1 receptors, we hypothesized that the application of Rimonabant can also affect the motor system. We tested the effects of a single dose of 20mg of Rimonabant on the excitability of motor cortex and of spinal motor neurons in order to detect a possible drug action on motor system at cortical and spinal levels. For this purpose we use classical protocols of transcranial magnetic and electrical stimulation (TMS and TES). Single and paired pulse TMS and TES were used to assess a number of parameters of cortical inhibition and cortical excitability as well as of the excitability of spinal motor neurons. We demonstrated that a single oral dose of 20mg of Rimonabant can increase motor system excitability at cortical and spinal levels. This opens new avenues to test the CB1R antagonists/inverse agonists for the treatment of a number of neurological dysfunctions in which can be useful to increase the excitability levels of motor system. Virtually all the disorders characterized by a reduced output of the motor cortex can be included in the list of the disorders that can be treated using CB1 antagonists/reverse agonists (e.g. stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, fatigue syndromes, parkinsonisms, etc.).

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways.

BACKGROUND AND PURPOSE: The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB(1), CB(2) and CB(1)/CB(2) receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity. EXPERIMENTAL APPROACH: Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB(1), CB(2) and CB(1) /CB(2) receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB(1) receptor) and AM630 (CB(2) receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation. KEY RESULTS: ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation. CONCLUSIONS AND IMPLICATIONS: Activation of cannabinoid CB(1) or CB(2) receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

CB2 cannabinoid receptors as an emerging target for demyelinating diseases: from neuroimmune interactions to cell replacement strategies.

Amongst the various demyelinating diseases that affect the central nervous system, those induced by an inflammatory response stand out because of their epidemiological relevance. The best known inflammatory-induced demyelinating disease is multiple sclerosis, but the immune response is a common pathogenic mechanism in many other less common pathologies (e.g., acute disseminated encephalomyelitis and acute necrotizing haemorrhagic encephalomyelitis). In all such cases, modulation of the immune response seems to be a logical therapeutic approach. Cannabinoids are well known immunomodulatory molecules that act through CB1 and CB2 receptors. While activation of CB1 receptors has a psychotropic effect, activation of CB2 receptors alone does not. Therefore, to bypass the ethical problems that could result from the treatment of inflammation with psychotropic molecules, considerable effort is being made to study the potential therapeutic value of activating CB2 receptors. In this review we examine the current knowledge and understanding of the utility of cannabinoids as therapeutic molecules for inflammatory-mediated demyelinating pathologies. Moreover, we discuss how CB2 receptor activation is related to the modulation of immunopathogenic states.

El sistema cannabinoide en situaciones de neuroinflamación: perspectivas terapéuticas en la esclerosis múltiple / Cannabinoid system and neuroinflammation: therapeutic perspectives in multiple sclerosis

Introducción. El sistema endocannabinoide está constituidopor los receptores cannabinoides, los ligandos endógenos y loselementos enzimáticos implicados en su síntesis y degradación.Objetivo. Describir el estado actual de conocimiento sobre la funcióndel sistema como modulador de los procesos neuroinflamatoriosasociados con enfermedades crónicas como la esclerosis múltiple.Desarrollo. Los cannabinoides se sintetizan y se liberan en demanda y su producción aumenta en situaciones de neuroinflamacióny de daño neural. En este contexto, sus acciones en la microglía y enlos astrocitos se caracterizan por una disminución en la expresión demediadores inflamatorios y de citocinas proinflamatorias. Además,los cannabinoides pueden ejercer acciones neuroprotectoras a travésde diferentes tipos de mecanismos y en modelos experimentalesde esclerosis múltiple atenúan la sintomatología, disminuyen lainflamación y pueden favorecer la remielinización. Conclusiones. Eluso clínico de cannabinoides o agentes farmacológicos que incidenen el sistema endógeno cannabinoide durante la inflamación del sistemanervioso central y en la esclerosis múltiple está actualmentesometido a consideración y debate. El análisis detallado de los resultadosobtenidos en la última década ha permitido establecer que sonmúltiples los mecanismos de actuación de los cannabinoides en patologíasdel sistema nervioso central que cursan con inflamación crónicay ponen de manifiesto el interés del sistema cannabinoide comonueva herramienta terapéutica

Introduction. The endocannabinoid system consists of cannabinoid receptors, endogenous ligands and the enzymaticelements involved in their synthesis and breakdown. Aim. To report on currently held knowledge about the functioning of thesystem as a modulator of the neuroinflammatory processes associated with chronic diseases such as multiple sclerosis.Development. Cannabinoids are synthesised and released on demand and their production increases in times of neuroinflammationand neural damage. In this context then, their actions in the microglial cells and in the astrocytes arecharacterised by a lowered expression of inflammatory mediators and pro-inflammatory cytokines. Furthermore,cannabinoids can play a role as neuroprotectors by means of different types of mechanisms and, in experimental models ofmultiple sclerosis, they slow down the symptoms, reduce inflammation and can favour remyelination. Conclusions. Theclinical use of cannabinoids or pharmacological agents that affect the endogenous cannabinoid system during inflammationof the central nervous system and in multiple sclerosis is currently under consideration and subject to debate. Detailedanalysis of the results obtained over the past decade has made it possible to establish the existence of several mechanisms ofaction of cannabinoids in pathologies affecting the central nervous system that are accompanied by chronic inflammation.Likewise, they also clearly show that the cannabinoid system is an interesting proposal as a new therapeutic tool

[Cannabinoid system and neuroinflammation:therapeutic perspectives in multiple sclerosis]. / El sistema cannabinoide en situaciones de neuroinflamación: perspectivas terapéuticas en la esclerosis múltiple.

INTRODUCTION: The endocannabinoid system consists of cannabinoid receptors, endogenous ligands and the enzymatic elements involved in their synthesis and breakdown. AIM: To report on currently held knowledge about the functioning of the system as a modulator of the neuroinflammatory processes associated with chronic diseases such as multiple sclerosis. DEVELOPMENT: Cannabinoids are synthesised and released on demand and their production increases in times of neuroinflammation and neural damage. In this context then, their actions in the microglial cells and in the astrocytes are characterised by a lowered expression of inflammatory mediators and pro-inflammatory cytokines. Furthermore, cannabinoids can play a role as neuroprotectors by means of different types of mechanisms and, in experimental models of multiple sclerosis, they slow down the symptoms, reduce inflammation and can favour remyelination. CONCLUSIONS: The clinical use of cannabinoids or pharmacological agents that affect the endogenous cannabinoid system during inflammation of the central nervous system and in multiple sclerosis is currently under consideration and subject to debate. Detailed analysis of the results obtained over the past decade has made it possible to establish the existence of several mechanisms of action of cannabinoids in pathologies affecting the central nervous system that are accompanied by chronic inflammation. Likewise, they also clearly show that the cannabinoid system is an interesting proposal as a new therapeutic tool.

[Theiler's virus encephalomyelitis infection as a model for multiple sclerosis: cytokines and pathogenic mechanisms]. / Encefalomielitis por infección con el virus de Theiler como modelo experimental de esclerosis múltiple: citocinas y posibles mecanismos patogénicos.

Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral inoculation of TMEV, a member of picornavirus family, in susceptible animals. The pathogenesis of paralytic syndrome is associated with a chronic progressive demyelinating disease characterized by perivascular of immune inflammatory cells. Although TMEV induced demyelinating disease (TMEV IDD) is initiated by virus specific CD4+ T cells targeting CNS persistent virus, CD4+ T cell responses against self myelin epitopes activated via epitope spreading contribute to chronic disease pathogenesis. In the present report we delineated possible pathogenic mechanisms related with inflammatory process, leading to demyelination and axonal loss. The importance of proinflammatory cytokines in sustaining the inflammatory process and cause direct oligodendrotoxicity is emphasized. Different approaches in therapeutic strategies affecting cytokines are also presented.

Encefalomielitis por infección con el virus de Theilercomo modelo experimental de esclerosis múltiple:citocinas y posibles mecanismos patogénicos / Theiler's virus encephalomyelitis infection as a model for multiple sclerosis: cytokines and pathogenic mechanisms

La esclerosis múltiple (EM) es la enfermedad desmielinizante más frecuente en adultos jóvenes, y aunque su etiología es desconocida, estudios epidemiológicos apuntan a una infección vírica como posible causa de la enfermedad. La inoculación de cepas susceptibles de ratones con el virus de la encefalomielitis murina de Theiler (TMEV, del inglés Theiler's Murine Encephalomyelitis Virus) conduce al desarrollo de una enfermedad crónica desmielinizante caracterizada por lesiones multifocales en la sustancia blanca, con infiltrados inflamatorios similares a la patología observada en la EM en su fase crónica progresiva. Los oligodendrocitos o la mielina son diana de factores solubles citotóxicos liberados por linfocitos CD4+ del subtipo Th1, en respuesta a un fenómeno de hipersensibilidad retardada contra el virus, así como por la microglía y astroglía cerebral activada. En los estadios más avanzados de la infección, se produce una respuesta de inmunidad específica contra antígenos de la mielina, debido al fenómeno de la expansión de epítopos. En la presente revisión, tratamos de delinear los mecanismos patogénicos que conducen a la desmielinización y daño axonal en este modelo. Se destaca la importancia de las citocinas proinflamatorias para el mantenimiento del proceso inflamatorio, y se describen estrategias terapéuticas relacionadas con el sistema de citocinas pro y antiinflamatorias (AU)

Pharmacological and functional characterization of muscarinic receptor subtypes in developing oligodendrocytes.

This study focused on the molecular and pharmacological characterization of muscarinic acetylcholine receptors expressed by progenitors and differentiated oligodendrocytes. We also analyzed the role of muscarinic receptors in regulating downstream signal transduction pathways and the functional significance of receptor expression in oligodendrocytes. RT-PCR analysis revealed the expression of transcripts for M3, and to a lesser extent M4, followed by M1, M2 and M5 receptor subtypes in both progenitors and differentiated oligodendrocytes. Competition binding experiments using [(3)H]N-methylscopolamine and several antagonists, as well as inhibition of carbachol-mediated phosphoinositide hydrolysis, showed that M3 is the main subtype expressed in these cells. In progenitors the activation of p42/44-mitogen-activated protein kinase (MAPK) and cAMP-response element binding protein (CREB) as well as c-fos mRNA expression were blocked by the M3 relatively selective antagonist, 4-DAMP, and its irreversible analogue, 4-DAMP-mustard. Carbachol increased proliferation of progenitors, an effect prevented by atropine and 4-DAMP, as well as by the MAPK kinase inhibitor PD98059. These results indicate that carbachol modulates oligodendrocyte progenitor proliferation through M3 receptors, involving activation of a MAPK signaling pathway. Receptor density and phosphoinositide hydrolysis are down-regulated during oligodendrocyte differentiation. Functional consequences of these events are a reduction in carbachol-stimulated p42/44(MAPK) and CREB phosphorylation, as well as induction of c-fos.

Re-evaluation of nestin as a marker of oligodendrocyte lineage cells.

Maturation of oligodendrocyte progenitors (O2A) is characterized by morphological changes and the sequential expression of specific antigens leading to the formation of myelin membrane. Monoclonal antibodies A2B5, A007, anti-vimentin, and anti-galactocerebroside, recognize oligodendroglia at different stages of development. The neuroepithelial precursor marker nestin is also expressed by the oligodendroglial lineage; we have used enriched populations of progenitors isolated from neonatal rat brain cultures to further examine the cellular distribution of this intermediate filament protein. The phenotypic distribution of nestin positive cells among the oligodendrocyte lineage showed that 65% reacted with A2B5, whereas only 5% were A007(+), and 4% galactocerebroside(+). The remaining 25% of the cells were not labeled and had small cellular bodies devoid of processes, characteristic of the pre-O2A progenitor. Further analysis of the nestin(+) population showed that the majority of the cells were also vimentin(+). Antibody-dependent complement mediated cytolysis of A2B5(+) (O2A cells) and galactocerebroside(+) (mature oligodendrocytes) cells left a population of nestin(+) cells that were induced to proliferate in the presence of growth factors and to differentiate into A2B5(+) and galactocerebroside(+) cells. Proliferating cells maintained in the presence of platelet-derived growth factor or basic fibroblast growth factor retained nestin expression along with A2B5. By contrast, in serum-free medium nestin expression decreased while postmitotic cells acquired A007 and galactocerebroside. Our results suggest that nestin expression is a marker of pre-O2A cells that is maintained in proliferating glial progenitors, but is quickly down-regulated in postmitotic oligodendrocytes (A007(+)/galacto-cerebroside(+)) along with A2B5 and vimentin. However, other glial cells including type 2 astrocytes and some amoeboid microglia also share nestin expression.

LPS/IFN-gamma cytotoxicity in oligodendroglial cells: role of nitric oxide and protection by the anti-inflammatory cytokine IL-10.

Proinflammatory mediators have been implicated in demyelinating disorders, including multiple sclerosis, whereas it has been proposed that the anti-inflammatory cytokines interleukin- (IL-) 4 and IL-10 participate in disease recovery. The present study analysed the effect of interferon-gamma (IFN-gamma) and bacterial endotoxin (lipopolysaccharide, LPS) on proliferation and survival of progenitors and differentiated oligodendrocytes. We also investigated the presence of receptors for IL-4 and IL-10 in oligodendroglial cells and explored a possible protective action of IL-4 and IL-10 in cultures following LPS/IFN-gamma. Finally, the role of endogenous nitric oxide (NO) on cell viability and the modulatory action of IL-4 and IL-10 on inducible nitric oxide synthase (iNOS) expression were also analysed. We report that LPS and/or IFN-gamma reduced proliferation and viability of oligodendroglial cells. Cell death, presumably by apoptosis as evidence by TUNEL and Annexin V binding, was observed following LPS/IFN-gamma, progenitors being more sensitive than differentiated cells. At both developmental stages, LPS/IFN-gamma-treated cultures expressed iNOS protein and released micromolar concentrations of NO. In progenitors, LPS/IFN-gamma-mediated cell damage was partially dependent on endogenous NO production, whereas NO was fundamental for cytotoxicity of differentiated oligodendrocytes. Both cell types expressed mRNA for IL-4 and IL-10 receptors and expression of IL-10 receptors at the protein level was also demonstrated. Treatment with either cytokine inhibited the expression of iNOS resulting from the proinflammatory stimulation. IL-10 was more effective than IL-4 in suppressing iNOS expression and, interestingly, IL-10 conferred protection against oligodendroglial death evoked by LPS/IFN-gamma. Our data raise the question of whether IL-10 may play a protective role in demyelinating diseases, not only downregulating the function of inflammatory cells but also promoting survival of progenitors and differentiated oligodendrocytes.

Induction of COX-2 and PGE(2) biosynthesis by IL-1beta is mediated by PKC and mitogen-activated protein kinases in murine astrocytes.

Interleukin-1 (IL-1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E(2) (PGE(2)) production after IL-1beta stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL-1beta or the phorbol ester, PMA significantly increased PGE(2) secretion. The stimulatory action of IL-1beta on PGE(2) production was totally abolished by NS-398, a specific inhibitor of cyclo-oxygenase-2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL-1beta induced the expression of COX-2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL-1 beta treatment also induced the expression of COX-2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H-7, bisindolylmaleimide and calphostin C) inhibited IL-1beta stimulation of PGE(2). In addition, PKC-depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL-1. The ablation of the effects of PMA and IL-1beta on PGE(2) production, likely results from down-regulation of phorbol ester sensitive-PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC-alpha from cytosol to membrane following treatment with IL-1beta. In addition, IL-1beta treatment led to activation of extracellular signal-regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL-1beta-induced PGE(2) release. ERK1/2 activation by IL-1beta was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE(2), likely by regulating the induction of cyclo-oxygenase-2, in IL-1beta-stimulated astroglial cells.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage.

Effects of cannabinoids on the immune system and central nervous system: therapeutic implications.

This review aims to improve understanding of the modulatory effects that cannabinoids exert on the immune system and CNS. Cannabinoids possess immunomodulatory activity, are neuroprotective in vivo and in vitro and can modify the production of inflammatory mediators, such as nitric oxide, prostanoids and cytokines, that are expressed by, and act on, the immune system and the brain. The mechanisms of cannabinoid actions are not fully understood, but appear to involve complex interactions between cannabinoid receptors and a number of signal transduction pathways. Endogenous cannabinoid ligands appear to act as local modulators of immune/inflammatory reactions. Cannabinoid-induced immunosuppression may have implications for the treatment of neurological disorders that are associated with excess immunological activity, such as multiple sclerosis and Alzheimer's disease. There is anecdotal evidence that cannabis use improves the symptoms of multiple sclerosis, and studies with animal models are beginning to provide evidence for the mechanism of such effects. The development of nonpsychotropic cannabinoid analogues and modulators of the metabolism of endogenous cannabinoid ligands may lead to novel approaches to the treatment of neurodegenerative disorders.

The endogenous cannabinoid anandamide potentiates interleukin-6 production by astrocytes infected with Theiler's murine encephalomyelitis virus by a receptor-mediated pathway.

Theiler's murine encephalomyelitis virus (TMEV) infection of a susceptible strain of mice results in virus persistence in the brain and chronic primary immune-mediated demyelination, which resembles multiple sclerosis. Recent attention has focused on the anti-inflammatory and immunosuppressive properties of interleukin-6, a pleiotropic cytokine involved in the regulation of immunological responses, acute phase protein production and hematopoiesis. Anandamide (arachidonoyl ethanolamine) is a natural brain constituent that binds a specific brain cannabinoid receptor. In this study we investigated whether anandamide can modify interleukin-6 production by primary cultures of murine brain cortical astrocytes infected with TMEV. Astrocytes from susceptible (SJL/J) and resistant (BALB/c) strains of mice infected with TMEV (10(5)PFU/well) increased IL-6 release over a period of 24 h. Anandamide caused an enhancement of the release of IL-6 by TMEV-infected astrocytes in a concentration-dependent manner (1-25 microM). Treatment of TMEV-infected astrocytes with 10 microM arachidonyl trifluoromethyl ketone, a potent inhibitor of the amidase that degrades anandamide, was found to potentiate the effects of anandamide on IL-6 release. A novel and selective cannabinoid receptor antagonist, SR 141617A, blocked the enhancing effects of anandamide on IL-6 release by TMEV-infected astrocytes, suggesting a cannabinoid receptor-mediated pathway. The physiological implications of these results are unknown, but may be related to the hypothesis of the protective effects of cannabinoids on neurological disorders like multiple sclerosis.

Glutamate-stimulated production of inositol phosphates is mediated by Ca2+ influx in oligodendrocyte progenitors.

The effect of glutamate on the accumulation of [3H]inositol phosphates was examined in oligodendrocyte progenitor cultures prepared from rat brains. Glutamate, and the analogues alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate, caused a concentration- and time-dependent increase in [3H]inositol trisphosphate (IP3) formation and the effect was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a competitive AMPA and kainate receptor antagonist. Similarly, the more selective, noncompetitive antagonist of AMPA receptors, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), significantly reduced the effect of both AMPA and kainate. In contrast, antagonists of N-methyl-D-aspartate (NMDA) receptor, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5, 10-imine (MK-801) and R(-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and antagonists of metabotropic receptors, L(+)-2-amino-3-phosphono-propanoic acid (L-AP3) and alpha-methyl-4-carboxyphenylglycine (MCPG), were ineffective. These results suggest that the effect of glutamate on [3H]IP3 accumulation is mediated through ionotropic AMPA receptors. Cyclothiazide, an inhibitor of AMPA receptor desensitization, strongly potentiated the AMPA and kainate-stimulated [3H]IP3 formation as well as the uptake of 45Ca2+ in line with the previous findings. 45Ca2+ uptake evoked by AMPA or kainate, in combination with cyclothiazide, was also prevented by both CNQX and GYKI 52466. Glutamate-stimulated [3H]IP3 accumulation was prevented by EGTA, suggesting a requirement for extracellular calcium. Pre-incubation with the voltage-gated Ca2+ channel blockers, diltiazem, nifedipine and CdCl2, partially prevented the glutamate-induced [3H]IP3 accumulation as well as 45Ca2+ uptake. Similarly, the Na+/Ca2+ exchanger blockers benzamil and 3,4-dichlorobenzamil reduced significantly kainate-stimulated 45Ca2+ uptake. These data indicate that glutamate-induced [3H]IP3 accumulation is triggered by calcium influx via AMPA receptors, voltage-gated calcium channels and the Na+/Ca2+ exchanger operating in reverse mode.

Carbachol stimulates c-fos expression and proliferation in oligodendrocyte progenitors.

To determine if muscarinic receptor-activation plays a role in oligodendrocyte development, the effect of carbachol a stable acetylcholine analog, on gene expression and proliferation was investigated. Using Northern blot analysis we showed that carbachol caused a time and concentration-dependent increase in c-fos mRNA. This effect was blocked by atropine, a non-selective muscarinic antagonist. In addition, the muscarinic-stimulated c-fos increase was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), but not by N-2-(p-bromocinnamylamino)-ethyl-5-isoquinoline-sulfonamide (H-89), a potent inhibitor of protein kinase A, suggesting the involvement of PKC in mediating the response. Down-regulation of PKC by overnight pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) blocked only the phorbol ester-stimulated c-fos accumulation while no effect was observed in the carbachol-induced response. These results suggested that carbachol stimulated an H-7 sensitive PKC pathway which may be different than that activated by TPA. Further evidence for two separate mechanisms of proto-oncogene induction was provided by the additive effect of carbachol and TPA. Induction of c-fos mRNA by carbachol was dependent on both influx of extracellular Ca2+ and release from intracellular stores, as both EDTA and BAPTA blocked the response. Since activation of muscarinic receptors can affect cell division in other cellular systems, the effect of carbachol on [3H]thymidine and bromodeoxyuridine incorporation into oligodendrocyte DNA was measured. Carbachol stimulated DNA synthesis in oligodendrocyte progenitors. This effect was mediated by muscarinic receptors as [3H]thymidine incorporation was prevented or significantly reduced by the addition of atropine. In conclusion, the present findings suggest that, the neurotransmitter, acetylcholine may act as a trophic factor in developing oligodendrocytes, regulating their growth and development in the central nervous system.

Changes in extracellular levels of dopamine metabolites in somatosensory cortex after peripheral denervation.

This study examined the effects of a nerve transection on monoamine release from primary somatosensory cortex. The technique of microdialysis was employed to sample extracellular levels of norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the barrel field of freely moving rats following the surgical transection of the contralateral infraorbital nerve. Microdialysates obtained 3, 4, and 5 days after deafferentation were analyzed using high-performance liquid chromatography with electrochemical detection. We found a significant increase in the release of the dopamine metabolites, DOPAC and HVA from the deafferented cortex. Three days after deafferentation the release of DOPAC was three-fold higher in the deafferented than in the control animals, and remained about 100% higher in the next two days in this group of animals. The release of HVA showed a gradual increase following the deafferentation procedure, since a 92% larger value on day 3 increased to a 338% difference on day 5. On the other hand, the release rate of NE and the levels of the serotonin metabolite 5-HIAA were not significantly affected by the deafferentation procedure. These results are discussed in the context of the possible participation of dopamine in the reorganization of the deafferented somatosensory cortex.

Dopamine receptor alterations correlate with increased GABA levels in adult rat neostriatum: effects of a neonatal 6-hydroxydopamine denervation.

The effects of neonatal intracerebroventricular 6-hydroxydopamine (6-OHDA) injection on the densities of dopamine (DA) receptors and GABA levels were determined in the rostral neostriatum of adult rats. Measurement of GABA turnover indicated that increased tissue GABA in the DA-lesioned neostriatum is a consequence of higher GABA synthesis rate (205%). Binding experiments with [3H]SCH23390 (D1 receptors) and [3H]raclopride (D2 receptors) point to a correlation between tissue GABA content and altered DA receptors. Three months after the lesion there was a 27% decrease in D1 receptors and a 22% increase in D2 receptors. In control neostriatum, GABA levels were inversely related to D2 receptors and this relationship was reversed after 6-OHDA treatment. In contrast, the positive correlation between GABA and D1 receptors remained unchanged after the lesion. Irreversible blockade of DA receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) decreased both D1 and D2 sites (73-87%) in both control and lesioned neostriatum, but increased GABA levels by 25% only in animals which have received 6-OHDA just after birth. Following acute inhibition of DA synthesis or of DA catabolism, GABA levels remained unchanged. The present results indicated that DA depletion by itself is not the cause for the increase in GABA levels. The augmented GABAergic activity following neonatal 6-OHDA is seemingly influenced primarily by DA receptor status; presumably, changes in D2 receptor properties during maturation may be a principal cause for an increase in neostriatal GABA content.