Esclerodermia localizada en Coup de Sabre: a propósito de un caso / Localized Scleroderma en Coup de Sabre: a case report

INTRODUCCIÓN: La esclerodermia localizada o morfea corresponde a una patología idiopática autoinmune que produce cambios escleróticos subcutáneos, que presenta diferencias con respecto a la esclerosis sistémica o esclerodermia. Un tipo de morfea lineal es la morfea "En Coup de Sabre" que consiste en la contracción y rigidez de la piel que culmina con una depresión de parte de la mitad del rostro, que puede asociarse a síntomas oftalmológicos y neurológicos. Aquí se describe un caso en un hombre joven con este tipo de morfea lineal. PRESENTACIÓN DEL CASO: Hombre de 23 años presenta lesión cutánea de morfología triangular en región frontal izquierda, por lo que decide consultar a dermatología, dónde se maneja con corticoides tópicos. Dos años después, la lesión sigue creciendo y se asocia a cefalea occipital, sin otros síntomas sistémicos. Se decide estudiar con biopsia, ecografía de cuero cabelludo y resonancia nuclear magnética (RNM) cerebral con gadolinio. Se diagnostica morfea en coup de sabre e indica tratamiento inmunosupresor. DISCUSIÓN: Dado que la Morfea en Coup de Sabre es una patología que compromete el rostro, es relevante realizar una derivación al oftalmólogo para evaluación de compromiso ocular y realizar una RNM para evaluación neurológica, en este caso ambos estudios resultaron negativos. El estudio serológico no es siempre necesario y debemos ser cautelosos en el uso de esta herramienta. Cuando existen dudas diagnósticas, se puede recurrir a una biopsia del tejido comprometido, la que debe incluir grasa subcutánea. La biopsia también ayuda para ver el grado de compromiso cutáneo que presenta el paciente. Con respecto al manejo, los corticoides tópicos son la elección para el manejo de lesiones agudas. El Metotrexato ha demostrado ser útil en lesiones agudas y profundas, asociado o no a corticoides.

INTRODUCTION: Localized scleroderma or morphea is an idiopathic autoimmune disorder that causes subcutaneous sclerotic changes and is different from systemic sclerosis or scleroderma. The morphea in "coup de Sabre" is a subtype of linear morphea that usually involves the forehead and scalp causing contraction and stiffness of the skin that culminates in a depression and that may be associated with ocular and neurological symptoms. We present a case of a young male patient with morphea in coup de sabre. CASE PRESENTATION: A 23 years old male patient presents with a skin lesion of triangular morphology in the left-frontal region. He was initially treated with topical corticosteroids, but had persistent growing of the skin lesion associated with new onset occipital headache. Ultrasound of the lesion as well as skin biopsy were performed confirming morphea in coup de sabre. Brain magnetic resonance imaging with gadolinium was normal. Inmunosuppresive tratment was started. DISCUSSION: Morphea in Coup de sabre is an rare disease. It is more frequent in women and children. Because it involves the deep tissues of the face and forehead, it is relevant to rule out any ocular or neurological involvement. The serological study is usually not necessary and results are of uncertain interpretation. When the diagnosis is unclear, a biopsy of the compromised tissue may help to identify inflammation and/or atrophy and to evaluate the degree of activity of the lesion. Ultrasound is also an useful tool for evaluation of the activity of the skin lesion, comparable to biopsy. Regarding treatment, topical corticosteroids are the first line therapy for acute lesions. Methotrexate has proven to be useful in deeper active lesions, with or without corticosteroids. Finally, there is an important asociation between this type of lineal morphea and progressive hemifacial atrophy (Parry Romberg syndome), which may involve the brain and needs to be referred to the specialist as soon as possible.

ß-Adrenergic Receptors/Epac Signaling Increases the Size of the Readily Releasable Pool of Synaptic Vesicles Required for Parallel Fiber LTP.

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca2+-induced increase in presynaptic cAMP that is mediated by Ca2+-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by ß-adrenergic receptors (ßARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that ßARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these ßARs occluded PF-PC LTP, while the ß1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2-/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the ßAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP.SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to ß-adrenergic receptor (ßAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by ßARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this ßAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of ßARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.

Predictors of Indwelling Pleural Catheter Removal and Infection: A Single-center Experience With 336 Procedures.

BACKGROUND: Indwelling pleural catheters (IPCs) offer ambulatory management of symptomatic persistent pleural effusions, but their widespread use is somewhat hampered by the risk of pleural infection and the inconvenience of carrying a catheter for a prolonged period of time. Factors associated with these 2 limitations were analyzed in this study. METHODS: Retrospective review of consecutive patients who had undergone IPC placement over a 5 ½-year period. Time to IPC removal was analyzed with the Fine and Gray competing risks survival model, with competing risk being death. A binary logistic regression method was used to evaluate factors influencing IPC-related pleural infections. RESULTS: A total of 336 IPCs were placed in 308 patients, mostly because of malignant effusions (83%). IPC removal secondary to pleurodesis was achieved in 170 (51%) procedures at a median time of 52 days. Higher rates of IPC removal were associated with an Eastern Cooperative Oncology Group (ECOG) grade of 0 to 2 [subhazard ratio (SHR)=2.22], an expandable lung (SHR=1.93), and development of a multiseptated pleural space (SHR=1.37). IPC-related pleural infections occurred in 8% of the cases, and were more often seen in hepatic hydrothoraces [odds ratio (OR)=4.75] and pleural fluids with a C-reactive protein <15 mg/L before the IPC insertion (OR=4.42). CONCLUSION: IPC removal is more likely to occur in patients with good performance status whose lungs fully expand after drainage. Hepatic hydrothorax is the most significant predictor of IPC-related infections.

Análisis de lesionología de una muestra de 353 autopsias de suicidios, Departamento de Medicina Legal, Costa Rica del 2010 al 2016 / Lesionology analysis of a sample of 353 suicide autopsies, Department of Legal Medicine, Costa Rica from 2010 to 2016

Resumen El presente trabajo corresponde a una investigación de la lesionología y antecedentes personales de las personas que han muerto de manera suicida en Costa Rica de 2010 a 2016. De los 2174 autopsias de suicidios que se realizaron en la Sección de Patología Forense del Departamento de Medicina Legal del Organismo de Investigación Judicial de Costa Rica, tomando como fuente de información la base de datos del Sistema de Automatización de Patología Forense del Departamento de Medicina Legal, se extrajo una muestra de 353 y a estos se les investigó información adicional en expediente y otros registros de poder judicial para obtener información relacionada con antecedentes patológicos personales, antecedentes sociales y mayores detalles del suicidio. Además, según el tipo de lesión que produjo la muerte, se caracteriza el tipo de asfixia, la localización del nudo, el lugar de ingreso del proyectil de arma de fuego o el tipo de sustancia usada para el suicidio.

Abstract The present work corresponds to research of the lesionology and personal antecedents of people that have died in a suicidal way in Costa Rica from 2010 to 2016. From the 2174 autopsies of suicides that were realized in the Section of Forensic Pathology of the Department of Legal Medicine of the Judicial Investigation Organization of Costa Rica, taking as a source of information the database of the Forensic Pathology Automation System of the Department of Legal Medicine, a sample of 353 was extracted and additional information was researched in the forensic file and other records of judicial system to obtain information related to personal pathological background, social background and more details of suicide. In addition, depending on the type of injury that caused the death, the type of asphyxia, the location of the knot, the place of entry of the firearm projectile or the type of substance used for suicide are characterized.

Suicidio en Costa Rica: Análisis de autopsias realizadas en el Departamento de Medicina Legal del 2010 al 2016 / Suicide in Costa Rica: Autopsy analysis performed in the Department of Legal Medicine from 2010 to 2016

Resumen El suicidio es un problema importante de salud pública. La presente investigación corresponde a un análisis estadístico descriptivo de los aspectos sociodemográficos de las personas que fallecen de manera suicida en Costa Rica durante el períodos de 2010 a 2016. Se toman los casos del Sistema de Automatización de Patología Forense del Departamento de Medicina Legal. Se hace un agrupamiento de datos según lugar de ocurrencia, edades, causa de muerte y ocupación. Se realiza la estadística descriptiva, se hace cruces de variables y se analizan las proporciones obtenidas mediante pruebas de Chi cuadrado de Pearson y Pruebas de Z con corrección de Bonferroni. Hubo 2174 casos, distribuidos usualmente en más de 300 casos por año. El suicidio es más frecuente en hombres, ente 19 y 44 años, solteros, no profesionales y la causa de muerte más común es asfixia por ahorcadura, luego intoxicación por agroquímicos y finalmente herida por arma de fuego.

Abstract Suicide is a major public health problem. The present investigation corresponds to a descriptive statistical analysis of the sociodemographic aspects of the people who died in a suicidal way in Costa Rica from 2010 to 2016. The cases were taken from the Forensic Pathology Automation System of the Department of Legal Medicine. A grouping of data is made according to place of occurrence, age, cause of death and occupation. Descriptive statistics are carried out, cross-data analysis are executed and the proportions obtained are analyzed by means of Pearson´s Chi-square test and Z-test with Bonferroni correction. There were 2174 cases, usually distributed in more than 300 cases per year. Suicide is more frequent in men, between 19 and 44 years of age, single, non-professional and the most common cause of death is asphyxia due to hanging, then intoxication by agrochemicals and finally wounding by firearm projectile.

Síndrome de Capgras: una revision de la literatura / Capgras syndrome: a review of the literature

Los síndromes de falsa identificación delirante (DMS, por su sigla en inglés) son trastornos neuropsiquiátricos poco frecuentes que se caracterizan por tener ideas delirantes respecto a la propia identidad y la de otras personas, animales o lugares conocidos por el paciente. Los principales DMS son el Síndrome de Capgras (SC), el de Fregoli, el de doble subjetivo y el de intermetamorfosis. Se presentan en contexto tanto de enfermedades psiquiátricas como en cuadros orgánicos. Distintos modelos han tratado de encontrar una explicación a los DMS, con aportes tanto desde la psicología como de las neurociencias. Entre las causas están enfermedades neurodegenerativas, cuadros psiquiátricos, alteraciones estructurales, efecto de drogas, y alteraciones metabólicas. El manejo depende de la patología de base y las características clínicas específicas. Esta revisión se centra específicamente en el SC, ya que dentro de los DMS, es el más frecuente y estudiado

Delusional misidentification syndromes (DMS) are rare neuropsychiatric disorders that are characterized by having delirious ideas regarding one's and other people, animals or places identity known by the patient. The main DMS are the Capgras syndrome, the Fregoli syndrome, the subjective double syndrome and the intermetamorphosis syndrome. They appear in context of both psychiatric illnesses and organic disorders. Different models have tried to find an explanation to the DMS, with contributions from both psychology and neurosciences. Among the causes are neurodegenerative diseases, psychiatric symptoms, structural alterations, drug effects, and metabolic alterations. Management depends on the underlying pathology and the specific clinical characteristics. This review focuses specifically on the SC, since within the DMS, it is the most frequent and studied.

CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.

Intoxicación alcohólica / Alcohol intoxication

Resumen:La intoxicación se considera como una manifestación patológica definida por los signos y síntomas que secundarios a la acción de una o varias dosis de un agente tóxico y su evolución puede llevar al sujeto a un estado irreversible e incluso a la muerte. Cada año mueren alrededor de un millón de personas a consecuencia de diversos envenenamientos. La intoxicación alcohólica es causada por alcoholes, compuestos orgánicos que se derivan de los hidrocarburos y están formados por grupos hidroxilos. El etanol es el alcohol que con más frecuencia produce intoxicaciones ya que es el más común y el que más al alcance de la población se encuentra, este produce múltiples alteraciones a nivel del sistema nervioso y de otros sistemas del organismo.

Abstract:Intoxication is considered a pathological manifestation defined by the signs and symptoms secondary to the action of one or more doses of a toxic agent and its evolution may lead to an irreversible subject to state and even to death.Every year about a million people as a result of various poisonings. Alcohol intoxication is caused by alcohols, organic compounds derived hydrocarbons and consist of hydroxyl groups. Ethanol is the alcohol intoxication occurs more frequently because it is the most common and the most accessible to the population is, this results in multiple abnormalities in the nervous system and other body systems.

Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals.

The co-existence of presynaptic G protein coupled receptors, GPCRs, has received little attention, despite the fact that interplay between the signaling pathways activated by such receptors may affect the neurotransmitter release. Using immunocytochemistry and immuhistochemistry we show that mGlu7 and ß-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. mGlu7 receptors readily couple to pathways that inhibit glutamate release. We found that when mGlu7 receptors are also coupled to pathways that enhance glutamate release by prolonged exposure to agonist, and ß-adrenergic receptors are also activated, a cross-talk between their signaling pathways occurs that affect the overall release response. This interaction is the result of mGlu7 receptors inhibiting the adenylyl cyclase activated by ß adrenergic receptors. Thus, blocking Gi/o proteins with pertussis toxin provokes a further increase in release after receptor co-activation which is also observed after activating ß-adrenergic receptor signaling pathways downstream of adenylyl cyclase with the cAMP analog Sp8Br or 8pCPT-2-OMe-cAMP (a specific activator of the guanine nucleotide exchange protein directly activated by cAMP, EPAC). Co-activation of mGlu7 and ß-adrenergic receptors also enhances PLC-dependent accumulation of IP1 and the translocation of the active zone protein Munc13-1 to the membrane, indicating that release potentiation by these receptors involves the modulation of the release machinery.

Altered neuronal and endothelial nitric oxide synthase expression in the bladder and urethra of cyclophosphamide-treated rats.

Increased nitric oxide (NO) production seems to play a key role in cyclophosphamide (CYP)-induced cystitis, although the underlying mechanisms and the relative involvement of the different NO synthase (NOS) isoforms remain to be elucidated. Moreover, the role of the urethra in this process is also unclear. In this study, we have analyzed the changes in the expression and distribution of the inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms of NOS, and the alterations in nerve-mediated contractility in the bladder and urethra of CYP-treated rats. Accordingly, Wistar rats were treated with 150 mg kg(-1) CYP for 4 (acute treatment) or 48 h (intermediate treatment), or with 70 mg kg(-1) CYP every 3 days for 10 days (chronic treatment), and the changes in protein expression were assessed by immunohistofluorescence and in Western blots, while mRNA expression was assessed by conventional and quantitative PCR. Similarly, nerve-mediated contractility was analyzed in vitro. Unexpectedly, no iNOS expression was detected in CYP-treated animals, while a transient downregulation of nNOS expression and a progressive upregulation of eNOS was observed, although the eNOS accumulated was not in the active phosphorylated form. Qualitative changes in mRNA expression were also observed in the bladder and urethra, although contractility only diminished in the bladder and this change was not dependent on NOS activity. These findings suggest that spatiotemporal alterations in NO production by constitutive NOS may be involved in the pathogenicity of CYP. Further studies will be necessary to understand the contribution of eNOS to the increases in NO associated with bladder inflammation, or that of free radicals.

Cannabinoid type 1 receptors transiently silence glutamatergic nerve terminals of cultured cerebellar granule cells.

Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals.

ß-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1α interaction to potentiate glutamate release at cerebrocortical nerve terminals.

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the ß-adrenergic receptor (ßAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of ßARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that ßARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.

Cannabinoid type 2 receptor activation downregulates stroke-induced classic and alternative brain macrophage/microglial activation concomitant to neuroprotection.

BACKGROUND AND PURPOSE: Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation. METHODS: Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide (MIP) -1α, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor ß (TGF-ß), arginase I, and Ym1. RESULTS: Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion-induced gene expression of both classic (IL-6, TNF-α, MCP-1, MIP-1α, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-ß, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion. CONCLUSIONS: The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.

The metabotropic glutamate receptor mGlu7 activates phospholipase C, translocates munc-13-1 protein, and potentiates glutamate release at cerebrocortical nerve terminals.

At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca(2+) influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca(2+) ionophore ionomycin, suggesting a mechanism that is independent of Ca(2+) channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function.

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors.

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca2+ channels and adenylyl cyclase. Through Ca2+ imaging and immunocytochemistry, we have recently shown that adenosine A1, GABAB and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca2+ channels by analysing this process in nerve terminals from mice lacking the alpha1B subunit (Cav 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca2+ channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABAB and adenosine A1. Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A1, GABAB and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca2+ channel type but rather, it is associated to functional mGlu7 receptors.

The coexistence of multiple receptors in a single nerve terminal provides evidence for pre-synaptic integration.

Excitatory synaptic transmission is inhibited by G protein coupled receptors, including the adenosine A(1), GABA(B), and metabotropic glutamate receptor 7. These receptors are present in nerve terminals where they reduce the release of glutamate through activating signaling pathways negatively coupled to Ca(2+) channels and adenylyl cyclase. However, it is not clear whether these receptors operate in distinct subpopulations of nerve terminals or if they are co-expressed in the same nerve terminals, despite the functional consequences that such distributions may have on synaptic transmission. Applying Ca(2+) imaging and immunocytochemistry, we show that these three G protein coupled receptors coexist in a subpopulation of cerebrocortical nerve terminals. The three receptors share an intracellular signaling pathway through which their inhibitory responses are integrated and coactivation of these receptors produced an integrated response. Indeed, this response was highly variable, from a synergistic response at subthreshold agonist concentrations to an occluded response at high agonist concentrations. The presence of multiple receptors in a nerve terminal could be responsible for the physiological effects of neurotransmitter spillover from neighboring synapses or alternatively, the co-release of transmitters by the same nerve terminal.

mGluR7 inhibits glutamate release through a PKC-independent decrease in the activity of P/Q-type Ca2+ channels and by diminishing cAMP in hippocampal nerve terminals.

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in hippocampal nerve terminals from adult rats, the inhibition of glutamate release by the group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) is largely mediated by mGluR7. In this preparation, P/Q-type Ca(2+) channels support the major component of glutamate release while the remaining release is supported by N-type Ca(2+) channels. The release associated with P/Q channels was modulated by mGluR7, either in the presence of omega-conotoxin-GVIA or after decreasing the extracellular Ca(2+) concentration [Ca(2+)](o) to abolish the contribution of N-type Ca(2+) channels. Under these conditions, L-AP4 (1 mm) reduced the evoked glutamate release by 35 +/- 2%. This inhibition was largely prevented by pertussis toxin, but it was insensitive to inhibitors of protein kinase C (bisindolylmaleimide) and protein kinase A (H-89). Furthermore, this inhibition was associated with a reduction in the Ca(2+) influx mediated by P/Q channels in the absence of any detectable change in cAMP levels. However, L-AP4 decreased the levels of cAMP in the presence of forskolin. The activation of this additional signalling pathway was very efficient in counteracting the facilitation of glutamate release induced by forskolin. Thus, mGluR7 mediates the inhibition of glutamate release at hippocampal nerve terminals primarily by inhibiting P/Q-type Ca(2+) channels, although augmenting the levels of cAMP reveals the ability of the receptor to decrease cAMP.

NMDA induces post-transcriptional regulation of alpha2-guanylyl-cyclase-subunit expression in cerebellar granule cells.

Activation of N-methyl-D-aspartate (NMDA) glutamate receptors commonly affects gene expression in different neurons. We reported previously that chronic treatment of rat cerebellar granule cells with NMDA (24 hours) upregulates the expression of mRNA encoding the alpha2 subunit of the nitric-oxide-sensitive guanylyl cyclase. However, the molecular mechanisms involved in this process remained to be elucidated. Here, we have performed mRNA-decay experiments using the transcriptional inhibitor actinomycin D, providing evidence that the half-life of alpha2 mRNA is significantly prolonged in cells exposed to NMDA. The role of the 3' untranslated region of the alpha2 transcripts in NMDA-induced mRNA stabilisation was examined and an association between the RNA-binding proteins AUF1 and ELAV-like protein 1 (HuR/HuA), and endogenous alpha2 mRNA was demonstrated in vivo, as revealed by coimmunoprecipitation experiments with specific antibodies against AUF1 and HuR. Further studies indicated that stimulation of the NMDA receptor induces a downregulation in AUF1 levels stabilising the alpha2 mRNA transcripts. These events are triggered through a mechanism that depends on formation of nitric oxide, and on the subsequent activation of guanylyl cyclase and cGMP dependent protein kinases.

The modulation of Ca2+ and K+ channels but not changes in cAMP signaling contribute to the inhibition of glutamate release by cannabinoid receptors in cerebrocortical nerve terminals.

While cannabinoid receptors activate multiple signaling pathways in the brain, it remains unclear what influence the inhibition of adenylylcyclase has on the inhibition of glutamate release. In cerebrocortical nerve terminals, the cannabinoid receptor agonist WIN55,212-2 reduced KCl-evoked glutamate release through a mechanism that restricted the rise of cytoplasmic free Ca2+, but not the changes in plasma membrane depolarization. These effects were consistent with the inhibition of Ca2+ channels. Furthermore, WIN55,212-2 reduced 4-aminopyridine (4AP) evoked glutamate release to a larger extent by modulating the behavior of both Ca2+ and K(+)-channels. The inhibition of 4AP-evoked release was associated with a decrease in cytoplasmic free Ca2+ and in plasma membrane depolarization that was reverted by the potassium channel blocker, tetraethylammonium. Interestingly, the reduction of KCl- and 4AP-evoked release by WIN55,212-2 was independent of adenylylcyclase activity and did not affect cAMP. Forskolin and the beta-adrenergic receptor increase intrasynaptosomal cAMP and promote a PKA-dependent tetrodotoxin (TTX)-sensitive increase in the spontaneous release of glutamate. These two responses were reduced by WIN55,212-2. However, the glutamate release induced by Sp-8-Br-cAMPS, which directly activated PKA without affecting cAMP, was also similarly reduced by WIN55,212-2. Hence, we conclude that the inhibition of glutamate release by WIN55,212-2 is unrelated to changes in cAMP and that the inhibition of release that a decrease in cAMP might produce is occluded by the activation of additional pathways such as the inhibition of Ca2+ channels and/or the activation of K(+)-channels that strongly depress glutamate release.

Co-activation of PKA and PKC in cerebrocortical nerve terminals synergistically facilitates glutamate release.

Protein kinase A and protein kinase C are involved in processes that enhance glutamate release at glutamatergic nerve terminals. However, it is not known whether these two kinases co-exist within the same nerve terminal, nor is it clear what impact their simultaneous activation may have on neurotransmitter release. In cerebrocortical nerve terminals, co-application of forskolin, which increases cAMP levels and activates protein kinase A, and 4beta-phorbol dibutyrate, a direct activator of protein kinase C, synergistically enhanced the spontaneous release of glutamate. This enhancement exhibited both tetrodotoxin-sensitive and tetrodotoxin-resistant components. Interestingly, the tetrodotoxin-resistant component of release was not observed when cyclic AMP-dependent protein kinase (PKA) and calcium- and phospholipid-dependent protein kinase (PKC) were activated separately, but developed slowly after the co-activation of the two kinases, accounting for 50% of the facilitated release. This release component was dependent on voltage-dependent Ca2+ channels that opened spontaneously after PKA and PKC activation and occurred in the absence of Na+ channel firing. These data provide functional evidence for the co-existence of PKA- and PKC-signalling pathways in a subpopulation of glutamatergic nerve terminals.