RESUMEN

Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.

Asunto(s)

Transporte Axonal , Técnicas de Transferencia de Gen , Lentivirus/genética , Virus de la Rabia/genética , Médula Espinal/citología , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Células Cultivadas , Vectores Genéticos/genética , Glicerol Quinasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Médula Espinal/metabolismo , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Genetic deletion of the plasma membrane calcium ATPase type 2 (PMCA2), a calcium transporter protein, is associated with an overtly ataxic phenotype in mice. PMCA2 is expressed at high levels in cerebellar Purkinje neurons (PNs) where functional integrity is essential for normal cerebellar function. Indeed, loss of PN function accompanies cerebellar ataxia in humans and mouse models. In the ataxic PMCA2 knockout (PMCA2-/-) mouse the ability of the PNs to control their cytosolic calcium levels was severely impaired; basal calcium levels were high and calcium recovery kinetics slow. Whole cell patch clamp recordings from PMCA2-/- PNs revealed that they possessed hyperpolarised membrane potentials, reduced frequency and increased irregularity of spontaneous action potential firing, curtailed complex spikes and sustained calcium-dependent outward K+ currents. We propose that these alterations limit pathological excursions in PN cytosolic calcium as an aid to survival but that they are insufficient to prevent loss of functional cerebellar output.

Asunto(s)

Cerebelo/citología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/fisiología , Células de Purkinje/fisiología , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Biofisica , Calcio/metabolismo , Estimulación Eléctrica , Humanos , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp/métodos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/deficiencia , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética

RESUMEN

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.

Asunto(s)

Cerebelo/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Sinapsis/metabolismo , Animales , Western Blotting , Homólogo 4 de la Proteína Discs Large , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , Proteómica , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Sinaptosomas/metabolismo , Sintaxina 1/metabolismo

RESUMEN

In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation. We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and -insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network excitability during epileptiform discharges.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio , Animales , Apamina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo N/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Epilepsia/inducido químicamente , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio

RESUMEN

Chlormethiazole has sedative, hypnotic, anticonvulsant and neuroprotective properties. Using in vitro grease-gap recordings, we show that it inhibits epileptiform activity in neocortical slices superfused with Mg(2+)-free medium (IC(50) approximately 200 microM). At an antiepileptic concentration (300 microM), chlormethiazole potentiated the action of exogenously applied GABA (1 mM) but did not affect responses to the glutamate receptor agonists N-methyl-D-aspartate (10 microM) or L-quisqualic acid (3 microM). The GABA(A) receptor antagonist N-methyl-bicuculline (50 microM) reduced chlormethiazole's potency to inhibit the epileptiform activity. These results indicate that chlormethiazole's anticonvulsant action is likely mediated by potentiating GABA(A)ergic inhibition rather than by antagonising glutamatergic excitation.

Asunto(s)

Corteza Cerebral/efectos de los fármacos , Clormetiazol/farmacología , Epilepsia/tratamiento farmacológico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores de GABA-A/efectos de los fármacos , Valina/análogos & derivados , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Bicuculina/farmacología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Relación Dosis-Respuesta a Droga , Epilepsia/metabolismo , Epilepsia/fisiopatología , Masculino , N-Metilaspartato/farmacología , Neuronas/metabolismo , Neuronas/patología , Ácido Quiscuálico/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Tetrodotoxina/farmacología , Valina/farmacología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Depolarising GABA(A) receptor-mediated responses recorded from the optic nerve using a grease gap technique were modulated by classical potentiators of GABA(A) receptors. The benzodiazepine, chlordiazepoxide, the barbiturate, pentobarbitone and the widely used anaesthetic, propofol, all potentiated gamma-aminobutyric acid (GABA) responses. They did so with different maximal efficacies, propofol>pentobarbitone>chlordiazepoxide, and potencies on the basis of EC(50) estimates, chlordiazepoxide>propofol>pentobarbitone. The greater than expected GABA potentiating properties of propofol were explained by a direct hyperpolarising action that occurred in the same concentration range as its action at the GABA(A) receptor but that was unlikely to be mediated by GABA(A) receptors.

Asunto(s)

Nervio Óptico/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Clordiazepóxido/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Electrofisiología , Técnicas In Vitro , Masculino , Nervio Óptico/fisiología , Pentobarbital/farmacología , Propofol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología

RESUMEN

Mutations in the presenilin-1 (PS1) gene cause approximately 50% of cases of early onset familial Alzheimer's disease. The function of this protein remains unknown. We have made an electrophysiological study of hippocampal slices from transgenic mice expressing either a normal human PS1 transgene (WT) or one of two human PS1 transgenes bearing pathogenic mutations at codon M146 (M146L and M146V). Medium and late afterhyperpolarizations in CA3 pyramidal cells were larger in mice expressing either mutant form compared with WT and nontransgenic controls. Calcium responses to depolarization were larger in M146L mice compared with nontransgenic littermates; synaptic potentiation of the CA3 to CA1 projection was also stronger. These results demonstrate disruption of the control of intracellular calcium and electrophysiological dysfunction in PS1 mutant mice.

Asunto(s)

Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Proteínas de la Membrana/genética , Animales , Regulación de la Expresión Génica , Hipocampo/fisiopatología , Técnicas In Vitro , Ratones , Ratones Transgénicos , Mutación/genética , Mutación/fisiología , Fenotipo , Presenilina-1 , Células Piramidales/fisiopatología

RESUMEN

1. The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5-HT-receptors at high density. Using extra- and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC. 2. Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry. 3. Serotonin (5-HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0. 1 - 100 microM; 59%-depression by 10 microM serotonin), while the antidromic response was not significantly changed by up to 50 microM 5-HT. Depression of field potential responses by serotonin was associated with a significant increase in paired-pulse facilitation from 1.15 to 1.88. 4. The effects of serotonin on field potential responses were mimicked by 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and partially prevented by the 5-HT1A-receptor antagonist (S-UH-301). Moreover, the 5-HT1A-receptor antagonist WAY100635 reduced the effect of 5-CT. 5. Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus-evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation. 6. Depression of isolated AMPA-receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action. 7. We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.

Asunto(s)

Corteza Entorrinal/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Neuronas/efectos de los fármacos , Serotonina/farmacología , Animales , Corteza Entorrinal/citología , Corteza Entorrinal/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Fenfluramina/farmacología , Antagonistas del GABA/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Factores de Tiempo

RESUMEN

Ku is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). This enzyme plays a role in DNA repair, recombination, and transcription. It is composed of a large catalytic subunit (p460), and a regulatory heterodimer, the Ku protein, which consists of 86-kDa and 70-kDa subunits. These various components of the enzyme have been found in both eggs and embryos of the sea urchin. When variable amounts of a specific monoclonal antibody to the Ku protein (Ku 162) were injected into one cell of a 2-cell embryo of Lytechinus pictus, they caused a dose-dependent developmental arrest of the injected cell. The non-injected cell continued to develop normally. In contrast, injection of an antibody (N3H10) raised against the 70-kDa subunit of the Ku protein had no effect on development when injected into 2-cell-stage embryos. Co-injection of purified DNA-PK with the antibody reversed the antibody-mediated inhibition of development. In the fertilized egg and during the early stages of development, the DNA-PK was localized largely in the cytoplasm, but in later developmental stages, it assumed a nuclear location. On the basis of these results, we postulate that the injection of the Ku antibody either prevents the translocation of the DNA-PK into the nucleus or interferes with its enzymatic activity either in the nucleus or in the cytoplasm. In either case, the results suggest that DNA-PK plays an important role in regulating the early stages of embryogenesis in this primitive organism.

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Antígenos Nucleares , ADN Helicasas , Proteínas de Unión al ADN/fisiología , Desarrollo Embrionario , Microinyecciones , Proteínas Nucleares/fisiología , Erizos de Mar/embriología , Animales , División Celular , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Humanos , Autoantígeno Ku , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/administración & dosificación , Proteínas Serina-Treonina Quinasas/fisiología

RESUMEN

The superficial cells of the entorhinal cortex (EC), main input to the hippocampus, receive a serotonergic input from the raphe nuclei and express 5-hydroxytryptamine creatine sulfate complex (5-HT) receptors at high density. With the use of intracellular recordings, we investigated the effects of serotonin on synaptic inhibition of layer II and III neurons of the EC. Serotonin reduced both polysynaptic fast and slow inhibitory postsynaptic potentials (IPSPs) in projection neurons of the superficial EC. Polysynaptic fast and slow IPSPs were depressed by serotonin in a dose-dependent manner (0.1-100 microM). Serotonin in a concentration of 1 microM reduced the amplitudes of polysynaptic fast and slow IPSPs by approximately 40 and 50%, respectively. To identify the subtype of the 5-HT-receptor mediating the effects on polysynaptic IPSPs, we applied various 5-HT-receptor agonists and antagonists. Although the serotonin agonists for the 5-HT1B,2C,3 receptors were ineffective, the effects were mimicked by the 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and prevented by the 5-HT1A-receptor antagonist NAN-190. To look at the direct effects of 5-HT on inhibitory interneurons, we elicited monosynaptic IPSPs in the absence of excitatory synaptic transmission. In contrast to the polysynaptic IPSPs, monosynaptic IPSPs were not significantly affected by serotonin. Recordings from putative inhibitory interneurons revealed that their excitatory postsynaptic potentials (EPSPs) were reversibly reduced by serotonin. We conclude that serotonin suppresses polysynaptic inhibition in projection neurons of layers II and III of the EC by depression of EPSPs on inhibitory interneurons via 5-HT1A receptors.

Asunto(s)

Corteza Entorrinal/química , Depuradores de Radicales Libres/farmacología , Receptores de Serotonina/fisiología , Serotonina/farmacología , Sinapsis/efectos de los fármacos , 2-Amino-5-fosfonovalerato/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Corteza Entorrinal/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Técnicas de Cultivo de Órganos , Piperazinas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Receptores de Serotonina 5-HT1 , Ritanserina/farmacología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología

RESUMEN

1. The superficial layers II and III of the entorhinal cortex, which form the main cortical input to the hippocampus, receive a large serotonergic projection from the raphe nuclei and express 5-HT receptors at high density. Here, we studied the effects of serotonin on the intrinsic properties and excitatory synaptic transmission of the superficial medial entorhinal cortex. 2. Intracellular and patch clamp recordings revealed that serotonin hyperpolarized only one-third of the cells, approximately, through a potassium conductance via a GTP-dependent process. 3. Serotonin depressed mixed as well as isolated alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid receptor (AMPAR)- and N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic potentials/currents (EPSPs/EPSCsapproximately 40 % reduction with 1 microM serotonin). 4. The effect of serotonin on EPSPs/EPSCs was similar in whole-cell versus intracellular recordings; it did not require intracellular GTP and was not visible in glutamate applications to excised patches. Miniature EPSCs recorded in the presence of tetrodotoxin and bicuculline were reduced in frequency, but not altered in amplitude. 5. The effects of serotonin on intrinsic properties and EPSPs were partially mimicked by 5-HT1A receptor agonists (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and 5-carboxamido-tryptamine maleate (5-CT), and reduced by 5-HT1A receptor antagonists S-(-)-5-fluoro-8-hydroxy-DPAT hydrochloride (S-UH-301), 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) and spiperone. 6. We conclude that serotonin potently suppresses excitatory synaptic transmission via 5-HT1A receptors in layers II and III of the medial entorhinal cortex by a presynaptic mechanism.

Asunto(s)

Corteza Entorrinal/fisiología , Receptores de Serotonina/fisiología , Serotonina/farmacología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Bicuculina/farmacología , Corteza Entorrinal/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Guanosina Trifosfato/metabolismo , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Técnicas de Placa-Clamp , Piperazinas/farmacología , Canales de Potasio/fisiología , Ratas , Ratas Wistar , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Serotonina/análogos & derivados , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Espiperona/farmacología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacología

RESUMEN

Among the molecular, cellular, and systemic events that have been proposed to modulate the function of the hippocampus and the entorhinal cortex (EC), one of the most frequently cited possibilities is the activation of the serotonergic system. Neurons in the hippocampus and in the EC receive a strong serotonergic projection from the raphe nuclei and express serotonin (5-HT) receptors at high density. Here we review the various effects of 5-HT on intrinsic and synaptic properties of neurons in the hippocampus and the EC. Although similar membrane-potential changes following 5-HT application have been reported for neurons of the entorhinal cortex and the hippocampus, the effects of serotonin on synaptic transmission are contrary in both areas. Serotonin mainly depresses fast and slow inhibition of the principal output cells of the hippocampus, whereas it selectively suppresses the excitation in the entorhinal cortex. On the basis of these data, we discuss the possible role of serotonin under physiological and pathophysiological circumstances.

Asunto(s)

Corteza Entorrinal/fisiología , Hipocampo/fisiología , Neuronas/fisiología , Serotonina/fisiología , Transmisión Sináptica/fisiología , Animales , Corteza Entorrinal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Humanos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Serotonina/farmacología , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos

RESUMEN

Intracellular recordings were performed to study the effects of 5-HT on membrane properties and EPSP/IPSP responses of subicular neurons in rat combined hippocampal-entorhinal cortex slices. Application of 5-HT induced in 76% of the investigated subicular cells a hyperpolarization and a reduction of membrane resistance. In bursting neurons, 5-HT caused a reduction of the depolarizing envelope underlying burst discharges and attenuated the subsequent afterhyperpolarization. While 5-HT decreased isolated AMPA/kainate and NMDA receptor-mediated responses as well as slow IPSPs, we could not find a consistent effect on isolated fast IPSPs. Since in approximately 25% of subicular neurons EPSPs and slow IPSPs were reduced without any increase of membrane conductance, we conclude that 5-HT has in addition to membrane effects also effects on synaptic currents.

Asunto(s)

Corteza Entorrinal/fisiología , Potenciales Evocados/efectos de los fármacos , Hipocampo/fisiología , Neuronas/fisiología , Serotonina/farmacología , Sinapsis/fisiología , Animales , Corteza Entorrinal/efectos de los fármacos , Potenciales Evocados/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Neuronas/efectos de los fármacos , Ácidos Fosfínicos/farmacología , Propanolaminas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos

RESUMEN

Ca2+ release from intracellular stores can be activated in neurons by influx of Ca2+ through voltage-gated Ca2+ channels. This process, called Ca2+-induced Ca2+ release, relies on the properties of the ryanodine receptor and represents a mechanism by which Ca2+ influx during neuronal activity can be amplified into large intracellular Ca2+ signals. In a differentiated neuroblastoma cell line, we show that caffeine, a pharmacological activator of the ryanodine receptor, released Ca2+ from intracellular stores in a Ca2+-dependent and ryanodine-sensitive manner. The pyridine nucleotide, cyclic ADP-ribose, thought to be an endogenous modulator of ryanodine receptors also amplified Ca2+-induced Ca2+ release in these neurons. Cyclic ADP-ribose enhanced the total cytoplasmic Ca2+ levels during controlled Ca2+ influx through voltage gated channels, in a concentration-dependent and ryanodine-sensitive manner and also increased the sensitivity with which a small amount of Ca2+ influx could trigger additional release from the ryanodine-sensitive intracellular Ca2+ stores. Single cell imaging showed that following the Ca2+ influx, cyclic ADP-ribose enhanced the spatial spread of the Ca2+ signal from the edge of the cell into its center. These powerful actions suggest a role for cyclic ADP-ribose in the functional coupling of neuronal depolarization, Ca2+ entry, and global intracellular Ca2+ signaling.

Asunto(s)

Adenosina Difosfato Ribosa/análogos & derivados , Calcio/metabolismo , Neuronas/fisiología , Adenosina Difosfato Ribosa/fisiología , Animales , Cafeína/farmacología , Canales de Calcio/fisiología , Compartimento Celular , Células Cultivadas , ADP-Ribosa Cíclica , Células Híbridas , Inositol 1,4,5-Trifosfato/farmacología , Activación del Canal Iónico , Potenciales de la Membrana , Ratones , Proteínas Musculares/fisiología , Ratas , Canal Liberador de Calcio Receptor de Rianodina

RESUMEN

Cyclic ADP-ribose (cADPR) is a putative second messenger that has been demonstrated to mobilize Ca2+ in many cell types. Its postulated role as the endogenous regulator of ryanodine-sensitive Ca2+ release channels has been greatly supported by the advent and use of specific cADPR receptor antagonists such as 8-NH2-cADPR (Walseth, T. F., and Lee, H. C. (1993) Biochim. Biophys. Acta 1178, 235-242). However, investigations of the role of cADPR in physiological responses, such as fertilization, stimulus-secretion coupling, and excitation-contraction coupling, have been hindered by the susceptibility of cADPR receptor antagonists to hydrolysis and the need to introduce these molecules into cells by microinjection or patch clamp techniques. We have recently reported on the discovery of a poorly hydrolyzable analogue of cADPR, 7-deaza-cADPR (Bailey, V. C., Sethi, J. K., Fortt, S. M., Galione, A., and Potter, B. V. L. (1997) Chem. Biol. 4, 41-51) but this, like cADPR, is an agonist of ryanodine-sensitive Ca2+ release channels. We therefore explored the possibility of combining antagonistic activity with that of hydrolytic resistance and now report on the biological properties of the first hydrolysis-resistant cADPR receptor antagonist, 7-deaza-8-bromo-cADPR. In addition this compound has the advantage of being membrane-permeable. Together these properties make this hybrid molecule the most powerful tool to date for studying cADPR-mediated Ca2+ signaling in intact cells.

Asunto(s)

Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/antagonistas & inhibidores , Adenosina Difosfato Ribosa/farmacología , Animales , Calcio/metabolismo , ADP-Ribosa Cíclica , Hidrólisis , Erizos de Mar , Relación Estructura-Actividad

RESUMEN

Entorhinal cortex layer III cells send their axons into hippocampal area CA1, forming the less well studied branch of the perforant path. Using electrophysiological and morphological techniques within a slice preparation, we can classify medial entorhinal cortex layer III cells into four different types. Type 1 and 2 cells were projection cells. Type 1 cells fired regularly and possessed high input resistances and long membrane time constants. Electrical stimulation of the lateral entorhinal cortex revealed a strong excitation by both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials. Type 2 cells accommodated strongly, had lower input resistances, faster time constants and featured prominent synaptic inhibition. Type 1 and 2 cells responded to repetitive synaptic stimulation with a prolonged hyperpolarization. We identified the two other, presumed local circuit, cell types whose axons remained within the entorhinal cortex. Type 3 cells were regular firing, had high input resistances and slow membrane time constants, while type 4 cells fired at higher frequencies and possessed a faster time constant and lower input resistance than type 3 neurons. Type 3 cells presented long-lasting excitatory synaptic potentials. Type 4 neurons were the only ones with different responses to stimulation from different sites. Upon lateral entorhinal cortex stimulation they responded with an excitatory postsynaptic potential, while a monosynaptic inhibitory postsynaptic potential was evoked from deep layer stimulation. In contrast to type 1 and 2 neurons, none of the local circuit cells could be antidromically activated from deep layers, and prolonged hyperpolarizations following synaptic repetitive stimulation were also absent in these cells. Together, the complementing morphology and the electrophysiological characteristics of all the cells can provide the controlled flexibility required during the transfer of cortical information to the hippocampus.

Asunto(s)

Corteza Entorrinal/citología , Corteza Entorrinal/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Neuronas/citología , Neuronas/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Bicuculina/análogos & derivados , Bicuculina/farmacología , Estimulación Eléctrica , Femenino , Técnicas In Vitro , Potenciales de la Membrana , Neuronas/efectos de los fármacos , Compuestos Organofosforados/farmacología , Ácidos Fosfínicos/farmacología , Propanolaminas/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Receptores de GABA-A/fisiología , Receptores de GABA-B/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

Low Mg2+-induced epileptiform activity in the entorhinal cortex is characterized by an initial expression of seizure-like events followed by late recurrent discharges. Both these forms of activity as well as the transition between them were blocked by serotonin. In contrast, serotonin had little effect upon the epileptiform activity in areas CA3 and CA1 of the hippocampus. Both forms of epileptiform activity in the entorhinal cortex are sensitive to N-methyl-D-aspartate receptor antagonists and it is shown here that serotonin blocked both types of epileptiform activity through an effective concentration-dependent reduction of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials in deep layer entorhinal cortex cells. Serotonin also prolonged or even prevented the transition between the two types of epileptiform activity and we suggest that this may be through activation of the Na+/K+-ATPase. The resistance of epileptiform activity in CA1 and CA3 to serotonin was most likely related to the inability of serotonin to reduce Schaffer collateral-evoked excitatory postsynaptic potentials. Given the strong serotonergic inputs to both the hippocampus and entorhinal cortex, the differential sensitivity of the two regions to serotonin suggests functional differences. In addition since the late recurrent discharges in the entorhinal cortex are resistant to all clinically used anticonvulsants, serotonin may open new avenues for the development of novel anticonvulsant compounds.

Asunto(s)

Corteza Entorrinal/fisiopatología , Epilepsia/fisiopatología , Hipocampo/fisiopatología , Deficiencia de Magnesio/fisiopatología , Serotonina/farmacología , Animales , Electroencefalografía , Femenino , Potenciales de la Membrana/fisiología , Ratas , Ratas Wistar , Convulsiones/fisiopatología , Convulsiones/psicología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología

RESUMEN

Storage and retrieval of information in the hippocampus is dependent on information transfer from the entorhinal cortex (EC). We studied how the separate pathways from layer II and III of the EC to the hippocampus are selected for information transfer during repetitive synaptic stimulation. Intracellular recordings were made from EC layer II and III projection cells in horizontal combined EC-hippocampal slices. Synaptic responses to stimulation of deep layers or the lateral EC with stimulus intensities approximately 70% of that required to elicit an action potential were analyzed during short trains of repetitive stimulation. The threshold intensities for induction of action potentials were in layer II cells 8.2 +/- 3.8 (SE) V, significantly larger than 4.4 +/- 1.5 V in type 1, and 5.2 +/- 3.3 V in type 2 layer III cells, respectively. During repetitive subthreshold stimulation with frequencies below 5 Hz the pathway from the EC layer II remained quiet and was preferentially activated with stimulation frequencies above 5 Hz. In contrast the EC layer III cells responded preferentially to low stimulus frequencies (<10 Hz) and became strongly inhibited when synaptically stimulated with frequencies above 10 Hz. Interestingly during stimulus frequencies between 5 and 10 Hz the likelihood that both layer II and III cells fire was large. Thus a frequency switch operates in the entrohinal cortex regulating output of layer II and III cells to the hippocampus. We suggest that such frequency dependent regulation of information flow presents a new principle of neuronal information processing.

Asunto(s)

Corteza Entorrinal/fisiología , Hipocampo/fisiología , Sinapsis/fisiología , Potenciales de Acción/fisiología , Animales , Estimulación Eléctrica , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Vías Nerviosas/fisiología , Ratas , Ratas Wistar

RESUMEN

The electrophysiological properties of 46 bursting cells and 39 regular firing cells were studied in the subiculum of rat combined hippocampal-entorhinal cortex slices. In bursting cells we found a significantly higher resting membrane potential than in regular firing cells. Upon hyperpolarization both cell types expressed a delayed inward rectification with a subsequent afterdepolarization. While in regular firing cells longer lasting depolarizing current injection caused a train of action potentials with a rather marked decline of discharge frequency, bursting cells displayed only little frequency accommodation. Regular firing cells usually displayed a fast and a slow afterhyperpolarization following a train of action potentials, while bursting neurons present only a slow afterhyperpolarization.

Asunto(s)

Hipocampo/fisiología , Potenciales de la Membrana/fisiología , Animales , Neuronas/fisiología , Ratas , Ratas Wistar

RESUMEN

Cyclic ADP ribose (cADPR) is a potent Ca(2+)-releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. beta-NAD+, cGMP and nitric oxide (NO) have been reported to mobilize Ca2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca(2+)-mobilizing action of beta-NAD+, cGMP and NO, but has no effect on cADPR-induced Ca2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca2+, we have shown that nicotinamide inhibits cADPR-mediated Ca2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca2+ release by beta-NAD+, cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.

Asunto(s)

Adenosina Difosfato Ribosa/análogos & derivados , Calcio/metabolismo , Niacinamida/farmacología , Erizos de Mar/metabolismo , Transducción de Señal/efectos de los fármacos , Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/farmacología , Animales , ADP-Ribosa Cíclica , Microsomas/metabolismo , Niacinamida/metabolismo , Erizos de Mar/embriología