RESUMEN
In adult cortical tissue, recruitment of GABAergic inhibition prevents the progression of synchronous population discharges to epileptic activity. However, at early developmental stages, GABA is excitatory and thus unable to fulfill this role. Here, we report that retrograde signaling involving endocannabinoids is responsible for the homeostatic control of synaptic transmission and the resulting network patterns in the immature hippocampus. Blockade of cannabinoid type 1 (CB1) receptor led to epileptic discharges, whereas overactivation of CB1 reduced network activity in vivo. Endocannabinoid signaling thus is able to keep population discharge patterns within a narrow physiological time window, balancing between epilepsy on one side and sparse activity on the other, which may result in impaired developmental plasticity. Disturbing this delicate balance during pregnancy in either direction, e.g., with marijuana as a CB1 agonist or with an antagonist marketed as an antiobesity drug, can have profound consequences for brain maturation even in human embryos.
Asunto(s)
Encéfalo/efectos de los fármacos , Moduladores de Receptores de Cannabinoides/farmacología , Hipocampo/efectos de los fármacos , Neuronas/metabolismo , Piperidinas/farmacología , Pirazoles/farmacología , Transducción de Señal , Animales , Encéfalo/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Cannabis , Electrodos , Electrofisiología , Femenino , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Microscopía Electrónica , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo , Receptores de GABA-A/metabolismo , Rimonabant , Trastornos Relacionados con Sustancias , Factores de TiempoRESUMEN
We have determined the properties of seizures induced in vitro during the first postnatal days using intact rat cortico-hippocampal formations (CHFs) and extracellular recordings. Two main patterns of activity were generated by nominally Mg2+-free ACSF in hippocampal and cortical regions: ictal-like events (ILEs) and late recurrent interictal discharges (LRDs). They were elicited at distinct developmental periods and displayed different pharmacological properties. ILEs were first observed in P1 CHFs 52 +/- 7 min after application of low-Mg2+ ACSF (frequency 1.5 +/- 0.3 h-1, duration 86 +/- 3 s). There is a progressive age-dependent maturation of ILEs characterized by a decrease in their onset and an increase in their frequency and duration. ILEs were abolished by d-APV and Mg2+ ions. From P7, ILEs were followed by LRDs that appeared 89 +/- 8 min after application of low-Mg2+ ACSF (frequency approximately 1 Hz, duration 0.66 s, amplitude 0.31 +/- 0.03 mV). LRDs were no longer sensitive to d-APV or Mg2+ ions and persisted for at least 24 h in low-Mg2+ or in normal ACSF. ILEs and LRDs were synchronized in limbic and cortical regions with 10-40 ms latency between the onsets of seizures. Using a double chamber that enables independent superfusion of two interconnected CHFs, we report that ILEs and LRDs generated in one CHF propagated readily to the other one that was being kept in ACSF. Therefore, at a critical period of brain development, recurrent seizures induce a permanent form of hyperactivity in intact brain structures and this preparation provides a unique opportunity to study the consequences of seizures at early developmental stages.
Asunto(s)
Epilepsia/metabolismo , Epilepsia/fisiopatología , Sistema Límbico/metabolismo , Sistema Límbico/fisiopatología , Magnesio/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Electrofisiología , Corteza Entorrinal/metabolismo , Corteza Entorrinal/fisiopatología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Sistema Límbico/crecimiento & desarrollo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/metabolismo , Convulsiones/fisiopatologíaRESUMEN
Although several investigations have shown that the local GABAergic circuit in the rat hippocampus is functional very early in development, this result has not been yet completed by the investigation of the full dendritic and axonal arborization of the neonatal interneurones. In the present study, intracellular injection of biocytin was used to assess the branching pattern of interneurones in the hippocampal CA3 region of rat between 2 and 6 days of age. Based on their dendritic morphology, the biocytin-filled interneurones were divided into four classes: bipolar, stellate, pyramidal-like and fusiform interneurones. About half of the biocytin-filled neonatal interneurones exhibited dendritic or somatic filopodial processes. The axonal arbors of the filled-interneurones were widely spread into the CA3 region, and in four out of nine cases extended beyond the CA3 region to branch into the CA1 region. These results show that, despite immature features, the filopodial processes, the hippocampal interneurones are well developed early in development at a time when their target cells, the pyramidal neurones, are still developing. These observations are consistent with a trophic role that GABA may play early in development.
Asunto(s)
Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Interneuronas/ultraestructura , Animales , Animales Recién Nacidos , Axones , Tamaño de la Célula , Dendritas , Masculino , Técnicas de Placa-Clamp , Células Piramidales , Ratas , Ratas WistarRESUMEN
N-Methyl-D-aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A-NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT-PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of theses findings is discussed.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Cerebelo/metabolismo , Variación Genética , ARN Mensajero/genética , Receptores de N-Metil-D-Aspartato/genética , Secuencia de Aminoácidos/genética , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Secuencia de Bases/genética , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Cerebelo/citología , Clonación Molecular , ADN Recombinante , Electrofisiología , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/fisiología , TransfecciónRESUMEN
Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/embriología , Hipocampo/fisiología , Células Piramidales/fisiología , Receptores AMPA/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Vías Aferentes/efectos de los fármacos , Vías Aferentes/embriología , Vías Aferentes/fisiología , Envejecimiento , Animales , Animales Recién Nacidos , Benzodiazepinas/farmacología , Bicuculina/farmacología , Cationes Bivalentes/farmacología , Estimulación Eléctrica , Desarrollo Embrionario y Fetal , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Colorantes Fluorescentes , Hipoxia , Lisina/análogos & derivados , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Receptores AMPA/antagonistas & inhibidores , Sinapsis/fisiología , Tetrodotoxina/farmacologíaRESUMEN
We have performed a morphofunctional analysis of CA1 pyramidal neurons at birth to examine the sequence of formation of GABAergic and glutamatergic postsynaptic currents (PSCs) and to determine their relation to the dendritic arborization of pyramidal neurons. We report that at birth pyramidal neurons are heterogeneous. Three stages of development can be identified: (1) the majority of the neurons (80%) have small somata, an anlage of apical dendrite, and neither spontaneous nor evoked PSCs; (2) 10% of the neurons have a small apical dendrite restricted to the stratum radiatum and PSCs mediated only by GABA(A) receptors; and (3) 10% of the neurons have an apical dendrite that reaches the stratum lacunosum moleculare and PSCs mediated both by GABA(A) and glutamate receptors. These three groups of pyramidal neurons can be differentiated by their capacitance (C(m) = 17.9 +/- 0.8; 30.2 +/- 1.6; 43.2 +/- 3.0 pF, respectively). At birth, the synaptic markers synapsin-1 and synaptophysin labeling are present in dendritic layers but not in the stratum pyramidale, suggesting that GABAergic peridendritic synapses are established before perisomatic ones. The present observations demonstrate that GABAergic and glutamatergic synapses are established sequentially with GABAergic synapses being established first most likely on the apical dendrites of the principal neurons. We propose that different sets of conditions are required for the establishment of functional GABA and glutamate synapses, the latter necessitating more developed neurons that have apical dendrites that reach the lacunosum moleculare region.
Asunto(s)
Dendritas/fisiología , Ácido Glutámico/fisiología , Hipocampo/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Animales Recién Nacidos , Biomarcadores , Senescencia Celular , Electrofisiología , Hipocampo/citología , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Biochemical and electrophysiological approaches were used to assess possible changes in 5-HT1A receptors in the rat brain after long-term treatment with an anxiolytic benzodiazepine. Rats were treated with diazepam (2 mg/kg i.p. daily) during 14 days and then untreated for 1 day (protocol A) or 5 days (protocol C) until they were killed for in vitro investigations on 5-HT1A receptors. In addition, other rats (protocol B) received the same 14-day treatment with diazepam, followed by 1 mg/kg of the drug on days 15 and 16, and 0.5 mg/kg on days 17 and 18, and were killed 24 h after the last injection. In vitro binding and quantitative autoradiographic experiments with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) showed that the characteristics of 5-HT1A receptor binding sites in the hippocampus and the dorsal raphe nucleus were not significantly altered by the administration of diazepam under the treatment protocols A, B and C. Furthermore, in vitro electrophysiological recordings of serotoninergic neurons in the dorsal raphe nucleus of brain stem slices revealed no modification in the sensitivity of somatodendritic 5-HT1A autoreceptors in rats treated with diazepam according to the protocols A and B. However, under the conditions of protocol C, the potency of 8-OH-DPAT to depress the firing rate of serotoninergic neurons was significantly enhanced, as expected of a hypersensitivity of somatodendritic 5-HT1A autoreceptors. These data support the hypothesis that some functional changes in these receptors could occur during benzodiazepine withdrawal. However, they do not support the idea of a reduced anxiolytic efficacy of 5-HT1A receptor agonists as a result of prior treatment with a benzodiazepine.
Asunto(s)
Ansiolíticos/farmacología , Encéfalo/efectos de los fármacos , Diazepam/farmacología , Receptores de Serotonina/efectos de los fármacos , 8-Hidroxi-2-(di-n-propilamino)tetralin/metabolismo , Potenciales de Acción , Animales , Autorradiografía , Encéfalo/metabolismo , Buspirona/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/metabolismo , Ratas , Ratas Wistar , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Agonistas de Receptores de Serotonina/metabolismoRESUMEN
N-methyl-D-aspartate (NMDA) receptor function can be regulated by direct binding of calmodulin to a low and high affinity (C1 exon cassette) site in the C-terminal region of the NR1 subunit. To evaluate the involvement of the high affinity binding site in the transient inactivation of the NMDA receptor-channels by intracellular calcium, several splice variants of the NR1 subunit have been individually co-transfected with the NR2A subunit in HEK 293 cells. The transient Ca2+ induced inactivation (40-50%) of the heteromeric receptors was similar whether the NR1 variants contained (NR1-1a, 1b) or lacked (NR1-2a, 2b, 4a, 4b) the C1 exon cassette bearing the high affinity binding site for calmodulin. This demonstrates that this site is not involved in the Ca2+ dependent transient inactivation of NMDA receptors.
Asunto(s)
Calcio/farmacología , Calmodulina/metabolismo , Canales Iónicos/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Unión Competitiva , Línea Celular Transformada , Humanos , Técnicas de Placa-Clamp , Proteínas Recombinantes , Factores de TiempoRESUMEN
Effects of redox reagents on excitatory and inhibitory synaptic responses as well as on the bidrectional plasticity of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses were studied in CA1 pyramidal neurons in rat hippocampal slices. The oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM) did not affect AMPA, GABAA or GABAB receptor-mediated synaptic responses or the activation of presynaptic metabotropic receptors. However, DTNB irreversibly decreased (by approximately 50%) currents evoked by focal application of NMDA. DTNB also decreased the NMDA component of the EPSC. The reversal potential of NMDA currents and the Mg2+ block were not modified. In the presence of physiological concentrations of Mg2+ (1.3 mM), DTNB did not affect the NMDA receptor-dependent induction of long-term potentiation (LTP) or long-term depression (LTD) expressed by AMPA receptors. In contrast, DTNB fully prevented LTP and LTD induced and expressed by NMDA receptors. Plasticity of NMDA receptor-mediated synaptic responses could be reinstated by the reducing agent tris-(2-carboxyethyl) phosphine (TCEP, 200 microM). These results suggest that persistent, bidirectional changes in synaptic currents mediated by NMDA receptors cannot be evoked when these receptors are in an oxidized state, whereas NMDA-dependent LTP and LTD are still expressed by AMPA receptors. Our observations raise the possibility of developing therapeutic agents that would prevent persistent excitotoxic enhancement of NMDA receptor-mediated events without blocking longterm modifications of AMPA receptor-mediated synaptic responses, thought to underlie memory processes.
Asunto(s)
Ácido Ditionitrobenzoico/farmacología , Hipocampo/fisiología , Plasticidad Neuronal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Simultaneous extracellular recordings were performed in stratum radiatum and stratum pyramidale of hippocampal slices 7 days following unilateral intracerebroventricular injections of kainic acid. In this ex vivo experimental model of human temporal lobe epilepsy, stimulation of the surviving commissural fibres in stratum radiatum produced graded epileptiform activity in the CA1 area. The oxidizing reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acting at NMDA receptors redox sites decreases NMDA receptor-mediated responses by half and suppresses evoked epileptiform discharges. We have examined the effect of DTNB on NMDA-dependent bidirectional synaptic plasticity and EPSP/spike coupling. DTNB treatment did not prevent either long-term potentiation induced by tetanic stimulation or long-term depression induced by low frequency stimulation of field EPSPs. Application of DTNB alone did not induce EPSP/spike dissociation. However, both high and low frequency stimulations induced EPSP/spike potentiation indicating that neurons had a high probability to discharge in synchrony. These results suggest that oxidizing reagents may provide novel antiepileptic treatments since they decrease NMDA-dependent evoked epileptiform activity but do not interfere with either NMDA-dependent synaptic plasticity or the probability of synchronous discharge.
Asunto(s)
Ácido Ditionitrobenzoico/farmacología , Epilepsia del Lóbulo Temporal/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Factores de TiempoRESUMEN
The contributions of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and gamma-aminobutyric acid (GABA[A]) receptors in the induction of long-term potentiation (LTP) have been studied in the CA1 region of the rat hippocampus. The results suggest that: (1) in physiological conditions, AMPARs are necessary for the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent LTP since LTP cannot be elicited in the presence of the AMPAR antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Although a NMDAR-dependent LTP occurs in the presence of a GABA(A) antagonist and high concentrations of divalents cations, blockade of AMPARs leads to a voltage-dependent calcium channels (VDCC)-dependent LTP since its induction is blocked by nifedipine and not by APV. (2) The bicarbonate-induced GABA(A) receptor-mediated depolarizing response is not necessary in the induction of NMDAR-dependent or VDCC-dependent LTP since induction of these two types of LTP were not blocked by acetazolamide or in a nominally bicarbonate-free solution.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Receptores AMPA/fisiología , Receptores de GABA-A/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Bicuculina/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Nifedipino/farmacología , Ratas , Ratas WistarRESUMEN
1. Graded N-methyl-D-aspartate receptor (NMDAR)-dependent epileptiform discharges were recorded from ex vivo hippocampal slices obtained from rats injected a week earlier with an intracerebroventricular dose of kainic acid. Intracellular recordings from pyramidal cells of the CA1 area showed that glutamate NMDAR actively participated in synaptic transmission, even at resting membrane potential. When NMDAR were pharmacologically isolated, graded burst discharges could still be evoked. 2. The oxidizing reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM, 15 min) suppressed the late part of the epileptiform burst that did not recover after wash but could be reinstated by the reducing agent tris (2-carboxyethyl) phosphine (TCEP, 200 microM, 15 min) and again abolished with the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV). 3. Pharmacologically isolated NMDAR-mediated responses were decreased by DTNB (56 +/- 10%, mean +/- SD, n = 6), an effect reversed by TCEP. 4. When only the fast glutamateric synaptic component was blocked, NMDA-dependent excitatory postsynaptic potentials (EPSPs) could be evoked despite the presence of underlying fast and slow inhibitory postsynaptic potentials (IPSPs). DTNB decreased EPSPs to 48 +/- 12% (n = 5) of control. 5. Since a decrease of the NMDAR-mediated response by +/- 50% is sufficient to suppress the late part of the burst, we suggest that epileptiform activity can be controlled by manipulation of the redox sites of NMDAR. Our observations raise the possibility of developing new anticonvulsant drugs that would spare alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-R (AMPAR)-mediated synaptic responses and decrease NMDAR-mediated synaptic transmission without blocking it completely.
Asunto(s)
Ácido Ditionitrobenzoico/farmacología , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Oxidantes/uso terapéutico , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Agonistas de Aminoácidos Excitadores , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Ácido Kaínico/antagonistas & inhibidores , Masculino , Oxidación-Reducción , Ratas , Ratas Wistar , Transmisión Sináptica/efectos de los fármacosRESUMEN
Although considerable progress has been made in characterising the 5-HT1A receptor using agonists, partial agonists or non-selective antagonists, further studies of 5-HT1A receptor function have been hindered by the lack of highly selective antagonists. The term 'silent' antagonist has been used for such compounds in order to distinguish them unequivocally from several 5-HT1A receptor partial agonists which were initially designated 'antagonists'. In this report we provide a comprehensive review of the biochemical, pharmacological and behavioural properties of the first potent, selective and silent 5-HT1A receptor antagonist, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). WAY-100635 had an IC50 (displacement of specific [3H]8-OH-DPAT binding to 5-HT1A receptors in the rat hippocampus) of 1.35 nM and was > 100-fold selective for the 5-HT1A site relative to a range of other CNS receptors. [3H]WAY-100635 was also characterised as the first 5-HT1A antagonist radioligand, displaying the same regional distribution of binding sites as [3H]8-OH-DPAT in rat brain. As would be expected for the binding of an antagonist to a G-protein-coupled receptor, the Bmax of [3H]WAY-100635 specific binding was consistently 50-60% greater than that of the agonist radioligand, [3H]8-OH-DPAT. Mn2+, but not guanine nucleotides, inhibited [3H]WAY-100635-specific binding. [3H]WAY-100635 was also shown to bind selectively to brain 5-HT1A receptors in vivo, following intravenous administration to mice. In vitro electrophysiological studies demonstrated that WAY-100635 had no 5-HT1A receptor agonist actions, but dose-dependently blocked the effects of agonists at both the postsynaptic 5-HT1A receptor in the CA1 region of the hippocampus, and the somatodendritic 5-HT1A receptor located on dorsal raphe 5-HT neurones. In vivo, WAY-100635 also dose-dependently blocked the ability of 8-OH-DPAT to inhibit the firing of dorsal raphe 5-HT neurones, and to induce the '5-HT syndrome', hypothermia, hyperphagia and to elevate plasma ACTH levels. In the mouse light/dark box anxiety model, WAY-100635 induced anxiolytic-like effects. WAY-100635 had no intrinsic effect on cognition in the delayed-matching-to-position model of short-term memory in the rat, but reversed the disruptive effects of 8-OH-DPAT on motor motivational performance. These data clearly demonstrate that WAY-100635 is the first potent, selective and silent 5-HT1A receptor antagonist. Furthermore, [3H]WAY-100635 is the first antagonist radioligand to become available for 5-HT1A receptor binding studies both in vitro and in vivo. The positive effects of WAY-100635 in an anxiety model also indicate that a postsynaptic 5-HT1A receptor antagonist action may contribute to the anxiolytic properties of 5-HT1A receptor partial agonists.
Asunto(s)
Conducta Animal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Neurotransmisores/metabolismo , Piperazinas/farmacología , Piridinas/farmacología , Antagonistas de la Serotonina/farmacología , Hormona Adrenocorticotrópica/sangre , Animales , Cognición/efectos de los fármacos , Electrofisiología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hiperfagia/inducido químicamente , Hipotermia Inducida , Masculino , Ratones , Células Piramidales/efectos de los fármacos , Núcleos del Rafe/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
NMDA receptors play a central role in neuronal plasticity and in several pathological situations. Transient activation of this receptor triggers long-term potentiation, whereas sustained activation leads to cell death. Evidence for control of this activity by a redox site in cell cultures, brain tissues and in recombinant NMDA receptors are discussed by Henri Gozlan and Yehezkel Ben-Ari. The characteristics of this modulation and the consequences of redox state modifications on NMDA-mediated events are examined in vitro under physiological and pathological conditions. Since metabolic disorders enhance NMDA receptor function, the redox site could constitute a new target for selectively preventing in vivo the deleterious consequences of overactivation without blocking neuronal plasticity mediated by NMDA receptors.
Asunto(s)
Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Compuestos de Sulfhidrilo/farmacología , Reactivos de Sulfhidrilo/farmacología , Alquilación , Animales , Ácido Ascórbico/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Epilepsia/metabolismo , Radicales Libres , Técnicas In Vitro , Isquemia/metabolismo , Oxidación-Reducción , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Proteínas Recombinantes , Compuestos de Sulfhidrilo/uso terapéutico , Reactivos de Sulfhidrilo/uso terapéutico , Transmisión Sináptica/efectos de los fármacos , Xenopus laevisRESUMEN
1. Using extracellular recording techniques in the CA1 region of the rat hippocampus, we have evaluated the effects of the redox reagents 5,5O-dithiobis-2-nitrobenzoic acid (DTNB) and tris (carboxyethyl) phosphine (TCEP) on long-term potentiation (LTP) expressed by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. In physiological conditions a high-frequency stimulation (HFS) of Schaffer collateral-commissural fibers induced a LTP expressed by a persistent increase (73 +/- 13%, mean +/- SE, n = 8/10) of AMPA field potentials (LTPA). In the presence of 10 microM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced concentration of Mg2+ (0.1 mM) to boost NMDA receptors, the HFS induced LTP of NMDA field potentials (LTPN; 62 +/- 11%, n = 8/10). 2. The thiol-oxidizing reagent DTNB (200 microM) reduced, by 46 +/- 5% (n = 24), NMDA-receptor field potentials (NMDA-FP), and this effect could not be reversed by extensive washing. The disulfide-reducing agent TCEP (200 microM) slightly increased AMPA-FP and reversed the DTNB-induced inhibition of NMDA-FP. 3. DTNB (200 microM, 10 min), and TCEP (200 microM, 20 min), had no effect on AMPA-FP (98 +/- 3% and 101 +/- 5%, respectively, n = 12). 4. DTNB (200 microM, 15 min) did not prevent the induction or expression of LTPA (-12 and -5%, respectively, n = 8/8). Similar results were observed with TCEP (200 microM, 20 min).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácido Ditionitrobenzoico/farmacología , Hipocampo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Masculino , Oxidación-Reducción , Fosfinas/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Long-term potentiation (LTP) is a form of synaptic plasticity thought to be involved in learning and memory. Although extensively studied, mainly in the CA1 region of the hippocampus, the mechanisms underlying the induction and expression of LTP are poorly elucidated. This is probably due to the fact that LTP is not a unique process and indeed recent studies have shown that several forms of LTP could be generated depending on the experimental conditions. Furthermore, LTP is generally associated with a long-lasting increase of the synaptic efficacy of AMPA receptors but an increasing number of data also suggested that NMDA receptors could be potentiated as well. NMDA receptor responses are modulated by a large number of extracellular and intracellular events, providing additional possibilities for the generation of LTP. The role of these different modulatory sites of the NMDA receptor and their relation with LTP are reviewed with a particular attention to the redox site which seems to be a selective target to distinguish between AMPA and NMDA-LTP.
Asunto(s)
Potenciación a Largo Plazo , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Hipocampo/fisiología , Técnicas In Vitro , Oxidación-Reducción , Receptores AMPA/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The tritiated derivative of the novel silent 5-HT1A receptor antagonist WAY 100635 [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide] was tested as a potential radioligand of 5-HT1A receptors in the rat brain. Binding assays with membranes from various brain regions showed that [3H]WAY 100635 specifically bound to a homogeneous population of sites, with a Kd of 0.10 nM. The regional distribution of [3H]WAY 100635 specific binding sites, as assessed in membrane binding assays and by autoradiography of labelled brain sections, superimposed exactly over that of 5-HT1A receptors specifically labelled by [3H]8-hydroxy-2-(di-n-propylamino) tetralin ([3H]8-OH-DPAT). Furthermore, the positive correlation (r = 0.96) between the respective pKi values of a large series of ligands as inhibitors of the specific binding of [3H]WAY 100635 and [3H]8-OH-DPAT in hippocampal membranes indicated that their pharmacological properties were similar. Nevertheless, marked differences also existed between [3H]8-OH-DPAT and [3H]WAY 100635 specific binding, as the former was inhibited by 1-100 microM GTP and GppNHp, whereas the latter was enhanced by these guanine nucleotides. In contrast, Mn2+ (1-10 mM) increased the specific binding of [3H]8-OH-DPAT, but inhibited that of [3H]WAY 100635. Treatment of membranes with N-ethylmaleimide (1-5 mM) markedly reduced their capacity to specifically bind [3H]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [3H]WAY 100635 specific binding capacity. Finally, the Bmax of [3H]WAY 100635 specific binding sites was regularly 50-60% higher than that of [3H]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that whereas [3H]8-OH-DPAT only binds to G-protein-coupled 5-HT1A receptors, [3H]WAY 100635 is a high affinity ligand of both G-protein-coupled and free 5-HT1A receptor binding subunits in brain membranes.
Asunto(s)
Química Encefálica/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Piperazinas/farmacología , Piridinas/farmacología , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/metabolismo , Animales , Autorradiografía , Unión Competitiva/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/efectos de los fármacos , Serotonina/metabolismoRESUMEN
1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.
Asunto(s)
Antidepresivos/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Animales , Antieméticos/farmacología , Neoplasias Encefálicas/metabolismo , Cisplatino/farmacología , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/metabolismo , Técnicas In Vitro , Masculino , Ratones , Neuroblastoma/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Reflejo/efectos de los fármacos , Serotonina/metabolismo , Antagonistas de la Serotonina/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Porcinos , Células Tumorales CultivadasRESUMEN
1. The effects of redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP), on anoxia-induced long-term potentiation (LTP) were investigated in CA1 hippocampal neurons using extracellular recording techniques. Experiments were performed in the presence of 0.1 mM MgCl2 and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA) receptor-mediated responses. 2. DTNB (200 microM), a thiol oxidizing reagent, reduces by 52 +/- 9% (mean +/- SE) (n = 9/9) NMDA-receptor field potentials evoked by electrical stimulation of Schaffer collaterals and this effect could not be reversed by extensive washing. Nearly the same reduction of the initial response was obtained with different concentrations of DTNB (100 and 500 microM), but the time required to reach the maximal inhibition was concentration-dependent. 3. In keeping with an earlier study oxygen and glucose deprivation for 2-3 min induced a long-term potentiation (LTP) of the NMDA receptor response (+65 +/- 16%, n = 4/6). This potentiation was reversed by DTNB (100-500 microM) (-47 +/- 18%; n = 4/4) and the initial LTP could not be restored upon extensive washing of the drug. 4. TCEP (200 microM), a reagent which reduces S-S bond, amplified the electrically evoked NMDA-receptor EPSP (+27 +/- 12%; n = 3). In addition, TCEP (200 microM), nearly completely reversed the effect of DTNB (200 microM) on anoxia-induced LTP (+56 +/- 19%; n = 3/3). Preliminary results also indicate that TCEP occlude anoxic-LTP (n = 3/4). 5. Following DTNB (200 microM) treatment, oxygen and glucose deprivation did not generate anoxic LTP and extensive washing did not restore a potentiated NMDA field potential. 6. These observations strongly suggest that the redox site of the NMDA receptor is involved in the induction and the maintenance of the anoxic LTP of the NMDA receptor-mediated response in CA1.