RESUMEN
KEY POINTS: To understand how a network operates, its elements must be identified and characterized, and the interactions of the elements need to be studied in detail. In the present study, we describe quantitatively the connectivity of two classes of inhibitory neurons in the hippocampal CA3 area (parvalbumin-positive and cholecystokinin-positive interneurons), a key region for the generation of behaviourally relevant synchronous activity patterns. We describe how interactions among these inhibitory cells and their local excitatory target neurons evolve over the course of physiological and pathological activity patterns. The results of the present study enable the construction of precise neuronal network models that may help us understand how network dynamics is generated and how it can underlie information processing and pathological conditions in the brain. We show how inhibitory dynamics between parvalbumin-positive basket cells and pyramidal cells could contribute to sharp wave-ripple generation. ABSTRACT: Different hippocampal activity patterns are determined primarily by the interaction of excitatory cells and different types of interneurons. To understand the mechanisms underlying the generation of different network dynamics, the properties of synaptic transmission need to be uncovered. Perisomatic inhibition is critical for the generation of sharp wave-ripples, gamma oscillations and pathological epileptic activities. Therefore, we aimed to quantitatively and systematically characterize the temporal properties of the synaptic transmission between perisomatic inhibitory neurons and pyramidal cells in the CA3 area of mouse hippocampal slices, using action potential patterns recorded during physiological and pathological network states. Parvalbumin-positive (PV+) and cholecystokinin-positive (CCK+) interneurons showed distinct intrinsic physiological features. Interneurons of the same type formed reciprocally connected subnetworks, whereas the connectivity between interneuron classes was sparse. The characteristics of unitary interactions depended on the identity of both synaptic partners, whereas the short-term plasticity of synaptic transmission depended mainly on the presynaptic cell type. PV+ interneurons showed frequency-dependent depression, whereas more complex dynamics characterized the output of CCK+ interneurons. We quantitatively captured the dynamics of transmission at these different types of connection with simple mathematical models, and describe in detail the response to physiological and pathological discharge patterns. Our data suggest that the temporal propeties of PV+ interneuron transmission may contribute to sharp wave-ripple generation. These findings support the view that intrinsic and synaptic features of PV+ cells make them ideally suited for the generation of physiological network oscillations, whereas CCK+ cells implement a more subtle, graded control in the hippocampus.
Asunto(s)
Región CA3 Hipocampal/fisiología , Colecistoquinina/fisiología , Interneuronas/fisiología , Parvalbúminas/fisiología , Células Piramidales/citología , Células Piramidales/fisiología , Transmisión Sináptica/fisiología , Potenciales de Acción/fisiología , Animales , Colecistoquinina/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/fisiología , Proteínas Luminiscentes/genética , Masculino , Ratones Transgénicos , Modelos Neurológicos , Parvalbúminas/genética , Regiones Promotoras Genéticas , Proteína Fluorescente RojaRESUMEN
Cannabinoid ligands show therapeutic potential in a variety of disorders including anxiety. However, the anxiety-related effects of cannabinoids remain controversial as agonists show opposite effects in mice and rats. Here we compared the effects of the cannabinoid agonist WIN-55,212 and the CB1 antagonist AM-251 in CD1 mice and Wistar rats. Special attention was paid to antagonist-agonist interactions, which had not yet been studied in rats. In mice, WIN-55,212 decreased whereas AM-251 increased anxiety. The antagonist abolished the effects of the agonist. In contrast, WIN-55,212 increased anxiety in rats. Surprisingly, the antagonist potentiated this effect. Cannabinoids affect both GABAergic and glutamatergic functions, which play opposite roles in anxiety. We hypothesized that discrepant findings resulted from species differences in the relative responsiveness of the two transmitter systems to cannabinoids. We investigated this hypothesis by studying the effects of WIN-55,212 on evoked hippocampal inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs). IPSCs were one order of magnitude more sensitive to WIN-55,212 in mice than in rats. In mice, IPSCs were more sensitive than EPSCs to WIN-55,212. This is the first study showing that the relative cannabinoid sensitivity of GABA and glutamate neurotransmission is species-dependent. Based on behavioural and electrophysiological findings, we hypothesize that WIN-55,212 reduced anxiety in mice by affecting GABA neurotransmission whereas it increased anxiety in rats via glutamatergic mechanisms. In rats, AM-251 potentiated this anxiogenic effect by inhibiting the anxiolytic GABAergic mechanism. We suggest that the anxiety-related effects of cannabinoids depend on the relative cannabinoid responsiveness of GABAergic and glutamatergic neurotransmission.
Asunto(s)
Ansiedad/tratamiento farmacológico , Cannabinoides , Ácido Glutámico/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Ansiolíticos/farmacología , Ansiolíticos/uso terapéutico , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Cannabinoides/agonistas , Cannabinoides/antagonistas & inhibidores , Cannabinoides/farmacología , Cannabinoides/uso terapéutico , Clordiazepóxido/farmacología , Clordiazepóxido/uso terapéutico , Potenciales Postsinápticos Excitadores/fisiología , Conducta Exploratoria/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Ligandos , Masculino , Ratones , Morfolinas/farmacología , Morfolinas/uso terapéutico , Naftalenos/farmacología , Naftalenos/uso terapéutico , Técnicas de Placa-Clamp , Piperidinas/farmacología , Piperidinas/uso terapéutico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/metabolismoRESUMEN
The aim of this study was to compare and contrast the properties of gamma oscillations induced by activation of muscarinic acetylcholine or metabotropic glutamate receptors in the CA3 region of rat hippocampal slices. Both carbachol and the group I metabotropic glutamate receptor agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), induced network oscillations in the gamma-frequency range (30-100 Hz). The M1 muscarinic receptor antagonist, pirenzepine, blocked carbachol-, but enhanced DHPG-induced oscillations, whereas LY 341495, an antagonist at metabotropic glutamate receptors, abolished DHPG-, but left carbachol-induced oscillations unchanged. There were significant differences in the peak frequency, maximal power, and spectral width of the two oscillations. Pharmacological experiments showed that both types of oscillation depend on fast excitatory and inhibitory synaptic transmission. Interestingly, activation of neurokinin-1 receptors by substance P fragment or enhancement of inhibitory synaptic currents by the benzodiazepine ligand, zolpidem, boosted DHPG-, but reduced the power of carbachol-induced oscillations. These results suggest that, although carbachol and DHPG might activate similar conductances in individual pyramidal cells, the oscillations they induce in slices involve different network mechanisms, most likely by recruiting distinct types of GABAergic interneuron.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Hipocampo/efectos de los fármacos , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Benzodiazepinas/farmacología , Bicuculina/farmacología , Diaminas/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Antagonistas de Aminoácidos Excitadores/farmacología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacología , Pirenzepina/farmacología , Piridinas/farmacología , Ratas , Análisis Espectral , Sustancia P/farmacología , Xantenos/farmacología , ZolpidemRESUMEN
A subset of GABAergic neurons projecting to the medial septum has long been described in the hippocampus. However, the lack of information about their local connectivity pattern or their correspondence with any of the well-established hippocampal interneuron types has hampered the understanding of their functional role. Retrograde tracing combined with immunostaining for neurochemical markers in the adult rat hippocampus showed that nearly all hippocampo-septal (HS) neurons express somatostatin (>95%) and, in the hilus and CA3 stratum lucidum, many contain calretinin (>45%). In contrast, in stratum oriens of the CA1 and CA3 subfields, the majority of HS neurons contain somatostatin (>86%) and calbindin (>73%), but not calretinin. Because somatostatin-positive hippocampal interneurons have been most extensively characterized in the stratum oriens of CA1, we focused our further analysis on HS cells found in this region. In 18-20-day-old rats, intracellularly filled CA1-HS cells had extensive local axon collaterals crossing subfield boundaries and innervating the CA3 region and the dentate gyrus. Electron microscopic analysis provided evidence that the axon terminals of CA1-HS cells form symmetrical synapses selectively on GABAergic interneurons, both locally and in the CA3 region. In addition, double retrograde labelling experiments revealed that many CA1-HS neurons of the dorsal hippocampus also have collateral projections to the ventral hippocampus. Thus, CA1-HS cells innervate inhibitory interneurons locally and in remote hippocampal regions, in addition to targeting mostly GABAergic neurons in the medial septum. This dual projection with striking target selectivity for GABAergic neurons may be ideally suited to synchronize neuronal activity along the septo-hippocampal axis.
Asunto(s)
Hipocampo/fisiología , Interneuronas/fisiología , Inhibición Neural/fisiología , Núcleos Septales/fisiología , Animales , Hipocampo/ultraestructura , Interneuronas/ultraestructura , Masculino , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Ratas , Ratas Wistar , Núcleos Septales/ultraestructuraRESUMEN
NMDA receptors participate in the glutamatergic excitation of both principal cells and GABAergic interneurons. The features of NMDA channels on specific interneurons, however, are not known. Therefore, we obtained direct measurements of single NMDA receptor channels on anatomically identified oriens/alveus interneurons projecting to stratum lacunosum-moleculare (O-LM cells) and compared them to those found on hippocampal principal cells using cell-attached recordings in in vitro slice preparations. The recorded channels could be blocked by ketamine, a membrane-permeable NMDA channel inhibitor. In the absence of Mg(2+), all O-LM cells had NMDA channels with a comparable slope conductance (approximately 60pS) to those measured on CA1 pyramidal cells or dentate granule cells. In addition, NMDA channels with smaller conductance (43-45 pS) were also found on two O-LM cells but not on principal cells. These results suggest that at least two types of NMDA channels are expressed on O-LM cells likely reflecting distinct subunit composition.
Asunto(s)
Hipocampo/fisiología , Interneuronas/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Hipocampo/citología , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Ratas , Ratas Wistar , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB(1) cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona, I. et al., Journal of Neuroscience 19 (1999) 4544; Hájos, N. et al., European Journal of Neuroscience 12 (2000) 3239; Hájos, N. et al., Neuroscience 106 (2001) 1). The novel receptor does not correspond to CB(2), since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB(1) receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC(50) values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC(50) value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 microM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 microM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB(1). We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 microM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 microM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs. These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB(1) than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB(1) (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.
Asunto(s)
Hipocampo/química , Fibras Nerviosas/fisiología , Receptor Cannabinoide CB2 , Receptores de Droga/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Benzoxazinas , Capsaicina/farmacología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Fibras Nerviosas/efectos de los fármacos , Piperidinas/farmacología , Células Piramidales/efectos de los fármacos , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , Receptores de Droga/aislamiento & purificación , Reflejo Monosináptico/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Cannabinoids are the most popular illicit drugs used for recreational purposes worldwide. However, the neurobiological substrate of their mood-altering capacity has not been elucidated so far. Here we report that CB1 cannabinoid receptors are expressed at high levels in certain amygdala nuclei, especially in the lateral and basal nuclei, but are absent in other nuclei (e.g., in the central nucleus and in the medial nucleus). Expression of the CB1 protein was restricted to a distinct subpopulation of GABAergic interneurons corresponding to large cholecystokinin-positive cells. Detailed electron microscopic investigation revealed that CB1 receptors are located presynaptically on cholecystokinin-positive axon terminals, which establish symmetrical GABAergic synapses with their postsynaptic targets. The physiological consequence of this particular anatomical localization was investigated by whole-cell patch-clamp recordings in principal cells of the lateral and basal nuclei. CB1 receptor agonists WIN 55,212-2 and CP 55,940 reduced the amplitude of GABA(A) receptor-mediated evoked and spontaneous IPSCs, whereas the action potential-independent miniature IPSCs were not significantly affected. In contrast, CB1 receptor agonists were ineffective in changing the amplitude of IPSCs in the rat central nucleus and in the basal nucleus of CB1 knock-out mice. These results suggest that cannabinoids target specific elements in neuronal networks of given amygdala nuclei, where they presynaptically modulate GABAergic synaptic transmission. We propose that these anatomical and physiological features, characteristic of CB1 receptors in several forebrain regions, represent the neuronal substrate for endocannabinoids involved in retrograde synaptic signaling and may explain some of the emotionally relevant behavioral effects of cannabinoid exposure.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Receptores de Droga/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Analgésicos/farmacología , Animales , Benzoxazinas , Moduladores de Receptores de Cannabinoides , Cannabinoides/farmacología , Colecistoquinina/biosíntesis , Ciclohexanoles/farmacología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Interneuronas/ultraestructura , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Noqueados , Morfolinas/farmacología , Naftalenos/farmacología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Especificidad de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/agonistas , Receptores de GABA-A/metabolismoRESUMEN
Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.
Asunto(s)
Cannabinoides/farmacología , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Inhibición Neural/efectos de los fármacos , Receptores de Droga/deficiencia , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Analgésicos/farmacología , Animales , Benzoxazinas , Cannabinoides/metabolismo , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Ratones Noqueados , Morfolinas/farmacología , Naftalenos/farmacología , Inhibición Neural/fisiología , Piperidinas/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Pirazoles/farmacología , Receptores de Cannabinoides , Receptores de Droga/efectos de los fármacos , Receptores de Droga/genética , Receptores de GABA/efectos de los fármacos , Receptores de GABA/metabolismo , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Rimonabant , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Using a new antibody developed against the C-terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N-terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212-2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential-driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+-independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1-/- knock-out mice to confirm the specificity of the antibody and of the agonist (WIN55,212-2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+-dependent GABA release, and thereby reduces the power of hippocampal network oscillations.
Asunto(s)
Cannabinoides/metabolismo , Hipocampo/metabolismo , Inhibición Neural/fisiología , Receptores de Droga/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Analgésicos/farmacología , Animales , Anticuerpos , Benzoxazinas , Ciclohexanoles/farmacología , Electrofisiología , Epítopos/análisis , Epítopos/inmunología , Hipocampo/química , Interneuronas/efectos de los fármacos , Interneuronas/fisiología , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Microscopía Electrónica , Morfolinas/farmacología , Naftalenos/farmacología , Vías Nerviosas/fisiología , Periodicidad , Piperidinas/farmacología , Terminales Presinápticos/química , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/análisis , Receptores de Droga/inmunología , Rimonabant , Transmisión Sináptica/fisiologíaRESUMEN
The degree of postsynaptic type A gamma-aminobutyric acid receptor (GABAA receptor) occupancy was investigated by using the benzodiazepine agonist zolpidem. This drug increases the affinity of GABAA receptors for gamma-aminobutyric acid (GABA) at room temperature (Perrais & Ropert 1999, J. Neurosci., 19, 578) leading to an enhancement of synaptic current amplitudes if receptors are not fully occupied by the released transmitter. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from eight different cell types in three brain regions of rats and mice. Receptors in every cell type were benzodiazepine sensitive, as 10-20 microM zolpidem prolonged the decays of mIPSCs (151-184% of control). The amplitude of the GABAA receptor-mediated events was significantly enhanced in dentate granule cells, CA1 pyramidal cells, hippocampal GABAergic interneurons, cortical layer V pyramidal cells, cortical layer V interneurons, and in cortical layer II/III interneurons. An incomplete postsynaptic GABAA receptor occupancy is thus predicted in these cells. In contrast, zolpidem induced no significant change in mIPSC amplitudes recorded from layer II/III pyramidal cells, suggesting full GABAA receptor occupancy. Moreover, different degrees of receptor occupancy could be found at distinct GABAergic synapses on a given cell. For example, of the two distinct populations of zolpidem-sensitive mIPSCs recorded in olfactory bulb granule cells, the amplitude of only one type was significantly enhanced by the drug. Thus, at synapses that generate mIPSCs, postsynaptic receptor occupancy is cell type and synapse specific, reflecting local differences in the number of receptors or in the transmitter concentration in the synaptic cleft.
Asunto(s)
Encéfalo/citología , Neuronas/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Animales , Bicuculina/farmacología , Encéfalo/ultraestructura , Estimulación Eléctrica , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Ácido Quinurénico/farmacología , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Piridinas/farmacología , Ratas , Ratas Wistar , Sinapsis/ultraestructura , Tetrodotoxina/farmacología , ZolpidemRESUMEN
Vasoactive intestinal polypeptide-immunoreactive interneurons are known to form three anatomically and neurochemically well-characterized neuron populations in the hippocampus. Two of these establish synaptic contacts selectively with other GABAergic cells (interneuron-selective cells), whereas the third type innervates pyramidal cell bodies and proximal dendrites like a conventional basket cell. Our aim was to examine which of the vasoactive intestinal polypeptide-containing interneuron populations are among the targets of GABAergic septohippocampal and serotonergic raphe-hippocampal pathways. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with double immunocytochemistry for vasoactive intestinal polypeptide was used at the light and electron microscopic levels. Our results show that both interneuron-selective cells and vasoactive intestinal polypeptide-containing basket cells receive synaptic input from the medial septum and median raphe nucleus. The GABAergic component of the septohippocampal pathway establishes multiple contacts on both cell types. In the case of the raphe-hippocampal projection, single or double contacts were more frequent on vasoactive intestinal polypeptide-positive interneuron selective cells (76%), whereas multiple contacts predominated on basket cells (83%). The extrinsic GABAergic innervation of interneuron-selective cells in the hippocampus indicates a complex interaction among GABAergic systems, which might ensure the timing and rhythmic synchronization of inhibitory processes in the hippocampus. On the other hand, our results suggest that the serotonergic effect on perisomatic inhibition is exerted via vasoactive intestinal polypeptide-containing basket cells that are functionally distinct from their parvalbumin-positive relatives, which appear to escape control of serotonergic as well as local interneuron-selective cells.
Asunto(s)
Hipocampo/fisiología , Interneuronas/fisiología , Núcleos del Rafe/fisiología , Tabique Pelúcido/fisiología , Sinapsis/fisiología , Péptido Intestinal Vasoactivo/análisis , Vías Aferentes/citología , Vías Aferentes/fisiología , Animales , Transporte Axonal , Dendritas/fisiología , Dendritas/ultraestructura , Hipocampo/anatomía & histología , Inmunohistoquímica , Interneuronas/citología , Masculino , Microscopía Inmunoelectrónica , Fitohemaglutininas , Núcleos del Rafe/citología , Núcleos del Rafe/ultraestructura , Ratas , Tabique Pelúcido/citología , Sinapsis/ultraestructura , Ácido gamma-Aminobutírico/análisisRESUMEN
Changes in synaptic efficacy are essential for neuronal development, learning and memory formation and for pathological states of neuronal excitability, including temporal-lobe epilepsy. At synapses, where there is a high probability of opening of postsynaptic receptors, all of which are occupied by the released transmitter, the most effective means of augmenting postsynaptic responses is to increase the number of receptors. Here we combine quantal analysis of evoked inhibitory postsynaptic currents with quantitative immunogold localization of synaptic GABA(A) receptors in hippocampal granule cells in order to clarify the basis of inhibitory synaptic plasticity induced by an experimental model of temporal-lobe epilepsy (a process known as kindling). We find that the larger amplitude (66% increase) of elementary synaptic currents (quantal size) after kindling results directly from a 75% increase in the number of GABA(A) receptors at inhibitory synapses on somata and axon initial segments. Receptor density was up by 34-40% and the synaptic junctional area was expanded by 31%. Presynaptic boutons were enlarged, which may account for the 39% decrease in the average number of released transmitter packets (quantal content). Our findings establish the postsynaptic insertion of new GABA(A) receptors and the corresponding increase in postsynaptic responses augmenting the efficacy of mammalian inhibitory synapses.
Asunto(s)
Hipocampo/fisiología , Inhibición Neural/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Animales , Giro Dentado/citología , Giro Dentado/fisiología , Electrofisiología , Potenciales Evocados , Hipocampo/citología , Hipocampo/ultraestructura , Técnicas para Inmunoenzimas , Excitación Neurológica/fisiología , Plasticidad Neuronal , Neuronas/fisiología , Ratas , Ratas WistarRESUMEN
The Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin (CN) participates in several Ca2+-dependent signal transduction cascades and, thus, contributes to the short and long term regulation of neuronal excitability. By using a specific antibody to CN, we demonstrate its absence from hippocampal interneurons and illustrate a physiological consequence of such CN deficiency. Consistent with the lack of CN in interneurons as detected by immunocytochemistry, the CN inhibitors FK-506 or okadaic acid significantly prolonged N-methyl-D-aspartate channel openings recorded in the cell-attached mode in hippocampal principal cells but not those recorded in interneurons. Interneurons were also devoid of Ca2+/calmodulin-dependent protein kinase IIalpha, yet many of their nuclei contained the cyclic AMP-responsive element binding protein. On the basis of the CN and Ca2+/calmodulin-dependent protein kinase IIalpha deficiency of interneurons, entirely different biochemical mechanisms are expected to govern Ca2+-dependent neuronal plasticity in interneurons versus principal cells.
Asunto(s)
Calcio/metabolismo , Hipocampo/fisiología , Interneuronas/fisiología , Proteínas del Tejido Nervioso/deficiencia , Plasticidad Neuronal/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Calcineurina/deficiencia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/deficiencia , Calmodulina/deficiencia , Compartimento Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/aislamiento & purificación , Electrofisiología/métodos , Hipocampo/citología , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de SeñalRESUMEN
In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the hippocampal formation. Only calretinin and somatostatin showed an appreciable degree of co-localization with m2 (20% and 15%, respectively). Using retrograde tracing, some of the m2-positive cells in stratum oriens were shown to project to the medial septum, accouting for 38% of all projection neurons. The present results demonstrate that there is a differential distribution of m2 receptor immunoreactivity on the axonal vs the somadendritic membranes of distinct interneuron types and suggest that acetylcholine via m2 receptors may reduce GABA release presynaptically from the terminals of perisomatic inhibitory cells, while it may act to increase the activity of another class of interneuron, which innervates the dendritic region of pyramidal cells.
Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Interneuronas/metabolismo , Terminales Presinápticos/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Axones/fisiología , Axones/ultraestructura , Dendritas/ultraestructura , Hipocampo/citología , Hipocampo/ultraestructura , Inmunohistoquímica , Interneuronas/ultraestructura , Masculino , Parvalbúminas/metabolismo , Perfusión , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The neuropeptide calcitonin gene-related peptide (CGRP) was localized in the hippocampus and dentate gyrus of the rat by immunocytochemistry at the light and electron microscopic levels. Without colchicine treatment only faint neuropil labelling was found in the inner molecular layer of the dentate gyrus. Following colchicine treatment, a large number of neurons with numerous complex spines along the proximal dendrites were visualized in the hilus of the dentate gyrus, particularly in the ventral areas, and, in addition, staining of the inner molecular layer became stronger. Several CA3c pyramidal cells located adjacent to the hilar region in the ventral hippocampus also appeared to be faintly positive, although in most cases only their axon initial segments were labelled. Outside this region, the subicular end of the CA1 subfield contained occasional CGRP-positive non-pyramidal cells. The hilar CGRP-positive neurons were negative for parvalbumin, calretinin, cholecystokinin and somatostatin, whereas most of them were immunoreactive for GluR2/3 (the AMPA-type glutamate receptor known to be expressed largely by principal cells). Correlated electron microscopy showed that the spines along the proximal dendritic shafts indeed correspond to thorny excrescences engulfed by large complex mossy terminals forming asymmetrical synapses. Pre-embedding immunogold staining demonstrated that CGRP immunoreactivity in the inner molecular layer was confined to axon terminals that form asymmetrical synapses, and the labelling was associated with large dense-core vesicles. The present data provide direct evidence that CGRP is present in mossy cells of the dentate gyrus and to a lesser degree in CA3c pyramidal cells of the ventral hippocampus. These CGRP-containing principal cells terminate largely in the inner molecular layer of the dentate gyrus, and may release the neuropeptide in conjunction with their 'classical' neurotransmitter, glutamate.
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Péptido Relacionado con Gen de Calcitonina/análisis , Giro Dentado/química , Fibras Musgosas del Hipocampo/química , Animales , Biomarcadores/química , Proteínas de Unión al Calcio/análisis , Giro Dentado/citología , Inmunohistoquímica , Masculino , Microscopía Electrónica , Neuropéptidos/análisis , Ratas , Ratas WistarRESUMEN
The properties of spontaneous IPSCs (sIPSCs) recorded with whole-cell patch-clamp techniques were investigated in various anatomically identified hippocampal CA1 interneurons and were compared with those recorded in pyramidal cells. Neurons labeled with biocytin or neurobiotin were classified on the basis of their dendritic and axonal arborizations, leading to the identification of previously unknown interneuron types projecting to the dendritic region of pyramidal cells. In most interneurons, the average sIPSCs decayed slower than did those observed in pyramidal cells. The properties of sIPSCs were homogeneous within a given morphologically identified neuron type. Many interneurons had comparable somatic size, location, and dendritic arbor but displayed extremely different axonal projections paralleled by distinct sIPSC properties. Thus, physiological comparisons are only meaningful after the complete morphological identification of the recorded cells. The decay of sIPSCs matched for amplitudes and rise times could vary over 10-fold in a given interneuron, consistent with electrotonic filtering and possibly with different GABAA receptor subunit assemblies present at distinct synapses. Our findings demonstrate an extensive connectivity among hippocampal interneurons through GABAA synapses of various properties that may underlie complex network oscillations at different frequencies.
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Hipocampo/fisiología , Interneuronas/fisiología , Plasticidad Neuronal , Receptores de GABA-A/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción , Animales , Dendritas/ultraestructura , Vías Eferentes/citología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/citología , Ácido Quinurénico/farmacología , Masculino , Técnicas de Placa-Clamp , Células Piramidales/fisiología , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacosRESUMEN
Vasoactive intestinal polypeptide (VIP) has been shown to be present in a morphologically heterogeneous subpopulation of interneurons in the dentate gyrus, but the relationship between their input and output characteristics and neurochemical features has not been established. Three types of VIP-immunoreactive cells have been identified on the basis of these criteria: (i) cells forming a dense axonal plexus in the hilus have always coexisted with the calcium binding protein calretinin (CR), but never with the neuropeptide cholecystokinin (CCK). The postsynaptic targets of these VIP-positive cells were neurons visualized by immunostaining for substance P receptor, which is known to label different hilar non-principal cells. (ii) VIP-immunoreactive basket cells, innervating predominantly the somata and proximal dendrites of granule cells, were found in the striatum moleculare and stratum granulosum. They contained CCK, but not CR. (iii) Cells projecting to the stratum moleculare were found to have dendrites and axons restricted to this layer. In 75% of these cells VIP coexisted with CR but not with CCK, and they established multiple contacts largely with non-principal cells. GABA was shown to be present but the calcium-binding proteins calbindin D28K and parvalbumin were absent in all three types of VIP-containing interneuron. On the basis of these observations we conclude that three different types of VIP-positive neuron are present in this area, and are likely to subserve different inhibitory functions, cells with a hilar projection as well as those projecting to the stratum moleculare may synchronize the activity of hilar and other interneurons, or disinhibit granule cells by specific interneuron-to-interneuron connections. In contrast, basket cells control the activity of granule cells directly, via perisomatic inhibition.
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Giro Dentado/química , Interneuronas/química , Sinapsis/ultraestructura , Péptido Intestinal Vasoactivo/análisis , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Giro Dentado/citología , Giro Dentado/ultraestructura , Inmunohistoquímica , Interneuronas/ultraestructura , Masculino , Vías Nerviosas/ultraestructura , Ratas , Ratas WistarRESUMEN
Spine-free calretinin-immunoreactive (CR-IR) interneurons form a subpopulation of GABAergic cells in the rat hippocampus. A characteristic feature of these cells--located in all areas and layers--is the frequent dendro-dendritic and axo-dendritic contacts they form with each other. In this study we examined in detail the connectivity of these neurons by reconstructing their dendritic and axonal arbor and by identifying their postsynaptic targets. Radially running dendrites of CR-IR cells, located in different layers, intermingled into long braids. An average cell was in contact with dendrites of three to seven other CR-IR cells. Reconstruction of the dendritic trees from six consecutive sections demonstrated that at least 15 cells may participate in a dendro-dendritically connected cluster. Electron microscopical examination revealed that regularly spaced zonula adherentia connect the touching dendrites. The postsynaptic targets of CR-IR neurons have been examined using postembedding immunogold staining for GABA. CR-containing GABA-immunoreactive axons of local origin formed multiple symmetrical synaptic contacts (two to five) exclusively on GABAergic dendrites (CR-negative as well as CR-positive). Two to 10 CR-IR axons may converge onto a single CR-IR neuron, often from cells belonging to the same dendro-dendritically connected cluster. Using double immunocytochemistry, CR-IR cells were shown to heavily innervate calbindin D28k-containing interneurons and VIP-containing basket cells but avoided the parvalbumin-containing basket and axo-axonic cells. The unique connectivity of CR-IR cells may enable them to play a crucial role in the generation of synchronous, rhythmic hippocampal activity by controlling other interneurons terminating on different dendritic and somatic compartments of principal cells.
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Hipocampo/anatomía & histología , Interneuronas/fisiología , Animales , Axones/ultraestructura , Calbindina 2 , Hipocampo/ultraestructura , Inmunohistoquímica , Interneuronas/ultraestructura , Masculino , Microscopía Electrónica , Ratas , Ratas Wistar , Proteína G de Unión al Calcio S100/análisis , Ácido gamma-Aminobutírico/análisisRESUMEN
Hippocampal synaptic inhibition is mediated by distinct groups of inhibitory cells. Some contact pyramidal cells perisomatically, while others terminate exclusively on their dendrites. We examined perisomatic and dendritic inhibition by recording from CA3 inhibitory and pyramidal cells and injecting biocytin to visualize both cells in light and electron microscopy. Single perisomatic inhibitory cells made 2-6 terminals clustered around the soma and proximal pyramidal cell processes. Dendritic cells established 5-17 terminals, usually on different dendrites of a pyramidal cells. Perisomatic terminals were larger than those facing dendritic membrane. Perisomatic inhibitory cells initiated the majority of simultaneous IPSPs seen in nearby pyramidal cells. Single IPSPs initiated by perisomatic sodium-dependent action potentials. Activation of inhibitory fibers terminating on dendrites could suppress calcium-dependent spikes. Thus, distinct inhibitory cells may differentially control dendritic electrogenesis and axonal output of hippocampal pyramidal cells.
Asunto(s)
Comunicación Celular , Dendritas/fisiología , Hipocampo/fisiología , Células Piramidales/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Axones/fisiología , Axones/ultraestructura , Calcio/farmacología , Electrodos , Cobayas , Hipocampo/ultraestructura , Técnicas In Vitro , Lisina/análogos & derivados , Microscopía Electrónica , Sodio/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Non-pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea-pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo-axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritic region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritic segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum-moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo-axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo-axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramidale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons--often in conjunction with particular excitatory afferents (Han et al., Eur. J. Neurosci., 5, 395-410, 1993)--underlies functional differences in inhibitory synaptic actions.