RESUMO
Reversal learning measures the ability to form flexible associations between choice outcomes with stimuli and actions that precede them. This type of learning is thought to rely on several cortical and subcortical areas, including the highly interconnected orbitofrontal cortex (OFC) and basolateral amygdala (BLA), and is often impaired in various neuropsychiatric and substance use disorders. However, the unique contributions of these regions to stimulus- and action-based reversal learning have not been systematically compared using a chemogenetic approach particularly before and after the first reversal that introduces new uncertainty. Here, we examined the roles of ventrolateral OFC (vlOFC) and BLA during reversal learning. Male and female rats were prepared with inhibitory designer receptors exclusively activated by designer drugs targeting projection neurons in these regions and tested on a series of deterministic and probabilistic reversals during which they learned about stimulus identity or side (left or right) associated with different reward probabilities. Using a counterbalanced within-subject design, we inhibited these regions prior to reversal sessions. We assessed initial and pre-/post-reversal changes in performance to measure learning and adjustments to reversals, respectively. We found that inhibition of the ventrolateral orbitofrontal cortex (vlOFC), but not BLA, eliminated adjustments to stimulus-based reversals. Inhibition of BLA, but not vlOFC, selectively impaired action-based probabilistic reversal learning, leaving deterministic reversal learning intact. vlOFC exhibited a sex-dependent role in early adjustment to action-based reversals, but not in overall learning. These results reveal dissociable roles for BLA and vlOFC in flexible learning and highlight a more crucial role for BLA in learning meaningful changes in the reward environment.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Ratos , Masculino , Feminino , Animais , Incerteza , Complexo Nuclear Basolateral da Amígdala/fisiologia , Ratos Long-Evans , Córtex Pré-Frontal/fisiologia , Reversão de Aprendizagem/fisiologiaRESUMO
Alcohol use disorder (AUD) is characterized by loss of intake control, increased anxiety, and susceptibility to relapse inducing stressors. Both astrocytes and neurons contribute to behavioral and hormonal consequences of chronic intermittent ethanol (CIE) exposure in animal models. Details on how CIE disrupts hypothalamic neuro-glial communication, which mediates stress responses are lacking. We conducted a behavioral battery (grooming, open field, reactivity to a single, uncued foot-shock, intermittent-access two-bottle choice ethanol drinking) followed by Ca2+ imaging in ex-vivo slices of paraventricular nucleus of the hypothalamus (PVN) from male rats exposed to CIE vapor or air-exposed controls. Ca2+ signals were evaluated in response to norepinephrine (NE) with or without selective α-adrenergic receptor (αAR) or GluN2B-containing N-methyl-D-aspartate receptor (NMDAR) antagonists, followed by dexamethasone (DEX) to mock a pharmacological stress response. Expectedly, CIE rats had altered anxiety-like, rearing, grooming, and drinking behaviors. Importantly, NE-mediated reductions in Ca2+ event frequency were blunted in both CIE neurons and astrocytes. Administration of the selective α1AR antagonist, prazosin, reversed this CIE-induced dysfunction in both cell types. Additionally, the pharmacological stress protocol reversed the altered basal Ca2+ signaling profile of CIE astrocytes. Signaling changes in astrocytes in response to NE were correlated with anxiety-like behaviors, such as the grooming:rearing ratio, suggesting tripartite synaptic function plays a role in switching between exploratory and stress-coping behavior. These data show how CIE exposure causes persistent changes to PVN neuro-glial function and provides the groundwork for how these physiological changes manifest in behavioral selection.
RESUMO
Recent studies have shown that infraorbital nerve constriction (IoNC)-induced mechanical allodynia has been attenuated by administration of highly purified 150-kDa Botulinum neurotoxin type A (BoNT/A). Here, we extend these studies to determine whether BoNT/A could attenuate IoNC-induced symptoms of thermal hyperalgesia. Instead of testing head withdrawal thresholds, a thermal operant assay was used to evaluate cortical processing of sensory input following IoNC. In this assay, a fasted rat's desire to obtain a food reward (sweetened condensed milk) is coupled to its ability to tolerate facial contact with a warm (45 °C) thermode. Bilateral IoNC decreased the ratio of thermode contact duration/event, which is an indicative of thermal hyperalgesia. BoNT/A injection intradermally in the area of infraorbital nerve (IoN) innervation 7 days after IoNC resulted in decreased number of facial contacts and increased the ratio of contact duration/event (measured at 14 days after IoNC). The BoNT/A (2-200 pg) effects were dose dependent and statistically significant at 100 and 200 pg (P < 0·05). Complete reversal of thermal hyperalgesia symptoms was obtained with a 200-pg dose, without affecting sham rat behaviour. Off-site (neck) injection of BoNT/A did not relieve thermal hyperalgesia, while co-injection of BoNT/A with a neutralising antibody in the area of IoN innervation prevented relief of thermal hyperalgesia. Neither IoNC nor BoNT/A injection affected operant assay parameters with a 24 °C thermode, indicating selectivity of thermal hyperalgesia measurements. These results strongly suggest that intradermal injection of BoNT/A in the area of IoN innervation alleviates IoNC-induced thermal hyperalgesia in an operant assay.
Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Hiperalgesia/tratamento farmacológico , Neurotoxinas/administração & dosagem , Órbita/inervação , Limiar da Dor/efeitos dos fármacos , Animais , Antitoxina Botulínica/administração & dosagem , Estudos de Casos e Controles , Constrição Patológica/complicações , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Face , Temperatura Alta/efeitos adversos , Hiperalgesia/etiologia , Injeções Intradérmicas , Masculino , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
We report the characterization of the first successful treatment of neuronal ischemic injury in vivo by cell-permeant Ca2+ chelators. The chelators attenuated glutamate-induced intracellular Ca2+ increases and neurotoxicity in neuronal explant cultures. When infused intravenously in rats, permeant fluorescent BAPTA analogs accumulated in neurons in several brain regions. BAPTA-AM, infused in vivo, reduced Ca(2+)-dependent spike frequency adaptation and post-spike train hyperpolarizations in CA1 neurons taken from treated animals. This effect was reproduced by direct injections of BAPTA into untreated neurons. The effects of three different chelators (BAPTA, 5,5'-difluoro BAPTA, and 4,4'-difluoro BAPTA) on Ca(2+)-dependent membrane excitability varied with their Ca2+ affinity. When the chelators' permeant forms were used to treat rats prior to the induction of focal cortical ischemia, they were highly neuroprotective, as gauged by significant reductions in cortical infarction volumes and neuronal sparing. The chelators' protective effects in vivo also reflected their affinity for Ca2+. This report provides the most direct evidence to date that intracellular Ca2+ excess triggers early neurodegeneration in vivo and contributes a novel therapeutic approach to neuronal ischemia of potential clinical utility.
Assuntos
Isquemia Encefálica/patologia , Cálcio , Quelantes/farmacologia , Neurônios/patologia , Neurotoxinas/farmacologia , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Quelantes/farmacocinética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Glutamatos/farmacologia , Ácido Glutâmico , Camundongos , Camundongos Endogâmicos , Neurônios/efeitos dos fármacos , Neurotoxinas/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Medula Espinal/citologiaRESUMO
Mammalian nociceptors have been classified into subclasses based on differential neurotrophin sensitivity and binding of the plant isolectin B4 (IB4). Most of the nerve growth factor-responsive IB4-negative (IB4 (-)) nociceptors contain neuropeptides such as substance P and calcitonin gene-related peptide, whereas the glial-derived neurotrophic factor-responsive IB4-positive (IB4 (+)) neurons predominantly lack such neuropeptides. We hypothesized that the differences in neuropeptide content between IB4 (+) and (-) neurons might be reflected in differences in stimulated exocytosis and/or endocytosis. To address this, we monitored the secretory activity of acutely dissociated neurons from adult rat trigeminal ganglia (TRG) using cell membrane capacitance (Cm) measurements and the fluorescent membrane-uptake marker N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). Cm measurements were performed under whole-cell voltage clamp and neurons were depolarized from -75 mV to +10 mV to elicit exocytosis. Both types of TRG neurons showed similarly-sized, calcium-dependent increases in Cm, demonstrating that both IB4 (+) and (-) TRG neurons are capable of stimulated exocytosis. However, the peak Cm of IB4 (+) neurons decayed faster toward baseline than that of IB4 (-) neurons. Also, IB4 (+) neurons had stable Cm responses to repeated stimuli whereas IB4 (-) neurons loss their secretory response during repeated stimulation. These data suggested that the IB4 (+) neurons possess a faster rate of endocytosis and vesicle replenishment than IB4 (-) neurons. To test this, we measured vesicle trafficking with the fluorescent membrane dye FM4-64. FM4-64 staining showed that IB4 (-) neurons exhibit a larger pool of endocytosed vesicles than IB4 (+) neurons because the peak fluorescence increases in IB4 (-) neurons were larger but slower than in IB4 (+) neurons. However, the recycled vesicles were released faster in IB4 (+) compared with IB4 (-) neurons. Taken together these data suggest that the IB4 (+) TRG neurons have faster exocytosis and endocytosis than the IB4 (-) neurons.
Assuntos
Lectinas/metabolismo , Neurônios/classificação , Neurônios/fisiologia , Neurotransmissores/metabolismo , Gânglio Trigeminal/citologia , Animais , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Cloreto de Potássio/farmacologia , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos , Fatores de TempoRESUMO
Mounting evidence indicates that ethanol (EtOH) exposure activates neuroimmune signaling. Alterations in pro-inflammatory cytokines after acute and chronic EtOH exposure have been heavily investigated. In contrast, little is known about the regulation of neurotransmission and/or modulation by anti-inflammatory cytokines in the brain after an acute EtOH exposure. Recent evidence suggests that interleukin-10 (IL-10), an anti-inflammatory cytokine, is upregulated during withdrawal from chronic EtOH exposure. In the present study, we show that IL-10 is increased early (1 h) after a single intoxicating dose of EtOH (5 g/kg, intragastric) in Sprague Dawley rats. We also show that IL-10 rapidly regulates GABAergic transmission in dentate gyrus neurons. In brain slice recordings, IL-10 application dose-dependently decreases miniature inhibitory postsynaptic current (mIPSC) area and frequency, and decreases the magnitude of the picrotoxin sensitive tonic current (Itonic), indicating both pre- and postsynaptic mechanisms. A PI3K inhibitor LY294002 (but not the negative control LY303511) ablated the inhibitory effects of IL-10 on mIPSC area and Itonic, but not on mIPSC frequency, indicating the involvement of PI3K in postsynaptic effects of IL-10 on GABAergic transmission. Lastly, we also identify a novel neurobehavioral regulation of EtOH sensitivity by IL-10, whereby IL-10 attenuates acute EtOH-induced hypnosis. These results suggest that EtOH causes an early release of IL-10 in the brain, which may contribute to neuronal hyperexcitability as well as disturbed sleep seen after binge exposure to EtOH. These results also identify IL-10 signaling as a potential therapeutic target in alcohol-use disorders and other CNS disorders where GABAergic transmission is altered.
Assuntos
Encéfalo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Interleucina-10/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipnóticos e Sedativos/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Sono/efeitos dos fármacos , Sono/fisiologia , Transmissão Sináptica/fisiologia , Técnicas de Cultura de TecidosRESUMO
Cell-permeant Ca2+ chelators such as 1,2-bis-(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) have been reported to protect neurons in experimental focal cerebral ischemia. However, their in vivo actions are uncertain, and their protective efficacy is proven only in brief cerebral ischemia paradigms. Here we examine their mechanism of action in vitro and duration of efficacy in vivo. Electrophysiological studies were made in CA1 neurons in rat hippocampal slices. When superfused with BAPTA-AM (30-50 microM), CA1 somatic field potential recordings showed attenuation of the population spike amplitude, and intracellular recordings showed reduced excitatory postsynaptic potentials, indicating inhibition of excitatory synaptic transmission. Also, Ca(2+)-dependent accommodation and post-spike-train hyperpolarizations were reduced, indicating Ca2+ chelation hear the internal cell membrane surface. To determine whether Ca2+ chelators reduce the size of cerebral infarction rather than simply delaying its evolution, we studied the effects of BAPTA-AM treatment on infarction size 24 h after permanent middle cerebral artery occlusion. Fischer rats (n = 8 per group) were pretreated with saline, BAPTA-AM (20 mg/kg), or MK-801 (0.5 mg/kg). Infarction volumes in animals treated with BAPTA-AM were reduced by 50.5% compared with controls (p = 0.018), whereas animals treated with MK-801 experienced a statistically insignificant infarct volume reduction (26%; p = 0.27). These data show a persistence of neuroprotection by the Ca2+ chelator at 24 h and indicate that it may act by attenuating synaptic transmission and subplasma membrane Ca2+ excess.
Assuntos
Encéfalo/efeitos dos fármacos , Cálcio , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/fisiopatologia , Permeabilidade da Membrana Celular , Infarto Cerebral/patologia , Quelantes/farmacocinética , Maleato de Dizocilpina/farmacologia , Ácido Egtázico/farmacologia , Eletrofisiologia , Técnicas In Vitro , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.
Assuntos
Dendritos/fisiologia , Fibras Musgosas Hipocampais/patologia , Estado Epiléptico/patologia , Animais , Dendritos/ultraestrutura , Epilepsia do Lobo Temporal/patologia , Agonistas de Aminoácidos Excitatórios , Ácido Caínico , Masculino , Microscopia Eletrônica , Fibras Musgosas Hipocampais/ultraestrutura , Agonistas Muscarínicos , Pilocarpina , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamenteRESUMO
The membrane electrical properties of neurons and their responses to endogenous compounds or other neuroactive substances were investigated in vitro with intracellular recording techniques in slices of trigeminal root ganglia of guinea-pigs. The mean resting membrane potential of these neurons was -60 mV. Intracellular injections of hyperpolarizing current pulses evoked time-dependent rectification with varying degrees of dependence on membrane voltage in 107 of 110 neurons. Membrane potential oscillations were observed following the termination of the hyperpolarizing pulses and after similar injections of depolarizing current. This phenomenon appeared to be voltage-dependent at levels that were subthreshold for spike genesis; the more pronounced oscillations were evident at the more depolarized levels and were insensitive to tetrodotoxin applications. Two groups of neurons could be distinguished on the basis of certain characteristics in their action potentials. The majority exhibited short duration (0.6 ms) spikes with mean amplitude of 72 mV in response to intracellular depolarizing current. The brief (3 ms) afterhyperpolarizations that followed such spikes were blocked by intracellular injections of Cs+ or by bath applications of tetraethylammonium. Action potentials in the minority group exhibited a hump in their repolarization phase. The humped spikes had a mean peak amplitude of 78 mV and a longer duration (2 ms). Both the duration (6 ms) and the amplitude (16 mV) of the afterhyperpolarization were significantly greater in this latter group of neurons. Some fast spikes were easily blocked whereas others, including humped spikes, were resistant to tetrodotoxin (10(-6) M). Spikes which were resistant, were also not affected by perfusion with Co2+ (10(-3) M) and were reduced in amplitude during perfusion with Na+-deficient solution. Bath applications of S-glutamate (10(-4)-10(-2) M) depolarized only two of ten neurons by less than 3 mV. Similarly, 5-hydroxytryptamine produced a small depolarization in only two of thirteen neurons. Perfusion of gamma-aminobutyrate (10(-5)-10(-2) M) resulted in an increase in input conductance that waned despite continued application and was associated with a depolarization (2-14 mV) in 44/50 neurons. In some neurons, gamma-aminobutyrate application enhanced their repetitive firing ability, possibly as a result of the increased oscillatory behavior of the membrane at certain depolarized potentials. The effects of gamma-aminobutyrate were blocked by the GABAA-receptor antagonist, bicuculline (10(-4) M) but were unaffected by the GABAB-receptor agonist, baclofen (10(-4) M).(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Gânglio Trigeminal/fisiologia , Nervo Trigêmeo/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cátions/farmacologia , Estimulação Elétrica , Feminino , Cobaias , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neurotransmissores/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Compostos de Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia , Gânglio Trigeminal/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Applications of histamine to neurons in slices of trigeminal root ganglia (guinea-pig) produced slow changes in the steady-state membrane potentials and input resistances. Several types of response to histamine could be distinguished: (i) depolarizations accompanied by an increase, a decrease or no change in input resistance; (ii) small hyperpolarizations associated with a decreased or unchanged input resistance; and (iii) combined hyper- and depolarizations. The amplitudes of all response types waned during prolonged applications of histamine. The depolarizing responses to histamine appeared to depend on the presence of outward rectification in the region of the initial resting potential; neurons which possessed linear current-voltage relationships near the initial resting potential were depolarized by > 10 mV, whereas neurons with outward rectification near rest showed smaller depolarizing responses. Histamine also reduced the magnitude of the long-duration spike afterhyperpolarizations which had been attributed in the ganglionic neuron to a Ca(2+)-activated K+ conductance mechanism. Application of substance P, another possible neuromodulator in the trigeminal system, had depolarizing, desensitizing actions similar to those of histamine. Substance P and histamine did not cross-desensitize during prolonged applications. Histamine-induced depolarizations were unchanged under zero Mg2+ extracellular conditions, in contrast to a dependency of the substance P-induced effects on external Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Histamina/farmacologia , Neurônios/fisiologia , Gânglio Trigeminal/fisiologia , Animais , Cálcio/farmacologia , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Cobaias , Técnicas In Vitro , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Substância P/farmacologia , Gânglio Trigeminal/efeitos dos fármacosRESUMO
The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3-7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 microM producing significantly less release. The effective concentration for 50% of response (EC(50)) for capsaicin, calculated from its stimulatory phase, was 2.3 microM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC(50)=0.37 microM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC(50)=1.9 microM) and lamina IV (EC(50)=14.5 microM), suggesting that neurokinins released in laminae I-II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I (R(2)=0.82) and III (R(2)=0.78), but it was poor (R(2)=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.
Assuntos
Capsaicina/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Receptores da Neurocinina-1/metabolismo , Medula Espinal/efeitos dos fármacos , Substância P/metabolismo , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados/farmacologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Técnicas In Vitro , Microscopia Confocal/instrumentação , Células do Corno Posterior/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/anatomia & histologia , Medula Espinal/química , Medula Espinal/metabolismo , Fatores de TempoRESUMO
Calcium chelators are commonly used for fluorescence and electrophysiological studies of neuronal Ca2+ signalling. Recently, they have also been used as neuroprotectants. Since they buffer calcium ions, these agents also modify the same signals which are being studied. These properties may be used to modulate Ca2+ signals such as those involved in synaptic transmission, and may explain their neuroprotective mechanism. To define factors which govern the modulation of synaptic transmission by Ca2+ chelators, we examined their actions on synaptic responses evoked in CA1 neurons of rat hippocampal slices. We used a spectrum of cell-permeant Ca2+ chelators having different structures, Ca(2+)-binding kinetics and Ca2+ affinities, as well as an impermeant, intracellularly perfused chelator salt. Application of the cell-permeant 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate acetoxymethyl ester (50 microM) markedly attenuated evoked synaptic responses. This application produced an intracellular chelator accumulation of 79-125 microM, as estimated using 14C-labelled chelator. The actions of a Ca2+ chelator on synaptic responses were dependent on the chelator's Ca2+ affinity, Ca(2+)-binding rate and Ca2+ selectivity, because 1,2-bis(2-amino-5-nitrophenoxy)ethane-N,N,N',N'-tetra-acetate acetoxymethyl ester (a low Ca2+ affinity analogue), ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetate acetoxymethyl ester (a slow buffer with similar Ca2+ affinity to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate) and the selective Zn2+ chelator, tetrakis(2-pyridylmethyl)ethylenediamine, were ineffective. The intrinsic cell membrane properties, including the post-spike train afterhyperpolarization, were not significantly affected by any of the Ca2+ chelators used in this study. Intracellular perfusion of 100-200 microM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate salt through patch pipettes into postsynaptic cells did not affect synaptic potentials, suggesting a presynaptic action of cell-permeant Ca2+ chelators on transmitter release. Other cell-permeant, fast Ca(2+)-binding chelators reduced synaptic responses according to their Ca2+ affinities, and not their chemical structure: those chelators with Kd values < or = 25 microM attenuated synaptic responses, whereas chelators of lesser affinity did not. These data support the ideas that [Ca2+]i rises to high (micromolar) levels during transmitter release, and that Ca2+ chelators may be used to attenuate excitotoxicity by attenuating excitatory neurotransmission without affecting Ca2+ signalling in the submicromolar [Ca2+]i range.
Assuntos
Cálcio/metabolismo , Quelantes/farmacologia , Hipocampo/fisiologia , Fármacos Neuroprotetores/farmacologia , Transmissão Sináptica/fisiologia , Animais , Autorradiografia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quelantes/metabolismo , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Cinética , Masculino , Potenciais da Membrana/fisiologia , Microeletrodos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacosRESUMO
Mossy fibre sprouting and re-organization in the inner molecular layer of the dentate gyrus is a characteristic of many models of temporal lobe epilepsy including that induced by perforant-path stimulation. However, neuroplastic changes on the dendrites of granule cells have been less-well studied. Basal dendrites are a transient morphological feature of rodent granule cells during development. The goal of the present study was to examine whether granule cell basal dendrites are generated in rats with epilepsy induced by perforant-path stimulation. Adult Wistar rats were stimulated for 24 h at 2 Hz and with intermittent (1/min) trains (10 s duration) of single stimuli at 20 Hz (20 V, 0.1 ms) delivered 1/min via an electrode placed in the angular bundle. The brains of these experimental rats and age- and litter-matched control animals were processed for the rapid Golgi method. All rats with perforant-path stimulation displayed basal dendrites on many Golgi-impregnated granule cells. These basal dendrites mainly originated from their somata at the hilar side and then extended into the hilus. Quantitative analysis of more than 800 granule cells in the experimental and matched control brains showed that 6-15% (mean=8.7%) of the impregnated granule cells have spiny basal dendrites on the stimulated side, as well as the contralateral side (mean=3.1%, range=2.9-3.9%) of experimental rats, whereas no basal dendrites were observed in the dentate gyrus from control animals. The formation of basal dendrites appears to be an adaptive morphological change for granule cells in addition to the previously described mossy fibre sprouting, as well as dendritic and somatic spine formation observed in the dentate gyrus of animal and human epileptic brains. The presence of these dendrites in the subgranular region of the hilus suggests that they may be postsynaptic targets of the mossy fibre collaterals.
Assuntos
Dendritos/fisiologia , Giro Denteado/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Via Perfurante/fisiopatologia , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Dendritos/patologia , Dendritos/ultraestrutura , Giro Denteado/patologia , Giro Denteado/fisiologia , Modelos Animais de Doenças , Estimulação Elétrica , Eletrofisiologia , Epilepsia do Lobo Temporal/patologia , Humanos , Técnicas In Vitro , Masculino , Fibras Nervosas/fisiologia , Via Perfurante/fisiologia , Ratos , Ratos WistarRESUMO
Cultures were prepared from ventral spinal cord tissue from 8-11-week gestational human fetuses and grown for a period of up to 6 months. These cultures were studied by morphological, immunocytochemical and intracellular electrophysiological techniques. From 2 weeks in vitro and onward, small bipolar cells were found in outgrowths of spinal cord explants and were identified as neurons by positive immunoreactions with an antibody specific for neurofilament protein. In addition, a large population of glial fibrillary acidic protein-positive astrocytes and a smaller number of galactocerebroside-positive oligodendrocytes were recognized in these cultures. The development of synaptic terminals was also studied by electron microscopy. The first appearance of synaptic terminal was found in a 3-week culture and was an axo-dendritic synapse. During the next 2 months, there was a steady increase in number and structural maturation of synaptic profiles. In addition to axo-dendritic synapses, which were most common, axo-somatic and axo-axonic synapses were demonstrated. After 3 months in culture, the occurrence of large neurons possessing the characteristic features of mature neurons was also noted. Although the occurrence of oligodendrocytes in these cultures was confirmed, no myelination of axons was demonstrated by electron microscopy. Intracellular recordings were obtained from the cultured spinal cord cells, and these cells were identified clearly as neurons by their action potential responses to depolarizing current pulses. The average input resistance of these neurons was 31 M omega with resting membrane potential of -52 +/- 2.3 mV.
Assuntos
Técnicas de Cultura/métodos , Neurônios/citologia , Medula Espinal/embriologia , Eletrofisiologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Microscopia EletrônicaRESUMO
SP is involved in sensory transmission as a mediator of excitation at target tissues following its release from the terminals of certain primary afferent fibers. In view of the pronounced SP effects on the perikarya of sensory neurons, it seems reasonable to suggest that its actions may extend to autoreceptors and to the enhancement of the excitabilities of sensory neurons that do not synthesize the peptide. This hypothesis would be strengthened by the demonstration of nonsynaptic release of SP within sensory ganglia, which would provide another site for interaction of SP with the various component cells of the sensory system.
Assuntos
Neurônios Aferentes/fisiologia , Substância P/farmacologia , Animais , Potenciais Evocados/efeitos dos fármacos , Técnicas In Vitro , Modelos Neurológicos , Neurônios Aferentes/efeitos dos fármacos , Neurotransmissores/farmacologia , Receptores da Neurocinina-1 , Receptores de Neurotransmissores/efeitos dos fármacos , Receptores de Neurotransmissores/fisiologiaRESUMO
GABA(A) receptor-mediated function was studied in rats treated with chronic intermittent ethanol (CIE). Rats were given 60 doses of 6g/kg ethanol every 24 h by gastric intubation, with repeated intoxicating and withdrawal episodes leading to a kindling-like increase in seizure susceptibility (Kokka et al., Alcohol: Clin. Exp. Res., 17 (1993) 525-531). Efflux of 36Cl-, evoked by application of muscimol, a measure of GABA(A) receptor function, was examined in 300 mu m slices obtained from frontal, parietal, and temporal cortex, hippocampus, and inferior colliculus, one day after the last administration of ethanol. Compared to controls, the 36Cl- efflux in hippocampal slices of CIE rats was significantly reduced by 29%, while there were no changes in the other brain regions studied. In hippocampal slices, paired-pulse inhibition in CA1 pyramidal neurons, measured extracellularly using homosynaptic orthodromic stimulation at an interval of 10 ms, was significantly reduced in CIE rats. A significant decrease by 40% both at 2 and 40 days after 60 doses of ethanol was found, implying a persistent decrease in GABA(A) receptor-mediated inhibition in CIE rats. These reductions in paired-pulse inhibition are consistent with the decrease in the pentylenetetrazol (PTZ) seizure threshold which was previously observed in CIE rats. Therefore, we suggest that this reduction of GABA(A) receptor-mediated inhibition contributes to the persistent increase in seizure susceptibility of CIE rats.
Assuntos
Etanol/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Receptores de GABA-A/fisiologia , Animais , Encéfalo/metabolismo , Cloretos/metabolismo , Esquema de Medicação , Estimulação Elétrica , Eletrofisiologia , Etanol/efeitos adversos , Etanol/farmacologia , Agonistas GABAérgicos/farmacologia , Masculino , Muscimol/farmacologia , Ratos , Ratos Sprague-Dawley , Valores de Referência , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia , Distribuição TecidualRESUMO
Neurons within sensory ganglia have been proposed to communicate via non-synaptic release of a diffusible chemical messenger, but the identity of the chemical mediator(s) remains unknown [J. Neurosci. 16 (1996) 4733-4741]. The present study addressed the possibility of co-released ATP and substance P (SP) within sensory ganglia to further advance the hypothesis of non-synaptic communication between sensory neurons. Microdialysis probes inserted into trigeminal ganglia (TRGs) of anesthetized guinea pigs were perfused with artificial cerebrospinal fluid and the collected perfusate analyzed for ATP and SP content using the firefly luciferin-luciferase (L/L) assay and radioimmunoassay, respectively. Significant reversible increases in ATP and SP levels were observed after infusion of 100 mM KCl or 1 mM capsaicin. Ca(2+)-free ACSF produced an eightfold increase in ATP levels, interpreted as a decrease in activity of Ca(2+)-dependent ecto-nucleotidases that degrade ATP. In contrast, KCl-induced release of ATP in the presence of normal Ca(2+) was blocked by Cd(2+), a voltage-gated Ca(2+) channel blocker, illustrating Ca(2+)-dependence of evoked ATP release. Since ganglionic release of ATP could arise from several neuronal and non-neuronal sources we directly tested acutely dissociated TRG neuron somata for ATP release. Neuron-enriched dissociated TRG cells were plated onto glass tubes and tested for ATP release using the L/L assay. Robust ATP release was evoked with 5 microM capsaicin. These data suggest that ATP is released concurrently with SP from the somata of neurons within sensory ganglia.
Assuntos
Trifosfato de Adenosina/metabolismo , Substância P/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Cloreto de Cádmio/farmacologia , Capsaicina/farmacologia , Cobaias , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cloreto de Potássio/farmacologia , Gânglio Trigeminal/efeitos dos fármacosRESUMO
We studied the effect of ZnCl2 application on GABAA receptor-mediated 36CI- flux in microsacs prepared from whole rat hippocampus and in region-specific hippocampal slices. Slices were obtained from the dentate gyrus (DG), which contains the zinc-enriched hilar region, and from the CA1 region which contains lower levels of endogenous zinc. Muscimol (10 microM)-evoked 36Cl- flux was significantly reduced by ZnCl2 (100 microM) in hippocampal microsacs. In hippocampal slices, muscimol (50 microM)-evoked 36Cl- efflux was higher in CA1 (112.5 +/- 27.9% above basal efflux rate) than in DG slices (29.7 +/- 5.6%). In the presence of ZnCl2, the muscimol effect on efflux rate in CA1 and DG regions was decreased to 10.6 +/- 5.4% and 6.9 +/- 4.9%, respectively. Preincubation with the zinc chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 20 microM), caused a significant increase in muscimol-evoked 36Cl- efflux only in DG slices (57.2 +/- 7.0%), suggesting that GABAA receptors in the DG of rat hippocampus under physiological conditions may function under the inhibitory influence of endogenous chelatable zinc. In intracellular recordings, ZnCl2 (100 microM) application had no effect on the responses to GABA applied perisomatically or in the dendritic region of CA1 neurons. The lack of Zn2+ effect on the postsynaptic GABAA receptor-mediated responses suggests that the decreases of the 36Cl- efflux observed in the biochemical assays may be due to zinc action on neurons other than the principal pyramidal CA1 cells, and possibly the non-neuronal cell populations.
Assuntos
Cloretos/metabolismo , Cloretos/farmacologia , Giro Denteado/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Compostos de Zinco/farmacologia , Animais , Quelantes/farmacologia , Giro Denteado/citologia , Giro Denteado/metabolismo , Etilenodiaminas/farmacologia , Técnicas In Vitro , Masculino , Muscimol/antagonistas & inibidores , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We examined the effects of the neuroprotective cell-permeant Ca2+ buffer, 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester (APTRA-AM, 20-40 mg/kg), on synaptically evoked potentials in the dentate gyrus of awake rats. Intravenous APTRA-AM (20 mg/kg) decreased the evoked potentials with peak effects approximately 6 h after infusion, and recovery to control levels by 24 h. Peak decrease in the population spike (PS) amplitude was by 72+/-17% of control, and the excitatory postsynaptic potential (EPSP) slope was decreased by 31+/-12%. APTRA-AM (40 mg/kg), decreased the PS amplitude and EPSP slope by 58+/-7% and 31+/-6% of pre-drug levels, respectively. These effects were qualitatively similar to the presynaptically mediated decreases in synaptic potentials previously demonstrated in vitro with APTRA-AM. These results indicate that the cell-permeant Ca2+ buffer, APTRA-AM, attenuates hippocampal excitability in vivo, most likely by decreasing synaptic neurotransmission.
Assuntos
Soluções Tampão , Cálcio/fisiologia , Giro Denteado/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Injeções Intravenosas , Masculino , Degeneração Neural/fisiopatologia , Ratos , Ratos WistarRESUMO
Substance P (SP) is synthesized in a subset of nociceptive sensory neurons and is released from their peripheral and central terminals. Here we demonstrate with the use of in vivo microdialysis and radioimmunoassay techniques that SP is also released within trigeminal ganglia following intraganglionic application of KCl, veratridine or capsaicin, and after electrical stimulation of peripheral afferent fibers. Both the basal and KCl-evoked release of SP are shown to be dependent on extracellular calcium. Using the turpentine-induced model of unilateral orofacial inflammation we also show that both the basal and KCl-evoked release of SP within trigeminal ganglia are greatly increased on the inflamed side 48 h after induction of inflammation. Coupled with previous demonstrations of excitatory effects of SP on sensory neurons, these results suggest that SP fulfils the role of a non-synaptically released diffusible chemical messenger that may modulate the somatic excitability of neurons within sensory ganglia in inflammatory pain states.