Excitatory synaptic input to granule cells increases with time after kainate treatment.

Temporal lobe epilepsy is usually associated with a latent period and an increased seizure frequency following a precipitating insult. After kainate treatment, the mossy fibers of the dentate gyrus are hypothesized to form recurrent excitatory circuits between granule cells, thus leading to a progressive increase in the excitatory input to granule cells. Three groups of animals were studied as a function of time after kainate treatment: 1-2 wk, 2-4 wk, and 10-51 wk. All the animals studied 10-51 wk after kainate treatment were observed to have repetitive spontaneous seizures. Whole cell patch-clamp recordings in hippocampal slices showed that the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) in granule cells increased with time after kainate treatment. This increased excitatory synaptic input was correlated with the intensity of the Timm stain in the inner molecular layer (IML). Flash photolysis of caged glutamate applied in the granule cell layer evoked repetitive EPSCs in 10, 32, and 66% of the granule cells at the different times after kainate treatment. When inhibition was reduced with bicuculline, photostimulation of the granule cell layer evoked epileptiform bursts of action potentials only in granule cells from rats 10-51 wk after kainate treatment. These data support the hypothesis that kainate-induced mossy fiber sprouting in the IML results in the progressive formation of aberrant excitatory connections between granule cells. They also suggest that the probability of occurrence of electrographic seizures in the dentate gyrus increases with time after kainate treatment.

Whole-cell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice.

Central control of reproduction is governed by a neuronal pulse generator that underlies the activity of hypothalamic neuroendocrine cells that secrete GnRH. Bursts and prolonged episodes of repetitive action potentials have been associated with hormone secretion in this and other neuroendocrine systems. To begin to investigate the cellular mechanisms responsible for the GnRH pulse generator, we used transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons. Whole-cell recordings were obtained from 21 GnRH neurons, visually identified in 200-microm preoptic/hypothalamic slices, to determine whether they exhibit high frequency bursts of action potentials and are electrically coupled at or near the somata. All GnRH neurons fired spontaneous action potentials, and in 15 of 21 GnRH neurons, the action potentials occurred in single bursts or episodes of repetitive bursts of high frequency spikes (9.77 +/- 0.87 Hz) lasting 3-120 sec. Extended periods of quiescence of up to 30 min preceded and followed these periods of repetitive firing. Examination of 92 GnRH neurons (including 32 neurons that were located near another green fluorescent protein-positive neuron) revealed evidence for coupling in only 1 pair of GnRH neurons. The evidence for minimal coupling between these neuroendocrine cells suggests that direct soma to soma transfer of information, through either cytoplasmic bridges or gap junctions, has a minor role in synchronization of GnRH neurons. The pattern of electrical activity observed in single GnRH neurons within slices is temporally consistent with observations of GnRH release and multiple unit electrophysiological correlates of LH release. Episodes of burst firing of individual GnRH neurons may represent a component of the GnRH pulse generator.

Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology.

GnRH neurons form the final common pathway for central control of reproduction, with regulation achieved by changing the pattern of GnRH pulses. To help elucidate the neurobiological mechanisms underlying pulsatile GnRH release, we generated transgenic mice in which the green fluorescent protein (GFP) reporter was genetically targeted to GnRH neurons. The expression of GFP allowed identification of 84-94% of immunofluorescently-detected GnRH neurons. Conversely, over 99.5% of GFP-expressing neurons contained immunologically detectable GnRH peptide. In hypothalamic slices, GnRH neurons could be visualized with fluorescence, allowing for identification of individual GnRH neurons for patch-clamp recording and subsequent morphological analysis. Whole-cell current-clamp recordings revealed that all GnRH neurons studied (n = 23) fire spontaneous action potentials. Both spontaneous firing (n = 9) and action potentials induced by injection of depolarizing current (n = 17) were eliminated by tetrodotoxin, indicating that voltage-dependent sodium channels are involved in generating action potentials in these cells. Direct intracellular morphological assessment of GnRH dendritic morphology revealed GnRH neurons have slightly more extensive dendrites than previously reported. GnRH-GFP transgenic mice represent a new model for the study of GnRH neuron structure and function, and their use should greatly increase our understanding of this important neuroendocrine system.

GABAA-mediated local synaptic pathways connect neurons in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) in mammals functions as the biological clock controlling circadian rhythms, but the synaptic circuitry of the SCN is largely unexplored. Most SCN neurons use the neurotransmitter gamma-aminobutyric acid (GABA), and anatomic studies indicate many GABAergic synapses and local axon collaterals; however, physiological evidence for synaptic communication among SCN neurons is indirect. We have used three approaches to investigate local circuitry in the SCN in acute hypothalamic slices from rat. First, tetrodotoxin was used to block action-potential-dependent synaptic release, which resulted in a decrease in the frequency of spontaneous synaptic currents in SCN neurons, suggesting that spontaneously active neurons in the slice connect synaptically to SCN neurons. Postsynaptic currents in SCN neurons were also evoked by the selective stimulation of other SCN neurons with glutamate, which avoids direct activation of axons that might originate outside the SCN. Two different methods of glutamate microapplication (i.e., pressure ejection and ultraviolet photolysis of caged glutamate) indicated that SCN neurons receive GABAA-receptor-mediated synaptic input from other SCN neurons. In contrast, glutamate-receptor-mediated synaptic connections between SCN neurons were not detected. The GABAergic synapses that comprise the network described here could conceivably be a substrate for the synchronization and amplification of the circadian rhythm of SCN firing. Alternatively, this circuitry might mediate other aspects of clock function such as the integration of environmental and physiological information.

Glutamate microstimulation of local inhibitory circuits in the supraoptic nucleus from rat hypothalamus slices.

The hypothesis of a local inhibitory input to the hypothalamic supraoptic nucleus was tested with combined glutamate microstimulation and whole cell patch-clamp recordings in slices from rat hypothalamus. Synaptic activity in supraoptic magnocellular neuroendocrine cells (MNCs) was monitored and glutamate microdrops were applied in the perinuclear region of the supraoptic nucleus to evoke firing of action potentials in putative presynaptic inhibitory cells. The effect of glutamate microdrops applied in the perinuclear region was tested on 57 supraoptic MNCs. In control conditions, spontaneous excitatory (EPSCs) and inhibitory (IPSCs) postsynaptic currents were observed at resting membrane potential in all MNCs tested. Glutamate microstimulation evoked an abrupt increase in the frequency and size of spontaneous IPSCs in eight MNCs. Forty-nine MNCs did not show any change in the inhibitory synaptic input. Microapplication of glutamate in the periphery of the supraoptic nucleus did not modify the amplitude or the frequency of spontaneous EPSCs in any of the 57 MNCs tested. In the group of eight MNCs that responded to glutamate microstimulation by an increase in inhibitory input, two types of responses were observed. Four MNCs showed an increase in both size and frequency of spontaneous IPSCs through the entire range of amplitude. In the other four MNCs, local glutamate stimulation produced a dramatic increase in the size of IPSCs and a lesser increase in the frequency of the smaller IPSCs. The potential effect of the glutamate-evoked increase in inhibitory input on the firing activity of MNCs was tested in current-clamp conditions. Intracellular current injection was applied to evoke firing of action potentials in six MNCs that had responded to local glutamate microstimulation by an increase in inhibitory input. Glutamate microdrop applications inhibited the evoked action potential firing in all six cells. These results suggest 1) that local inhibitory interneurons are present in the periphery of the supraoptic nucleus, 2) that they contain functional glutamate receptors, 3) that they form inhibitory synapses with supraoptic MNCs, and 4) that activation of these interneurons inhibits firing in MNCs. These results support the hypothesis that local inhibitory interneurons play a important role in the firing activity of supraoptic MNCs.

Electrographic seizures and new recurrent excitatory circuits in the dentate gyrus of hippocampal slices from kainate-treated epileptic rats.

Mossy fiber sprouting has been proposed to lead to new excitatory connections between dentate granule cells, which in turn cause electrographic seizures. We tested this hypothesis in hippocampal slices from rats made epileptic-by kainate injections. The Timm's histological method revealed intense staining of the inner molecular layer in slices from all kainate-treated rats. In bicuculline (10 microM) and 6 mM [K +]o, antidromic stimulation of the granule cells evoked bursts of population spikes superimposed on long-lasting negative shifts in all slices tested from all kainate-treated rats. Long-duration (2-47 sec), seizure-like bursts with tonic and clonic components occurred spontaneously (53%) or in response to antidromic stimulation (81%). Under identical conditions, prolonged bursts were never seen in slices from controls or from kainate-injected rats 2-4 d after treatment. Glutamate microdrops applied in the granule cell layer evoked abrupt increases in the frequency of excitatory postsynaptic potentials (EPSPs) in two thirds of the cells tested. Glutamate microstimulation was effective at several sites in the granule cell layer but ineffective in the hilus. The proportion of granule cells responding to local application of glutamate by an increase in EPSPs was higher in slices with long bursts (80% with bursts of > 3 sec) than in slices with shorter bursts (33% with bursts of < 3 sec). Glutamate microstimulation did not affect EPSPs in granule cells from control preparations. These results support the hypothesis that kainate-induced mossy fiber sprouting forms new excitatory connections between granule cells and can lead to increased seizure susceptibility in the dentate gyrus.

Neurophysiology of neocortical slices resected from children undergoing surgical treatment for epilepsy.

The recent emergence of surgical treatment of childhood epilepsy has led to the accessibility of young human cerebral tissue for electrophysiological studies of the mechanisms involved in epileptogenesis. Intracellular recordings were obtained from neurons in slices prepared from neocortical tissue resected from children (3 months to 15 years) with catastrophic epilepsy. Data from 'least abnormal' versus 'most abnormal' tissue were compared; the evaluation of the degree of abnormality was based on several clinical criteria. Hypotheses concerning NMDA receptors, local synaptic circuits, and epileptiform bursts were tested. The NMDA receptor-mediated component of synaptic responses, which was isolated pharmacologically, had a voltage dependence that was functionally mature by 8-10 months of age and did not appear to be altered even in the most abnormal tissue. Local inhibitory and excitatory synaptic circuits were present as early as 11 months and 8 months, respectively. Local excitatory circuits were sufficiently extensive in young children to initiate and sustain epileptiform activity when synaptic inhibition was suppressed. Bicuculline-induced epileptiform bursts were similar to those in adult human or animal neocortical slices. Burst duration and the presence of after-discharges were unrelated to patient age or tissue abnormality. These data demonstrate that (1) the electrophysiological properties of human neocortical neurons are very similar to those observed in animal experiments, (2) the mechanisms of neuronal communication are qualitatively mature within the first year of life, and (3) synaptic transmission and local neuronal circuits appear qualitatively normal, even in the most abnormal tissue from children with catastrophic epilepsy.

Whole-cell recordings of spontaneous synaptic currents in medial preoptic neurons from rat hypothalamic slices: mediation by amino acid neurotransmitters.

Whole-cell recordings in hypothalamic slices from immature rats were used to test the hypothesis that inhibitory and excitatory amino acid neurotransmitters mediate fast synaptic currents in the medial preoptic area (MPOA). Bicuculline methiodide reversibly blocked spontaneous inhibitory postsynaptic currents (IPSCs), and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) blocked spontaneous excitatory postsynaptic currents (EPSCs). These competitive antagonists act at gamma-aminobutyric acid (GABA)A and non-N-methyl-D-aspartate (NMDA) glutamate receptors, respectively, thus supporting the hypothesis that these amino acid receptors activate most if not all fast synaptic currents in the MPOA.

Membrane properties of identified guinea-pig paraventricular neurons and their response to an opioid mu-receptor agonist: evidence for an increase in K+ conductance.

Intracellular recordings were obtained from neurons in the paraventricular nucleus (PVN) of guinea-pig hypothalamic slices. Passive and active properties of the neurons were determined, and when possible, recorded neurons were injected with biocytin. The effects of a mu-receptor opioid agonist [D-Ala2, Nme-Phe4, Gly5-ol]enkephalin (DAGO) were studied in order to determine which types of cells have mu receptors and to test the hypothesis that an increase in K+ conductance causes mu-receptor-mediated inhibition in the PVN. The recorded cells inside the PVN were divided into two groups, primarily on the basis of the presence or absence of a low threshold Ca2+ spike (LTS). In one group of neurons, LTS potentials could not be evoked (non-LTS cells, n = 42). In another group of neurons (n = 35), LTS potentials with one or two Na+ spikes could be initiated with depolarizing pulses superimposed on steady hyperpolarizing currents; however, these neurons did not fire robust bursts (i.e. non-bursting LTS cells). The mean time constant of non-LTS cells (19.9 +/- 1.6 ms; mean +/- SEM) was significantly shorter than that of non-bursting LTS cells (26.7 +/- 2.1 ms). There were no differences in the mean resting membrane potential, spike amplitude, spike duration, input resistance, spike threshold and pattern of synaptic inputs between the two groups. Intracellular labeling with biocytin combined with cresyl violet counter-staining demonstrated that the two types of cells were located in the PVN.(ABSTRACT TRUNCATED AT 250 WORDS)

Patch-clamp analysis of spontaneous synaptic currents in supraoptic neuroendocrine cells of the rat hypothalamus.

Spontaneous synaptic currents were recorded in supraoptic magnocellular neurosecretory cells using whole-cell patch-clamp techniques in the rat hypothalamic slice preparation. Numerous spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were observed in the 27 cells recorded. The rate of occurrence of the spontaneous currents and the relative proportion of EPSCs versus IPSCs varied significantly from cell to cell. Miniature EPSCs and IPSCs were clearly distinguished from background noise in TTX (n = 3 cells at 0.5 micrograms/ml). The frequency of EPSCs and IPSCs decreased by approximately 70% and the largest events were blocked in TTX, but the peaks of the amplitude histograms were shifted by only a few picoamperes. Bicuculline (n = 10 cells at 10 microM and 2 cells at 20 microM) blocked completely all the IPSCs without any detectable effect on the frequency or amplitude of the EPSCs. No slow spontaneous outward currents, indicative of a K+ current from activation of GABAB receptors, were observed. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX; n = 7 cells at 10 microM) consistently blocked all EPSCs without any apparent effect on the frequency or amplitude of the IPSCs. No synaptic events could be detected when CNQX was applied in combination with bicuculline (n = 4). The decay phase of averaged spontaneous IPSCs and EPSCs recorded at resting membrane potential could be well fitted by single exponential functions in most cells. The time constants ranged from 0.92 to 3.0 msec for EPSCs (five cells) and from 5.3 to 6.6 msec for IPSCs (four cells). A second, slower time constant of 4-15 msec was found in the largest averaged EPSCs (> or = 40 pA). The amplitude of this slow component was -2 to -4 pA. These results suggest that, in the in vitro slice preparation, glutamate mediates all the spontaneous EPSCs in magnocellular neurosecretory cells by acting primarily on AMPA/kainate-type receptors at resting membrane potential and that activation of GABAA receptors mediates most if not all IPSCs.

Single-electrode voltage-clamp analysis of the N-methyl-D-aspartate component of synaptic responses in neocortical slices from children with intractable epilepsy.

1. Synaptic transmission mediated by the N-methyl-D-aspartate (NMDA)-receptor type was studied in neocortex from children undergoing surgical treatment for intractable epilepsy. Intracellular recordings from pyramidal cells were obtained in slices of neocortical tissue by use of microelectrodes. Synaptic responses were induced by electrical stimulation and studied with current-clamp and single-electrode voltage-clamp techniques. The NMDA-receptor-mediated component of the synaptic responses was isolated by addition of 10 microM bicuculline and 30 microM 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) in the perfusion solution. 2. In the presence of bicuculline and CNQX, electrical stimulation evoked an excitatory postsynaptic potential (EPSP) in every recorded cell. The amplitude of this EPSP increased when membrane potential was depolarized with injected current. 3. All cells studied in voltage clamp were recorded with microelectrodes containing Cs+ and QX 314. To avoid contamination of the responses from voltage-dependent Ca2+ conductances, membrane potential was held at depolarized potentials until Ca2+ spiking inactivated completely. The evoked excitatory postsynaptic currents (EPSCs) measured at resting membrane potential ranged from 100 to 400 pA. The NMDA receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP-5) reversibly decreased the current amplitude by 60% for 10 microM and 80% for 30 microM. 4. The current-voltage (I-V) relation showed a region of negative slope conductance between -100 and -20 mV. The largest currents (-250 to -900 pA) were recorded in the range of -45 to -20 mV and reversed between -10 and +10 mV. Removing Mg2+ from the perfusion solution decreased the negativity of the slope, which is consistent with a reduction in the voltage-dependent Mg2+ block of the NMDA-receptor channel. 5. The I-V plots obtained from cells recorded in the most abnormal tissue were averaged and compared with those from the least abnormal tissue. No significant difference was found between these two groups. The averaged plots from the youngest patients (8 and 10 mo old) and those from the oldest (5-15 yr old) patients were also compared, and the results from these two groups were not significantly different.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid antagonists inhibit synaptic responses in the guinea pig hypothalamic paraventricular nucleus.

1. The effects of specific excitatory amino acid (EAA) antagonists on evoked excitatory synaptic responses were studied in the hypothalamic paraventricular nucleus (PVN) of the guinea pig, by the use of the in vitro slice preparation. Intracellular recordings were obtained from paraventricular neurons, and excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were induced by perifornical electrical stimulation. To reduce the influence of a potential gamma-aminobutyric acidA (GABAA) inhibitory component on the synaptic responses, all experiments were performed in the presence of 50 microM picrotoxin. 2. Of 20 cells tested, 13 had electrophysiological characteristics similar to magnocellular neuropeptidergic cells (MNCs) and 7 displayed low-threshold Ca2+ spikes (LTSs). No difference was detected in the effect of the antagonists on the synaptic responses of cells with or without LTS potentials. 3. The broad-spectrum EAA antagonist kynurenic acid decreased the amplitude of the EPSPs and EPSCs in a dose-dependent manner: the mean decrease was 5% for 100 microM, 43% for 300 microM, and 70% for 1 mM. 4. The quisqualate/kainate-receptor-selective antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) induced a dose-dependent decrease of the EPSPs and EPSCs: 1 microM had no detectable effect, 3 and 10 microM caused 30 and 70% decreases, respectively, and 30 microM blocked the response almost completely. This effect was not accompanied by a change in resting membrane potential or input resistance and was slowly reversible. 5. The N-methyl-D-aspartate (NMDA)-receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP5), applied at 30 and 300 microM, reduced slightly the amplitude of the decay phase of the EPSP but did not significantly affect the peak amplitude. In some cells, the current-voltage relationship of the decay phase of the EPSC revealed a region of negative slope conductance between -70 and -40 mV. 6. These results suggest that 1) glutamate or a related EAA is responsible for the fast excitatory input to magnocellular and parvocellular neurons in the PVN and probably also for cells around PVN, 2) a quisqualate/kainate receptor type is responsible for the rising phase and peak amplitude of the synaptic current, and 3) an NMDA receptor contributes to the late part of the synaptic response.

In vivo Intracellular Recording of Neurons in the Supraoptic Nucleus of the Rat Hypothalamus.

Abstract Intracellular recordings were made from cells in the hypothalamic supraoptic nucleus in the urethane-anaesthetized male rat using the ventral surgical approach. Impalements lasted from 5 min to 1 h and recorded cells had an input resistance of 55 to 170 megohms. Spikes of over 50 mV were recorded from 14 cells which could be antidromically activated by stimulation of the neural stalk. The spikes showed a hyperpolarizing afterpotential and the broadening characteristic of rapidly firing magnocellular neurons, which recovered rapidly (<200 ms). When depolarized, the cells showed evidence of a transient potassium current. Recurrent synaptic coupling between the recorded cell and adjacent cells would be expected to alter the hyperpolarizing afterpotential of an antidromic spike as compared with a spontaneous spike; no perceptible difference in the waveforms of the different types of spike could be detected in 11 spontaneously active cells. Application of just subthreshold stimuli to the neural stalk did not evoke depolarizing or hyperpolarizing potentials. Suprathreshold shocks to the neural stalk, when the antidromic spike was prevented by collision, also had no discernible effect on membrane potential. Thus intracellular recordings from magnocellular neurons in vivo revealed electrophysiological properties similar to those seen in vitro. No evidence for synaptic interconnection between magnocellular neurons was found in male rats.

Glutamate, the dominant excitatory transmitter in neuroendocrine regulation.

Glutamate has been found to play an unexpectedly important role in neuroendocrine regulation in the hypothalamus, as revealed in converging experiments with ultrastructural immunocytochemistry, optical physiology with a calcium-sensitive dye, and intracellular electrical recording. There were large amounts of glutamate in boutons making synaptic contact with neuroendocrine neurons in the arcuate, paraventricular, and supraoptic nuclei. Almost all medial hypothalamic neurons responded to glutamate and to the glutamate agonists quisqualate and kainate with a consistent increase in intracellular calcium. In all magnocellular and parvocellular neurons of the paraventricular and arcuate nuclei tested, the non-NMDA (non-N-methyl-D-aspartate) glutamate antagonist CNQX (cyano-2,3-dihydroxy-7-nitroquinoxaline) reduced electrically stimulated and spontaneous excitatory postsynaptic potentials, suggesting that the endogenous neurotransmitter is an excitatory amino acid acting primarily on non-NMDA receptors. These results indicate that glutamate plays a major, widespread role in the control of neuroendocrine neurons.

Synaptic transmission in human neocortex removed for treatment of intractable epilepsy in children.

Synaptic transmission to pyramidal cells was studied in slices of neocortex resected from infants and children (n = 10, age 8 months to 13 years) undergoing surgical treatment for intractable epilepsy. Most specimens were from the least abnormal area of the resection. Stable intracellular recordings could be obtained for up to 8 hours. Most of the recorded neurons had electrophysiological characteristics similar to those of regular-firing pyramidal cells and were in layers III to V, which was confirmed by intracellular staining with Lucifer yellow. Local extracellular stimulation evoked a sequence of excitatory and inhibitory postsynaptic potentials. After application of the gamma-aminobutyric acid antagonist, bicuculline (10-30 microM), extracellular stimulation induced large excitatory postsynaptic potentials and epileptiform bursts. Spontaneous bursts occasionally occurred in bicuculline. This effect of bicuculline was observed in all the tissue samples, even those from infant patients (n = 4, age 8-16 months). Kynurenic acid depressed or abolished both spontaneous and stimulation-induced bursts. The competitive antagonist for N-methyl-D-aspartate receptors, DL-2-amino-5-phosphonopentanoic acid decreased the duration of bicuculline-induced bursts. These data provide evidence that, similar to rat and cat neocortex, excitatory and inhibitory amino acids are important transmitters to pyramidal cells in immature human neocortex.

Direct effects of an opioid peptide selective for mu-receptors: intracellular recordings in the paraventricular and supraoptic nuclei of the guinea-pig.

Responses to [D-Ala2, MePhe4, Gly-ol5]enkephalin, a selective agonist for mu-receptors, were recorded intracellularly from 26 neurons in slices of guinea-pig hypothalamus. Of eight cells tested in the supraoptic nucleus, all of which had electrical properties characteristic of magnocellular neuroendocrine cells, four were sensitive to the agonist applied in the perfusion bath or with microdrops. The main effect was a decrease or suppression of spontaneous firing. In the paraventricular nucleus, seven of 18 cells tested also had electrophysiological characteristics similar to magnocellular neurons: two of them were sensitive to the mu-agonist and the effect was similar to that observed in the supraoptic nucleus. The remaining paraventricular neurons displayed low-threshold Ca2+ spikes, and thus had electrophysiological characteristics different from putative magnocellular neurons. Ten of 11 cells with low-threshold Ca2+ spikes were hyperpolarized by more than 10 mV by the mu-agonist, and showed a 33 +/- 1.9% (S.E.M.) decrease in input resistance. In both types of cells, when synaptic transmission was blocked with tetrodotoxin, the mu-agonist could still induce a hyperpolarization, suggesting that the effect was in part direct. Hyperpolarization was also obtained when the Cl- reversal potential was shifted to more positive values by using KCl electrodes, thus excluding a Cl- conductance mechanism. These results provide evidence that opioid peptides can directly inhibit hypothalamic neurons, that the mechanism is an increase in K+ conductance, and that two types of hypothalamic neurons appear to have different sensitivities to a mu-agonist.

Intrinsic and synaptic mechanisms of hypothalamic neurons studied with slice and explant preparations.

The use of slice and explant preparations has allowed major advances in our understanding of the membrane physiology of mammalian hypothalamic neurons. This article will review intracellular electrophysiological studies of neurons in or immediately surrounding the supraoptic and paraventricular nuclei. Considerable information is now available on the intrinsic membrane mechanisms that control action potential generation and burst firing in magnocellular neuroendocrine cells (MNCs) within these nuclei. Neurons surrounding the paraventricular nucleus have different electrical properties than the MNCs, including low-threshold Ca2+ spikes and pronounced anomalous rectification. Bicuculline and kynurenic acid strongly depress fast IPSPs and EPSPs in MNCs, thus suggesting that inhibitory and excitatory amino acids mediate fast synaptic transmission in the hypothalamus. The effects of neuromodulators, such as noradrenaline and opioid peptides, have also been examined. Noradrenaline excites supraoptic neurons and leads to phasic firing through an alpha-1 mechanism and decreased K+-conductance. Opioid peptides act directly on mu-receptors to hyperpolarize about half of the neurons through an increased K+-conductance. In conclusion, using the magnocellular neuroendocrine system as a model, in vitro slice and explant preparations have allowed the characterization of electrophysiological properties, the identification of neurotransmitters for synaptic events, and studies on the mechanism of action of neuromodulators.

Effect of opioid peptides on the paraventricular nucleus of the guinea pig hypothalamus is mediated by mu-type receptors.

The effects of opioid peptides on paraventricular neurones were investigated by using intracellular recordings from hypothalamic slices of the guinea pig. Forty-eight out of 128 neurones were hyperpolarized by DAGO, a synthetic structural analogue of enkephalin selective for mu-receptors. This effect was concentration-dependent and reversibly suppressed by naloxone. DPLPE, a selective delta-agonist, and U-50,488, a selective kappa-agonist, had no effect. The localization and the size of the recorded perikarya were assessed following injection of the fluorescent dye Lucifer yellow. Most of the DAGO-responsive neurones were located within the paraventricular nucleus, some of them in the region of the nucleus which is rich in vasopressin-containing cells, as shown by immunocytochemistry. DAGO-sensitive cells were found among magnocellular as well as parvocellular neurones.