Biomedical subjects

F E Dudek

Publications and source records attributed to F E Dudek.

At least 91 records · Page 5Linked to original sources

PubMed1991

Immunohistochemical differentiation of electrophysiologically defined neuronal populations in the region of the rat hypothalamic paraventricular nucleus.

Intracellular recording and labeling were combined with neurophysin immunohistochemistry to study neurons in the paraventricular nucleus region of the rat hypothalamus. Neuronal membrane properties were examined in hypothalamic slices, and cells were labeled by injecting biocytin or Lucifer yellow. Slices were then embedded, sectioned, and immunohistochemically processed for neurophysin. Immunoreactivity patterns, and in some cases counterstaining, enabled determinations of the cytoarchitectonic positions of recorded cells to be made. Recorded cells were divided into three types according to their electrophysiological characteristics. The first type lacked low-threshold Ca2+ spikes and displayed linear current-voltage relations, a short time constant, and evidence for an A current. These were relatively large cells that were typically immunoreactive for neurophysin and were situated near other neurophysin-positive neurons. The second type had relatively small low-threshold potentials that did not generate bursts of Na+ spikes. These cells had heterogeneous current-voltage relations and intermediate time constants. They did not label for neurophysin, and most were located in the parvicellular subregion of the paraventricular nucleus. The third type had large low-threshold Ca2- spikes that generated bursts of Na+ spikes, and these cells had nonlinear current-voltage relations and long time constants. These neurons were dorsal or dorsolateral to the paraventricular nucleus and were not immunoreactive for neurophysin. These results indicate that paraventricular magnocellular neurons lack low-threshold potentials, whereas paraventricular parvicellular neurons display low-threshold potentials that generate one or two action potentials. Neurons that fire spike bursts from low-threshold potentials are adjacent to the paraventricular nucleus, confirming earlier reports.

Action Potentials↗

PubMed1991

Electrophysiology of GABA-mediated synaptic transmission and possible roles in epilepsy.

Epileptogenic conditions come about from a disequilibrium between excitatory and inhibitory mechanisms, creating a state of neuronal hypersynchrony. From experimental studies in animal models of epilepsy it appears that several mechanisms, alone or in combination, could be responsible for this imbalance. An alteration of GABA-mediated inhibition has long been considered to be one of the most likely candidates. We review recent data on the synaptic physiology of GABA-mediated inhibition, with emphasis on GABAA and GABAB receptors and their conductances. We describe the integrative role of GABAergic local-circuit neurons in the normal control of recurrent excitation. We then discuss possible alterations in GABAA-mediated inhibition in two chronic animal models of epilepsy, the kindled rat and the kainate-treated rat. Finally, we review studies on GABA inhibition in human epileptic cortex resected for the treatment of intractable epilepsy.

Animals↗

PubMed1991

Comparison of three intracellular markers for combined electrophysiological, morphological and immunohistochemical analyses.

Hypothalamic paraventricular and supraoptic neurons were recorded intracellularly in coronal slices and injected with Lucifer yellow, ethidium bromide or biocytin. Electrical properties, morphological staining and neurophysin immunohistochemistry were compared among the 3 markers. Lucifer yellow electrodes had a high resistance and frequently blocked during experiments. Neurons recorded with Lucifer yellow electrodes had low input resistances and low-amplitude, broad spikes. Lucifer yellow labeling in whole mount was highly fluorescent, revealing distal dendrites and axons. Of cells injected with Lucifer yellow, 64% were recovered but were faint after immunohistochemical processing. Recordings with ethidium bromide electrodes were similar to controls, although electrode blockage sometimes occurred. Only somata and proximal dendrites of ethidium bromide-filled neurons were visible in whole-mount. Forty percent of cells injected with ethidium bromide were recovered after immunohistochemical processing; these were invariably faint. Recordings with biocytin-filled electrodes were similar to control recordings. Biocytin-filled, HRP-labeled cells showed distal dendrites and often dendritic spines and axons in 50-75-microns sections. Seventy percent of biocytin-injected cells labeled with fluorescent markers were recovered and remained strongly labeled after immunohistochemical processing. Biocytin had the best electrical and staining properties for combined electrophysiological and anatomical studies.

Animals↗

PubMed1991

Electrophysiological properties of neurones in the region of the paraventricular nucleus in slices of rat hypothalamus.

1. Neurones in the region of the hypothalamic paraventricular nucleus (PVN) of the rat were studied with intracellular recording in the coronal slice preparation. Three types of hypothalamic neurones were distinguished according to their membrane properties and anatomical positions. Lucifer Yellow or ethidium bromide was injected intracellularly to determine the morphology of some recorded cells. 2. The most distinctive electrophysiological characteristic was the low-threshold depolarizing potentials which were totally absent in type I neurones, present but variable in type II neurones and very conspicuous in type III neurones. Type II neurones generally showed relatively small low-threshold depolarizations (26.5 +/- 2.2 mV) which generated at most one to two action potentials. Type III neurones, on the other hand, generated large low-threshold potentials (40.3 +/- 2.8 mV) which gave rise to bursts of three to six fast action potentials. Deinactivation of the low-threshold conductance in both type II and type III neurones was voltage- and time-dependent. Low-threshold potentials persisted in TTX (1-3 microM) but were blocked by solutions containing low Ca2+ (0.2 mM) and Cd2+ (0.5 mM), suggesting they were Ca(2+)-dependent. 3. Type I neurones had a significantly shorter membrane time constant (14.5 +/- 1.7 ms) than those of type II (21.6 +/- 1.7 ms) and type III neurones (33.8 +/- 4.4 ms). Input resistance and resting membrane potential did not differ significantly among the cell groups. 4. Current-voltage (I-V) relations were significantly different among the three cell types. Type I neurones had linear I-V relations to -120 mV, while type III neurones all showed marked anomalous rectification. I-V relations among type II neurones were more heterogeneous, although most (75%) had linear I-V curves to about -90 to -100 mV, inward rectification appearing at more negative potentials. 5. Type I neurones generated fast action potentials of relatively large amplitude (64.2 +/- 1.1 mV, threshold to peak) and long duration (1.1 +/- 0.1 ms, measured at half-amplitude); the longer duration was due to a shoulder on the falling phase of the spike. Type II neurones had large spikes (66.5 +/- 1.6 mV) of shorter duration (0.9 +/- 0.1 ms) with no shoulder. Type III neurones had relatively small spikes (56.1 +/- 2.2 mV) of short duration (0.8 +/- 0.1 ms) with no shoulder. 6. The three cell populations showed different patterns of repetitive firing in response to depolarizing current pulses. Type I neurones often generated spike trains with a delayed onset and little spike-frequency adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1991

Intracellular electrophysiological study of suprachiasmatic nucleus neurons in rodents: excitatory synaptic mechanisms.

1. To study the synaptic mechanisms of excitatory transmission in the suprachiasmatic nucleus (SCN), we assessed the effects of excitatory amino acid receptor antagonists on excitatory postsynaptic potentials (EPSPs) recorded from SCN neurons in horizontal and parasagittal hypothalamic slice preparations from rats and guinea-pigs. The EPSPs were evoked by electrical stimulation of either optic nerve or a site near the SCN. 2. When evoked at membrane potentials between -60 and -100 mV, the EPSPs from optic nerve stimulation were conventional in shape; they rose to the peak quickly (6.2 +/- 0.5 ms, mean +/- S.E.M.; n = 45) and decayed gradually over 50-250 ms. When evoked at membrane potentials between -20 and -55 mV after blockade of outward K+ currents and fast Na+ spikes by intracellular injection of Cs+ and QX-314 (n = 5 neurons), a slow depolarizing potential emerged near the fast peak of the EPSP. This slow potential, unlike the fast peak, was not linearly related to membrane potential. 3. An antagonist for kainate- and quisqualate-type excitatory amino acid receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX 1-10 microM), depressed in a concentration-dependent and reversible manner the EPSPs evoked by optic nerve stimulation at membrane potentials between -60 and -100 mV (n = 9). The effects of DNQX were not associated with any significant changes in the baseline input resistance or membrane potential of the postsynaptic neurons. The selective N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5, 50-100 microM), did not affect significantly and consistently the EPSPs evoked at these membrane potentials (n = 7). On the other hand, AP5 (50 microM) blocked or depressed the slow depolarizing component of the EPSPs evoked at membrane potentials between -20 and -55 mV (n = 4). No significant changes in baseline input resistance or membrane potential accompanied the effects of AP5. 4. Stimulation of a site lateral or dorsocaudal to the SCN evoked EPSPs distinct from those evoked by optic nerve stimulation. Again, DNQX (0.3-10 microM) depressed the EPSPs evoked at membrane potentials between -60 and -100 mV (n = 4) whereas AP5 (50 microM) had no effect (n = 5). When evoked at less negative membrane potentials (i.e. -20 to -55 mV) after intracellular injection of Cs+ and QX-314, the EPSPs had a slow depolarizing potential, similar to the EPSPs from optic nerve stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1991

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.

2-Amino-5-phosphonovalerate↗

PubMed1990

Osmolality-induced changes in extracellular volume alter epileptiform bursts independent of chemical synapses in the rat: importance of non-synaptic mechanisms in hippocampal epileptogenesis.

The contribution of non-synaptic mechanisms to the seizure susceptibility of rat CA1 hippocampal pyramidal cells was examined in vitro by testing the effects of osmolality on synchronous neuronal activity, using solutions which blocked chemical synaptic transmission both pre- and post-synaptically. Decreases in osmolality, which shrink the extracellular volume, caused or enhanced epileptiform bursting. Increases in osmolality with membrane-impermeant solutes, which expand the extracellular volume, blocked or greatly reduced epileptiform discharges. Reductions in the extracellular volume, therefore, can enhance synchronization among CA1 hippocampal neurons through non-synaptic mechanisms. Since similar osmotic treatments are known to modify epileptiform discharges in several models of epilepsy, non-synaptic mechanisms are probably more important in hippocampal epileptogenesis than previously realized and may contribute to the high susceptibility of this brain region to epileptic seizures in animals and humans. These data also provide a possible explanation for the observation in humans that decreased plasma osmolality, which can be associated with a wide range of clinical syndromes, leads to seizures.

Action Potentials↗

PubMed1990

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1990

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.

2-Amino-5-phosphonovalerate↗

PubMed1990

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.

Animals↗

PubMed1990

Effects of excitatory amino acid antagonists on synaptic responses of supraoptic neurons in slices of rat hypothalamus.

1. Intracellular recordings from magnocellular neurons in the supraoptic nucleus (SON) were obtained from rat hypothalamic slices to determine the effects of specific transmitter antagonists on evoked postsynaptic potentials (PSPs), action potential after-discharge, and spontaneously occurring PSPs. 2. Broad-spectrum excitatory amino acid (EAA) antagonists, kynurenic acid (KYN) and gamma-d-glutamylglycine (DGG), significantly diminished or eliminated electrically evoked depolarizing PSPs and spike discharges. These compounds also greatly reduced the amplitude and frequency of spontaneous PSPs. 3. The specific N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), did not significantly reduce these measures of synaptic activation under these experimental conditions. 4. The gamma-aminobutyric acid (GABA) antagonist, bicuculline methiodide (BIC), partially antagonized some PSPs when the cells were hyperpolarized (-75 to -80 mV) with steady injected currents; KYN antagonized BIC-resistant PSPs. 5. The involvement of a hypothetical cholinergic input to the SON in the responses to stimulation of the region dorsolateral to the SON was tested by bath application of nicotinic cholinergic antagonists, particularly d-tubocurarine (dTC). Nicotinic cholinergic antagonists, even after prolonged exposure to high concentrations, did not block the responses of SON cells to dorsolateral stimulation. 6. These findings strongly suggest that EAAs mediate fast excitatory synaptic responses of SON neurons to stimulation of cells and axons in the region dorsolateral to the SON. The blockade of almost all spontaneous EPSPs by broad-spectrum EAA antagonists likewise argues that EAAs are responsible for the majority of ongoing fast excitatory input. These responses appear to involve an interaction with kainate- and/or quisqualate-type EAA receptors.

Animals↗

PubMed1989

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.

Action Potentials↗

PubMed1988

Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats.

Kainic acid (KA) injections destroy hippocampal pyramidal cells, induce recurrent collateral sprouting of the hilar mossy fibers (MFs), and lead to chronic seizures. In the present study, rats were injected systemically with KA (14 mg/kg) to determine whether the subsequent occurrence of seizures was correlated with MF sprouting, as indicated by Timm's staining of proximal dendrites. At 4 weeks, 56% of the KA-treated rats had MF sprouting in the temporal (ventral) hippocampus, and 52% had shown seizures between 10 and 28 days. Both seizures and sprouting were seen in 32% of the treated animals. While Timm's staining of MFs in the inner molecular layer was not directly correlated with seizure scores, animals which exhibited chronic seizures had significantly more sprouting than animals which did not have seizures.

Animals↗

PubMed1988

The effects of the excitatory amino acid antagonist kynurenic acid on synaptic transmission to supraoptic neuroendocrine cells.

The magnocellular neuroendocrine system of the mammalian hypothalamus regulates numerous physiological functions by releasing the peptide hormones oxytocin and vasopressin from the neurohypophysis. Although several substances are known to promote hormone release, relatively little research has been aimed at testing the hypothesis that excitatory amino acids (EAAs) mediate synaptic transmission in this neuroendocrine system. In the present experiments, intracellular recordings in slices of rat hypothalamus revealed that kynurenic acid, a specific antagonist of EAA receptors, strongly and reversibly blocked excitatory postsynaptic potentials (EPSPs) in supraoptic neurons. The profound antagonism of EPSPs by kynurenic acid strongly suggests that EAAs may be an important class of fast excitatory neurotransmitter within this central regulatory nucleus.

Action Potentials↗

PubMed1988

Reduction of dye coupling in glial cultures by microinjection of antibodies against the liver gap junction polypeptide.

Intracellular injection of antibodies to the 27-kDa liver gap junction polypeptide have been shown previously to uncouple pairs of cultured mammalian hepatocytes, cardiac myocytes, and ganglionic neurons (Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2412-2416). In confluent primary cultures of astrocytes, similar injections significantly reduced dye coupling for cells closer than 80 micron to the injected glial cell. Western blots identified a 27-kDa protein in extracts of the astrocyte cultures that cross-react with the gap junction-specific antibodies. These results suggest that homologous gap junction polypeptides exist in liver and glial cells.

Animals↗

PubMed1988

Cholinergic excitation of supraoptic neurons in hypothalamic slices of rat.

The actions of the cholinergic agonists acetylcholine (ACh), nicotine (NIC) and carbamylcholine (CARB) were studied in the supraoptic nucleus with extracellular recordings from coronal slices of the hypothalamus of the rat. Agonists were either applied to the bath or pressure-ejected from micropipettes. Acetylcholine, NIC and CARB produced short-latency increases in discharge rate of about one-half of the cells of the supraoptic nucleus, particularly if the cell was spontaneously active. Excitations produced by NIC were typically brief and the firing rate often declined during prolonged exposure to the agonist. A vigorous excitation frequently occurred in normal medium immediately after cessation of prolonged application of NIC. Local application of glutamate (GLUT) reliably excited cells of the supraoptic nucleus. In some of the cells that were unresponsive to cholinergic agonists, ACh and NIC enhanced responses to electrical stimulation in the dorsolateral area. The nicotinic cholinergic antagonists, hexamethonium (HEX) and d-tubocurarine (dTC), reduced or blocked excitations induced by NIC, but not those from GLUT. This study provides further evidence that nicotinic receptors can mediate cholinergic influences on cells of the supraoptic nucleus. However, these effects were not observed in all cells in the region of the supraoptic nucleus and the excitations by NIC showed densensitization. Acetylcholine may subserve a modulatory role to other transmitters in the supraoptic nucleus.

Acetylcholine↗

PubMed1988

Electrophysiological evidence from glutamate microapplications for local excitatory circuits in the CA1 area of rat hippocampal slices.

1. Evidence for local excitatory synaptic connections in CA1 of the rat hippocampus was obtained by recording excitatory postsynaptic potentials (EPSPs) intracellularly from pyramidal cells during local microapplications of glutamate. 2. Experiments were performed in hippocampal slices cut parallel to (transverse slice) or perpendicular to (longitudinal slice) alvear fibers. In normal solutions, glutamate microdrops (10-20 mM, 10-20 micron diam) applied in CA1 within 400 micron of recorded cells sometimes increased the frequency of inhibitory postsynaptic potentials for 5-10 s in both transverse and longitudinal slices. Increases in EPSP frequency were also occasionally observed, but only in transverse slices. Tetrodotoxin (1 microgram/ml) blocked glutamate-induced increases in PSP frequency, thus indicating that they were not caused by subthreshold effects on presynaptic terminals. Increases in PSP frequency were interpreted to result from glutamate activation of hippocampal neurons with inhibitory and excitatory connections to recorded neurons. 3. In both slice orientations, local excitatory circuits were studied in more isolated conditions by surgically separating CA1 from CA3 (transverse slices) and by blocking GABAergic inhibitory synapses with picrotoxin (5-10 microM). Microdrops were systematically applied at 200 and 400 micron on each side of the recording site. Significant glutamate-induced increases in EPSP frequency were observed in neurons from both slice orientations to microdrops in at least one of the locations. This provided evidence that excitatory synapses are present in both transverse and longitudinal slices. 4. Substantial increases in EPSP frequency only occurred in neurons from longitudinal slices when glutamate was microapplied 200 micron or less from the recording site. In transverse slices, however, large increases in EPSP frequency were observed to glutamate microapplications at 200 or 400 micron. These data suggest that CA1 local excitatory connections project for longer distances in the transverse than in the longitudinal plane of section. 5. Increases in EPSP frequency, averaged across cells, did not differ significantly in the four microapplication sites in either transverse or longitudinal slices. Thus local excitation in CA1 does not appear to be asymmetrically arranged in the way suggested for CA3. 6. The densities of local excitatory circuits in CA1 versus CA3 were studied by quantitatively comparing glutamate-induced increases in EPSP frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices.

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗