Frequency Of Spontaneous Bursts Research Articles

µ-Opioid Receptor Activation Reduces Glutamate Release in the PreBötzinger Complex in Organotypic Slice Cultures.

The inspiratory rhythm generator, located in the brainstem preBötzinger complex (preBötC), is dependent on glutamatergic signaling and is affected profoundly by opioids. Here, we used organotypic slice cultures of the newborn mouse brainstem of either sex in combination with genetically encoded sensors for Ca2+, glutamate, and GABA to visualize Ca2+, glutamatergic and GABAergic signaling during spontaneous rhythm and in the presence of DAMGO. During spontaneous rhythm, the glutamate sensor SF-iGluSnFR.A184S revealed punctate synapse-like fluorescent signals along dendrites and somas in the preBötC with decay times that were prolonged by the glutamate uptake blocker (TFB-TBOA). The GABA sensor iGABASnFR showed a more diffuse fluorescent signal during spontaneous rhythm. Rhythmic Ca2+- and glutamate transients had an inverse relationship between the spontaneous burst frequency and the burst amplitude of the Ca2+ and glutamate signals. A similar inverse relationship was observed when bath applied DAMGO reduced spontaneous burst frequency and increased the burst amplitude of Ca2+, glutamate, and GABA transient signals. However, a hypoxic challenge reduced both burst frequency and Ca2+ transient amplitude. Using a cocktail that blocked glutamatergic, GABAergic, and glycinergic transmission to indirectly measure the release of glutamate/GABA in response to an electrical stimulus, we found that DAMGO reduces the release of glutamate in the preBötC but has no effect on GABA release. This suggest that the opioid mediated slowing of respiratory rhythm involves presynaptic reduction of glutamate release, which would impact the ability of the network to engage in recurrent excitation, and may result in the opioid-induced slowing of inspiratory rhythm.SIGNIFICANCE STATEMENT Opioids slow down breathing rhythm by affecting neurons in the preBötzinger complex (preBötC) and other brainstem regions. Here, we used cultured slices of the preBötC to better understand this effect by optically recording Ca2+, glutamate, and GABA transients during preBötC activity. Spontaneous rhythm showed an inverse relationship between burst frequency and burst amplitude in the Ca2+ and glutamate signals. Application of the opioid DAMGO slowed the rhythm, with a concomitant increase in Ca2+, glutamate, and GABA signals. When rhythm was blocked pharmacologically, DAMGO reduced the presynaptic release of glutamate, but not GABA. These data suggest the mechanism of action of opioids involves presynaptic reduction of glutamate release, which may play an important role in the opioid-induced slowing of inspiratory rhythm.

Electrophysiological Evidence for Intrinsic Pacemaker Currents in Crayfish Parasol Cells.

I used sharp intracellular electrodes to record from parasol cells in the semi-isolated crayfish brain to investigate pacemaker currents. Evidence for the presence of the hyperpolarization-activated inward rectifier potassium current was obtained in about half of the parasol cells examined, where strong, prolonged hyperpolarizing currents generated a slowly-rising voltage sag, and a post-hyperpolarization rebound. The amplitudes of both the sag voltage and the depolarizing rebound were dependent upon the strength of the hyperpolarizing current. The voltage sag showed a definite threshold and was non-inactivating. The voltage sag and rebound depolarization evoked by hyperpolarization were blocked by the presence of 5–10 mM Cs2+ ions, 10 mM tetraethyl ammonium chloride, and 10 mM cobalt chloride in the bathing medium, but not by the drug ZD 7288. Cs+ ions in normal saline in some cells caused a slight increase in mean resting potential and a reduction in spontaneous burst frequency. Many of the neurons expressing the hyperpolarization-activated inward potassium current also provided evidence for the presence of the transient potassium current IA, which was inferred from experimental observations of an increased latency of post-hyperpolarization response to a depolarizing step, compared to the response latency to the depolarization alone. The latency increase was reduced in the presence of 4-aminopyridine (4-AP), a specific blocker of IA. The presence of 4-AP in normal saline also induced spontaneous bursting in parasol cells. It is conjectured that, under normal physiological conditions, these two potassium currents help to regulate burst generation in parasol cells, respectively, by helping to maintain the resting membrane potential near a threshold level for burst generation, and by regulating the rate of rise of membrane depolarizing events leading to burst generation. The presence of post-burst hyperpolarization may depend upon IA channels in parasol cells.

Novel Actions of Oxazolidinones: In vitro Screening of a Triazolyloxazolidinone for Anticonvulsant Activity

Objective: To test the hypothesis that a triazolyloxazolidinone (PH084) has anticonvulsant activity by examining its effects on in vitro seizure models in the rat hippocampus. Materials and Methods: Whole-cell synaptic currents, action potentials and extracellular population spikes (PS) were recorded in the cell body area of rat hippocampal CA1 region in acutely prepared slices. Chemical [picrotoxin (100 µ<smlcap>M</smlcap>) and zero magnesium] and electrical seizures were induced and the effect of PH084 (10 µ<smlcap>M</smlcap>) was tested on cellular responses, multiple spikes and spontaneous bursting frequencies. Results: PH084 depressed evoked excitatory postsynaptic currents, action potential firing frequency and PS amplitude. All of these responses did not recover to baseline after 15-20 min washout of PH084. Perfusion with zero magnesium ion (Mg²⁺)-containing buffer converted a single PS to multiple PS (mPS) accompanied by spontaneous burst. PH084 suppressed the mPS and the spontaneous burst frequency and it also suppressed the picrotoxin-induced mPS number. However, it did not affect the frequency of stimulus train-induced after discharge or bursts. Furthermore, 8-10 min pretreatment with PH084 did not affect the ability of zero Mg²⁺ buffer, picrotoxin or stimulus train to induce epileptiform activity. Conclusions: Thus, while PH084 may have potential for anticonvulsant activity against chemically induced seizures, it has little or no potential against electrically induced seizures or in preventing epileptiform discharge.

Past as prologue

Baclofen is used clinically to treat spasticity, but has received little attention as a potential antiepileptic agent. To explore the antiepileptic potential of baclofen further, we tested its effect on stimulus train-induced bursting, an in vitro model of hippocampal epileptiform activity. In hippocampal slices prepared from male rats, extracellular field potentials were recorded in stratum pyramidale of CA3, and electrical stimuli were delivered to s. radiatum of CA3. After stable responses to single stimuli were established, stimulus trains were delivered every 5 min until stable triggered and spontaneous population bursting were elicited. (+/-)-Baclofen was bath-applied to the slices at varying concentrations to study its ability to suppress synaptic transmission and epileptiform activity. EC50 values for suppression of orthodromic population spike amplitude, of triggered burst duration and of spontaneous burst frequency were 2300, 355 and 26.9 nM, respectively; all statistically significantly different. These findings suggest that baclofen suppresses epileptiform electrical activity in the hippocampus at concentrations well below those which suppress normal synaptic transmission, and support renewed consideration of baclofen as an antiepileptic agent.

Anticonvulsant enaminone E139 suppresses epileptiform activity in rat hippocampal slices

Some enaminones are reported to have in vivo anticonvulsant activity. We asked if methyl 4-(4'-bromophenyl)aminocyclohex-3-en-6-methyl-2-oxo-1-oate (E139), one of such enaminones produced in vitro effects that may underlie or explain these in vivo anticonvulsant actions by testing if E139 suppressed in vitro seizures. In vitro seizures were generated chemically in hippocampal slices using picrotoxin and zero Mg(2+) buffer and electrically by high frequency stimulation (HFS). E139 (10 microM) depressed evoked field population spike (PS) amplitude by -28.6+/-4.5% (n=5), an effect that was blocked by 1 microM CGP55845 (2.7+/-5.5%, n=6). Picrotoxin (100 microM) transformed single PS into multiple PS (4.5+/-0.2, n=5) and E139 reversibly reduced the number of these multiple PS by -23.4+/-1.8% (n=5). Similarly, zero Mg(2+) buffer produced multiple spikes (3.6+/-0.6, n=5) that were suppressed by E139 (-54.8+/-9.7%, n=5). This effect was also blocked by CGP55845 (2.3+/-5.7%, n=6). Furthermore, E139 suppressed the frequency of spontaneous bursts (SB) that were recorded in zero Mg(2+) by -65.8+/-10.5% (n=12). CGP55845 significantly reduced this E139-induced SB suppression (-21.7+/-9.6%, n=6). In the electrical model, afterdischarges (AD) and SB recorded in area CA3 after a pattern of HFS (100Hz) were suppressed by E139 (-48.6+/-14.3% and -66.7+/-6.7%, respectively, n=6). These E139 effects on AD and SB were reduced, but not completely blocked, by CGP55845 (-32.1+/-5.3% and -44.4+/-9.7%, respectively, n=7). Finally, pretreatment of slices with E139 did not prevent zero Mg(2+)-induced multiple spikes and SB. We conclude that E139 suppresses in vitro seizures in the hippocampus by synaptic and non-synaptic mechanisms. These actions on network activity may underlie their reported in vivo anticonvulsant effects.

Modulation of epileptiform burst frequency by the metabotropic glutamate receptor subtype mGluR3

Spontaneous epileptiform burst activity occurs in acute hippocampal slice dentate granule cells perfused with 10 mM potassium and 0.5 mM calcium [J. Neurophys. 68 (1992) 2016]. We report that activation of the group II metabotropic glutamate receptor subtype 3 (mGluR3) induces an increase in spontaneous burst duration, whereas inhibition of mGluR3 reversibly reduces spontaneous burst frequency. Neither activation, nor inhibition, of group II mGluR had any effects on spontaneous negative dc shifts, or the number of spikes per burst, as compared to control. We conclude that mGluR3 can modulate high potassium, low calcium-induced spontaneous epileptiform burst activity in acute rat hippocampal slice dentate granule cells.

Metabotropic glutamate 1 (mGlu1) receptor antagonists enhance GABAergic neurotransmission: a mechanism for the attenuation of post-ischemic injury and epileptiform activity?

Selective antagonists of mGlu1 metabotropic glutamate receptors attenuate neuronal death in models of cerebral ischemia. Because GABAergic mechanisms have recently been proposed to contribute to these neuroprotective effects, we examined the effects of selective mGlu1 antagonists characterized in our laboratory on GABAergic transmission in three different models of neuropathology. In rat organotypic hippocampal slices exposed to oxygen–glucose deprivation, the mGlu1 antagonists AIDA, CBPG and 3-MATIDA reduced CA1 pyramidal cell loss when added to the medium during the insult and the subsequent recovery period. This effect was mimicked by the GABA A and GABA B agonists muscimol and baclofen and partially prevented by the antagonists bicuculline and CGP 55845. In gerbils subjected to global ischemia, protection of CA1 pyramidal cells by transdialytic perfusion of AIDA and CBPG was associated with a significant increase in the basal and ischemic output of GABA and minor changes in the output of glutamate. In a mouse cortical wedge model, both muscimol and 3-MATIDA reduced the frequency of spontaneous bursts induced by 4-aminopyridine and this reduction was prevented by co-perfusion with bicuculline. Taken together, our results suggest that the release of GABA, and the subsequent activation of GABA receptors, may contribute to the attenuation of post-ischemic neuronal damage and epileptiform activity induced by mGlu1 receptor antagonists.

Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity.

Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.

Characterization of carbachol-induced rhythmic bursting discharges in neurons from guinea pig lateral septum slices.

A brain slice preparation from guinea pigs and intracellular recording techniques were used to examine the effects of carbachol application on the three classes (A, B, and C) of neurons (n = 68, 40 of class A, 12 of class B, 16 of class C) within the mediolateral part of the lateral septum (LSml). Bath application of carbachol elicited a sustained depolarization associated with an increase in membrane input resistance, action-potential firing and triggered rhythmic bursting discharges in 59% of recorded neurons. According to the configuration of these bursts, LSml neurons were classified into type I, II, and III neurons with reference to their response to carbachol. The frequency of spontaneous bursts was increased by depolarization caused by applied DC current in the three types of neurons. Bursts in type II and III neurons were voltage and dose dependent. These dependences were responsible for a continuum of variation in carbachol responses in these two types of neurons. As the neuron depolarized in the presence of carbachol, spontaneous action potentials increased in frequency and slow afterdepolarizing potentials (sADPs) appeared and preceded the occurrence of the first burst. These sADPs from adjacent action potentials appeared to progressively increase to initiate a burst. In the presence of carbachol, sADPs and bursts were also observable after action potentials evoked by depolarizing current pulses at the resting membrane potential (RMP) in LSml neurons. Evoked sADPs and bursts were associated with an apparent increase in input conductance. Application of low Na+ medium blocked both the sADP and bursts. Application of zero Ca2+ medium either 1) blocked completely the generation of sADPs and bursts (n = 16), or 2) did not block bursts (n = 14). Evoked sADPs and bursts were blocked by tetraethylammonium but were resistant to external Cs+. The results indicate that the activation of cholinergic receptors does not differentially affect the three classes of LSml neurons. The responses to carbachol in type II and III neurons form a continuum of variation, whereas these of type I neurons constitute a discrete entity. The selective cholinergic induction of a sADP, and the progressive activation of these sADPs in LSml neurons are thought to be responsible for the onset of the three types of rhythmic bursting discharges. We propose that sADPs and bursts induced by carbachol are generated by a cationic conductance largely permeable to Na+. In a subpopulation of LSml neurons (n = 16), the bursts are dependent on the presence of Ca2+ in the medium.

The influence of electric fields on the epileptiform bursts induced by high potassium in CA3 region of rat hippocampal slice

The effects of pulsed direct current (de) electric fields on the frequency of spontaneous bursting in a model epileptic focus were studied. The high potassium hippocampal slice model was used to generate spontaneous burst firing activity similar to interictal spikes in the pyramidal cell layer of CA3. Electric fields were generated from platinum subdural electrodes placed in the perfusion bath. Three hundred and seventy-eight experimental trials were performed on 10 hippocampal slices from 10 rats and the effects of field polarity, field strength and duration of stimuli on firing frequency was examined. Hippocampal slices were oriented horizontally with the CA3 layer towards the positive electrodel the average interburst interval did not correlate significantly with polarity of the delivering pulses (one-way ANOVA, p = 0.96). Average interburst interval showed a significant correlation with pulse duration of200 and 400msec (p = 0.030 and p = 0.004, respectively). As a function of field strength, there were significant average interval changes for fields of 33, 46, and 73 mV/mm (p = 0.024, P = 0.001 and p = 0.001, respectively). In conclusion, CA3 burst firing activity in high potassium concentration can therefore be altered by electric fields. [Neural Res 1998; 20: 542-548]

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors.

1. When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 microM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5'-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity. 2. ATP can be metabolized by ecto-nucleotidases to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine. 3. We now show that both AMP and ATP (50 microM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5'Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml(-1)), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 microM), AMP deaminase prevented the depressant effect of AMP on discharge rate. 4. AMP deaminase, at a concentration of 0.2 u ml(-1) which annulled the effect of AMP (50 microM), prevented the inhibitory activity of ATP (50 microM). A higher concentration of ATP (200 microM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase. 5. Superfusion of the slices with 5'-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges. 6. The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the effect of ATP. A corollary to this is that, when examining the pharmacology of ATP, care must be taken to inactivate AMP with AMP deaminase, as well as adenosine with adenosine deaminase, before a direct action of ATP on P1 receptors can be postulated. Failure to do so may have led to erroneous conclusions in some previous studies of nucleotide activity on nucleotide receptors.

Modification of parallel activity elicited by propagating bursts in developing networks of rat cortical neurones.

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity-dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.

Effects of in vivo perforant path kindling on O-Mg 2+-induced CA1 burst activity in rat hippocampal slices

Previous studies have reported persistent alterations in the electrophysiological characteristics of the CA1 region of the hippocampus after kindling. The present study examined the effects of perforant path kindling on O-Mg 2+-induced epileptiform bursting in the CA1 region of hippocampal slices. The duration of evoked bursting was significantly longer in slices taken from kindled animals as compared to those taken from implanted, non-stimulated controls. No significant differences were found in spontaneous burst frequency between slices taken from kindled and control animals. These data suggest that perforant path kindling causes a persistent increase in hyperexcitability in the CA1 region of the hippocampus leading to a facilitation of evoked burst activity, perhaps through an enhancement in NMDA-related excitatory neurotransmission.

Relationship between frequency of spontaneous bursting and tonotopic position in the developing avian auditory system

Neural activity in the developing brainstem auditory pathway of the chick embryo is dominated by a rhythmic pattern of spontaneous discharge. Neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL), second and third order auditory nuclei, discharge spontaneously in synchronous bursts at periodic intervals. Rhythmic bursting is present as early as embryonic day 14 (E14), shortly after the onset of functional synaptogenesis, and gives way to an adult-like, steady level of firing on E19, two days prior to hatching. In the present experiment, multiple-unit recording techniques were used in E17 and E18 embryos to examine the relationship between rate of rhythmic bursting and tonotopic position in NM and NL. The mean rate of rhythmic bursting ranged from 0.21-0.71 Hz. Bursting rate varied systematically as a function of position, being faster at progressively higher frequency regions of the nuclei at both E17 ( r = 0.75) and E18 ( r = 0.86). In addition, the rate of bursting at a given location in the nuclei increased during development. The presence of a systematic relationship between the rate of rhythmic bursting and tonotopic location suggests that the spatio-temporal pattern of spontaneous discharges could provide developmental cues for the spatial ordering of auditory projections.

Role of metabotropic glutamate receptor subtypes in the patterning of epileptiform activities in vitro.

1. Epileptiform activities were elicited from the in vitro guinea pig hippocampus by the addition of picrotoxin. Modification of the picrotoxin-induced activities by agents active at metabotropic glutamate receptors (mGluRs) was examined using intracellular and extracellular recordings. 2. Picrotoxin typically elicited synchronized discharges (epileptiform bursts) in CA3 neurons. These spontaneously occurred at regular intervals. In the presence of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 700-1,000 microM), an antagonist at multiple mGluR subtypes, the frequency of spontaneous epileptiform bursts decreased. In contrast, when the mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 5 microM) or (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 10 microM) were added to the incubating medium, the frequency of epileptiform bursts increased. No consistent change in membrane potential, burst duration, nor burst afterhyperpolarization was associated with the changes in burst frequency. 3. When spontaneous burst frequency was reduced in MCPG, stimulation at a higher frequency entrained bursts without failure. Bursts evoked in MCPG were similar in waveform and amplitude to those evoked in the control state. 4. (S)-4-carboxyphenylglycine (S-4CPG) and (R,S)-4-carboxy-3-hydroxyphenylglycine (RS-4C3HPG) are antagonists at mGluR subtypes 1 and 5 but agonists at mGluRs 2 and 3. Addition of either of these agents increased the frequency of epileptiform bursts. 5. These results suggest that sufficient glutamate is released during epileptiform activities to activate mGluRs. The overall effect is to increase the frequency of synchronized discharges. This modulatory action on burst frequency is probably mediated via the mGluR 2 and 3 receptor subclass.

Combined administration of growth-hormone-releasing hormone and clonidine restores defective growth hormone secretion in old dogs.

We have studied in old dogs the effects of short-term administration of growth hormone (GH)-releasing hormone (GHRH) alone or co-administered with clonidine (CLO), an alpha 2-adrenergic agonist, on the GH secretory pattern (cluster analysis), and GH responsiveness to an acute GHRH or GHRH + CLO challenge and plasma somatomedin C (SMC) levels. Dogs were given either GHRH alone twice daily for 10 days (treatment 1) or combined GHRH + CLO both given twice daily (treatment 2) or GHRH + CLO given once daily (treatment 3). Animals were sampled from 09.00 to 15.00 h, at 10-min intervals, both before and 14 h after treatments. At the end of the 6-hour sampling period, dogs were challenged with simultaneous administration of GHRH and CLO, while they were tested with GHRH alone on the morning of the following day. In dogs undergoing treatment 1, acute administration of GHRH or GHRH + CLO elicited mean GH peak responses higher than before treatment, but none of the GH secretory indices were modified during the 6-hour sampling period, except for the increase in mean GH peak amplitude. In dogs undergoing treatment 2, acute administration of GHRH elicited a mean GH peak response higher than that before treatment, whereas administration of GHRH + CLO induced a mean GH peak response not different from that elicited by GHRH + CLO before treatment or by GHRH alone after treatment. However, this treatment significantly augmented the frequency of spontaneous bursts of GH secretion, the mean GH peak amplitude and the total peak area.(ABSTRACT TRUNCATED AT 250 WORDS)

The epileptiform activity induced by 4-aminopyridine in rat amygdala slices: antagonism by non- N-methyl- d-aspartate receptor anatagonists

The effects of excitatory amino acid receptor antagonists kynuretic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on epileptiform activity induced by 4-aminopyridine (4-AP) were studied in rat amygdala slices using intracellular recording techniques. Five to 10 min after switching to 4-AP-containing solution, spontaneous epileptiform bursts were observed in 35 out of 45 slices studied. The spontaneous epileptiform events consisted of an initial burst followed by a number of afterdischarges. Superfusion with kynuretic acid, a broad-spectrum excitatory amino acid receptor antagonist, reversibly reduced the duration of the spontaneous bursting discharges in a dose-dependent manner. The frequency of spontaneous bursting was also decreased. The IC 50, estimated from the graph of the concentration-response relationship, was approximately 130 μM. In addition, CNQX which is a specificnon-N-methyl- d-aspartate (NMDA) receptor antagonist blocked the spontaneous and evoked epileptiform bursting. In 11 out of 15 neurons tested, there was a residual depolarizing component remained. This depolarizing component was reversibly blocked by specific NMDA receptor antagonist, d,l-2-amino-5-phosphonovaleate ( d,l-APV). Relative to the CNQX-sensitive component, the d,l-APV-sensitive component is much smaller in amplitude and shorter in duration indicating that NMDA receptor plays only a minor role in this process. These data suggest that the generation of propagation of spontaneous epileptiform events induced by 4-AP in the neurons of basolateral amygdala is mediated by excitatory amino acids and that activation of non-NMDA receptors is of primary importance.

Electrical and mechanical responses to inhibition of cell respiration in vascular smooth muscle of the rat portal vein.

Metabolic regulation of contractility in vascular smooth muscle was studied in the spontaneously active rat portal vein using respiratory depression by cyanide (0.2-2.0 mM) as a model for tissue hypoxia. Intracellular recordings of electrical activity were done with concomitant registration of force development. Average membrane potential in the absence of cyanide was -61 +/- 1 mV (n = 27). Addition of cyanide to normal Krebs solution resulted in a reduction of force amplitude and the number of action potentials per burst, with a relatively more pronounced effect on the mechanical activity. At moderate levels of inhibition of force amplitude the frequency of spontaneous bursts of action potentials transiently increased concomitant with a slight depolarization, but after prolonged (15-20 min) exposure to cyanide the membrane repolarized to the level prior to cyanide addition and the burst frequency decreased to be equal to or lower than that in the absence of cyanide. Higher concentrations of cyanide totally inhibited spontaneous mechanical and electrical activity. In contrast to the results with glucose, it was found that when beta-hydroxybutyrate was used as substrate the addition of 2 mM cyanide led to a marked hyperpolarization (13 +/- 1 mV) after total inhibition of spontaneous activity. The hyperpolarization was not prevented by administration of 4-aminopyridine (2.5 mM) or tetraethylammonium (4-6 mM) prior to the addition of cyanide. To investigate the effects of increased metabolic demand on the relation between force and membrane potential in cyanide-treated muscle, high-K+ (40 mM) contractures were studied. Contractures were associated with depolarization of 34 +/- 3 mV (n = 5). 1 mM cyanide reduced the amplitude of the contractures to about 9% of control with a moderate reduction in the amount of depolarization (28 +/- 1 mV, n = 5). It is concluded that the decrease of mechanical activity during respiratory inhibition may partly reflect a reduction in the number of spikes per burst but that other mechanisms, independent of membrane activity, also contribute to the inhibition. The increase of glycolysis during respiratory inhibition seems to prevent more pronounced changes in membrane potential.

GABAergic inhibition and the induction of spontaneous epileptiform activity by low chloride and high potassium in the hippocampal slice

Intracellular recordings from CA3b/c neurons in rat hippocampal slices showed that reduction of the extracellular Cl − concentration from 136 to 53 mM produced a positive (+ 10 mV) shift in the reversal potential of GABAergic inhibitory postsynaptic potentials (IPSPs). This shift was not significantly different from the shift produced by raising K + from 3.5 to 8.5 mM. Spontaneous interictal bursting occurred in both low Cl − and high K +. Extracellular recordings from the pyramidal cell layer in the CA3b/c region of hippocampal slices showed that bursts in 56 mM Cl − were of the same waveform and intensity as bursts produced by high K +. However the frequency of spontaneous bursting was much lower (6.6 ± 1.2/min, n = 10) in low Cl − compared to high K + (42.2 ± 3.0/min, n = 33). Burst frequency was a linear function of the shift in IPSP reversal potential produced by high K +, but not low Cl −. Replacing 60% of the Cl − with methylsulfate or isethionate was sufficient to produce spontaneous bursting, whereas it was necessary to replace 80% of the Cl − when propionate was used as a substitute. All 3 Cl − substitutes lowered the ionized Ca 2+ concentration, but raising the extracellular Ca 2+ concentration back to normal did not change the burst frequency. Since the amplitude of IPSPs is reduced to a similar extent in low Cl − and high K + solutions, whereas bursting is much faster in high K +, we suggest that impaired GABAergic inhibition is insufficient to fully account for spontaneous interictal bursting that is produced in hippocampal slices by raised extracellular K +.

Potassium-induced epileptiform activity in area CA3 varies markedly along the septotemporal axis of the rat hippocampus

Hippocampal slices are generally treated as equivalent regardless of their site of origin along the septotemporal axis. In this study, spontaneous epileptiform bursting was induced in area CA3 of rat hippocampal slices by bathing them in 7 mM potassium. The frequency of spontaneous bursting was measured in all viable slices from 12 hippocampi. Burst frequency was found to vary markedly and in a consistent fashion with site of slice origin along the septotemporal axis. Burst frequency was maximal in slices from near the temporal end and declined progressively toward the septal end. This finding was independent of slicing angle. These results demonstrate that site of slice origin along the septotemporal axis is an important confounding variable in in vitro studies of hippocampal neuronal activity. Furthermore, they support the notion that the temporal portion of the hippocampus may be more prone to seizure activity than the septal hippocampus, possibly because of factors intrinsic to the hippocampus.