Effects of quinidine and quinine on the excitability of pyramidal neurons in guinea-pig hippocampal slices

Effects of quinidine (25M–1mM) and its stereoisomer, quinine (1–5 mM), on the excitability of CA3 pyramidal neurons were investigated in guinea-pig hippocampal slices using intracellular recording techniques. At concentrations of quinidine higher than 100 M (and higher than 1 mM for quinine), 1) the resting potential shifted to the depolarizing direction with an increase of the input resistance, 2) the spike duration was prolonged, 3) the spike amplitude was decreased, 4) the late component of the afterhyperpolarization (AHP) (caused by the activity of the Ca²⁺-mediated K conductance) were suppressed, and 5) finally, neurons became inexcitable. The results indicate that the blocking action of quinidine and quinine is not specific to the Ca²⁺-mediated K conductance in mammalian hippocampal neurons, and that this conductance is much less sensitive to the drugs in comparison with other preparations.     

Epileptogenic effect of cyclosporine in guinea-pig hippocampal slices

Cyclosporine A (CsA) neurotoxicity is a common cause of seizures in transplant patients and others receiving immunosuppressive therapy. CsA at concentrations higher than the levels estimated for cerebrospinal fluid of the patients suffering from seizure attacks was ineffective to induce epileptiform field potentials (EFP) in in vitro brain-slice preparation. The aim of this study was to test the effect of CsA at lower concentrations on neuronal activity. Guinea-pig hippocampal slices were exposed to artificial cerebrospinal fluid containing CsA (0.1-2 microM). Furthermore, the effects of CsA (0.25-10 microM) were tested on EFP elicited by omission of Mg2+ from superfusate. Low concentrations of CsA (0.1-0.25 microM) induced EFP while higher doses (0.5-2 microM) failed to decrease the seizure threshold. CsA at concentrations of 0.25 and 1 microM had no significant effect on the low Mg2+-induced EFP. Higher CsA concentration (10 microM) strongly suppressed EFP. The results indicate that CsA at doses that are probably clinically relevant increases the neuronal excitability.     

Post-inhibitory excitation of adenosine on neurotransmission in guinea pig hippocampal slices.

The postsynaptic field potential (population spike potential; PS) was recorded from the granule cell layer of guinea pig hippocampal slices. Adenosine at low concentrations ranging from 10 nM to 1 microM enhanced the amplitude of PS, whereas at concentrations over 10 microM it inhibited the neurotransmission. There appeared to be a rebound phenomenon after the removal of adenosine at inhibitory concentrations and the amplitude of the PS overshot the initial amplitude (we called this post-inhibitory excitation; PIE). Neither depressants such as gamma-aminobutyric acid (GABA; 1 mM) nor sodium pentobarbital (100 microM) by itself induced PIE. After application of GABA or sodium pentobarbital together with adenosine (0.1 microM), however, removal of all agents could induce the PIE. PIE as well as the excitatory effect of adenosine at low concentrations was counteracted by application of H-7 (100 microM), melittin or polymyxin B, potent protein kinase C (PKC) inhibitors, suggesting that the excitatory effect of adenosine is mediated by a metabolic process involving PKC. These results indicate that PIE induced by adenosine at high concentrations is due to a mechanism similar to the excitatory effect induced by adenosine at low concentrations, and that during application of adenosine at high concentrations the excitation is masked by its potent inhibitory effect.     

Temperature dependence of extracellular ionic changes evoked by anoxia in hippocampal slices

1. Extracellular [K] and [Ca] were measured with ion-selective microelectrodes in CA1 pyramidal cell layer of rat hippocampal slices in an interface chamber. 2. Near room temperature (21-22 degrees C), brief periods of anoxia (3- to 4-min substitution of 95% N2-5% CO2 for 95% O2-5% CO2) produced very small changes in [K]o [-0.022 +/- 0.10 (SE) mM] or [Ca]o (-0.030 +/- 0.0029 mM) and were associated with only minor depression of population spikes (-22.5 +/- 11%). 3. Stratum radiatum (SR) stimulation (0.2-5 Hz) could evoke substantial increases in [K]o (by 0.2-2 mM); although variable, they were consistent in any one slice. The same stimulation regularly caused only small depressions of [Ca]o (by less than 0.1 mM, typically). 4. Also at 21-22 degrees neither stimulation nor anoxia generated more than minimal reductions in extracellular space [by 2.3 +/- 0.94%, as measured by the tetramethylammonium (TMA) method], and spreading depression (SD) occurred in only 1 out of 20 slices. 5. At 33-34 degrees C, anoxia (also for 3-4 min) consistently produced more substantial increases in [K]o (0.83 +/- 0.18 mM); but the apparent changes in [Ca]o at 33 degrees C (0.058 +/- 0.12 mM) could not with certainty be distinguished from thermoelectric artifacts. There was a severe depression of population spikes (-76 +/- 10%). 6. Although electrical stimulation evoked greater reductions in [Ca]o, increases in [K]o were 50% smaller. 7. During anoxia at 33-34 degrees C, the extracellular space was significantly reduced, by 6.1 +/- 0.9%. Moreover, in 37% of the slices, either stimulation or anoxia triggered massive increases in [K]o (greater than 10 mM) and large reductions in [Ca]o (less than 1 mM), associated with SD-like swings in focal potential. 8. It is concluded that the extracellular ionic changes evoked by brief anoxia do not contribute in a major way to the depression of synaptic transmission.     

Effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum

The effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum were investigated. The resting membrane potential of the duodenal smooth muscle cells was reduced by neurotensin at the concentration of 3 X 10(-8) M or more. The amplitude of the non-adrenergic inhibitory potentials was decreased by neurotensin (1-3 X 10(-8) M). In the Ca2+-free solution, the amplitude of the inhibitory potentials was also decreased. The increased amplitude of the non-adrenergic inhibitory potentials evoked in the high calcium solution (Ca2+, 5 mM) was decreased by neurotensin (10(-8) M). Neither atropine (1.4 X 10(-6) M) nor propranolol (3.4 X 10(-6) M) blocked the inhibitory action of neurotensin (10(-8) M) on the inhibitory potential. The frequency of the spontaneous action potentials of the duodenal smooth muscle cells was strongly increased with accompanying depolarization by neurotensin 8 X 10(-8) M. The tonic contraction of the duodenal smooth muscles was produced by neurotensin (3-8 X 10(-8) M). The results obtained suggest that neurotensin relates to the non-adrenergic inhibitory pathway in the control mechanism on intestinal motility.     

Studies on substances that induce long-term potentiation in guinea-pig hippocampal slices.

Fluids were collected from the rabbit neocortex during a tetanic stimulation of the cortical surface. When these samples from the neocortex were applied on the guinea-pig hippocampal slices, only those fractions containing substances with molecular weights less than 3000, 3000-10,000 and greater than 50,000 and not with other molecular weights, could induce long-term potentiation of population spikes in the CAI area in response to stratum radiatum stimulation. Intracellular recordings from the CAI neurons revealed that the long-term potentiation-inducing substances increased the excitatory postsynaptic potential without changing the membrane potential and the input resistance of these cells. A pretreatment of the rabbits with MK-801 prevented the release of the long-term potentiation-inducing substances. 2-Amino-5-phosphonovalerate was unable to block the long-term potentiation-inducing action of the substances from the rabbit neocortex. Gel-electrophoresis of the substances collected from the rabbit neocortex revealed the presence of an acidic peptide with a molecular weight of about 69,000. These results indicate that tetanic stimulation of rabbit neocortex results in a release of substances with molecular weights of less than 3000, 3000-10,000 and greater than 50,000 that could induce long-term potentiation in guinea-pig hippocampal slices. The release, but not the long-term potentiation-inducing action, of these substances appears to depend on the activation of N-methyl-D-aspartate receptors. The long-term potentiation-inducing substance in the greater than 50,000 mol. wt fraction may be an acidic peptide with a molecular weight of about 69,000.     

Excitatory effect of adenosine on neurotransmission is due to increase of transmitter release in the hippocampal slices.

Adenosine has dose-dependent biphasic excitatory and inhibitory effects on neurotransmission in the hippocampus. The mechanism of the excitatory action is not known although that of the inhibitory action has been well analyzed. Here we report on the mechanism of excitatory action of adenosine, using hippocampal slices. Studies of intracellular recordings of CA3 pyramidal neurons showed that the amplitude of EPSP was dramatically enhanced by application of adenosine at low concentration (0.1 microM) without changing resting membrane potentials, membrane conductance or the threshold for spike generation by injecting current pulses. On the other hand, the presence of adenosine at a concentration of 0.1 microM during electrical stimulation to the slices increased 1.7 times the release of glutamate, an excitatory neurotransmitter in the hippocampus. These results indicate that the excitatory action of adenosine at low doses is due to the increase of transmitter release.     

Effects of temperature on the oxygen consumption in thin slices from different brain regions

The oxygen consumption of brain slices from 12 different regions of the guinea pig brain was measured and the effects of temperature on the consumption was investigated. At 37 degrees C the oxygen consumption in cerebral gray matter and inferior colliculus was higher than that of other regions, whilst the consumption in cerebral white matter and brainstem was approximately half of that in cerebral gray matter. In any region lowering the tissue temperature from 37 to 28 degrees C reduced the oxygen consumption by 6-8% of the value obtained at 37 degrees C per one degree. At 21 degrees C the consumption was decreased to 15% of the value obtained at 37 degrees C. It is notable that the consumption in hippocampal slices tested was remarkably increased by raising the temperature up to 50 degrees C and no consumption was observed at 55 degrees C.     

Effects of 4-aminopyridine on the field potentials of hippocampal slices

In addition to promoting long-lasting negative (and positive) dendritic field potentials elicited by stimulation of Schaffer collaterals in hippocampal slices, 4-aminopyridine causes a slow negative wave which may have another origin than the dendritic potentials.     

Sprouting and functional recovery in co-cultures between old and young hippocampal organotypic slices

We developed a model of lesion of Schaffer collaterals in hippocampal organotypic slice cultures to analyse the capacity for sprouting and functional recovery expressed in young (one week old) and old (four week old) slice cultures. Slice cultures were sectioned at different ages of maturation in two separate half-slices and maintained in co-culture. Functional recovery was assessed by measuring synaptic responses elicited across the lesion seven days after the lesion and sprouting was evaluated by biocytin labeling of the regenerating fibers seen under the same conditions. Sprouting and functional recovery were found to be markedly reduced and delayed in old vs young cultures. Preparation of co-cultures between young CA3 and old CA1 half-slices resulted in a significant reduction in the capacity for sprouting and regeneration of the young CA3 neurons. Conversely, co-cultures prepared between old CA3 and young CA1 half-slices showed a markedly enhanced capacity for sprouting and functional recovery of old CA3 neurons.

These results indicate that the age-dependent impairment in sprouting and regeneration expressed in cortical regions can be improved by and depends upon the presence of a favourable environment.     

Rapid preconditioning neuroprotection following anoxia in hippocampal slices: role of the K+ ATP channel and protein kinase C

Sublethal cerebral anoxic/ischemic insults may "precondition" and thereby protect brain from subsequent anoxic/ischemic insults. We tested two hypotheses in hippocampal slices: (i) that short periods of anoxia, each followed by reoxygenation, precondition and thereby improve recovery of synaptic activity following "lethal" anoxic insults; and (ii) that the ATP-sensitive potassium channel [K+ ATP] or protein kinase C mediates anoxic preconditioning neuroprotection in hippocampal slices. Hippocampal slices were subjected to three short periods of anoxia, each separated by 10 min of reoxygenation. These anoxic insults were prolonged only until the onset of anoxic depolarization. Thirty minutes following these insults, slices underwent a "test" anoxic insult, which was characterized by an anoxic insult that lasted 1 min of anoxic depolarization. Recovery of evoked potential amplitudes was followed for 30 min of reoxygenation. The beneficial effects of preconditioning was shown by the significant recovery of evoked potentials after "test" anoxic insults in preconditioned slices, when compared to controls that only underwent a "test" anoxic insult. In control slices, transient superfusion with an ATP-sensitive potassium channel agonist (10 microM pinacidil) 30 min prior to "test" anoxia markedly improved evoked potential recovery. Administration of 5 microM of the sulfonylurea tolbutamide, an ATP-sensitive potassium channel antagonist during preconditioning insults, blocked the protection afforded by preconditioning. Transient superfusion of a protein kinase C activator (500 nM phorbol 12-myristate 13-acetate) did not improve evoked potential recovery. Administration of 50 nM chelerythrine, a protein kinase C inhibitor during preconditioning insults did not block the protection afforded by preconditioning. These data support the hypothesis that the ATP-sensitive potassium channel is involved in the neuroprotection afforded by anoxic preconditioning in hippocampal slices. However, protein kinase C activation does not appear to play a role in this neuroprotection.     

Effects of polyvalent cations and dihydropyridine calcium channel blockers on recovery of CNS white matter from anoxia

The effects of anoxic injury on the functional integrity of mammalian central white matter were studied electrophysiologically using the rat optic nerve model. Previous studies on this model have shown that extracellular Ca2+ is critical to the production of irreversible anoxic injury, and suggest that during anoxia Ca2+ crosses the membrane to enter the intracellular compartment. We attempted to elucidate the mechanism by which this damaging Ca2+ influx occurs. The inorganic Ca2+ channel blockers Mn2+ (1 mM), Co2+ (1 mM) or La3+ (0.1 mM) had no effect on recovery of the area under the compound action potential after a standard 60 min period of anoxia; only Mg2+ (10 mM) significantly improved recovery (54.9 +/- 8.9% vs. 28.7 +/- 10.1%, P less than 0.005). Treatment with organic Ca2+ channel blockers of the dihydropyridine class, nifedipine (1-10 microM) or nimodipine (1-40 microM), also had no effect on recovery from anoxia. We conclude that Ca2+ influx during anoxia does not occur via conventional Ca2+ channels sensitive to polyvalent cations or dihydropyridines.     

The effect of GABA on neurotransmission in frog tectal slices

The regional distribution of gamma-aminobutyric acid (GABA) was determined in the central nervous system of the frog. The highest amount of GABA was found in the tectum (35.7 +/- 2.3 mmol/kg protein). A thin tectal slice (400-600 microns thick) preparation was developed in which the postsynaptic potential (PSP) could be elicited. The addition of GABA to the perfusion medium depressed the PSP in a dose-dependent manner in the concentration range 1-12.5 mM at 28 degrees C. Nipecotic acid, a GABA uptake inhibitor, at a concentration of 0.5 mM shifted the dose-response curve for GABA to the left. These results from the frog, a cold-blooded animal, were in good agreement with those for hippocampal slices from the guinea-pig.     

The effects of vasopressin on hippocampal slices from trained rats

The effects of vasopressin on hippocampal slices from control and pretrained rats were compared. Prolonged presentation of both combined and noncombined conditioned stimuli (CS) and unconditioned stimuli (US) increases the sensitivity of hippocampal neurons to vasopressin. The effect of this was that responses to application of DG-AVP after training and active control treatment was biphasic, consisting of initial excitation and subsequent inhibition; similar effects were seen in control slices when the peptide concentration was increased. Increases in the excitatory phase occurred with presentation of the CS and US and were independent of the training factor. The inhibitory phase increased to a greater extent on presentation of combinations of the CS and US. It is suggested that increases in depression in response to application of vasopressin in trained animals, as compared with active controls, are due to a higher level of free intracellular calcium occurring as a result of the training procedure. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti, Vol. 48, No. 2, pp. 222–228, March–April, 1998.     

Effects of noradrenaline on potassium reflux, membrane potential and electrolyte levels in tissue slices prepared from guinea-pig liver

1. Some effects of noradrenaline on potassium efflux, electrolyte levels, membrane potential and current distribution in guinea-pig liver slices have been examined.2. The slices (thickness ca. 300 mum) were prepared from the median lobe of the liver and incubated at 38 degrees C in a mammalian Ringer fluid containing 2 mM pyruvate. After an initial recovery period, the ionic composition of the tissue remained stable for several hours.3. The steady-state contents of sodium, potassium and chloride were 296, 266 and 272 m-equiv/kg dry tissue respectively. The inulin space was 29 ml./100 g wet tissue.4. Most if not all of the tissue potassium was exchangeable. The rate constant for (42)K efflux was 0.019 min(-1).5. Noradrenaline (1 muM) markedly increased the efflux of (42)K and within 2 min caused tissue potassium to fall by 8%. At the same time the sodium content rose.6. Traverses of the slices with micro-electrodes showed many negative-going deflexions of 30-40 mV in amplitude. The evidence suggests that these correspond to the membrane potentials of the parenchymal cells.7. Noradrenaline (1 muM) caused a reversible hyperpolarization of about 10 mV. The response became larger on replacing external chloride by isethionate or methylsulphate, but was little affected by a reduction in external potassium.8. After slices had been bathed in potassium and chloride-free solutions for several min, restoration of external potassium caused the membrane potential to increase by up to 10 mV. This hyperpolarization, but not that caused by noradrenaline, was abolished by ouabain.9. Noradrenaline reduced the amplitude and quickened the time course of electrotonic potentials set up by current pulses from another microelectrode, suggesting that the membrane conductance had risen.10. Although certain mechanisms based on electrogenic active transport processes with unusual properties have not been excluded, the present findings are more simply explained by supposing that noradrenaline increases the potassium permeability of the parenchymal cell membrane.     

Requirement of NMDA receptor/channels for intracellular high-energy phosphates and the extent of intraneuronal calcium buffering in cultured mouse hippocampal neurons

Whole-cell patch-clamp recordings were undertaken in cultured mouse hippocampal neurons in order to investigate time-dependent changes in: (i) currents evoked by L-aspartic acid (Asp) and kainic acid (KAI), two excitatory amino acids active at N-methyl-D-aspartic acid (NMDA) and KAI receptor sites respectively, and (ii) tetrodotoxin (TTX) resistant voltage-dependent inward currents carried by Ca2+. Consistent with previous observations, Ca2+ currents gradually run down unless a support system containing Mg-ATP, phosphocreatine and creatinine phosphokinase is added to the intracellular medium. Here we report that, in addition to suppressing the rundown of currents through voltage-gated Ca2+ channels, such a support system is also necessary to prevent rundown of ionic currents through excitatory amino acid-gated channels of the NMDA type. When this support system was omitted from the recording pipette, currents induced by Asp, but not KAI, progressively declined over a period of 20 min and stabilized at values of about 50% of the initial. This progressive decline occurred regardless of the extent of intraneuronal Ca2+ buffering, indicating that it was not due to accumulation of cytosolic Ca2+. After the rundown, reversal potentials of ASP-induced currents were the same whether recorded with or without the intracellular support system and the Asp induced currents could be blocked by the specific NMDA channel blocker ketamine. We conclude that ionic currents through NMDA gated channels have two components: one requires high-energy phosphates and will run down if these are not supplied; the other requires no such supply and remains steady.