John C. Szerb

The effect of cholinergic drugs on [3H]acetylcholine release from slices of rat hippocampus, striatum and cortex.

Slices from rat hippocampus, striatum or cortex were incubated with l mum [3H] choline and following 75 min superfusion with Krebs solution the efflux of radioactivity was measured. The slices were stimulated either electrically (1 Hz) or with 25 mM potassium and the rate constant of the evoked release and the size of the releasable pool were estimated. The spontaneous efflux of radioactivity and the releasable pool but not the rate of evoked release correlated with the reported endogenous ACh content of the 3 areas. Raised potassium released radioactivity at a lower rate but from a larger pool than electrical stimulation from all 3 areas. In all 3 areas atropine alone potentiated while physostigmine, oxotremorine and carbamylcholine decreased the rate of evoked release. This depression was fully antagonized by atropine. The drugs had no effect on the size of the releasable pool. Findings suggest that muscarinic receptors located on cholinergic axons or terminals have a physiological role in the autoregulation of ACh release from these 3 areas.

Effect of ammonium ions on synaptic transmission in the mammalian central nervous system.

It is not surprising that a compound with such unique properties as NH3/NH4+, should have a large variety of biochemical and neurological effects and to find itself implicated in many pathological conditions. Its undissociated (NH3) or dissociated (NH4+) forms, having different physicochemical properties, enter neurons and other cells through differing pathways. These two forms then change internal pH in opposite directions, and initiate a variety of regulatory processes that attempt to overcome these pH changes. In addition, ammonia has a central role in normal intermediary metabolism, and when present in excess, it can disturb reversible reactions in which it participates. The challenge in interpreting these various observations lies in the difficulty in assigning to them a role in the generation of symptoms seen in experimental and clinical hyperammonemias. In this review we have attempted to summarize information available on the effects of ammonium ions on synaptic transmission, a central process in nervous system function. Evidence has been presented to show that ammonium ions, in pathologically relevant concentrations, interfere with glutamatergic excitatory transmission, not by decreasing the release of glutamate, but by preventing its action on post-synaptic AMPA receptors. Furthermore, NH4+ depolarizes neurons to a variable degree, without consistently changing membrane resistance, probably by reducing [K+]i. A decrease in EK+ may also be responsible for decreasing the effectiveness of the outward chloride pump, thus explaining the well known inhibitory effect of NH4+ on the hyperpolarizing IPSP. There is a consensus of opinion that chronic hyperammonemia increases 5HT turnover and this may be responsible for altered sleep patterns seen in hepatic encephalopathy. There does not seem to be a consistent effect on catecholaminergic transmission in hyperammonemias. However, chronic hyperammonemia causes pathological changes in perineuronal astrocytes, which may lead to a reduced uptake of released glutamate and a decreased detoxification of ammonia by the brain. Chronic moderate increase in extracellular glutamate results in a down-regulation of NMDA receptors, while the decreased detoxification of ammonia makes the central nervous system more vulnerable to a sudden hyperammonemia, due, for instance, to an increased dietary intake of proteins or to gastrointestinal bleeding in patients with liver disease. Clearly, data summarized in this review represent only the beginning in the elucidation of the mechanism of ammonia neurotoxicity. It should help, we hope, to direct future investigations towards some of the questions that need to be answered.

THE RELEASE OF LABELLED ACETYLCHOLINE AND CHOLINE FROM CEREBRAL CORTICAL SLICES STIMULATED ELECTRICALLY

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [3H]‐choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation. 2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [3H]‐ACh which was greatly enhanced by the addition of atropine. 3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [3H]‐ACh which declined asymptotically. This evoked release appeared to follow first‐order kinetics with a rate constant which remained stable over the course of prolonged stimulation. 4 The rate constant for the evoked release of [3H]‐ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [3H]‐ACh was released was nearly identical under these two conditions. 5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [3H]‐choline in the samples. 6 Reduction of [3H]‐ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [3H]‐ACh without affecting the release of [3H]‐choline. 7 Results suggest that electrical stimulation of cortical slices which had been incubated with [3H]‐choline causes the release of only [3H]‐ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [3H]‐ACh.

Modification of neocortical acetylcholine release and electroencephalogram desynchronization due to brainstem stimulation by drugs applied to the basal forebrain.

Acetylcholine released from the cerebral cortex was collected using microdialysis while stimulating the region of the pedunculopontine tegmentum in urethane-anesthetized rats. Electrical stimulation in the form of short trains of pulses delivered once per minute produced a 350% increase in acetylcholine release and a desynchronization of the electroencephalogram, as measured by relative power in the 20-45 Hz range (low-voltage fast activity). Perfusion of the region of cholinergic neurons believed to be responsible for the cortical release of acetylcholine, the nucleus basalis magnocellularis, was carried out using a second microdialysis probe. Exposure of the nucleus basalis magnocellularis to blockers of neural activity (tetrodotoxin or procaine) or to blockers of synaptic transmission (calcium-free solution plus magnesium or cobalt) produced a substantial decrease in the release of acetylcholine and desynchronization evoked by brainstem stimulation. Exposure of the nucleus basalis magnocellularis to the glutamate antagonist, kynurenate, resulted in a decrease in evoked acetylcholine release and electroencephalogram desynchronization similar in magnitude to that produced by nonspecific blockers, whereas application of muscarinic or nicotinic cholinergic blockers to the nucleus basalis magnocellularis did not reduce acetylcholine release or electroencephalogram desynchronization. Application of tetrodotoxin to the collection site in the cortex abolished the stimulation-evoked acetylcholine release, but not the low baseline release indicating that cholinergic nucleus basalis magnocellularis neurons have a low spontaneous firing rate in urethane-anesthetized animals. The results of this study suggest that the major excitatory input to the cholinergic neurons of the nucleus basalis magnocellularis from the pedunculopontine tegmentum is via glutamatergic and not cholinergic synapses.

Release of [3H]acetylcholine from rat hippocampal slices: Effect of septal lesion and of graded concentrations of muscarinic agonists and antagonists

To establish the existence and sensitivity of presynaptic muscarinic receptors on central cholinergic neurons, the electrically evoked release of [3H]ACh from hippocampal slices was measured after medial septal lesion or in the presence of graded concentrations of muscarinic agonists and antagonists. One week after septal lesion, the evoked release of [3H]ACh was abolished, indicating that septo-hippocampal cholinergic fibres are the source of this release. The muscarinic agonists, Oxotremorine, carbamylcholine and arecoline reduced the rate of evoked release of [3H]ACh with an ED50 similar to the ED50 required to displace specific [3H]quinuclidinyl benzilate (QNB) binding as found by Yamamura and Snyder. However, the antagonists QNB, antropine and scopolamine were 10 times weaker in increasing the rate of [3H]ACh release than in displacing [3H]QNB binding. Results suggest that the lower affinity of muscarinic antagonists to presynaptic receptors prevents the demonstration of the specific labelling of these receptors with [3H]QNB.

DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [3H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC‐3) increased the spontaneous efflux of [3H]choline. Electrical stimulation at 4/s increased the efflux of [3H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [3H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [3H]choline evoked by stimulation probably originates from stores of [3H]ACh synthetized during incubation.

THE OUTPUT PER STIMULUS OF ACETYLCHOLINE FROM CEREBRAL CORTICAL SLICES IN THE PRESENCE OR ABSENCE OF CHOLINESTERASE INHIBITION

1 The release of endogenous acetylcholine (ACh) from cerebral cortical slices stimulated at 0.25, 1, 4, 16 and 64 Hz was measured in the presence either of physostigmine or of physostigmine and atropine. 2 Atropine potentiated the evoked release of endogenous ACh especially at low frequencies resulting in an output per stimulus which sharply declined with increasing frequency of stimulation, while in the absence of atropine the output of ACh per stimulus was low and fairly constant. 3 The evoked release of [3H]‐ACh per stimulus following the incubation of the slices with [3H]‐choline, as estimated by means of rate constants of the evoked release of total radioactivity, showed a frequency dependence similar to endogenous ACh when the two were tested under identical conditions. 4 In the absence of an anticholinesterase the evoked release of [3H]‐ACh per stimulus was dependent on frequency of stimulation in a similar way to that in the presence of physostigmine and atropine. 5 Results suggest that under physiological conditions, i.e. in the absence of an anticholinesterase, the release of ACh per stimulus decreases with increasing frequency of stimulation and that this decrease is due to a lag in the mobilization of stored ACh rather than in the synthesis of new ACh.

Frequency-dependent increase in cortical acetylcholine release evoked by stimulation of the nucleus basalis magnocellularis in the rat ☆

Acetylcholine was collected from the somatosensory cortex of anesthetized rats, using the microdialysis technique. Electrical stimulation of the nucleus basalis magnocellularis (NBM) with trains of 10 pulses at 100 Hz delivered every second produced a 3-4-fold increase in acetylcholine release. Stimulation with an intratrain frequency of 10, 50, 100 or 200 Hz demonstrated that 100 Hz trains produced the greatest increase, while the other frequencies were about half as effective. The cortical release of acetylcholine in this paradigm supports the hypothesis that the previously demonstrated enhancement by NBM stimulation of cortical sensory inputs is due to cholinergic activation.

RELATIONSHIP BETWEEN Ca2+‐DEPENDENT AND INDEPENDENT RELEASE OF [3H]GABA EVOKED BY HIGH K+, VERATRIDINE OR ELECTRICAL STIMULATION FROM RAT CORTICAL SLICES

Abstract— It has been reported that the release of GABA by high K+ is Ca2+‐dependent while release induced by veratridine or electrical stimulation has been frequently found to be independent of Ca2+. To see the source of Ca2+‐dependent and independent release of GABA, cortical slices which had accumulated [3H]GABA were exposed to 50 mm‐K+ or 50 μm‐veratridine for 48min. In the presence of Ca2+ the 2 agents released approx the same amount of [3H]GABA but tetrodotoxin (TTX) abolished release induced only by veratridine, while omission of Ca2+ reduced release induced only by 50mm‐K+. Pre‐exposure of the slices for 48min to 50mm‐K+ in the presence of Ca2+ reduced the second release by 50mm‐K+ by 77% and that by veratridine by 74%, suggesting that in the presence of Ca2+ the 2 depolarizing agents release [3H]GABA from the same pool. Pre‐exposure to 50mm‐K+ in the absence of Ca2+ reduced the second release by 50mm‐K+ or by veratridine only by 37 and 27% respectively, indicating that most of the reduction in release was the result of a depletion of releasable [3H]GABA stores. The second exposure to 50mm‐K+ in the absence of Ca2+ reduced the evoked release further, while exposure to veratridine in the absence of Ca2+, after depletion of the stores, enhanced release 2.7 times. Electrical stimulation (64 Hz, 2 ms, 40 mA, alternating polarity) during 24min in the presence of Ca” caused an initial 5‐fold increase in efflux, which declined subsequently. In the absence of Ca2+, instead of a rapid increase, a slow but smaller increase in the efflux of [3H]GABA was found. TTX almost completely abolished the electrically evoked increase in release. Pre‐treatment with 50mm‐K+ reduced the electrically evoked release by 94% but electrical stimulation in the absence of Ca2+ after depletion of releasable stores doubled this release. Results suggest that in the presence of Ca2+, high K+, veratridine and electrical stimulation release [3H]GABA from the same Ca2+‐dependent store, but in the absence of Ca2+ veratridine and electrical stimulation enhance the release from a Ca2+‐independent store, probably as a result of an increased influx of Na+.

Neurochemical and electrophysiological studies on the inhibitory effect of ammonium ions on synaptic transmission in slices of rat hippocampus: Evidence for a postsynaptic action

To elucidate the mechanisms involved in the inhibition of synaptic transmission by ammonium ions, the effects of NH4Cl on glutamate release and on synaptic transmission from Schaffer collaterals to CA1 pyramidal cells were measured in fully submerged slices of rat hippocampus. The large, Ca(2+)-dependent release of glutamate evoked by electrical-field stimulation or by 56 mM K+ was not reduced by 5 mM NH4Cl. In contrast, 5 mM NH4Cl decreased the smaller, field stimulation-induced release of glutamate observed in the presence of low concentrations of Ca2+ (0.1 mM), as well as the spontaneous release of glutamate both in normal and low Ca2+. Unlike the Ca(2+)-dependent release of glutamate, synaptic transmission was reversibly depressed even by 1 mM NH4 Cl. Firing of CA1 pyramidal cells evoked by iontophoretically applied glutamate was significantly inhibited by 2 or 5 mM NH4Cl. This depression was increased in the presence of 25 microM bicuculline. Results suggest that ammonium ions do not depress the Ca(2+)-dependent release of glutamate originating from synaptic vesicles, which is involved in synaptic transmission. Rather, ammonium ions inhibit synaptic transmission by a postsynaptic action, a conclusion strengthened by the inhibitory effect of NH4Cl on glutamate-induced firing. However, NH4Cl may inhibit the formation of cytoplasmic glutamate, the source of spontaneous and Ca(2+)-independent release.