Effects of high-potassium-induced depolarization on amino acid chemistry of the dorsal cochlear nucleus in rat brain slices.

High K+ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, gamma-aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K+, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K+, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K+. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K(+)-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.

Metabolism of the dorsal cochlear nucleus in rat brain slices.

In vitro brain slices of the cochlear nucleus have been used for electrophysiological and pharmacological studies. More information is needed about the extent to which the slice resembles in vivo tissue, since this affects the interpretation of results obtained from slices. In this study, some chemical parameters of the dorsal cochlear nucleus (DCN) in rat brain slices were measured and compared to the in vivo state. The activities of malate dehydrogenase and lactate dehydrogenase were reduced in some DCN layers of incubated slices compared to in vivo brain tissue. The activities of choline acetyltransferase and acetylcholinesterase were increased or unchanged in DCN layers of slices. Adenosine triphosphate (ATP) concentrations for in vivo rat DCN were similar to those of cerebellar cortex. Compared with in vivo values, ATP concentrations were decreased in the DCN of brain slices, especially in the deep layer. Vibratome-cut slices had lower ATP levels than chopper-cut slices. Compared with the in vivo data, there were large losses of aspartate, glutamate, glutamine, gamma-aminobutyrate and taurine from incubated slices. These amino acid changes within the slices correlated with the patterns of release from the slices.

Reduced spontaneous activity in the dorsal cochlear nucleus of Scn8a mutant mice.

Spontaneous activity was recorded in the dorsal cochlear nucleus of brain slices from mice homozygous for the med-J and jolting mutations in the neuronal sodium channel alpha-subunit Scn8a. Densities of spontaneously active neurons in slices from both mutants were significantly lower than in control slices. Spontaneous firing patterns with bursts of action potentials were recorded from approximately 50% of the neurons in control slices, but the typical bursting patterns were not observed in neurons of med-J and jolting mouse slices. The results suggest that this voltage-gated sodium channel is essential for the spontaneous bursting firing of cochlear nucleus cartwheel neurons. This mutant animal model may be useful for the study of the functional roles of cochlear nucleus neurons.

Glutamergic transmission of neuronal responses to carbachol in rat dorsal cochlear nucleus slices.

This study found that glutamate receptor antagonists block the excitatory effects of carbachol, a cholinergic agonist, on bursting neurons in the dorsal cochlear nucleus of rat brain slices. Among antagonists for glutamate receptor subtypes, those for non-N-methyl-D-aspartate ionotropic glutamate receptors were more potent than those for N-methyl-D-aspartate receptors. The glutamate receptor antagonists did not block the effects of carbachol on regularly firing neurons in the dorsal cochlear nucleus of the same slices. Antagonists for GABA or glycine receptors did not alter the effects of carbachol on bursting neurons. Effects of carbachol on bursting activity could be mimicked by application of glutamate or its agonist, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, whose effects were not blocked by synaptic blockade. During carbachol application, increased release of glutamate and glycine from the dorsal cochlear nucleus part of brain slices was measured using high-performance liquid chromatography. Release of other amino acids showed no significant change. The results suggest that, in rat dorsal cochlear nucleus, cholinergic effects on regular and bursting spontaneous firing occur through different mechanisms. Cholinergic effects on regular neurons (which include fusiform cells) are direct, through muscarinic receptors. Cholinergic effects on bursting neurons (which include cartwheel cells) are indirect and involve glutamatergic neurotransmission, mostly via non-N-methyl-D-aspartate ionotropic receptors. The granule cell-parallel fiber pathway may be involved in this glutamatergic transmission.

Effects of endogenous acetylcholine on spontaneous activity in rat dorsal cochlear nucleus slices.

We have examined the contribution of endogenous acetylcholine (ACh) release to the spontaneous firing of both regular (probably fusiform cells) and bursting neurons (probably cartwheel cells) in the dorsal cochlear nucleus (DCN) in rat brainstem slices. The muscarinic antagonists atropine, scopolamine, and tropicamide (1-2 microM) caused substantial decreases of firing rates in a majority of the neurons. Reversible acetylcholinesterase (AChE) inhibitors typically caused large transient increases in firing that decayed more slowly than responses to carbachol. The irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) usually caused a sustained increase, with an initial peak followed by a gradual change to a final level higher than before DFP. Tropicamide caused large decreases in firing after DFP, confirming sustained ACh release. Both neostigmine and DFP applied after AChE inhibition by DFP sometimes elicited a transient response. We conclude that the level of sustained response to DFP is determined by the rate of endogenous ACh release, and that DFP and reversible AChE inhibitors exert an initial transient agonist effect that overlaps the initial effect of acetylcholinesterase inhibition. The slice experiments provide a model for cholinergic mechanisms in vivo, confirm that the release of endogenous ACh increases the firing rates of regular and bursting neurons in superficial DCN, and support the hypothesis that spontaneous firing of DCN neurons is sustained in part by cholinergic inputs.

Effects of parallel fiber stimulation on neurons of rat dorsal cochlear nucleus.

We have compared the effects of parallel fiber stimuli on extracellularly recorded neurons showing regular or bursting spontaneous activity patterns in the dorsal cochlear nucleus of rat brainstem slices. Ninety percent of regular neurons failed to respond to stimulus currents (1.4 +/- 0.28 mA, mean +/- SEM) significantly greater than those (0.4 +/- 0.07 mA) that elicited responses from 96% of bursting neurons. Responses of bursting neurons were elicited from widely separated loci along the molecular layer. Kynurenic acid and CNQX or DNQX blocked both spontaneous firing and responses to parallel fiber stimuli of bursting neurons. The same agents also blocked responses of regular neurons but had little or no effect on their spontaneous firing rates. AP-5 caused small decreases in spontaneous rates of both bursting and regular neurons but did not appear to affect responses to stimuli. The data support the hypothesis that the responses of both regular and bursting neurons to parallel fiber stimulation are mediated by glutamate, acting mainly through non-NMDA receptors. Spontaneous activity of bursting, but not regular, neurons also requires non-NMDA glutamatergic transmission, suggesting that the spontaneous firing of bursting neurons, consisting largely of cartwheel cells, may depend upon granule cell activity.

Muscarinic receptor subtypes in rat dorsal cochlear nucleus.

We previously reported that responses of spontaneously active rat dorsal cochlear nucleus (DCN) neurons to cholinergic agonists are mediated predominantly by muscarinic receptors. We have now tested the effects of 7 antagonists with differing affinities for the muscarinic receptor subtypes M1-M4 on the responses to constant, submaximal doses of carbachol in rat brainstem slices. Each slice was exposed to one or more concentrations of one antagonist applied during extracellular recording of a DCN neuron. The concentrations yielding 50% reduction of test responses (IC50) of regular and bursting neurons were estimated for each antagonist. Correlation coefficients were calculated between log(IC50) values and log(Ki) values of the drugs for the receptor subtypes. Correlation coefficients for both regular and bursting neurons were not significant (P > 0.05) for M1 and M3, but were significant (P < 0.02) for M4. Bursting but not regular neurons also showed a significant correlation for M2 (P < 0.05). Our results suggest that (1) M4 contributes to the cholinergic responses in DCN and M2 may also contribute to the responses of bursting neurons, but the contribution of other subtypes cannot be completely excluded; (2) muscarinic subtypes in DCN probably differ from those reported for cochlea and some brain regions.

Cholinergic modulation of spontaneous activity in rat dorsal cochlear nucleus.

Extracellular recordings were made from brain stem slices to test the effects of bath application of cholinergic agonists and antagonists on the firing rates of spontaneously active dorsal cochlear nucleus neurons. About 90% of neurons responded to carbachol. A higher proportion responded to muscarine than to nicotine. Muscarine elicited larger responses at lower concentrations than nicotine. Responses to either carbachol or muscarine were always blocked by atropine or scopolamine. The nicotinic antagonists d-tubocurarine, hexamethonium, and mecamylamine blocked the responses to nicotine, but did not decrease the responses to carbachol. Regularly firing neurons showed only increases of firing rate during exposure to cholinergic agonists. About half of responsive bursting neurons showed increased firing; half showed increased followed by decreased firing to 10 microM carbachol or muscarine. All phases of the responses of most bursting neurons were greatly decreased or abolished in low calcium, high magnesium medium, while responses of regular neurons were not detectably affected. Thus, cholinergic agonists appear to act directly on regularly firing neurons, while their actions on bursting neurons may require synaptic activity. The data suggest that cholinergic transmission in the dorsal cochlear nucleus is predominantly muscarinic, and that most regularly firing spontaneously active neurons have muscarinic receptors.

Functional characteristics of spontaneously active neurons in rat dorsal cochlear nucleus in vitro.

1. The cochlear nucleus of rat brain stem slices was explored with extracellular microelectrodes to determine the distribution and characteristics of spontaneously active neurons. 2. In mapping experiments few spontaneously active neurons were found in anteroventral or posteroventral divisions of the cochlear nucleus. In contrast, spontaneously active neurons (N = 648) were widely distributed in the dorsal cochlear nucleus (DCN), especially its more superficial part. The density (neurons per penetration) was greatest 100-400 microns from the lateral surface of DCN, corresponding approximately to the fusiform soma layer and closely adjacent portions of the molecular and deeper regions. In penetrations with active neurons as many as 13 were found, with a mean of 4.3 neurons per penetration. Activity was found along the entire dorsomedial-ventrolateral extent of the nucleus, across the tonotopic representation. 3. Most neurons were readily categorized according to the spike interval pattern as regular (40%), bursting (30%), or irregular (30%). Regular and bursting patterns were highly stable, but few bursting neurons were found in relatively inactive slices. Although there was extensive overlap in location, bursting neurons were significantly closer to the lateral edge of the slice. Also, they were more likely to have initially negative action potentials than regular or irregular neurons. 4. A high density of spontaneous firing, including regular, bursting, and irregular patterns, was observed in slices containing only DCN and adjacent fiber tracts, with other nuclear structures trimmed away. 5. When the K+ concentration of the perfusion medium was decreased from 6.25 to 3.25 mM firing rates of regular neurons decreased moderately without changes in pattern. In contrast, firing rates of most bursting and irregular neurons showed large increases, and bursts were prolonged. 6. When the K+ concentration was increased from 6.25 to 9.25 or 12.25 mM regular neurons showed moderate increases in rate without changes in pattern. Effects on firing rates differed among bursting and irregular neurons, but bursts usually increased in frequency and decreased in duration, and irregular neurons showed some burst firing. 7. When Ca2+ was decreased to 0.2 mM and Mg2+ increased to 3.8 or 7.8 mM regular neurons did not change in pattern of firing although firing rates increased or decreased moderately. Bursting neurons showed large increases in the durations of the bursts. Firing rates of bursting neurons usually increased during 0.2 mM Ca2+ -3.8 mM Mg2+ but typically decreased, after an initial rise, during 0.2 mM Ca2+ -7.8 mM Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Sampling fluid from slice chambers by microsiphoning.

We have developed a simple technique that continuously samples perfusion fluid from an in vitro slice chamber and permits simultaneous electrophysiological recordings from a slice. This procedure uses a microsiphon that diverts approximately 1.5% of the total effluent of artificial cerebrospinal fluid from an interface slice chamber into a collection tube at a lower level outside the chamber. The tip of the microsiphon may be moved anywhere within the chamber without disturbing the slice. For optimum sampling of the release of substances from one area of a slice, the tip is positioned immediately downstream from the location being studied. Concentrations of several amino acids were measured to identify significant spatial and temporal characteristics of the technique and to demonstrate that spontaneous and induced release of amino acids from slices can be measured with adequate sensitivity and time resolution.

Somatosensory thalamic neurons: effects of cortical depression.

Deafferented somatosensory thalamic neurons showed hyperactivity, followed by greatly reduced activity, after initiation of cortical spreading depression; local cooling of sensorimotor cortex was followed only by the inactive phase. Stimulation of contralateral midbrain reticular formation during the inactive phase failed to induce the typical increase in discharge rate of somatosensory thalamic neurons, but produced desynchronization in unaffected cortex. These results indicate that corticothalamic discharge is necessary for sustaining the ongoing activity of deafferented somatosensory thalamic neurons and for maintaining their responsiveness to stimulation of the reticular formation.