Dominik Felix

Stimulation of nigrostriatal dopamine neurones by nicotine

In rats anaesthetized with urethane, firing of neurones of the substantia nigra zona compacta was accelerated after subcutaneous or iontophoretic administration of nicotine or after iontophoretic application of acetylcholine. The excitation was prevented by iontophoretic application of dihydro-beta-erythroidine, but not by atropine. The units were identified by antidromic stimulation as neurones of the nigrostriatal system; their activity was depressed by iontophoretically applied dopamine (DA). Under the same conditions of anaesthesia, a subcutaneous injection of nicotine produced an increase in DA turnover and in homovanillic acid levels in the striatum. The effect of nicotine on striatal DA turnover was comparable to that of electrical stimulation of the nigrostriatal pathway at the average frequency seen in the firing of zona compacta neurones after systemic administration of nicotine. These observations corroborate the idea that nicotine exerts an excitatory action at the level of nigral DA nerve cells. Observations made after electrical stimulation or haloperidol under urethane anaesthesia and after nicotine in unanaesthetized rats suggest that the relatively modest effect of nicotine on striatal DA turnover is due mainly to the short duration of drug action rather than to effects of the anaesthetic on DA metabolism.

Angiotensin-sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III

Angiotensin-activated neurons were examined using microiontophoretic methods in the paraventricular nucleus (PNV) of the rat. In all cases angiotensin III (AIII) was more potent than angiotensin II (AII). This greater sensitivity to AIII was manifested by lower thresholds, shorter latencies, and higher spike frequencies/amplitudes of applied current. The superior potency of AIII was further exaggerated in the spontaneously hypertensive rat (SHR) compared with normotensive Wistar Kyoto (WKY) rats. Postactivity for both AII and AIII was greatly prolonged in SHR. This appeared specific since no prolongation in acetylcholine postactivity was seen in SHR. These data support the notion that AIII may be the centrally active form of angiotensin and are consistent with an obligatory conversion of AII to AIII prior to activation. The selective enhancement of postactivity observed in SHR following angiotensin application suggests a possible defect in signal termination.

A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine

In order to investigate the possible relationship between neuronal activity and cellular fluorescence intensity, extracellular recordings of single unit activity and determinations of fluorescence intensity of dopamine (DA) neurones by histochemical microfluorimetry were performed in the same (rostral) part of zona compacta of substantia nigra in male rats. In urethane anaesthesia, zona compacta neurones characteristically showed a slow and fairly regular type of firing. Nicotine (1 mg/kg s.c.) induced a transient decrease in unit activity for 1 min followed by a sustained increase in firing rate. During that stage, 4-5 neurones/rat were recorded at different anteroposterior levels, each during 200 sec. Microfluorimetric examination of the fluorescence intensity developed at the end of the 30-min observation period by the DA neurones of the same area revealed a marked rise in cellular fluorescence intensity. Similar results were obtained with a lower dose of nicotine and/or a shorter observation period. Additional microiontophoretic experiments supported the view that extracellular recordings of the correlative electrophysiological-microfluorimetric investigation belonged to DA neurones. Release of DA from terminals was indicated by an increase in HVA concentration of caudate-putamen in rats subjected to the same nicotine treatment. When tested on one cell during a prolonged period of time, physostigmine (0.25 mg/kg i.p.) caused an initial increase in firing rate of zona compacta neurones (5-10 min) followed by a decrease of unit activity (15-23 min). In agreement with previous observations in mice, fluorescence intensity of nigral DA neurones likewise showed a biphasic change with an initial rise and subsequent decrease (examined at 9.5 and 22-23.5 min, respectively). When mean unit activity and mean fluorescence intensity of individual rats out of various experimental groups were related to each other, a highly significant positive correlation between neuronal fluorescence intensity and firing rate was found. The results obtained with physostigmine demonstrate that mean intensity closely paralleled mean unit activity in time, so that this correlation was maintained. These findings indicate that cellular fluorescence intensity of DA neurone groups can be used as an index of the level of neuronal activity, except for cases where a drug treatment interferes directly with catecholamine synthesis or storage mechanisms.

Specific angiotensin II receptive neurons in the cat subfornical organ

To test if neurons in the subfornical organ (SFO) are specifically sensitive to angiotensin II (AII) we have applied the AII analog sar1-ala8-AII (P113) directly on to cells in the SFO by microiontophoresis. Adult cats were anesthetized and the SFO exposed for penetration by a 5-barreled micropipette electrode. Of 22 units which responded positively to AII, 7 were also positive to acetylcholine. P113 alone produced either no effect or a decrease in unit firing. P113 plus AII produced antagonism in 17 of 18 units. P113 plus acetylcholine produced antagonistic effects in 5 of 14 cases. Only two units were completely antagonized and 5 units showed agonistic interaction. The most sensitive antagonism with respect to dose of P113 was on neurons responsive only to AII and not to both AII and acetylcholine. We conclude that there are specific AII neurons in the SFO.

Angiotensin receptive neurones in the subfornical organ. Structure-activity relations.

A microiontophoretic study was performed of the actions of angiotensin II and angiotensin fragments on neurones of the subfornical organ (SFO). Adult cats were anaesthetized and the SFO exposed for penetration by a multibarrelled micropipette. We found that angiotensin II-[2--8]-heptapeptide shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin II-[5--8]-tetrapeptide still produced an excitatory action on a single units. Both the action of the heptapeptide and the tetrapeptide were blocked by [sar1, Ala8]-angiotensin II (P 113). In contrast, angiotensin II-[6--8]-tripeptide failed to enhance the firing rate of the same neurones. Our data indicate that angiotensin II and some shorter chain peptide fragments can directly affect neurones of the SFO. The study may give new insight in structure-activity relations for angiotensin II. The results support the hypothesis that the subfornical organ is a receptor site which is available to this peptide.

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.

The efferent modulation of mammalian inner hair cell afferents.

The results of immunocytochemical, enzymatic and electrophysiological studies have indicated that acetylcholine and GABA may act as neurotransmitters in lateral olivocochlear efferent endings on inner hair cell afferent dendrites. Since spike activity can be recorded in the dendritic region of inner hair cells, microiontophoretic techniques were used testing the possible neurotransmitter candidates, acetylcholine and GABA, on spontaneous and induced firing of the afferent dendrites. The experiments were carried out in anaesthetised guinea-pigs, the third and fourth turns of the cochlea being exposed for electrode penetration. Ejection of acetylcholine resulted in a pronounced dose-dependent increase in subsynaptic spiking activity. Furthermore, acetylcholine enhanced glutamate-induced activity. In contrast, even at high doses, GABA had very little effect on the spontaneous cochlear firing rate. When the firing rate had first been enhanced by glutamate or N-methyl-D-aspartate, however, this activation could be reduced by the ejection of GABA. A similar reduction was observed when the firing rate had been enhanced with acetylcholine. The results of our studies support the hypothesis that these substances are involved in efferent neurotransmission on inner hair cell afferent fibres. It should be pointed out, however, that besides acetylcholine and GABA, several opioids such as enkephalins and dynorphins seem to be involved in efferent cochlear innervation.

Receptor Pharmacological Models for Inner Ear Therapies with Emphasis on Glutamate Receptors: A Survey

With the aid of microiontophoretic techniques we evaluated the action of different postsynaptic glutamate receptor subtypes that mediate neurotransmission between the inner hair cell and the afferent neuron. The sensory input is modulated by axodendritic efferents. In the central nervous system, excessive activation of glutamate receptors is thought to be responsible for a wide variety of neurotoxic actions, and calcium is involved in the etiology of glutamate-induced cell damage. Glutamatergic neurotoxicity may form an appropriate pathophysiological model to explain a variety of inner ear diseases characterized by acute or progressive hearing loss and tinnitus. In clinical trials, three sites of action are thought to attenuate glutamatergic otoneurotoxicity: presynaptically, via the reduction of excessive transmitter release; postsynaptically, via competitive or noncompetitive receptor antagonism; and intracellularly, via blockage of glutamate receptor-dependent calcium stores. The drugs discussed in this paper are currently available clinically and have only recently been found to attenuate glutamate toxicity. Magnesium and the quinoxaline derivative Caroverine, which have already been tested in humans, exhibit a statistically significant otoneuroprotective action in noise-induced hearing loss and tinnitus. The intensive search for further drugs that enhance the survival of cochlear afferents without disrupting acoustic signal processing is one of the main goals of research in clinical otoneuropharmacology in the near future.

Localization of angiotensinogen in multiple cell types of rat brain

Angiotensinogen was localized in 3 cell types in brain using immunohistochemical methods. These locations included subpopulations of neurons in nuclei that co-stain for angiotensin II, subpopulations of astrocytes that make putative contacts with brain microvessels, and cells of the choroid plexus. These findings are consistent with multiple functions for brain angiotensinogen as a precursor for neuronal angiotensin II and as a potential source for angiotensin II that is locally produced in the brain.

Angiotensin II-sensitive neurons in septal areas of the rat

Abstract Angiotensin II, injected intravenously or by microiontophoresis, was tested on identified neurons in the rat septal area. About 35% of the cells tested were excited by ionophoreticaly applied angiotensin II. A low percentage of neurons (15%), regularly distributed over the whole septum, decreased their activity in reaction to the peptide. The excitatory action varied between a small change of the baseline activity to a pronounced increase of the discharge frequency exceeding 100% of the control values. This action was blocked by [Sar 1 , Ala 8 ]-angiotensin II. Activation of neurons was confined to the lateral part of the septum. Neurons reacting to microiontophoretically administered angiotensin II showed no effect to i.v. angiotensin II injection, indicating that the blood-brain barrier remained intact. Angiotensin II weakly modulated the orthodromic activation of lateral septal cells caused by fimbria stimulation, whereas the lengthy inhibition which follows the orthodromic activation remained unchanged. No changes on the synaptic response were observed by the administration of angiotensin II antagonist. Our data indicate that regions which have shown significant specific binding activity, and which were reported to be sensitive sites for dipsogenic response to angiotensin II, contain neurons with specific chemosensitivity to iontophoretically applied angiotensin octapeptide.