Daniel Eugène

Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra.

The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single‐channel and whole‐cell patch‐clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7‐P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19‐P22), both the GlyR main conductance state (46‐55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μm picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine‐evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α2 homomeric GlyRs in neonatal rats and α1/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 μm gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 μm strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co‐released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K+ and hyposmotic solutions were also unable to induce any strychnine‐sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc.

Incremental conductance levels of GABAA receptors in dopaminergic neurones of the rat substantia nigra pars compacta

1 Molecular and biophysical properties of GABAA receptors of dopaminergic (DA) neurones of the pars compacta of the rat substantia nigra were studied in slices and after acute dissociation. 2 Single‐cell reverse transcriptase‐multiplex polymerase chain reaction confirmed that DA neurones contained mRNAs encoding for the α3 subunit of the GABAA receptor, but further showed the presence of α4 subunit mRNAs. α2, β1 and γ1 subunit mRNAs were never detected. Overall, DA neurones present a pattern of expression of GABAA receptor subunit mRNAs containing mainly α3/4β2/3γ3. 3 Outside‐out patches were excised from DA neurones and GABAA single‐channel patch‐clamp currents were recorded under low doses (1‐5 μM) of GABA or isoguvacine, a selective GABAA agonist. Recordings presented several conductance levels which appeared to be integer multiples of an elementary conductance of 4‐5 pS. This property was shared by GABAA receptors of cerebellar Purkinje neurones recorded in slices (however, with an elementary conductance of 3 pS). Only the 5‐6 lowest levels were analysed. 4 A progressive change in the distribution of occupancy of these levels was observed when increasing the isoguvacine concentration (up to 10 μM) as well as when adding zolpidem (20‐200 nM), a drug acting at the benzodiazepine binding site: both treatments enlarged the occupancy of the highest conductance levels, while decreasing that of the smallest ones. Conversely, Zn2+ (10 μM), a negative allosteric modulator of GABAA receptor channels, decreased the occupancy of the highest levels in favour of the lowest ones. 5 These properties of α3/4β2/3γ3‐containing GABAA receptors would support the hypothesis of either single GABAA receptor channels with multiple open states or that of a synchronous recruitment of GABAA receptor channels that could involve their clustering in the membranes of DA neurones.

α2-Adrenoceptor activation increases a cationic conductance and spontaneous GABAergic synaptic activity in dopaminergic neurones of the rat substantia nigra

Abstract Noradrenaline (NA) plays an important role in compensating for the loss in dopaminergic (DA) function following lesions of the DA neurones of the substantia nigra (SN). α 2 -Adrenoceptors are largely expressed in these neurones, but the cellular response to their activation is unknown. Whole-cell patch-clamp recordings were made from DA neurones of rat SN. At a holding potential of −60 mV, bath application of NA (50 μM) induced an inward current (−20.3±10.0 pA) in 50% of the recorded neurones. This effect was mimicked by UK-14304 (50 μM), a specific α 2 -adrenoceptor agonist, whereas α 1 -adrenoceptor and β-adrenoceptor agonists failed to induce a response. Surprisingly, α 2 -adrenoceptor antagonists (idazoxan, RX-811059, SKF-86466 and yohimbine) also induced an inward current that could occlude the one induced by UK-14304, suggesting that they may act as α 2 -adrenoceptor agonists. The inward current results from an increase in cationic conductance identical to the one previously described in these neurones, as neurotensin (1 μM), known to activate it, occluded the inward current induced by UK-14304. In addition, GABAergic miniature inhibitory postsynaptic current frequency was increased by activation of presynaptic α 2 -adrenoceptors. We conclude that the effects of NA on α 2 -adrenoceptors can contribute to the previously described composite action of NA on DA neurone firing and can be pharmacologically differentiated from the effect of NA on DA and neighbouring neurones known to be mediated through α 1 -adrenoceptors.

Fast non-cholinergic depolarizing postsynaptic potentials in neurons of rat superior cervical ganglia

After the blockade of cholinergic transmission, stimulation of the preganglionic sympathetic trunk elicited fast depolarizing postsynaptic potentials (PSPs) in rat superior cervical ganglia. At 50 min, their amplitude measured intracellularly was 6.9 +/- 1.7 mV and their duration 25.9 +/- 7.6 ms (mean +/- S.D., n = 9 ganglia). The extracellular electrical activity recorded from the postganglionic internal carotid nerve was monophasic and equal to 4.0 +/- 2.2% of the normal activity (mean +/- S.D., n = 12 ganglia). The effects on these PSPs of some postsynaptic receptor antagonists have been tested. Bicuculline decreased the amplitude of the PSPs as well as that of the monophasic extracellular activity, suggesting that GABA could mediate these non-cholinergic synaptic potentials.

Synaptic organization of amphibian sympathetic ganglia

The synaptic organization of the amphibian sympathetic ganglia was studied, especially in the last two abdominal paravertebral ganglia of the frog. These ganglia appear to form a monosynaptic relay, not containing interneurons. They consist of two systems working in parallel: the principal neurons, by far the most numerous, and a small number of chromaffin (i.e., SIF) cells, usually arranged in clusters. Each principal neuron is innervated by a preganglionic branch forming a set of cholinergic synapses which exhibit classical ultrastucture. The only peculiarity is the presence of a subsynaptic apparatus in a variable percentage of synaptic complexes. Electro‐physiological studies have demonstrated that synaptic transmission is due to ACh release and involves several postsynaptic potentials. Moreover, the principal neurons are of two types, B and C, whose preganglionic axons and their own axons have different conduction velocities. C neurons tend to be small in diameter, and B neurons are larger, but the size distribution of the two populations overlaps. More recently, it was demonstrated that these two neuronal systems have different immunocytochemical features. The C preganglionic fibers contain an LHRH‐like peptide, which is responsible for late synaptic events. The B preganglionic fibers contain CGRP, whose role has not yet been established. The principal neurons all contain adrenaline, but neuropeptide Y is also present in C neurons and could be a second transmitter at peripheral junctions. SP‐containing fibers also pass through the ganglia, but give rise to intraganglionic synapses only rarely, except in the celiac plexus. Galanin can coexist with neuropeptide Y in certain C neurons. Numerous principal neurons are immunoreactive for VIP. Chromaffin cells contain noradrenaline and metenkephalin, and some contain SP or LHRH; they are endocrine cells controlled by preganglionic fibers and can have a modulatory effect on principal neurons endowed with appropriate receptors. The accessibility of frog abdominal ganglia and the anatomical separation of B and C preganglionic fiber pathways provide interesting systems in which to carry out experimentation on the stability and specificity of synaptic contacts. After postganglionic axotomy, the majority of synapses disappear by disruption of synaptic contacts. There is a certain discrepancy between the recovery of synaptic transmission and the reappearance of morphologically identifiable synapses, suggesting that a certain amount of transmission is possible at contacts devoid of synaptic complexes. The selective deafferentation of B or C neurons showed that the subsynaptic apparati are mainly found at B neuron synapses. The course of reinnervation following selective deafferentation reveals the existence of different specificities at B and C synapses: C neurons are easily reinnervated by B preganglionic fibers, whereas C fibers appear fairly ineffective at reinnervating B neurons, even after a long interval. Attempts were made to reinnervate ganglionic neurons with somatic motor nerve fibers. Reinnervation was achieved only rarely, and it is concluded that the ganglionic synapses in the frog have a higher specificity and lower plasticity than in mammals.

Furosemide modulation of GABAA receptors in dopaminergic neurones of the rat substantia nigra

Abstract Furosemide is a diuretic which has been shown to decrease recombinant GABAA receptor (GABAAR)-mediated currents and also to block epileptiform discharges. Here, we show that furosemide actions on GABAARs of rat substantia nigra dopaminergic neurones depend on both furosemide and GABAAR agonist concentrations. The whole-cell currents induced by low concentrations of GABA (5 μM) or by the selective GABAAR agonist isoguvacine (7–25 μM) were enhanced by 200 μM furosemide. However, furosemide did not affect GABAAR currents induced by 60 μM isoguvacine and even decreased those induced by 200 μM isoguvacine. At the single-channel level, furosemide had comparable effects. It increased the open time proportion with 7 μM isoguvacine but had no significant effect on the open time proportion with 60 μM isoguvacine. These effects resulted from a differential action on the multiple conductance levels activated by GABAAR agonists. The concentration-response relationship to isoguvacine in the whole-cell mode revealed the presence of a high and a low apparent affinity GABAAR population (EC50 4.8 vs 89 μM). These two populations of receptors coexist in the same dopaminergic neurone. They are both furosemide-sensitive and may represent different GABAAR subunit assemblies.

Reinnervation of frog sympathetic ganglia after selective denervation of B or C neurons.

SummarySelective transection of the B or C preganglionic nerve fibres respectively innervating the B and C sympathetic neurons was carried out on the last two ganglia of the sympathetic chain of the frogRana esculenta. At different times thereafter, the crossreinnervation of one type of denervated neuron by nerve endings sprouting within the ganglia from intact fibres innervating the other type was investigated by both the quantitative morphology of the synaptic contacts and related structures and electrophysiological recordings of ganglionic transmission. As there are no fine ultrastructural criteria for distinguishing B from C neurons, the overall density of synapse, simple contact, and ‘vacated’ postsynaptic differentiation profiles was measured in the two cases of selective section and compared with the values for normal ganglia, therefore permitting the progress of cross-reinnervation with time for each type of neuron to be followed. At ten days after section of the C preganglionic fibres, immunocytochemistry showed that there were no anti-LH-RH-like peptide containing fibres within the ganglia. The B myelinated preganglionic fibres were able to reinnervate the denervated C neurons, with return to normal values of synaptic density and fully efficient transmission at two months in all tested C neurons. However, the latency of orthodromic action potentials was close to that of normally innervated B neurons. In contrast, the C non-myelinated preganglionic fibres reinnervated the denervated B neurons with limited efficiency, the synaptic density being two-thirds the normal value after five months, while subthreshold excitatory postsynaptic potentials or action potentials were only recorded in 44% of the tested B neurons. The latency of these orthodromic responses was close to that of normally innervated C neurons. It is postulated that the poor cross-reinnervation of B neurons could be due to insufficient sprouting of C fibres and/or lack of ‘affinity’ betwen C fibres and B neurons. In addition, these experiments demonstrated that the subsynaptic apparatus, fairly characteristics of frog ganglionic synapses, is present in both types of sympathetic neurons, although predominantly in B neurons.

Ultrastructural changes in the shape of neurons and related structures in frog sympathetic ganglia after axotomy

Summary— Changes in the shape of neuronal perikarya and other ganglionic structures were observed by electron microscopy in the frog sympathetic ganglia at different times after axotomy. Degenerating and hypertrophic profiles appeared to reflect a remodelling process affecting preganglionic fibres. The shape of neuronal perikarya was modified by the formation of infoldings occupied by preganglionic fibres and/or by that of short winding dendrites often bearing a synapse. The origin of these changes is discussed. In frog sympathetic ganglia, the period of recovery after axotomy was marked by specific reactions which affected neuronal shape and preganglionic fibres, and are not known to occur in the ganglia of mammals.