Laurent Massotte

Galanin decreases the activity of locus coeruleus neurons in vitro

A brain slice preparation was used to examine the effects of galanin on the spontaneous firing rate of locus coeruleus noradrenergic neurons. Galanin (10(-9)-10(-7) M), added to the bath, inhibited the firing of 14 out of 19 neurons in a concentration-dependent manner. The observed effect was quite variable, ranging from 20 to 100% at 10(-7) M. Experiments performed in low-Ca2+, high-Mg2+ medium also showed a significant inhibition by galanin (10(-7) M) in three out of five neurons, which suggests that the peptide acts directly.

Electrophysiological effects of neurotensin on dopaminergic neurones of the ventral tegmental area of the rat in vitro

The effects of neurotensin on the spontaneous firing rate of presumed dopaminergic neurones of the ventral tegmental area of the rat, were studied in a slice preparation of brain by extracellular single-cell recordings. Bath-applied neurotensin excited all cells which were studied (N = 25). This effect was concentration-dependent; the threshold was 10(-10) M and maximal activation (about 30 spikes/10 sec) was obtained with 10(-6) M. The EC50 (half-maximal effective concentration) was roughly estimated at 35 nM. The action of neurotensin was mimicked by neurotensin 8-13 (N = 6), but not neurotensin 1-8 (N = 6). It persisted in low-calcium, high-magnesium solutions (N = 5) and therefore probably resulted from a direct activation of neurotensin receptors. The responses to neurotensin were long-lasting (30-60 min after a 10 min 10(-7) M infusion) and exhibited little tachyphylaxis. Dose-response curves to the dopaminergic agonist BHT920 showed that, during the infusion of 10(-7) M neurotensin, dopaminergic autoreceptors of some neurones were less sensitive than in control conditions. This was not a non-specific effect produced by the excitation, since it was not observed during the infusion of another excitant, N-methyl-D-aspartate (NMDA). These results show that neurotensin potently activates presumed dopaminergic neurones in the ventral tegmental area in vitro; it may also decrease the effectiveness of the autoreceptors of some neurones.

Evidence for the presence of N-methyl-D-aspartate receptors in the ventral tegmental area of the rat : an electrophysiological in vitro study

Extracellular recordings were obtained from spontaneously active, presumed dopaminergic neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied N-methyl-D-aspartate (NMDA) (1-20 microM) activated all neurons tested (n = 36). This effect was clearly concentration-dependent (n = 14), quickly reversible and reproducible. No bursting type of discharge was observed during NMDA infusion. The NMDA receptor blocker DL-2-amino-5-phosphonovaleric acid (50 microM) reversibly antagonized the increase in cell firing produced with 10 microM NMDA by 83.5 +/- 3% (mean +/- S.E.M.) (n = 8, P less than 0.05). Lowering the Mg2+ concentration of the perfusion fluid to one-third of its normal value significantly enhanced the excitatory effect of 5 microM NMDA (n = 7, P less than 0.05), but not of 500 nM carbachol (n = 6). Finally, NMDA did not modify the sensitivity of dopaminergic autoreceptors of VTA neurons (n = 8), when compared to controls (n = 10). These observations strongly support the presence of specific NMDA receptors in the VTA.

SK channels control the firing pattern of midbrain dopaminergic neurons in vivo

A vast body of experimental in vitro work and modelling studies suggests that the firing pattern and/or rate of a majority of midbrain dopaminergic neurons may be controlled in part by Ca2+‐activated K+ channels of the SK type. However, due to the lack of suitable tools, in vivo evidence is lacking. We have taken advantage of the development of the water‐soluble, medium potency SK blocker N‐methyl‐laudanosine (CH3‐L) to test this hypothesis in anaesthetized rats. In the lateral ventral tegmental area, CH3‐L iontophoresis onto dopaminergic neurons significantly increased the coefficient of variation of their interspike intervals and the percentage of spikes generated in bursts as compared to the control condition. The effect of CH3‐L persisted in the presence of a specific GABAA antagonist, suggesting a direct effect. It was robust and reversible, and was also observed in the substantia nigra. Control experiments demonstrated that the effect of CH3‐L could be entirely ascribed to its blockade of SK channels. On the other hand, the firing pattern of noradrenergic neurons was much less affected by CH3‐L. We provide here the first demonstration of a major role of SK channels in the control of the switch between tonic and burst firing of dopaminergic neurons in physiological conditions. This study also suggests a new strategy to develop modulators of the dopaminergic (DA) system, which could be of interest in the treatment of Parkinsons disease, and perhaps other diseases in which DA pathways are dysfunctional.

Evidence for a modulatory role of Ih on the firing of a subgroup of midbrain dopamine neurons.

A previous investigation has suggested that the hyperpolarization-activated cation current Ih does not contribute to the spontaneous firing of midbrain dopaminergic neurons. This conclusion was reached using Cs+. We have re-examined this question with extracellular recordings in slices using the more specific blocker ZD7288. In two-thirds of the cells, low concentrations of ZD7288 induced a decrease of the spontaneous firing. The maximal inhibition was about 40% and the mean IC50 was 1.6 μM. This effect was probably direct because it persisted in the presence of antagonists of various receptors. These concentrations of ZD7288 had no effect in the remaining one third of the examined cells. However, the highest concentration of ZD7288 (300 μM) abolished the firing of all dopaminergic neurons, probably by a mechanism unrelated to the blockade of Ih. We conclude that Ih controls to a certain extent the firing of a majority of midbrain dopaminergic neurons.

How Modeling Can Reconcile Apparently Discrepant Experimental Results: The Case of Pacemaking in Dopaminergic Neurons

Midbrain dopaminergic neurons are endowed with endogenous slow pacemaking properties. In recent years, many different groups have studied the basis for this phenomenon, often with conflicting conclusions. In particular, the role of a slowly-inactivating L-type calcium channel in the depolarizing phase between spikes is controversial, and the analysis of slow oscillatory potential (SOP) recordings during the blockade of sodium channels has led to conflicting conclusions. Based on a minimal model of a dopaminergic neuron, our analysis suggests that the same experimental protocol may lead to drastically different observations in almost identical neurons. For example, complete L-type calcium channel blockade eliminates spontaneous firing or has almost no effect in two neurons differing by less than 1% in their maximal sodium conductance. The same prediction can be reproduced in a state of the art detailed model of a dopaminergic neuron. Some of these predictions are confirmed experimentally using single-cell recordings in brain slices. Our minimal model exhibits SOPs when sodium channels are blocked, these SOPs being uncorrelated with the spiking activity, as has been shown experimentally. We also show that block of a specific conductance (in this case, the SK conductance) can have a different effect on these two oscillatory behaviors (pacemaking and SOPs), despite the fact that they have the same initiating mechanism. These results highlight the fact that computational approaches, besides their well known confirmatory and predictive interests in neurophysiology, may also be useful to resolve apparent discrepancies between experimental results.

Hydrogen peroxide hyperpolarizes rat CA1 pyramidal neurons by inducing an increase in potassium conductance.

It has been suggested that hydrogen peroxide is involved in cascades of pathological events affecting neural cells. The aim of this study was therefore to examine whether this molecule is able by itself to modify membrane properties of pyramidal neurons in the CA1 region of the rat hippocampus. Intracellular recordings in the slice preparation showed that 3.3 mM hydrogen peroxide hyperpolarized all neurons tested (n = 41) by 11 +/- 3 mV. This effect persisted in the presence of tetrodotoxin. It developed slowly, was reversible and reproducible. In the presence of tetrodotoxin, the extrapolated reversal potential of this effect was -95 +/- 5 mV in 2.5 mM external potassium. This value was not significantly different from the one obtained with the GABAB agonist baclofen (10 microM) (-98 +/- 5 mV). It shifted when the concentration of external potassium was increased to 10.5 mM (from -96 +/- 5 to -62 +/- 4 mV), in close agreement with the Nernst equation potassium ions. The hyperpolarization was significantly reduced (by 65 +/- 22%) by the potassium channel blocker barium (100 microM). We suggest that hydrogen peroxide is able to induce an increase in potassium conductance in rat CA1 pyramidal neurons. The exact mechanism by which it produces this effect (direct action on channels or indirect effect) remains to be determined.

Acute amphetamine-induced subsensitivity of A10 dopamine autoreceptors in vitro.

Extracellular recordings were obtained from spontaneously active, presumed dopamine (DA) neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied (+)-amphetamine (AMPH) (1-30 microM) induced a concentration-dependent decrease in the firing rate of these neurons, which tended to saturate with the highest concentrations used (n = 11). This inhibitory effect was dependent on the activation of D2 receptors since it was reversed by the D2 antagonist sulpiride (n = 8). However, the most striking effect of AMPH was the induction of a prominent subsensitivity of DA autoreceptors: whereas in 18 out of 20 control neurons, the D2 agonist BHT 920 (100 nM) produced a rapid and complete inhibition of the firing, this was observed in none out of 11 neurons 10 min after the end of the application of AMPH (1-30 microM) (P less than 0.001). In these cells, the mean percent inhibition produced by BHT 920 was only 47 +/- 8%. This subsensitivity remained unchanged after 20 min and declined after one hour. This effect was specific, since the sensitivity of GABAB receptors to baclofen (500 nM-1 microM) was not modified by the application of AMPH (n = 12). These results suggest that AMPH-induced DA autoreceptor subsensitivity can be produced acutely and may be the first step in a cascade of events leading to behavioral sensitization to this compound.

SK Channel blockade promotes burst firing in dorsal raphe serotonergic neurons

Previous in vivo studies have shown that blockade of small‐conductance Ca2+‐activated potassium (SK) channels enhances burst firing in dopaminergic neurons. As bursting has been found to be physiologically relevant for the synaptic release of serotonin (5‐HT), we investigated the possible role of SK channels in the control of this firing pattern in 5‐HT neurons of the dorsal raphe nucleus. In these cells, bursts are usually composed of doublets consisting of action potentials separated by a small interval (< 20 ms). Both in vivo and in vitro extracellular recordings were performed, using anesthetized rats and rat brain slices, respectively. In vivo, the specific SK blocker UCL 1684 (200 μm) iontophoresed onto presumed 5‐HT neurons significantly increased the production of bursts in 13 out of 25 cells. Furthermore, the effect of UCL 1684 persisted in the presence of both the GABAA antagonist SR 95531 (10 mm) and the GABAB antagonist CGP 35348 (10 mm), whereas these agents by themselves did not significantly influence the neuronal firing pattern. In vitro, bath superfusion of the SK channel blocker apamin (300 nm) induced bursting in only three out of 18 neurons, although it increased the coefficient of variation of the interspike intervals in all the other cells. Our results suggest that SK channel blockade promotes bursting activity in 5‐HT neurons via a direct action. An input which is present only in vivo seems to be important for the induction of this firing pattern in these cells.

Effect of BHT 920 on monoaminergic neurons of the rat brain: an electrophysiological in vivo and in vitro study

SummaryBHT 920 was originally described as a dopamine autoreceptor agonist. In this study, the effect of this compound on the firing rate of noradrenergic locus coeruleus, serotonergic dorsal raphe and dopaminergic ventral tegmental area neurons was examined both in the anaesthetized rat and in rat brain slices. Extracellular recordings were performed in cells whose identity was determined by electrophysiological, pharmacological and histological criteria. In vivo, BHT 920 inhibited the firing of locus coeruleus neurons (ID 50: 14.5 ± 4.7 μg/kg, mean ± SEM) and ventral tegmental area neurons (ID50 7 ± 3 μg/kg) at very low doses. As a comparison, the ID50 of clonidine on locus coeruleus cells was 5.5 ± 0.6 μg/kg and the ID50 of apomorphine on ventral tegmental area neurons was 13 ± 3 μg/kg. BHT 920 also decreased the firing of dorsal raphe cells, but this effect was obtained at higher doses (ID50: 57 ± 11 μg/kg).The in vitro study confirmed the results obtained in vivo. BHT 920 potently inhibited the firing of locus coeruleus cells (IC50: 71 ± 28 nM) and was less potent than clonidine (IC50: 5.3 ± 0.98 nM). The compound also inhibited the firing of ventral tegmental area neurons at very low concentrations (IC50: 21 ± 3.3 nM), being more potent than apomorphine (IC50: 56 ± 29 nM).BHT 920 only slightly decreased the firing rate of dorsal raphe neurons at 50 gM, showing that the drug has little direct effect on these cells.A pharmacological analysis performed in vitro showed that the effect of BHT 920 was specifically inhibited by the D2 antagonist sulpiride (1 μM) in the ventral tegmental area and by the alpha2 antagonist idazoxan (1 μM) in the locus coeruleus.This electrophysiological study shows that BHT 920 is a potent D2 and alpha2 agonist in the rat brain.