Vincent Seutin

Bicuculline methiodide potentiates NMDA-dependent burst firing in rat dopamine neurons by blocking apamin-sensitive Ca2+-activated K+ currents

Apamin, a bee venom toxin which blocks a Ca2+-dependent K+ current, potentiates N-methyl-D-aspartate (NMDA)-induced burst firing in dopamine neurons. We now report that burst firing is also potentiated by an apamin-like effect of bicuculline methiodide (BMI) at the same concentration (30 microM) which blocks GABA(A) receptors in vitro. Using microelectrodes to record intracellularly from rat dopamine neurons in the midbrain slice, BMI reduced the apamin-sensitive afterhyperpolarization in all cells tested. BMI also mimicked apamin (100 nM) by potentiating burst firing produced by a concentration of NMDA (10 microM) which is too low to evoke burst firing when perfused alone. When recording under voltage-clamp, both BMI and apamin reduced a depolarization-activated outward current which was also sensitive to perfusate containing no-added Ca2+. Although picrotoxin (100 microM) and bicuculline free base (30 microM) blocked the inhibition of firing produced by the GABA(A) agonist isoguvacine (100 microM), neither had apamin-like effects. We conclude that BMI potentiates burst firing by blocking an apamin-sensitive Ca2+-activated K+ current.

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.

Recent advances in the pharmacology of quaternary salts of bicuculline

We thank Prof. A. Dresse for helpful comments on this manuscript. This work was supported in part by a grant from the National Fund for Scientific Research (F.N.R.S., Belgium) (VS).

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.

Modulation of Small Conductance Calcium-Activated Potassium (SK) Channels: A New Challenge in Medicinal Chemistry

Small conductance calcium-activated potassium (SK) channels are found in many types of neurons as well as in some other cell types. These channels are selective for K(+) and open when intracellular Ca(2+) rises to omega 500 nM. In neurons, this occurs during and after an action potential. Activation of SK channels hyperpolarizes the membrane, thus reducing cell excitability for several tens or hundreds of milliseconds. This phenomenon is called a afterhyperpolarization (AHP). Three subtypes of SK channels (SK1, SK2, SK3) have been cloned and exhibit a differential localization in the brain. SK channels may play a role in physiological and pathological conditions. They may be involved in the control of memory and cognition. Moreover, they are heavily expressed in the basal ganglia (in particular in the substantia nigra, pars compacta) and in the limbic system, suggesting that they may modulate motricity and emotional behaviour. Based on these facts, SK channel subtypes may be a suitable target for developing novel therapeutic agents, but more work is needed to validate these targets. Hence, there is a great need for selective ligands. Moreover, although the risk of peripheral side-effects for SK channel modulators appears to be low, some questions remain to be investigated. Currently, different molecules are known as SK channel modulators. Apamin is a very potent peptidic agent; it produces a strong blockade of these targets which is only very slowly reversible and it has limited selectivity. Dequalinium was found to be an effective blocker. Different chemical modulations on the dequalinium structure led to the discovery of highly potent bis-quinolinium derivatives such as UCL 1684. Other bis-(2-amino-benzimidazole) derivatives are in development. On the other hand, quaternary salts of bicuculline were reported to be effective in inhibiting AHPs. More recent developments on structurally-related molecules revealed that methyl-laudanosine is a new interesting tool for exploring SK channel pharmacology. Finally, a family of compounds has been shown to facilitate SK channel opening. Such compounds may be useful in treating disorders involving neuronal hyperexcitability.

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.

Disrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function

In order to investigate the physiological properties of the melanin‐concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1Neo/Neo mice). Complementary experimental approaches were used to investigate alterations in the learning and memory processes of our transgenic model. The ability of the knockout strain to carry out the inhibitory passive avoidance test was found to be considerably impaired although no significant differences were observed in anxiety levels. This impaired cognitive property prompted us to explore modifications in N‐methyl d‐aspartate (NMDA) responses in the hippocampus. Intracellular recordings of CA1 pyramidal neurons in hippocampal slices from the MCHR1Neo/Neo mice revealed significantly decreased NMDA responses. Finally, using in situ hybridization we found a 15% reduction in NMDAR1 subunit in the CA1 region. These results show for the first time a possible role for MCH in the control of the function of the NMDA receptor.