Jean-François Liégeois

Nitric-oxide - a New Messenger in the Brain

The important role played by nitric oxide (NO) in the central nervous system has largely been emphasized in the recent literature. It can originate at least from four different sources: the endothelium of cerebral vessels, the immunostimulated microglia and astrocytes, the nonadrenergic noncholinergic nerve, and the glutamate neuron. NO has been implicated in a large number of pathologies (such as neurotoxicity in Alzheimers disease and Huntingtons disease, cerebral ischemia, stroke, and anxiety) and also in normal physiological functions (such as memory and learning, regulation of the cerebrovascular system, modulation of the wakefulness, mediation of nociception, olfaction, food intake and drinking, regulation of noradrenaline, and dopamine release). The aim of this paper is to review and to integrate the most recent advances in our understanding of the roles of NO in the brain.

5-HT2A receptor antagonism potentiates haloperidol-induced dopamine release in rat medial prefrontal cortex and inhibits that in the nucleus accumbens in a dose-dependent manner

Combined serotonin (5-HT)2A and dopamine (DA) D2 blockade has been shown to contribute to the ability of atypical antipsychotic drugs (APDs) to increase DA release in rat medial prefrontal cortex (mPFC). We provide additional support for this hypothesis by examining the effect of the selective 5-HT2A antagonist M100907 plus haloperidol, a potent D2 antagonist APD, on DA release in the mPFC and nucleus accumbens (NAC). Haloperidol (0.01–1.0 mg/kg) produced an inverted U-shaped increase in DA release in the mPFC, with a significant increase only at 0.1 mg/kg. Haloperidol (0.1 and 1.0 mg/kg) significantly increased DA release in the NAC. M100907 (0.1 mg/kg) by itself had no effect on DA release in either region. This dose of M100907 potentiated the ability of low (0.01–0.1 mg/kg), but not high dose (0.3–1.0 mg/kg) haloperidol to increase mPFC DA release, whereas it abolished the effect of both 0.1 and 1.0 mg/kg haloperidol on NAC DA release. These results suggest that the relatively higher ratio of 5-HT2A to D2 antagonism may contribute to the potentiation of haloperidol-induced mPFC DA release, whereas 5-HT2A antagonism can diminish haloperidol-induced NAC DA release, even when combined with extensive D2 antagonism, which may not be synergistic with 5-HT2A antagonism in the mPFC.

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.

The inflammatory reaction induced by formalin in the rat paw

The involvement of bradykinin and some other inflammatory mediators in formalin-induced oedema and plasma extravasation was examined. Formalin was injected in rat paws at two doses, 1.75% or 5%. The lower dose induced the development of an immediate oedema associated with a progressive accumulation of 125I-labelled albumin in the paws. These changes were suppressed by pretreatment with capsaicin or xylocaine. They were abolished by RP67580, a NK1 receptor antagonist, and increased by phosphoramidon or diprotin A. They were not affected by HOE140, a bradykinin B2 antagonist, captopril, methysergide, mepyramine, indomethacin, ketoprofen or l-NG-nitroarginine. The higher dose of formalin induced a swelling of the paws which took place in two phases associated with two periods of increase in vascular permeability. This oedema was reduced by pretreatment with capsaicin but not with xylocaine. It was reduced by RP67580 injected before or 30 min after formalin. It was inhibited by mepyramine, methysergide, indomethacin and NS-398, a cyclooxy-genase-2 inhibitor. It was not modified by HOE140. Its development was similar in normal and kininogen-deficient rats. We concluded that formalin administered at a low dose induces an oedema which mainly results from a neurogenic inflammation mediated by neuropeptides such as substance P. At higher doses, formalin induces an oedema which mainly depends on the release of substance P, prostanoids, 5-hydroxytryptamine and histamine. Bradykinin plays no significant role in the vascular changes whereas this peptide has been reported to participate in the stimulation of nociceptive afferent neurons. This discrepancy could be explained by a difference in the threshold of stimulation of the nociceptive neurons and that of the cells of the vascular walls, or by a formation of kinins in close contact of the neurons.

Anxiolytic potential of sulpiride, clozapine and derivatives in the open-field test

Recently acquired data question the sharp dichotomy between anxiolytics and neuroleptics, since disinhibitory effects have been measured in the rat with very low doses of haloperidol and higher doses of atypical neuroleptics in FI and DRL schedules, but also in the open-field test. That the DA transmission in certain brain regions is involved in some aspects of anxiety has recently been suggested. The present study confirms this hypothesis particularly with high doses of sulpiride (80 mg/kg) and clozapine (24 mg/kg) when tested in the open-field test. Moreover, the results show how a slight chemical modification of clozapine can give a direction to pharmacological activity with one derivative still resembling clozapine and the second one resembling haloperidol. As neuroleptics do not seem to influence the synthesis and utilization of GABA, the higher entry score observed with them would seem to depend above all on DA antagonism in the mesolimbic system.

Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels

Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.

Ligand effects in the hydrogenation of methacycline to doxycycline and epi-doxycycline catalysed by rhodium complexes. Molecular structure of the key catalyst [closo-3,3-(η2,3-C7H7CH2)-3,1,2-Rh C2B9H11]

Abstract The catalytic reduction of the exocyclic methylene group of methacycline (A) leads to the formation of two diastereoisomers, doxycycline (B, the α-epimer) and 6-epi-doxycycline (C, the β-epimer), with a selectivity which markedly depends on the nature of hydrocarbon and carborane ligands of closo-(π-cyclodienyl)rhodacarborane catalysts. Neutral norbornadienyl complexes with unsubstituted carborane ligands [closo-3,3-(η2,3-C7H7CH2)-3,1,2-RhC2B9H11] (1) and [closo-2,2-(η2,3-C7H7CH2)-2,1,7-RhC2B9H11] (7) are more active and afford higher selectivity in the formation of doxycycline than those having mono- or di-substituents at the carborane cage, [closo-3,3-(cyclodienyl)-1-R-2-R′-3,1,2-RhC2B9H9] (R = H, R′ = Me, PhCH2; R = R′ = Me; cyclodienyl = η2,3-C7H7CH2 or η-C10H13) as well as those from the closely related series of η5-cyclopentadienyl complexes [(η2,3-C7H7CH2)Rh(η5-C5Rn)]+PF6− (Rn = H5, Me5, or H2-1,2,4-Ph3). Mechanistic aspects of the hydrogenation reaction of methacycline are sketched. The results of the X-ray diffraction study of the best catalyst 1 are reported.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (KCa2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both KCa2.2 and KCa2.3 with the same high affinity (KD ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC50 values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks KCa2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

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.