E.S. Vizi

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.

Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions : role of glutamic acid

We studied the effect of local administration of nicotine on the release of monoamines in striatum, substantia nigra, cerebellum, hippocampus, cortex (frontal, cingulate), and pontine nucleus and on the release of glutamic acid in striatum of rats in vivo, using microdialysis for nicotine administration and for measuring extracellular amine and glutamic acid levels. Following nicotine administration the extracellular concentration of dopamine, increased in all regions except cerebellum; serotonin increased in cingulate and frontal cortex; and norepinephrine increased in substantia nigra, cingulate cortex, and pontine nucleus. Cotinine, the major nicotine metabolite, had no effect at similar concentrations. The cholinergic antagonists mecamylamine and atropine, the dopaminergic antagonists haloperidol and sulpiride, and the excitatory amino acid antagonist kynurenic acid all inhibited the nicotine-induced increase of extracellular dopamine in the striatum. The fact that kynurenic acid almost completely prevented the effects of nicotine, and nicotine at this concentration produced a 6-fold increase of glutamic acid release, suggests that the effect of nicotine is mainly mediated via glutamic acid release.

Characterization of nicotinic receptors involved in the release of noradrenaline from the hippocampus.

The pharmacological features of putative nicotinic acetylcholine receptor sites involved in the release of [3H]noradrenaline were assessed in rat hippocampus. The effect of nicotinic agonists to induce [3H]noradrenaline release was examined in superfused slices. The nicotinic agonists (-)-epibatidine, (+)-anatoxin-a, dimethylphenylpiperazinium, (-)-nicotine and (-)-lobeline released [3H]noradrenaline. The dose-response curves to nicotinic agonists were bell shaped, and indicated that their functional efficacies and potency vary across agonists. Maximal efficacy was seen with dimethyl-phenylpiperazinium and lobeline (Emax values two to three times higher than other agonists). The rank order of potency for the agonists to release [3H]noradrenaline was (-)-epibatidine > (+)- anatoxin-a > dimethylphenylpiperazinium > cytisine > nicotine > (-)-lobeline. The nicotinic acetylcholine receptor antagonists (n-bungarotoxin > mecamylamine > (+)-tubocurarine > hexamethonium > alpha-bungarotoxin = dihydro-beta-erythroidine) and tetrodotoxin antagonized the effect of dimethylphenylpiperazinium to release [3H]noradrenaline. The results, based on these pharmacological profiles, suggest the possible involvement of alpha 3 and beta 2 nicotinic acetylcholine receptor subunits in the control of [3H]noradrenaline release from hippocampal slices. The absence of effect of alpha-bungarotoxin and alpha-conotoxin-IMI excludes the possible involvement of nicotinic acetylcholine receptors containing the alpha 7 subunit. The release of [3H]noradrenaline by dimethylphenylpiperazinium was Ca2+ dependent. Nifedipine failed to prevent the dimethylphenylpiperazinium-induced release of [3H]noradrenaline, but Cd2+, omega-conotoxin and Ca(2+)-free conditions significantly reduced the dimethylphenylpiperazinium-induced release, suggesting that N-type voltage-sensitive Ca2+ channels are involved in the nicotinic acetylcholine receptor response. These voltage-sensitive Ca2+ channels are activated by the local depolarization produced by sodium influx through the nicotinic channels activated by dimethylphenylpiperazinium. Thus, the observed tetrodotoxin sensitivity of dimethylphenylpiperazinium-induced release of [3H]noradrenaline can be explained either by local depolarization and subsequent generation of action potentials at the preterminal area or that these nicotinic acetylcholine receptors are located on interneurons rather than directly on noradrenergic terminals.

Non-synaptic receptors and transporters involved in brain functions and targets of drug treatment

Beyond direct synaptic communication, neurons are able to talk to each other without making synapses. They are able to send chemical messages by means of diffusion to target cells via the extracellular space, provided that the target neurons are equipped with high‐affinity receptors. While synaptic transmission is responsible for the ‘what’ of brain function, the ‘how’ of brain function (mood, attention, level of arousal, general excitability, etc.) is mainly controlled non‐synaptically using the extracellular space as communication channel. It is principally the ‘how’ that can be modulated by medicine. In this paper, we discuss different forms of non‐synaptic transmission, localized spillover of synaptic transmitters, local presynaptic modulation and tonic influence of ambient transmitter levels on the activity of vast neuronal populations. We consider different aspects of non‐synaptic transmission, such as synaptic–extrasynaptic receptor trafficking, neuron–glia communication and retrograde signalling. We review structural and functional aspects of non‐synaptic transmission, including (i) anatomical arrangement of non‐synaptic release sites, receptors and transporters, (ii) intravesicular, intra‐ and extracellular concentrations of neurotransmitters, as well as the spatiotemporal pattern of transmitter diffusion. We propose that an effective general strategy for efficient pharmacological intervention could include the identification of specific non‐synaptic targets and the subsequent development of selective pharmacological tools to influence them.

Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices : origin and modulation by α2-adrenoceptors

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [3H]noradrenaline was diminished by more than 80% under Ca2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [3H]noradrenaline, however, it was less effective in reducing the release of [3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 microg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 microM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (-)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [3H]noradrenaline release. While CH 38083, a non subtype-selective alpha2-antagonist and BRL44408, the subtype-selective alpha2AD antagonist augmented the evoked release of [3H]noradrenaline, ARC239, a selective alpha2BC antagonist was without effect. In contrast, neither of the alpha2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and alpha1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [3H]noradrenaline is inhibited by endogenous noradrenaline via alpha2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.

Prejunctional modulation of acetylcholine release from the skeletal neuromuscular junction: link between positive (nicotinic)- and negative (muscarinic)-feedback modulation.

1 Presynaptic receptor‐mediated modulation of stimulation‐evoked [3H]‐acetylcholine([3H]‐ACh) release from the neuromuscular junction was studied in the region of the mouse hemidiaphragm which contains the motor endplates, and which can easily be loaded with [3H]‐choline. This method made it possible to detect exclusively the [Ca2+]o‐dependent, quantal release of [3H]‐ACh in response to axonal stimulation. 2 Atropine enhanced, and non‐depolarizing muscle relaxants ((+)‐tubocurarine, pancuronium and pipecuronium) reduced, the release of [3H]‐ACh evoked by high frequency trains of stimulation (50 Hz, 40 shocks) of the phrenic nerve. The effect of (+)‐tubocurarine was frequency‐dependent as at 5 Hz (40 shocks) it was less effective than at 50 Hz. The resting release of [3H]‐ACh was not affected by these compounds. These findings indicate that ACh released into the synaptic gap by axonal firing reaches a concentration sufficient to influence its own release by a prejunctional effect. 3 The anticholinesterase, physostigmine sulphate, enhanced the release of [3H]‐ACh in a concentration‐dependent manner. This effect was mediated via prejunctional nicotinic receptor stimulation: (+)‐tubocurarine, pancuronium and pipecuronium completely prevented the effect of physostigmine. 4 When the prejunctional nicotinic and muscarinic receptors were stimulated by a high concentration of extracellular ACh which had accumulated in the junctional gap in the presence of physostigmine, atropine did not influence the evoked release of [3H]‐ACh. However, when the effect of endogenous ACh on nicotinic receptors was prevented by (+)‐tubocurarine, atropine enhanced the release. 5 It is concluded that quantally‐released ACh from motor endplates is subject to prejunctional automodulation: (a) ACh facilitates its own release via an effect on prejunctional nicotinic receptors (positive feedback), (b) ACh release is reduced by an action on muscarinic receptors. When the nicotinic receptor‐mediated facilitation is fully operative, the muscarinic receptor‐mediated negative feedback is much less effective. It is supposed that there is a link between the two feedback mechanisms possibly at the level of the second messenger system(s).

The effect of lithium on acetylcholine release and synthesis

Abstract When sodium concentration in Krebs solution was replaced by different concentrations of lithium to maintain isosmolarity, the acetylcholine release during resting conditions in rat cortex slices was increased depending on the lithium concentration. The maximal augmentation in ACh resting release was reached at a lithium concentration of 117.9 mM. This acetylcholine release proved to be tetrodotoxin (2.5 × 10−7 g ml ) and noradrenaline (10−6 g ml ) resistant. However, when sodium was partly replaced by lithium (117.9 mM LiCl and 25 mM NaCl) the synthesis was blocked completely. This inhibition can be attributed to the lithium ions present in the nerve terminals, since the acetylcholine release was unaffected at low sodium concentration (25 mM) when isosmolarity was maintained by glucose. Furthermore lithium (117.9 mM) reduced the acetylcholine output due to stimulation from the nerve terminals of guinea-pig ileum longitudinal muscle strip. It is suggested that lithium accumulating inside the nerve terminals inhibited the rate of synthesis of acetylcholine. The reduction of freshly synthetized acetylcholine led to a reduction of acetylcholine output due to stimulation.

The mechanism of activity-dependent sodium channel inhibition by the antidepressants fluoxetine and desipramine.

The effect of monoamine uptake inhibitor-type antidepressants on sodium channels of hippocampal neurons was investigated. Members of the tricyclic group of antidepressants are known to modify multiple targets, including sodium channels, whereas selective serotonin-reuptake inhibitors (SSRIs) are regarded as highly selective compounds, and their effect on sodium channels was not investigated in detail. In this study, a representative member of each group was chosen: the tricyclic antidepressant desipramine and the SSRI fluoxetine. The drugs were roughly equipotent use-dependent inhibitors of sodium channels, with IC50 values ∼100 μMat -150 mV holding potential, and ∼1 μMat -60 mV. We suggest that therapeutic concentrations of antidepressants affect neuronal information processing partly by direct, activity-dependent inhibition of sodium channels. As for the mechanism of inhibition, use-dependent inhibition by antidepressants was believed to be due to a preferential affinity to the fast-inactivated state. Using a voltage and perfusion protocol by which relative affinities to fast-versus slow-inactivated states could be assessed, we challenged this view and found that the affinity of both drugs to slowinactivated state(s) was higher. We propose a different mechanism of action for these antidepressants, in which slow rather than fast inactivation plays the dominant role. This mechanism is similar but not equivalent with the novel mechanism of usedependent sodium channel inhibition previously described by our group (Neuroscience 125:1019-1028, 2004; Neuroreport 14:1945-1949, 2003). Our results suggest that different drugs can produce use-dependent sodium channel inhibition by different mechanisms.

Prejunctional nicotinic receptors involved in facilitation of stimulation-evoked noradrenaline release from the vas deferens of the guinea-pig

1 In guinea‐pig prostatic vas deferens loaded with [3H]‐noradrenaline ([3H]‐NA), nicotinic receptor agonists, nicotine and dimethylphenylpiperazinium (DMPP) enhanced the resting and facilitated the stimulation‐evoked release of [3H]‐NA in a concentration‐dependent fashion. The effect of nicotine on both contraction of vas deferens and release of NA in response to field stimulation was stereospecific in favour of the naturally occurring (−)‐enantiomer. Prolonged (15 min) exposure to (−)‐nicotine resulted in a cessation of the facilitatory effect on NA release and on responses of the vas deferens to field stimulation. 2 The rank order of agonist potency in facilitating NA release was DMPP = (−)‐nicotine > (+)‐nicotine. Cytisine had no agonistic activity. The dissociation constants (KD) of antagonists were 9.3 ± 0.6 and 31.4 + 2.4 μm for (+)‐tubocurarine and hexamethonium, respectively, when (−)‐nicotine was used as agonist. α‐Bungarotoxin had no antagonistic activity. These findings suggest that nicotinic receptors located on noradrenergic axon terminals are different from those located postsynaptically in striated muscle or ganglia but seem similar to those present on cholinergic axon terminals at the neuromuscular junction. 3 Cotinine, the breakdown product of nicotine failed to have any agonistic activity indicating that nicotine itself is responsible for the effects observed on axon terminals. 4 Stimulation of presynaptic muscarinic receptors by oxotremorine prevented the nicotine‐induced facilitation of [3H]‐NA release, indicating the presence of both inhibitory muscarinic and facilitatory nicotinic receptors on noradrenergic axon terminals.

Neurochemical evidence of dopamine release by lateral olivocochlear efferents and its presynaptic modulation in guinea-pig cochlea

In this study, using an in vitro superfusion technique for the first time, we provide direct neurochemical evidence of the transmitter role of dopamine at the level of lateral olivocochlear efferent fibres of the guinea-pig cochlea. Our results revealed that nerve terminals are able to take up and release dopamine upon axonal stimulation. Since dopamine is thought to protect the afferent nerve fibres from damage due to acoustic trauma or ischaemia, enhancement of the release of dopamine, a potential therapeutic site of these injuries, was investigated. Positive modulation of dopamine release has been shown by a D1 dopamine receptor agonist, an antagonist and piribedil. Furthermore, negative feedback on the stimulation-evoked release of dopamine via D2 dopamine receptors has been excluded. Electrical stimulation of the cochlear tissue produced a significant and reproducible release of [3H]dopamine, which could be blocked by tetrodotoxin (1 microM) and cadmium (100 microM), proving that axonal activity releases dopamine and its dependence on Ca2+ influx verifies its neuronal origin. Nomifensine, a high-affinity dopamine uptake blocker, prevented the tissue from taking up [3H]dopamine from the bathing solution, also indicating the neural origin of dopamine released in response to stimulation. SKF-38393 (a selective D1 agonist) increased both the resting and electrically evoked release of dopamine. Piribedil (a D3/D2/D1 agonist), a drug under investigation, known to prevent acoustic trauma or ischaemia-induced hearing loss, had a similar and concentration-dependent increasing effect on both resting and evoked release of dopamine. The effect of both drugs on stimulation-evoked release could be prevented by SKF-83566 (a selective D1 antagonist). However, SKF-83566 alone enhanced the resting and axonal conduction-associated release of dopamine. D2 agonists and antagonists failed to modulate the release of dopamine, indicating the lack of negative feedback modulation of dopamine release. Our results suggest that the release of dopamine was subjected to modulation by a D1 receptor agonist and an antagonist. In addition, it is concluded that D2 receptors are not involved in the modulation of dopamine release. This observation may have clinical relevance in the prevention or therapy of particular types of hearing loss, because enhanced dopaminergic input into the primary auditory neuron may inhibit the (over)excitation of this neuron by glutamatergic input from inner hair cells.