The tricyclic antidepressant desipramine inhibited the neurotoxic, kainate-induced [Ca(2+)]i increases in CA1 pyramidal cells in acute hippocampal slices.

Kainate (KA), used for modelling neurodegenerative diseases, evokes excitotoxicity. However, the precise mechanism of KA-evoked [Ca(2+)]i increase is unexplored, especially in acute brain slice preparations. We used [Ca(2+)]i imaging and patch clamp electrophysiology to decipher the mechanism of KA-evoked [Ca(2+)]i rise and its inhibition by the tricyclic antidepressant desipramine (DMI) in CA1 pyramidal cells in rat hippocampal slices and in cultured hippocampal cells. The effect of KA was dose-dependent and relied totally on extracellular Ca(2+). The lack of effect of dl-2-amino-5-phosphonopentanoic acid (AP-5) and abolishment of the response by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggested the involvement of non-N-methyl-d-aspartate receptors (non-NMDARs). The predominant role of the Ca(2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the initiation of the Ca(2+) response was supported by the inhibitory effect of the selective AMPAR antagonist GYKI 53655 and the ineffectiveness of 1-naphthyl acetylspermine (NASPM), an inhibitor of the Ca(2+)-permeable AMPARs. The voltage-gated Ca(2+) channels (VGCC), blocked by ω-Conotoxin MVIIC+nifedipine+NiCl2, contributed to the [Ca(2+)]i rise. VGCCs were also involved, similarly to AMPAR current, in the KA-evoked depolarisation. Inhibition of voltage-gated Na(+) channels (VGSCs; tetrodotoxin, TTX) did not affect the depolarisation of pyramidal cells but blocked the depolarisation-evoked action potential bursts and reduced the Ca(2+) response. The tricyclic antidepressant DMI inhibited the KA-evoked [Ca(2+)]i rise in a dose-dependent manner. It directly attenuated the AMPA-/KAR current, but its more potent inhibition on the Ca(2+) response supports additional effect on VGCCs, VGSCs and Na(+)/Ca(2+) exchangers. The multitarget action on decisive players of excitotoxicity holds out more promise in clinical therapy of neurodegenerative diseases.

Chemical neuroprotection in the cochlea: the modulation of dopamine release from lateral olivocochlear efferents.

The prevalence of sensorineural hearing loss is increasing worldwide, mainly due to ageing, increased noise exposure and cardiovascular risk factors. Several papers dealt with the mechanisms underlying the primary causes of impaired hearing and eventual deafness, including the damage and loss of auditory hair cells; however, very little is known about the protective mechanisms that exist for hearing. Several recent investigations have implicated dopamine (DA) in a neuroprotective circuit for the cochlea. The lateral olivocochlear (LOC) efferents provide axonal innervation of the inner hair cell afferent synapses and release DA and other substances in response to different stimuli. Under ischemic conditions or during noise exposure, DA has been proven to play a neuroprotective role against glutamate excitotoxicity. This review summarises what is currently known about the modulation of DA release in the cochlea, using primarily in vitro experimental data. Based on recent knowledge, there could be two functional subgroups within the LOC fibres, i.e., the DA- and GABA-containing projections. In this review, we attempt to show the neurochemical interactions between these two subsystems. Other aspects of cochlear neurotransmission are also discussed to provide a complete picture of cochlear dopaminergic function in physiological and pathophysiological cases with particular reference to excitotoxicity.

The effect of antiparkinsonian drugs on oxidative stress induced pathological [3H]dopamine efflux after in vitro rotenone exposure in rat striatal slices.

An in vitro model of mitochondrial dysfunction with subsequent oxidative stress was elaborated and utilized to study the effect of drugs, currently used for the treatment of Parkinson's disease, on pathological H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of toxic dopamine metabolites in rat striatal slices. 60 min rotenone (0.1-10 muM) pretreatment decreased dopamine content and [(3)H]dopamine uptake, as well as ATP level and energy charge of the slices. In addition, a robust potentiation of H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of dopamine quinone in the effluent was detected. l-DOPA (200 muM) markedly elevated resting but not 100 muM H(2)O(2)-evoked and electrically-induced [(3)H]dopamine efflux. Furthermore, l-DOPA promoted the formation of dopamine quinone. Ropinirole (100 nM) did not affect resting and H(2)O(2)-evoked [(3)H]dopamine efflux and inhibited the electrically evoked release only in untreated slices. l-deprenyl, at concentration of 0.01 muM potentiated, whilst between 1 and 50 muM diminished H(2)O(2)-evoked [(3)H]dopamine efflux. Rasagiline (0.01-50 muM) slightly inhibited H(2)O(2)-evoked [(3)H]dopamine efflux, and it was able to prevent the generation of dopamine quinone. Neither of the drugs was able to suppress both the pathological H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of dopamine quinone with simultaneous augmentation of electrically evoked [(3)H]dopamine release what should be a future concept of antiparkinsonian drug-design.

Mechanism of the persistent sodium current activator veratridine-evoked Ca elevation: implication for epilepsy.

Although the role of Na(+) in several aspects of Ca(2+) regulation has already been shown, the exact mechanism of intracellular Ca(2+) concentration ([Ca(2+)](i)) increase resulting from an enhancement in the persistent, non-inactivating Na(+) current (I(Na,P)), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na(+) current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na(+) concentration ([Na(+)](i)) and biphasic [Ca(2+)](i) increase in CA1 pyramidal cells in acute hippocampal slices. The Ca(2+) response was tetrodotoxin- and extracellular Ca(2+)-dependent and ionotropic glutamate receptor-independent. The first phase of [Ca(2+)](i) rise was the net result of Ca(2+) influx through voltage-gated Ca(2+) channels and mitochondrial Ca(2+) sequestration. The robust second phase in addition involved reverse operation of the Na(+)-Ca(2+) exchanger and mitochondrial Ca(2+) release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non-inactivating Na(+) current and [Ca(2+)](i) regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I(Na,P). Describing the magnitude, temporal pattern and sources of Ca(2+) increase induced by I(Na,P) may provide novel targets for antiepileptic drug therapy.

Neurochemical evidence that stimulation of CB1 cannabinoid receptors on GABAergic nerve terminals activates the dopaminergic reward system by increasing dopamine release in the rat nucleus accumbens.

We examined the effect of cannabinoid receptor activation on basal and electrical field simulation-evoked (25 V, 2 Hz, 240 shocks) [(3)H]dopamine efflux in the isolated rat nucleus accumbens in a preparation, in which any effect on the dendrites or somata of ventral tegmental projection neurons was excluded. The cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2, 100 nM) significantly enhanced stimulation-evoked [(3)H]dopamine release in the presence of the selective dopamine transporter inhibitor 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride (GBR12909, 100 nM). GBR12909 (100 nM-1 microM), when added alone, increased the evoked [(3)H]dopamine efflux in a concentration-dependent manner. The stimulatory effect of WIN55,212-2 on the evoked tritium efflux was inhibited by the selective CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 100 nM) and by the GABA(A) receptor antagonist bicuculline (10 microM). Repeated application of N-methyl-d aspartate (1 mM) under Mg(2+)-free conditions, which directly acts on dopaminergic terminals, reversibly increased the tritium efflux, but WIN55,212-2 did not affect N-methyl-d aspartate-evoked [(3)H]dopamine efflux, indicating that WIN55,212-2 has no direct action on dopaminergic nerve terminals. AM251 (100 nM) alone also did not have an effect on electrical stimulation-evoked [(3)H]dopamine efflux. Likewise, the selective CB2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630, 0.3 microM) and the anandamide transport inhibitor (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11, 10 microM) had no significant effect on electrically evoked [(3)H]dopamine release. This is the first neurochemical evidence that the activation of CB1 cannabinoid receptors leads to the augmentation of [(3)H]dopamine efflux via a local GABA(A) receptor-mediated disinhibitory mechanism in the rat nucleus accumbens.

Modulation of neurotransmitter release by P2X and P2Y receptors in the rat spinal cord.

In this study, the P2 receptor-mediated modulation of [3H]glutamate and [3H]noradrenaline release were examined in rat spinal cord slices. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and 2-methylthioadenosine 5'-diphosphate (2-MeSADP) decreased the electrical stimulation-evoked [3H]glutamate efflux with the following order of potency: ADP>2-MeSADP>ATP. The effect of ATP was antagonized by suramin (300microM), the P2Y12,13 receptor antagonist 2-methylthioadenosine 5'-monophosphate (2-MeSAMP, 10microM), and partly by 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS, 30microM) and the P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179, 10muM). ATP, ADP and 2-MeSADP also decreased evoked [3H]noradrenaline outflow; the order of agonist potency was ADP> or =2-MeSADP>ATP. The effect of ATP was reversed by 2-MeSAMP (10microM), and partly by MRS 2179 (10microM). By contrast, 2-methylthioadenosine-5'-triphosphate (2-MeSATP, 10-300microM) increased resting and electrically evoked [3H]glutamate and [3H]noradrenaline efflux, and this effect was prevented by the P2X1 receptor selective antagonist 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis (carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt (NF449, 100nM). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNAs encoding P2Y12 and P2Y13 receptors are expressed in the brainstem, whereas P2Y13 but not P2Y12 receptor mRNA is present in the dorsal root ganglion and spinal cord. P2Y1 receptor expression in the spinal cord is also demonstrated at the protein level. In conclusion, inhibitory P2Y and facilitatory P2X1-like receptors, involved in the regulation of glutamate (P2Y13 and/or P2Y1) and noradrenaline (P2Y13 and/or P2Y1, P2Y12) release have been identified, which provide novel target sites for analgesics acting at the spinal cord level.

Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors.

The function of nicotinic acetylcholine receptors in the main central systems has been documented in the past decade. These studies focused mostly on the synaptic functions, although acetylcholine is released dominantly into the extrasynaptic space and the majority of nicotinic acetylcholine receptors on remote neurons are found on extrasynaptic membranes. Here, we show further evidence for the role of nonsynaptic nicotinic functions in the cognitive and the reward system. Dendrites of gamma-amino-n-butyric acid (GABA)-containing interneurons of the hippocampus are densely equipped with nicotinic acetylcholine receptors. These cells play an important role in memory processing. We analysed the effects of nicotinic acetylcholine receptor stimulation on the Ca(2+) dynamics of interneurons in different dendritic compartments. We also investigated the role of nicotinic receptors in the nucleus accumbens where nicotine stimulated vesicular dopamine release via activation of receptors located on varicosities. Nicotine produced comparable effects with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) on dopamine release. These examples demonstrate that nonsynaptic nicotinic acetylcholine receptors can effectively influence activity pattern of neural networks in key structures of central systems.

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves.

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.

Effect of noradrenaline and vanadate on sodium, potassium-activated ATPase of human brain cortical homogenate.

Human brain cortical homogenate derived from surgical operations exhibited Na(+), K(+)-ATPase and K-p-nitrophenylphosphatase activity values of 2.12 +/- 0.08 ?mol P(i)/mg protein/15 min and 0.38 +/- 0.01 ?mol p-nitrophenol/mg protein/15 min, respectively which is in the range of those observed in rat brain cortical homogenates. Vanadate concentration dependently inhibited the activity of both enzymes. Noradrenaline, dopamine and isoprenaline reversed the inhibitory effect of vanadate in the presence of EDTA (0.2 mM). When Mg(2+) concentration was enhanced from 4 to 24 mM, the inhibitory effect of vanadate (1 ?M) was significantly potentiated. Evidence has been obtained that the effect of catecholamines is not a receptor mediated process; antagnoists such as phentolamine, phenoxybenzamine, propranolol, haloperidol failed to prevent the effect of adrenoceptor agonists. It is concluded that there is an interaction between vanadate and noradrenaline on human brain Na, K-ATPase.