Naoyuki Todo

Inhibition by a putative antipsychotic quinolinone derivative (OPC-14597) of dopaminergic neurons in the ventral tegmental area

The effects of the newly synthesized quinolinone derivative, OPC-14597 (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy}-3, 4-dihydro-2(1 H)-quinolinone), on dopaminergic neuronal activity in the ventral tegmental area were examined using both in vivo microiontophoretic methods in chloral hydrate-anesthetized rats and the tight-seal whole-cell patch-clamp technique in thin-slice preparations of the rat brain. Neurons in the ventral tegmental area were classified as type I or type II according to their responses to antidromic stimulation of the nucleus accumbens, probably corresponding to dopaminergic and non-dopaminergic neurons, respectively. Antidromic spikes elicited by nucleus accumbens stimulation were inhibited by microiontophoretic application of dopamine and OPC-14597 in type I, but not in type II neurons. Although the OPC-14597-induced inhibition was antagonized by simultaneous application of domperidone (5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1 H-benzimidazo-1-yl)-propy]-4-piperidinyl]-1,3-dihydro-2H- benzimidazol-2-one; dopamine D2 receptor antagonist), SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1 H-3-benzazepine hydrochloride; dopamine D1 receptor antagonist) had no such effect. Spontaneous firing of type I neurons was also inhibited by iontophoretically applied OPC-14597 and dopamine, whereas that of type II neurons was unaffected. The inhibitory effect of OPC-14597 on the spontaneous firing of type I neurons was antagonized by domperidone, but not by SCH 23390. In a whole-cell patch-clamp study using a thin-slice preparation of the rat brain, bath application of OPC-14597 induced hyperpolarization accompanied by inhibition of spontaneously occurring action potentials in the large neurons (> 20 microns in diameter) in a concentration-dependent manner. These results suggest that OPC-14597 acts on dopaminergic neurons in the ventral tegmental area as a dopamine D2 receptor agonist to inhibit neuronal activities, probably by increasing membrane potassium conductance.

A mechanism underlying dopamine D1 and D2 receptor‐mediated inhibition of dopaminergic neurones in the ventral tegmental area in vitro

1 An intracellular recording study was performed to elucidate the mechanism underlying D1 and D2 receptor‐mediated inhibition of neuronal activities of dopaminergic neurones in the ventral tegmental area (VTA) using slice preparations of the rat brain. 2 VTA neurones were classified into type I and type II neurones according to the shape of the action potential, which correspond to dopaminergic and non‐dopaminergic neurones, respectively. 3 Addition of dopamine (10 μm) and quinpirole (1–100 μm) to the bath hyperpolarized the membrane of the type I neurones concomitantly with an increase in membrane conductance and an inhibition of action potentials which occurred spontaneously and were elicited by depolarizing pulses applied to the cell. However, quinpirole (10 μm) had no effect on the threshold for action potentials induced by a depolarizing pulse. 4 These quinpirole (10 μm)‐induced effects were antagonized by simultaneous application of domperidone (5 μm), a D2 receptor antagonist. 5 The amplitude of quinpirole (10 μm)‐induced hyperpolarization was decreased by increasing the potassium concentration in the perfusing fluid or simultaneous application of tetraethylammonium (10 μm). 6 SKF 38393 (10 or 100 μm), a D1 receptor agonist, had no effect on the resting membrane potential or action potential firing induced by a depolarizing pulse applied to the cell. However, when SKF 38393 (10 μm) was applied simultaneously with quinpirole (10 μm), the threshold for action potential generation was elevated by 5–6 mV, although there was no enhancement of hyperpolarization induced by quinpirole. 7 The elevation of the threshold for action potentials induced by SKF 38393 in the presence of quinpirole was antagonized by simultaneous application of SCH 23390 (5 μm), a D1 receptor antagonist. 8 Dopamine (10 μm), quinpirole (10 or 100 μm) and SKF 38393 (10 or 100 μm) had no effect on the resting membrane potential or spontaneously occurring action potentials in type II neurones. 9 These findings suggest that activation of dopamine D2 receptors of dopaminergic neurones in the VTA increases potassium conductance, thereby hyperpolarizing the membrane and eventually inhibiting neuronal activities. They also suggest that simultaneous activation of both D1 and D2 receptors enhances the D2 receptor‐mediated inhibitory effects by elevation of the threshold for action potential generation.

5-HT1A receptor-mediated inhibition of lateral vestibular nucleus neurons projecting to the abducens nucleus.

Electrophysiological studies were performed using cats anesthetized with alpha-chloralose, to elucidate the 5-hydroxytryptamine (5-HT) receptor subtypes involved in the 5-HT-induced inhibition of the lateral vestibular nucleus (LVN) neurons projecting to or through the abducens nucleus. The effects of 5-HT receptor subtype agonists and antagonist were examined in polysynaptic neurons activated by stimulation of the ipsilateral abducens nucleus (IAN) antidromically, since these neurons are sensitive to 5-HT as shown in our previous study. Iontophoretic application of 5-HT and 8-hydroxy-2-(di-n-propylamino)tetrain (8-OH-DPAT), a selective 5-HT1A agonist, inhibited orthodromic spikes elicited by vestibular nerve stimulation in the majority of polysynaptic neurons activated by stimulation of ipsilateral IAN antidromically. There was a good correlation between the effects of 5-HT and 8-OH-DPAT. Iontophoretically applied 5-HT and 8-OH-DPAT also inhibited glutamate-induced firing in these neurons. Simultaneous application of 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190), a 5-HT1A agonist/antagonist, significantly antagonized the 8-OH-DPAT-induced inhibition of glutamate-induced firing, although NAN-190 alone also caused weak suppression of glutamate-induced firing. Microiontophoretically applied 1-(3-chlorophenyl)piperazine (mCPP), a 5-HT1B agonist inhibited the orthodromic spike elicited by vestibular nerve stimulation and glutamate-induced firing in only a small number of the LVN neurons. 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2 agonist, rarely affected these neurons. We postulate that postsynaptically located 5-HT1A receptors are mainly involved in the 5-HT-induced inhibition of polysynaptic neurons projecting in the region of the IAN.

Antagonizing effects of a novel antipsychotic quinolinone derivative (OPC-14597) on dopaminergic inhibition of neuronal activities in the nucleus accumbens.

1. The effects of a newly synthesized quinolinone derivative, 7-(4-[4-(2,3-dichlorophenlyl)-1-piperazinyl]butyloxy)-3,4-di hydro-2-(1H)- quinolinone (OPC-14597), an antipsychotic drug, on neuronal activities of the nucleus accumbens (Acc) were investigated in rats anesthetized with chloral hydrate using a microiontophoretic method. 2. Spikes elicited by stimulation of the parafascicular nucleus (Pf) of the thalamus were extracellularly recorded in the Acc neuron of chloral hydrate-anesthetized adult Wistar rats using a glass microelectrode attached along a seven-barreled micropipette, each of which was filled with dopamine, OPC-14597, SKF 38393 (D1 receptor agonist), quinpirole (D2 receptor agonist) and 2M NaCl. The drugs were microiontophoretically applied to the target neurons recorded. 3. Effects of the drugs on the Acc neurons activated monosynaptically by stimulation of the Pf were examined. Spikes elicited by Pf stimulation were inhibited by iontophoretic application of dopamine, SKF 38393 and quinpirole in a dose-dependent manner. 4. Microiontophoretic application of OPC-14597 alone affected the spikes elicited by the Pf stimulation in none of 26 neurons tested. However, the dopamine-, SKF 38393- and quinpirole-induced inhibition of the spike generation in the Acc neurons was antagonized during simultaneous application of OPC-14597. 5. The firing induced by iontophoretically applied glutamate was inhibited by dopamine, SKF 38393 and quinpirole, but not by OPC-14597. However, the dopamine-, SKF 38393- and quinpirole-induced inhibition of the glutamate-induced firing was also antagonized during simultaneous application of OPC-14597 in a dose-dependent manner in all neurons tested. 6. These findings suggest that OPC-14597 blocks dopaminergic inhibition of the Acc neurons receiving input from the Pf by acting on both D1 and D2 receptors located on the neurons.

Antiepileptic effects of CNK-602A, a novel thyrotropin-releasing hormone analog, on absence-like and tonic seizures of spontaneously epileptic rats

The effects of CNK-602A (N-[(6-methyl-5-oxo-3-thiomorpholinyl) carbonyl]-L-histidyl-L-prolinamide), a novel thyrotropin-releasing hormone related analog, were investigated on absence-like seizure and tonic convulsion in the spontaneously epileptic rat (SER), which is a genetically defined double-mutant. When CNK-602A of 0.2-1 mg/kg was given intravenously to the animal, there were no changes in the background EEG except for an increase in low-voltage fast waves concomitant with behavioral alertness. However, CNK-602A suppressed absence-like seizure and tonic convulsion in a dose-dependent manner for over 1 h. These antiepileptic effects of CNK-602A on both seizures were antagonized by pretreatment with haloperidol (1 mg/kg, i.p.). It was found, using a brain in vivo microdialysis method, that CNK-602A at a dose of 1 mg/kg, which inhibits the seizures, increased the release of dopamine in the caudate nucleus. These results suggest that CNK-602A inhibits the seizures of SER in a similar manner to thyrotropin-releasing hormone (TRH), probably by increasing the release of dopamine in the central nervous system. In addition, the antiepileptic effects of CNK-602A were more potent and lasted longer than those of TRH.

Membrane depolarization by activation of prostaglandin E receptor EP3 subtype of putative serotonergic neurons in the dorsal raphe nucleus of the rat

A whole-cell current-clamp study using a thin slice preparation of the rat brain was carried out to elucidate the function of prostaglandin E (PGE) receptor EP3, subtype in the dorsal raphe nucleus (DR), where mRNA of this subtype is highly expressed. Bath application of PGE2 or M&B 28767, an EP3 agonist, depolarized the membrane of the large DR neurons in a concentration-dependent manner between 10−9 and 10−6 M. These neurons showed hyperpolarization of membrane potential to 10 or 50 μM serotonin. Neither an EP2 receptor agonist, butaprost, an EP2/EP4 receptor agonist, 11-deoxy-PGE1, nor an EP1 receptor agonist, 17-phenyl-PGE2, had any effect on large DR neurons between 10−9 and 10−6 M. The M&B 28767-induced depolarization was observed in a Ca2+-free, high Mg2+ (5 mM) solution containing 0.3 μM tetrodotoxin, and occurred equally well when intracellular Cl− was replaced by gluconate. These results suggest that direct agonist-activation of EP3 receptor depolarizes the membrane by a cationic conductance, leading to excitation of DR neurons, and indicate a physiological implication that EP3 receptor may modulate the serotonergic inhibition of neuronal activities.

Excitation by talipexole, a dopamine D2 agonist, of caudate nucleus neurons activated by nigral stimulation

An electrophysiological study using cats anesthetized with alpha-chloralose was performed to elucidate whether or not talipexole (B-HT 920 CL2: 6-allyl-2-amino -5, 6, 7, 8-tetrahydro-4H-thiazolo [4, 5 -d] -azepine-dihydrochroride), a dopamine D2 agonist, acts on postsynaptic dopamine receptors in the caudate nucleus (CN) neurons receiving excitatory input from the pars compacta of substantia nigra (SN). Extracellular neuron activities were recorded in the CN using a glass-insulated silver wire microelectrode attached along a seven-barreled micropipette, each of which was filled with talipexole, quinpirole (dopamine D2 agonist), domperidone (dopamine D2 antagonist), glutamate and 2M NaCl. These drugs were microiontophoretically applied to the immediate vicinity of the target neuron. In the same neurons in which the spikes elicited by the SN stimulation were blocked by microiontophoretically applied domperidone, microiontophoretic application of talipexole and quinpirole induced a dose-dependent increase in spontaneous firing. This increase in firing by talipexole and quinpirole was blocked during simultaneous application of domperidone, although glutamate-induced firing remained unaffected by domperidone. In the CN neurons, in which the SN stimulation-induced spikes were not blocked by domperidone, spontaneous firing was not affected by talipexole or quinpirole. These findings suggest that talipexole activates CN neurons receiving a dopaminergic input from SN via D2 receptors, as does quinpirole.

Pharmacological Application of Spontaneously Epileptic Rat(SER).

The spontaneously epileptic rat (SER) is a double mutant (zi/zi, tm/tm) obtained by mating zitter homozygote (zi/zi) with tremor heterozygote (tm/ + ) . This animal exhibits both tonic convulsion and absence-like seizures characterized by a sudden appearance of 5-7 Hz spikes and wave complex in cortical and hippocampal EEG. This study was performed to elucidate the usefulness of SER in evaluating of acute and long-term effects of antiepileptic drugs. Moreover, activities of the central inhibitory system and abnormal excitability of neurons related to the epileptic seizures were investigated. (1) From recordings of implanted electrode in cerebral cortex and hippocampus, phenytoin and carbamazepine (agents for tonic-clonic grand mal seizure) inhibited tonic convulsion without affecting absence-like seizures, whereas trimethadione and ethosuximide (agents effective for absence seizure, petit mal epilepsy) suppressed absence-like seizures without any effects on tonic convulsion. Both seizures were inhibited by phenobarbital, valproate and diazepam, agents which are effective for grand and petit mal epilepsy. This antiepileptic profile suggests that tonic and absence-like seizures in SER correspond to human grand and petit mal seizures. Therefore, SER is a useful model for evaluating the acute effects of novel antiepileptics. (2) The group of SER, which were orally administered with 1 % phenobarbital-containing pellet displayed a marked increase in body weight and longer a life span than the control group with a plasma concentration of 35-50 μg/ml. Similar to the futile results of chronic administration of phenobarbital in human absence epilepsy, absence-like seizures in SER were not affected by this drug, although tonic convulsion was inhibited. Long-term effects of valproate (1 and 5 %) were similar to those of phenobarbital except for its efficacy on absence-like seizures. Therefore, SER is also useful for the evaluation of long-term effects of antiepileptics. (3) Both tonic and absence-like seizures in SER were inhibited by TRH and its analog, CNK-602A, and dopamine agonists, methamphetamine and apomorphine. These results suggest that dopaminergic pathways act as an inhibitory system on the seizure-producing mechanism. Further, SER can be used for investigations of the central inhibitory system involved in the regulation of seizures. (4) In hippocampal slice preparations, a single stimulus given to the mossy fiber induced a longlasting depolarization shift with repetitive firings in CA3 field pyramidal neurons of the SER but not zitter or wistar rats. This abnormal excitation of the neurons were completely blocked by verapamil, a calcium channel blocker. These findings suggest that abnormal calcium channel activities in the CA3 pyramidal neurons are involved in the seizures of SER. Thus, SER is a useful animal model for investigating abnormalities related to epileptic seizures.