Shigenori Watanabe

Effects of 5-HT1A receptor agonists on the circadian rhythm of wheel-running activity in hamsters

The effects of 5-HT1A receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and ipsapirone on wheel-running activity in hamsters were investigated in comparison with those of GABAA receptor agonist muscimol and benzodiazepine triazolam. Intraperitoneal administration of 8-OH-DPAT, buspirone, ipsapirone, muscimol and triazolam at circadian time (CT) 8 (CT 12; onset of activity) induced a significant phase advance of wheel-running activity under constant light conditions. However, administration of these drugs at other CT points did not induce phase changes. The administration of trifluoromethylphenylpiperazine (TFMPP), a 5-HT1B receptor agonist, at CT8 produced a small phase advance. The phase advance induced by 8-OH-DPAT was blocked by pretreatment with (-)-pindolol, a 5-HT1A receptor antagonist. In addition, 8-OH-DPAT, buspirone and SM3997 accelerated the rate of re-entrainment to an 8-h phase advance in the light-dark cycle. These observations suggest that 5-HT1A receptors in the brain participate in the regulation of the circadian rhythm of wheel-running activity in hamsters.

Phase-resetting efect of 8-OH-DPAT, a serotonin1A receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro

The 5-HTergic neurons in the mesencephalic raphe nuclei provide a robust projection to the hypothalamic suprachiasmatic nucleus (SCN), the site of a putative neuronal circadian pacemaker. Although it has been suggested that 5-HT neurons may play a role in the circadian timing system, this role has not yet been specified. Prosser et al. (Brain Res., 534 (1990) 336-339) reported that 1 h treatments with quipazine induce robust phase shifts in vitro, and that this effect depends upon the circadian time of treatment. However, quipazine is a non-specific 5-HT agonist. Besides, it is reported that the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetraline hydrobromide (8-OH-DPAT) affected a circadian rhythm of hamster wheel-running activity. In the present study we investigated whether the 5-HT1A agonist 8-OH-DPAT can reset the phase of the SCN clock when it is isolated in vitro. The present results show that 1 h treatments with 8-OH-DPAT induce robust phase advances in vitro when it was administered during the subjective day. This result suggests that 5HT1A receptor functioning may play a role in modulating the phase of SCN clock, especially during the subjective day.

Estrogen differentially regulates expression of Per1 and Per2 genes between central and peripheral clocks and between reproductive and nonreproductive tissues in female rats

Although it has long been established that estrogen alters circadian rhythms in behavior, physiology, and reproductive functions in mammals, the molecular mechanism for these effects remains unknown. To explore the possibility that estrogen affects circadian rhythms by changing the expression of clock‐related genes, we investigated the effects of chronic treatment with 17β‐estradiol (E2) on the expression of Per1 and Per2 genes in the brain (suprachiasmatic nucleus and cerebral cortex) and periphery (liver, kidney, and uterus) of ovariectomized rats by means of in situ hybridization and northern blotting. In the brain, E2 treatment advanced the peak of Per2 mRNA expression in the SCN; however, it failed to affect the rhythm of Per2 mRNA expression in the CX and Per1 mRNA expression in both the SCN and the CX. In nonreproductive peripheral tissues (liver and kidney), E2 delayed the phase and increased the amplitude of Per1 mRNA expression. In the reproductive tissues (uterus), biphasic rhythms in Per1 and Per2 mRNA were observed after E2 treatment. These findings suggest that the effects of estrogen are different between central and peripheral clock in the brain, and between reproductive and nonreproductive tissues in the periphery.

Endocannabinoid System Modulates Relapse to Methamphetamine Seeking: Possible Mediation by the Arachidonic Acid Cascade

We clarified the modulating action of the endocannabinoid system, and its possible mediation by the arachidonic acid cascade, on the reinstatement of methamphetamine (METH)-seeking behavior, using the intravenous self-administration paradigm in rats. Following 12 days of self-administration of METH, the replacement of METH with saline resulted in a gradual decrease in lever press responses (extinction). Under extinction conditions, METH-priming or re-exposure to cues previously paired with METH infusion markedly increased the responses (reinstatement of drug-seeking). The cannabinoid CB1 receptor antagonist, SR141716A, blocked this behavior. Although the cannabinoid agonist, Δ8-tetrahydrocannabinol (THC), had no effects by itself, coadministration of the agonist and METH at small doses reinstated the drug-seeking behavior. THC attenuated the effects of the reinstatement-inducing dose of METH, but enhanced the effect of cues. Either given repeatedly during the extinction or singly, 24 h before the first METH-priming or cues challenge, THC suppressed the reinstatement. In another set of experiments, we found that diclofenac, a cyclooxygenase inhibitor, also attenuated the reinstatement induced by exposure to cues or drug-priming. These results suggest that the endocannabinoid system, through possible mediation by the arachidonic acid cascade, serves as a modulator of the reinstating effects of METH-priming and cues. Extending the current view on the treatment of drug dependence, these results indicate that endocannabinoid-activating substances as well as cyclooxygenase inhibitors may be promising as antirelapse agents.

Blockade of hippocampal nicotinic receptors impairs working memory but not reference memory in rats

In a three-panel runway task, intrahippocampal injection of the nicotinic receptor antagonist, mecamylamine (10 and 18 micrograms/side), significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in a test of working memory. This increase in errors also occurred after rats were given IP mecamylamine (10 mg/kg). Mecamylamine did not affect the number of errors in a test of reference memory whether it was given at doses up to 18 micrograms/side intrahippocampally or up to 10 mg/kg IP. These results suggest that mechanisms mediated by hippocampal nicotinic receptors play a role in working memory but not in reference memory.

Proteases involved in long-term potentiation.

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.

Circadian rhythm of spontaneous neuronal activity in the suprachiasmatic nucleus of old hamster in vitro

The effects of aging on neuronal activity in the suprachiasmatic nucleus (SCN) were examined in hamsters kept under light-dark (LD) or constant light (LL) conditions. The free-running period in wheel-running rhythm of 24-month-old hamsters (24.2 +/- 0.04) was shorter than that of the 2-month-old hamsters (24.4 +/- 0.057). There was a significant difference in the mean firing rates of SCN neuron activity between old and young hamsters during subjective day (6.58 +/- 0.36 spikes/s in young and 5.63 +/- 0.24 in old hamsters), but not during subjective night (4.33 +/- 0.47 in young and 4.05 +/- 0.39 in old). Similar to LL condition, the firing activity during zeitgeber time 3-11 (4.33 +/- 0.27) in old hamsters kept under LD condition, was significantly lower than that of young hamsters (6.22 +/- 0.32). These results suggest that deterioration of SCN neuronal activity in old hamsters assessed as reduction of daytime activity may reflect changes in the interaction between SCN clocks and the overt behaviors and/or pacemaking properties of SCN cells.

Effect of substance P on circadian rhythms of firing activity and the 2-deoxyglucose uptake in the rat suprachiasmatic nucleus in vitro

The suprachiasmatic nuclei (SCN) have been identified as a pacemaker for many circadian rhythms in mammals. Although substance P (SP) fibers from retina are found to terminate the SCN, the physiological role of this peptide is uncertain. The 2-deoxyglucose (2-DG) uptake and firing activity in the SCN show a robust circadian change. SP causes an increase in 2-DG uptake by SCN during the subjective night but not during subjective day. SP-induced increase in 2-DG uptake is blocked by co-treatment with the SP receptor antagonist, spantide. Treatment with SP produces phase shifts of circadian rhythm in spontaneous neural activity in SCN neurons with a phase-response curve that is similar to the effect of light pulses to animals under constant darkness. SP-induced phase change is also blocked by pretreatment with spantide. SP-induced increase in 2-DG uptake and phase changes in firing activity occur only during subjective night, at circadian times when photic phase shifting of activity occurs. The present results suggest that SP may be an important transmitter for conveying environmental light-dark information from retina to the SCN.

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats

In order to clarify the roles of hippocampal M1 and M2 muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M1 muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 microgram/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 microgram/side) on working memory was attenuated by concurrent injection of 10 micrograms/side AF102B, the selective M1 muscarinic receptor agonist. Intrahippocampal injection of the M2 muscarinic receptor antagonist methoctramine at doses up to 1.0 microgram/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 micrograms/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 micrograms/side AF102B. Concurrent injection of 0.32 microgram/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 microgram/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 micrograms/side, respectively. These results suggest that processes mediated by M1 muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M2 muscarinic receptors ameliorates working memory deficits produced by M1 muscarinic blockade, possibly by increasing acetylcholine release.

Effects of nitric oxide synthase inhibitors on N-methyl-d-aspartate-induced phase delay of circadian rhythm of neuronal activity in the rat suprachiasmatic nucleus in vitro

Excitatory amino acid (EAA) receptors such as N-methyl-D-aspartate (NMDA) and non-NMDA receptors have been suggested to play an important role in the regulation of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated the role of glutamate as a retinohypothalamic transmitter by analyzing the phase-resetting effects of NMDA and a non-NMDA agonist, (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), on the circadian rhythm of SCN firing activity. Nitric oxide (NO) production is believed to be an essential intermediate in NMDA-induced cGMP production in the CNS. Thus, we examined the effects of blockers of NO production on NMDA- or AMPA-induced phase delay of SCN activity rhythm. N-nitro-L-arginine methylester (L-NAME) blocked NMDA- but not AMPA-induced phase shift, indicating the involvement of NO synthesis in NMDA-induced phase changes. L-arginine but not D-arginine caused a phase delay, and L-NAME blocked L-arginine-induced phase delay. In addition, cotreatment with NMDA and L-arginine did not have an additive effect. These results suggest that NO production itself is involved in the phase change of SCN neuron activity, and NMDA-induced phase changes are also mediated via activation of NO synthesis in this nucleus.