Kiyoharu Ukai

The intrinsic microglial molecular clock controls synaptic strength via the circadian expression of cathepsin S

Microglia are thought to play important roles in the maintenance of neuronal circuitry and the regulation of behavior. We found that the cortical microglia contain an intrinsic molecular clock and exhibit a circadian expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain. The genetic deletion of CatS causes mice to exhibit hyperlocomotor activity and removes diurnal variations in the synaptic activity and spine density of the cortical neurons, which are significantly higher during the dark (waking) phase than the light (sleeping) phase. Furthermore, incubation with recombinant CatS significantly reduced the synaptic activity of the cortical neurons. These results suggest that CatS secreted by microglia during the dark-phase decreases the spine density of the cortical neurons by modifying the perisynaptic environment, leading to downscaling of the synaptic strength during the subsequent light-phase. Disruption of CatS therefore induces hyperlocomotor activity due to failure to downscale the synaptic strength.

The role of circulating ghrelin in growth hormone (GH) secretion in freely moving male rats

To examine the physiological significance of plasma ghrelin in generating pulsatile growth hormone (GH) secretion in rats, plasma GH and ghrelin levels were determined in freely moving male rats. Plasma GH was pulsatilely secreted as reported previously. Plasma ghrelin levels were measured by both N-RIA recognizing the active form of ghrelin and C-RIA determining total amount of ghrelin. Mean +/- SE plasma ghrelin levels determined by N-RIA and C-RIA were 21.6 +/- 8.5 and 315.5 +/- 67.5 pM, respectively, during peak periods when plasma GH levels were greater than 100 ng / ml. During trough periods when plasma GH levels were less than 10 ng / ml, they were 16.5 +/- 4.5 and 342.1 +/- 29.8 pM, respectively. There were no significant differences in plasma ghrelin levels between two periods. Next, effect of a GH secretagogue antagonist, [D-Lys-3]-GHRP-6, on plasma GH profiles was examined. There were no significant differences in both peak GH levels and area under the curves of GH (AUCs) between [D-Lys-3]-GHRP-6-treated and control rats. These findings suggest circulating ghrelin in peripheral blood does not play a role in generating pulsatile GH secretion in freely moving male rats.

Enhancement of NMDA receptor-mediated synaptic potential evoked in rat medial-amygdala neuron following olfactory bulbectomy

Synaptic potentials evoked in the medial amygdala (m-AMG) neurons were studied in in vitro slice preparations obtained from normal and olfactory bulbectomized rats. Local stimulation induced a sequence of responses: a fast EPSP, a fast IPSP and a slow EPSP. The fast EPSP was suppressed by kynurenic acid (KYN) at a concentration of 1 mM but not by 3-[(+-)-2-carboxypiperazin-4-yl-]-propyl-1-phosphonic acid (CPP) at concentrations up to 20 microM. The slow EPSP was reversibly blocked by both KYN (1 mM) and CPP (5-10 microM). Addition of bicuculline methiodide (50 microM) to the bath suppressed the fast IPSP and augmented both the fast and slow IPSPs leading to burst discharges. In a small population of m-AMG neurons, the slow EPSP was followed by a slow IPSP. The slow IPSP was suppressed by phaclofen (500 microM) but not by bicuculline methiodide (up to 100 microM). In slice preparations obtained from olfactory bulbectomized rats, local stimulation evoked burst discharges, which were similar to those observed when bicuculline methiodide was applied to slice preparations obtained from normal rats. These results suggest that GABAA receptor mediating fast IPSP and N-methyl-D-aspartate (NMDA) receptors mediating slow EPSP regulate activities of m-AMG neurons and that the enhancement of NMDA receptor mediating slow EPSP is responsible for the hyperexcitability of m-AMG neurons following olfactory bulbectomy.

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D‐deficient mice

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D‐deficient (CD–/–) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD–/– mice at post‐natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild‐type mice, which was substantiated by the decreased level of γ‐aminobutyric acid (GABA) contents in the hippocampus measured by high‐performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD–/– mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD–/– mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67‐immunoreactive somata. In the hippocampus and the cortex of CD–/– mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD–/– mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD–/– mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild‐type and CD–/– mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD–/– mice.

Effects of dopaminergic agents on reversal of reserpine-induced impairment in conditioned avoidance response in rats

Male Slc:Wistar, Std:Wistar, and Slc:F344/N rats had good acquisition of the conditioned avoidance response (CAR), while that of the male Slc:Wistar/ST, Jcl:Wistar, and Crj:Wistar rats was bad. Reserpine-induced impairment (RII) in CAR was observed 2-72 h after administration of dopaminergic (DAergic) agents in male Slc:Wistar rats. Amitriptyline (5-80 mg/kg, P.O.), imipramine, desipramine, cis-dosulepine, and trans-dosulepine at dose of 40 mg/kg, P.O. showed no antagonism against RII in CAR 20-23 h after reserpine injection (1 mg/kg, S.C.). However, the atypical antidepressive agents sibutramine (5-10 mg/kg, P.O.), bupropion (40 mg/kg, P.O.), and nomifensine (10-40 mg/kg, P.O.) exhibited antagonism against RII in CAR. The calcium channel antagonists flunarizine, nimodipine, and KP-840 at dose of 10 and 100 mg/kg, P.O., the cerebral improving agent indeloxazine (20-80 mg/kg, P.O.), the anticholinergic agent atropine (5-40 mg/kg, P.O.), 5-hydroxy-L-tryptophan (5-HTP) (40 mg/kg, I.P.), a precursor of 5-hydroxytryptamine (5-HT), and (+/-)-threo-dihydroxyphenylserine [(+/-)-threo-DOPS] (20-200 mg/kg P.O.), a norepinephrine (NE) precursor, showed no antagonism against RII in CAR. The DAergic agents methamphetamine (5 mg/kg, P.O.) and amantadine (50-250 mg/kg, P.O.), L-DOPA (200 mg/kg, P.O.), and the DAergic D1/D2 receptor agonist apomorphine (0.1-1 mg/kg, S.C.) showed marked antagonism against RII in CAR. Although the DAergic D1-receptor agonist KF-38393 (0.3-30 mg/kg, I.P.) and the DAergic D2-receptor agonist quinpirole (0.3-10 mg/kg, I.P.) induced only a weak recovery of RII in CAR when they were administered alone, in contrast to a potent synergistic recovery of RII in CAR, which was observed when SKF-38393 (1 mg/kg, I.P.) and quinpirole (1 mg/kg, I.P.) were administered together. These results suggest that the DAergic nervous system rather than the adrenergic or 5-HT nervous system is involved in RII in CAR, and that both the DAergic D1- and D2-mediated nervous systems play important roles in this process.

ACTH releasing activity of KP-102 (GHRP-2) in rats is mediated mainly by release of CRF

KP-102 (GHRP-2: pralmorelin) is a synthetic growth hormone releasing peptide (GHRP) that powerfully stimulates the release of GH by acting (i.v.) at both hypothalamic and pituitary sites. Intravenous (i.v.) administration of KP-102 also elicits slight but significant release of adrenocorticotropic hormone (ACTH) in both animals and humans, as is seen with other GHRPs. GHRPs are thought to stimulate the hypothalamic-pituitary-adrenal axis by releasing endogenous ACTH secretagogues such as arginine vasopressin (AVP) and/or corticotropin releasing factor (CRF), though neither AVP nor CRF has been shown clearly to be involved significantly in GHRP-evoked ACTH release. In the present study, we investigated the effects of KP-102 on ACTH release in conscious rats under improved experimental conditions that minimized the influence of stress. Administration of KP-102 i.v. increased plasma ACTH significantly, but did not stimulate ACTH release from rat primary pituitary cells. Administration of KP-102 together with either AVP or CRF elicited significantly greater increases in plasma ACTH levels than any of the agonists alone. Notably, the combination of KP-102 and AVP produced a much greater increase in ACTH than KP-102 plus CRF, indicating that KP-102 augments the effect of exogenous CRF only weakly. Conversely, a CRF antagonist markedly inhibited KP-102-induced ACTH release in conscious rats, whereas an AVP antagonist or anti-AVP antiserum did not. Taken together, these findings suggest that KP-102 acts via the hypothalamus to stimulate ACTH release in rats, and that these effects are mediated mainly by the release of CRF.

Effects of the synthesized growth hormone releasing peptide, KP-102, on growth hormone release in sodium glutamate monohydrate-treated low growth rats

KP-102 (D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH2), a new second generation hexapeptide, has a potent growth hormone (GH)-releasing action in vivo and in vitro. Here, we evaluated the GH-releasing action of KP-102 under pentobarbital (PB) anesthesia in neonatally sodium-glutamate-monohydrate-treated low growth (NMSG-LG) rats. The plasma GH level in NMSG-LG rats after i.v. administration of KP-102 at 100 micrograms/kg was 1/6.7 (95% C.L. 1/14.7 - 1/3.0) of that in normal rats given the same dose (p < 0.01). However, the increase was significant compared with that in normal rats after saline administration (p < 0.01). The plasma GH releasing action of KP-102 at 100 micrograms/kg i.v. in rats with lesions in the bilateral hypothalamic arcuate nuclei (ARC), was about 1/6.3 (95% C.L. 1/12.4 - 1/3.2) of that in normal rats under PB anesthesia (p < 0.01). When KP-102 was injected into the ARC at doses of 0.0002, 0.02 and 2 micrograms/rat, GH release was dose-related (p < 0.01) under PB anesthesia. KP-102 at 2 micrograms i.c.v. also increased the plasma GH levels (p < 0.01) to about 1/8.3 (95% C.L. 1/22.7 - 1/3.1) of that by systematic administration, at the same potency as the ARC injection (1/13.7 and 95% C.L. 1/37.2 - 1/5.0). These findings suggest that KP-102 potently stimulates the GH release by a direct or indirect antagonism of somatostatin (SRIF) and growth hormone releasing hormone (GHRH) release in the hypothalamus and by a direct action on the pituitary. Furthermore, the GH-releasing action of KP-102 was similar and additive upon both regions in vivo at the maximum effective dose. Moreover, since the GH-release in response to KP-102 administration differed between NMSG-LG and normal rats, and since KP-102 increased the GH release even in NMSG-LG rats, it should be evaluated in the hypophysial GH secretion tests, and may be used to treat the hypophysial GH secretion insufficiency.

Effects of sibutramine on the central dopaminergic system in rodents.

The effects of sibutramine on central dopaminergic system in rats and mice were examined by neurochemical and behavioral pharmacological methods. Dopamine reuptake inhibition by sibutramine in brain synaptosomes was only 4–5 times stronger than those of amitriptyline and dosulepin, which do not exhibit dopamine uptake inhibition in vivo. Single treatment with sibutramine did not alter the brain content of dopamine and DOPAC. However, similar to methamphetamine and pargyline, sibutramine antagonized methyl-4-phenyl-l, 2,3,6-tetrahydro-pyridine (MPTP) induced dopamine depletion in mouse brain. In forced swimming tests of reserpinized mice, sibutramine shortened the immobilized time, similar to dopaminergic drugs including nomifensine, bupropion (dopamine-reuptake inhibitor), methamphetamine, SKF 38393 (dopamine D1 agonist), quinpirole (dopamine D2 agonist) and apomorphine (dopamine D1/D2 agonist). In addition, sibutramine caused rotational behavior toward the lesioned side in rats with unilateral lesions of the substantia nigra induced by 6-hydroxydopamine. These results suggest that sibutramine exhibits neurochemical and behavioral dopaminomimetic activity in vivo, which is mediated by dopamine reuptake inhibition by the active metabolites of sibutramine.

Novel Series of Highly Potent Non-peptide Growth Hormone Secretagogues with Improved Bioavailability

The discovery and the SAR of acylproline derivatives as highly potent growth hormone secretagogues (GHSs) with good oral bioavailability are described. One representative compound, N-[3-(2,2-dimethylpropylamino)-2-hydroxypropyl]-2(R)-[l-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2-ylpropionamide (4e), showed potent GHS activity (ED 50 =1 nM) and good oral bioavailability (BA=33.2%). Moreover, the optically pure N-[3-(2,2-dimethylpropylamino)-2(S)-hydroxypropyl]-2(R)-[1-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2-ylpropionamide ((2S)-4e) showed a good metabolic stability against in vitro clearance (human liver microsome) with potent GHS activity.