Eiko Kishi

The Anxiolytic Effect of Two Oriental Herbal Drugs in Japan Attributed to Honokiol from Magnolia Bark

An improved elevated plus‐maze test in mice revealed that seven daily treatments with two differnt traditional Chinese medicines, known as Kampo medicines in Japan, Hange‐koboku‐to (composed of extracts of 5 plants) and Saiboku‐to (composed of extracts of 10 plants), produced an anxiolytic effect, and the effect was mainly due to the presence of honokiol derived from magnolia. This study was carried out to evaluate the anxiolytic potential of honokiol, Hange‐koboku‐to and Saiboku‐to, which were prescribed with two different magnolia samples: Kara‐koboku (Magnoliae officinalis) (KA) or Wa‐koboku (Magnoliae obovata) (WA).

Effects of hepatic nerve stimulation on blood glucose and glycogenolysis in rat liver: studies with in vivo microdialysis.

In vivo microdialysis was applied to investigate the effects of hepatic nerve stimulation on glycogenolysis in rat liver under anesthesia. We analyzed the norepinephrine (NE) outflow and glucose output from the liver through the measurement of NE and glucose in the microdialysis dialyzate, as well as the plasma glucose level. Stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms, 20 s every minute) increased NE outflow and glucose output from the liver. The blood glucose level increased by 1.5-1.6 times over the basal level at the end of the 10 min intermittent stimulation. Bilateral adrenalectomy and pancreatectomy did not abolish the glycogenolysis that was induced by the nerve stimulation. Phentolamine an alpha-antagonist, reduced the effects of nerve stimulation on the glucose output and the plasma glucose level. Phentolamine caused an increase in the NE outflow. Quinacline, an inhibitor of phospholipase A2, inhibited the glycogenolytic nerve effects without any inhibition of the NE outflow. These data show that hepatic nerve stimulation produces glycogenolysis via alpha-adrenergic mechanism and partly mediated by eicosanoids, and that microdialysis is a useful and simple method for the study of liver metabolism in physiological conditions.

Application of the elevated plus-maze test in mice for evaluation of the content of Honokiol in water extracts of magnolia

In our previous study using an improved elevated plus‐maze in mice, the oriental herbal medicine Saiboku‐to prolonged the time spent in open arms, showing an anxiolytic effect, and the effect was mainly caused by honokiol derived from magnolia. This study was carried out to compare the anxiolytic potentials of honokiol and water extracts of three magnolia samples; two being Kara‐koboku (Magnolia officinalis) (KA: from Zhejiang‐sheng, China; honokiol 0.25% and magnolol 1.16%, and KB: from Sichuan‐sheng, China; honokiol 1.72% and magnolol 1.71%), and one being Wa‐koboku (Magnolia obovata) (WA: from Iwate‐ken, Japan; honokiol 0.32% and magnolol 0.81%). Seven daily treatments with 0.1–1 mg/kg honokiol, but not 0.2 and 1 mg/kg magnolol, revealed an anxiolytic effect with the peak potential at 0.2 mg/kg. The anxiolytic potentials of 40 and 80 mg/kg KA, which contained the highest amount of magnolol, were almost equivalent to those of 0.1 and 0.2 mg/kg honokiol, respectively. KB, at 11.6 mg/kg, and 62.5 mg/kg WA resulted in almost the same anxiolytic potential as that of 0.2 mg/kg honokiol. No significant change in the ambulatory activity was produced by any drug treatment. These results suggest that honokiol is the chemical responsible for the anxiolytic effect of the water extract of magnolia and that the other chemicals including magnolol in magnolia scarcely influence the effect of honokiol. It is also considered that the elevated plus‐maze test is applicable for evaluation of the content of honokiol in magnolia. Copyright

Role of preoptic and anterior hypothalamic cholinergic input on water intake and body temperature

To elucidate the role played by cholinergic mechanism in the preoptic area (POA) and anterior hypothalamus (AH) in the control of body temperature and water intake of rats, we used microdialysis without disturbing the behavior of unanesthetized animals. After microdialysis, we also investigated immunoreactivity for c-Fos protein in the hypothalamus. Stimulation with neostigmine, an acetylcholine esterase inhibitor, through microdialysis probe increased extracellular concentration of acetylcholine (ACh) in the POA and AH, and was accompanied by a dose-dependent fall in body temperature and increased water intake. Addition of atropine, a muscarinic receptor antagonist, to the dialysis medium containing neostigmine suppressed the neostigmine-induced changes in rectal temperature and water intake. Neostignime markedly increased c-Fos-like immunoreactivity (Fos-IR) in certain hypothalamic areas, including the paraventricular nucleus, supraoptic nucleus and median preoptic nucleus. This increase was also attenuated by atropine. These results suggest that cholinergic inputs and activation of muscarinic processes in POA and AH induced a decline in body temperature and increased water intake.

Effects of ventromedial hypothalamus stimulation on glycogenolysis in rat liver using in vivo microdialysis

In vivo microdialysis was applied to study the effects of ventromedial hypothalamus (VMH) stimulation on liver glycogenolysis under anesthesia. We examined glucose output and norepinephrine (NE) outflow from the liver through analysis of glucose and NE in the liver dialyzate. Stimulation of the VMH increased glucose output and NE outflow from the liver and increased the plasma glucose level. Similar results were obtained on hepatic nerve stimulation. Bilateral adrenalectomy did not abolish the glycogenolysis induced by VMH stimulation. NE outflow increased to a much greater extent in adrenalectomized rats. These data show that VMH stimulation causes glycogenolysis and glucose output from the liver mainly via the hepatic nerves, and that microdialysis is a simple and useful method for the study of liver metabolism in vivo.

Hypothalamic cholinergic regulation of body temperature and water intake in rats

Without disturbing the behavior of unanesthetized rats, the perfusion of neostigmine through microdialysis probe into the anterior hypothalamus (AH), paraventricular nucleus (PVN) and lateral ventricle (LV) decreased body temperature and increased water intake. On the other hand, the perfusion into the supraoptic nucleus (SON) increased the body temperature. The perfusion of neostigmine increased the extracellular concentration of acetylcholine in the perfusion sites except LV. Changes, both decrease and increase, in body temperature and increase in water intake were correlated with increases in c-fos-like immunoreactivity (Fos-IR) in the hypothalamus, pons and medulla. Distinct Fos-IR was found in the PVN, SON, median preoptic nucleus (MnPO), locus coeruleus (LC), area postrema and nucleus of the solitary tract (NTS). Co-administration of atropine with neostigmine completely suppressed the changes in the body temperature, water intake and Fos-IR, all of which were induced by the neostigmine perfusion into AH, PVN and SON. In the LV-perfused rats, on the other hand, co-administration of atropine and neostigmine only partially prevented body temperature reduction and still induced significant hypothermia. These results suggest that muscarinic receptor activation in specific regions of the hypothalamus and the activation of LC and NTS are implicated in the regulation of body temperature and water intake. Other receptor processes are involved in the LV-induced changes.

Hypothalamic cholinergic and noradrenergic neurons in hyperglycemia induced by 2-deoxyglucose

To investigate the contribution of the hypothalamic cholinergic and noradrenergic neurons in 2-deoxyglucose (2-DG) induced hyperglycemia, we after microwave irradiation analyzed the contents of the neurotransmitters and the metabolites in the microdissected hypothalamic nucleus, ventromedial hypothalamic nucleus (VMH), paraventricular nucleus (PVN) and lateral hypothalamus (LH). A dose dependent decrease in the acetylcholine (ACh) content and a corresponding increase in the choline content was observed in those hypothalamic nuclei 20 min after intravenous administration of 250 or 500 mg/kg 2-DG. The norepinephrine content decreased in 500 mg/kg 2-DG group but did not change significantly in the 250 mg/kg group. These results suggest the involvement and importance of the hypothalamic cholinergic system in 2-DG induced hyperglycemia and, furthermore, that the hyperglycemic response can not be solely attributed to the noradrenergic system.

Hypothalamic cholinergic activity associated with 2-deoxyglucose-induced hyperglycemia

In order to clarify the role of the hypothalamic cholinergic system in the regulation of peripheral glucose metabolism, we investigated hypothalamic cholinergic activity after administration of 2-deoxyglucose (2-DG). Intravenous administration of 2-DG (500 mg/kg) caused marked hyperglycemia; the level of plasma glucose increased by 2.1 times over the initial levels at 20 min. For evaluation of the cholinergic activity, we analyzed extracellular levels of acetylcholine (ACh) and choline using brain microdialysis, as well as measuring their tissue levels in the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamus (LH) of rats killed by microwave. In the microdialysis perfusate, extracellular levels of ACh and the metabolite choline were increased by 2-DG administration. In the tissue, a dose dependent decrease in the ACh content and a corresponding increase in the choline content were observed in both hypothalamic nuclei 20 min after administration of 2-DG. These data show that cholinergic activity is increased after 2-DG administration. Both the plasma glucose increment and the fluctuation of ACh and choline content were reduced in pentobarbital anesthetized rats. In 6-hydroxydopamine (6-OHDA) pretreated rats, the hypothalamic content of norepinephrine (NE) was reduced to one-third of that in controls, but there was no significant effect on the hyperglycemia or increase in hypothalamic tissue choline levels following 2-DG. Our results suggest the involvement of the hypothalamic cholinergic system in 2-DC-induced hyperglycemia.

Opposite regulation of body temperature by cholinergic input to the paraventricular nucleus and supraoptic nucleus in rats

Hypothalamic cholinergic system plays an important role in the regulation of body temperature and fluid balance. We have previously shown that cholinergic stimulation of the anterior hypothalamus and preoptic area was accompanied by a fall in body temperature, increased water intake, and increased Fos protein in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In the present study, to estimate the role played by cholinergic input to the PVN and SON in thermoregulation and water intake, we used microdialysis for cholinergic stimulation with neostigmine and analysis of the nucleus, and also investigated immunoreactivity for c-Fos protein in the brain. This stimulation increased extracellular concentration of acetylcholine in these nuclei. Stimulation of the PVN decreased body temperature and increased water intake. On the other hand, stimulation of the SON increased body temperature. Both in PVN-stimulated and SON-stimulated rats, c-Fos-like immunoreactivity (Fos-IR) was evident in the PVN, SON and certain regions including locus coeruleus (LC), area postrema and nucleus of the solitary tract (NTS). Addition of atropine to the dialysis medium attenuated the increase of Fos-IR and suppressed the cholinergic stimulation-induced responses in body temperature and water intake. These results suggest that cholinergic muscarinic mechanisms in PVN and SON play an opposite function in the regulation of body temperature. The same neuronal pathway including LC and NTS may participate in an advance both in hypothermia and in hyperthermia.

Stimulation of rat hypothalamus by microdialysis with K+: increase of ACh release elevates plasma glucose

The effects of stimulation of the ventromedial hypothalamus (VMH) or lateral hypothalamus (LH) with potassium chloride through a microdialysis probe were studied. The concentrations of ACh and norepinephrine (NE) in the dialysate obtained from the hypothalamic nuclei and plasma glucose concentration were measured. Stimulation of the hypothalamic nuclei, VMH and LH, with potassium increased the plasma glucose level as well as the extracellular concentrations of ACh and choline. Addition of atropine, a muscarinic ACh receptor antagonist, into the potassium solution reduced the increase in the level of plasma glucose. Cholinergic stimulation of these nuclei with neostigmine increased the extracellular concentrations of ACh and plasma glucose. Stimulation of the nuclei with potassium also increased the release of NE. However, stimulation of the VMH or LH with NE and/or pargyline, a monoamine oxidase inhibitor, through the dialysis probe membrane did not significantly increase the plasma glucose concentration. These results suggest that activation of the muscarinic cholinergic or ACh-receptive neurons in the hypothalamic nuclei, VMH and LH, contribute to the elevation of plasma glucose level.The effects of stimulation of the ventromedial hypothalamus (VMH) or lateral hypothalamus (LH) with potassium chloride through a microdialysis probe were studied. The concentrations of ACh and norepinephrine (NE) in the dialysate obtained from the hypothalamic nuclei and plasma glucose concentration were measured. Stimulation of the hypothalamic nuclei, VMH and LH, with potassium increased the plasma glucose level as well as the extracellular concentrations of ACh and choline. Addition of atropine, a muscarinic ACh receptor antagonist, into the potassium solution reduced the increase in the level of plasma glucose. Cholinergic stimulation of these nuclei with neostigmine increased the extracellular concentrations of ACh and plasma glucose. Stimulation of the nuclei with potassium also increased the release of NE. However, stimulation of the VMH or LH with NE and/or pargyline, a monoamine oxidase inhibitor, through the dialysis probe membrane did not significantly increase the plasma glucose concentration. These results suggest that activation of the muscarinic cholinergic or ACh-receptive neurons in the hypothalamic nuclei, VMH and LH, contribute to the elevation of plasma glucose level.