Immunocytochemical techniques locating neurotransmitter-synthsizing enzymes are currently being employed. to determine the nature of transmitters associated with individual neurons. The use of peroxidase-anti-peroxidase Fab (PAP Fab) complex modified from Sternberger's PAP method, among several other immunocyto-chemical methods is recommended for the visualization of antigens in cerebral tissues. The enzyme fixed. in nervous tissues is reacted with anti-enzyme produced in rabbits followed by incubation with goat-anti-rabbit serum. Subsequent application of PAP Fab complex prepared separately results in a formation of a complex composed of enzyme : anti-enzyme : goat-anti-rabbits : PAP-Fab. The enzymes can be visualized under light and electron microscope by the deposition produced by the action of peroxidase on 3, 3'-diaminobenzidine. Thus, the antibody to glutamate decarboxylase (GAD), the enzyme that synthesizes γ-aminobutyric acid (GABA) was employed to identify GABAergic neurons in central nervous system of rodents. Specific staining for GAD was highly localized in close association with synaptic vesicles in certain axon terminals including basket, Golgi and the Purkinje cell terminals in the cerebellum. The distribution of GAD observed in immunocytochemical preparations was consistent with indirect biochemical, physiological and morphological data dealing with the synaptic role of GABA neurons in the cerebellum. The correlation of the immunocytochemical distributionof GABA neurons in the spinal cord, substantia nigra, olfactory bulb, retina and Ammon's horn with physiological and biochemical results can also been obtained. The method has been successfully employed to visualizc dopamine-β-hydroxylase (DBH) and substance P. DBH, as an indicative enzyme for noradrenergic (NA) neurons, was highly localized in the neuronal soma of the locus coeruleus and in synaptic varicosities in the stria terminalis associated with synaptic vesicles. Association of substance P in probable primary afferent terminals with large vesicles also supports the synaptic function of the compound in the spinal cord.

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Effects of (±) -1- (3, 4, 5-trimethoxybenzyl) -1, 2, 3, 4-tetrahydroisoquinoline (TMI) derivatives with hydroxy groups at the A ring were investigated on the common carotid blood flow and cyclic 3', 5'-adenosine monophosphate phosphodiesterase (PDE) activity of the isolated carotid artery in dogs. Results were compared to those of isoproterenol and papaverine. When administered into the common carotid artery, isoproterenol produced the most potent vasodilating activity and the potency was followed by those of 6, 7-dihydroxyl, 5, 7-dihydroxyl, 5, 6, 7-trihydroxyl, 6, 7, 8-trihydroxyl, 7-hydroxyl, 5-hydroxyl, 6-hydroxyl, 5, 6-dihydroxyl, TMI, 8-hydroxyl, papaverine, 6, 8-dihydroxyl, 7, 8-dihydroxyl, 5, 8-dihydroxyl analogues in decreasing order. Vasodilating actions of 6, 7-dihydroxyl, 5, 7-dihydroxyl, 5, 6, 7-trihydroxyl, 6, 7, 8-trihydroxyl and 7-hydroxyl analogues were almost abolished by propranolol, while those of 8-hydroxyl, 6, 8-dihydroxyl and 7, 8-dihydroxyl analogues were little affected by propranolol. In the TMI derivatives, the inhibition caused by propranolol became more pronounced when the vasodilating activity was more potent. Inhibitory effects of the TMI derivatives on the PDE activity in the supernatant of homogenate of the common carotid artery were weaker than those of papaverine, but were stronger than those of isoproterenol. The TMI derivatives that exhibited potent inhibitory effects of PDE activity had a tendency to show weak vasodilating action. These results suggest that the vasodilating action of the TMI derivatives cannot be ascribed to their inhibitory action of PDE activity but rather is mainly due to their β-syrnpathomimetic action and that the hydroxy group at position 7 is important for manifestation of β-sympathomimetic action, which is attenuated when the hydroxy group is presented at position 8.

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The humoral immuno-response : A study was carried out to determine the effect ot α-MPG and DPA on humoral antibody formation. The following results were obtained : The 1st and 2nd immuno-responses in rabbits to bacterial α-amylase were potentiated by treatment with both α-MPG and DPA given i. p. for 10 days after the 1st immunization, but the treatment after the 2nd immunization did not affect the 2nd immunoresponse. Formation of hemolytic plaque forming cells (HPFC) in the spleen of mice immunized with 5×10⁸ sheep red blood cells (SRBC) was potentiated by the administration of the both drugs given i. p. for 5 days before or after the immunization. No appreciable formation of HPFC was observed by immunization with 5×10⁶ SRBC, and here α-MPG or DPA were without effects. Immunization with more than 5×107 SRBC resulted in an increase of HPFC in a dose-dependent fashion. Such an increase was further reinforced by treatment with both drugs. HPFC increased with a single dose of α-MPG at 48 hr before or 24-72 hr after the immunization and also increased with that of DPA at 24 hr before or immediately after the SRBC treatment.

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Effects of procaine and xylocaine on the lipolytic actions caused by catecholamines and methylxanthine derivatives in white adipose tissues from rats were investigated. Both procaine and xylocaine remarkably inhibited the lipolyses caused by norepinephrine, epinephrine, caffeine and theophylline. Xylocaine inhibited the lipolysis more strongly than procaine, and also inhibited the basal lipolysis. The inhibition by either procaine or xylocaine appeared 60 minutes after the addition of the norepinephrine-induced lipolytic action. The antilipolytic action of procaine was evident in medium containing 2 mM EDTA instead of Ca²⁺, and its antilipolytic action was accelerated by increasing Ca2+ concentration in the medium. From these positive results, we suggest that both procaine and xylocaine have an antilipolytic effect, and this effect is closely dependent on the Ca2+ concentration in the medium.

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N-5' shows a potent inhibitory action on the homologous passive cutaneous anaphylaxis (PCA) in rats mainly through the inhibition of histamine release from mast cells. The present experiment was an attempt to clarify in detail the pharmacological properties of N-5'. Inhibition of PCA was most potent at 30 or 60 min pre-treatment with N-5', and negligible at 240 min pretreatment. Given p.o., N-5' produced a dose-dependent, potent inhibitory action at 30-min pretreatment. On the other hand, disodium cromoglycate (DSCG) had little effect on PCA when given orally. On the case of i.v. administration, N-5' (20 mg/kg) and DSCG (5 mg/kg) showed a most potent inhibition of PCA at 5 min pretreatment. The inhibitory action of DSCG was, however, shorter lasting than that of N-5'. Median effective doses (ED50) of DSCG and N-5' on the PCA were estimated to be 0.79 and 8.8 mg/kg i.v., respectively. Inhibitory activity of N-5' in the adrenalectomized rat did not differ from that in sham operated animals. N-5' had a more potent inhibitory action on the PCA in infant rats than in adults. Inhibitory activity of N-5' in the case of 1, 2, 3 and 4 weeks of successive administration was equipotent to that with a single administration.

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The present experiment was an attempt to determine the anti-inflammatory activity of N-5'. The authors have already reported that when given orally, N-5' shows a dose-dependent, significant inhibition on the homologous passive cutaneous anaphylaxis (PCA) in rats mainly through an inhibition of histamine release from mast cells, nevertheless, the drug at a dose sufficient to inhibit the homologous PCA, shows only slight inhibition on the paw edema induced by carrageenin. In the present experiment, the inhibitory effect of N-5' on rat paw edema induced by carrageenin, dextran or egg white was only slight at a dose level of 200 mg/ kg or less which is sufficient to inhibit homologous PCA, but significant at 400 mg/kg of the drug. The potency of 400 mg/kg of N-5' was nearly equal to that of 200 mg/kg of phenylbutazone. N-5' at a dose of only 400 mg/kg showed a significant inhibition on the increased vascular permeability induced by histamine, hyaluronidase or acetic acid. The inhibitory effect was more potent than that of 200 mg/kg of phenylbutazone. The inhibitory effect of 400 mg/kg N-5' on the granuloma tissue formation was equipotent to that of 100 mg/kg of phenylbutazone. It was speculated from our findings that the moderate anti-inflammatory activity of N-5' should contribute to clinical application of the drug for allergic diseases which involve complications of inflammation.

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The allergic histamine release mediated by homocytotropic antibody (HTA) was significantly inhibited by 100 and 1, 000 μM N-5', but was little affected by the drug at concentrations of 1 and 10, μM. Disodium cromoglycate (DSCG) showed a potent inhibition of the histamine release at concentrations of 10 and 100 μM, in a concentration dependent manner. Although a significant inhibition of histamine release was elicited by 1, 000 μM DSCG, the effect was less potent than that by 100 μM of the drug. One μM DSCG had no effect. Allergic histamine release as well as a spontaneous one without antigen was significantly potentiated by addition of 1 μM phosphatidylserine. The inhibitory activity of N-5' was unchanged by phosphatidylserine, while the activity of DSCG was significantly reduced in the presence of phosphatidylserine. The inhibitory activity of N-5' on the allergic histamine release was unchanged by glucose, while the activity of papaverine was significantly enhanced in the absence of glucose. N-5' showed little effect on the histamine release induced by compound 48/80, while DSCG showed a concentration dependent inhibition of the release. Phosphatidylserine or dextran alon induced only slight release of histamine from peritoneal exudate cells. However, the release was significantly potentiated by the combined treatment with phosphatidylserine and dextran. N-5' (10 to 100 μM) showed a significant inhibition of the release, in a concentration dependent manner. Although the inhibitory action of DSCG was significant even at a concentration of 1 μM, the action did not increasewith an increase in concentration of the drug. N-5' was more potent than DSCG. Approximately 25% histamine release was induced by phospholipase-A (10, μg/ml). N-5' at concentrations of 100 and 1, 000 μMshowed a significant inhibition (42.0 and 62.6%, respectively) of the release. In the case of DSCG, the inhibition percent was calculated to be 30.5 and 37.9% at the same levels of concentration as N-5'. DSCG had no effect on the phospholipase-A activity at any concentration tested herein, while N-5' at a concentration of 1, 000 μM showed a significant inhibition (23%).

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The evoked potential in the lateral hypothalamus (LH) recorded by stimulation of basal medial amygdaloid nucleus (Abm) showed a triphasic pattern and diazepam (2 mg/kg, i. p.) decreased the late component. The evoked potential in the midbrain reticular formation (MRF) recorded by stimulation of Abm showed a fast component with a relatively short latency followed by a biphasic late component and diazepam decreased the late component. Though the evoked potential in the Abm recorded by stimulation of LH showed a triphasic pattern, diazepam had no influence on the amplitude. Diazepam increased markedly the amplitude of evoked potential in the MRF recorded by stimulation of LH. Diazepam was ineffective on the evoked potential in the Abm recorded by stimulation of MRF. Diazepam decreased markedly the late component of evoked potential in the LH recorded by stimulation of MRF. These results suggest that the depression of emotional behavior by diazepam may be particularly related to the fact that the evoked potential in the LH recorded by stimulation of Abm was decreased by diazepam.

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Effects of the typical psychotropic drugs such as neuroleptics, tricyclic antidepressants and benzodiazepines on the monosynaptic reflex (MSR) and polysynaptic reflex (PSR) were investigated using intact and spinal cats. Drugs used were chlorpromazine, levomepromazine, perphenazine, prochlorperazine, droperidol, haloperidol, amitriptyline, imipramine, diazepam and flurazepam. Neuroleptics depressed the ventral root reflexes markedly to slightly in both preparations. The inhibitory effects of these drugs on MSR of intact cats were stronger than those on MSR of spinal cats, and the activity was slightly higher than that of PSR depression in intact preparations. In intact cats, those depressant effects of neuroleptics on PSR produced various characteristic action patterns in comparison with MSR inhibition. Tricyclic antidepressants were similar in the mode of action to the dimethylamino side-chain groups in phenothiazines, but the inhibitory effects of ventral root reflexes were distinctly weaker than that of neuroleptics. Benzodiazepines depressed only PSR without MSR inhibition, especially in spinal cats. Thus, it is suggested that the structure-activity relationship considered, a classification of the psychotropic drugs can be given according to the inhibitory activities on spinal ventral root roflexes in intact and spinal cats.

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The inhibitory effects of γ-oryzanol and atropine on the gastric secretion were studied using insulin and 2-deoxy-D-glucose as vagal stimulants. Pretreatment with γ-oryzanol (100 mg/kg, s.c., once daily×5) dep-ressed the gastric secretion stimulated by insulin or 2-deoxy-D-glucose, but the potency was less than that with atropine (10 mg/kg, s.c.). γ-Oryzanol had no effect on decrease in the serum glucose level or on increase in the gastrin level induced by insulin injection, while atropine enhanced these responses. From these results, it is considered that the inhibitory action of γ-oryzanol on gastric secretion may be due to depression of the vagas system but the mode of action is different from that of atropine.

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The action of 5-hydroxytryptamine (5-HT) in the rat ileum was analysed pharmacologically. In the isolated rat ileum, either mono or biphasic contraction was induced depending on the concentrations of 5-HT. The former was induced by lower concentrations of 5-HT (<6.25 × 10^-7M) and the latter by higher concentrations of 5-HT (>2.5 × 10^-6M). The monophasic contraction and the tonic contraction in response to 5-HT were blocked by methysergide (MTG). The phasic contraction in response to 5-HT after inhibition of muscular receptor with MTG was potentiated by physostigmine and blocked by mecamylamine (MC) or tetrodotoxin (TTX). The response to 5-HT changed from a contraction to a relaxation by preincubation with MTG plus hyoscine. The relaxation was inhibited by TTX but not by MC and such was provoked even in the ileum from rats treated with 6-hydroxydopamine. Therefore, the inhibitory neuronal receptor for 5-HT does not appear to belong to the adrenergic nervous system. At lower concentrations of 5-HT, relaxation was induced with MTG which was only partially inhibited by TTX. These results indicate that the stimulatory responses to 5-HT were caused by activations of muscular receptors and of intramural parasympathetic ganglion cells while inhibitory responses were caused by activations of non-adrenergic inhibitory nerve terminals and of non-neuronal elements.

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