Radioimmunoassay for an analog of thyrotrophin-releasing hormone, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide (DN-1417).

A heterologous radioimmunoassay method was established to determine plasma levels of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide (DN-1417). As this compound is unstable in the incubation buffer, we introduced a conversion step. DN-1417 in the plasma was extracted with a solution of isopropanol-isobutylamine (4 : 1) and incubation was performed at room temperature for 2 h for the conversion of DN-1417 into N-[2-hydroxy-4-(isobutylcarbamoyl)butyryl]-L-histidyl-L-prolinamide (DN-isobutylamide). 125I-labeled 2-hydroxy-4-carboxybutyryl-L-histidyl-L-prolinamide and antisera, which was raised in the rabbit using an esterified derivative of DN-1417 conjugated with BSA as an antigen, were used for a sensitive radioimmunoassay of DN-isobutylamide. In this system, 0.2 ng DN-isobutylamide/ml plasma, equivalent to 0.16 ng DN-1417/ml, was detected and there was no apparent interference from its metabolites. The within-assay coefficients of variation were 7.6% at 7.73 ng/tube and 13.7% at 1.32 ng/tube. The between-assay coefficients of variation were 16.1% at 6.43 ng/tube and 12.2% at 1.20 ng/tube. The mean recovery rate of the assay system was 76.0 +/- 3.2% (S.E.M.).

A TRH analog (DN-1417): motor stimulation with rearing related to catecholaminergic mechanisms in rats.

The effect of an analog of TRH, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417) on motor activity was studied in rats. Peripheral administration of DN-1417 (0.2-20 mg/kg, i.p.) caused a significant, dose-dependent increase in total spontaneous motor activity, with a definite increase in rearing behaviour. Both increases in spontaneous motor activity and rearing behaviour were markedly inhibited by pretreatment with chlorpromazine (1, 5 mg/kg, i.p.), haloperidol (0.1, 0.5 mg/kg, i.p.), pimozide (1 mg/kg, i.p.) or alpha-methyltyrosine (250 mg/kg, i.p.). Only stimulation of rearing behaviour was selectively attenuated by phenoxybenzamine (5 mg/kg, i.p.) or FLA-63 (25 mg/kg, i.p.) at doses producing no significant effect on spontaneous motor activity. Although propranolol (10 mg/kg, i.p.) and methysergide (10 mg/kg, i.p.) had no effect, atropine (10 mg/kg, i.p.) and mecamylamine (10 mg/kg, i.p.) respectively potentiated and counteracted the effects of DN-1417. Concerning the stimulation of spontaneous motor activity, the nucleus accumbens and lateral hypothalamic area were most sensitive to DN-1417, and the lateral hypothalamic area was the most sensitive site for the stimulation of rearing. Furthermore, DN-1417 (5 X 10(-5) M) significantly enhanced the spontaneous release of [3H]dopamine from the rat nucleus accumbens slices in vitro. These findings indicate that the motor stimulatory action of DN-1417 appears to be mediated primarily via a dopaminergic mechanism by enhancing the release of dopamine from nerve terminals, including the nucleus accumbens in the mesolimbic dopamine system, and, in turn, the rearing may be mediated via noradrenergic mechanism.

Lesioning of the rat basal forebrain leads to memory impairments in passive and active avoidance tasks.

Effects of the bilateral electrolytic lesioning of the basal forebrain (BF), including the ventral globus pallidus, on passive or active avoidance tasks, were studied in male Wistar rats. A severe deficit in acquisition of passive avoidance response was produced by the lesioning in the posterior level of BF. The retention of the passive avoidance response was markedly disrupted with post-training lesioning. Time-dependent but only slight recovery from the memory impairments was observed in the passive avoidance task given 4, 8 or 16 weeks after BF lesions. The acquisition of active avoidance response using a two-way shuttle box was also disturbed by BF lesioning. Retention of active avoidance response was clearly impaired by post-training lesions of the BF. The BF lesioned rats gradually acquired the passive avoidance performance when trained repeatedly at 24- or 48-h intervals, by giving a foot shock in case of avoidance failure. Extinction of the acquired passive avoidance response rapidly occurred in the BF lesioned rats. Furthermore, neurotoxic lesions of BF with kainic acid produced a significant impairment in acquisition of passive avoidance response. These results suggest that bilateral BF lesions impair the acquisition and retention of passive or active avoidance response, and that these impaired rats may be useful as an experimental model for Alzheimer's disease and senile dementia.

Mesolimbic involvement in the locomotor stimulant action of thyrotropin-releasing hormone (TRH) in rats.

Thyrotropin-releasing hormone (TRH) injected i.p. in doses of 5 mg/kg and higher had a strong locomotor stimulant action with development of frequent rearing, mild sniffing, grooming, preening and other excitatory behaviours. The locomotor stimulation was also produced by bilateral injection of TRH and dopamine (DA) into the nucleus accumbens but not by bilateral injection of these substances into the caudate nucleus. Unilateral intracaudate injection of TRH provoked no behavioural changes in contrast to a distinct circling response to similarly injected DA. Either i.p. or bilateral intra-accumbens injection of haloperidol or pimozide on low doses effectively blocked the locomotor stimulant action of TRH. These results indicate that the DA system in the nucleus accumbens may be of importance in mediation of the locomotor stimulant action of TRH. Differential affinity of TRH to the two DA systems, the mesolimbic and nigrostriatal DA systems is also suggested.

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in oxygen consumption and cyclic AMP formation of rat cerebral cortex slices.

Effects of TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L- prolinamide ) and pentobarbital, alone or in combination, on oxygen consumption and cyclic AMP formation in rat cerebral cortex slices were investigated. The oxygen consumption of rat cerebral cortex slices as measured with a Warburg apparatus, increased linearly over time (0 to 60-min incubation at 37C). Addition of pentobarbital (1 to 7 x 10-4M) inhibited oxygen consumption, in a concentration-dependent manner, up to 45% of control. A concomitant application of DN-1417 (10-5M) or TRH (10-4M) and pentobarbital (5 x 10-4M) led to a partial recovery of the pentobarbital effect. The similar anti-pentobarbital effects were observed with the addition of carbachol (10-4M) or dibutyryl cyclic AMP (10-3M), but not norepinephrine (10-4M) or dopamine (10-4M). DN-1417, TRH, carbachol, norepinephrine or dopamine at 10-4M stimulated cyclic AMP formation in the cerebral cortex slices. Addition of pentobarbital (1 to 7 x 10-4M) inhibited the cyclic AMP formation, in a concentration-dependent manner. DN-1417, TRH or carbachol at 10-4M but not norepinephrine or dopamine at 10-4M significantly reversed the reduction of cyclic AMP formation induced by pentobarbital (5 x 10-4M). Atropine (10-4M) almost completely abolished DN-1417-, TRH- and carbachol-induced cyclic AMP formation in the presence and absence of pentobarbital.

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices.

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.

Separation of aromatic isomers on cyclophane-bonded stationary phases.

A cyclophane (CP66)-bonded silica gel stationary phase (CP66-SP) was prepared and the retention of water-insoluble hydrophobic compounds on it was investigated in comparison with that on the CP44-bonded stationary phase (CP44-SP) reported previously. Like CP44-SP, it retained aromatic compounds more strongly than the corresponding alicyclic compounds, as was expected by the cavity size of the cyclophane. The CP66-SP also showed isomer-selectivity for monosubstituted and disubstituted naphthalenes, but its selectivity was perfectly reversed to that of the CP44-SP. On the CP66-SP, isomers having methyl and ethyl groups at beta-position were eluted prior to those having groups at alpha-position, whereas on the CP44-SP beta-substituted naphthalenes were retained more strongly than alpha-substituted ones. Isomers of three- and four-ring aromatic compounds were also separated on these cyclophane-bonded stationary phases. The retention order on the CP66-SP was almost opposite to that on the CP44-SP; on the CP66-SP, the retention order was phenanthrene > anthracene, and chrysene > 1,2-benzanthracene > 2,3-benzanthracene, whereas on the CP44-SP, anthracene > phenanthrene, and 2,3-benzanthracene > chrysene > 1,2-benzanthracene. The retention mechanism of aromatic compounds is discussed on the basis of the structure of the cyclophane-involved complex.

Age-related changes in learning and memory in the senescence-accelerated mouse (SAM).

Age-related changes in learning ability were studied in senescence-accelerated mice (SAM) reared under specific pathogen-free (SPF) conditions. SAM-P/8/Ta (SAM-P/8, senescence-prone substrain) showed an age-associated increase in spontaneous motor activity (SMA) compared with SAM-R/1/Ta (SAM-R/1, senescence-resistant substrain) in a novel environment when the activity was measured in the light period, although there was no significant difference in the dark period. In observations of the circadian rhythm of SMA, SAM-P/8 showed a significant increase in diurnal SMA. In SAM-P/8 mice, the acquisition of passive avoidance response was slightly but significantly impaired even at 2 months of age, compared with SAM-R/1 control; the impairment became obvious with aging. In a one-way active avoidance task, SAM-P/8 did not show any impairment in the acquisition of avoidance response at 2 and 4 months of age. However, significant impairment was observed in SAM-P/8 at 12 months of age. The impairments of avoidance tasks were not due to a decrease in shock sensitivity, as indicated by no significant change in the flinch-jump threshold. In a water-filled multiple T-maze task, there was no difference in the number of errors between the two groups. With regard to the performance time to reach the goal, however, SAM-P/8 showed a mild prolongation at 2 months of age, and the prolongation became marked with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral embolization leads to memory impairment of several learning tasks in rats.

The effects of cerebral embolization, produced by injecting microspheres into the left internal carotid artery, on passive and active avoidance tasks and water filled multiple T-maze task, were studied in male Wistar rats. The rats with cerebral embolization were markedly impaired acquisition and retention of the one-trial passive avoidance response. The impairment depended on the number of microspheres injected and continued for 2 weeks. The cerebral embolized rats were also impaired acquisition of two-way active avoidance response in a shuttle box. These impairments are not due to decrease in shock sensitivity, because there was no significant change in the flinch-jump threshold. The embolized rats also exhibited a significant disturbance in performance of water filled multiple T-maze learning. These results suggest that rats with cerebral embolization are impaired in three different types of learning tasks, and may be useful as an animal model for the vascular type of dementia.

TRH and its novel analog (DN-1417): antipentobarbital action and involvement of cholinergic mechanisms.

Possible neuroanatomical loci and the mode of action of thyrotropin-releasing hormone (TRH) or its analog, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417), in reducing the pentobarbital-induced sleeping time were investigated by using an intracerebral microinjection technique in rats. Intravenous, intraperitoneal or intracerebroventricular (ICV) injection of TRH or DN-1417 produced a dose-related reduction of the sleeping time induced by pentobarbital. TRH or DN-1417 given into the posterior hypothalamic regions including the dorsal premammillary nucleus, lateral hypothalamic area and posterior nucleus of hypothalamus had a significant pentobarbital sleep shortening action in low doses. Injection of these peptides into the dorsomedial nucleus of thalamus, mesencephalic reticular formation, medial septal nucleus or hippocampus was also effective, in comparatively low doses. However, higher doses were required to elicit the effect when the injections were made into the nucleus accumbens, lateral preoptic area or caudate nucleus. In this respect, the parietal cortex was insensitive to TRH or DN-1417. The pentobarbital sleep shortening action of TRH or DN-1417 injected peripherally or into the hypothalamic regions was markedly antagonized by ICV or intrahypothalamic pretreatment with atropine methyl bromide. On the contrary, ICV injection of atropine methyl bromide had a weak or no antagonizing action on the effect of TRH injected ICV or into the reticular formation, medial septal nucleus or hippocampus. These results suggest that possible neuroanatomical sites mediating the pentobarbital sleep shortening action of TRH or DN-1417 may be posterior hypothalamic regions, dorsomedial nucleus of thalamus, reticular formation, medial septal nucleus or hippocampus. A cholinergic mechanism may also be involved in the effect of TRH on the hypothalamus.

Idebenone improves learning and memory impairment induced by cholinergic or serotonergic dysfunction in rats.

The effects of idebenone, a cerebral metabolic enhancer, on learning and memory impairment in two rat models with central cholinergic or serotonergic dysfunction were investigated using positively reinforced learning tasks. A delayed alternation task using a T maze was employed to test the effect of idebenone on short-term memory impairment induced by a cholinergic antagonist, scopolamine. A correct response, defined as a turn toward the arm opposite to that in the forced run, was rewarded with food pellets. Scopolamine (0.2 and 0.5 mg/kg, i.p.) significantly decreased the correct responses to the chance level in the 60-s-delayed alternation task. The scopolamine (0.2 mg/kg, i.p.)-induced impairment of short-term memory was improved by idebenone (3-30 mg/kg, i.p.) or an acetylcholinesterase inhibitor, physostigmine (0.1 and 0.2 mg/kg, i.p.), administered simultaneously. The central serotonergic dysfunction model was produced by giving rats a diet deficient in tryptophan, a precursor of serotonin. The rats fed on a tryptophan-deficient diet (TDD) showed a slower learning process in the operant brightness discrimination task (mult V115 EXT) than did rats fed on a normal diet. Idebenone (60 mg/kg/day) admixed with the TDD decreased the number of lever-pressing responses emitted during the extinction periods. The percentage of correct responses was significantly higher in the idebenone-treated group than in the control TDD group. These results suggest that idebenone may improve both the impairment of short-term memory induced by a decreased cholinergic activity and the retardation of discrimination learning induced by central serotonergic dysfunction.

Effects of idebenone on metabolism of monoamines and cyclic AMP formation in rats.

Idebenone, 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone, at a dose of 100 mg/kg (i.p.) markedly increased the level of 5-hydroxyindole-3-acetic acid (5-HIAA) in several brain regions without affecting monoamine contents in normal rats. In rats with cerebral ischemia, idebenone (10 mg/kg, i.p.) normalized the decreased levels of 5-HIAA in the cerebral cortex, hippocampus, diencephalon and brain stem. A 5-hydroxytryptamine (serotonin, 5-HT) biosynthesis inhibitor, DL-p-chlorophenylalanine (PCPA, 150 mg/kg, i.p.) decreased the levels of 5-HT to one-third of the control level 24 h after administration. Idebenone (10, 30, or 100 mg/kg, i.p.), administered 24 h after the treatment with PCPA, accelerated the PCPA-induced 5-HT decreased in the hippocampus, diencephalon and brain stem in a dose-dependent manner. Idebenone (100 mg/kg, i.p.) stimulated the release of 5-HT in the dorsal hippocampus as determined by in vivo differential pulse voltammetry. Idebenone, like p-chloroamphetamine (PCA), stimulated 5-HT release from slices of hippocampus and diencephalon, and the formation of cyclic AMP in a concentration-dependent manner in rat diencephalon slice. This stimulation was almost completely blocked by methysergide, a 5-HT receptor blocker. Idebenone slightly and PCA markedly inhibited 5-HT uptake into hippocampus slices. The mechanism of the 5-HT releasing actions of idebenone in the hippocampal slices may be mediated through endogenous calcium. These results suggest that idebenone has an enhancing effect on the turnover of 5-HT in the hippocampus, diencephalon, and brain stem of rats.

Effects of idebenone on memory impairment induced in ischemic and embolization models of cerebrovascular disturbance in rats.

Two rat models of memory impairment in passive avoidance learning induced by cerebrovascular disturbance, were established to estimate the effects of a cerebral metabolic enhancer, idebenone. Transient and global cerebral ischemia in rats, produced by 4-vessel occlusion for 200 s immediately after the acquisition trial of passive avoidance learning, shortened the latencies in the retention test trial performed 24 h later. This retrograde amnesia was reversed significantly by idebenone administered orally or intraperitoneally at the doses of 10 and 30 mg/kg before the retention test trial. Idebenone at a dose of 10 mg/kg, given intraperitoneally before or immediately after the ischemia, also markedly inhibited the appearance of amnesia. In the second model, permanent and cerebral hemisphere embolization produced by injecting 2,000 microspheres into the internal carotid artery, significantly impaired passive avoidance learning performed 7 days later. The repeated administration of idebenone (30 mg/kg, i.p.). once a day after the embolization, significantly improved the impairment of passive avoidance learning in the embolized rats. Furthermore, physostigmine and arginine-vasopressin as reference compounds improved the impairment of passive avoidance learning in these models. These findings suggest that idebenone ameliorates memory impairment induced by cerebral vascular disturbance in rats.

Modification of dopaminergic transmission by thyrotropin-releasing hormone.

TRH may act on the brain independently of its effects on the pituitary, and some of the CNS actions of TRH are probably closely related to brain catecholamines. TRH enhanced SMA by systemic injection (20 mg/kg, i.p.) or direct microinjection (10 micrograms) of TRH into the nucleus accumbens (ACB), one of the nerve terminal sites of the mesolimbic dopaminergic system, in rats. Prevention of this TRH effect by pretreatment with haloperidol or pimozide, a DA receptor blocker, or alpha-MT, an inhibitor of tyrosine hydroxylase, indicated that the SMA stimulatory action of TRH was the result of an enhancement of the release of DA from the nerve terminals. In rats lesioned unilaterally in the nigrostriatal DA pathway by 6-OHDA, high doses of TRH given i.p. (100 mg/kg) or into the nonlesioned caudate nucleus (50 micrograms) produced a circling toward the lesioned side which was suppressed by haloperidol or alpha-MT. The in vitro study demonstrated that TRH (5 X 10(-4)M) enhanced the release of preloaded [3H]DA from the slices of ACB after stable spontaneous DA release was established under superfusion, while a higher concentration (10(-2)M) was required to enhance the DA release from striatal slices. These in vitro studies supported the findings in investigation in vivo. In addition, TRH (2.5-20 mg/kg) markedly enhanced the circling behavior induced by L-DOPA or apomorphine in mice with unilateral caudate nucleus lesions induced by injection of 6-OHDA. In the 6-OHDA lesioned mice treated with TRH, DA-induced cyclic AMP formation was clearly enhanced in the striatal slices taken from the lesioned side but not from the intact side. In conclusion, TRH in low doses facilitates the DA presynaptic transmission by increasing the release of this amine from the ACB and also the DA postsynaptic transmission by increasing DA-stimulated cyclic AMP formation in striatum supersensitized with 6-OHDA. Endogenous TRH may play a physiological role as a modulator on DA transmission in CNS.

[TRH and its analog (DN-1417). Effects on central dopaminergic system-dependent behaviors in rats and mice].

Effects of TRH and its analog, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417), on circling, stereotyped or climbing behavior were investigated in rats and mice. High doses of TRH (100 mg/kg, i.p.) and DN-1417 (20-50 mg/kg, i.p.) produced a significant ipsilateral circling behavior in rats with the lesions of the unilateral dopamine (DA) pathway by 6-hydroxydopamine. In intact mice, stereotyped sniffing accompanying hyperactivity was caused at low doses of TRH or DN-1417 (1-20 mg/kg, i.p.), and stereotyped licking behavior was observed at high doses of TRH or DN-1417 (20-200 mg/kg, i.p.). Furthermore, TRH, DN-1417 or methamphetamine caused a dose-related climbing behavior in mice pretreated with L-DOPA and Ro 4-4602. Circling behavior, stereotyped sniffing or licking behavior induced by TRH or DN-1417 was markedly suppressed by pretreatment with pimozide or alpha-methyltyrosine. However, atropine and scopolamine potentiated the circling inducing action of TRH or DN-1417, in contrast with suppression of the licking behavior. These results suggest that TRH and DN-1417 produce circling, sniffing, licking and climbing behaviors via activation of the central DA system, and that cholinergic mechanisms may be also involved in licking behavior induced by TRH or DN-1417.

Effects of idebenone (CV-2619) on endogenous monoamine release and cyclic AMP formation in diencephalon slices from rats.

The addition of CV-2619 (10(-4) M) stimulated the release of endogenous 5-hydroxytryptamine (5-HT) markedly and that of norepinephrine (NE) slightly from slices of rat diencephalon, but it did not affect dopamine (DA) release. The addition of CV-2619 (10(-5)-10(-4) M) or 5-HT (10(-6)-10(-4) M) stimulated cyclic AMP formation in a concentration-dependent manner. Methysergide (10(-4) M), a 5-HT receptor blocker, almost completely blocked CV-2619 (10(-4) M)- and 5-HT (10(-4) M)-induced cyclic AMP formation. These results suggest that CV-2619 stimulates cyclic AMP formation via endogenous monoamine, particularly, 5-HT release.

[Behavioral and EEG alterations with brain stem compression and effect of thyrotropin-releasing hormone (TRH) in chronic cats (author's transl)].

Behavioral and EEG changes induced by brain stem compression and the effect of TRH were studied. The compression was given for 1 to 6 min by inflating a balloon chronically implanted on the dorsal surface of the cat brain stem in the 4th ventricle via cisterna magna. Within 10 to 36 sec after the start of the compression, the cats turned sideways and became motionless in a spastic extension of four legs, and thereafter all reverted to a normal position, after 45 to 120 min, although slight movements or head-up position was observed in some animals. The cortical EEG patterns observed after the compression were initially a brief rush of low amplitude-fast waves (EEG arousal) followed by a flattened and/or spike pattern, and subsequently these shifted to high amplitude-slow waves with or without an accompanying EEG arousal. These behavioral EEG alterations were remarkably improved by i.v. administration of TRH as follows: eight of 12 cats with 1 mg/kg and one of 4 cats with 0.5 mg/kg promptly changed from the lateral to a crouching or abdominal position, and thereafter never turned sideways again. Partial recovery such as movements of forelegs, struggling or head-up in the lateral position, rolling or slight shift of position was also observed within several min in three cats with 1 mg/kg as well as in two cats with 0.5 mg/kg. Furthermore, TRH induced a dose dependent, persistent EEG arousal in all cats. These results show that TRH ameliorates deterioration in behavior and the EEG, this deterioration being similar to clinical states of disturbance in consciousness induced by compressing the brain stem.