Microinjection of thyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus stimulates gastric contractility

Changes in gastric contractility following microinjection of thyrotropin-releasing hormone (TRH) into the paraventricular nucleus of the hypothalamus (PVN) were examined in fasted, urethane-anesthetized rats. Gastric contractility was measured with extraluminal force transducers and analysed by computer. Unilateral and bilateral PVN microinjections of TRH (0.5 and 1.0 μg) significantly increased the force index of gastric contractions from 0 to 60 min postinjection, when compared with animals microinjected with 0.1 μg TRH, 0.1% BSA or TRH (0.5 and 1.0 μg TRH) in sites adjacent to the PVN. The gastric force index was also significantly elevated from 61 to 120 min postinjection in rats receiving bilateral PVN microinjections of TRH (0.5 and 1.0 μg). Peak gastric responses occurred within 10–20 min postinjection and represented an approximately eight-fold increase over basal values. In the remaining groups, the force index was not significantly altered from preinjection values. The excitatory action of TRH (1.0 μg) on gastric contractility was completely abolished by subdiaphragmatic vagotomy. These results suggest that TRH acts within the PVN to stimulate gastric contractility via vagal-dependent pathways.     

Bombesin microinjected into the dorsal vagal complex inhibits TRH-stimulated gastric contractility in rats

The effects of centrally injected bombesin on central and peripheral stimulated gastric contractility were investigated in fasted urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and gastric contractility was analyzed by computer. Intracisternal injection of the stable thyrotropin-releasing hormone (TRH)-analog RX 77368 (77 pmol) induced a stimulation of gastric contractility for 40 min. Intracisternal injection of bombesin (62-620 pmol) followed 30 min later by that of RX 77368 resulted in a dose-related inhibition of the TRH-analog-induced gastric contractility. Intracisternal injection of bombesin (620 pmol) did not modify gastric contractility stimulated by intravenous carbachol. Stimulation of gastric contractility induced by TRH-analog microinjected into the dorsal vagal complex (DVC) was dose-related suppressed by concomitant injections of bombesin (6.2-620 pmol). Neither bombesin alone (6.2 pmol) nor vehicle modified basal gastric contractility. These results demonstrate that bombesin acts within the brain to inhibit vagally stimulated gastric contractility and that the DVC is a sensitive site for bombesin inhibitory action. These findings suggest a possible interaction between TRH and bombesin in the central vagal regulation of gastric contractility.     

Microinjection of TRH analogs into the raphe pallidus stimulates gastric acid secretion in the rat

The effects of microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, [pGlu-His-(3,3'-dimethyl)-Pro-NH2] into the raphe pallidus on gastric acid secretion were studied in urethane-anesthetized rats with gastric fistula. RX 77368 microinjected into the raphe pallidus at doses of 0.07, 0.7 and 7.7 pmol induced a dose-dependent net stimulation of gastric acid secretion (7 +/- 4, 50 +/- 7 and 61 +/- 12 mumol/h respectively). The peak acid response was reached within 30 min and returned to basal level 90 min post-injection. The stimulatory effect was abolished by bilateral cervical vagotomy and pirenzepine pretreatment (1 mg/kg, i.v.). RX 77368 (7.7 pmol) microinjected into the inferior olive or pyramidal tract induced smaller or no gastric acid secretory response. These results demonstrate that chemical stimulation of the raphe pallidus increases gastric acid secretion through vagal pathways and peripheral muscarinic receptors. These data suggest that the nucleus raphe pallidus may be involved in vagal modulation of gastric acid secretion in the rat.     

CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats

The effect of CRF microinjected into the dorsal vagal complex (DVC) on centrally-stimulated gastric contractility was investigated in fasted, urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and contractility was analyzed by computer. Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, (26 pmol) into the DVC induced a 12.2-fold stimulation of gastric contractility within 30 min. Corticotropin-releasing factor (CRF) (63-210 pmol) microinjected into the DVC concomitantly with RX 77368 (26 pmol) induced a dose-related inhibition of stimulated gastric contractility. Neither CRF alone (210 pmol) nor vehicle modified basal gastric contractility. Microinjection of kainic acid (141 pmol) into the raphe pallidus nucleus induced a 3.6-fold stimulation of gastric contractility after 45 min. This stimulation was suppressed by bilateral microinjection of CRF (105 pmol/site) into the DVC. These results demonstrate that CRF acts in the DVC to inhibit centrally-stimulated gastric contractility and suggest that TRH and CRF may interact in the DVC to regulate gastric motor function.     

Administration of thyrotropin-releasing hormone into the central nucleus of the amygdala induces gastric lesions in rats

Studies utilizing microinjections of thyrotropin-releasing hormone (TRH) were performed to determine potentially sensitive limbic brain sites to the ulcerogenic effect of TRH. Administration of TRH into the central nucleus of the amygdala (CEA) produced a high (80%) incidence of gastric lesions and also significantly stimulated acid secretion. Microinjections of TRH into other brain sites including superior colliculus, medial septum, substantia nigra and the hippocampus (CA1 area) were ineffective. Intra-CEA TRH-induced gastric lesions and acid secretion were prevented by vagotomy. These results indicate that the amygdala is of importance for mediating the ulcerogenic effect of brain TRH through peripheral vagal pathways.     

Microinjection of arginine vasopressin into the central nucleus of amygdala suppressed nociceptive jaw opening reflex in freely moving rats

This study was performed to examine the antinociceptive effect after microinjection of arginine vasopressin (AVP) into the central nucleus of amygdala. We recorded the jaw opening reflex in freely moving rats. After injection of 0.2 or 0.4 nM AVP into the central nucleus of amygdala, digastric electromyogram (dEMG) was suppressed to 55 +/- 5% or 88 +/- 3 of the control. Artificial cerebrospinal fluid had no effects on the basal dEMG activity. V(1) vasopressin receptor antagonist blocked the suppressive effect produced by microinjection of 0.4 nM AVP from 53 +/- 3 to 81 +/- 3% of the control. However, V(2) vasopressin receptor antagonist did not affect changes in dEMG. We observed dEMG activity after intracerebroventricular injection of naloxone, methysergide, or phentolamine. All drugs did not affect the basal dEMG activity at our dose. Naloxone blocked the suppressive effect of 0.4 nM AVP from 42 +/- 4 to 79 +/- 5% of the control. Methysergide also inhibited the suppression of dEMG from 44 +/- 3 to 83 +/- 6% of the control. However, phentolamine, an alpha-adrenergic receptor antagonist, did not affect the suppression of dEMG. These results indicate AVP in the central nucleus of amygdala has potent analgesic effects in the orofacial area. The antinociception of central AVP seems to be mediated by opioid and serotonergic pathways.     

Microinjection of RFRP-1 in the central nucleus of amygdala decreases food intake in the rat

Several members of the RFamide peptide family are known to have role in the regulation of feeding. For example, neuropeptide FF and prolactin-releasing peptide cause anorexigenic, while 26RFa and QRFP result in orexigenic effects in rodents. I.c.v. microinjection of neuropeptide RFRP-1 significantly reduced food and water intake in chicks. However, feeding related effects of RFRP-1 have not been studied in mammals yet. The central part of amygdala (CeA) is essentially involved in the regulation of feeding and body weight. RFRP-1 positive nerve cells were detected in the rat hypothalamus and RFRP-1 immunoreactive fibers were identified in the CeA. RFRP analogs bind with relatively high affinity to the NPFF1 and NPFF2 receptors (NPFF-R). RFRP-1 has potent activity for NPFF1. Significant expression of NPFF1 was detected in the CeA. To evaluate the role of RFRP-1 in feeding regulation rats were microinjected with different doses of RFRP-1 and their food intake were quantified over a 60min period. Liquid food intake of male Wistar rats was measured after bilateral intraamygdaloid administration of RFRP-1 (25, 50 or 100ng/side, RFRP-1 dissolved in 0.15M sterile NaCl/0.4μl, respectively). The 50ng dose of RFRP-1 microinjections resulted in significant decrease of food intake. The 25 and 100ng had no effect. Action of 50ng (37.8pmol) RFRP-1 was eliminated by 20ng (41.4pmol) RF9 NPFF-R antagonist pretreatment. In open-field test 50ng RFRP-1 did not modify spontaneous locomotor activity and general behavior of animals did not change. Our results are the first reporting that RFRP-1 injected to the CeA result in a decrease of liquid food consumption. This is a receptor-linked effect because it was eliminated by a NPFF-R selective antagonist.     

Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal

Previous studies have shown that spontaneous exploration of the Elevated Plus Maze provides a sensitive measure of ‘anxiety’ induced by pharmacological or behavioral stressors. In particular, the percent time spent exploring the open arms of the plus maze is decreased during ethanol withdrawal, and this effect is antagonized by intracerebroventricular administration of 25 μg of alpha-helical CRF, a corticotropin-releasing factor antagonist (H.A. Baldwin et al.,Psychopharmacology, 103 (1991) 227–232). The present study was designed to examine the effect of α-helical CRF infusion within the central nucleus of the amygdala during ethanol withdrawal. Rats were made dependent on ethanol by maintenance on an ethanol-containing liquid diet for 16 days, withdrawn from ethanol and tested on the elevated plus maze at 8 h post-ethanol access. In comparison with pair-fed control rats, ethanol withdrawn subjects spent significantly less percent time exploring the open arms of the plus maze. This decrease in open arm exploration was antagonized by administration of α-helical CRF (250 ng) bilaterally into the central nucleus of the amygdala, but not by intracerebroventricular administration of 250 ng of α-helical CRF. The ability of intra-amygdala α-helical CRF to antagonize decreased open arm exploration is unlikely to be due to changes in motor activity, since general activity on the maze was reduced in all EtOH withdrawal groups. These results suggest that the central nucleus of the amygdala may be an effective site for endogenous CRF systems to mediate anxious behavior associated with ethanol withdrawal.     

Corticosterone effects on corticotropin-releasing hormone mRNA in the central nucleus of the amygdala and the parvocellular region of the paraventricular nucleus of the hypothalamus

Using in situ hybridization histochemistry, we report differential expression of corticotropin-releasing hormone (CRH) mRNA in the central nucleus of the amygdala (CEA) and the parvocellular region of the paraventricular nucleus of the hypothalamus (PVN) following systemic treatment with corticosterone (CORT) in adrenally-intact rats. Both injection of low (1 mg/kg/day) and high (5 mg/day) CORT reduced CRH mRNA expression in the PVN in a dose-dependent manner, although it returned to normal at the low dose by 14 days. By contrast, the high dose of CORT increased CRH mRNA transiently in the CEA at 4 days, although the low dose of CORT decreased it at 14 days. In a second experiment, we implanted a slowly-releasing CORT pellet for 2 weeks (200 mg, 60 day release) subcutaneously. This treatment produced an elevation of CRH mRNA in the CEA both at 1 and 2 weeks, whereas CRH mRNA in the PVN was decreased to a large extent as seen in the high CORT group of the first experiment. These results suggest that glucocorticoids can facilitate CRH mRNA expression in the CEA, a site implicated in anxiety and fear, while restraining the hypothalamic-pituitary-adrenal axis as indicated by the reduction in CRH mRNA in the PVN.     

Excitatory effects of thyrotropin-releasing hormone on neurons within the nucleus ambiguus of adult guinea pigs

The electrophysiological effects of thyrotropin-releasing hormone (TRH) on neurons within the nucleus ambiguus (NA) of adult guinea pigs were studied using an in vitro brain stem slice preparation. In 0.01–1.0 μm TRH, NA neurons depolarized (25/39), expressed enhanced postinhibitory rebound (8/8 tested), or exhibited oscillations of the membrane potential (17/39). Because the amplitude of postinhibitory rebound in tetrodotoxin (TTX) at various membrane potentials was not altered by TRH, it suggests that TRH enhanced postinhibitory rebound indirectly by depolarizing the cell membrane. The membrane potential oscillations in NA neurons were persistent in TTX and their frequency was dependent on the membrane potential, suggesting that these oscillations were due to intrinsic membrane properties and not to synaptic inputs. The excitation of NA neurons in vitro by TRH suggests that endogenous TRH may modulate the activity of neurons involved in the regulation of respiratory and autonomic function.     

The thyrotropin-releasing hormone test in the diagnosis of unipolar depression

The establishment of criteria for a blunted thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) may prove useful in distinguishing patients with major unipolar depression from patients with nonmajor depressions and controls. To this end, we administered the TRH test to a group of depressed, euthyroid inpatients diagnosed by Research Diagnostic Criteria and 20 normal volunteer controls. The mean maximal TSH response (delta TSH) to infusion of 500 micrograms of TRH of 7.3 +/- SD 4.6 microIU/ml in the 105 patients with major depressive disorder, primary unipolar subtype was significantly lower than that of 13.4 +/- SD 4.4 in the 20 controls and 10.9 +/- SD 4.4 in the 40 patients with nonmajor depressions. The differences were not explainable by differences in baseline thyroid function, age, or sex. When a delta TSH less than or equal to 7.0 microIU/ml was used as a diagnostic test for unipolar depression, the sensitivity of the TRH test was 56%, the specificity 93%, and the predictive value 91%. These results suggest that the TRH test may be useful in confirming the diagnosis of major unipolar depression and hence identifying patients likely to respond to antidepressant medications or electroconvulsive therapy.     

Restraint stress increases corticotropin-releasing hormone mRNA content in the amygdala and paraventricular nucleus

Corticotropin-releasing hormone (CRH) neurons located in the paraventricular nucleus (PVN) of the hypothalamus are implicated in regulating the endocrine response to stress. The amygdala is an established component of the neural circuitry mediating the stress response. To obtain information concerning the effects of stress on amygdala CRH neurons, a time-course study was conducted to examine, in rats, whether a 1-h restraint period increases CRH mRNA levels. The effects of restraint were also measured in the PVN. Using a sensitive RNase protection assay, we found that CRH mRNA levels in both the amygdala and paraventricular nucleus were significatly elevated 1 h after cessation of restraint. CRH mRNA levels in the paraventricular nucleus, but not the amygdala, remained elevated at the 3-h post-stress interval. 48 h after the termination of restraint, CRH mRNA levels in both brain structures returned to control levels. These data provide the first direct evidence that stress activates amygdala CRH neurons.     

Psychological stress increased corticotropin-releasing hormone mRNA and content in the central nucleus of the amygdala but not in the hypothalamic paraventricular nucleus in the rat

The central administration of corticotropin-releasing hormone (CRH) to experimental animals sets into motion a coordinated series of physiological and behavioral events that promote survival during threatening situation. A large body of evidence suggest that CRH in the central nucleus of the amygdala (CEA) induces fear-related behaviors and is essential to fear conditioning; however, evidence of CRH-mediated activation of the amygdala under physiological situation is still limited. We report here a study of the impact of a psychological stressor on hypothalamic and amygdala CRH systems in the rat. Non-footshocked rats placed in a floored compartment surrounded by footshocked rats were defined as the psychological stress group. Rats were exposed to psychological stress for 15 min, and then sacrificed 1.5 and 3 h after cessation of stress. We found that our psychological stressor induced an increase in both CRH mRNA levels, as assessed by in situ hybridization histochemistry, and CRH content, as assessed by micropunch RIA, in the CEA. Exposure to the psychological stressor also caused a significant increase in CRH mRNA levels with a trend for an increase in CRH content in the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNST) which is anatomically associated with the CEA. In contrast, psychological stress induced a small, but significant increase in type-1 CRH receptor (CRHR-1) mRNA in the hypothalamic paraventricular nucleus (PVN), while it failed to elevate either PVN CRH mRNA levels or content, CRH content in the median eminence (ME), or levels of plasma ACTH or corticosterone (CORT). Thus, in the context of a psychological stressor, the activation of the amygdala CRH system can occur without robust activation of the hypothalamic CRH system. In the light of previous data that the psychological stress-induced loss of sleep was reversed by the central administration of a CRH antagonist, these data suggest that CRH in the CEA may contribute to the psychological stress-evoked fear-related behavior such as hyperarousal. These data also indicate that in response to a psychological stressor, the amygdala CRH system is much more sensitive than is the CRH system emanating from the PVN.     

Intrathecal thyrotropin-releasing hormone therapy of amyotrophic lateral sclerosis

Summary Six patients with amyotrophic lateral sclerosis were given from 800 to 4000 g of thyrotropin-releasing hormone (TRH) intrathecally for a period of 2–6 months. The progressive course of this disease, manifested by increasing atrophy, paralysis and disability score, was not altered. This supports the hypothesis that the decrease in TRH content in the anterior horn region is secondary to the cellular destruction. TRH appears to play no significant role in the pathogenesis of amyotrophic lateral sclerosis     

Immunocytochemistry of thyrotropin-releasing hormone in the rabbit medulla oblongata

The distribution and ultrastructure of thyrotropin-releasing hormone-like immunoreactive (TRH-LI) neurons were examined in rabbit medulla oblongata. TRH-LI cell bodies were located in the ventral region of the medulla oblongata: in the paraolivary and parapyramidal regions, regions in and around the pyramidal tract, the dorsolateral region of the lateral reticular nucleus, and the raphe nuclei. The paraolivary and parapyramidal regions contained most of the TRH-LI cell bodies in the medulla oblongata. TRH-LI neurons processes were densely distributed in the dorsal vagal complex and the area postrema. Electron-microscopic immunocytochemical studies revealed TRH-LI neurons at the obex level in the paraolivary region of rabbits. TRH-like immunoreactivity was localized in larger granular vesicles. TRH-LI somata and dendrites received synaptic inputs from both TRH-LI and unlabeled axon terminals. More than half of the TRH-LI axon terminals made synapses with somata or processes of TRH-LI neurons. These observations, together with previous reports that TRH causes respiratory facilitation, suggest that TRH-LI neurons in the paraolivary region in rabbits may be involved in respiratory functions.     

Immunological blockade of endogenous thyrotropin-releasing hormone impairs recovery from hyperglycemia in mice

Administration of antiserum to thyrotropin-releasing hormone (TRH) into the lateral cerebral ventricle of mice significantly attenuated recovery from hyperglycemia induced by treatment with 2-deoxyglucose but had no effect on the plasma glucose of saline-treated mice. TRH, injected centrally together with the anti-TRH antibody, reversed the effect of the antiserum and blocked the development of hyperglycemia. These findings suggest that activation of TRH neurons in the central nervous system may be a physiological event influencing recovery from hyperglycemia.     

Electrical stimulation of the medial amygdala facilitates gastric acid secretion in conscious rats

One hundred and twenty-seven conscious rats prepared with chronic gastric fistula were studied to investigate the effect of stimulation of the medial amygdala on gastric acid secretion. Gastric acid output was significantly increased by electrical stimulation of the medial amygdala in normal rats and the increase in acid secretion was completely abolished by vagotomy. Vagotomized rats, with or without amygdaloid stimulation, showed comparable levels of gastric acid output which were significantly lower than in controls. These results indicate that the amygdala effect on gastric acid secretion is carried via the vagus nerve. Subcutaneous injections of high doses of histamine increased gastric acid secretion which was further increased by amygdaloid stimulation. Plasma levels of gastrin were not significantly changed by stimulation of the medial amygdala with or without vagotomy. From the above results, we concluded that in conscious rats the medial amygdala plays a significant role in stimulating gastric acid secretion, the vagus nerve is involved in this process, but it is not mediated by release of either histamine or gastrin.     

GABA-ergic innervation of thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

To determine whether GABA-ergic axons are anatomically situated to directly influence TRH neurons in the PVN, double-labeling light- and electronmicroscopic immunocytochemistry was performed using antisera against glutamic acid decarboxylase (GAD) and prothyrotropin-releasing hormone (proTRH). In the anterior, periventricular and medial parvocellular subdivisions of the PVN, GAD-immunoreactive (IR) axon varicosities were closely apposed to all proTRH containing cell bodies and proximal dendrites. Ultrastucturally, GAD-IR nerve terminals established symmetric type synapses with both perikarya and dendrites of proTRH-IR neurons, indicating the inhibitory nature of the contacts. Since a subpopulation of neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus co-synthesize GABA, and NPY-containing neurons of arcuate nucleus origin densely innervate TRH neurons in the PVN, we performed triple labeling immunocytochemistry to elucidate the origin of the GAD-IR innervation of hypophysiotropic TRH neurons. While axons co-containing GAD and NPY were observed throughout the PVN, only approximately 10% of GAD-IR terminals in contact with TRH neurons were found to contain NPY-immunoreactivity. We conclude that GABA-ergic neurons are in position to act directly on hypophysiotropic TRH neurons and while this innervation arises partly from neurons in the arcuate nucleus that co-synthesize NPY, the majority of the GABA-ergic input arises from other neuronal groups.     

Microinjection of bicuculline into the central nucleus of the amygdala alters gustatory responses of the rat parabrachial nucleus

The central amygdaloid nucleus (CeA) receives projection from the parabrachial nucleus (PBN) gustatory neurons and descendingly projects to the PBN, and taste responses in the PBN are significantly affected by stimulation or lesion of the CeA. To examine whether the GABA receptors within the CeA are involved in this modulation, the effects of microinjection of bicuculline, a GABA(A)-selective antagonist, into the CeA on the activities of PBN taste neurons were observed by using extracellular recording technique. In general, after bicuculline was administered to ipsilateral CeA, the responses of PBN neurons to four tastants all increased, with the magnitudes significantly higher than those obtained before drug administration (P<0.01), respectively. However, after bicuculline was delivered into the contralateral CeA, only the responses to NaCl, HCl and QHCl increased. According to the best-stimulus category, 47% NaCl-best (8/17), 64% HCl-best (7/11), 80% QHCl-best (4/5), and 33% sucrose-best (1/3) increased their responses to at least one basic taste stimulus after GABA(A) receptors within the ipsilateral CeA were blocked. After contralteral CeA injection, more NaCl-best neurons (6/8) increased responses than that after ipsilateral CeA injection, but other best-stimulus units showed no differences before and after drug injection into the contralateral CeA. Analyses of across-unit patterns indicated that the correlation coefficient of responses between NaCl and sucrose was apparently higher after drug administration to the CeA. However, after drug injection into the contralateral CeA, the correlations between NaCl and the other three tastants were higher than those before. These results indicate that the GABA(A) receptors within the CeA play an important role in modulating the gustatory activities of PBN neurons.