Intrahypothalamic Microinjection Research Articles

Cannabidiol-induced panicolytic-like effects and fear-induced antinociception impairment: the role of the CB1 receptor in the ventromedial hypothalamus.

The behavioural effects elicited by chemical constituents of Cannabis sativa, such as cannabidiol (CBD), on the ventromedial hypothalamus (VMH) are not well understood. There is evidence that VMH neurons play a relevant role in the modulation of unconditioned fear-related defensive behavioural reactions displayed by laboratory animals. This study was designed to explore the specific pattern of distribution of the CB1 receptors in the VMH and to investigate the role played by this cannabinoid receptor in the effect of CBD on the control of defensive behaviours and unconditioned fear-induced antinociception. A panic attack-like state was triggered in Wistar rats by intra-VMH microinjections of N-methyl-D-aspartate (NMDA). One of three different doses of CBD was microinjected into the VMH prior to local administration of NMDA. In addition, the most effective dose of CBD was used after pre-treatment with the CB1 receptor selective antagonist AM251, followed by NMDA microinjections in the VMH. The morphological procedures demonstrated distribution of labelled CB1 receptors on neuronal perikarya situated in dorsomedial, central and ventrolateral divisions of the VMH. The neuropharmacological approaches showed that both panic attack-like behaviours and unconditioned fear-induced antinociception decreased after intra-hypothalamic microinjections of CBD at the highest dose (100nmol). These effects, however, were blocked by the administration of the CB1 receptor antagonist AM251 (100pmol) in the VMH. These findings suggest that CBD causes panicolytic-like effects and reduces unconditioned fear-induced antinociception when administered in the VMH, and these effects are mediated by the CB1 receptor-endocannabinoid signalling mechanism in VMH.

Effects of direct QRFP-26 administration into the medial hypothalamic area on food intake in rats.

The RFamide peptide family comprises a number of biologically active peptides sharing RF motif at their C-terminal end. These peptides are involved in the control of multiple physiological functions including regulation of metabolism and feeding behavior. QRFP-43 as well as its 26-aminoacid residue QRFP-26 are able to cause orexigenic effect when administered to the rodents' cerebral ventricles. QRFPs have been suggested as the endogenous ligands of the previously orphan GPR103 receptors. GPR103 receptors share amino acid identity with other receptors of neuropeptides involved in feeding (NPY, NPFF, galanin). QRFP-26 expressing neurons and binding sites are densely present in the rat medial hypothalamus (MHA), an area directly responsible for the regulation of feeding. QRFP-26 was delivered to the target area by direct intrahypothalamic microinjection, and the consumption of liquid food was measured over a 60 min period. Both doses (100 and 200 ng) significantly increased food intake. Non-specific receptor antagonist BIBP3226 eliminated the orexigenic effect caused by QRFP-26 administration. Effective doses of QRFP-26 did not modify general locomotor activity and behavioral patterns examined in the open-field test. This study is the first reporting feeding modulating effects following direct intrahypothalamic QRFP-26 administration.

Effect of p53 Inhibition by Pifithrin-Alpha on Functional Activity of Vasopressin Neurons in Rat Hypothalamus

In the present work we investigated the effects of p53 inhibition by pifithrin-alpha (PFT) in vitro and in vivo on functioning vasopressinergic magnocellular neurones of rat hypothalamus. In vivo treatments with PFT were done by intra-hypothalamic microinjections or by intra peritoneal injections. In in vitro experiments hypothalamic slices containing supraoptic nuclei and intact pituitary were incubated with or without PFT. In all experiments we observed accumulation of vasopressin (VP) in the cell perikarya after PFT injections, however expression of VP mRNA was not changed. Analysis of VP content in the posterior pituitary demonstrated that amount of VP was significantly decreased after PFT treatments. Additionally, long-term inhibition of p53 in experiments with intra-hypothalamic injections of PFT resulted in an increased diuresis rate. The obtained results demonstrated that in all experiments PFT treatments inhibited VP anterograde transport from the cells of supraoptic nuclei. Moreover, analysis of MEK/ERK activities revealed that phosphorylation levels of MEK1/2 and ERK1/2 were decreased after PFT treatments. Our findings provide new evidences that p53 could be involved in the control of VP secretion from hypothalamo-hypophyseal system and that this action probably can be mediated by ERK signalling pathway.

Paraventricular hypothalamic regulation of trigeminovascular mechanisms involved in headaches.

While functional imaging and deep brain stimulation studies point to a pivotal role of the hypothalamus in the pathophysiology of migraine and trigeminal autonomic cephalalgias, the circuitry and the mechanisms underlying the modulation of medullary trigeminovascular (Sp5C) neurons have not been fully identified. We investigated the existence of a direct anatomo-functional relationship between hypothalamic excitability disturbances and modifications of the activities of Sp5C neurons in the rat. Anterograde and retrograde neuronal anatomical tracing, intrahypothalamic microinjections, extracellular single-unit recordings of Sp5C neurons, and behavioral trials were used in this study. We found that neurons of the paraventricular nucleus of the hypothalamus (PVN) send descending projections to the superior salivatory nucleus, a region that gives rise to parasympathetic outflow to cephalic and ocular/nasal structures. PVN cells project also to laminae I and outer II of the Sp5C. Microinjections of the GABAA agonist muscimol into PVN inhibit both basal and meningeal-evoked activities of Sp5C neurons. Such inhibitions were reduced in acutely restrained stressed rats. GABAA antagonist gabazine infusions into the PVN facilitate meningeal-evoked responses of Sp5C neurons. PVN injections of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP38) enhance Sp5C basal activities, whereas the antagonist PACAP6-38 depresses all types of Sp5C activities. 5-HT1B/D receptor agonist naratriptan infusion confined to the PVN depresses both basal and meningeal-evoked Sp5C activities. Our findings suggest that paraventricular hypothalamic neurons directly control both spontaneous and evoked activities of Sp5C neurons and could act either as modulators or triggers of migraine and/or trigeminal autonomic cephalalgias by integrating nociceptive, autonomic, and stress processing mechanisms.

Serotonergic neural links from the dorsal raphe nucleus modulate defensive behaviours organised by the dorsomedial hypothalamus and the elaboration of fear-induced antinociception via locus coeruleus pathways

Decrease of γ-aminobutyric acid (GABA)-mediated neurotransmission in the dorsomedial hypothalamus (DMH) evokes instinctive fear-like responses. The aim of the present study was to investigate the involvement of the serotonin (5-HT)- and norepinephrine-mediated pathways of the endogenous pain inhibitory system, including the dorsal raphe nucleus (DRN) and the locus coeruleus (LC), in the defensive responses and antinociceptive processes triggered by the blockade of GABAergic receptors in the DMH. The intra-hypothalamic microinjection of the GABAA receptor antagonist bicuculline (40 ng/200 nL) elicited elaborate defensive behaviours interspersed with exploratory responses. This escape behaviour was followed by significantly increased pain thresholds, a phenomenon known as fear-induced antinociception. Furthermore, at 5 and 14 days after DRN serotonin-containing neurons were damaged using the selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), the frequency and duration of alertness and escape behaviour evoked by the GABAA receptor blockade in the DMH decreased, as well as fear-induced antinociception. Pre-treatment with the non-selective 5-HT receptor antagonist methysergide, the 5-HT2A/2C receptor antagonist ketanserin and the 5-HT2A receptor selective antagonist R-96544 in the LC also decreased fear-induced antinociception, without significant changes in the expression of defensive behaviours. These data suggest that the serotonergic neurons of the DRN are directly involved in the organisation of defensive responses as well as in the elaboration of the innate fear-induced antinociception. However, serotonin-mediated inputs from the NDR to the LC modulate only fear-induced antinociception and not the defensive behaviours evoked by GABAA receptor blockade in the DMH.

Hypothalamic angiotensinergic–noradrenergic systems interaction in fructose induced hypertension

Objective Several studies suggest the importance of the interaction between the renin angiotensin and sympathetic nervous systems in blood pressure control, especially in clinical situations such as the metabolic syndrome. Previously, we have demonstrated changes in noradrenergic hypothalamic control of blood pressure in an animal model of insulin resistance and hypertension. The aim of the present study was to evaluate the effects of the interaction between the noradrenergic and angiotensinergic systems on hypothalamic blood pressure regulation in fructose hypertensive rats. Methods In control (C) and fructose-fed hypertensive (F) rats, we studied: 1) the effects of hypothalamic perfusion of irbesartan (AT 1 angiotensin receptor antagonist, 50 and 500 μg ml − 1) and metoprolol (β 1 adrenergic receptor antagonist, 10 and 100 μg ml − 1) on blood pressure, heart rate and noradrenaline intrahypothalamic levels, by means of the microdialysis technique; and 2) the effects of intrahypothalamic microinjection of angiotensin II alone or after metoprolol pre-administration, on blood pressure and heart rate. Results Meanwhile irbesartan perfusion did not modify neither mean arterial pressure (MAP) nor heart rate or noradrenaline hypothalamic levels in the C group, its highest dose diminished MAP (ΔMAP: F: − 16.3 ± 1 mm Hg, p < 0.05) and noradrenaline levels (% of basal levels: 58 ± 7%, p < 0.05) in the F group, without affecting heart rate. Intrahypothalamic perfusion of metoprolol diminished MAP only in the F group (ΔMAP: F: − 12.1 ± 1.1 mm Hg, p < 0.05), but did not modify heart rate in both groups. On the other hand, it diminished noradrenaline hypothalamic levels in C (% of basal levels: 53 ± 6%, p < 0.05) but not in the F group. The pressor response to angiotensin II microinjection was increased in F rats (ΔMAP: F: 13.3 ± 1.5 mm Hg vs. C: 6.9 ± 1.8 mm Hg; p < 0.05). Previous administration of metoprolol markedly abolished this increment. Conclusions Our results suggest the existence of an increase in AT 1 and β 1 adrenergic receptors tone in the hypothalamus of F rats, which could be related to the increase in blood pressure present in this experimental model. On the other hand, considering that the enhanced pressor response to angiotensin II intrahypothalamic injection in F rats was abolished by previous administration of a β 1 adrenergic receptor antagonist, these results would indicate that β 1 adrenergic receptors activation participates in the pressor response to angiotensin II in this experimental model of insulin resistance and hypertension.

The effect of posterior hypothalamic injection of cholinergic agents on hippocampal formation theta in freely moving cat

The effect of intra-hypothalamic micro-injection of muscarinic (atropine sulphate, pirenzepine and gallamine) and nicotinic (hexamethonium) antagonists on spontaneous, sensory and electrically-induced hippocampal formation (HPC) theta EEG activity was investigated in the freely behaving cat. Administration of hexamethonium and gallamine failed to elicit a detectable effect on HPC theta activity. However, the injection of atropine sulphate and pirenzepine abolished the theta rhythm recorded from HPC. This effect was reversible. A substantial difference in the recovery time course between frequency versus amplitude (μV) and power (μV 2) of hippocampal theta was observed. While theta frequency exhibited a rapid reappearance with a shallow slope, the power and amplitude showed a gradual recovery with a steeper slope. The injection of carbachol into posterior hypothalamus (PH) produced almost a continuous HPC theta with increased power. These results demonstrate that cholinergic (M1) receptors localised in the posterior area of the hypothalamus are engaged in mechanisms responsible for generating hippocampal theta oscillations in the freely behaving cat. The contribution of posterior hypothalamic region to HPC theta frequency and amplitude is discussed.

Role of serotonin, gamma-aminobutyric acid, and glutamate in the behavioral thermoregulation of female tilapia during the prespawning phase.

The role of hypothalamic neurotransmitter systems in behavioral thermoregulation was investigated in the prespawning female tilapia, Oreochromis mossambicus. Intrahypothalamic microinjection with serotonin (5-HT, 3 microliters of 1.0 x 10(-6) M) resulted in a significant increase in the selected temperature. This effect was mimicked by the agonist of 5-HT1A, 1B, and 2C receptors, N-3-trifluoromethylphenyl piperazine. Intrahypothalamic microinjection of tilapia with gamma-aminobutyric acid (GABA) resulted in a biphasic effect of the temperature selection, whereas microinjection with muscimol, an agonist of GABAA receptor, had no effect on temperature selection. Both agonist and antagonist of glutamate (Glu), N-methyl-D-aspartate (NMDA), and MK-801 (1.0 x 10(-6) M), a noncompetitive blocker of NMDA receptor, significantly decreased the preferred temperature. These results indicate that the hypothalamic 5-HT, GABA, and Glu systems play a role in the temperature selection of prespawning female tilapia.

Effects of intrahypothalamic morphine and its interactions with oxytocin and vasopressin during food intake in rats

The effects of intra-hypothalamic microinjections of morphine (MOR) into the ventromedial (VMH) and lateral (LH) nucleus and its interactions with oxytocin (OXY) and vasopressin (AVP) were evaluated during food intake (FI) in rats. Acute MOR clearly enhanced FI during the dark phase (6–24 h) of the diurnal cycle, while no appreciable changes in the light phase (0–6 h) FI was seen after intra-VMH or LH injections. Long term MOR administration into both VMH and LH attenuated the enhancements in FI seen after acute MOR during the dark phase, while FI was enhanced during the light phase. Pretreatment with OXY or AVP clearly antagonized such tolerance or reverse tolerance/sensitization development to the orexic effect of MOR. These results are discussed in the light of complex peptidergic interactions in the hypothalamus during ingestive behavior.

Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monoamine metabolism

Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monoamine metabolism. The central hypothesis underlying this study is that there is a reciprocal relationship between food intake and sympathetic activity to IBAT. This hypothesis was tested by using intrahypothalamic microinjections of glucagon, a peptide that has been reported to decrease food intake. Sympathetic nerve activity to interscapular brown adipose tissue (IBAT) was measured as electrophysiological discharges of sympathetic nerves to IBAT. The microinjection of glucagon into the lateral hypothalamus (LH) increased sympathetic nerve activity by + 103.8 ± 35.0% (mean±S.E.M.) from pre-injection basal level by 30 min after injection. There was a gradual return to baseline. Micro-injection of glucagon into the LH depressed food intake. Monoamine metabolism was measured by using a microdialysis probe attached to a guide cannula for microinjection of glucagon into the LH. After microinjection of glucagon, the dialysates were collected over 30 min intervals and assayed for norepinephrine (NE), serotonin (5-HT), dopamine (DA) and their metabolites (3-methoxy-4-hydroxyphenylglyucol (MHPG); 5-hydroxyindole-3-acetic acid (5-HIAA); and 3,4-dihydroxyphenylacetic acid (DOPAC). Glucagon suppressed both NE and MHPG concentrations in the lateral hypothalamus (LH), and the concentration of DOPAC was also decreased. There was no change of 5-HT concentration but 5-HIAA levels were reduced by glucagon treatment. These data show that glucagon injected in the LH stimulates sympathetic activity and suggest that this may have occurred by suppression of norepinephrine, dopamine and serotonin turnover in the LH of freely moving rats. These data support the hypothesis of a reciprocal relationship between food intake and sympathetic activity.

Microdialysis: A system for localized drug delivery into the brain

To determine why intrahypothalamic microinjections of pyrogen-free saline (PFS) often induce core temperature (T co) rises, guide cannulas were implanted bilaterally into the preoptic-anterior hypothalamus (POA) of guinea pigs, 1 week later, injectors were inserted to 1 mm beyond the guides and either no injection or 1 μl PFS was administered. Injector insertion without injection evoked a 0.5°C T co rise within 40 min, culminating in 3.7 hr. PFS microinjection elicited a 0.9°C T co rise within 10 min, culminating in 3.8 hr. PFS injected 4 hr later caused a further T co rise. Indomethacin (10 mg/kg, IM), given 30 min before, prevented these effects. To determine whether microdialysis obviates them, a guide cannula was implanted unilaterally into the POA; 1 week later, a dialysis probe (nominal cutoff, 10 kD) was inserted to 1 mm beyond the guide. PFS or prostaglandin E 2 (PGE 2, 1 μg/μl) was perfused 2 days later (2 μl/min for 3 hr). T co was unchanged during PFS perfusion but increased during PGE 2 perfusion to 1.5°C in 1.6 hr, and plateaued until 2 hr after dialysis. These results indicate the T co rise induced by PFS microinjection is mediated by prostaglandins, probably released due to tissue puncture by the injectors and injury by the PFS droplet. Microdialysis prevents these effects. It should, therefore, be preferred over microinjection for intracerebral drug administration.

Reversibility of Para-chlorophenylalanine-induced insomnia by intrahypothalamic microinjection of l-5-hydroxytryptophan

Para-chlorophenylalanine, a blocker of serotonin biosynthesis by inhibiting tryptophan hydroxylase, induced total insomnia which was accompanied in cat by a permanent discharge of ponto-geniculo-occipital activity. l-5-Hydroxytryptophan microinjection (1–4 μg/0.5 μl) in the anterior hypothalamus 72 h after para-chlorophenylalanine administration, restored both slow wave sleep and paradoxical sleep with variable latencies for each state of sleep. On the contrary, ponto-geniculo-occipital activity was never suppressed. The hypnogenic effects of l-5-hydroxytryptophan were always followed by a return of the para-chlorophenylalanine-induced insomnia. On the other hand, the temperature recording did not show any alteration of the cerebral temperature after para-chlorophenylalanine treatment but the subsequent l-5-hydroxytryptophan microinjection was followed by hyperthermia. Using immunohistochemistry for serotonin after intrahypothalamic l-5-hydroxytryptophan microinjection in parachlorophenylalanine-pretreated cat, we defined a restricted region of the anterior hypothalamus possibly responsible for the hypnogenic effect. This region included the lateral hypothalamus and the anterior hypothalamic area. It is suggested that the reversible hypersomnia after l-5-hydroxytryptophan microinjection in the anterior hypothalamus in para-chlorophenylalanine-pretreated cat is due to a neurohormonal action of serotonin: serotonin could act upon the anterior hypothalamus which secondarily inhibits a waking system located in the ventrolateral hypothalamus leading to the appearance of paradoxical sleep.

Intrahypothalamic microinjection of calcitonin prevents stress-induced gastric lesions in rats

Injection of salmon calcitonin into the lateral ventricle or the cisterna magna was reported to potently inhibit gastric lesions induced by cold restraint stress. The forebrain sites of action were investigated using unilateral microinjection of salmon calcitonin prior to exposing conscious pylorus-ligated rats to cold restraint stress for 2 hr. Calcitonin (100 ng), microinjected in 100 nl volume by pressure ejection from glass micropipette positioned into the lateral or ventromedial hypothalamus or the paraventricular nucleus, prevented the development of gastric lesions whereas microinjections into the caudate putamen, the cerebral cortex or the hippocampus were ineffective. The antiulcerogenic effect of lateral hypothalamic injection was dose dependent and specific since calcitonin gene-related peptide, tested under the same conditions, had no effect. Microinjection of calcitonin at 100 ng dose into the ventromedial hypothalamus did not modify gastric secretion whereas microinjection into the lateral hypothalamus or the paraventricular nucleus induced 75–82% inhibition of gastric acid output in pylorus-ligated rats exposed to restraint stress. These results demonstrate that the hypothalamus including the lateral, ventromedial and paraventricular nuclei are responsive sites of action for calcitonin-induced inhibition of cold restraint stress ulcers. The antiulcerogenic effect may be related to suppression of gastric acid secretion along with other mechanisms that remain to be elucidated.

Evidence for GABAergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats

The hypothesis that endogenous γ-aminobutyric acid (GABA) suppresses the activity of a latent hypothalamic sympathoexcitatory mechanism was tested by examining the effects of stereotaxic intrahypothalamic microinjection of drugs influencing GABAergic inhibitionv in anesthetized rats. Bicuculline methiodide (BMI) 1–25 ng, a competitive antagonist at post-synaptic GABA receptors, as well as isoniazid (INH) 35 and 70 μg and 3-mercaptopropionic acid (3MP) 0.02 μl, inhibitors of GABA synthesis, all evoked marked increases in heart rate and modest pressor responses. However, while the effects of BMI appeared almost immediately and peaked within 10 min of injection, changes caused by INH or 3MP developed much more slowly, attaining a maximum 35–40 and 19 min after injection, respectively. The effects of BMI on heart rate were blocked by pretreatment with propranolol 2 mg/kg i.v. or hexamethonium 20 mg/kg i.v. plus atropine 2 mg/kg i.v. and were shown to be highly localized to the posterior hypothalamic nucleus and the adjacent lateral hypothalamus. In addition to the cardiovascular effects, BMI also elicited dose-related increases in respiratory rate which were independent of the heart rate changes although they followed a similar time course. The results support the notion that hypothalamic GABA inhibits a local mechanism capable of generating cardiorespiratory arousal.

Topographic studies of the effects of microinjections of muscimol on the hypothalamic control of feed intake in sheep.

Ten sheep were used to define the anatomical basis for the feeding systems sensitive to gamma-aminobutyric acid, by using intrahypothalamic microinjections of the gamma-aminobutyric acid agonist, muscimol. In satiated sheep, 1 microL of muscimol (0.5 nmol/microL) elicited feeding when injected into paraventricular, ventromedial, and anterior hypothalamic areas. Similar injections into 39 sites tested in 6-h fasted sheep failed to decrease feed intake. The data suggest that neurons sensitive to gamma-aminobutyric acid in medial hypothalamus may be involved in the initiation of feeding.

Ontogenetic, thermoadaptive and pharmacological changes in threshold and slope for thermoregulatory reactions in the guinea-pig ( Cavia aperea porcellus)

1. 1.|The threshold and slope of thermoregulatory responses to external cooling or heating were examined before and after pharmacological treatment in newborn and 4-week-old guinea-pigs adapted to different ambient temperatures. 2. 2.|Only threshold temperature for shivering ( T b-Sh ) increased during ontogeny and adaptation to heat; the threshold for respiratory evaporative heat loss ( T b-REHL ) remained relatively stable. The zone between T b-Sh and T b-REHL was narrowed in adult and in warm-adapted animals. 3. 3.|Intrahypothalamic microinjections of noradrenaline, narrowed whereas 6-hydroxydopamine and 5-hydroxytryptamine widened the width of the zone between T b-Sh and T b-REHL . 4. 4.|It is concluded that both noradrenergic and serotonergic inputs into the hypothalamus act as antagonists determining the width of the zone between T b-Sh and T b-REHL .

The role of protein synthesis in the hypothalamic mechanism mediating pyrogen fever

The effects of inhibition of protein synthesis by anisomycin on the pathogenesis of fever and normal thermoregulatory processes were investigated in the conscious and unrestrained cat. Subcutaneous administraton of 5.0–25.0 mg/kg of anisomycin prevented the fever normally evoked by an intravenous infusion of either 1.0 ml (10 8 organisms) of a 1:10 dilution of S. typhosa or 1.0–5.0 ml (3.5×10 5−2.1×10 7 cells/ml) of endogenous pyrogen. In addition, systemic pre-treatment with anisomycin delayed and/or blocked the fever typically elicited by a direct micro-injection into the anterior hypothalamic, preoptic area (AH/POA) at AP 12.5–16.0 of 1.0 μl of the endotoxin. Anisomycin did not alter the hyperthermic response to an anterior hypothalamic injection of either 1.0–7.0 μg/1.0 μl of serotonin (5-HT) or 100.0 ng/1.0 μl of prostaglandin (PGE). Inhibition of protein synthesis, furthermore, did not prevent the fall in body temperature usually produced by an intrahypothalamic micro-injection of 2.33–14.0 μg/1.0 μl of either norepinephrine (NE) or dopamine (DA). The thermoregulatory capacity of the cat was unaffected by the administration of comparable doses of anisomycin, i.e., the animal was able to maintain normal body temperature (±0.5°C) when exposed to an ambient temperature of either 10°C or 34°C. These results strongly suggest that the synthesis of new protein within the region of the AH/POA is a functional requisite for the development of a pyrogen-induced fever.

The site of action of corticosteroid antipyresis in the rabbit.

1. The antipyretic effects of corticosteroids on the fevers produced by bacterial and endogenous pyrogens in the rabbit were investigated. 2. Intravenous infusions of hydrocortisone and methyl prednisolone, when administered simultaneously with bacterial or endogenous pyrogens, failed to produce an antipyresis. 3. Pretreatment of rabbits with methyl prednisolone for 3 days diminished the febrile effect of both bacterial and endogenous pyrogens. 4. The fever produced by intrahypothalamic micro-injections of endogenous pyrogen was significantly attenuated by the simultaneous micro-injection of methyl prednisolone. 5. These results indicate that the antipyretic effect of steroids in the rabbit is the result not of a peripheral inhibition of endogenous pyrogen production, but rather of an action on the central nervous system.

Chemical coding of the hypothalamic neurones in metabolic control. I. Acetylcholine-sensitive neurones and glycogen synthesis in liver

To specify the lateral hypothalamic (LH) neurones concerned with regulation of liver glycogen metabolism, the changes in the activities of liver glycogen synthetase and phosphorylase were analyzed after microinjection of different neurotransmitters 3nto the LH of rats. Cholinergic stimulation of the LH with minute amounts (5 x 10 -9 - 5 × 10 -8mole) of acetylcholine or carbachol resulted in a marked increase of the active form of glycogen synthetase, but it did not affect the phosphorylase activity. Total activity of glycogen synthetase (active plus inactive forms) was not increased significantly, indicating that the effect of cholinergic stimulation was due to activation of the enzyme. Noradrenergic, dopaminergic, serotonergic and GABAergic stimulations of the LH, on the other hand, did not affect appreciably the activities of glycogen synthetase and phosphorylase in the liver. The effect of cholinergic stimulation of the LH on liver glycogen synthetase was blocked by previous intrahypothalamic microinjection of anticholinergic agents, such as atropine and scopolamine, or by previous intraperitoneal injection of N-methylatropine or hexamethonium. These results suggest that, among other neurones, cetylcholine-sensitive neurones in the LH are specifically concerned with regulation of glycogen synthetase, and that the cholinergic system in an LH-vagal pathway mediates the neural regulation of glycogen synthesis in the liver.

A dissociation between temperature regulation and fever in the rabbit.

1. The role of 5-hydroxytryptamine (5-HT) in temperature regulation and in fever in the rabbit has been investigated. 2. Intrahypothalamic microinjections of 5-HT in the conscious rabbit alters body temperature in a dose-dependent manner. 3. Low doses (5-5nmol) of 5-Ht and control saline injections produced a small, non-significant increase in temperature, with a long latency. 4. Doses of 14 nmol 5-HT produce a hyperthermia with a 45 min delay; while microinjections of 28 nmol result in a biphasic response; an initial short hypothermia is followed later by a hyperthermia. 5. Depleting the rabbit's brain of 5-HT by pretreatment with p-chlorophenylalanine (PCPA) fails to affect its body temperature at thermoneutral temperatures but significantly impairs the ability to thermoregulate against a cold stress. 6. PCPA pretreatment did not, however, impair the febrile response to bacterial pyrogen and prostaglandin E1. 7. These results reveal a dissociation between the effects of 5-HT depletion on temperature regulation, and on fever. The site of action of 5-HT in temperature regulation must be proximal to the fever input, but distal to the convengence of peripheral and hypothalamic temperature inputs.