Thyrotropin-releasing hormone effects in the central nervous system: dependence on arousal state.

Thyrotropin-releasing hormone was microinjected into the dorsal hippocampus of ground squirrels (Citellus lateralis) when they were at different levels of arousal, as assessed by electrophysiological and behavioral criteria. When administered to the awake animal, thyrotropin-releasing hormone produced dose-dependent decreases in body temperature accompanied by behavioral quieting and reductions in metabolic rate and electromyographic activity. The magnitude of these effects was greater when the peptide was microinjected during a period of behavioral activation. In contrast, administration of the peptide during slow wave sleep produced increased thermogenesis, an increase in electromyographic activity, and an increase in the amount of electroencephalographic desynchronization.

Physical dependence on morphine fails to develop during the hibernating state.

Physical dependence on morphine occurs in a typical fashion during the active state of the mammalian hibernator Citellus lateralis, but does not occur when morphine exposure is confined to the hibernating state. Morphine exposure during hibernation can produce stereotyped behavior, thus demonstrating partial responsiveness of the central nervous system to opioids during this natural state.

Seasonal variation in thyrotropin-releasing hormone (TRH) content of different brain regions and the pineal in the mammalian hibernator, Citellus lateralis.

We have measured the endogenous TRH concentration in the pineal and 9 brain regions of a seasonal hibernator, the golden-mantled ground squirrel, during euthermia and hibernation in order to investigate the possibility that changes in TRH concentration might occur in association with naturally-occurring changes in CNS-mediated physiological and behavioral processes. Regional TRH content was assessed by radioimmunoassay in adult animals that were killed during euthermia in the mid-portion of each season and during hibernation in mid-winter. No significant changes in TRH concentration related to season or to hibernation versus euthermic state were noted in the hippocampus, brainstem, or cerebellum. In the olfactory bulb, preoptic area, and pineal, seasonal variation within euthermic groups was evident. During hibernation, statistically significant decreases in TRH content occurred in the forebrain, olfactory bulb, hypothalamus, septum, preoptic area, and midbrain. Significant fluctuations during hibernation were also observed in the pineal. In this structure, TRH concentration varied in relation to the phase of the hibernation bout. TRH content in the last quarter of the bout was three times greater when compared to values observed in the first quarter of the bout. These results suggest that TRH may be involved in the control processes attributed to these regions and support a role for TRH in the neural control of hibernation.

Changes in body temperature and metabolic rate following microinjection of Met-enkephalinamide in the preoptic/anterior hypothalamus of rats.

The effects of Met-enkephalinamide (MET-ENKamide) on brain temperature (Tb) and metabolic rate (MR) were assessed following direct administration into the preoptic/anterior hypothalamus (PO/AH) of freely moving rats. Bilateral microinjections of saline or MET-ENKamide (1-25 micrograms/microliter) were delivered through cannula guide tubes previously implanted in nine animals. Thiorphan, an enkephalinase inhibitor, was microinjected into the PO/AH of two of the animals. All injections were made remotely at an ambient temperature of 22 +/- 1 degree C in a volume of 1 microliter. Measurements of Tb (via a brain-dwelling thermistor) and MR were recorded continuously. The ability of naloxone to antagonize the effects of MET-ENKamide was investigated by fashioning a double-barreled injection cannula to fit within each guide tube; 1 microliter of saline or naloxone (1-10 micrograms) was delivered bilaterally into the PO/AH followed by 1 microliter of MET-ENKamide (25 micrograms) 5-10 min later. PO/AH administration of MET-ENKamide (1-25 micrograms) produced dose-dependent increases in Tb preceded by dose-dependent increases in MR, with a characteristic time course of approximately 30 min. Naloxone antagonized the rise in Tb and MR, either partially or completely, depending on dose. When administered alone, naloxone had no effect on Tb or MR. Microinjection of thiorphan (10 micrograms) into the PO/AH evoked increases in Tb and MR that were similar to those responses induced by MET-ENKamide. These results support a role for endogenous Met-enkephalin in the regulation of Tb in the rat.

Thyrotropin-releasing hormone action in the preoptic/anterior hypothalamus decreases thermoregulatory set point in ground squirrels.

Earlier work has shown that thyrotropin releasing hormone (TRH) produces dose-dependent decreases in body temperature (Tb) and metabolic rate when microinjected into the dorsal hippocampus (HPC) or preoptic/anterior hypothalamus (PO/AH) of awake ground squirrels. This study employed a behavioral paradigm to investigate the possibility that TRH-induced hypothermia is associated with a decrease in thermoregulatory set point. Six animals were successfully trained to press a bar for radiant heat escape and cool air reinforcement in order to obtain a cooler ambient temperature (Ta). During experimental testing, the animals were microinjected remotely with TRH (10-1000 ng/microliters) or a control solution (sterile saline or TRH-OH) into the PO/AH. The micro-injections were delivered via bilateral injection cannulae inserted through chronic bilateral cannula guides that had been stereotaxically implanted under pentobarbital anesthesia. Cumulative and time-integrated bar presses were obtained on a computer generated display. Tb, measured in the brain via a bead-type thermistor, and chamber Ta were recorded continuously. Following TRH administration, a significant increase in mean bar-press rate was observed during the period in which Tb was falling, when compared to a comparable time period just prior to the microinjection. These findings complement results obtained from four animals that were trained to press a bar for heat reinforcement in a cold (- 10 degrees C) environment. In this alternative behavioral paradigm, microinjection of TRH into the PO/AH or HPC induced a decrease in mean bar-press rate as Tb was falling. The results support the hypothesis that TRH-induced hypothermia in golden-mantled ground squirrels is achieved by lowering thermoregulatory set point.

Prolongation of hibernation bout duration by continuous intracerebroventricular infusion of melatonin in hibernating ground squirrels.

Melatonin was infused intracerebroventricularly into hibernating golden-mantled ground squirrels (Citellus lateralis) maintained at 5 degrees C in darkness. Continuous infusion at a rate of 0.5 microliter/h was accomplished using an osmotic minipump. The effect of melatonin on hibernation bout duration was determined with reference to the natural trend in bout duration for each animal. At doses of 200 and 400 ng/h, melatonin produced a dose-related increase in bout duration. No effect was observed following control infusions of artificial cerebrospinal fluid or lower doses of melatonin.

Reduction of hibernation bout duration by intraventricular infusion of met-enkephalin.

The potential of brain met-enkephalin (met-enk) systems to modulate central nervous system (CNS) activity during periods of general depression (modeled by the mammalian hibernation state) was studied in the ground squirrel (Citellus lateralis). Following entrance into hibernation, continuous met-enk infusion into the lateral ventricle (1 microliter/h; 0.2, 1 and 5 micrograms/microliter) produced a dose-dependent reduction in bout duration ranging from 1.2 to 3.9 days (13.8-44.6% of baseline bout duration). We suggest that the activity of met-enk-releasing neurons may serve to increase the excitability of the depressed CNS, thus accelerating the termination of the hibernation bout.

The area of 2-[125I] iodomelatonin binding in the pars tuberalis of the ground squirrel is decreased during hibernation.

Quantitative receptor autoradiography was used to analyze 2-[125I]iodomelatonin binding sites in the brains of golden-mantled ground squirrels (Citellus lateralis). Specific binding appeared to be discretely localized to the pars tuberalis region of the pituitary that surrounds the medial basal hypothalamus. The total area of binding was significantly decreased in brains of hibernating squirrels as compared to those of awake, euthermic animals. These findings support a role for melatonin receptors of the pars tuberalis in seasonal behavior.

In vitro measurements of cholinergic activity in brain regions of hibernating ground squirrels.

High affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity were measured in synaptosomal P2 fractions from four brain regions in a mammalian hibernator, the golden-mantled ground squirrel. The 14CO2 evolution from [6-14C]glucose was also measured. Comparisons were made across the euthermic (not hibernating) and hibernation state in synaptosomes from cortex (CTX), preoptic area and hypothalamus (POA/HYP), olfactory apparatus (OA), and hippocampus (HPC). HACU was significantly increased in the CTX, from hibernating ground squirrels compared to euthermic animals. ChAT activity was significantly increased in the CTX and OA from hibernating animals. No change in either cholinergic marker was evident for the POA/HYP and HPC. The evolution of 14CO2 from [6-14C]glucose was generally, though not significantly, higher for synaptosomes from euthermic animals compared to hibernating animals. The results are discussed with reference to the involvement of cholinergic mechanisms in the control of hibernation.

Seasonal and state-dependent changes in brain TRH receptors in hibernating ground squirrels.

Quantitative autoradiography was used to localize and quantify thyrotropin-releasing hormone (TRH) receptors in the brain of hibernating (H), winter euthermic (WE), and summer euthermic (SE) animals to further explore the state-dependent physiological and behavioral effects of TRH in ground squirrels. The pattern of [3H]MeTRH binding (Kd 6.7 +/- 0.1 nM) was heterogeneous and highly concentrated in structures primarily associated with the limbic forebrain. Statistically significant seasonal changes (SE vs. WE) were reflected by decreases in TRH receptor binding in the arcuate nucleus, dorsomedial nucleus, and ventral pallidum of WE animals. Increased binding in WE animals was evident in the suprachiasmatic nucleus and choroid plexus of the lateral ventricles. Significant state-dependent changes (WE vs. H) were characterized by decreases in TRH receptor binding in the hypothalamic paraventricular nucleus, medial preoptic area, ventral tegmental area, and choroid plexus of the lateral ventricles of H animals. Increased binding occurred in the anterior cortical nucleus of the amygdala in H animals. The results suggest that naturally occurring changes in central TRH systems may be important in the mediation of physiological and behavioral processes that undergo seasonal and state-dependent adjustments in hibernating mammals.

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis.

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.

Differential effect of environmental temperature on morphine physical dependence and abstinence.

1) Ambient temperature (Ta) significantly influenced the display of 4 of the 14 naloxone-precipitated withdrawal signs (nesting, flat posture, vocalization, dyspnea) in morphine-dependent, non-hibernating ground squirrels (Citellus lateralis). 2) Analysis of variance performed on the six quantified signs revealed that Ta during withdrawal, but not during the development of physical dependence, was a significant factor in determining the expression of two signs (nesting and vocalization). 3) The interaction between the influence of Ta during the periods of morphine administration and abstinence was a significant factor in determining the expression of nesting behavior, a finding that is consistent with the natural role of nesting as a behavioral thermoregulatory response. 4) We conclude that environmental temperature modulates the expression of selected components of the naloxone-precipitated abstinence syndrome in C. lateralis without exerting a measurable influence on the development of morphine physical dependence itself.

Seasonal variation of morphine physical dependence.

The characteristics of morphine physical dependence in ground squirrels (Citellus lateralis) were examined during each of the four annual seasons. The results showed that this rodent hibernator exhibits a strong and characteristic naloxone-precipitated abstinence syndrome during its non-hibernation state, irrespective of the season. Although qualitatively unchanged throughout the year, the abstinence syndrome showed clear quantitative seasonal differences. These differences were evident in terms of both the number of occurrences of particular signs and the percentage of the morphine-dependent population exhibiting them.

Changes in striatal dopamine metabolism during the development of morphine physical dependence in rats: observations using in vivo microdialysis.

The present study examined the effect of prolonged morphine treatment on striatal dopamine (DA) release and metabolism, during the initial phase of the development of morphine dependence. Sprague-Dawley rats were implanted with chronic guides for microdialysis of the striatum. Morphine (two 75-mg pellets, subcutaneous implant) or placebo was given (12 hr) to pentobarbital anesthetized animals. Following recovery from anesthesia, morphine physical dependence was verified by the naloxone-evoked abstinence syndrome. Morphine produced significant increases in the dialysate level of DA nad its metabolites (DOPAC and HVA) above baseline compared to placebo treatment. HVA levels began to increase immediately following morphine administration, whereas DA and DOPAC levels began to increase after a latency of one and three hr, respectively. Morphine effects on striatal DA metabolism included changes in the metabolic disposition of DA. Increases in HVA concentration accompanied increases in DOPAC concentration up to a threshold value of DOPAC efflux; further increases in DOPAC level were associated with decreases in HVA level. These in vivo data suggest that morphine-induced changes in the regulation of striatal dopaminergic function may be an important component of the development of physical dependence.

Changes in CNS responsiveness during hibernation.

The ability of the midbrain reticular formation (MRF) to produce thermogenic responses and to trigger arousal from hibernation was tested during successive quarters of individual hibernation bouts. Golden-mantled ground squirrels (Citellus lateralis) were implanted with bilateral cannula guides into the MRF. Single, bilateral, 1-mul injections of acetylcholine (ACh) at concentrations of 50, 100, or 200 mug/mul were delivered in each quarter of the same bout or in selected quarters of different bouts. The results show that the magnitude of thermogenic responses evoked by ACh stimulation of the MRF was depressed during the early portion of the bout and increased as time in the bout elapsed. Furthermore, the magnitude of responses evoked during hibernation was depressed in comparison to those evoked during euthermia, indicating the influence of inhibition on the responsiveness of the CNS during hibernation. We suggest that during hibernation, a progressive change in responsiveness of the CNS, perhpas focused in the MRF, controls the duration of each hibernation bout.

Characterization of midbrain component of the trigger for arousal from hibernation.

The ability of the midbrain reticular formation (MRF) to trigger arousal from hibernation and to change body temperature (Tb) during euthermia was tested in golden-mantled ground squirrels (Citellus lateralis). During hibernation (ambient temperature 5-6 degrees C) microinjections of cholinomimetic compounds (acetylcholine and carbachol) triggered full arousal or produced transient increases in Tb in 15 out of 19 tests; MRF microinjections of norepinephrine (NE) and 5-hydroxytryptamine (5-HT), at the same concentrations that produced full arousal when microinjected into the preoptic/anterior hypothalamus (PO/AH) area in previous experiments, has no effect on Tb in 13 out of 16 tests. In experiments on euthermic ground squirrels tested at an ambient temperature of 25 degrees C, MRF microinjections of acetylcholine raised Tb, whereas the monoamines had no significant effects. These results show that cholinoceptive neurons in the MRF can trigger arousal from hibernation and increase Tb during euthermia. The failure of NE and 5-HT to produce any significant effects at the same doses that were reported to be effective in the PO/AH indicates that the functional organization of the MRF portion of the arousal mechanism is different from that reported for the PO/AH.

Spiramycin therapy for cryptosporidial diarrhoea in immunocompromised patients.

Spiramycin, 3 g daily, was used to treat immunocompromised patients with chronic diarrhoea due to Cryptosporidium. Most patients treated had acquired immune deficiency syndrome. Among 37 patients evaluated, 28 had a favourable response, defined as a reduction in the daily number of bowel movements to less than 50% of baseline and fewer than five. Twelve of these individuals had Cryptosporidium oocysts eradicated from stool. Spiramycin was well tolerated. These data suggest that spiramycin is effective in alleviating cryptosporidial diarrhoea in some immunocompromised patients and may lead to eradication of cryptosporidial oocysts.

Pineal melatonin: circadian rhythm and variations during the hibernation cycle in the ground squirrel, Spermophilus lateralis.

Variations in pineal melatonin content throughout a 24-hour period and during different phases of the hibernation bout cycle were studied in the golden-mantled ground squirrel (Spermophilus lateralis). In addition to pineal melatonin, the circadian variation in the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were also investigated in summer animals maintained at 22 +/- 2 degrees C, on a light:dark (L:D) schedule of 12:12 hr for 1 month (lights on at 08.00 hr). Pineal glands were collected from six animals in each group at 1200, 1600, 2000, 2400, 0200, 0400, and 0800 hr. Changes in pineal melatonin content during the hibernation bout cycle were investigated in ground squirrels housed at 4 +/- .05 degrees C in relative darkness (1.9-3.4 lux; 10:14 LD). Pineal glands were obtained between 12:00 and 18:00 hr from 30 animals during one of three phases of the cycle (deep hibernation, euthermic interbout, and entrance into hibernation). Pineal melatonin was also measured for comparison in six winter euthermic animals that were housed at 22 +/- 2 degrees C, on a L:D schedule of 10:14 hr. Melatonin was measured in individual pineal glands by radioimmunoassay. The daily melatonin rhythm in S. lateralis was characterized by a marked increase in pineal melatonin during the dark phase, in which peak nighttime values were nearly 20-fold greater than daytime basal levels. The daily rhythm for NAT activity paralleled the changes in melatonin, showing a peak activity at 0200 hr that was 45 times greater than mean daytime values.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the CNA trigger process for arousal from hibernation.

Ground squirrels (Citellus lateralis) produced three distinct types of thermogenic response during hibernation. These responses were evoked spontaneously as well as after stimulation produced by brief handling, or after microinjection of acetylcholine into the midbrain reticular formation. Type I responses were characterized by small magnitude and a slow (mean rate, 0.03 degrees C/min), variable rising phase. Type II responses were characterized by a smooth, rapid rising phase with a mean rate of increase of 0.11 degrees C/min and by an abrupt reversal of the rising phase within a restricted ceiling temperature band with a mean value of 9.4 degrees C. The third type of response, full arousal, was characterized by a return of body temperature to euthermic (nonhibernating) levels and by an early rising phase that was indistinguishable from the rising phase of type II responses. This indicates that the rising phase of type II responses and the duplicate portion of full arousals are produced by a common neuronal mechanism that functions as the trigger for arousal from hibernation, and that this mechanism can be spontaneously inhibited when increasing internal temperature reaches a hibernation ceiling level.