Regional changes in central monoamine and metabolite levels during the hibernation cycle in the golden-mantled ground squirrel

Regional changes in central monoamine and metabolite levels during the hibernation cycle in the golden-mantled ground squirrel

Brain Research, 563 (1991) 215-220 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391171245 215 BRES...

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Brain Research, 563 (1991) 215-220 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391171245

215

BRES 17124

Regional changes in central monoamine and metabolite levels during the hibernation cycle in the golden-mantled ground squirrel Laurel L. Haak 1" , Emmanuel Mignot 1, Thomas S. Kilduff 1'2, W.C. Dement 1 and H. Craig Heller 2 Sleep Disorders Center, Departments of I Psychiatry and 2Biological Sciences, Stanford University, Stanford, CA 94305 (U.S.A.) (Accepted 18 June 1991) Key words: Hibernation; Monoamine; Serotonin; Dopamine; Noradrenaline; Metabolite

We assayed various brain regions for levels of monoamines and their metabolites throughout the hibernation cycle of the golden-mantled ground squirrel Spermophilus lateralis. The tissue concentrations of serotonin, dopamine, norepinephrine and their metabolites were determined in the parietal cortex, striatum, midbrain, hippocampus, hypothalamus, and ports. Telencephalic regions exhibited the most significant variations in biogenic amine content. Cortical serotonin (5-HT) levels increased significantly at entrance (P < 0.0001) relative to other periods of the hibernation cycle, suggesting a role for 5-HT in the initiation of hibernation. Among striatal dopamine (DA) metabolites, 3-methoxytyramine was detectable only during euthermia and arousal; from entrance through arousal, homovanillic acid (HVA) levels were half that found during euthermia (P = 0.0001); and dihydroxyphenylacetic acid (DOPAC) levels increased during day 1 of hibernation (P < 0.0005). Midbrain DA (P = 0.0295) and hippocampal HVA (P = 0.0194) levels also changed significantly across the hibernation bout. The absence of a consistent change in any monoamine or metabolite throughout the brain precludes the possibility of preferential temperaturedependent impairment of an enzyme involved in biogenic amine synthesis or degradation and suggests that the levels observed reflect changes in neural activity specific to each brain region. Together with previous studies of brain 2-deoxyglucose uptake throughout the hibernation cycle, these data indicate that a transient change in afferent monoaminergic metabolism and neurotransmission in the forebrain is a necessary component for the entrance to hibernation. INTRODUCTION Hibernation is a physiological state in which extreme but regulated reductions in body temperature, metabolism, and other physiological systems occur to reduce energy expenditure. Although this process involves the central nervous system, the neurochemical mechanisms involved in hibernation are not understood. Central mononoaminergic systems have been implicated in the regulation of arousal states x° and thermoregulatory responses 7. Decreased noradrenergic (NE) activity in the whole brain and/or hypothalamus has been suggested as a prerequisite for hibernation 2. During entrance to hibernation there is a decrease in N E turnover in the brain, specifically in the hypothalamus 5'6'9. Furthermore, a 6-hydroxydopamine-induced lesion of N E cell bodies has been shown to facilitate hibernation in the ground squirrel z, while intracerebroventricular injections of NE trigger a rise in core body temperature and subsequent arousal from hibernation 1'9. Serotonergic (5-HT) mechanisms may be involved in controlling entrance to hibernation 3'15'1s, and ventricular

injection of 5-HT appears to suppress thermogenesis 7. Several reports demonstrate changes in 5-HT levels during entrance 2'15, although the direction of change varies between species examined. Depletion of 5-HT by electrolytic lesion of the median raphe or by injection of either para-chlorophenylalanine (p-CPA) or the neurotoxin 5,7-dihydroxytryptamine inhibits hibernation 3'1s. These results indicate that the anterior median raphe nucleus, in particular, may play a role in the initiation of hibernation 3. While N E and 5-HT systems have received much attention, relatively few studies have examined the role of dopamine (DA) in hibernation. Analysis of striatal perfusates in the golden-mantled ground squirrel revealed increased extracellular levels of both free and conjugated D A as well as decreased extracellular levels of both homovanillic acid (HVA) and dihydroxyphenylacetic acid ( D O P A C ) 16. In the same species, tissue levels of striatal D A and D O P A C did not change across the hibernation cycle, although both H V A levels and the number of striatal D 2 receptors declined significantly 1~, consistent with increased extracellular D A concentrations.

* Present address: Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, U.S.A. Correspondence: H.C. HeUer, Department of Biological Sciences, Stanford University, Stanford, CA 94305, U.S.A. Fax: (1) (415) 725-5356.

216 These studies suggest that differential activation/inhibition of central monoaminergic systems, especially in the hypothalamus, might induce and maintain hibernation. Most of these studies have compared only euthermic and hibernating animals, and many have focussed exclusively on the hypothalamus or non-specifically on the whole brain. In the present study, we have assayed monoamine and metabolite content in 6 brain regions in 5 phases of the hibernation cycle of Spermophilus lateralis. Monoamine levels were measured during euthermia, entrance, after one complete day of hibernation, after 4-5 days of hibernation, and during arousal from hibernation. Our results suggest that catecholamines, especially dopamine, undergo significant and meaningful changes in a number of brain regions and may therefore be involved in the regulation of hibernation. MATERIALS A N D M E T H O D S

phase B enabled us to detect NE content, and was used for the separation of midbrain, hippocampal, hypothalamic, and pontine samples. Concentrations of 3-MT and 5-HT were at times difficult to assess due to their late elution (ca. 1.5 and 1.8 tl). We used a modified version of mobile phase B, henceforth called mobile phase C, containing 12% methanol, to characterize a putative striatal 3-MT peak. Run time for C was 32 rain. Our detection and recording apparatus consisted of a Waters 460 Electrochemical Detector with a Waters flow cell including a glassy carbon working electrode and Ag/AgCI reference electrode. Applied potential was +0.85V. Chromatograms were recorded and integrated using a Spectra-Physics SP4290 integrator. The limit of detection was 5 pmol/g tissue. Prior to and following each set of runs, standards were injected to determine the retention time and to standardize the ratio of concentration to integration value of the monoamines and metabolites of interest. In any set of runs, tissue from each phase of hibernation was represented at least once, the order of the samples random. Monoamines in brain tissue were identified by their retention time as compared to standards. Retention times varied as much as 5% within and between runs. Tissue sample concentrations were calculated by integrating the area under the representative peak on the chromatogram of the tissue sample, and dividing this by the corresponding reference values, then adjusting for reference concentration and sample dilution.

Animals Golden-mantled ground squirrels (Spermophilus lateralis) were implanted with abdominal thermotelemeters (Mini-Mitter Co., Sun River, OR). Body temperatures were monitored continuously during the hibernation season while the animals were maintained at an ambient temperature of 5 °C in a constant 12:12 h light-dark cycle (lights on at 08.00 h). Animals were decapitated between 12.00 and 16.00 h during each of 5 phases of the hibernation cycle: euthermia (T b = 37 °C; n = 6); entrance to hibernation (T b = 20 °C; n = 5); day one of deep hibernation (T b < 8 °C; n = 6); day 4 or 5 of deep hibernation (Tb < 8 °C; n = 6); and arousal (T b = 20 °C; n = 7). Hibernation bout lengths were typically 10-14 days at this time of year. The brains were rapidly removed and dissected in a refrigerated room (4 °C) and immediately frozen on dry ice. Brain regions were weighed while frozen and stored in polyethylene tubes at -80 °C until analyzed.

Striatal 3-MT characterization Frozen reserve aliquots of euthermic (n = 5), day 1 (n = 5), and arousal (n = 5) striatal samples were thawed and used to verify the identification of a sample peak having the same retention time as reference 3-MT. These samples were analyzed within 24 h of each other first using mobile phase A and then C preparations. In addition, we established the voltage-response of the putative stri-

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Preparation of tissue and monoamine determination We used a procedure previously described by Mefford and Barchas 13. To frozen tissue, 4 vols. (14 vols. in the striatum) of liquid was added in the following proportions: 10% 4 N perchloric acid, 10% 10 #M ascorbic acid, and 80% double-distilled water. Tissue plus liquid were then sonicated at 4 °C for 10 s using a W-385 sonicator from Heat Systems Ultrasonics (50% duty cycle, output control at 5), and centrifuged for 3 min at 7100 g. Supernatant was collected and spun again in the same manner to generate the final sample. We injected 20/~1 of the recentrifuged supernatant into the liquid chromatography system and the remainder was aliquoted into 2 Eppendorf tubes and frozen at -20 °C. Such reserved aliquots were used to characterize a striatal sample peak having the same elution time as reference 3-methoxytyramine (3-MT).

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Liquid chromatography system The liquid chromatography equipment consisted of a pump (LKB-Bromma 2150), autoinjector with cooling unit (Waters WISP 712), and a C-18 column (Waters 27324 #Bondapak). Three variations of mobile phase were used at a flow rate of 1.0 ml/min. The pH of all mobile phase solutions was between 3.3 and 3.5. Mobile phase A (stock) was used for striatum and cortex analyses of monoamines and metabolites and was composed of 3 vols.:2 vols. 0.1 M citric acid (Sigma) and 0.1 M Na2HPO4*7H20 (Sigma), to which was added 7.5% HPLC grade methanol (Mallinckrodt). Run time averaged 30 min. Mobile phase B was similar to stock but included 2.70 mM octyl sulfate sodium salt (Sigma) and 4% methanol, which lengthened the run time to approximately 2 h. Mobile

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APPLIED POTENTIAL Fig. 1. Voltage-response curves for a 3-MT standard and putative 3-MT in a striatal sample. Electrode potential was stepped from 0.6 V to 1.10 V. See Materials and Methods for further details.

217 atal 3-MT peak (Fig. 1). Applied potential was stepped from 0.50 V to 1.10 V using 0.05 V or 0.10 V intervals. Using mobile phase A, 20/A of a 0.1/,M 3-MT standard was injected alternatively with 20 #1 of a pooled striatal euthermic sample. Peak areas were compared and graphed as a percentage of total signal at 1.10 V versus voltage applied to the glassy carbon electrode. Statistics In cases where monoamine or metabolite levels were within the limits of detection in 4 or more samples of each hibernation phase, data were analyzed using BMDP statistical software (1440

Sepulveda Blvd., Los Angeles, CA 90025, version 1988). Using the unweighted means of the groups, multivariate analysis of variance (MANOVA) was used to evaluate whether significant variation occurred in monamine/metabolite content among the brain regions across the hibernation cycle. Univariate ANOVA was then used to evaluate the significance of variation in specific monoamines and metabolites observed within a given brain region. In cases where tissue concentrations of monoamines or metabolites were below levels of detection in specific stages of hibernation, differences between hibernation phases were assessed using a Z2-test (G-statistic) 17.

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Fig. 2. Histograms of monoamines and metabolites that showed significant change across the hibernation cycle. A: cortical 5-HT levels showed a transient increase during entrance to hibernation. No change was found in the levels of the 5-HT metabolite 5-HIAA. B: in the striatum, D A metabolites 3-MT and HVA decreased during entrance and returned to euthermic values during arousal from hibernation. DOPAC levels increased on day 1 of hibernation, unlike HVA and 3-MT, suggesting a decrease in the activity of COMT, the enzyme that converts DOPAC to HVA and D A to 3-MT. C: day 1 hippocampal HVA increased from euthermic and entrance levels by a factor of 2.5. D: both DA and HVA levels decreased during hibernation in the midbrain, while DOPAC levels showed no significant difference among hibernation phases.

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218 RESULTS Of the 6 brain areas assayed, the hypothalamus and pons showed no statistically significant changes in monoamine or metabolite levels among hibernation phases. We measured DA, DOPAC, HVA, 3-MT, NE, 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT in these brain areas.

Cortex Cortical 5-HT levels showed a selective increase during entrance to hibernation (Fig. 2A). At other points in the hibernation cycle 5-HT levels were statistically equivalent. M A N O V A showed 5-HT to have significant variability among phases of hibernation (F = 11.080, df = 4,19; P = 0.0001). Although we also measured DA, DOPAC, HVA, 3-methoxy-4-hydroxyphenethanol (MOPET), 3-methoxy-4-hydroxymandelic acid (VMA), MHPG, and 5-HIAA in this brain area, no other monoamine or metabolite other than 5-HT changed significantly.

Striatum We m e a s u r e d t h e s a m e m o n o a m i n e s a n d metabolites in the striatum as in the cortex. Of these, levels of HVA (F = 9.25, df = 4,23; P < 0.0001), D O P A C (F = 7.48, df = 4,23; P < 0.0005), and 3-MT (F = 13.42, df = 4,23;

P < 0.0001) were significantly different among the phases of hibernation (Fig. 2B). HVA levels were over two times higher in euthermia than in any other hibernation phase. D O P A C levels increased during day 1 of hibernation. 3-MT levels fell below our level of detection (5 pmol/g tissue) during entrance, day 1, and days 4-5 of hibernation, while 3-MT was always detected during euthermia and arousal. Differences in 3-MT levels between hibernation phases were significant ( G h = 56.452; P < 0.001). To verify this striking result, we ran the same samples using another separation procedure (mobile phase C) and found once again that 3-MT was only detectable during euthermia and arousal (Fig. 3). Using mobile phase C, the oxidation potential of reference 3-MT and the sample peak having the same retention time were measured. The voltage-response curves generated yielded very similar potentials (Fig. I).

Hippocampus MANOVA revealed a significant variation among the biogenic amines in the hippocampus (F = 2.27, df = 16, 67.85; P < 0.01). As in the midbrain, both HVA (F = 3.58, df = 4,25; P = 0.0194) and, to a lesser degree, DA (F = 2.61, df = 4,25; P = 0.06) levels changed during the hibernation cycle. HVA levels for day 1 through arousal show a two-fold increase over euthermic and entrance levels (Fig. 2C). Tissue concentrations of D A fell from euthermic levels throughout the hibernation cycle.

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Fig. 3. Chromatogramsof a reference solution containing 6 pmol of a 3-MT standard and of ground squirrel striatal samples. Samples were separated using mobile phase C, as described in Materials and Methods. 3-MT was detected electrochemically at a potential of +0.85V. A: standard solution of 3-MT, which elutes at 31.28 min. B: striatum from an euthermic squirrel exhibits a peak corresponding to 3-MT at 30.42 rain. C: striatum collected after one complete day of hibernation has no peak corresponding to 3-MT. D: sample collected during arousal exhibits peak corresponding to 3-MT having a retention time of 30.34 min. All samples were analyzed on the same day.

219 In several runs, the levels of DA were below our levels of detection, and a ~2-test determined that differences existed in DA levels among the various hibernation states (Gh = 27.928; P < 0.001).

Midbrain Little variation among biogenic amines and their metabolites was observed across the phases of hibernation in the midbrain (F = 1.55, df = 16,49; P < 0.12). NE, DOPAC, HVA, and DA were assayed. Of these, both DA (F = 3.40, df = 4,19; P < 0.03) and HVA (F = 2.76, df = 4,19; P < 0.06) showed variation among hibernation phases. HVA levels were lowest during hibernation, and began to increase during arousal, while DA increased only during entrance (Fig. 2D). DISCUSSION In this study, we assayed tissue concentrations of monoamines and their metabolites in several regions of the golden-mantled ground squirrel brain throughout the hibernation cycle in order to evaluate whether systematic variations occur across the hibernation cycle. The brain regions with the most significant variations in monoamine and metabolite levels were telencephalic in origin: the cortex, striatum, and hippocampus. Only weak variation was found in the mesencephalon, with no systematic variation in the pons or hypothalamus. The concentration of any given neurotransmitter or metabolite reflects a combination of anabolic, catabolic, and release processes. Only by assaying metabolites in conjunction with their parent amines can one begin to assess catabolic and release processes. Each of the enzyme and transport pathways involved are temperaturedependent biochemical reactions. During hibernation, monoamine and metabolite levels might show complex changes resulting from differential inhibition of various metabolic reactions due to Q~0 effects. It is thus surprising that relatively few significant changes were observed in this study. In addition, the fact that no consistent change in any metabolite/monoamine was found throughout the brain suggests that there is no preferential impairment of a specific enzyme or pathway. Consequently, the differing levels of monoamines and metabolites across the hibernation cycle observed in the present study most likely reflect activity changes unique to each brain region. Among telencephalic regions, we observed that 5-HT levels in the parietal cortex selectively increased during entrance to hibernation. 5-HIAA, the primary 5-HT metabolite, did not show a concomitant increase (Fig. 2A). This elevation in 5-HT is consistent with previous observations of increased 5-HT levels in several brain regions

in Citellus erythrogenys 15, and suggests an increase in 5-HT synthesis and/or a decrease in 5-HT turnover. However, we found no changes in 5-HT or 5-HIAA levels in the striatum or other subcortical areas. These resuits indicate that the 5-HT fluctuations in the cortex do not reflect a global change in forebrain serotonergic activity. Previous investigators have found 5-HT to be an important factor in entrance to hibernation, although they have reported increases 2'14'15, decreases is, or no change 3'6 during entrance or hibernation as compared to euthermic 5-HT levels. Furthermore, blockade of 5-HT synthesis or transmission by intraperitoneal injection of p-CPA or median raphe lesions inhibits hibernation 3'5, indicating a physiological role for 5-HT neurons in the process of entrance to hibernation. Our results suggest that cortical 5-HT projections may be involved in the initiation of hibernation. The most pervasive neurochemical changes were observed in dopamine metabolism, which was significantly affected in the striatum, midbrain, and the hippocampus. As with 5-HT, the metabolic pattern observed appeared to reflect local rather than global changes. In the striarum, increased DOPAC levels, often correlated with increased nigrostriatal neurotransmission, were observed early in a hibernation bout (day 1) while HVA levels fell (Fig. 2B). This unexpected uncoupling of HVA and DOPAC, while confirming our earlier observations regarding striatal DA metabolites 11, indicates a complex change in the catabolism of dopamine in this structure, perhaps reflecting a reduction in the activity of catecholo-methyltransferase (COMT). In the hippocampus, we found rising HVA levels (Fig. 2C) and declining levels of DA during the same phase of the hibernation cycle. In midbrain, DA as well as HVA levels changed significantly across hibernation (Fig. 2D). Together, these observations suggest a transient change in nigrostriatal and mesolimbocortical DA transmission early in a hibernation bout. One of the most striking results of our study was the dramatic disappearance of striatal 3-MT during hibernation. Since a decrease in DA release is associated with a decrease in 3-MT levels 19'21, levels of 3-MT are considered good indicators of DA release. Thus, decreased 3-MT and HVA levels in the striatum during hibernation suggest decreased dopaminergic activity in this structure, although another explanation might be a decrease in COMT activity. Salzman et al) 6 have reported that in vivo DA turnover is affected during hibernation in a similar fashion. On the other hand, the increased striatal DOPAC levels we observed during day 1 of hibernation may be associated with increased nigrostriatal neurotransmission. This putative increase in nigrostriatal DA transmission would be transient, occurring early in

220 the hibernation bout and, along with the above-mentioned change in cortical 5-HT activity, could facilitate the entrance to hibernation. Several researchers have found that 3-MT, but not D A or its other metabolites, accumulates rapidly in the rat brain after decapitation, even with rapid dissection 4'8'19'2° and have proposed microwave irradiation as an alternative to decapitation to avoid this possibility. It is therefore possible that the absence of 3-MT during hibernation might be the result of faster post-mortem 3-MT accumulation at euthermic Tb than at the lower body temperatures of hibernating animals, due to temperature dependence of C O M T in this structure. As others have reported TM, no significant change in the D A content of the hypothalamus was observed during

several brain regions across the hibernation cycle, it has been proposed that the entrance to hibernation is mediated through two processes: (1) activation of a group of brain regions which include several hypothalamic nuclei, and (2) inhibition of neural activity in a group of cortical regions, including frontal cortex ~-~. Whereas the activation of hypothalamic regions increases as depth of hibernation increases, the inhibition of cortical activity was observed early in entrance to hibernation. Since both 5-HT and D A generally inhibit cortical neuron firing, the changes described here may be indicative of a transient modification of monoaminergic neurotransmission to the forebrain that underlies cortical inhibition of 2-deoxyglucose uptake and is a necessary c o m p o n e n t for the entrance to hibernation.

the hibernation cycle. There was a moderate but nonsignificant decrease in levels of NE during deep hibernation, as observed in previous studies 2'9, consistent with the proposal that NE turnover decreases in the hypothalamus during hibernation. Based on examination of 2-deoxyglucose uptake in

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Acknowledgements. We thank Carolyn Radeke for help with dissections, and Dr. S. Joe Miller, Malcolm Reid and Rand Wheatland for invaluable discussion and help in statistical matters. This work was supported by a grant from the Upjohn Company.

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