The Journal of Neuroscience, September 1967, 7(9): 2732-2736

Modulation of Activity of the Striatal Dopaminergic System During the Hibernation Cycle

Thomas S. Kilduff, S. Scott Bowersox, Kym F. Faull, Lori Zeller-DeAmicis, Carolyn M. Radeke,’ Roland D. Ciaranello, ii. Craig Heller,’ Jack D. Barchas, and William C. Dement Departments of Psychiatry and ‘Biological Sciences, Stanford University School of Medicine, Stanford, California 94305

To evaluate how the activity of a well-established neuro- thoxy-4-hydroxyphenethanol (MOPET) (Salzman et al., 1985). transmitter pathway is modulated by a behavioral state, 3H- On the basis of these findings, Salzman et al. concluded that spiperone binding sites and (DA) and DA metab- during hibernation higher extracellular levels of DA exist in the olite concentrations were measured in the striata of ground striatum and that DA degradation is shifted away from the squirrels in 5 phases of the hibernation cycle. Whereas levels aldehyde dehydrogenaseroute to metabolism by alcohol de- of striatal DA and its deaminated metabolite DOPAC did not hydrogenase.Moreover, it was suggestedthat the higher extra- change significantly, the concentrations of the O-methyl- cellular DA levels and the appearanceof a conjugated form of ated-deaminated metabolite, (HVA), de- DA may serve to prevent the development of receptor super- creased in all phases of hibernation relative to euthermia. sensitivity that might otherwise occur as a consequenceof the Striatal 3H-spiperone binding sites declined across the hi- prolonged periods of reduced neural activity characteristic of bernation cycle in parallel with the reduction of HVA con- hibernation. centration; receptor binding affinity was unchanged by The purpose of the present study was to assesswhether a arousal state. In conjunction with previously reported find- supersensitivity of postsynaptic striatal dopamine D, receptors ings, these results are consistent with the hypothesis that is present during hibernation, as Salzman and colleaguespos- hibernation is associated with a down-regulation of the post- tulated. To gain a more complete insight into the modulation synaptic D, receptors secondary to increased extracellular of activity that occurs in a well-establishedneurotransmitter DA concentration and reduced DA degradation. pathway during hibernation, an analysis of the concentrations of striatal DA and its metabolites was undertaken in parallel Mammalian hibernation is characterized by down-regulation of with measurementof receptor binding parameters.Our results multiple physiological systems(Heller et al., 1986). Thesephys- indicate that D, receptors decreasein concentration acrossthe iological adjustmentsat the systemiclevel are accompaniedby hibernation cycle as a consequenceof increased extracellular a generaldepression of CNS activity, as evidencedby decreased DA and decreasedextracellular DA degradation. responsivenessto external stimuli (Strumwasser, 1959a),atten- uated cortical EEG activity (Strumwasser, 1959b; South et al., Materials and Methods 1969; Shtark, 1970), and reduced sensitivity to neurochemical Animals. Golden-mantledground squirrels (Citellus lateralis) were im- stimulation (Beckmanand Stanton, 1982).Although there have olantedwith abdominaltelemeters (Mini-Mitter Co.. Sunriver.OR) to monitor body temperature (7’,) continuously during the hibernaiion beenno systematicstudies of the firing rate of singlecells during season.Animals were placed in-a constant-temperature environment at the hibernation cycle to this date, it is probable that unit activity 5°Cunder a light-dark 12:12 photoperiod. Four animalswere decapi- in most, if not all, brain regions is greatly attenuated during tatedbetween 1200-l 600 hr duringeach of 5 phasesof the hibernation entrance to and during deephibernation. Under suchconditions cycle:euthermia (T, = 37°C);entrance to hibernation(Tb = 20°C);day of “functional deafferentation,” the development of an up-reg- 1 of deen hibernation (T,. i 8°C): davs 4 or 5 of deeD hibernation (T,, < 8°C); aid arousal (T,‘ = 20”C):‘Thk brains were removed, dissected ulation or supersensitivity of postsynaptic receptors might be into subregions,and frozen on dry ice. Tissuesamples were weighed expected to occur. while still frozen and stored at - 80°C until they were analyzed (< 2 Recently, analysis of perfusatesextracted from the striatum months for D, receptors and 6 months for biogenic amines). One stria- of ground squirrelsduring hibernation revealed significantly in- turn from each animal was assigned to the receptor study and the con- tralateral striatum from each animal to analysis for biogenic amines. creasedlevels of both free and conjugated dopamine (DA), de- Biogenic amine analysis. Tissue samples (typically 60 mg) were pre- creasedconcentrations of DA metabolites homovanillic acid pared for analysis by sonication in 7 vol 0.4 N HCl at 0°C containing (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), and the a mixture of internal standards (caffeic acid and dihydroxybenzylamine appearanceof the unusual 0-methylated-deaminated metabo- [DHBA]) andcentrifuged (2000 x g, 15min). The pelletwas rehomoge- nized in 5 vol HCl and recentrifuged; the supematants were pooled. lite homovanillyl alcohol (HVOH), also referred to as 3-me- HVA waschromatographically separated from other compoundsby injection of the recentrifuged supematant (5 ~1) onto a reverse-phase Received Nov. 26, 1986; revised Mar. 23, 1987; accepted Mar. 23, 1987. microbore high-pressure liquid chromatography (HPLC)system (col- We thank Lauri Haak and Debby Egner for technical assistance.This work was umn C,,, 3~, 10 cm x 1.0 mm i.d.; eluan< 0.2 M NaH,PO,containing supported by NSF Grant BNS 8216918 (to H.C.H.), NIH MH23861 (to J.D.B.), 50 m&liter EDTA. 0.005% sodium octvl sulfate, and 5% CHXN. PH and NIH NS 23724 and MH05804 (to W.C.D.). 4.2,at 100pl/min)iCaliguri et al., 1985jand detectedampero&etri&ly Correspondence should be addressed to Dr. Kilduff, Sleep Research Center, (BASTL-8A glassycarbon electrode, Ag/AgCl reference,0.85 V). Quan- Department of Psychiatry, Stanford University School of Medicine, Stanford, CA titation was achieved using caffeic acid as the internal standard. The 94305. and DOPAC in the remaining supematant were ad- Copyright 0 1987 Society for Neuroscience 0270-6474/87/092732-05$02.00/O sorbed to alumina at pH 8.6 (1 ml, Tris-HCl, 3 M) andthen desorbed The Journal of Neuroscience, September 1987, 7(9) 2733

DAY 1 HIBERNATION: STRIATUM

n I z Figure 1. Saturation isotherm for ‘H- spiperone binding to striatum from a ground squirrel sacrificed on day 1 of hibernation (0, specific binding; 0, 200 300 400 500 600 nonspecific binding). Inset, Scatchard c3H] SPIPERONE (PM) plot calculated from these data. with HClO, (30 ~1, 0.4 M) and chromatographically separated by injec- mM NaCl as describedpreviously (Usdin et al., 1980). As found tion of the perchlorate solution onto a reverse-phase microbore HPLC in other rodent species,cinanserinPH-spiperone competition system, the same as described above with the eluant containing sodium octyl sulfate (99 mg/liter) and 0.4% acetone at pH 3.2. The compounds curves in the presenceof 4 mM MgSO, and 100 mM NaCl were were detected amperometrically (0.85 V), and quantitation was achieved biphasic (Boehme and Ciaranello, 1982; Hamblin et al., 1984), using DHBA as the internal standard (Mefford, 198 1). with a plateau between 0.1 and 500 nM. A concentration of 40 Receptor binding studies. Tissues were homogenized with a Polytron nM cinanserin was thus chosen to block 5-HT receptors. tissue grinder in 6 ml of ice-cold 50 mM Na-K phosphate buffer, pH A typical saturation isotherm and Scatchard plot are illus- 7.4. Homogenates were centrifuged (14,000 x g, 15 min, 4°C) and pellets were washed twice by resuspension and recentrifugation. The trated in Figure 1. All binding data were best describedby a final pellet was resuspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.4 1-site-binding model. Scatchard plots were uniformly linear, at 30°C) containing 4 mM MgSO,, 1 mM EDTA, and 100 mM NaCl, with regressioncoefficients ranging from 0.94 to 0.99. Further and then preincubated for 30 min at 30°C to destroy endogenous DA. evidence of receptor homogeneity wasprovided by Hill analyses D, receptors were measured by ‘H-spiperone (22.0 Ci/mmol; New En- gland Nuclear) binding in the presence of 40 nM cinanserin to mask of binding data, which yielded coefficients near unity (0.92- 5-HT receptors (optimal cinanserin concentration was determined in 1.10). preliminary competition studies). Nonspecific binding was defined as Receptor binding affinity (IQ did not differ amongthe 5 groups that remaining in the presence of 300 nM butaclamol. Total binding (Table 1); however, the number of 3H-spiperonebinding sites was measured in triplicate and nonspecific binding in duplicate. Binding (B,,,) varied consistently throughout the hibernation cycle (Fig. was initiated by adding 900 ~1 of tissue suspension (approximately 0.21 mg protein) to tubes containing receptor ligands made up in 1 mM HC1; 2). The B,,, values of day 1 and day 4/5 of deep hibernation final assay volume was 1 ml. Tissues were incubated for 20 min at 30°C were significantly reduced with respect to the euthermic B,,, with 6-7 increasing concentrations of)H-spiperone; final concentrations values (p < 0.01). B,,, values of both entrance and arousal ranged from 15 to 600 PM. Incubation was terminated by rapid vacuum filtration with subsequent rinsing (ice-cold 50 mM Tris-HCl, pH 7.4, at 4°C) over glass-fiber filters (Whatman GF/C) presoaked in 0.1% poly- ethylinimine (Sigma). Quantitation of receptor parameters was by Scat- chard analysis of radioligand binding data using the weighted nonlinear, Table 1. Parameters of 3H-spiperone binding to striata from ground least-squares curve-fitting program LIGAND, modified for use on the squirrels in different phases of hibernation IBM-XT. Data were analyzed according to both I- and 2-site-binding models. The statistical goodness-of-fit to computed curves was defined K* B Inax by the weighted residual variance. The binding model best describing Phase of cycle (PM) (pmol/g protein) the distribution of a given data set was determined by statistical com- parisons of residual variances (F test). Group comparisons of binding Euthermia 104.0 + 4.7 113.3 + 3.8 data were 2-tailed t tests corrected for multiple contrasts (Games, 1977). Entrance 117.5 * 1.2 90.4 k 8.4 Hibernation-Day 1 91.6 + 10.9 62.9 k 7.4 Results Hibernation-Day 4/5 76.8 k 11.6 77.0 + 5.6’ Receptor binding Arousal 102.4 ? 10.4 83.6 + 12.2 3H-spiperonebinding to squirrel striatum was linear in the range Values are means -t SEM. of 1.0-l .5 mg tissue/tube and reached equilibrium after 20 min Up < 0.0 1 compared with the euthermic (nonhibernation) condition. Corrected incubation. Equilibrium was facilitated by the presenceof 100 for multiple contrasts as per Games (1977). 2734 Kilduff et al. - Striatal Dopamingeric System During Hibernation

D2 RECEPTORS - STRIATUM

* p 4 0.0 1 vs. Euthermia 120 _

Figure 2. Concentrationsof ‘H-spi- EUTHERMIA ENTRANCE HlBEDRANyATlON HIBERNATION AROUSAL peronebinding sites (B,,,) in ground DAY 415 squirrelstriata acrossthe hibernation cycle (*, p < 0.05;**, p < 0.01). PHASE OF HIBERNATION CYCLE

groups were intermediate between the euthermic and hibema- tion values.

Biogenic amine analysis Analysis of the homologouscontralateral striata did not reveal any systematic changesin tissue concentrations of DA or DO- PAC acrossthe hibernation cycle. Figure 3 illustrates a typical chromatogram identifying peaks correspondingto specificbio- genie amines. Although the response obtained for authentic HVOH suggesteda conservative estimate of the sensitivity for the detection of this metabolite in tissue extracts to be 70 ng/ gm (typically, 4 @sample), we were unable to identify HVOH in any of the 20 samples.In contrast, the levels of HVA were significantly reduced in all phasesof the hibernation cycle with respectto euthermia (Fig. 4). Discussion The objective of the present study was to evaluate whether striatal 3H-spiperonebinding sites increasedin number (B,,,) during hibernation, as would be expected under conditions of “functional deafferentation,” or are “protected” from supersen- sitivity, as suggestedby Salzman and colleagues(1985). Our data (Fig. 2) demonstrateyet another result: 3H-spiperonebind- ing sitesclearly decreasein concentration acrossthe hibernation cycle. During stable deep hibernation, this reduction reaches statistical significance. During the transitional stages(entrance to and arousal from hibernation), B,,, values intermediate be- Figure 3. Chromatographyof extractsof striatafrom groundsquirrels tween those observed in euthermia and deep hibernation are sacrificedduring 2 stagesof hibernation.A, Chromatogramof the cate- present. cholamineextract from an animalentering hibernation. NE, norepi- The concentration of the DA metabolite HVA also decreased nephrine;DHBA (internalstandard), 3,4-dihydroxybenzylamine; DO- acrossthe hibernation cycle in parallel with reduced receptor PAC, 3,4-dihydroxyphenylaceticacid; DA, dopamine.B, Chromatogram concentrations (Fig. 4). Values in all 4 experimental groups of the unpurifiedtissue extract from an animalarousing from hiber- nation. SHT, ;5-HIAA, Shydroxyindoleaceticacid; HVA, underwent significant decreasesrelative to euthermic values. In homovanillicacid; CA (internalstandard), caffeic acid; HVOH, homo- contrast, the concentrations of DA itself and the other major vanillyl alcohol. DA metabolite DOPAC did not significantly change.These data The Journal of Neuroscience, September 1987, 7(g) 2735

STRIATUM

Dopamine 0 Dopac q 10,000 _ HVA I c5 P 0 8000. 5 p 1 T 2 6000 -

2

2000 -

Figure 4. Concentrationsof DA, EUTHERMIA ENTRANCE HIBERNATION AROUSAL DOPAC, and HVA in striatal ho- DAY 1 DAY 4/5 mogenatesof groundsquirrels sacri- ficed acrossthe hibernationcycle (*, PHASE OF HIBERNATION CYCLE p < 0.05; **, p < 0.01). suggestthat the degradation of DA is shifted away from HVA nation may be related to the increasedextracellular DA levels during deep hibernation to some other mode of metabolism. describedpreviously for this state(Salzman et al., 1985). Rather In an analysis of perfusates obtained from the striatum of than serving to “protect” postsynaptic striatal receptors from squirrels,Salzman et al. (1985) also documenteddecreased HVA developing supersensivity due to “functional deafferentation” levels during hibernation. However, in these perfusates, DA during hibernation, increasedextracellular DA may actually re- levels were also significantly elevated, DOPAC was not detect- sult in D, receptor down-regulation (Fig. 2). Alternatively, D, able and an atypical metabolite, MOPET, was detected during receptorsmay be degradedbut not replacedduring hibernation hibernation. We were unable to confirm any of theselatter find- becauseof decreasedanabolic activity. However, the fact that ings (Fig. 4). Although both studiesemployed HPLC with elec- the value for D, receptor concentration during arousal was in- trochemical detection, Salzman et al. analyzed striatal perfusates termediate between the hibernating and euthermia levels sug- collected in vivo, whereasour study was a post-mortem analysis gestsrapid initiation of receptor synthesis, if the latter hypoth- of striatal homogenates.Thus, our resultsare measurementsof esisis correct. total tissue concentrations of DA and its metabolites, whereas The mechanism by which extracellular DA is elevated in Salzman et al. presumably reported data obtained from extra- hibernation remains to be elucidated. At least 2 possibilities cellular fluid in the striatum. exist: Either presynaptic DA releaseis increasedduring hiber- Taken together, the results of Salzman et al. (1985) and the nation or the degradation of extracellular DA is decreasedsub- present study suggestthe following interpretation: (1) Although stantially greater than any decrement in presynaptic DA release. the total tissue concentration of DA in the striatum doesnot The former possibility is unlikely due to the profound depres- changeacross the hibernation cycle, a greater proportion of DA sion of electroencephalographicactivity documented in various is to be found extracellularly during hibernation than during brain regionsduring hibernation (Shtark, 1970). To substantiate euthermia; (2) the net metabolism of DA to DOPAC is not whether the striatum is truly subjected to functional deaffer- significantly changedduring hibernation, although extracellular entation during hibernation as expected, it would be desirable DOPAC decreases;(3) the total tissueconcentration of HVA is to record from cells, for example, in the nigrostriatal system decreaseddespite increasedextracellular DA levels and, pos- acrossthe hibernation cycle. Becausethe location of the source sibly, the appearanceof a conjugated form of DA (Salzman et of the D, receptorswithin the striatum is still unclear (Trugman al., 1985). This decreaseof extracellular HVA is consistentwith et al., 1986) selection of the precise cells to record is difficult. the idea of a shift of metabolism of the intermediate dopamine It would also be useful to measurestriatal DA releasedirectly metabolite, 3-methoxy-4-hydroxy-phenylacetaldehyde, from using in vivo voltammetry, as has been done in freely moving aldehyde dehydrogenaseto alcohol dehydrogenase(Salzman et euthermic animals (Braze11et al., 1984; Marsden et al., 1984). al., 1985). However, the failure to detect significant quantities Furthermore, measurementof the presumedpresynaptic D, re- of the alcohol dehydrogenasemetabolite HVOH in the present ceptor would provide an indirect index of DA releasein this study challengesthis interpretation. state (Seeman, 1980). The decreasedlevels of striatal D, receptors during hiber- The latter possibility of decreasedDA metabolism is a more 2736 Kilduff et al. * Striatal Dopamingeric System During Hibernation likely explanation for the increased extracellular DA levels dur- douamine and serotonin release after morphine. Life Sci. 36: 2269- ing hibernation. The decreased levels of HVA found in the 2275. Caliguri, E. J., P. Capella, L. Bottari, and I. N. Mefford (1985) High- current study as well as that by Salzman et al. are consistent sneed microbore liauid chromatoaranhv using C 18 packing material. with this hypothesis. The proposed shunting of DA degradation Anal. Neurochem. 37: 2423-242s. - _ - - - from aldehyde dehydrogenase to alcohol dehydrogenase could Chesselet, M. F., A. Cheramy, T. D. Reisine, and J. Glowinski (198 1) best be addressed by direct measurement of the activities of Morphine and b-opiate agonists locally stimulate in vivo dopamine these enzymes in the euthermic and hibernating states. Our release in cat caudate nucleus. Nature 291: 320-322. Games, P. A. (1977) An improved t table for simultaneous control results do not support the shunting hypothesis because of the on g contrasts. J. Am. Stat. Assoc. 72: 53 l-534. failure to detect significant quantities of the metabolite MOPET Hamblin, M. W., S. E. Leff, and I. Creese (1984) Interactions of ag- during hibernation. onists with D-2 dopamine receptors: Evidence for a single receptor These changes in striatal DA metabolism and 3H-spiperone population existing in multiple agonist affinity-states in rat striatal membranes. Biochem. Pharmacol. 33: 877-887. binding may reflect alteration in endogeneous opiates during Heller, H. C., X. J. Musacchia, and L. C. H. Wang (1986) Living in the hibernation cycle. It is now well established that opiates the Cold, Physiological and Biochemical Adaptations, Elsevier, New affect DA release (Chesselet et al., 198 1; Yonehara and Clouet, York. 1984; Broderick, 1985) and turnover (Yonehara and Clouet, Kilduff, T. S., F. R. Sharp, and H. C. Heller (1982) [%]2-deoxyglucose 1984) in several species. Alteration of endogenous opiate levels uptake in ground squirrel brain during hibernation. J. Neurosci. 2: 143-157. in the CNS during hibernation has been implicated by the ob- Kramarova, L. I., S. H. Kolaeva, R. Y. Yukhanov, and V. V. Rozhanets servations that hibernating animals administered morphine do (1983) Content of DSIP, enkephalins and ACTH in some tissues of not develop physical dependence (Beckman et al., 198 I), where- active and hibernating ground squirrels (Citellus suslicus). Comp. as the abstinence syndrome develops in nonhibernating squir- Biochem. Physiol. 74C: 3 l-33. Marsden, C. A., M. P. Brazell, and N. T. Maidment (1984) An intro- rels at all times of year (Beckman et al., 1982). Whole brain duction to in vivo voltammetry. In Measurement of levels of met-enkephalin have been found to undergo a 2-fold Release, C. A. Marsden, ed., pp. 127-15 1, Wiley, Chichester, U.K. increase in hibernating ground squirrels (Kramarova et al., 1983), Mefford, I. N. (198 1) Application of high performance liquid chro- whereas leu-enkephalin does not change significantly. Last, in- matography with electrochemical detection in neurochemical analy- tracerebroventricular infusions of naloxone cause a dose-de- sis: Measurement of catecholamines, serotonin, and metabolites in rat brain. J. Neurosci. Methods 3: 207-224. pendent reduction of hibernation bout length (Beckman and Salzman. S. K.. C. Llados-Eckman. and A. L. Beckman (1985) In vivo Llados-Eckman, 1985). These observations are suggestive of anal&s of d&amine and its metabolites in the caudatenucleus during widespread alterations of the activity of neurotransmitter path- euthkrmia and hibernation. Brain Res. 343: 95-103. ways, as well as neural metabolism (Kilduff et al., 1982), during Seeman. P. (1980) Brain dooamine receptors. Pharmacol. Rev. 32: 229-313. ’ ’ the hibernating state. Shtark, M. B. (1970) The brain of hibernating animals. NASA Tech- nical Translation TTF-6 19. (Translation of Mozg Zimnespyashchekt, References Nauka Press, Siberian Branch, Novosibrisk.) Beckman, A. L., and C. Llados-Eckman (1985) Antagonism of brain South, F. E., J. E. Breazile, H. S. Dellman, and A. D. Epperly (1969) opioid peptide action reduces hibernation bout duration. Brain Res. Sleep, hibernation and hypothermia in the yellow-bellied marmot (M. 328: 201-205. j7aviventris). In Depressed Metabolism, X. J. Musacchia and J. F. Beckman, A. L., and T. L. Stanton (1982) Properties ofthe CNS during Saunders, eds., pp. 277-3 12, Elsevier, New York. hibernation. In The Neural Basis of Behavior, A. L. Beckman, ed., Strumwasser, F. (1959a) Thermoregulatory brain and behavioral pp. 19-45, Spectrum, New York. mechanism during entrance into hibernation in the squirrel Citellus Beckman, A. L., C. Llados-Eckman, T. L. Stanton, and M. W. Adler beecheyi. Am. J. Physiol. 196: 15-22. (198 1) Physical dependence on morphine fails to develop during the Strumwasser, F. (1959b) Regulatory mechanisms, brain activity and hibernating state. Science 212: 1527-l 529. behavior during deep hibernation in the squirrel, Citellus beecheyi, Beckman, A. L., C. Llados-Eckman, T. L. Stanton, and M. W. Adler Am. J. Physiol. 196: 23-30. (1982) Seasonal variation of morphine physical dependence. Life Trugman, J. M., W. A. Geary II, andG. F. Wooten (1986) Localization Sci. 30: 147-154. of D-2 receptors to intrinsic striatal neurones by quantitative auto- Boehme, R. E., and R. D. Ciaranello (1982) Genetic control of do- radiography. Nature 323: 267-269. pamine and serotonin receptors in brain regions of inbred mice. Brain Usdin, T. B., I. Creese, and S. H. Snyder. (1980) Regulation by cations Res. 266: 51-65. of [‘HI spiperidol binding associated with dopamine receptors of rat Brazell, M. P., N. T. Maidment, and C. A. Marsden (1984) Microelec- brain. J. Neurochem. 34: 669-676. trodes for in vivo neuroelectrochemistry. In Charge and Field Effects Yonehara, N., and D. H. Clouet (1984) Effects of delta and mu opio- in Biosystems, M. J. Allen and P. N. R. Usherwood, eds., pp. 499- peptides on the turnover and release of dopamine in rat striatum. J. 505, Abacus Press, Tunbridge Wells, Kent, U.K. Pharmacol. Exp. Ther. 231: 38-42. Broderick, P. A. (1985) In vivo electrochemical studies of rat striatal