THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276~262-269(1996) Circadian Rhythm in Brain Gamma Aminobutyric Acid Levels in the Cockroach, Leucophuea muderae J . McCAY, K. ROMERO, J. GIBSON, J. NEWTON, L. WILSON, JENNIFER WRIGHT, D.B. DAHL, AND B.R. FERRELL Llepartments of Biology and Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101 ABSTRACT Brain levels of y-aminobutyric acid (GABA) were determined using high performance liquid chroinatography coupled with fluorometeric detection at 3-h intervals throughout a 24-h time period in roaches acclimated to a photoperiodic cycle of 12 h of light and 12 h of darkness at 25 1?: 2°C to establish whether or not the levels of this inhibitory neurotransmitter vaned temporally. Brain levels of GABA did vary temporally, with highest levels occurring a t midday. Therefore, an additional experiment was conducted to determine if this rhythm persisted on a circadian basis. Brain levels of GABA were determined a t two circadian times (CT)that corresponded t o the times during the daily cycle when levels were highest and lowest in animals held under constant darkness. The differences in GABA levels observed at these two times of day in animals held under a lightldark cycle persisted on the second cycle under conditions of constant darkness, indicating that the rhythm of brain GABA levels may persist on a circadian basis. Q 1996 WiIey LISS, Inc. Invertebrates offer excellent models in which to study circadian mechanisms involved in timing the occurrence of bioch,emical, physiological, and behavioral events and how the circadian system is organized. Cockroaches, particularly Leucophaea maderae, have keen studied extensively in this regard (for reviews see Page, '89a, b, '90). Circadian rhythms in lcicomotor activity (Nishiitsutsuji-Uwo and Pittendrigh, '68), eye sensitivity t o light (Wills et al., 'G),retinula cell morphology (Ferrell and Reitcheck, '931, cytochrome oxidase activity (Lavialle el; al., '891, and neural output from optic lobes measured in vitro and in vivo (Colwell and Page, '90) have been characterized in this species. The paceniaker(s) that regulates the expression of locomotor (activity(Nishiitsutsuji-Uwo and Pittendrigh, '68; Page, '82, '83) and eye sensitivity to light (Wills et al., '85) rhythms consists of a pair of oscillators, each of which has been localized t o the lobula neuropil within each optic lobe. These oscillators are mutually coupled and together regulate the expression of locomotor activity and eye sensitivity to light rhythms via neural pathways. In addition, this pacemaker(s) is (are) photically entrained via a neural pathway from the compound eyes (for review see Page, '90). That a single pacemalker locus can regulate a number of separate processes is consistent with data from mammals (Rusak and Zucker, '79; Moore, '79). The supratchiasmatic nucleus (SCN) 0 1996 WILEY-LISS, INC. has been implicated in regulating a wide range of rhythmic processes in mammals, for example. One explanation for the endogenous mechanism controlling the temporal organization of circadian rhythms involves synchronized neural output via efferent pathways from the optic lobe pacemakers t o broad areas of the nervous system. It is envisioned that altered neural output temporally organizes the expression of behavioral, physiological, and metabolic rhythms via its widespread influence on central nervous system activity (Page, '89a, '90). The specific responses to this output at intermediate neural and local effector tissue levels might determine the temporal expression of behavioral and physiological events. Evidence in support of this idea comes from experiments in which neural activity was monitored from the cut end of the optic tract of cultured optic lobes isolated from L. maderae (Page, '89a,b). Electrical recording data from efferent neuronal pools indicate that output from the oscillators controlling rhythms of locomotor activity is greatest during subjective day and lowest during subjective night. Received January 24, 1996; revision accepted June 28, 1996. Address reprint requests to Blaine R. Ferrell, Department of Biology, Western Kentucky University, 1 Big Red Way, Bowling Green, KY 42101. CIRCADIAN RHYTHM OF BRAIN GABA LEVELS IN A ROACH 263 It would seem that the neural output from the tory neurotransmitter prevalent in the central neroptic lobe oscillators measured in L. maderae vous system of vertebrates (Moore and Speh, '93) might be inhibitory in light of the fact that increased neural output occurs at a time coincident with decreased locomotor activity (Page, '89a). Additional evidence that optic lobe neural output is inhibitory comes from electrical recordings taken from cervical connectives in intact roaches. Peak impulse activity occurred during the animals' subjective night. Severing the optic tract, thus preventing neural output from the optic lobe oscillator from influencing the cervical connectives, abolished this rhythm and the impulse frequency increased tenfold over subjective daytime levels. These findings are consistent with an inhibitory role for neural output from optic lobe oscillators (Colwell and Page, '90). These findings are also consistent with results from studies in nocturnal and diurnal mammals (Inouye and Kawamura, '79; Kurumiya and Kawamura, '88), in which neural activity in the suprachiasmatic nucleus, the putative circadian pacemaker, is greatest during subjective daytime, whereas the neural activity is lowest at this time of day elsewhere in the brain. The suggestion that neural output from the oscillators is general and inhibitory is tempered by the findings in Limulus polyphemus (Kass and Barlow, '92) and a cricket (Tomioka and Chiba, '92) that increased efferent neural activity recorded from excised brains occurs on a circadian basis with heightened activity during subjective night. Furthermore, there may be different neural pools conveying different circadian information, as found in the cricket, Gryllus bimaculatus, where increased neural activity occurred during subjective days or subjective nights in different neuronal pools monitored simultaneously (Tomioka and Chiba, '92). Evidence from electrical recordings from single interneurons associated with the visual system in another cockroach, Blaberus giganteus (Bult and Mastebroek, '93) indicates that three visual interneuron types express circadian activity, each with their maximal activity occurring at a different time of day. Although neural output from the pacemaker has been examined in L . maderae (Colwell and Page, '901 and other arthropods (Kass and Barlow, '92; Tomioka and Chiba, '86, '92; Bult and Mastebroek, '93), its effect on the temporal pattern of specific neurosecretions, which could have broad effects on neural and general tissue activity, has not been investigated in this model organism. Gamma aminobutyric acid (GABA) in an inhibi- and invertebrates (Takeuchi, '76; Pfeiffer-Linn and Glantz, '91), with high levels in the optic lobes (Pfeiffer-Linn and Glantz, '91; Garcia and Arechiga, '86). GABA occurs at high levels in nuclei associated with the circadian pacemaker and appears to be the principal neurotransmitter of the circadian system in mammals (Moore and Speh, '93). Furthermore, pharmacological studies have implicated GABA in the photic entrainment mechanism of the clock in mammals (Ralph and Menaker, '89). Because of the analogous organization of the circadian system in mammals and L. maderae and the presence of high levels of GABA in the central nervous systems of both groups, characterization of a daily and circadian rhythm in brain GABA levels seemed a good place to begin in determining whether or not GABA plays a role in the circadian system of this model organism. Results of this study indicate GABA does play a role in that it varies temporally on a circadian basis indicative of endogenous control by a pacemaker. MATERIALS AND METHODS Animals Adult male L. maderae were transferred from a rearing colony in our laboratory maintained under a 12-h light/l2-h dark cycle at room temperature to environmental chambers set at the same photoperiod conditions and a constant temperature of 25 -c 2OC. Light onset occurred at 0600 h. Roaches from which brains were to be excised were placed in clear plastic containers (19.0 cm [Ll x 14.0 cm [HI x 10.5 cm [WI). Representative roaches were placed in running wheels equipped with magnetic reed switches wired to an EsterlineAngus event recorder. Each revolution of the wheel produced two dashes on a paper strip-chart moving at a constant rate. These animals were used t o monitor entrainment and circadian times during conditions of constant darkness. All animals were acclimated at least 10 days prior t o brain removal. Surgery Animals were removed from plastic cages in the environmental chambers either under room light conditions during daytime hours or under dim red light (i.e., safelight) during nighttime or constant darkness conditions. At each sampling time, three t o four animals were flash frozen in -70°C petro- 264 J. McCAY ET AL. leum ether in order to immediately stop any bioPreparation of standards chemical reactions. The animals were then removed Standards were 0.2, 0.5, 1.0, or 2.0 pg/mL from the petroleum ether and placed in a freezer. GABA 4.5 pg/mL AVA in methanol. Standards Brains were maintained frozen until they were were stored in a -70°C freezer until HPLC analyhomogenized. A frozen roach was taped t o a Petri sis could be carried out. dish with its head protruding through an opening. The Petri dish was placed under a dissecting HPLC, column, and fluorometric microscope and a flap oT cuticle encompassing both detection conditions antennae was removed using a razor blade scalThe assay for GABA was a slight modification pel and iridectomy scissors, The exposed brain was of that used in vertebrates (Sunol et al., '88). A then removed by sectioning the left optic nerves, Varian (5000) HPLC and a CI8reverse phase collifting the left optic lobe with forceps, and peeling umn with dimensions of 22 cm x 4.6 mm and 5 the brain toward the right side while transecting pm particle size (Supelco) was used for the assay. all tissues connected to the brain. The column was coupled to a Shimadzu fluorometric detector (RF-540) operated at an excitation Tissue preparation for injection onto the wavelength of 355 nm, emission wavelength of 440 HPLC column nm, and a bandwith of 10 nm. The mobile phase, Each excised brain was placed in an Eveljham 45% acetonitrile (by volume) and 55% phosphate tissue homogenizer chilled by ice (approximately buffer at a pH of 2.8, was delivered isocratically. 4"C), and 200 pL of methanol containing the surThe mobile phase was filtered and degassed using rogate standard, aminovaleric acid (AVA, Sigma, a section filter and moderate warming to encourSt. Louis, MO), was added, and the tissue was age the removal of gas prior to HPLC analysis. A homogenized for 30 sec. The homogenate was flow rate of 1.5 mL/min and temperature setting transferred t o a 1 mL inicrofuge tube and centriof 25°C was also used. fuged at 15,366g for 30 min. The supernatant was collected using a 1 cc: tuberculin syringe, the Analysis of endogenous GABA levels needle was replaced with a 45 pm syringe filter, Peak heights were measured using calipers for and the supernatant filtered into a clean 1.5 mL centrifuge tube. The filtered extract was then both GABA and AVA on graphs generated by an maintained frozen at --70°C until it was diluted integratodrecorder. A standard curve was gener1:4 (v/v, sample extract/mobile phase) in mobile ated from triplicate measurements of each stanphase immediately prior to injection onto the dard (Fig. 1)throughout each assay run and the equation of the regression line determined. This HPLC column. Samples and standards were derivatized by curve plotted the GABAAVA ratio (y-axis) against placing 10 pL of dilutedl sample (or standard) with the known GABA levels in the standard (x-axis). 50 FL of Borate (0.1 M boric acid solution, pH For brain samples (Fig. 21, the GABA:AVA ratio 10.51, 100 pL OPA (5.5 mg o-phthaldialdehyde, 40 was used to calculate the levels of GABA in the pL ethanethiol, and 4 mL methanol, Sigma) in a brain. Levels were determined in six brains at polypropylene microfuge tube, and allowed to re- each sampling interval during Experiment 1.Samact for 90 sec on a vortex mixer. The derivatization pling occurred at 3-h intervals throughout a 24-h reaction was stopped bly adding 100 pL of glacial time period in which roaches were acclimated to acetic acid. Immediately following the addition of a 12L:12D photoperiod cycle. A brain sample from the glacial acetic acid. an aliquot of derivatized each of the eight sampling times was analyzed sample (or standard) was injected into the HPLC under each assay run in order to ensure that any system using a 25 pL injection loop. Each sample temporal variance in GABA levels was not the re(standard) was injected in triplicate and the in- sult of assay variability. The existence of a daily jector loop rinsed with mobile phase several times variation in brain levels was determined using a between injections. A d erivatized methanol blank one-way analysis of variance and Student-Newmanwas injected each day prior t o the injection of Keul's range test at the 95% confidence interval. In Experiment 2, endogenous levels of GABA samples t o check for contamination. Several brain samples were spiked with an were determined in seven brains at each sampling equal volume of the high GABA standard and in- time. Sampling times coincided with the times of jected onto the HPLC to check for interference day that GABA levels were determined to be high and low in Experiment 1.Brain samples were obfrom other analytes. CIRCADIAN RHYTHM OF BRAIN GABA LEVELS IN A ROACH 12.5 ng GABA: 112.5 ng AVA BRAIN 80.0 80.0 AVA al U c al U 60.0 al k 0 1 d L, al + 265 40.0 .rl A 4 a rl al 20.0 0.0 I 2 4 6 Minutes Fig. 1. Representative chromatogram of a derivatized standard with GABA and AVA (aminovaleric acid). tained at 1200 h and 1800 h from roaches acclimated to a 12L:12D photoperiodic schedule (light onset at 0600 h) and at corresponding circadian times from roaches during their second cycle under constant dark conditions. Data were analyzed using a two-way analysis of variance and differences were identified using Student-NewmanKeul's range test. u E: Q) 60.t u m L El 0 -2 40.0 c, a L 2 20.0 0.0 7 2 4 6 Minutes Fig. 2. Example of a chromatogram of a brain sample. temperature conditions in Experiment 2 were similar t o those determined in Experiment 1. In addition, this difference persisted between measurements at circadian time (CT) 0600 h and CT 1200 h on the second day aRer light conditions were changed to constant darkness (Fig. 4). Circadian time 1200 h was the time of onset of activity in animals maintained under constant conditions of darkness (DD) and temperature, and was assumed RESULTS to be the equivalent of 1800 h (i.e., onset of activBrain GABA levels did vary with time of day in ity) in animals held under LD 12:12. The period roaches acclimated t o LD 12:12 photoperiodic con- length of the locomotor activity cycle recorded unditions and 25 2 2°C in Experiment 1. Levels of der constant dark conditions was divided into 24 brain GABA at 1200 h were 896 36 nghrain, equal intervals in order to establish other circawhereas they were 544 2 13 nghrain at 1800 h dian times, such as CT 0600 h (i.e., subjective (Fig. 3). Levels at other times of day sampled were midday). similar to those measured at 1800 h. Based on locomotor activity recordings of repThe difference in brain GABA levels determined resentative animals housed under the same envibetween 1200 h and 1800 h in animals acclimated ronmental conditions, it armears that roaches had under a LD 12:12 photoperiodic cycle and constant been acclimated t o the light-dark cycle and did J. McCAY ET AL. 266 Daily P a t t e r n of B r a i n GABA Levels 900a 800.rl m 700- $4 t S.E.M. I/ '\ II I 6005000 400- 1 \ '\ 4' $ 300'"O] 100 I o! : I I I -300 600 I I 1 I I 900 1200 1600 1800 2100 2400 HOUR Fig. 3. Daily pattern of brain GABA levels in cockroaches acclimated to a 12 h lighUl2 h dark cycle (horizontal bar) with light onset at 0600 h. "ean levels of brain GABA (dark squares) were determined from measurements in six roaches at each sampling time, each run in triplicate (S.E.M. = standard error of the mean). free run under conditions of constant darkness (Fig. 5). GABA in other arthropods, brain levels of GABA in L. maderae could be expected to range between 147 and 693 nghrain, values which include the levels determined in this study. We are unaware of any studies which have looked at the daily rhythm of GABA in arthropods. The temporal variation in brain GABA levels appeared to continue on a circadian basis. Differences between the maximum and minimum levels determined in roaches held under LD 12:12 conditions persisted during the second cycle of constant dark conditions. The fact that GABA levels peaked during subjective daytime, a time of day when locomotor activity and eye sensitivity to light are low, is consistent both with data regarding neural output from the optic lobe oscillators (Page, '89b; Colwell and Page, '90) and its inhibitory role in the central nervous system of arthropods (Takeuchi, '76; Pfeiffer-Linn and Glantz, '91). Interestingly, GABAergic neurons have been found in the major brain nuclei associated with the generation and control of circadian rhythms in mammals (Hendrickson et al., '83; Card and Moore, '84; Van den Pol and Tsujimoto, '85). Interaction of the SCN with areas they innervate is largely one of cyclic levels of inhibitory control (Moore and Speh, '93). In that several correlates between the circadian systems in mammals and L. maderue DISCUSSION Brain levels of GABA in L. maderae do vary with time of day, with highest levels occurring at midday. Levels found at other times of day in L. maderae (e.g., 544 to 689 ng) with a brain wet weight of approximately 2.5 mg (Garrett et al., '91) are comparable to those found in mouse brain cerebral cortex (i.e., 4.96 ng/2 mg wet weight; Kapetanovjc et al., '87; Sunol et al., '88). Comparisons with brain 'levels reported in other arthropods were difficulit to make in that reported levels vaned widely and levels were reported relative to protein levels (Breer and Heilgenberg, '85; Clarke and Donellan, '82; Fuchs et al., '89). Nevertheless, levels of GAB.A previously reported were substantially higher compared with other neurotransmitter levels mentioned (Fuchs et al., '89) consistent with the data reported herein. The ratio of 5-HT (5-hydroxyt~yptamine):GABA reported for other arthropods ranged from 1:35 to 1:165. The level of 5-HT measured in another cockroach, Periplaneta americana (Evans, '80) was approximately 4.2 nghrain consistent with the level of 5HT reported in L. maderae (Garrett et al., '91). Based on the range of ratios between 5-HT and CIRCADIAN RHYTHM OF BRAIN GABA LEVELS IN A ROACH 267 1400 El200 lsID 400 E 200 - o m I I 1200 1800 Time (h) CT CT 06001200 Fig. 4. Mean brain levels of GABA measured at midday (1200) and light offset (1800) in cockroaches acclimated t o a 12 h light/l2 h dark cycle (LD) and at subjective midday (CT 0600) and subject lights off (CT 1200) on day two after animals were transferred to constant dark conditions. Circadian times (CT)0600 and 1200 h in animals maintained under constant darkness (DD) were judged t o be times equivalent to 1200 and 1800 h in animals acclimated to a 12 h light/l2 h dark cycle based on examination of locomotor activity pat- terns of representative animals. The light and dark conditions are represented by the unshaded and shaded horizontal bar above the X axis. The zigzag line indicates the number of cycles in brain GABA levels. The range of values is given by the vertical lines and one standard deviation of the mean by the shaded boxes. Means with different letters were found to be significantly different at the 95%confidence level by a two-way analysis of variance and Student-Newman-Keul's range test. have been found, the role of GABA in the circadian system certainly warrants further pharmacological and anatomical investigation. Although the possibility that other analytes interfered with our GABA peaks exists, the fact that peak shoulders were not produced by spiking brain samples with known concentrations of GABA, that the levels determined from spiking brain samples fell within predicted levels, and that this assay (Sunol et al., '88) had been verified using GC-MS in vertebrates t o include no other analytes gave us confidence that we did in fact measure endogenous GABA levels and that temporal variances are real. Freezing and thawing experienced during our analysis should have had no effect on GABA levels in that no effect on determinations of GABA levels in cerebrospinal fluid samples that were analyzed using an HPLC/fluorometric assay (Manyam and Hare, '83) were observed due to repeated freezing and thawing of samples. The fact that maximum brain GABA levels occur shortly after the time of maximum neural output measured electrophysiologically from isolated optic lobes in this species (Colwell and Page, '90) is intriguing. In fact, the daily pattern of brain GABA levels is similar to the pattern of multiunit neural activity recordings from the optic J. McCAY ET AL. 268 TIME (hours) 1200 1800 0 600 1200 Fig. 5. Locomotor activity pattern of a representative cockroach used to verify that the animals were entrained and that brain samples were taken from cockroaches at the appropriate times under freerunning conditions. Lights were left off beginning with the clay indicated by DD>. The dark- ened regions on the lines for each day represent periods of locomotor activity. Prior to the transition to constant darkness, animals were acclimated to a LD 1212 photoperiod schedule with lights off a t 1800 h. tracts of optic lobes in vitro (Page, '89). However, the phase of the GABA rhythm is slightly delayed compared with phase of the rhythm in neural activity. Neural activity (Page, '89) and brain GABA levels peaked approximately 4 and 6 h after the onset of light under LD 12:12 conditions, respectively. Both neural and GABA peaks were short in duration and declined rapidly thereafter. Based on the neural activity pattern, the pacemakerb) function, and possibly GABA's, may not be through continued suppression of neural activity throughout the day, but rather as a master temporal signal that resets rhythms of neurons elsewhere that are responsible for the expression of individual physiological, behavioral, and biochemical rhythms. The coincidence between patterns in neural activity from the pacemaker and brain GABA levels does not argue for a causal relationship between neural output from the optic lobe pacemaker and GABA levels, however, especially in light of the fact that GABA levels were determined for whole brains not just optic lobes. Therefore, caution should be exercised in thawing conclusions regarding functional significance of varying GABA levels based on measurements of GABA in this study. Furthermore, it is impossible from data in the present study to know whether or not the elevated GABA levels represenl; an increase in the release of GABA from neurons or an accumulation of GABA in inactive neurons through increased synthesis and a decreased release for example. However, based on comparisons of the patterns of neural activity arid brain GABA levels, it seems more plausible to suggest that elevated GABA levels represent the accumulation of GABA in less active neurons and not increased release. Pharmacological and anatomical studies are currently in progress to explore the role of GABA in the circadian system in this model organism. Studies involving labeling of GABAergic neurons should help t o determine if they are localized in the lobula neuropil, site of the putative pacemaker in L. maderue, as GABAergic neurons are in mammals (Moore and Speh, '93). According to results of studies employing agonistic and antagonistic pharmacological agents carried out in hamsters, GABA is involved in the regulation of circadian responses to light and this regulation is mediated by both GABA, and GABABreceptors (Ralph and Menaker, '89; Smith et al., '90). A GABAergic systems involving both types of receptors can reset the mammalian circadian clock. However, GABAergic neural pathways may not be involved in the circadian timing system (Smith et al., '90). Similar pharmacological work needs to be carried out in L. maderue in order t o determine the role of GABA in its circadian system. 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