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Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro
Neuroscience Letters, 34 (1982) ?.83-288 Elsevier Scientific Publishers Ireland Ltd.
283
CIRCADIAN RHYTHMS IN ELF~TRICAL DISCHARGE OF RAT SUPRACHIASMATIC NEURONF~ RECORDED IN VITRO
GERARD GROOS* and JAN HENDRIKS**
National Institute o f Mental Health, Clinical Psychobiology Branch, Bethesda, MD 20~3.5 (U.S.A.) (Received October 29th, 1982; Accepted November 1lth, 1982)
Key words: arcuate nucleus - circadian rhythms - in vitro recordings - retrochiasmatic area suprachiasmatic nucleus
The discharge of suprachiasmatic (SCN), retrochiasmatic (RCA) and arcuate (A ~C) neurones was recorded in brain slices incubated for periods up to 30 h. The firing rate of cells in the SCN, but not in the RCA and ARC, exhibited a circadian rhythm similar to that reported for the SCN in freely moving animals. This rhythm cannot be ascribed to subtle exogenous diurnal variations in the incubation conditions. It :s co~lcluded that the SCN explant is capable of endogenous generation of at least one circadian cycle in vit;o
It is widely assumed that mammalian circadian rhythms are endogenously generated by self-sustaining pacemakers that are susceptible to entrainm,mt by the daily light-dark cycle in the environment [5, 8, 10]. Initial evidence that ~t least one such pacemaker exists in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus was based on lesion studies: it has been demonstrated that selective lesions or surgical isolation of the SCN in rodents result in the permanent elimination of various circadian rhythms [8]. Several recent investigations support the interpretation of the lesion studies that the mammalian SCN contain a circadian pacemaker. Mild electrical stim~alation of the SCN in blinded rodents results in phase shifts of thor free-running behavioural activity rhythms [7], and local pharmacological stimulation of the SCN affects normal circadian timekeeping in rats [5]. In vivo studies have,, identified visually responsive SCN cells that are optimally suited to code the environmental luminance changes that are responsible for photic entrainment of circadian rhythms [4, 5]. Furthermore, it has been demonstrated that the rat SCN exhibit circadian rhythms in glucose utilization and electrical activity [6, 9]. The rhyth~rJ of electrical multiple unit activity in the SCN persists when the • Address for ccrre;pondence: National Institute of Mental Health, Clinical Psychobiology Branch, Buildin~ 10, Room 4S-239, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A. • " Present address: U.N.D.P. - Wcrld Health Orga~zation, P.O. Box 280, Kingston, Jamaica.
284
animal is housed in constant environmental conditions and when the SCN is surgically isolated from the rest of the brain in a 'hypoth~amic island' preparation -6]. Significantly, in such a preparation electrical activity rhythms elsewhere in ~he brain are absent [6]. This finding demonstrates that the rhythm of the SCN is not neurona!ly driven by a circadian pacemaker out~ide the island. However, it does not exclude the possibility that the rhythmic activity of the SCN is driven by circadia,': ~emperature or humoral rhythms generated outsiae the island [5]. More stringent conditions for the demonstration of endogenous rhythmicity of the putative SCN pacemaker can be achieved in in vitro SCN preparations. This in vitro approach to the study of circadian pacemakers has been successfully.applied in the case of the molluscan eye and the avian and reptiliaal pineal gland [10]. Recently, several investigators have developed conditions that allow for electrical recording ~rom single cells in in vitro explants of the SCN [1, 2, 3]. It was found that the d;scharge rate of single SCN neurones in vitro resembles the multiple unit activity rhythm that has been recorded in the SCN in vivo [1, 2, 6]. The present paper reports short-term and long-term recordings in in vitro explants of the rat SCN, retrc~chiasmatic area (RCA) and arcuate nucleus (ARC). Evidence is presented that cells in the SCN, but not in the RCA and ARC, are capable of sustaining at least one circadian cycle in vitro. Adu!t male Wistar rats were kept in light- and climate-controlled rooms. The ah "-als were exposed to daily light-dark cycles of 12 h of light alternating with 12 h of darkness. For one group of animals the lights were on from 09.00-21.00 h (LD group). A secoJ~d group was exposed to a lighting regimen with lights on from 23.00-11.00 h (DL group). ,~dl animals were entrained to the LD or DL cycle for ~tt least 3 weeks. For each animal 'he phase of its circadian cycle was expressed in hours circa.di;m time (CT) witi3 CT 12 h defined as the beginmng of the dark portion of the LD or DL cycle. For the expe~-iments, individual rats in the LD group were taken from their cage between 0~.00 and 16.00 h (corresponding to CT 0--CT 7 h). Animals in the DL group wer,; taken cut between 08.00 and 11.00 h (corresponding to CT 9 - C T 12 h). The rats were t,l.en lightly anaesthetized with l~lothane and decapitated. The brain was rapidly removed, rinsed with saline at 37°C, and a 2 mm coronal section was cut at the level of the posterior optic chiasm. This section was gent l>' rinsed and positioned cn a guide glass with a 300 ~tm deep well. Using a sharp r~tzor blade 300-400 ~n~ thici~ sections were cut and those sections containing the S CN at their largest me dio-h~teral extent, the RCA or the ARC were immediately transferred to a dish containing oxygenated incubation medium (see below) kept at Y'°C. Here all tissue dorsal to the anterior commissure and lateral to the supraoptic ntlclei was dissected away from the SCN and RCA sliee~. From the ARC slice tissue dorsal to tbe third ventricle and lateral to the optic tracts was removed. Following dissection, the slices were transferred to an incubation chamber. The time from decapitation to incubation was less than 5 min. For all procedures, sterile media and in.,;truments were used.
285 In t~-: incubation chamber the stic.es rested on a P t - l r grid and were bathed in a glucose/saline bicarbonate buffered medium (perfusion rate: 1 . 0 - i . 2 ml. m i n - ~). The composition of tile l:,erfusate was as follows: NaCI, 124 mM; KCI, 5 mM; KHzPO4, 1.24 raM; MgSO4, 1.3 raM; CaClz, 1 mM; NaHCO3, 26 raM; glucose, 10 mM [31. Penicillin (60 m g . l - ' ) a n ( streptomycin (100 r a g . l - ~ ) were ~dded to the medium which was subsequently pa~sed through a bacterial filter. Both measures effectively served to prevent bacteri~ growth in the cour:~e of the 6 - 3 0 h recording period. The medium was equilibrated with a 95010 O z - 5~/9 CO2 gas mixture to giv(, a pH of 7 . 2 - 7 . 4 at 37°C. The temperatti,'e of the medium bathing :he slices was c o n trolled ,*. 37 ± 0.1 °C. The O2-COz gas mixture was humidified in the temoeraturecontrolled water bath surrounding the incubation chamber and directed over the surface of the slice to prevent it from superficial cooling and drying. For extracellular recording from single neuroo.es glass microelectrodes with a tip diameter of approximately 1 ttm and filled with 3 M KCt were used. Electrode impedance ranged from 0.5 to 2 M r at 1 kHz. The P t - l r supporting the slice served as a ground reference electrode. The extracellular signals were amplified and filtered and action potentials were electronically discriminated. The spikes and the window discriminator levels were continuously monitored on an oscilloscope set at a 10 msec time base. The spikes were counted in 1, 5 or 10 sec time bins and the counts recorded on a pen recorder. Mean discharge rates and spike interval distributions of the spike trains were computed on-line with a PDP 11-10 computer. At the end of each experiment the recording sites were marked electrolytically and their locations examined in crystal violet/Luxol fast blue stained sections cf the slices.
A
0
I000
0
I000
0
B
A
1000 ms C
Ss"
0
10
ttme (minutes)
i
170' Lux
'--I
Fig. 1. Recordirg from a rat SCN cell in vitro under two different illumination intensities. The discharge of this cell is shown in the lower record. The slice was exposed to darkness and to brief direct illumination of 170 lux, as indicated below the record. The upper panels show the similarity in the interspike interval distributions corresponding to the recording intervals marked A, B artd C.
?86
We have previously retorted that cells in the in vitro S C N may exhibit beating, bursting and random firing patterns [3]. In the present paper we report on SCI~ RCA and ARC cells exhibith~ random discharge ~ (e.g, Fig. 1), which are representative of the nmjority of ~ in vitgo as well as in vivo [3~ 4]. ~:de,recOr, dings for periods exceeding 10 mitt were obtained ~rom a total n u n ~ r iof 336 neurones in 57 slices. Histological examination verified that 99 cells were r¢~onl~ t
15 q
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Fig. 2. Mean discharge rates of rat SCN, R C A and A R C neurone~ recorded in vitro. A: long-term (2-14.3 h) r~.',:dings from s;nIle SCN ,'ells pl~ted as a function of circadian time. Calls ~¢m'ded in slices from LD rats are represented by open circles, cells from DL slices by filled circles. The mean firing rate was determined for each cell in 20-rain time bins. The circles for ~¢h individual cells are in[ercon. r,ected. B: the mean discharge lares of SCN cells recorded for short ~riods o.t time (10-120 minutes) plotted versus circadian time. All cells recorded in each part~dar 2-h bin along the time axis are h~nped ~ogether. The horizontal bars represent the ~amda~ ¢ deviatio~ of the mean discharge rates of those cells ,~cord~l during the corres~ionding 2-h interval. C: simii~r to that in B but illustratingthe mean firing ,'ares or" R C A (triangles) and A R C (circles)cells computed for 4-h time bins.
287
in the SCN o f L D n~s (16 slices), 47 in the SCN of DL r~,ts (12 slices), 41 in the RCA of LD rats (6 slices), 32 in the RCA of DL rats (5 slices), 53 in the ARC of L D r a t s (8 slices) and 64 in the ARC of DL rats (10 slices). Recordings exceedir, l~, 2 h were obtained from 9 a n d 5 SCN ~lis in LD and DL ~lices, respectively. The longest :recording period in any single cell was 14.3 h. Spontaneous aqaivity cou~td be observed forover 2 6 h but never within the first 30 min after incubation. The recordingsites were located in the ventral and medial areas of the SCN, RCA ~,nd ARC, bordering the third ventricle, Since it was initially es~.~._blished that the discharge rate of the cells in each area was independent of the illumination inte~sity of the slices (Fig. l ) , all subsequent recordings ~rere carried out in constant ille ruination. In the SCN a marked difference in mean discharge rate was observed between '~he subjective day (CT0-CTI2) and the subjective night (CTt2-CT24). Thi,~ is illustrated in Fig. 2A for SCN cells that were recorded for an extensive length of time (2-14.3 h) and in Fig, 2B for cells studied ~or shorter periods (0.17-2 h). The mean discharge rates for cells studied between CT4-CT8 and CTI6-CT20 were 8.3 and 3.8 sec-z, respectively, these frequencies were significantly different (one-tailed ttest, P<0,001). The circadian variation in firing of SCN cells was observed regardless of the actual time of incubation or whether the slices were prepared from LD or DL rats (Fig. 2A). This rules out the possibility that the discharge rate reflected uncontrolled diurnal fluctuations in the conditions of incubation and supports the conclusion that the circadian variation is endogenous. The discharge rate in the in vitro SCN ranged between 0.4 and 17.2 sec-~, which is cc,mparable to values reported both for in vitro and for in vivo studies of the rat SCN [1-4]. The time course of the in vitro SCN rhythm, moreover, qualitatively parallels that recorded in the SCN of conscious rats, although the typical in vivo u ltradian variations [6] were absent in our recordings (Fig. 2A). As in the SCN of the intact rat, the highest discharge rate was observed around CT6, while the lowest values occurred between CTI5 and CT20. In some of the long-duration recordings it was noted that single cells decreased their firing rate gradually between CT6 and CTI8 or exhibited a slowly increasing discharge between CTI8 and CT6 (Fig. 2A). In contrast to the SCN, electrical activity of RCA and ARC neurones did not exhibit significant variations with circadian time. Fig. 2C illustrates this observation for cells in both these areas that were recorded for short periods of time (10-30 m;,n). Analysis of variance (factorial blocks design) confirmed that the mean discharge rates of RCA and ARC neurones were not significantly different (overall mean rate: 5.2 ± 3.7 sec - ~) and did not vary with circadian time. The lack of circadian variation in the RCA ~nd ARC ne~rones lends further support to the conclusion that the SCN rh~l~hm was not induced by diurnal variations in the recording conditions. It can I:e expected that such exogenous influences, if present, would also be leflected in the AKC and RCA re(ordings. There is general consensus that the SCN of the mammalian brain represent ama-
288
jor circadian pacemaker [5, 8, 10]. However, the best available evidence for this view is based on ,%sion studies and chronic recording in the ncurally isolated SCN [5, 6, 8]. A primary requirement for conclading that the SCN are in fact a pacemaker is to demonstrate that these nuclei can sustain a circadian rhythm in absolute isolation from neural as well as from hmnoral, thermal and other influences . The study of the SCN under in vitro conditions can be expected to supply adirect answer to the question whether the S,.,~ is capable of generating circadian rhythms. We previously reported that rat ~prachiasmatic neurones exhibit firing rates in vitro that vary rl:vthmically with circadian time [2]. In the present study we have extended these prelimirmry fiadings with additional short-term recordings. Moreover, we have shown that in long-term recordings SCN cells may show spontaneous gradual transitions in firi~g rate from a high to a low level (and vice versa) at the appropriate circadian times. In contrast, circadian rhythmicity wss no* observed in the RCA and ARC. This finding suggests that circadian rhythms can be uniquely generated in the SCN and, possibly, in other as yet unidentified circadian pacemaker.,. Extending the in vitro recording time would enable us to deter. mine if the SCN ~ e capable of generating more than one ~-qa'cadian cycle in vitro. This experiment is a necessary :~ext step in evaluating whether the SCN contain a self-sustaining, rather than a damped, circadian oscillator. The major part of this work was completed when the authors were at the Department of Physiology and Physiological Physics, University of Leiden, Leiden, The Netherlands. We are grateful to Mr. J. den Hoed for his histotechnical assistance. 1 Green, J.D. and Gillette, R., Circadian rhythm of firinll rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 2 Groos, G., Circadian rhythms in rat hypothalamic neurones in vivo and in vitro, Chronobiologia, 6 (1979) 104. 3 Groom, G. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia, 35 (I 979) 1597-1598. 4 Groos, G. and Mason, R., The visual properties of rat and cat suprachiasmatic neurones, J. comp. Physiol., 135 (1980) 3~9-356. 5 Groos, G.~ Mason, R. and Meijer, J.H., Electrical and pharmacological properties of the suprachiasmatic auclei, Fed. Proc., in press. 6 lnouye, S.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic rmcleus, J. comp. Physiol., 146 (1982) 153-160. 7 Rusak, B. and Groos, G., Suprachlasmatic stimulation phase shifts rodents circadian rhythms, Science, 215 (1982) 1407-1~t09. 8 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 9 Schwartz, W.J., Davidsen, L.C. and Smith, J., In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus, J. comp. Neurol., 189 (1980) 157-167. 10 Takahashi, J.S. and Zatz, M., R~,!ation of circadian rhythmicity, Science.., 217 (1982) 1104-111 I.
283
CIRCADIAN RHYTHMS IN ELF~TRICAL DISCHARGE OF RAT SUPRACHIASMATIC NEURONF~ RECORDED IN VITRO
GERARD GROOS* and JAN HENDRIKS**
National Institute o f Mental Health, Clinical Psychobiology Branch, Bethesda, MD 20~3.5 (U.S.A.) (Received October 29th, 1982; Accepted November 1lth, 1982)
Key words: arcuate nucleus - circadian rhythms - in vitro recordings - retrochiasmatic area suprachiasmatic nucleus
The discharge of suprachiasmatic (SCN), retrochiasmatic (RCA) and arcuate (A ~C) neurones was recorded in brain slices incubated for periods up to 30 h. The firing rate of cells in the SCN, but not in the RCA and ARC, exhibited a circadian rhythm similar to that reported for the SCN in freely moving animals. This rhythm cannot be ascribed to subtle exogenous diurnal variations in the incubation conditions. It :s co~lcluded that the SCN explant is capable of endogenous generation of at least one circadian cycle in vit;o
It is widely assumed that mammalian circadian rhythms are endogenously generated by self-sustaining pacemakers that are susceptible to entrainm,mt by the daily light-dark cycle in the environment [5, 8, 10]. Initial evidence that ~t least one such pacemaker exists in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus was based on lesion studies: it has been demonstrated that selective lesions or surgical isolation of the SCN in rodents result in the permanent elimination of various circadian rhythms [8]. Several recent investigations support the interpretation of the lesion studies that the mammalian SCN contain a circadian pacemaker. Mild electrical stim~alation of the SCN in blinded rodents results in phase shifts of thor free-running behavioural activity rhythms [7], and local pharmacological stimulation of the SCN affects normal circadian timekeeping in rats [5]. In vivo studies have,, identified visually responsive SCN cells that are optimally suited to code the environmental luminance changes that are responsible for photic entrainment of circadian rhythms [4, 5]. Furthermore, it has been demonstrated that the rat SCN exhibit circadian rhythms in glucose utilization and electrical activity [6, 9]. The rhyth~rJ of electrical multiple unit activity in the SCN persists when the • Address for ccrre;pondence: National Institute of Mental Health, Clinical Psychobiology Branch, Buildin~ 10, Room 4S-239, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A. • " Present address: U.N.D.P. - Wcrld Health Orga~zation, P.O. Box 280, Kingston, Jamaica.
284
animal is housed in constant environmental conditions and when the SCN is surgically isolated from the rest of the brain in a 'hypoth~amic island' preparation -6]. Significantly, in such a preparation electrical activity rhythms elsewhere in ~he brain are absent [6]. This finding demonstrates that the rhythm of the SCN is not neurona!ly driven by a circadian pacemaker out~ide the island. However, it does not exclude the possibility that the rhythmic activity of the SCN is driven by circadia,': ~emperature or humoral rhythms generated outsiae the island [5]. More stringent conditions for the demonstration of endogenous rhythmicity of the putative SCN pacemaker can be achieved in in vitro SCN preparations. This in vitro approach to the study of circadian pacemakers has been successfully.applied in the case of the molluscan eye and the avian and reptiliaal pineal gland [10]. Recently, several investigators have developed conditions that allow for electrical recording ~rom single cells in in vitro explants of the SCN [1, 2, 3]. It was found that the d;scharge rate of single SCN neurones in vitro resembles the multiple unit activity rhythm that has been recorded in the SCN in vivo [1, 2, 6]. The present paper reports short-term and long-term recordings in in vitro explants of the rat SCN, retrc~chiasmatic area (RCA) and arcuate nucleus (ARC). Evidence is presented that cells in the SCN, but not in the RCA and ARC, are capable of sustaining at least one circadian cycle in vitro. Adu!t male Wistar rats were kept in light- and climate-controlled rooms. The ah "-als were exposed to daily light-dark cycles of 12 h of light alternating with 12 h of darkness. For one group of animals the lights were on from 09.00-21.00 h (LD group). A secoJ~d group was exposed to a lighting regimen with lights on from 23.00-11.00 h (DL group). ,~dl animals were entrained to the LD or DL cycle for ~tt least 3 weeks. For each animal 'he phase of its circadian cycle was expressed in hours circa.di;m time (CT) witi3 CT 12 h defined as the beginmng of the dark portion of the LD or DL cycle. For the expe~-iments, individual rats in the LD group were taken from their cage between 0~.00 and 16.00 h (corresponding to CT 0--CT 7 h). Animals in the DL group wer,; taken cut between 08.00 and 11.00 h (corresponding to CT 9 - C T 12 h). The rats were t,l.en lightly anaesthetized with l~lothane and decapitated. The brain was rapidly removed, rinsed with saline at 37°C, and a 2 mm coronal section was cut at the level of the posterior optic chiasm. This section was gent l>' rinsed and positioned cn a guide glass with a 300 ~tm deep well. Using a sharp r~tzor blade 300-400 ~n~ thici~ sections were cut and those sections containing the S CN at their largest me dio-h~teral extent, the RCA or the ARC were immediately transferred to a dish containing oxygenated incubation medium (see below) kept at Y'°C. Here all tissue dorsal to the anterior commissure and lateral to the supraoptic ntlclei was dissected away from the SCN and RCA sliee~. From the ARC slice tissue dorsal to tbe third ventricle and lateral to the optic tracts was removed. Following dissection, the slices were transferred to an incubation chamber. The time from decapitation to incubation was less than 5 min. For all procedures, sterile media and in.,;truments were used.
285 In t~-: incubation chamber the stic.es rested on a P t - l r grid and were bathed in a glucose/saline bicarbonate buffered medium (perfusion rate: 1 . 0 - i . 2 ml. m i n - ~). The composition of tile l:,erfusate was as follows: NaCI, 124 mM; KCI, 5 mM; KHzPO4, 1.24 raM; MgSO4, 1.3 raM; CaClz, 1 mM; NaHCO3, 26 raM; glucose, 10 mM [31. Penicillin (60 m g . l - ' ) a n ( streptomycin (100 r a g . l - ~ ) were ~dded to the medium which was subsequently pa~sed through a bacterial filter. Both measures effectively served to prevent bacteri~ growth in the cour:~e of the 6 - 3 0 h recording period. The medium was equilibrated with a 95010 O z - 5~/9 CO2 gas mixture to giv(, a pH of 7 . 2 - 7 . 4 at 37°C. The temperatti,'e of the medium bathing :he slices was c o n trolled ,*. 37 ± 0.1 °C. The O2-COz gas mixture was humidified in the temoeraturecontrolled water bath surrounding the incubation chamber and directed over the surface of the slice to prevent it from superficial cooling and drying. For extracellular recording from single neuroo.es glass microelectrodes with a tip diameter of approximately 1 ttm and filled with 3 M KCt were used. Electrode impedance ranged from 0.5 to 2 M r at 1 kHz. The P t - l r supporting the slice served as a ground reference electrode. The extracellular signals were amplified and filtered and action potentials were electronically discriminated. The spikes and the window discriminator levels were continuously monitored on an oscilloscope set at a 10 msec time base. The spikes were counted in 1, 5 or 10 sec time bins and the counts recorded on a pen recorder. Mean discharge rates and spike interval distributions of the spike trains were computed on-line with a PDP 11-10 computer. At the end of each experiment the recording sites were marked electrolytically and their locations examined in crystal violet/Luxol fast blue stained sections cf the slices.
A
0
I000
0
I000
0
B
A
1000 ms C
Ss"
0
10
ttme (minutes)
i
170' Lux
'--I
Fig. 1. Recordirg from a rat SCN cell in vitro under two different illumination intensities. The discharge of this cell is shown in the lower record. The slice was exposed to darkness and to brief direct illumination of 170 lux, as indicated below the record. The upper panels show the similarity in the interspike interval distributions corresponding to the recording intervals marked A, B artd C.
?86
We have previously retorted that cells in the in vitro S C N may exhibit beating, bursting and random firing patterns [3]. In the present paper we report on SCI~ RCA and ARC cells exhibith~ random discharge ~ (e.g, Fig. 1), which are representative of the nmjority of ~ in vitgo as well as in vivo [3~ 4]. ~:de,recOr, dings for periods exceeding 10 mitt were obtained ~rom a total n u n ~ r iof 336 neurones in 57 slices. Histological examination verified that 99 cells were r¢~onl~ t
15 q
_
,
i
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,
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Fig. 2. Mean discharge rates of rat SCN, R C A and A R C neurone~ recorded in vitro. A: long-term (2-14.3 h) r~.',:dings from s;nIle SCN ,'ells pl~ted as a function of circadian time. Calls ~¢m'ded in slices from LD rats are represented by open circles, cells from DL slices by filled circles. The mean firing rate was determined for each cell in 20-rain time bins. The circles for ~¢h individual cells are in[ercon. r,ected. B: the mean discharge lares of SCN cells recorded for short ~riods o.t time (10-120 minutes) plotted versus circadian time. All cells recorded in each part~dar 2-h bin along the time axis are h~nped ~ogether. The horizontal bars represent the ~amda~ ¢ deviatio~ of the mean discharge rates of those cells ,~cord~l during the corres~ionding 2-h interval. C: simii~r to that in B but illustratingthe mean firing ,'ares or" R C A (triangles) and A R C (circles)cells computed for 4-h time bins.
287
in the SCN o f L D n~s (16 slices), 47 in the SCN of DL r~,ts (12 slices), 41 in the RCA of LD rats (6 slices), 32 in the RCA of DL rats (5 slices), 53 in the ARC of L D r a t s (8 slices) and 64 in the ARC of DL rats (10 slices). Recordings exceedir, l~, 2 h were obtained from 9 a n d 5 SCN ~lis in LD and DL ~lices, respectively. The longest :recording period in any single cell was 14.3 h. Spontaneous aqaivity cou~td be observed forover 2 6 h but never within the first 30 min after incubation. The recordingsites were located in the ventral and medial areas of the SCN, RCA ~,nd ARC, bordering the third ventricle, Since it was initially es~.~._blished that the discharge rate of the cells in each area was independent of the illumination inte~sity of the slices (Fig. l ) , all subsequent recordings ~rere carried out in constant ille ruination. In the SCN a marked difference in mean discharge rate was observed between '~he subjective day (CT0-CTI2) and the subjective night (CTt2-CT24). Thi,~ is illustrated in Fig. 2A for SCN cells that were recorded for an extensive length of time (2-14.3 h) and in Fig, 2B for cells studied ~or shorter periods (0.17-2 h). The mean discharge rates for cells studied between CT4-CT8 and CTI6-CT20 were 8.3 and 3.8 sec-z, respectively, these frequencies were significantly different (one-tailed ttest, P<0,001). The circadian variation in firing of SCN cells was observed regardless of the actual time of incubation or whether the slices were prepared from LD or DL rats (Fig. 2A). This rules out the possibility that the discharge rate reflected uncontrolled diurnal fluctuations in the conditions of incubation and supports the conclusion that the circadian variation is endogenous. The discharge rate in the in vitro SCN ranged between 0.4 and 17.2 sec-~, which is cc,mparable to values reported both for in vitro and for in vivo studies of the rat SCN [1-4]. The time course of the in vitro SCN rhythm, moreover, qualitatively parallels that recorded in the SCN of conscious rats, although the typical in vivo u ltradian variations [6] were absent in our recordings (Fig. 2A). As in the SCN of the intact rat, the highest discharge rate was observed around CT6, while the lowest values occurred between CTI5 and CT20. In some of the long-duration recordings it was noted that single cells decreased their firing rate gradually between CT6 and CTI8 or exhibited a slowly increasing discharge between CTI8 and CT6 (Fig. 2A). In contrast to the SCN, electrical activity of RCA and ARC neurones did not exhibit significant variations with circadian time. Fig. 2C illustrates this observation for cells in both these areas that were recorded for short periods of time (10-30 m;,n). Analysis of variance (factorial blocks design) confirmed that the mean discharge rates of RCA and ARC neurones were not significantly different (overall mean rate: 5.2 ± 3.7 sec - ~) and did not vary with circadian time. The lack of circadian variation in the RCA ~nd ARC ne~rones lends further support to the conclusion that the SCN rh~l~hm was not induced by diurnal variations in the recording conditions. It can I:e expected that such exogenous influences, if present, would also be leflected in the AKC and RCA re(ordings. There is general consensus that the SCN of the mammalian brain represent ama-
288
jor circadian pacemaker [5, 8, 10]. However, the best available evidence for this view is based on ,%sion studies and chronic recording in the ncurally isolated SCN [5, 6, 8]. A primary requirement for conclading that the SCN are in fact a pacemaker is to demonstrate that these nuclei can sustain a circadian rhythm in absolute isolation from neural as well as from hmnoral, thermal and other influences . The study of the SCN under in vitro conditions can be expected to supply adirect answer to the question whether the S,.,~ is capable of generating circadian rhythms. We previously reported that rat ~prachiasmatic neurones exhibit firing rates in vitro that vary rl:vthmically with circadian time [2]. In the present study we have extended these prelimirmry fiadings with additional short-term recordings. Moreover, we have shown that in long-term recordings SCN cells may show spontaneous gradual transitions in firi~g rate from a high to a low level (and vice versa) at the appropriate circadian times. In contrast, circadian rhythmicity wss no* observed in the RCA and ARC. This finding suggests that circadian rhythms can be uniquely generated in the SCN and, possibly, in other as yet unidentified circadian pacemaker.,. Extending the in vitro recording time would enable us to deter. mine if the SCN ~ e capable of generating more than one ~-qa'cadian cycle in vitro. This experiment is a necessary :~ext step in evaluating whether the SCN contain a self-sustaining, rather than a damped, circadian oscillator. The major part of this work was completed when the authors were at the Department of Physiology and Physiological Physics, University of Leiden, Leiden, The Netherlands. We are grateful to Mr. J. den Hoed for his histotechnical assistance. 1 Green, J.D. and Gillette, R., Circadian rhythm of firinll rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 2 Groos, G., Circadian rhythms in rat hypothalamic neurones in vivo and in vitro, Chronobiologia, 6 (1979) 104. 3 Groom, G. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia, 35 (I 979) 1597-1598. 4 Groos, G. and Mason, R., The visual properties of rat and cat suprachiasmatic neurones, J. comp. Physiol., 135 (1980) 3~9-356. 5 Groos, G.~ Mason, R. and Meijer, J.H., Electrical and pharmacological properties of the suprachiasmatic auclei, Fed. Proc., in press. 6 lnouye, S.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic rmcleus, J. comp. Physiol., 146 (1982) 153-160. 7 Rusak, B. and Groos, G., Suprachlasmatic stimulation phase shifts rodents circadian rhythms, Science, 215 (1982) 1407-1~t09. 8 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 9 Schwartz, W.J., Davidsen, L.C. and Smith, J., In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus, J. comp. Neurol., 189 (1980) 157-167. 10 Takahashi, J.S. and Zatz, M., R~,!ation of circadian rhythmicity, Science.., 217 (1982) 1104-111 I.