158
Brain Research, 5t 1 (1990) 158-162
Elsevier BRES 24010
Short Communications
Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasrnatic nucleus Nico P.A. Bos and Majid Mirmiran Netherlands Institute for Brain Research, Amsterdam (The Netherlands)
(Accepted 5 December 1989) Key words: Circadian rhythm; Suprachiasmatic nucleus; Brain slice; Electrophysiology; Biological clock
The suprachiasmatic nucleus (SCN) is believed to play a major role in the generation and control of circadian rhythms in mammals. In order to obtain further evidence 60ncerning this, single and multiple neuronal discharges were continuously recorded over a period of several days in neonatal rat SCN explants. These organotypic explants, which had been cultured for several weeks in a chemically defined medium, showed alternating high and low levels of spontaneous neuronal discharges with a periodicity around 24 h. Such explants can serve as a useful model to study the neuronal mechanisms underlying the generation of mammalian circadian rhythms. Although the neuronal mechanisms underlying the generation of circadian rhythms in m a m m a l s are not well understood, there is a large body of evidence indicating that the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is involved in generating circadian rhythms 9J3'2°'21. This is supported by the fact that circadian rhythms have been found in electrical discharges of SCN neurons 4'5'7, SCN deoxyglucose uptake ~5' ~7.24,27 and release of vasopressin from SCN explants 2'3. Furthermore, electrical stimulation of SCN resulted in a phase-dependent way in phase shifts of the free-running circadian rhythms 22. Moreover, fetal SCN tissue transplanted into the brain of the previously SCN-lesioned rats resulted in restoration of circadian rhythms in the otherwise arrhythmic animals s. It has recently been possible to breed two new strains of golden hamsters, both resulting from a mutation in a single gene, with distinct circadian rhythms of 20 and 22 h 23. The SCN in both naturally and specially bred hamsters was destroyed. Upon recovery all animals received SCN from fetal hamsters known to have inherited different circadian rhythms; each transplanted animal began to live according to the cycle of the donor SCN 23. In vivo studies in which multi-neuronal activity was recorded in the SCN, have demonstrated a high level of activity during the subjective day with significantly lower activity in the subjective night 7. However, it was not possible to discriminate single neurons in these recordings. This is inherent in the small diameter of SCN neurons, the small size of this nucleus and the type of recording electrodes that were used. Moreover, the SCN
is situated 8-9 mm deep in the rat brain which m a k e s approaching it dorsally with a bundle of microwires very difficult. In order to circumvent these difficulties many investigators have begun using in vitro preparations. In vitro studies using acute SCN slices have shown circadian rhythms in SCN neuronal activities 4'5'16'25. A high level of neuronal firing was found in the SCN when recorded in animals sacrificed during the subjective day in comparison with those killed during the subjective night. In some of these experiments, however, continuous but short-term recordings were carried out and in others the data of different SCN slices were crosssectionally compared. To overcome these problems we have developed a long-term cultured SCN preparation. We have hypothesized that, if SCN neurons are capable of endogenously generating circadian rhythms, they should maintain such rhythms in their firing rate when cultured for several weeks and recorded continuously for more than one circadian cycle. Furthermore, the minimal deterioration during recordings in cultured SCN, in comparison with acute slices, would increase the significance of any finding of circadian rhythmicity. Such rhythm in the spontaneous activity within SCN would provide firm evidence in favor of the SCN being a biological clock 9'10'13'21 .
In each series of cultures, anterior hypothalamic tissues were obtained from 6-8 12-day-old Wistar rat pups, sacrificed during the middle of their maternal light phase. By postnatal day 12, a certain level of maturity is reached within the rat S C N 14"26. The procedure for maintaining such slices in vitro was similar to the one
Correspondence: M. Mirmiran, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
159 routinely used in our laboratory for cerebral cortex explants zsA9. The anterior hypothalamus of each pup was coronally chopped into 400/~m thick slices at the level of the optic chiasm, using a Mcllwain tissue chopper. The sections were transferred to a petri-dish containing glucose-supplemented Hanks balanced salt solution (BBS; 6.5 mg/ml final concentration). The slices were examined under a dissecting microscope and those containing the middle part of the SCN were selected, using the optic chiasm and the third ventricle as landmarks. Each explant was trimmed at the level of the anterior commissure and the supra-optic nucleus, and then left for 1 h at room temperature. All explants were then individually transferred on a specially prepared polyamide gauze (grid), freely floating in a standard 35 mm petri-dish filled with 1.2 ml R16 medium (pH 7.4 at 5% CO2). The R16 chemically defined serum free bicarbonate buffered medium is developed in our institute for long-term neuronal culturing. The chemical composition of this medium is described extensively in ref. 18. For long-term culturing, the explants were given several hours to attach to the grid, whereafter the culture dishes were placed in stainless steel box on a rocking device inside an automatically regulated incubator (set for 5% CO 2 and 35 °C)19. Medium was changed every 2 or 3 days, and those slices which were used for recording were given an additional 2.4 ml medium, before they were brought into the recording chamber. The recording chamber was especially designed to maintain a constant and sterile environment for longterm electrophysiological recordings, using several features of previously developed slice chambers TM. In short, it consists of a small heated perspex container filled with approximately 500 ml sterile aquadest. A small plateau above the bath was made in which a standard 35 mm culture dish is pressed into a recessed holder. The lid of the culture dish is then replaced by a chimney-like plexiglass cover with openings through which the recording and a platinum reference electrodes were passed. In order to maintain the internal climate and sterile environment of the chamber, these openings are covered with double isolated plexiglass plates. A heating ring around the chimney completed the assembly and prevented condensation on the electrodes and on plexiglass plates. The reference electrode was also used to hold and anchor the preparation to the bottom of the dish. All recordings were made in non-perfused R16 medium to prevent movement artefacts and infections. The light intensity and temperature throughout the recording session were kept constant at 5 lux and 35 °C, respectively. During the recording, a pH of 7.4 and a humidity of 100% was maintained using a gas mixture of 5% CO 2 and 95% air, at a flow rate of 2 ml/min bubbled through the water
bath. The recording electrode consisted of a standard glass pipette (Clark GC150F-15) pulled together with a 10 /zm carbon fiber, broken to an approximate tip diameter of 13/zm and sealed with ultraviolet glue. The pipette was filled with 3 M NaCI covered with liquid paraffin. The electrode impedance was in the range of 2-5 MJ2 at 150 Hz, and was sterilized before use. The search for neuronal activity was started an hour after transfer of the explant into the recording chamber. Under the dissecting microscope a more densely packed neuronal area was located which was, in most cases, clearly recognizable as SCN due to its unique bilateral location along the third ventricle immediately above the degenerated optic chiasm. In addition to the postrecording morphological identification, a surprisingly high level of background spontaneous activity (compared with anterior lateral hypothalamic area) could distinguish SCN in these cultured slices. Extracellular SCN action potentials, filtered at 300-3000 Hz bandpass (Neurolog: NL 102), were discriminated using two Mentor 750 amplitude-window discriminator coupled with an ANDGATE, NL 501. The windows were set at both positive and negative phases of the selected unit in order to rule
A
ILJ_L_J 1 L_.[.L L.I t_LLIJ_.IIJ J TW
i
J,J ._L
T--I r-r r llec
J5 O p V
Fig. 1. A: action potentials recorded in two different SCN explants showing the quality of units analyzed in the present study. B: a 20-day-oldSCN explant stained with an antibody raised against VIP.
160 TABLE I Slice name
Days in culture
Recording time (h)
P. V.A. (%)
Period length
Maximum freq. (Hz)
Minimum freq. (Hz)
Number units
S12JAN S16JAN S26JAN S09FEB S13FEB S21FEB S23FEB S25FEB S01MRT S15AUG S15SEP S21OCT S05NOV S09NOV S21NOV S05DEC S12DEC
9 13 23 15 19 27 29 20 7 5 9 17 16 20 7 22 29
60 66 54 46 66 36 38 44 60 58 66 48 60 60 36 46 54
26 47 35 76 59 37 18 73 59 49 27 63 55 19 69 17 43
28 25 24 32 27 20 22 25 20 25 26 21 25 18 20 16 21
2.6 3.5 4.0 4.5 11.0 9.0 7.0 11.0 6.5 13.0 0.6 42.0 21.0 10.0 0.9 4.0 4.5
1.0 2.0 2.5 1.5 6.0 3.0 3.8 5.0 3.5 6.0 0.2 20.0 8.0 4.0 0.2 1.0 3.5
1 1 1 1 1 l 1 1 1 1 1 2-3 2-3 1 1 1 1
out artifacts. Discriminated spikes were fed into an
analysis and display of the time histogram. Mainly single
intelligent interface ( C a m b r i d g e Electronic Device: C E D 1401) connected to a T a n d o n (PC-AT) for on-line
neurons were r e c o r d e d , in rare cases 2 or 3 neurons (which could not be individually discriminated) were simultaneously analyzed. Fig. 1A shows examples of single and multi SCN n e u r o n s that were recorded in this study. Only one continuous recording was obtained per explant. Additional controls were made by 24 h recording of neuronal activity from the lateral hypothalamic area and from neocortical explants cultured u n d e r identical conditions. No circadian rhythms were found in the n e u r o n a l activity of these structures. For statistical analysis of the periodicity of the recordings, a cosine-fit periodogram program written for small computers was used 12. The proportion of the variability accounted for (P.V,A.), derived from this program, indicates the magnitude of rhythmicity. The m a x i m u m P.V.A. values
L) uJ u) (n i,u a. u)
12
24
12
24
TIME
6
12
18 PERIOD
12
24
12
( H )
24
LENGTH
30
M
(H)
Fig. 2. Time histogram (top) and the cosine-fit periodogram of a single SCN neuron showing the maximum proportion of variability accounted (P.V.A.) for a period length of 20 h.
6
12
18 PERIOD
24 LENGTH
30
36
(H)
Fig. 3. Mean P.V.A. values (the dashed bars indicate S.E.M.) for all SCN neurons (n --- 17) that had a significant periodicity between 16 and 32 h.
161 vivo (see also ref. 28). The presence of a circadian rhythm in firing rate of SCN neurons cultured over a long period of time in the absence of serum, cerebrospinal fluid and many brain areas that normally innervate this structure, provides direct evidence for SCN as a circadian oscillator. However, we have to admit that only 17 out of 22 explants showed circadian rhythms. Furthermore, although the mean (S.E.M.) P.V.A. of these neurons showed a maximum peak at 23 h (Fig. 3), a large scattered periodicity (ranging from mean period length of 16 h to 32 h; Table I) was found. Such large variations in spontaneous activity of SCN neurons were also reported in earlier studies using acute slices4'5'25. Apart from technical difficulties (see above) and the limited duration of continuous recording (maximum 66 h), we have the following two explanations for the relatively low percentage of active SCN explants each with a different period length: (i) the mammalian SCN, contrary to the pacemaker cells recorded in the Bulla eye 1, consists of a minority of pacemaker cells remaining active after longterm culturing in vitro; and/or (ii) not every individual rat SCN neuron, but a combination of an orchestrated group of neurons (each spontaneously firing with a periodicity different from 24 h, see Table I) are able to generate a precise circadian rhythm. Simultaneous recording from different single SCN neurons is required to get more insight into the SCN network. The cultured SCN preparation developed in this study is useful for studying central neuronal mechanisms underlying the mammalian circadian system, similar to the Bulla eye that is commonly used in studying circadian rhythms in invertebrates 1.
of the smoothed data were tested for statistical significance at the 1% level. Following the recordings, the explants were stained by conventional Nissl and by immunocytochemical techniques for vasointestinal polypeptide (VIP) and vasopressin (VP). Fig. 1B illustrates a 20-day-old SCN explant that was immunocytochemically stained with an antibody raised against VIP. The firing frequency of different SCN neurons varied from 0.2 and 15 Hz (Table I). The time histogram and the cosine periodogram output of a typical SCN neuron are shown in Fig. 2. High and low levels of spontaneous firing rate are visible in this unit, although the rhythm amplitude diminishes as a function of recording time. Out of 98 cultured explants only 22 showed spontaneous activity lasting for at least 36 h, to make the analysis of the circadian rhythm meaningful. This low percentage of success was due to several factors: e.g., the cessation of spontaneous activity, the development of infections, changes in amount and concentration of the medium during the recording and the mechanical neuronal damage caused by the recording electrode. In 5 out of the 22 successfully recorded explants no circadian rhythm was found (judged on the basis of the non-significant P.V.A. values using the Cosine-fit periodogram12). The remaining 17 showed clear periodic high and low levels of activity with a significant P.V.A. for periods ranging from 16 to 32 h (Table I). Despite large differences in the period length of different SCN explants, the averaged mean (S.E.M.) P.V.A. showed a maximum peak at 23 h (Fig. 3). The present experiment demonstrates that it is possible to continuously record neuronal discharges of single SCN neurons over several days in vitro. These SCN explants, which were cultured for several weeks, showed a characteristic morphology comparable to that found in
Acknowledgements. We are grateful to B.M. de Jong, J.M. Ruijter, R.E. Baker and H.J. Romijn for their assistance and advice in culturing and to R. Buijs for immunocytochemicalstaining.
1 Block, G.D. and McMahon, D.G., Cellular analysis of the Bulla ocular circadian pacemaker system, J. Comp. Physiol. A, 155 (1984) 387-395. 2 Earnest, D.J. and Sladek, C.D., Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro, Brain Research, 382 (1986) 129-133. 3 Gillette, M.U. and Reppert, S.M., The hypothalamic suprachiasmatic nuclei: circadian patterns of vasopressin secretion and neuronal activity in vitro, Brain Res. Bull., 19 (1987) 135-139. 4 Green, D. and Gillette, R., Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Research, 245 (1982) 198-200. 5 Groos, G. and Hendriks, J., Circadian rhythms in electrical discharges of rat suprachiasmatic neurons recorded in vitro, Neurosci. Lett., 34 (1982) 283-288. 6 Ince, C., Ypey, D., Diesselhoff-Den Dulk, M.M.C., Visser, J.A.M., de Vos, A. and van Furth, R., Micro-CO2-incubatorfor use on a microscope, J. Immunol. Methods, 60 (1983) 269-275. 7 Inouye, S.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. Comp. Physiol., 146 (1982) 153-160. 8 Lehman, M.N., Silver, R., Gladstone, W.R., Kahn, R.M.,
Gibson, M. and Bittman, E.L., Circadian rhythmicity restored by neural transplant. Immunocytochemical characterization of the graft and its integration with the host, J. Neurosci., 7 (1987) 1626-1638. 9 Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev., 69 (1989) 671-707. 10 Mirmiran, M., Swaab, D.F., Witting, W., Honnebier, M.B.O.M., Van Gool, W.A. and Eikelenboom, P., Biological clocks in development, aging and Alzheimer's disease, Brain Dysfunct., 2 (1989) 57-66. 11 Mollevanger, W.J., Nijland, R. and Tielen, A.M., A multicup brain slice perfusion system, Progress Report Inst. Med. Phys. TNO, Utrecht, 7 (1980) 57-60. 12 Monk, T.H. and Fort, A., Cosine: a cosine curve fitting program suitable for small computers, Int. J. Chronobiol., 8 (1983) 193-222. 13 Moore, R.Y., Organization and function of central nervous system oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. 14 Moore, R.Y. and Bernstein, M.E., Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy
162
15
16
17
18
19
20
21
and synapsin I immunoreactivity, J. Neurosci., 9 (1989) 21512162. Newman, G.C. and Hospod, EE., Rhythm of suprachiasmatic nucleus 2-deoxyglucose uptake in vitro, Brain Research, 381 (1986) 345-350. Prosser, R.A. and Gillette, M.U., The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP, J. Neurosci., 9 (1989) 1073-1081. Reppert, S.M. and Schwartz, W.J., The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using C-labeled deoxyglucose, J. Neurosci., 4 (1984) 1677-1682. Romijn, H.J., Development and advantage of serum-free, chemically defined media for culturing of nerve tissue, Biology of the Cell 63 (1988) 263-268. Romijn, H.J., de Jong, B.M. and Ruiter, J.M., A procedure for culturing rat neocortex explants in a serum-free nutrient medium, J. Neurosci. Methods, 23 (1988) 75-83. Rosenwasser, A.M. and Adler, N.T., Structure and function in circadian timing system: evidence for multiple coupled oscillators, Neurosci. Biobehav. Rev., 10 (1987) 431-448. Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526.
22 Rusak, B. and Groos, G., Suprachiasmatic stimulation phase shifts rodent circadian rhythms, Science, 215 (1982) 1407-1409. 23 Ralph, M.R., The rhythm maker, Proc. N.Y. Acad. Sci., 29 Nov. (1989) 40-44. 24 Schwartz, W.J., Reppert, S.M., Eagan, S.M. and Moore-Ede, M.C., In vivo metabolic activity of the suprachiasmatic nuclei: a comparative study, Brain Research, 274 (1983) 184-187. 25 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Research, 247 (1982) 154-158. 26 Shibata, S. and Moore, R.Y., Development of neuronal activity in the rat suprachiasmatic nucleus, Brain Research, 34 (1987) 311-315. 27 Shibata, S. and Moore, R.Y., Electrical and metabolic activity of suprachiasmatic nucleus neurons in hamster hypothalamic slices, Brain Research, 438 (1988) 374-378. 28 Sofroniew, M.V., Dreifuss, J.J. and Gahwiler, B.H., Slice cultures of rat hypothalamus examined by immunohistochemical staining for neurohypophyseal peptides and GFAP, Brain Res. Bull., 20 (1988) 669-674.
Brain Research, 5t 1 (1990) 158-162
Elsevier BRES 24010
Short Communications
Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasrnatic nucleus Nico P.A. Bos and Majid Mirmiran Netherlands Institute for Brain Research, Amsterdam (The Netherlands)
(Accepted 5 December 1989) Key words: Circadian rhythm; Suprachiasmatic nucleus; Brain slice; Electrophysiology; Biological clock
The suprachiasmatic nucleus (SCN) is believed to play a major role in the generation and control of circadian rhythms in mammals. In order to obtain further evidence 60ncerning this, single and multiple neuronal discharges were continuously recorded over a period of several days in neonatal rat SCN explants. These organotypic explants, which had been cultured for several weeks in a chemically defined medium, showed alternating high and low levels of spontaneous neuronal discharges with a periodicity around 24 h. Such explants can serve as a useful model to study the neuronal mechanisms underlying the generation of mammalian circadian rhythms. Although the neuronal mechanisms underlying the generation of circadian rhythms in m a m m a l s are not well understood, there is a large body of evidence indicating that the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is involved in generating circadian rhythms 9J3'2°'21. This is supported by the fact that circadian rhythms have been found in electrical discharges of SCN neurons 4'5'7, SCN deoxyglucose uptake ~5' ~7.24,27 and release of vasopressin from SCN explants 2'3. Furthermore, electrical stimulation of SCN resulted in a phase-dependent way in phase shifts of the free-running circadian rhythms 22. Moreover, fetal SCN tissue transplanted into the brain of the previously SCN-lesioned rats resulted in restoration of circadian rhythms in the otherwise arrhythmic animals s. It has recently been possible to breed two new strains of golden hamsters, both resulting from a mutation in a single gene, with distinct circadian rhythms of 20 and 22 h 23. The SCN in both naturally and specially bred hamsters was destroyed. Upon recovery all animals received SCN from fetal hamsters known to have inherited different circadian rhythms; each transplanted animal began to live according to the cycle of the donor SCN 23. In vivo studies in which multi-neuronal activity was recorded in the SCN, have demonstrated a high level of activity during the subjective day with significantly lower activity in the subjective night 7. However, it was not possible to discriminate single neurons in these recordings. This is inherent in the small diameter of SCN neurons, the small size of this nucleus and the type of recording electrodes that were used. Moreover, the SCN
is situated 8-9 mm deep in the rat brain which m a k e s approaching it dorsally with a bundle of microwires very difficult. In order to circumvent these difficulties many investigators have begun using in vitro preparations. In vitro studies using acute SCN slices have shown circadian rhythms in SCN neuronal activities 4'5'16'25. A high level of neuronal firing was found in the SCN when recorded in animals sacrificed during the subjective day in comparison with those killed during the subjective night. In some of these experiments, however, continuous but short-term recordings were carried out and in others the data of different SCN slices were crosssectionally compared. To overcome these problems we have developed a long-term cultured SCN preparation. We have hypothesized that, if SCN neurons are capable of endogenously generating circadian rhythms, they should maintain such rhythms in their firing rate when cultured for several weeks and recorded continuously for more than one circadian cycle. Furthermore, the minimal deterioration during recordings in cultured SCN, in comparison with acute slices, would increase the significance of any finding of circadian rhythmicity. Such rhythm in the spontaneous activity within SCN would provide firm evidence in favor of the SCN being a biological clock 9'10'13'21 .
In each series of cultures, anterior hypothalamic tissues were obtained from 6-8 12-day-old Wistar rat pups, sacrificed during the middle of their maternal light phase. By postnatal day 12, a certain level of maturity is reached within the rat S C N 14"26. The procedure for maintaining such slices in vitro was similar to the one
Correspondence: M. Mirmiran, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
159 routinely used in our laboratory for cerebral cortex explants zsA9. The anterior hypothalamus of each pup was coronally chopped into 400/~m thick slices at the level of the optic chiasm, using a Mcllwain tissue chopper. The sections were transferred to a petri-dish containing glucose-supplemented Hanks balanced salt solution (BBS; 6.5 mg/ml final concentration). The slices were examined under a dissecting microscope and those containing the middle part of the SCN were selected, using the optic chiasm and the third ventricle as landmarks. Each explant was trimmed at the level of the anterior commissure and the supra-optic nucleus, and then left for 1 h at room temperature. All explants were then individually transferred on a specially prepared polyamide gauze (grid), freely floating in a standard 35 mm petri-dish filled with 1.2 ml R16 medium (pH 7.4 at 5% CO2). The R16 chemically defined serum free bicarbonate buffered medium is developed in our institute for long-term neuronal culturing. The chemical composition of this medium is described extensively in ref. 18. For long-term culturing, the explants were given several hours to attach to the grid, whereafter the culture dishes were placed in stainless steel box on a rocking device inside an automatically regulated incubator (set for 5% CO 2 and 35 °C)19. Medium was changed every 2 or 3 days, and those slices which were used for recording were given an additional 2.4 ml medium, before they were brought into the recording chamber. The recording chamber was especially designed to maintain a constant and sterile environment for longterm electrophysiological recordings, using several features of previously developed slice chambers TM. In short, it consists of a small heated perspex container filled with approximately 500 ml sterile aquadest. A small plateau above the bath was made in which a standard 35 mm culture dish is pressed into a recessed holder. The lid of the culture dish is then replaced by a chimney-like plexiglass cover with openings through which the recording and a platinum reference electrodes were passed. In order to maintain the internal climate and sterile environment of the chamber, these openings are covered with double isolated plexiglass plates. A heating ring around the chimney completed the assembly and prevented condensation on the electrodes and on plexiglass plates. The reference electrode was also used to hold and anchor the preparation to the bottom of the dish. All recordings were made in non-perfused R16 medium to prevent movement artefacts and infections. The light intensity and temperature throughout the recording session were kept constant at 5 lux and 35 °C, respectively. During the recording, a pH of 7.4 and a humidity of 100% was maintained using a gas mixture of 5% CO 2 and 95% air, at a flow rate of 2 ml/min bubbled through the water
bath. The recording electrode consisted of a standard glass pipette (Clark GC150F-15) pulled together with a 10 /zm carbon fiber, broken to an approximate tip diameter of 13/zm and sealed with ultraviolet glue. The pipette was filled with 3 M NaCI covered with liquid paraffin. The electrode impedance was in the range of 2-5 MJ2 at 150 Hz, and was sterilized before use. The search for neuronal activity was started an hour after transfer of the explant into the recording chamber. Under the dissecting microscope a more densely packed neuronal area was located which was, in most cases, clearly recognizable as SCN due to its unique bilateral location along the third ventricle immediately above the degenerated optic chiasm. In addition to the postrecording morphological identification, a surprisingly high level of background spontaneous activity (compared with anterior lateral hypothalamic area) could distinguish SCN in these cultured slices. Extracellular SCN action potentials, filtered at 300-3000 Hz bandpass (Neurolog: NL 102), were discriminated using two Mentor 750 amplitude-window discriminator coupled with an ANDGATE, NL 501. The windows were set at both positive and negative phases of the selected unit in order to rule
A
ILJ_L_J 1 L_.[.L L.I t_LLIJ_.IIJ J TW
i
J,J ._L
T--I r-r r llec
J5 O p V
Fig. 1. A: action potentials recorded in two different SCN explants showing the quality of units analyzed in the present study. B: a 20-day-oldSCN explant stained with an antibody raised against VIP.
160 TABLE I Slice name
Days in culture
Recording time (h)
P. V.A. (%)
Period length
Maximum freq. (Hz)
Minimum freq. (Hz)
Number units
S12JAN S16JAN S26JAN S09FEB S13FEB S21FEB S23FEB S25FEB S01MRT S15AUG S15SEP S21OCT S05NOV S09NOV S21NOV S05DEC S12DEC
9 13 23 15 19 27 29 20 7 5 9 17 16 20 7 22 29
60 66 54 46 66 36 38 44 60 58 66 48 60 60 36 46 54
26 47 35 76 59 37 18 73 59 49 27 63 55 19 69 17 43
28 25 24 32 27 20 22 25 20 25 26 21 25 18 20 16 21
2.6 3.5 4.0 4.5 11.0 9.0 7.0 11.0 6.5 13.0 0.6 42.0 21.0 10.0 0.9 4.0 4.5
1.0 2.0 2.5 1.5 6.0 3.0 3.8 5.0 3.5 6.0 0.2 20.0 8.0 4.0 0.2 1.0 3.5
1 1 1 1 1 l 1 1 1 1 1 2-3 2-3 1 1 1 1
out artifacts. Discriminated spikes were fed into an
analysis and display of the time histogram. Mainly single
intelligent interface ( C a m b r i d g e Electronic Device: C E D 1401) connected to a T a n d o n (PC-AT) for on-line
neurons were r e c o r d e d , in rare cases 2 or 3 neurons (which could not be individually discriminated) were simultaneously analyzed. Fig. 1A shows examples of single and multi SCN n e u r o n s that were recorded in this study. Only one continuous recording was obtained per explant. Additional controls were made by 24 h recording of neuronal activity from the lateral hypothalamic area and from neocortical explants cultured u n d e r identical conditions. No circadian rhythms were found in the n e u r o n a l activity of these structures. For statistical analysis of the periodicity of the recordings, a cosine-fit periodogram program written for small computers was used 12. The proportion of the variability accounted for (P.V,A.), derived from this program, indicates the magnitude of rhythmicity. The m a x i m u m P.V.A. values
L) uJ u) (n i,u a. u)
12
24
12
24
TIME
6
12
18 PERIOD
12
24
12
( H )
24
LENGTH
30
M
(H)
Fig. 2. Time histogram (top) and the cosine-fit periodogram of a single SCN neuron showing the maximum proportion of variability accounted (P.V.A.) for a period length of 20 h.
6
12
18 PERIOD
24 LENGTH
30
36
(H)
Fig. 3. Mean P.V.A. values (the dashed bars indicate S.E.M.) for all SCN neurons (n --- 17) that had a significant periodicity between 16 and 32 h.
161 vivo (see also ref. 28). The presence of a circadian rhythm in firing rate of SCN neurons cultured over a long period of time in the absence of serum, cerebrospinal fluid and many brain areas that normally innervate this structure, provides direct evidence for SCN as a circadian oscillator. However, we have to admit that only 17 out of 22 explants showed circadian rhythms. Furthermore, although the mean (S.E.M.) P.V.A. of these neurons showed a maximum peak at 23 h (Fig. 3), a large scattered periodicity (ranging from mean period length of 16 h to 32 h; Table I) was found. Such large variations in spontaneous activity of SCN neurons were also reported in earlier studies using acute slices4'5'25. Apart from technical difficulties (see above) and the limited duration of continuous recording (maximum 66 h), we have the following two explanations for the relatively low percentage of active SCN explants each with a different period length: (i) the mammalian SCN, contrary to the pacemaker cells recorded in the Bulla eye 1, consists of a minority of pacemaker cells remaining active after longterm culturing in vitro; and/or (ii) not every individual rat SCN neuron, but a combination of an orchestrated group of neurons (each spontaneously firing with a periodicity different from 24 h, see Table I) are able to generate a precise circadian rhythm. Simultaneous recording from different single SCN neurons is required to get more insight into the SCN network. The cultured SCN preparation developed in this study is useful for studying central neuronal mechanisms underlying the mammalian circadian system, similar to the Bulla eye that is commonly used in studying circadian rhythms in invertebrates 1.
of the smoothed data were tested for statistical significance at the 1% level. Following the recordings, the explants were stained by conventional Nissl and by immunocytochemical techniques for vasointestinal polypeptide (VIP) and vasopressin (VP). Fig. 1B illustrates a 20-day-old SCN explant that was immunocytochemically stained with an antibody raised against VIP. The firing frequency of different SCN neurons varied from 0.2 and 15 Hz (Table I). The time histogram and the cosine periodogram output of a typical SCN neuron are shown in Fig. 2. High and low levels of spontaneous firing rate are visible in this unit, although the rhythm amplitude diminishes as a function of recording time. Out of 98 cultured explants only 22 showed spontaneous activity lasting for at least 36 h, to make the analysis of the circadian rhythm meaningful. This low percentage of success was due to several factors: e.g., the cessation of spontaneous activity, the development of infections, changes in amount and concentration of the medium during the recording and the mechanical neuronal damage caused by the recording electrode. In 5 out of the 22 successfully recorded explants no circadian rhythm was found (judged on the basis of the non-significant P.V.A. values using the Cosine-fit periodogram12). The remaining 17 showed clear periodic high and low levels of activity with a significant P.V.A. for periods ranging from 16 to 32 h (Table I). Despite large differences in the period length of different SCN explants, the averaged mean (S.E.M.) P.V.A. showed a maximum peak at 23 h (Fig. 3). The present experiment demonstrates that it is possible to continuously record neuronal discharges of single SCN neurons over several days in vitro. These SCN explants, which were cultured for several weeks, showed a characteristic morphology comparable to that found in
Acknowledgements. We are grateful to B.M. de Jong, J.M. Ruijter, R.E. Baker and H.J. Romijn for their assistance and advice in culturing and to R. Buijs for immunocytochemicalstaining.
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