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Administration of carbachol into the lateral ventricle and suprachiasmatic nucleus (SCN) produces dose-dependent phase shifts in the circadian rhythm of locomotor activity
Neuroscience Letters, 137 (1992)211 215
211
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08498
Administration of carbachol into the lateral ventricle and suprachiasmatic nucleus (SCN) produces dose-dependent phase shifts in the circadian rhythm of locomotor activity Beth E, F. Wee a, K e i t h D. A n d e r s o n b'*, N i c k S. K o u c h i s b a n d F r e d W. T u r e k b aDepartment of Psychology, Tulane University, New Orleans, LA 70118 (USA) and bDepartment of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208 (USA)
(Received 11 September 1991;Revised version received16 December 1991;Accepted20 December 1991) Key words: Carbachol; Circadian rhythm; Suprachiasmatic nucleus; Locomotor activity; Phase shift; Dose response curve; Lateral ventricle;
Acetylcholine The cholinergicagonist, carbachol,inducesphase-dependentshiftsin the timing of the circadian rhythm of locomotoractivity(CRLA). The effects of carbachol injectionsinto the lateral ventriclesof hamsters were compared betweencircadian times that produce phase delays vs. phase advances in the CRLA. The shape of the dose response curves and the EDs0 for carbachol injectionswere similar for the two injection times. The second experiment demonstrated the dose
When an animal is placed in constant darkness, rhythms, such as the locomotor activity rhythm, free-run with a period of about 24 h and can be phase advanced or phase delayed by exposure to a brief pulse of light if the light is presented at appropriate phases of the rhythm [3]. Such light-induced phase shifts are thought to underlie entrainment of the activity rhythm to the daily lightdark cycle. Pharmacological studies have revealed a number of chemical agents that phase shift and/or entrain circadian rhythms in rodents [see reviews in refs. 10 and 14], but only one agent, the cholinergic agonist, carbachol, has been shown to mimic both the phase shifting and entraining effects of light on the circadian system. Injections of carbachol into the lateral ventricles (LV) at certain phases of the circadian cycle produced phase shifts in the circadian rhythm of pineal N-acetyltransferase activity in rats [18] and the circadian rhythm of locomotor activity (CRLA) in mice [19] and golden hamsters [6] that were similar to those produced by light pulses given at the same circadian phase. Daily injections of carbachol to hamsters entrained the C R L A and main*Presentaddress: RegeneronPharmaceuticals,777 Old Saw Mill River Rd., Tarrytown,NY 10591,USA. Correspondence." B. Wee, Dept. of Psychology, 2007 Percival Stern Hall, Tulane University,New Orleans, LA 70118, USA.
tained gonadal function in a manner similar to that of daily light pulses [6]. Furthermore, intraventricular injection of the nicotinic cholinergic antagonist, mecamylamine, shortly before exposure of hamsters to brief light pulses reduced or blocked light-induced phase shifts in the C R L A [9]. Several additional studies have investigated the possible role of acetylcholine in the transmission of photic information to the suprachiasmatic nucleus (SCN), the major neural pacemaker responsible for the generation of most mammalian circadian rhythms [12, 15] (see reviews in refs. 10 and 14). Two experiments were performed to examine further the phase-shifting effects of carbachol on the C R L A in the hamster. In Experiment 1, the dose-response relationships were determined for phase advances and delays in the C R L A induced by lateral ventricular administration of carbachol to hamsters free-running in constant darkness. A comparison of these dose-response curves is an important step in addressing the possibility that light input to the circadian clock is mediated by different neurochemical pathways at phase advance and phase delay time points. In Experiment 2, the dose-response curve was determined for the phase-advancing effects of carbachol injections into the area of the SCN. Following stereotaxic surgery, adult male golden hamsters (Mesocricetus auratus Lak: LVG (SYR)) were
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Fig.1. Dose-response relationships for phase shifts produced by carbachol injections into the lateral ventricles of golden hamsters freerunning in constant darkness. A: mean phase shift (_+ S.E.M.) vs. dose of carbachol (5-11 hamsters/dose)injected at either CT 14 (c;) or CT 22 (@). The data were statistically fit to the curves generated by the program ALLFIT [4], and the EDs0values were determined to be 15.1 _+ 1.0 nmol for phase delays at CT 14 and 11.0 _+0.2 nmol for phase advances at CT 22. In some cases, standard error bars are enclosed within the circles. B: percent of the maximum mean phase shift (the mean phase shift at 63.2 nmol) vs. dose of carbachol. With constraints set at 0 and 100 for the minimum and maximum percent, the EDso determined by ALLFIT was 12.9 _+1.2 nmol for injectionsat CT 14 and 10.6 + 0.7 nmol for injections at CT 22. housed in individual polypropylene cages equipped with a running wheel and were maintained in constant darkness. The running wheel cages were connected to an event recorder (Esterline-Angus, Indianapolis) for continuous recording of the hamster's locomotor activity. The phase-shifting effects of the vehicle or carbachol injections were assessed by visual inspection of the chart record of each animal for 7-10 days before and after each injection, as described previously [17]. The onset of locom o t o r activity is defined as circadian time (CT) 12. Carbachol (Sigma, St. Louis) was dissolved in artifi-
cial cerebrospinal fluid vehicle in Expt. 1 and Krebs Ringer's Phosphate vehicle in Expt. 2, p H 7.4. Cannulae used for administration of the vehicle or carbachol were stereotaxically implanted unilaterally into the lateral ventricle (Expt. 1) or the area of the SCN (Expt. 2) while the animal was anesthetized with sodium pentobarbital (100 mg/kg b.wt.) (Sigma, St. Louis). In both experiments, the outer guide cannula was fit with an inner stylette that extended 1 m m beyond the cannula tip. Placement of each cannula was verified histologically at the end of each experiment. The order of injections was assigned randomly. In Expt. 1, the dose-response relationships of phaseadvancing and phase-delaying carbachol injections were examined in two groups of hamsters. Hamsters in one group received an injection of vehicle or a different dose of carbachol (2.0, 6.3, 11.2, 20.0, or 63.2 nmol) 10 h after activity onset (CT 22), and hamsters in the second group received the same doses of carbachol or the vehicle 2 h after activity onset (CT 14). Carbachol was injected into the lateral ventricle in a vol. of 2/zl through a 21-gauge stainless steel guide cannula. Some of the 24 animals received more than one injection, and consecutive injections were separated by at least two weeks. Injections were performed with the aid of a dim red light (Kodak safelight filter no. I A) while hamsters were lightly anesthetized with ether. Exposure of hamsters maintained in constant darkness to a red safelight for the brief time necessary to perform an LV injection does not induce a phase shift in the C R L A [6]. In Expt. 2, the dose response relationships of carbachol-induced phase advances were examined in 28 hamsters by administering vehicle or a different dose of carbachol (1.0, 3.2, 10.0, 32.0, 56.0, or 100 nmol) at CT 22. Carbachol was injected into the area of the SCN in a vol. of 200 nl through a 23-gauge stainless steel cannula. Each animal received up to 3 injections, and consecutive injections were separated by at least two weeks. Injections were performed with the aid of an infrared viewer (FJW Industries, Elgin, IL) while hamsters were lightly anesthetized with ether. D a t a were analyzed using Analysis of Covariance using injection number (first, second . . . . etc.) as the covariate to determine if phase shifts induced by the drug were affected by the number of injections the animal received. This covariate was not significant for any of the dose-response curves. Post hoc comparisons were performed with Dunnett's test to determine the effect of dose on phase shift. Best fit curves for dose-response date were generated by the program A L L F I T [4]. Injections of carbachol into the lateral ventricle at CT 22 induced clear phase advances in the C R L A that varied in magnitude depending upon the dose. Using the
213 statistical program ALLFIT, the data were fit to a sigmoidal curve (Fig. 1A). The calculated dose that would produce a half-maximal response (EDs0) was 11.0 + 0.2 nmol, and the calculated maximal phase shift was 256 + 3 min. Injections of carbachol at CT 14 induced dosedependent phase delays in the CRLA (Fig. 1A). The EDs0 at CT 14 was 15.1 + 1.0 nmol, and the calculated maximal phase shift was -73 + 3 min. Analysis of Variance revealed that phase shift was affected significantly by dose of carbachol (CT 14 F(5.45) = 6.39, P<0.001; CT 22 F(5.5]) = 6.88, P<0.001). Phase shifts produced by injections of either 20 or 63.2 nmol were significantly greater than phase shifts produced by the vehicle at CT 14 and CT 22 (Dunnett's, P<0.03). In contrast, phase shifts produced by carbachol injections of less than 20 nmol were not significantly different from vehicle-induced phase shifts at either circadian phase. Although the doses of carbachol yielding the halfmaximal response were similar for phase delays and advances, the magnitude of the phase shifts differed between the two phases. The maximum mean phase delay was only 28% of the maximum mean phase advance. Thus, for each circadian phase, the data were normalized by calculating the mean phase shift at a given dose as a percent of the maximum mean phase shift, which was obtained at a dose of 63.2 nmol. The ALLFIT-generated curves for normalized data illustrate the similarities in the dose-response relationships for advances and delays (Fig. 1B). The calculated EDs0 for these curves, setting constraints at 0 and 100 for minimum and maximum percent, were 12.9 + 1.2 nmol for injections at CT 14 and 10.6 + 0.7 nmol for injections at CT 22. Injections of carbachol into the area of the SCN at CT 22 induced permanent phase shifts in the CRLA which were dose-dependent (Figs. 2 and 3). In contrast, injections of the vehicle at the same circadian time induced little or no phase shift. Carbachol-induced phase shifts were primarily phase advances, although some phase delays were observed at doses of 1.0 and 3.2 nmol. All phase shifts were completed within 2-3 cycles of activity. The ALLFIT-calculated values indicate that the full dose-response curve was not measured, because the maximal phase shift was determined by ALLFIT to be 384 + 288 rain, and the mean phase shift obtained at the highest dose (100 nmol) was only 222 + 23.1 min. Using 384 min as the maximal response, the ALLFIT program calculated that a dose for a half-maximal response was 56.9 nmol. Analysis of Variance revealed that phase shift was affected significantly by dose of carbachol (F(6" 57) = 10.56, P<0.001). Phase shifts produced by injections of 32 nmol or larger were significantly greater than phase shifts produced by the vehicle (Dunnett's, P<0.01). Failure of the SCN curve to reach a plateau may have
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Fig. 2. Carbachol injections into the area of the suprachiasmatic nucleus (SCN) induce dose-dependent phase shifts. Portions of wheelrunning activity records from two representative hamsters free-running in constant darkness. Each horizontal line depicts the activity pattern over a 24-h period. Successive days are plotted from top to bottom. On the days indicated, each hamster was given an injection into the area of the SCN of a dose (in nanomoles) of carbachol or the KRP vehicle 10 h after the onset of activity (CT 22). The precise times of injection are denoted by the asterisks.
been due to the toxicity of the higher doses, which, when administered to the SCN area, resulted in disruption of the activity rhythm. To investigate the possibility that carbachol was acting directly on the SCN, a series of comparisons were made between the total estimated distance of the cannula tip to the SCN and the phase shift induced by carbachol at that distance. The correlation coefficients of the relationships
#
214
between phase shift and distance from the SCN were 0.310, -0.141, -0.270, -0.439, -0.521, 0.063, and -0.1 for the carbachol doses of 100, 56, 32, 10, 3.2, 1.0, and 0 nmol, respectively. None of these correlation coefficients were statistically significant. In addition, no significant correlations were found between phase shift and estimated distance in any of the 3 planes (dorsal-ventral, anterior-posterior, medial-lateral) for any of the carbachol doses. These results indicate that dose-dependent phase delays or phase advances of the CRLA in hamsters freerunning in constant darkness are induced by carbachol administration in the early or late subjective night, respectively. The similarities between dose-response curves at CT 14 and CT 22 suggests that carbachol's mechanism of action is similar for phase delays and phase advances. Our findings that carbachol injections into the LV or SCN induce phase advances in the late subjective night confirm and extend the findings of Earnest and Turek [6] but do not agree with the results reported by Meijer et al. [11] who observed phase advances in response to carbachol administration during the subjective day but not in response to treatment during the late subjective night. Meijer and coworkers attribute this difference, in part, to their use of a different strain of hamster. However, phase advances in response to carbachol injections in the late subjective night also have been observed for the C R L A in mice [19] and the circadian rhythm of pineal N-acetyltransferase activity in rats [18]. In addition, the dose-response relationships obtained for constant darkness (this study) differed somewhat from those reported for constant light [11]. The specific neural site(s) at which cholinergic stimulation may induce phase shifts in the C R L A is unknown, but because carbachol induces permanent phase shifts of the circadian clock, it must be acting directly on the clock itself or on an input pathway to the clock. Although the evidence for cholinergic involvement in the circadian system has been controversial [see reviews in refs. i0 and 14], recent evidence suggests that cholinergic neurons may project to the SCN [1, 5, 7, 16]. In the present study, phase advances produced by carbachol injections to the area of the SCN were dose-dependent, but the amplitude of these phase shifts was not significantly correlated with the distance between the cannula tip and the SCN. A relationship in which the amplitude of the phase shift is a function of the proximity of the injection site to the border of the SCN, as has been observed for injections of the protein synthesis inhibitor, anisomycin [8], would support the hypothesis that the phase-shifting effects are due to a direct action of the drug on the SCN. However, in the present study no such relationship existed for data points taken from animals whose cannulae
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Carbachol (nmol) Fig. 3. Dose-response curve for phase advances produced by carbachol injections at CT 22 into the SCN region of golden hamsters free-running in constant darkness. Mean phase shift (_+ S.E.M.) vs. dose of carbachol (8 10 hamsters/dose). The data were fit to the ALLFITgenerated curve, and the E D s , , value for SCN injections was determined to be 56.9 nmol.
were located up to 600/2m from the border of the SCN, as shown by the non-significant correlations between phase shift and distance from the SCN for each dose of carbachol. This finding suggests that carbachol may be acting at a site other than the SCN to induce phase shifts. A second way to examine the site of carbachol's actions would be to compare the dose-response curves for injections into the lateral ventricle vs. injections into the area of the SCN. If carbachol is acting at the level of the SCN, then one would expect phase shifts of a given magnitude to be induced by lower doses following SCN injections than following LV injections. This approach was used by Inouye et al. [8] to show that injections of anisomycin directly into the SCN are more effective than subcutaneous injections of anisomycin. In the present study, the dose-response curves for SCN vs. LV injections overlapped each other (comparison not shown), a finding that does not support the hypothesis that carbachol is acting at the level of the SCN itself to induce phase shifts in the CRLA. However, because different vehicles were used for the two experiments, definitive conclusions from these data can not be made. In conclusion, carbachol has been shown to mimick the phase-shifting effects of light on the CRLA in golden hamsters free-running in constant darkness in a dosedependent manner. These phase-shifting effects of light probably also involve other neurotransmitters such as the excitatory amino acids, the putative neurotransmitters of the retinohypothalamic tract [2, 13]. The exact site of carbachol's action remains to be determined.
215 The a u t h o r s t h a n k J a n e t Joy, Dave Millie, Rick Smith, a n d Joe T a k a h a s h i for their c o n t r i b u t i o n s to this project. This w o r k was s u p p o r t e d b y a p o s t d o c t o r a l research fellowship MH-09653 (B.E.F.W.), a p r e d o c t o r a l fellowship f r o m N I H t r a i n i n g G r a n t HD-07068 (K.D.A.), P H S G r a n t s HD-09885, MH-41211, a n d HD-21921 (F.W.T.) a n d the N S F C e n t e r for Biological Timing. 1 Bina, K.G., Semba, K. and Rusak, B., Localization of cholinergic and nerve growth factor-receptor neurons projecting to the suprachiasmatic nucleus of the rat, Neurosci. Abstr., 16 (1990) 601. 2 Colwell, C.S., Ralph, M.R. and Menaker, M., Do NMDA receptors mediate the effects of light on circadian behavior?, Brain Res., 523 (1990) 117-120. 3 Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemakers. II. The variability of phase response curves, J.Comp. Physiol., 105 (1976) 253-266. 4 DeLean, A., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiologicaldose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 5 Dwyer, M.R., Harrington, M.E. and Rahmani, T., Localization of choline acetyltransferase immunoreactive (CAT-I) cells afferent to the suprachiasmatic nucleus (SCN) in the golden hamster, Neurosci. Abstr., 16 (1990) 602. 6 Earnest, D.J. and Turek, F.W., Neurochemical basis for the photic control of circadian rhythms and seasonal reproductive cycles: roles for acetylcholine,Proc. Natl. Acad. Sci. USA, 82 (1985) 42774281. 7 Fuller, C.A., Nayduch, R. and Murakami, D.M., AChE positive neurons that project to the SCN, Neurosci Abstr., 15 (1989) 1059. 8 Inouye, S.-I.T., Takahashi, J.S., Wollnik, F. and Turek, F.W., Inhibitor of protein synthesis phase shifts a circadian pacemaker in mammalian SCN, Am. J. Physiol., 255 (1988) R1055-R1058.
9 Keefe, D.L., Earnest, D.J., Nelson, D., Takahashi, J.S. and Turek, F.W., A cholinergic antagonist, mecamylamine, blocks the phaseshifting effects of light on the circadian rhythm of locomotor activity in the golden hamster, Brain Res., 403 (1987) 308-312. 10 Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev., 69 (1989) 671-707. 11 Meijer, J.H., van der Zee, E. and Dietz, M., The effects of intraventricular carbachol injections on the free-running activity rhythm of the hamster, J. Biol. Rhythms, 3 (1988) 333-348. 12 Moore, R.Y., Organization and function of a central nervous system oscillator: the suprachiasmatic nucleus, Federation Proc., 42 (1983) 2783-2789. 13 Ohi, K., Takashima, M., Nishikawa, T. and Takahashi, K., Nmethyl-D-aspartate receptor participates in neuronal transmission of photic information through the retinohypothalamic tract, Neuroendocrinology, 53 (1991) 344-348. 14 Rusak, B. and Bina, K.G., Neurotransmitters in the mammalian circadian system, Annu. Rev. Neurosci., 13 (1990) 387401. 15 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 16 van der Zee, E.A., Streefland, C., Strosberg, A.D., Schroder, H., and Luiten, P.G.M., Colocalization of muscarinic and nicotinic receptors in cholinoceptive neurons of the suprachiasmatic region in young and aged rats, Brain Res., 542 (1991) 348-352. 17 Wee, B.E.F. and Turek, F.W., Midazolam, a short-acting benzodiazepine, resets the circadian clock of the hamster, Pharm. Biochem. Behav., 32 (1989) 901-906. 18 Zatz, M. and Brownstein, M.J., Intraventricular carbachol mimics the effects of light on the circadian rhythm in the rat pineal gland, Science, 203 (1979) 358-361. 19 Zatz, M. and Herkenham, M.A., Intraventricular carbachol mimics the phase-shifting effect of light on the circadian rhythm of wheelrunning activity, Brain Res., 212 (1981) 234-238.
211
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08498
Administration of carbachol into the lateral ventricle and suprachiasmatic nucleus (SCN) produces dose-dependent phase shifts in the circadian rhythm of locomotor activity Beth E, F. Wee a, K e i t h D. A n d e r s o n b'*, N i c k S. K o u c h i s b a n d F r e d W. T u r e k b aDepartment of Psychology, Tulane University, New Orleans, LA 70118 (USA) and bDepartment of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208 (USA)
(Received 11 September 1991;Revised version received16 December 1991;Accepted20 December 1991) Key words: Carbachol; Circadian rhythm; Suprachiasmatic nucleus; Locomotor activity; Phase shift; Dose response curve; Lateral ventricle;
Acetylcholine The cholinergicagonist, carbachol,inducesphase-dependentshiftsin the timing of the circadian rhythm of locomotoractivity(CRLA). The effects of carbachol injectionsinto the lateral ventriclesof hamsters were compared betweencircadian times that produce phase delays vs. phase advances in the CRLA. The shape of the dose response curves and the EDs0 for carbachol injectionswere similar for the two injection times. The second experiment demonstrated the dose
When an animal is placed in constant darkness, rhythms, such as the locomotor activity rhythm, free-run with a period of about 24 h and can be phase advanced or phase delayed by exposure to a brief pulse of light if the light is presented at appropriate phases of the rhythm [3]. Such light-induced phase shifts are thought to underlie entrainment of the activity rhythm to the daily lightdark cycle. Pharmacological studies have revealed a number of chemical agents that phase shift and/or entrain circadian rhythms in rodents [see reviews in refs. 10 and 14], but only one agent, the cholinergic agonist, carbachol, has been shown to mimic both the phase shifting and entraining effects of light on the circadian system. Injections of carbachol into the lateral ventricles (LV) at certain phases of the circadian cycle produced phase shifts in the circadian rhythm of pineal N-acetyltransferase activity in rats [18] and the circadian rhythm of locomotor activity (CRLA) in mice [19] and golden hamsters [6] that were similar to those produced by light pulses given at the same circadian phase. Daily injections of carbachol to hamsters entrained the C R L A and main*Presentaddress: RegeneronPharmaceuticals,777 Old Saw Mill River Rd., Tarrytown,NY 10591,USA. Correspondence." B. Wee, Dept. of Psychology, 2007 Percival Stern Hall, Tulane University,New Orleans, LA 70118, USA.
tained gonadal function in a manner similar to that of daily light pulses [6]. Furthermore, intraventricular injection of the nicotinic cholinergic antagonist, mecamylamine, shortly before exposure of hamsters to brief light pulses reduced or blocked light-induced phase shifts in the C R L A [9]. Several additional studies have investigated the possible role of acetylcholine in the transmission of photic information to the suprachiasmatic nucleus (SCN), the major neural pacemaker responsible for the generation of most mammalian circadian rhythms [12, 15] (see reviews in refs. 10 and 14). Two experiments were performed to examine further the phase-shifting effects of carbachol on the C R L A in the hamster. In Experiment 1, the dose-response relationships were determined for phase advances and delays in the C R L A induced by lateral ventricular administration of carbachol to hamsters free-running in constant darkness. A comparison of these dose-response curves is an important step in addressing the possibility that light input to the circadian clock is mediated by different neurochemical pathways at phase advance and phase delay time points. In Experiment 2, the dose-response curve was determined for the phase-advancing effects of carbachol injections into the area of the SCN. Following stereotaxic surgery, adult male golden hamsters (Mesocricetus auratus Lak: LVG (SYR)) were
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Fig.1. Dose-response relationships for phase shifts produced by carbachol injections into the lateral ventricles of golden hamsters freerunning in constant darkness. A: mean phase shift (_+ S.E.M.) vs. dose of carbachol (5-11 hamsters/dose)injected at either CT 14 (c;) or CT 22 (@). The data were statistically fit to the curves generated by the program ALLFIT [4], and the EDs0values were determined to be 15.1 _+ 1.0 nmol for phase delays at CT 14 and 11.0 _+0.2 nmol for phase advances at CT 22. In some cases, standard error bars are enclosed within the circles. B: percent of the maximum mean phase shift (the mean phase shift at 63.2 nmol) vs. dose of carbachol. With constraints set at 0 and 100 for the minimum and maximum percent, the EDso determined by ALLFIT was 12.9 _+1.2 nmol for injectionsat CT 14 and 10.6 + 0.7 nmol for injections at CT 22. housed in individual polypropylene cages equipped with a running wheel and were maintained in constant darkness. The running wheel cages were connected to an event recorder (Esterline-Angus, Indianapolis) for continuous recording of the hamster's locomotor activity. The phase-shifting effects of the vehicle or carbachol injections were assessed by visual inspection of the chart record of each animal for 7-10 days before and after each injection, as described previously [17]. The onset of locom o t o r activity is defined as circadian time (CT) 12. Carbachol (Sigma, St. Louis) was dissolved in artifi-
cial cerebrospinal fluid vehicle in Expt. 1 and Krebs Ringer's Phosphate vehicle in Expt. 2, p H 7.4. Cannulae used for administration of the vehicle or carbachol were stereotaxically implanted unilaterally into the lateral ventricle (Expt. 1) or the area of the SCN (Expt. 2) while the animal was anesthetized with sodium pentobarbital (100 mg/kg b.wt.) (Sigma, St. Louis). In both experiments, the outer guide cannula was fit with an inner stylette that extended 1 m m beyond the cannula tip. Placement of each cannula was verified histologically at the end of each experiment. The order of injections was assigned randomly. In Expt. 1, the dose-response relationships of phaseadvancing and phase-delaying carbachol injections were examined in two groups of hamsters. Hamsters in one group received an injection of vehicle or a different dose of carbachol (2.0, 6.3, 11.2, 20.0, or 63.2 nmol) 10 h after activity onset (CT 22), and hamsters in the second group received the same doses of carbachol or the vehicle 2 h after activity onset (CT 14). Carbachol was injected into the lateral ventricle in a vol. of 2/zl through a 21-gauge stainless steel guide cannula. Some of the 24 animals received more than one injection, and consecutive injections were separated by at least two weeks. Injections were performed with the aid of a dim red light (Kodak safelight filter no. I A) while hamsters were lightly anesthetized with ether. Exposure of hamsters maintained in constant darkness to a red safelight for the brief time necessary to perform an LV injection does not induce a phase shift in the C R L A [6]. In Expt. 2, the dose response relationships of carbachol-induced phase advances were examined in 28 hamsters by administering vehicle or a different dose of carbachol (1.0, 3.2, 10.0, 32.0, 56.0, or 100 nmol) at CT 22. Carbachol was injected into the area of the SCN in a vol. of 200 nl through a 23-gauge stainless steel cannula. Each animal received up to 3 injections, and consecutive injections were separated by at least two weeks. Injections were performed with the aid of an infrared viewer (FJW Industries, Elgin, IL) while hamsters were lightly anesthetized with ether. D a t a were analyzed using Analysis of Covariance using injection number (first, second . . . . etc.) as the covariate to determine if phase shifts induced by the drug were affected by the number of injections the animal received. This covariate was not significant for any of the dose-response curves. Post hoc comparisons were performed with Dunnett's test to determine the effect of dose on phase shift. Best fit curves for dose-response date were generated by the program A L L F I T [4]. Injections of carbachol into the lateral ventricle at CT 22 induced clear phase advances in the C R L A that varied in magnitude depending upon the dose. Using the
213 statistical program ALLFIT, the data were fit to a sigmoidal curve (Fig. 1A). The calculated dose that would produce a half-maximal response (EDs0) was 11.0 + 0.2 nmol, and the calculated maximal phase shift was 256 + 3 min. Injections of carbachol at CT 14 induced dosedependent phase delays in the CRLA (Fig. 1A). The EDs0 at CT 14 was 15.1 + 1.0 nmol, and the calculated maximal phase shift was -73 + 3 min. Analysis of Variance revealed that phase shift was affected significantly by dose of carbachol (CT 14 F(5.45) = 6.39, P<0.001; CT 22 F(5.5]) = 6.88, P<0.001). Phase shifts produced by injections of either 20 or 63.2 nmol were significantly greater than phase shifts produced by the vehicle at CT 14 and CT 22 (Dunnett's, P<0.03). In contrast, phase shifts produced by carbachol injections of less than 20 nmol were not significantly different from vehicle-induced phase shifts at either circadian phase. Although the doses of carbachol yielding the halfmaximal response were similar for phase delays and advances, the magnitude of the phase shifts differed between the two phases. The maximum mean phase delay was only 28% of the maximum mean phase advance. Thus, for each circadian phase, the data were normalized by calculating the mean phase shift at a given dose as a percent of the maximum mean phase shift, which was obtained at a dose of 63.2 nmol. The ALLFIT-generated curves for normalized data illustrate the similarities in the dose-response relationships for advances and delays (Fig. 1B). The calculated EDs0 for these curves, setting constraints at 0 and 100 for minimum and maximum percent, were 12.9 + 1.2 nmol for injections at CT 14 and 10.6 + 0.7 nmol for injections at CT 22. Injections of carbachol into the area of the SCN at CT 22 induced permanent phase shifts in the CRLA which were dose-dependent (Figs. 2 and 3). In contrast, injections of the vehicle at the same circadian time induced little or no phase shift. Carbachol-induced phase shifts were primarily phase advances, although some phase delays were observed at doses of 1.0 and 3.2 nmol. All phase shifts were completed within 2-3 cycles of activity. The ALLFIT-calculated values indicate that the full dose-response curve was not measured, because the maximal phase shift was determined by ALLFIT to be 384 + 288 rain, and the mean phase shift obtained at the highest dose (100 nmol) was only 222 + 23.1 min. Using 384 min as the maximal response, the ALLFIT program calculated that a dose for a half-maximal response was 56.9 nmol. Analysis of Variance revealed that phase shift was affected significantly by dose of carbachol (F(6" 57) = 10.56, P<0.001). Phase shifts produced by injections of 32 nmol or larger were significantly greater than phase shifts produced by the vehicle (Dunnett's, P<0.01). Failure of the SCN curve to reach a plateau may have
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Fig. 2. Carbachol injections into the area of the suprachiasmatic nucleus (SCN) induce dose-dependent phase shifts. Portions of wheelrunning activity records from two representative hamsters free-running in constant darkness. Each horizontal line depicts the activity pattern over a 24-h period. Successive days are plotted from top to bottom. On the days indicated, each hamster was given an injection into the area of the SCN of a dose (in nanomoles) of carbachol or the KRP vehicle 10 h after the onset of activity (CT 22). The precise times of injection are denoted by the asterisks.
been due to the toxicity of the higher doses, which, when administered to the SCN area, resulted in disruption of the activity rhythm. To investigate the possibility that carbachol was acting directly on the SCN, a series of comparisons were made between the total estimated distance of the cannula tip to the SCN and the phase shift induced by carbachol at that distance. The correlation coefficients of the relationships
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between phase shift and distance from the SCN were 0.310, -0.141, -0.270, -0.439, -0.521, 0.063, and -0.1 for the carbachol doses of 100, 56, 32, 10, 3.2, 1.0, and 0 nmol, respectively. None of these correlation coefficients were statistically significant. In addition, no significant correlations were found between phase shift and estimated distance in any of the 3 planes (dorsal-ventral, anterior-posterior, medial-lateral) for any of the carbachol doses. These results indicate that dose-dependent phase delays or phase advances of the CRLA in hamsters freerunning in constant darkness are induced by carbachol administration in the early or late subjective night, respectively. The similarities between dose-response curves at CT 14 and CT 22 suggests that carbachol's mechanism of action is similar for phase delays and phase advances. Our findings that carbachol injections into the LV or SCN induce phase advances in the late subjective night confirm and extend the findings of Earnest and Turek [6] but do not agree with the results reported by Meijer et al. [11] who observed phase advances in response to carbachol administration during the subjective day but not in response to treatment during the late subjective night. Meijer and coworkers attribute this difference, in part, to their use of a different strain of hamster. However, phase advances in response to carbachol injections in the late subjective night also have been observed for the C R L A in mice [19] and the circadian rhythm of pineal N-acetyltransferase activity in rats [18]. In addition, the dose-response relationships obtained for constant darkness (this study) differed somewhat from those reported for constant light [11]. The specific neural site(s) at which cholinergic stimulation may induce phase shifts in the C R L A is unknown, but because carbachol induces permanent phase shifts of the circadian clock, it must be acting directly on the clock itself or on an input pathway to the clock. Although the evidence for cholinergic involvement in the circadian system has been controversial [see reviews in refs. i0 and 14], recent evidence suggests that cholinergic neurons may project to the SCN [1, 5, 7, 16]. In the present study, phase advances produced by carbachol injections to the area of the SCN were dose-dependent, but the amplitude of these phase shifts was not significantly correlated with the distance between the cannula tip and the SCN. A relationship in which the amplitude of the phase shift is a function of the proximity of the injection site to the border of the SCN, as has been observed for injections of the protein synthesis inhibitor, anisomycin [8], would support the hypothesis that the phase-shifting effects are due to a direct action of the drug on the SCN. However, in the present study no such relationship existed for data points taken from animals whose cannulae
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Carbachol (nmol) Fig. 3. Dose-response curve for phase advances produced by carbachol injections at CT 22 into the SCN region of golden hamsters free-running in constant darkness. Mean phase shift (_+ S.E.M.) vs. dose of carbachol (8 10 hamsters/dose). The data were fit to the ALLFITgenerated curve, and the E D s , , value for SCN injections was determined to be 56.9 nmol.
were located up to 600/2m from the border of the SCN, as shown by the non-significant correlations between phase shift and distance from the SCN for each dose of carbachol. This finding suggests that carbachol may be acting at a site other than the SCN to induce phase shifts. A second way to examine the site of carbachol's actions would be to compare the dose-response curves for injections into the lateral ventricle vs. injections into the area of the SCN. If carbachol is acting at the level of the SCN, then one would expect phase shifts of a given magnitude to be induced by lower doses following SCN injections than following LV injections. This approach was used by Inouye et al. [8] to show that injections of anisomycin directly into the SCN are more effective than subcutaneous injections of anisomycin. In the present study, the dose-response curves for SCN vs. LV injections overlapped each other (comparison not shown), a finding that does not support the hypothesis that carbachol is acting at the level of the SCN itself to induce phase shifts in the CRLA. However, because different vehicles were used for the two experiments, definitive conclusions from these data can not be made. In conclusion, carbachol has been shown to mimick the phase-shifting effects of light on the CRLA in golden hamsters free-running in constant darkness in a dosedependent manner. These phase-shifting effects of light probably also involve other neurotransmitters such as the excitatory amino acids, the putative neurotransmitters of the retinohypothalamic tract [2, 13]. The exact site of carbachol's action remains to be determined.
215 The a u t h o r s t h a n k J a n e t Joy, Dave Millie, Rick Smith, a n d Joe T a k a h a s h i for their c o n t r i b u t i o n s to this project. This w o r k was s u p p o r t e d b y a p o s t d o c t o r a l research fellowship MH-09653 (B.E.F.W.), a p r e d o c t o r a l fellowship f r o m N I H t r a i n i n g G r a n t HD-07068 (K.D.A.), P H S G r a n t s HD-09885, MH-41211, a n d HD-21921 (F.W.T.) a n d the N S F C e n t e r for Biological Timing. 1 Bina, K.G., Semba, K. and Rusak, B., Localization of cholinergic and nerve growth factor-receptor neurons projecting to the suprachiasmatic nucleus of the rat, Neurosci. Abstr., 16 (1990) 601. 2 Colwell, C.S., Ralph, M.R. and Menaker, M., Do NMDA receptors mediate the effects of light on circadian behavior?, Brain Res., 523 (1990) 117-120. 3 Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemakers. II. The variability of phase response curves, J.Comp. Physiol., 105 (1976) 253-266. 4 DeLean, A., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiologicaldose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 5 Dwyer, M.R., Harrington, M.E. and Rahmani, T., Localization of choline acetyltransferase immunoreactive (CAT-I) cells afferent to the suprachiasmatic nucleus (SCN) in the golden hamster, Neurosci. Abstr., 16 (1990) 602. 6 Earnest, D.J. and Turek, F.W., Neurochemical basis for the photic control of circadian rhythms and seasonal reproductive cycles: roles for acetylcholine,Proc. Natl. Acad. Sci. USA, 82 (1985) 42774281. 7 Fuller, C.A., Nayduch, R. and Murakami, D.M., AChE positive neurons that project to the SCN, Neurosci Abstr., 15 (1989) 1059. 8 Inouye, S.-I.T., Takahashi, J.S., Wollnik, F. and Turek, F.W., Inhibitor of protein synthesis phase shifts a circadian pacemaker in mammalian SCN, Am. J. Physiol., 255 (1988) R1055-R1058.
9 Keefe, D.L., Earnest, D.J., Nelson, D., Takahashi, J.S. and Turek, F.W., A cholinergic antagonist, mecamylamine, blocks the phaseshifting effects of light on the circadian rhythm of locomotor activity in the golden hamster, Brain Res., 403 (1987) 308-312. 10 Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev., 69 (1989) 671-707. 11 Meijer, J.H., van der Zee, E. and Dietz, M., The effects of intraventricular carbachol injections on the free-running activity rhythm of the hamster, J. Biol. Rhythms, 3 (1988) 333-348. 12 Moore, R.Y., Organization and function of a central nervous system oscillator: the suprachiasmatic nucleus, Federation Proc., 42 (1983) 2783-2789. 13 Ohi, K., Takashima, M., Nishikawa, T. and Takahashi, K., Nmethyl-D-aspartate receptor participates in neuronal transmission of photic information through the retinohypothalamic tract, Neuroendocrinology, 53 (1991) 344-348. 14 Rusak, B. and Bina, K.G., Neurotransmitters in the mammalian circadian system, Annu. Rev. Neurosci., 13 (1990) 387401. 15 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 16 van der Zee, E.A., Streefland, C., Strosberg, A.D., Schroder, H., and Luiten, P.G.M., Colocalization of muscarinic and nicotinic receptors in cholinoceptive neurons of the suprachiasmatic region in young and aged rats, Brain Res., 542 (1991) 348-352. 17 Wee, B.E.F. and Turek, F.W., Midazolam, a short-acting benzodiazepine, resets the circadian clock of the hamster, Pharm. Biochem. Behav., 32 (1989) 901-906. 18 Zatz, M. and Brownstein, M.J., Intraventricular carbachol mimics the effects of light on the circadian rhythm in the rat pineal gland, Science, 203 (1979) 358-361. 19 Zatz, M. and Herkenham, M.A., Intraventricular carbachol mimics the phase-shifting effect of light on the circadian rhythm of wheelrunning activity, Brain Res., 212 (1981) 234-238.