Intraventricular carbachol mimics the phase-shifting effect of light on the circadian rhythm of wheel-running activity

234

Brain Research, 212 (1981) 234-238 © Elsevier/North-Holland Biomedical Press

Intraventricular carbachol mimics the phase-shifting effect of light on the circadian rhythm of wheel-running activity

MARTIN ZATZ and MILES A. HERKENHAM (M.Z.) Laboratory of Clinical Science and (M.A.H.) Laboratory of Neurophysiology, National Institute of Mental Health, Bethesda, Md. 20205 (U.S.A.)

(Accepted December 25th, 1980) Key words: circadian rhythm - - acetylcholine - - pbotoentrainment

Intraventricular injections of carbachol, a cholinergic agonist, into free-running mice can cause phase dependent phase shifts, in both directions, in the circadian rhythm of wheel-running activity. These effects mimic the action of light in entraining the circadian pacemaker, and suggest that acetylcholine may play a role in photoentrainment. Numerous behavioral, physiologic and biochemical functions display circadian rhythms z,10. The rhythmicity of these functions in mammals is thought to derive from one or more endogenous, self-sustained oscillators in the central nervous system1,6,11. In view of the complex regulation of individual circadian functions (e.g. motor activity, pineal indoleamine metabolism, corticosteroid secretion), an agent's effect upon the central oscillatory mechanism is revealed only through a change in the freerunning period or phase of a rhythmic functionL Period and phase are profoundly affected by environmental lighting (photoentrainment). The effects of light on mammalian circadian rhythms are mediated by the retinohypothalamic projection to the suprachiasmatic nucleus of the hypothalamus v. It should therefore be possible to mimic or block the effects of light using neuropharmacologic agents4,13. Such drugs should be useful in identifying the neurotransmitters involved in photoentrainment and the mechanisms regulating circadian rhythms. We showed previously that intraventricular injections of carbachol could mimic the effects of light on the circadian rhythm of rat pineal indoleamine metabolismlL In this paper we show that intraventricular injections of carbachol can mimic the effects of light on the circadian rhythm of wheel-running activity in the mouse. The ability of this drug, which specifically mimics the neurotransmitter acetylcholine, to mimic the effects of light on different rhythms demonstrates that it can affect the circadian pacemaker and raises the possibility that acetylcholine is involved in photoentrainment. Adult male C57B16J mice were purchased from Jackson Laboratories (Bar Harbor, Me.). They were individually housed in activity-wheel cages with free access to food and water in a r o o m with controlled artifical lighting. Activity wheels were

235 connected through microswitches to an Intel 8080 microprocessor, which recorded 24 h wheel-running activity onto digital tape cassettes. Data were plotted by a PDP 11/40 system with a Versatec plotter. There were 4 experiments, between July, 1979 and May, 1980. In each experiment mice were entrained to an LD 12:12 cycle (12 h light, 12 h dark), then permitted to free-run (without light cues) for at least two weeks, then injected intraventricularly at specific phase points with carbachol or saline and, finally, permitted to resume their free-run. The first experiment differed somewhat from the others in that the animals free-ran in dim red light and received two injections 25 days apart. In the others, the mice free-ran after blinding and received only one injection. In all, there were 37 animals that completed the experiments and 19 that did not, because of mortality or technical failure in the data acquisition. No animals were excluded for any other reason. Animals were blinded by bilateral orbital enucleation under ether anesthesia in the middle of the light period. Holes were drilled bilaterally in the skull using bregma as a landmark. Injections were made bilaterally into the lateral cerebral ventricles with a 10 /~1 Hamilton syringe fitted with a stainless steel sleeve to limit depth of penetration. Appropriate needle position had been determined in preliminary experiments using trypan-blue. In the experimental animals, intraventicular site of injections was confirmed by histological examination at the end of the experiments. Circadian periods were calculated from the slope of the lines best fitting activity onset over at least 12 days preceding or following injections, as determined by eye. Phase shifts were measured by the distance between these lines on the day after injection. In the first experiment, mice received 6 nmol of carbachol (Sigma Chemicals, St. Louis, Mo.) in 2 #1 of sterile phosphate buffered saline, into each lateral ventricle after 10 days free-run in dim red light. Some animals injected 3 h after activity onset (circadian time (c.t.) 15) showed phase delays, and one animal injected 10 h after activity onset (c.t. 22) showed a phase advance, but these phase shifts were small (all under 30 min). Animals injected with saline alone showed no change. The mice were injected again 25 days later with a higher dose, 20 nmol of carbachol on each side. A greater proportion of animals showed phase shifts and these shifts were larger than those seen with the lower dose. These preliminary results suggested that intraventricular injections of carbachol could cause phase-dependent phase shifts and that the effect was dose dependent. The higher dose was used in subsequent experiments. Data from the second injection only are included below. In order to demonstrate that the initial results were not due to the animals' exposure to light (injections were performed close to the 60 W Westinghouse red light), animals were blinded in subsequent experiments. Blinded animals also showed phase-delays in response to intraventricular carbachol injected 3 h after activity onset (c.t. 15). Data from two such animals are illustrated in Fig. 1A, B. Some mice injected at c.t. 22 showed phase advances. Data from one such animal are illustrated in Fig. IC. None of the 11 mice injected with saline alone at either c.t. 15 or c.t. 22 showed either phase delays or phase advances. Results of these experiments are summarized in Table I. Free running periods varied between 23.35 and 23.93 h and averaged about 23.57 h, which is consistent with

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Fig. I. In each experiment shown, a mouse was entrained to an LD 12:12 cycle (lights offat noon) for at least 12 days and then blinded. After at least two weeks free-run, each received bilateral intraventricular injections of carbachol either 3 h after onset of activity (c.t. 15) (A and B) or 10 h after onset of activity (e.t. 22) (C). The day of injection is indicated by an asterisk. The actograms shown plot one 24 h period per horizontal line with total daily activity to the right of each line. Scales above and below the actogram indicate solar time.

237 previous reports 9. The average free-running period did not differ between groups and was unaffected by the injection of carbachol. The period did change in about half the animals after injection (averaging 0.08 h) and such changes seemed greater, though not more frequent, after carbachol injection as compared to saline injection. However, there was no correlation between the size or direction of the change in period and the presence, size, or direction of phase shift. Six of eleven mice injected with carbachol 3 h after activity onset (c.t. 15) showed clear phase delays in their free-running activity rhythms. These delays averaged about 100 min. The remaining animals showed no clear phase delay - - none showed a phase advance. Five of fifteen mice injected with carbachol 10 h after activity onset (c.t. 22) showed phase advances. None of the remaining 10 animals injected at c.t. 22 showed a phase delay. The phase shifts obtained using intraventricular carbachol varied in direction as a function of the time the drug was given. This indicates the presence of a biphasic phase-response curve and implies that carbachol could entrain the circadian oscillator. The phase points used were chosen on the basis of the phase-response curve of response to pulses of light. Using light, Daan and Pittendrigh 5, found an asymmetric phase-response curve with maximal phase delays of about 3 h at about c.t. 16 and maximal phase advances of less than 1 h at about c.t. 23. Carbachol also mimicked the phase-shifting effect of light in that phase delays were more often obtained and clearly larger than phase advances. We have previously shown that intraventricular injections of carbachol can mimic the effects of light on the circadian rhythm of pineal serotonin N-acetyltransferase activity in grouped rats la. Here we have extended this finding to the circadian rhythm of wheel-running activity in individual mice. Taken together these results suggest that carbachol acts on a circadian pacemaker common to both these rhythms. They also raise the possibility that acetylcholine is involved in the circadian effects of light. The anatomical site of action of carbachol has not been defined. It may, like light, act on the suprachiasmatic nucleus of the hypothalamus. Carbachol may affect the circadian pacemaker 'directly' or, like light, 'indirectly.' The identity of the neurotransmitter used by the retinohypothalamic projection is unknown. The effect of carbachol on blinded animals speaks against a presynaptic action oa the terminals of TABLE I Effect o f intraventricular carbachol on period and phase of free-running mouse activity cycles Injection time

c.t. 15

c.t. 22

Preinjection period (h) Postinjection period (h)

23.58 4- 0.04l (11) 23.64 4- 0.027 (11)

23.52 ± 0.044 (15) 23.56 ~ 0.043 1.15)

Phase shifts

6/11

5/15

--101 4- 10 (6)

+33 ± 8 (5)

--55 4- 16.8 (11)

+11 4- 4.9 (15)

Average phase shift when seen (rain) Average phase shift, all animals (rain)

238 the r e t i n o h y p o t h a l a m i c projection. C h o l i n e acetyltransferase, which synthesizes acetylcholine, is present in the s u p r a c h i a s m a t i c nucleus 3 as are cholinergic receptors 1~. I t should be noted, however, t h a t even if acetylcholine is involved in the effects o f light on the c i r c a d i a n p a c e m a k e r , it need n o t be the n e u r o t r a n s m i t t e r released by the nerves f r o m the retina. It might be used b y an ' i n t e r n e u r o n ' a n d be involved in the processing o f the i n f o r m a t i o n c o n c e r n i n g light. T h e r e need n o t be a u n i q u e n e u r o t r a n s m i t t e r ' m e d i a t i n g ' the effects o f light; several m a y p a r t i c i p a t e in p h o t o e n t r a i n m e n t . W e t h a n k Dr. E. Evarts for the use o f his facilities. W e t h a n k M r . W i l l i a m V a u g h n for developing the c o m p u t e r p r o g r a m s a n d his advice on their use.

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