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Perturbations of locomotor activity rhythms following suprachiasmatic bungarotoxin infusion
Physiology & Behuvior, Vol. 43, pp. 859-865. Copyright
0 Pergamon
Press plc, 1988. Printed
0031-9384/88
in the U.S.A.
$3.00 + .OO
BRIEF COMMUNICATION
Perturbations of Locomotor Activity Rhythms Following Suprachiasmatic Bungarotoxin Infusion’ JAMES R. PAULYz AND NELSON Department
of Biology,
Marquette
D. HORSEMAN3
University,
Received 23 October
Milwaukee,
WI 53233
1987
PAULY, J. R. AND N. D. HORSEMAN. Perturbations of locomotor activity rhythms following supruchiasmatic bungarotoxin infusion. PHYSIOL BEHAV 43(6) 859-865, 1988.-The actions of intracranial injections of alpha bungarotoxin (BTX) on locomotor activity rhythms were examined in male rats. The hypothalamic suprachiasmatic region is known to be a locus of high affinity BTX binding although the potential roles of this receptor system are unknown. Rats were stereotaxitally implanted with cannulae aimed just dorsal to the SCN (or cortex for control injections). Free-running locomotor activity rhythms in darkness were constantly monitored. Seventy-eight percent (78%) of the animals injected with BTX in the SCN region had phase shifts that were outside the 9% confidence limits of control animals. Infusion of either saline (into the SCN) or BTX into cortex were without effects. Doses of BTX varied from 125 fmol to 600 pmol. At the highest dose a substantial fraction of the animals showed both period changes and loss of rhythmicity as well as phase shifts. Although nearly all of the animals injected with BTX experienced phase shifts the direction of the shifts were not consistently correlated with the circadian time of injection. However, the sensitivity of the animals varied systematically with the smallest shifts resulting after injection at CT12 and CT16. These results argue that BTX does not influence the SCN pacemaker as an entraining signal but does potently perturb the circadian system. Rats
Biological rhythms
Bungarotoxin
Cholinergic systems
ALPHA bungarotoxin (BTX) binds densely and with high affinity in the area of the hypothalamic suprachiasmatic nuclei (SCN) [l, 5, 7, 8, 14, 181, which function as circadian pacemakers in mammals. The functions of central nervous system (CNS) receptors that bind BTX with high affinity are largely unknown. Although BTX has been presumed in many studies to act as a nicotinic cholinergic antagonist in the CNS, its pharmacology and physiology appear now to be quite different from its classical mechanism of action on peripheral cholinergic receptors. Several autoradiographic and biochemical studies have in fact shown that there is little overlap between brain areas that bind nicotine (and acetylcholine) and those that are labeled by BTX [2, 11, 121. Previously, BTX has been used as a probe for putative involvement of cholinergic systems in light/dark synchronization of circadian systems [21]. However, no studies have carefully examined what effects BTX alone might have on circadian timekeeping systems. Because the SCN have such a dominant role in the regulation of circadian systems [lo] and also bind BTX with high
affinity, we have examined the effects of BTX infusion on locomotor activity rhythms. BTX injections consistently perturbed the locomotor activity rhythms but in complex ways that suggest it might interact with neuromodulatory or other neuronal systems. METHOD
Animals and Surgery Male Sprague-Dawley rats (n=75) were obtained from Charles River Breeding Laboratories (Wilmington, MA). Adults weighing between 300-350 g at the beginning of the experiment were used for all studies. The animals were housed individually in 38x 38 x 15 cm translucent plastic cages and had free access to water and pelleted food throughout the experiments. Routine animal care was performed every 6-8 days at irregular times during the circadian cycle. After a two week period of laboratory acclimation and photoperiod entrainment (L,D: 12,12), animals were anesthetized with Chloropent (3.0 mg/kg; Fort Dodge Labs,
‘Portions of this work were presented in the program of the 15th and 16th Annual Meetings of the Society for Neuroscience, November, 1985 and Washington, DC, November, 1986. *Present address: Institute For Behavioral Genetics, University of Colorado, Campus Box 447, Boulder, CO 80309. 3Requests for reprints should be addressed to Dr. Nelson D. Horseman.
859
Dallas, TX.
860
PAULY AND HORSEMAh
0
24 I
48
FIG. 1. Effects of BTX after infusion in cortex and the hypothalamic suprachiasmatic nucleus. The activity rhythm is graphed in double plot form and successive days are plotted below one another. The time of day in hours is represented by the horizontal line at the top of the figure. Active intervals (those with activity greater than the daily mean for all intervals) are indicated by dark marks. At CT20 on the day marked “A” this animal (No. 219) received a 1 ~1 injection of BTX (250 fmol) into the cortex. Effects on phase and period were not detectable. When BTX was injected into the suprachiasmatic region at the same phase and dose, a phase advance (+ 1.7 hr) and an increase in free-running period resulted.
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FIG. 2. BTX induces phase advances in activity rhythms. (A) BTX (2.50fmol) was administered at CT0 and induced a phase advance of +3.5 hr (animal No. 229). (B) BTX (600 pmol) at CT20 (+ 1.8 hr) (animal No. 177). (C) BTX (600 pmol) at CT4 (+2.2 hr) (animal No. 185). Legend as in Fig. 1.
SCN BTX INFUSION Fort Dodge, IA) and stereotaxically implanted with stainless steel cannulae assemblies. Each assembly consisted of a chronic 26 gauge guide cannula and a removable 33 gauge insert. Cannulae were lowered through a small hole in the skull which had been made with a dental burr. The cannulae were implanted medially into the region just dorsal to the SCN (the flat skull coordinates were: 1.3 mm behind bregma, 9.0 mm below the surface of the dura and on the midline). Several animals were also implanted with cannulae assemblies in visual cortex (coordinates: -5.8, -2.0, +2.0). All cannula placements were made with reference to the atlas of Paxinos and Watson [ 151. After a two week recovery period under L,D: 12,12 the animals were placed on activity monitors and free-run in constant darkness. Animal care during the free-run was performed with the aid of a red safelight (<0.8 lux). Activity
Rhythm
Measurement
Locomotor activity rhythms were monitored continuously using Plexiglas activity monitors which registered ambulatory measurements photoelectrically. The components of the microcomputer-based system have been described in detail elsewhere [3,13]. Movements were recorded in 144 ten-minute bins per day and saved for subsequent analysis. Activity counts in each ten-minute interval were converted into a percentage of the daily total activity. Each interval was then designated as active or-&active depending on whether its percentage was above or below that day’s mean. In double plots, active intervals are presented by a dark mark. Phase shifts in activity rhythms were calculated by a modification of the linear regression model of Daan and Pittendrigh [4]. For rat total locomotor activity rhythms we find that the beginning of the inactive phase (definable as 0 hr circadian time) is the most precise and convenient phase reference point in the circadian cycle. Therefore, regressions were calculated for the rest onset interval (first interval after the active phase followed by at least 1 hr of inactivity). Times of rest onset were determined for the 15 days prior to treatments and for days 2-17 following treatments. For most animals these intervals represented the most stable patterns for analysis. Interpolation of the expected value for the injection day from each regression yielded values which were compared to calculate phase shifts. Period changes were determined by comparing the slopes of the two regression lines. Data from the day of the injection and the following day were not used for any calculations since on these days many animals showed disrupted rhythms. Injections
BTX was obtained from Sigma Chemical Company (St. Louis, MO). In all experiments, animals were given 1 ~1 intracerebral infusions (saline vehicle, pH 7.4) at specific phases of their free-running circadian cycles. Prior to the infusions, inserts were removed and replaced with 33 gauge injection cannulae that extended into the infusion site. The infusion cammlae were connected by polyethylene tubing to a 1 ~1 Hamilton syringe which was mounted on a Harvard microinfusion pump (Harvard Apparatus Company, South Nattick, MA). Drugs were infused for 50 seconds and the cannulae were left in the place for an additional minute to allow for diffusion away from the tip. No anesthesia was needed for these treatments which were all made under a dim red safelight. Infusions of BTX were delivered into the striate
861
cortex for area controls and physiological saline (pH 7.4) was infused into the suprachiasmatic area for drug controls. Initially, effects of 600 pmol of BTX were measured after injections at six phases of the cycle. Six lower doses of BTX were then screened for their potency. For these experiments, two additional groups of animals were used. The first group received 100 pmol, 10 pmol or 1 pmol doses of BTX and the second group received 500 fmol, 250 fmol or 125 fmol. The order of presentation of the doses was randomized and although all animals did not receive every dose of BTX, no animals received the same dose twice. At least 3 weeks separated sequential injections given to an animal. After these doses were screened for phase-shifting potency, a fourth group of animals were given 250 fmol infusions of BTX at various circadian phases. The times at which the injections were given were CT (circadian time) 0, CT4, CT8, CT12, CT16, and CT20 (CT0 represents the beginning of the rest phase for this nocturnal species). At the end of each expermental sequence, the animals were overanesthetized with Chloropent and perfused pericardially with saline and buffered formalin. The brains were sectioned at 50 pm and stained with thionin to verify cannula placement. Only data from animals that had cannula placements in the suprachiasmatic region were used in the analysis. In general, the injections caused little or no tissue damage. RESULTS
A total of 192 BTX injections were administered to freerunning animals at various doses and phases of the day. The mean phase shift for saline-treated animals was 0.16+0.15 hr (s.e.m. ; n = 18). Confidence limits (99%) were determined for these animals. Phase changes outside these 99% confidence limits (-0.28 to +0.60 hr) were defined as significant. Using this criterion, 77.6% of the animals injected with BTX had significant phase changes. When BTX was infused into the cortex, the mean phase changes were essentially identical to those for saline (0.15+0.12 hr; n=8). Figure 1 represents the activity record of an animal that received 2 sequential infusions of BTX (250 fmol). The first injection (A) was given into cortex and there was no significant phase or period change. The second injection (B) of BTX was delivered into the SCN and resulted in both phase and period changes in this animal. The various locomotor activity perturbations that were induced by BTX included phase advances (Fig. 2), phase delays (Fig. 3), changes in the period (tau) or the length of the active interval (alpha), and loss of integrated rhythmic activity (dyscronism) (Fig. 4). Phase effects after 600 pmol infusions of BTX are shown in Table 1. The mean shifts were not significantly different among phases. However, individual animals experienced large phase changes ranging from +3.16 hr to -4.44 hr. The magnitudes of individual phase shifts were highest during the late subjective night and early subjective day (CT20 to CT8) and most animals injected at CT12 and CT16 did not show phase changes which were outside the 99% confidence limits of controls. Also, several animals (5148) had highly disrupted activity patterns with no observable rhythmicity following infusion of BTX at this dose. We suspect that this dose of BTX was high enough that its effects were not confined to discrete phases. Thus we screened 6 lower doses of BTX for their phase-shifting potency tested at CT20. This time point was chosen because a significantly larger number (85%) of animals phase-shifted at
I’AULY AND HORSEMAN
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I
__ ._ _
FIG. 3. BTX induces phase delays in activity rhythms. (A) BTX (125 fmol) was infused at CT20 and caused a phase delay of - 1.5 hr (animal No. 185). (B) BTX (125 fmol) at CT20 (-2.4 hr) (animal No. 177).(C) BTX (600 pmol) at CT4 (-2.4 hr) (animal No. 171). Legend same as in Fig. I.
time point when compared with different phases (p
DISCUSSION
These data represent the first report of behavioral alterations induced by exogenously administered BTX, a ligand which has been extensively used in the identification and/or characterization of putative nicotinic cholinergic receptors in the CNS. The circadian system effects of BTX appear to be discrete and not secondary to toxic or indirect actions on the
circadian regulatory system. In no cases did intracranial BTX injections in this study result in mortality or morbidity. Histological evaluation of brain slices did nor reveal any cellular damage (necrosis, etc.) as a result of the BTX injections. Although intracranial injections of BTX did in some cases cause a short-term increase in locomotor activity and/or seizure-like conditions, it has been reported that rats readily survive central injections of up to 75 pg of BTX [ 111. At each dose and time of the circadian cycle tested, BTX infusion caused both phase advances and phase delays. Since at any given phase the direction of shifts incurred by BTX was variable, it is unlikely that this drug could entrain a circadian oscillator. The sensitivity of animals to the injections did, however, vary with phase. Rats were least responsive to BTX infusions from CT12 through CT16 and these times coincide with times when rats are insensitive to the phase-shifting actions of light pulses [4,18]. The incidence of BTX-induced dyschronisms, “alpha” and “tau” effects decreased as the dose of BTX administered decreased. The reasons for the dyschronisms are not known but they may be due to either residual drug presence or permanent biochemical alterations in the target cells. The potency of BTX effects on the circadian system are
SCN BTX INFUSION
863
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FIG. 4. Miscellaneous circadian perturbations induced by BTX infusions. (A) BTX (600 pmol) at CT20 caused the active interval of this animal’s (No. 176) to shorten by 1.3 hr (alpha effect). (B) BTX (600 pmol) at CT8 caused permanent dyschronism when administered to this animal (No. 172). (C) BTX (600 pmol) at CT20 caused this animal’s period to lengthen by 20.0 min and also a phase advance (+ 1.6 hr).
TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Circadian Time n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
INFUSED
1
WITH 600 pmol BTX AT VARIOUS
CIRCADIAN
PHASES
CT0
CT4
CT8
CT12
CT16
CT20
12
11
12
10
11
12
-0.40 k 0.68
-0.88 k 0.37
0.20 2 0.46
0.08 + 0.22
0.24 + 0.26
0.67 2 0.45
6 0.09-2.94
4 0.461.19
6 0.35-3.16
6 0.24-1.22
6 0.21-1.94
8 0.33-2.84
6 0.88-4.44
7 0.77-3.31
6 0.07-2.66
4 0.52-0.85
4 0.31-0.87
5 0.12-1.81
X64
PAULY TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Dose
(pmol)
n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
AND HORSEMAN
2
INFUSED
WITH VARlOUS
DOSES OF BTX Al’ CT20
600
100
10.0
1.0
0.500
0.250
(I. i !i
12
10
10
11
12
Y
10
0.67 2 0.45
-0.22 t 0.31
0.55 + 0.47
-0.54 t 0.43
0.28 + 0.30
0.67 t 0.24
- I.00 i- o.I%*
8 0.33-2.84
4 0.27-1.03
7 0.63-2.03
4 0.39-l. 13
8 0.03-1.69
8 0.07-2.26
2 0.0.5-0.75
4 0.12-1.81
6 0.08-1.30
3 0.1 l-3.08
7 0.14-3.48
4 0.23-2.02
0.12
1
8 0.14-2.43
*Mean significantly different from that of other doses (p
TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Circadian Time n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
INFUSED
3
WITH 250
fmol BTX
AT VARIOUS
CIRCADIAN
PHASES
CT0
CT4
CT8
CT12
CT16
CT20
9
8
8
8
8
9
1.38 + 0.52
-0.38 ? 0.70
0.45 + 0.38
0.44 _t 0.43
0.57 2 0.30
0.65 + 0.48
7 0.04-3.48
4 0.07-3.06
5 0.42-2.44
5 0.15-2.52
6 0.82-1.44
6 0.21-2.93
2 0.24-0.38
4 1.19-2.33
3 0.14-0.87
3 0.3kO.96
2 0.48-1.02
3 0.59-1.24
particularly interesting in light of the distribution of BTX receptors in the suprachiasmatic area which we and others have investigated [2, 5, 7, 9, 121. The most dense area of BTX binding in the suprachiasmatic region occurs in a band that runs dorsal and lateral to the SCN nuclear boundary. A major SCN efferent neuronal pathway runs dorsal and lateral
to the nucleus projecting to the periventricular/medial paraventricular area of the hypothalamus [20]. This pathway also projects caudal to the SCN as does BTX binding. When BTX is injected into the suprachiasmatic region it may induce circadian alterations by binding to SCN efferents in the areas dorsolateral and caudal to the nuclei. Previous experiments on BTX effects on pineal circadian function have concluded that this toxin may act by blocking light effects [21]. Our results show that the circadian effects of BTX are very complex and probably not explainable by a single interpretation. Based on the dominant role of the SCN in circadian timekeeping, we presume, but have not proven, that the
of the phase-shifting effects of exogenously administered BTX is specific for neurons in this area. Because these effects of BTX on locomotor activity occur at extremely low doses, it is likely that they are mediated by the high affinity BTX receptors present in the CNS. This receptor does not correlate with the binding of other nicotinic cholinergic ligands [2, 11, 121. An endogenous peptide-like ligand for high affinity BTX receptors has been indicated in experiments by Quik [16,17] but has not been isolated. These studies indicate that circadian locomotor activity rhythms in rats can be potently perturbed by a receptor system that binds BTX with high affinity. The complexity of the effects suggests that BTX might act through more than one input to the circadian system. These results emphasize the need to cautiously interpret other experiments using BTX or other putative cholinergic drugs. Further understanding the physiology of this system will depend on biochemical proof and characterization of any endogenous drugs that might bind to the central nervous system BTX receptor. locus
REFERENCES 1. Block, G. A.; Billiar, R. B. Properties and regional distribution of nicotinic cholinergic receptors in the rat hypothalamus. Brain Res. 212:152-158; 1981.
2.
Clarke, P. B. S.; Schwartz, R. D.; Paul, 8.. M.; Pert, C. B.; Pert, A. Nicotinic binding in rat brain: autoradiographic comparison of [3H]-acetylcholine, [sH]-nicotine and [‘*sI]-o-bungarotoxin. J. Neurosci. 5:1307-1315; 1985.
SCN BTX INFUSION
3. Czech, D. A. A versatile integrated circuit activity monitor for small animals. Physiol. Behav. 32871879; 1984. 4. Daan, S.; Pittendrigh, C. S. A function analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J. Comp. Physiol. [A] 106:253-266; 1976. 5. Fuchs, J. L.; Hoppens, K. S. cY-Bungarotoxin binding in relation to functional organization the rat suprachiasmatic nucleus. Brain Res. 4079-16; 1987. 6. Honma, K.; Homna, S.; Hiroshige, T. Response curve, freerunning period and activity time in circadian locomotor rhythms of rats. Jpn. J. Physiol. 35643-658; 1985. 7. Hunt, S. P.; Schmidt, J. The electron microscope autoradiographic localization of a-bungarotoxin binding sites within the central nervous system of the rat. Brain Res. 142: 152-159; 1978. 8. Meeker, R. B.; Michels, K. M.; Libber, M. T.; Hayward, J. N. Characteristics and distribution of high- and low-afftnity alphabungarotoxin binding sites in the rat hypothalamus. J. Neurosci. 6:18661875; 1986. 9. Miller, M. M.; Biliiar, R. B. A quantitative and morphometric evaluation of ‘%cY-bungarotoxin binding in the rat hypothalamus. Brain Res. Bull. 16:681-688; 1986. IO. Moore, R. Y. Organization and function of a central nervous system circadian oscillatory: the suprachiasmatic hypothalamic nucleus. Fed. Proc. 42:2783-2789; 1983. 11. Morley, B. J.; Kemp, G. E. Characterization of a putative nicotinic acetylcholine receptor in mammalian brain. Brain Res. Rev. 1:81-104; 1981. 12. Oswald, R. E.; Freeman, J. A. Alpha bungarotoxin and central nervous system nicotinic acetylcholine receptors. Neuroscience 6: l-14: 1981.
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13. Pauly, J. R.; Horseman, N. D. Anticholinergic agents do not block liaht-induced circadian chase shifts. Brain Res. 348:162167; 1985. 14. Pauly, J. R.; Horseman, N. D. Autoradiographic localization of cY-bungarotoxin binding sites in the suprachiasmatic region of rat brain. Brain Res. 452:105-112; 1988. 15. Paxinos, G.; Watson, C.; The rat brain in stereotaxic coordinates. New York: Academic Press: 1982. 16. Quik, M. Presence of an endogenous factor which inhibits the binding of a-bungarotoxin 2.2 to its receptor. Brain Res. 24557-67; 1982. 17. Quik, M.; Garfalo, L. Contrasting effects of a brain supematant extract on neuronal vs. neuromuscular ry-bungarotoxin receptors. Brain Res. 374:395-398; 1986. 18. Segal, M.; Dudai, Y.; Amsterdam, A. Distribution of an a-bungarotoxin binding cholinergic nicotinic receptor in rat brain. Brain Res. 148: 105-l 19; 1978. 19. Summer, T. L.; Ferraro, J. S.; McCormack, C. E. Phaseresponse and Aschoff illuminance curves for locomotor activity rhythms of the rat. Am. J. Physiol. 246:R299-304; 1984. 20. Van Den Pol, A. N. The hypothalamic suprachiasmatic nucleus of the rat: intrinsic anatomy. J. Comp. Neurol. 191:661-672; 1980. 21 Zatz, M.; Brownstein, M. J. Injection of cY-bungarotoxin near the suprachiasmatic nucleus blocks the effects of light on nocturnal pineal enzyme activity. Brain Res. 213:438-442; 1981.
0 Pergamon
Press plc, 1988. Printed
0031-9384/88
in the U.S.A.
$3.00 + .OO
BRIEF COMMUNICATION
Perturbations of Locomotor Activity Rhythms Following Suprachiasmatic Bungarotoxin Infusion’ JAMES R. PAULYz AND NELSON Department
of Biology,
Marquette
D. HORSEMAN3
University,
Received 23 October
Milwaukee,
WI 53233
1987
PAULY, J. R. AND N. D. HORSEMAN. Perturbations of locomotor activity rhythms following supruchiasmatic bungarotoxin infusion. PHYSIOL BEHAV 43(6) 859-865, 1988.-The actions of intracranial injections of alpha bungarotoxin (BTX) on locomotor activity rhythms were examined in male rats. The hypothalamic suprachiasmatic region is known to be a locus of high affinity BTX binding although the potential roles of this receptor system are unknown. Rats were stereotaxitally implanted with cannulae aimed just dorsal to the SCN (or cortex for control injections). Free-running locomotor activity rhythms in darkness were constantly monitored. Seventy-eight percent (78%) of the animals injected with BTX in the SCN region had phase shifts that were outside the 9% confidence limits of control animals. Infusion of either saline (into the SCN) or BTX into cortex were without effects. Doses of BTX varied from 125 fmol to 600 pmol. At the highest dose a substantial fraction of the animals showed both period changes and loss of rhythmicity as well as phase shifts. Although nearly all of the animals injected with BTX experienced phase shifts the direction of the shifts were not consistently correlated with the circadian time of injection. However, the sensitivity of the animals varied systematically with the smallest shifts resulting after injection at CT12 and CT16. These results argue that BTX does not influence the SCN pacemaker as an entraining signal but does potently perturb the circadian system. Rats
Biological rhythms
Bungarotoxin
Cholinergic systems
ALPHA bungarotoxin (BTX) binds densely and with high affinity in the area of the hypothalamic suprachiasmatic nuclei (SCN) [l, 5, 7, 8, 14, 181, which function as circadian pacemakers in mammals. The functions of central nervous system (CNS) receptors that bind BTX with high affinity are largely unknown. Although BTX has been presumed in many studies to act as a nicotinic cholinergic antagonist in the CNS, its pharmacology and physiology appear now to be quite different from its classical mechanism of action on peripheral cholinergic receptors. Several autoradiographic and biochemical studies have in fact shown that there is little overlap between brain areas that bind nicotine (and acetylcholine) and those that are labeled by BTX [2, 11, 121. Previously, BTX has been used as a probe for putative involvement of cholinergic systems in light/dark synchronization of circadian systems [21]. However, no studies have carefully examined what effects BTX alone might have on circadian timekeeping systems. Because the SCN have such a dominant role in the regulation of circadian systems [lo] and also bind BTX with high
affinity, we have examined the effects of BTX infusion on locomotor activity rhythms. BTX injections consistently perturbed the locomotor activity rhythms but in complex ways that suggest it might interact with neuromodulatory or other neuronal systems. METHOD
Animals and Surgery Male Sprague-Dawley rats (n=75) were obtained from Charles River Breeding Laboratories (Wilmington, MA). Adults weighing between 300-350 g at the beginning of the experiment were used for all studies. The animals were housed individually in 38x 38 x 15 cm translucent plastic cages and had free access to water and pelleted food throughout the experiments. Routine animal care was performed every 6-8 days at irregular times during the circadian cycle. After a two week period of laboratory acclimation and photoperiod entrainment (L,D: 12,12), animals were anesthetized with Chloropent (3.0 mg/kg; Fort Dodge Labs,
‘Portions of this work were presented in the program of the 15th and 16th Annual Meetings of the Society for Neuroscience, November, 1985 and Washington, DC, November, 1986. *Present address: Institute For Behavioral Genetics, University of Colorado, Campus Box 447, Boulder, CO 80309. 3Requests for reprints should be addressed to Dr. Nelson D. Horseman.
859
Dallas, TX.
860
PAULY AND HORSEMAh
0
24 I
48
FIG. 1. Effects of BTX after infusion in cortex and the hypothalamic suprachiasmatic nucleus. The activity rhythm is graphed in double plot form and successive days are plotted below one another. The time of day in hours is represented by the horizontal line at the top of the figure. Active intervals (those with activity greater than the daily mean for all intervals) are indicated by dark marks. At CT20 on the day marked “A” this animal (No. 219) received a 1 ~1 injection of BTX (250 fmol) into the cortex. Effects on phase and period were not detectable. When BTX was injected into the suprachiasmatic region at the same phase and dose, a phase advance (+ 1.7 hr) and an increase in free-running period resulted.
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FIG. 2. BTX induces phase advances in activity rhythms. (A) BTX (2.50fmol) was administered at CT0 and induced a phase advance of +3.5 hr (animal No. 229). (B) BTX (600 pmol) at CT20 (+ 1.8 hr) (animal No. 177). (C) BTX (600 pmol) at CT4 (+2.2 hr) (animal No. 185). Legend as in Fig. 1.
SCN BTX INFUSION Fort Dodge, IA) and stereotaxically implanted with stainless steel cannulae assemblies. Each assembly consisted of a chronic 26 gauge guide cannula and a removable 33 gauge insert. Cannulae were lowered through a small hole in the skull which had been made with a dental burr. The cannulae were implanted medially into the region just dorsal to the SCN (the flat skull coordinates were: 1.3 mm behind bregma, 9.0 mm below the surface of the dura and on the midline). Several animals were also implanted with cannulae assemblies in visual cortex (coordinates: -5.8, -2.0, +2.0). All cannula placements were made with reference to the atlas of Paxinos and Watson [ 151. After a two week recovery period under L,D: 12,12 the animals were placed on activity monitors and free-run in constant darkness. Animal care during the free-run was performed with the aid of a red safelight (<0.8 lux). Activity
Rhythm
Measurement
Locomotor activity rhythms were monitored continuously using Plexiglas activity monitors which registered ambulatory measurements photoelectrically. The components of the microcomputer-based system have been described in detail elsewhere [3,13]. Movements were recorded in 144 ten-minute bins per day and saved for subsequent analysis. Activity counts in each ten-minute interval were converted into a percentage of the daily total activity. Each interval was then designated as active or-&active depending on whether its percentage was above or below that day’s mean. In double plots, active intervals are presented by a dark mark. Phase shifts in activity rhythms were calculated by a modification of the linear regression model of Daan and Pittendrigh [4]. For rat total locomotor activity rhythms we find that the beginning of the inactive phase (definable as 0 hr circadian time) is the most precise and convenient phase reference point in the circadian cycle. Therefore, regressions were calculated for the rest onset interval (first interval after the active phase followed by at least 1 hr of inactivity). Times of rest onset were determined for the 15 days prior to treatments and for days 2-17 following treatments. For most animals these intervals represented the most stable patterns for analysis. Interpolation of the expected value for the injection day from each regression yielded values which were compared to calculate phase shifts. Period changes were determined by comparing the slopes of the two regression lines. Data from the day of the injection and the following day were not used for any calculations since on these days many animals showed disrupted rhythms. Injections
BTX was obtained from Sigma Chemical Company (St. Louis, MO). In all experiments, animals were given 1 ~1 intracerebral infusions (saline vehicle, pH 7.4) at specific phases of their free-running circadian cycles. Prior to the infusions, inserts were removed and replaced with 33 gauge injection cannulae that extended into the infusion site. The infusion cammlae were connected by polyethylene tubing to a 1 ~1 Hamilton syringe which was mounted on a Harvard microinfusion pump (Harvard Apparatus Company, South Nattick, MA). Drugs were infused for 50 seconds and the cannulae were left in the place for an additional minute to allow for diffusion away from the tip. No anesthesia was needed for these treatments which were all made under a dim red safelight. Infusions of BTX were delivered into the striate
861
cortex for area controls and physiological saline (pH 7.4) was infused into the suprachiasmatic area for drug controls. Initially, effects of 600 pmol of BTX were measured after injections at six phases of the cycle. Six lower doses of BTX were then screened for their potency. For these experiments, two additional groups of animals were used. The first group received 100 pmol, 10 pmol or 1 pmol doses of BTX and the second group received 500 fmol, 250 fmol or 125 fmol. The order of presentation of the doses was randomized and although all animals did not receive every dose of BTX, no animals received the same dose twice. At least 3 weeks separated sequential injections given to an animal. After these doses were screened for phase-shifting potency, a fourth group of animals were given 250 fmol infusions of BTX at various circadian phases. The times at which the injections were given were CT (circadian time) 0, CT4, CT8, CT12, CT16, and CT20 (CT0 represents the beginning of the rest phase for this nocturnal species). At the end of each expermental sequence, the animals were overanesthetized with Chloropent and perfused pericardially with saline and buffered formalin. The brains were sectioned at 50 pm and stained with thionin to verify cannula placement. Only data from animals that had cannula placements in the suprachiasmatic region were used in the analysis. In general, the injections caused little or no tissue damage. RESULTS
A total of 192 BTX injections were administered to freerunning animals at various doses and phases of the day. The mean phase shift for saline-treated animals was 0.16+0.15 hr (s.e.m. ; n = 18). Confidence limits (99%) were determined for these animals. Phase changes outside these 99% confidence limits (-0.28 to +0.60 hr) were defined as significant. Using this criterion, 77.6% of the animals injected with BTX had significant phase changes. When BTX was infused into the cortex, the mean phase changes were essentially identical to those for saline (0.15+0.12 hr; n=8). Figure 1 represents the activity record of an animal that received 2 sequential infusions of BTX (250 fmol). The first injection (A) was given into cortex and there was no significant phase or period change. The second injection (B) of BTX was delivered into the SCN and resulted in both phase and period changes in this animal. The various locomotor activity perturbations that were induced by BTX included phase advances (Fig. 2), phase delays (Fig. 3), changes in the period (tau) or the length of the active interval (alpha), and loss of integrated rhythmic activity (dyscronism) (Fig. 4). Phase effects after 600 pmol infusions of BTX are shown in Table 1. The mean shifts were not significantly different among phases. However, individual animals experienced large phase changes ranging from +3.16 hr to -4.44 hr. The magnitudes of individual phase shifts were highest during the late subjective night and early subjective day (CT20 to CT8) and most animals injected at CT12 and CT16 did not show phase changes which were outside the 99% confidence limits of controls. Also, several animals (5148) had highly disrupted activity patterns with no observable rhythmicity following infusion of BTX at this dose. We suspect that this dose of BTX was high enough that its effects were not confined to discrete phases. Thus we screened 6 lower doses of BTX for their phase-shifting potency tested at CT20. This time point was chosen because a significantly larger number (85%) of animals phase-shifted at
I’AULY AND HORSEMAN
0
24
1
48
I
__ ._ _
FIG. 3. BTX induces phase delays in activity rhythms. (A) BTX (125 fmol) was infused at CT20 and caused a phase delay of - 1.5 hr (animal No. 185). (B) BTX (125 fmol) at CT20 (-2.4 hr) (animal No. 177).(C) BTX (600 pmol) at CT4 (-2.4 hr) (animal No. 171). Legend same as in Fig. I.
time point when compared with different phases (p
DISCUSSION
These data represent the first report of behavioral alterations induced by exogenously administered BTX, a ligand which has been extensively used in the identification and/or characterization of putative nicotinic cholinergic receptors in the CNS. The circadian system effects of BTX appear to be discrete and not secondary to toxic or indirect actions on the
circadian regulatory system. In no cases did intracranial BTX injections in this study result in mortality or morbidity. Histological evaluation of brain slices did nor reveal any cellular damage (necrosis, etc.) as a result of the BTX injections. Although intracranial injections of BTX did in some cases cause a short-term increase in locomotor activity and/or seizure-like conditions, it has been reported that rats readily survive central injections of up to 75 pg of BTX [ 111. At each dose and time of the circadian cycle tested, BTX infusion caused both phase advances and phase delays. Since at any given phase the direction of shifts incurred by BTX was variable, it is unlikely that this drug could entrain a circadian oscillator. The sensitivity of animals to the injections did, however, vary with phase. Rats were least responsive to BTX infusions from CT12 through CT16 and these times coincide with times when rats are insensitive to the phase-shifting actions of light pulses [4,18]. The incidence of BTX-induced dyschronisms, “alpha” and “tau” effects decreased as the dose of BTX administered decreased. The reasons for the dyschronisms are not known but they may be due to either residual drug presence or permanent biochemical alterations in the target cells. The potency of BTX effects on the circadian system are
SCN BTX INFUSION
863
48 1
24 I
0
1
FIG. 4. Miscellaneous circadian perturbations induced by BTX infusions. (A) BTX (600 pmol) at CT20 caused the active interval of this animal’s (No. 176) to shorten by 1.3 hr (alpha effect). (B) BTX (600 pmol) at CT8 caused permanent dyschronism when administered to this animal (No. 172). (C) BTX (600 pmol) at CT20 caused this animal’s period to lengthen by 20.0 min and also a phase advance (+ 1.6 hr).
TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Circadian Time n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
INFUSED
1
WITH 600 pmol BTX AT VARIOUS
CIRCADIAN
PHASES
CT0
CT4
CT8
CT12
CT16
CT20
12
11
12
10
11
12
-0.40 k 0.68
-0.88 k 0.37
0.20 2 0.46
0.08 + 0.22
0.24 + 0.26
0.67 2 0.45
6 0.09-2.94
4 0.461.19
6 0.35-3.16
6 0.24-1.22
6 0.21-1.94
8 0.33-2.84
6 0.88-4.44
7 0.77-3.31
6 0.07-2.66
4 0.52-0.85
4 0.31-0.87
5 0.12-1.81
X64
PAULY TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Dose
(pmol)
n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
AND HORSEMAN
2
INFUSED
WITH VARlOUS
DOSES OF BTX Al’ CT20
600
100
10.0
1.0
0.500
0.250
(I. i !i
12
10
10
11
12
Y
10
0.67 2 0.45
-0.22 t 0.31
0.55 + 0.47
-0.54 t 0.43
0.28 + 0.30
0.67 t 0.24
- I.00 i- o.I%*
8 0.33-2.84
4 0.27-1.03
7 0.63-2.03
4 0.39-l. 13
8 0.03-1.69
8 0.07-2.26
2 0.0.5-0.75
4 0.12-1.81
6 0.08-1.30
3 0.1 l-3.08
7 0.14-3.48
4 0.23-2.02
0.12
1
8 0.14-2.43
*Mean significantly different from that of other doses (p
TABLE MEAN PHASE SHIFT DATA FOR ANIMALS
Circadian Time n Mean Phase Shift (hr) Phase Advances: n range Phase Delays: n range
INFUSED
3
WITH 250
fmol BTX
AT VARIOUS
CIRCADIAN
PHASES
CT0
CT4
CT8
CT12
CT16
CT20
9
8
8
8
8
9
1.38 + 0.52
-0.38 ? 0.70
0.45 + 0.38
0.44 _t 0.43
0.57 2 0.30
0.65 + 0.48
7 0.04-3.48
4 0.07-3.06
5 0.42-2.44
5 0.15-2.52
6 0.82-1.44
6 0.21-2.93
2 0.24-0.38
4 1.19-2.33
3 0.14-0.87
3 0.3kO.96
2 0.48-1.02
3 0.59-1.24
particularly interesting in light of the distribution of BTX receptors in the suprachiasmatic area which we and others have investigated [2, 5, 7, 9, 121. The most dense area of BTX binding in the suprachiasmatic region occurs in a band that runs dorsal and lateral to the SCN nuclear boundary. A major SCN efferent neuronal pathway runs dorsal and lateral
to the nucleus projecting to the periventricular/medial paraventricular area of the hypothalamus [20]. This pathway also projects caudal to the SCN as does BTX binding. When BTX is injected into the suprachiasmatic region it may induce circadian alterations by binding to SCN efferents in the areas dorsolateral and caudal to the nuclei. Previous experiments on BTX effects on pineal circadian function have concluded that this toxin may act by blocking light effects [21]. Our results show that the circadian effects of BTX are very complex and probably not explainable by a single interpretation. Based on the dominant role of the SCN in circadian timekeeping, we presume, but have not proven, that the
of the phase-shifting effects of exogenously administered BTX is specific for neurons in this area. Because these effects of BTX on locomotor activity occur at extremely low doses, it is likely that they are mediated by the high affinity BTX receptors present in the CNS. This receptor does not correlate with the binding of other nicotinic cholinergic ligands [2, 11, 121. An endogenous peptide-like ligand for high affinity BTX receptors has been indicated in experiments by Quik [16,17] but has not been isolated. These studies indicate that circadian locomotor activity rhythms in rats can be potently perturbed by a receptor system that binds BTX with high affinity. The complexity of the effects suggests that BTX might act through more than one input to the circadian system. These results emphasize the need to cautiously interpret other experiments using BTX or other putative cholinergic drugs. Further understanding the physiology of this system will depend on biochemical proof and characterization of any endogenous drugs that might bind to the central nervous system BTX receptor. locus
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2.
Clarke, P. B. S.; Schwartz, R. D.; Paul, 8.. M.; Pert, C. B.; Pert, A. Nicotinic binding in rat brain: autoradiographic comparison of [3H]-acetylcholine, [sH]-nicotine and [‘*sI]-o-bungarotoxin. J. Neurosci. 5:1307-1315; 1985.
SCN BTX INFUSION
3. Czech, D. A. A versatile integrated circuit activity monitor for small animals. Physiol. Behav. 32871879; 1984. 4. Daan, S.; Pittendrigh, C. S. A function analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J. Comp. Physiol. [A] 106:253-266; 1976. 5. Fuchs, J. L.; Hoppens, K. S. cY-Bungarotoxin binding in relation to functional organization the rat suprachiasmatic nucleus. Brain Res. 4079-16; 1987. 6. Honma, K.; Homna, S.; Hiroshige, T. Response curve, freerunning period and activity time in circadian locomotor rhythms of rats. Jpn. J. Physiol. 35643-658; 1985. 7. Hunt, S. P.; Schmidt, J. The electron microscope autoradiographic localization of a-bungarotoxin binding sites within the central nervous system of the rat. Brain Res. 142: 152-159; 1978. 8. Meeker, R. B.; Michels, K. M.; Libber, M. T.; Hayward, J. N. Characteristics and distribution of high- and low-afftnity alphabungarotoxin binding sites in the rat hypothalamus. J. Neurosci. 6:18661875; 1986. 9. Miller, M. M.; Biliiar, R. B. A quantitative and morphometric evaluation of ‘%cY-bungarotoxin binding in the rat hypothalamus. Brain Res. Bull. 16:681-688; 1986. IO. Moore, R. Y. Organization and function of a central nervous system circadian oscillatory: the suprachiasmatic hypothalamic nucleus. Fed. Proc. 42:2783-2789; 1983. 11. Morley, B. J.; Kemp, G. E. Characterization of a putative nicotinic acetylcholine receptor in mammalian brain. Brain Res. Rev. 1:81-104; 1981. 12. Oswald, R. E.; Freeman, J. A. Alpha bungarotoxin and central nervous system nicotinic acetylcholine receptors. Neuroscience 6: l-14: 1981.
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13. Pauly, J. R.; Horseman, N. D. Anticholinergic agents do not block liaht-induced circadian chase shifts. Brain Res. 348:162167; 1985. 14. Pauly, J. R.; Horseman, N. D. Autoradiographic localization of cY-bungarotoxin binding sites in the suprachiasmatic region of rat brain. Brain Res. 452:105-112; 1988. 15. Paxinos, G.; Watson, C.; The rat brain in stereotaxic coordinates. New York: Academic Press: 1982. 16. Quik, M. Presence of an endogenous factor which inhibits the binding of a-bungarotoxin 2.2 to its receptor. Brain Res. 24557-67; 1982. 17. Quik, M.; Garfalo, L. Contrasting effects of a brain supematant extract on neuronal vs. neuromuscular ry-bungarotoxin receptors. Brain Res. 374:395-398; 1986. 18. Segal, M.; Dudai, Y.; Amsterdam, A. Distribution of an a-bungarotoxin binding cholinergic nicotinic receptor in rat brain. Brain Res. 148: 105-l 19; 1978. 19. Summer, T. L.; Ferraro, J. S.; McCormack, C. E. Phaseresponse and Aschoff illuminance curves for locomotor activity rhythms of the rat. Am. J. Physiol. 246:R299-304; 1984. 20. Van Den Pol, A. N. The hypothalamic suprachiasmatic nucleus of the rat: intrinsic anatomy. J. Comp. Neurol. 191:661-672; 1980. 21 Zatz, M.; Brownstein, M. J. Injection of cY-bungarotoxin near the suprachiasmatic nucleus blocks the effects of light on nocturnal pineal enzyme activity. Brain Res. 213:438-442; 1981.