Further Evaluation of the Tetrodotoxin-Resistant Circadian Pacemaker in the Suprachiasmatic Nuclei William J. Schwartz
Department of Neurology, University of Massachusetts Medical School,
55 Lake Avenue,
North, Worcester, Massachusetts 01655 Abstract
We previously reported the results of an experimental paradigm in which tetrodotoxin (TTX) chronically infused by miniosmotic pump into the rat suprachiasmatic nuclei (SCN) (Schwartz et al., 1987). Although TTX reversibly blocked photic entrainment and overt expression of the circadian drinking rhythm, the circadian pacemaker in the SCN continued to oscillate unperturbed by the toxin, and we concluded that Na -dependent action potentials are not a part of the SCN pacemaker’s internal + timekeeping mechanism. In the research reported in the present paper, we used our paradigm to chronically infuse other agents, in order to evaluate the validity of this interpretation further. ) Infusion of 50% procaine into the SCN of blinded rats resulted in a disorganized circadian 1 ( drinking rhythm during the infusion, after which behavioral rhythmicity returned without apparent phase shift. In intact rats, procaine reduced the phase-resetting action of a reversed light-dark cycle + channel blockade by a local imposed during the infusion. Thus, the effects of voltage-dependent Na anesthetic resemble those produced by TTX. + or 100 μM veratridine into the SCN of blinded rats resulted in (2) Infusion of high (20 mM) K an apparent phase advance of the circadian drinking rhythm by over 4 hr. The phase-shifting effect of veratridine was blocked by simultaneous infusion of 1 μM TTX. Thus, membrane depolarization or direct activation of voltage-dependent Na + channels can affect the pacemaker’s oscillation. Our infusion paradigm can detect alterations of rhythm phase, and the lack of phase shift after TTX or procaine infusion is not an artifact of an insensitive method. was
There is considerable evidence to indicate that the suprachiasmatic nuclei (SCN) in the anterior hypothalamus are the site of an endogenous circadian pacemaker in mammals (see Ralph et al., 1990). Much current investigation is now directed to the cellular and molecular processes that might constitute the pacemaker’s oscillatory mechanism, and one of the obvious early objectives of this work will be a better understanding of the role played by SCN intercellular interactions. We recently presented evidence suggesting that Na+-dependent action potentials in the SCN are not a part of the internal timekeeping mechanism of the circadian pacemaker, although such spike activity is required for photic entrainment and overt expression of behavioral rhythmicity (Schwartz et al., 1987). Figure 1 outlines the results of our experimental paradigm, in which 1 pLM tetrodotoxin (TTX) or artificial cerebrospinal fluid (CSF) was chronically infused by miniosmotic pump into the SCN of unanesthetized, unrestrained albino rats while their free-running circadian rhythms of drinking activity were monitored. We first showed (Figs. lA and 1B) that TTX resulted in temporary behavioral arrhythmicity during its 14-day infusion, after which rhythmic drinking activity returned without apparent phase shift (i.e., the phase of the restored rhythm was that predicted by extrapolation of the free149 Downloaded from jbr.sagepub.com at NORTH CAROLINA STATE UNIV on May 1, 2015
FIGURE 1. (A and B) Actograms of two blinded rats with cannulae located in the SCN. Artificial CSF (A) or TTX (B) was continuously infused for the 14 days bracketed by arrows. (C and D) Actograms of two intact rats with cannulae located in the SCN and exposed to an LD 12:12 cycle progressively advanced by three successive 4-hr advances until a completely reversed cycle was attained. Artificial CSF (C) or TTX (D) was continuously infused for the 14 days bracketed by arrows.
running period [T] of the original [preinfusion] rhythm). We then showed (Figs. 1C and ID) that TTX prevented the phase-resetting action of a reversed light-dark (LD) cycle imposed during the infusion (i.e., the phase of the restored rhythm was what would have been expected had the rhythm continued its free run despite application of the light). Thus, the circadian pacemaker in the SCN continued to oscillate unperturbed by TTX treatment, although the 150 Downloaded from jbr.sagepub.com at NORTH CAROLINA STATE UNIV on May 1, 2015
reversibly inactivate the pacemaker’s input pathway for photic entrainment and its output pathway for overt expression of drinking rhythmicity. This paper reports the results of additional experiments using our infusion paradigm, in order to test the validity of this interpretation further. First, we infused 50% procaine to determine whether inhibition of SCN action potentials by another agent would reproduce the TTX results. Procaine blocks voltage-dependent Na+ channels and suppresses hypothalamic discharge activity (Harlan et al., 1983), but unlike TTX-which appears to bind to a receptor at the extracellular opening of the ion-conducting pore-the anesthetic is believed to inhibit action potential generation by binding to an internal receptor with differing affinities for the resting, active, and inactivated states of the channel (see Catterall, 1987). Second, we infused high (20 mM) K+ or 100 yvt veratridine to determine whether membrane depolarization would alter the pacemaker’s ongoing oscillation. Veratridine is an alkaloid that causes persistent activation of the Na+ channel by blocking its inactivation and by shifting the voltage dependence of its activation to more negative membrane potentials (see Catterall, toxin did
1987). z
MATERIALS AND METHODS
._
’
z
ANIMALS Adult male Sprague-Dawley rats were obtained from Charles River (Wilmington, MA) and housed individually in clear plastic cages contained within well-ventilated, light-proof end vironmental compartments as previously described (Schwartz et al., 1987). Light was provides by 15-W cool-white fluorescent tubes delivering an intensity of 600 lux at the midcage level. During darkness, 15-W safe lights with dark red (series 2) filters remained on in the facility to allow for routine care. Chow and water were freely available and replenished once every 6-7 days at irregular hours.
Some of the rats were blinded by bilateral orbital enucleation. Animals were fully anesthetized with ether; periorbital tissue was dissected carefully with iris scissors; optic nerves were cut; and eyes were removed. The entire procedure was accomplished in 50%) SCN damage. In the other 5 rats (Fig. 2A), postinfusion drinking rhythms resumed with T’s unaltered from those expressed preinfusion (24.13 ± 0.03 hr preinfusion vs. 24.16 ± 0.01 hr postinfusion). The phase of these restored rhythms was essentially that predicted by extrapolation of the T’s of the original (preinfusion) rhythms, with a phase shift of only 0.1± 0.5 hr for the population of animals. These results are similar to those previously reported with TTX (Schwartz et al., 1987). When
procaine
=
was
In order to test whether procaine treatment would uncouple the circadian pacemaker’s input pathway for photic entrainment, rats were first entrained to an LD 12:12 schedule for at least 2 weeks, after which cannula-pump assemblies were implanted. Rodents were maintained in constant environmental darkness for the first 8 days of the 14-day infusion period; during the last 6 days of the infusions, animals were exposed to an LD 12:12 cycle progressively delayed by three successive 4-hr delays until a completely reversed cycle was attained. At the end of the infusions, rats were placed in constant darkness, and free-running circadian rhythms of drinking activity were recorded for approximately 1 month. Drinking rhythms were successfully phase-shifted by the imposed LD cycle in rats with procaine infusions in the hypothalamus missing the SCN (n 5), to a degree (7.4 ± 0.6 hr) comparable to that previously reported in rats (Schwartz et al., 1987) with TTX infusions outside the SCN (6.2 ± 0.8 hr) or artificial CSF infusions into the SCN (6.6 ± 0.3 hr) (Fig. 2C). When procaine was infused into the SCN (n 5), a complete lesion of the nuclei by an abscess resulted in post infusion arrhythmicity in 1 animal. In the remaining 4 animals (Fig. 2B), procaine reduced the phase-resetting action of light, with an apparent phase shift of only 2.1 ± 0.5 hr for the population of animals-a value close to that previously reported with TTX (1.9 ± 0.7 hr) (Schwartz et al., 1987). This estimated 2.1-hr phase shift may be artifactually low, however, because procaine treatment resulted in a lengthened postinfusion T (24.30 ± 0.01 hr after procaine vs. 24.21 ± 0.01 hr after CSF). We calculate that this 0.09-hr T difference would lead to a 1.3-hr underestimate of the light-induced phase shift during procaine infusion; even with this corrected value, however, the phase shift during procaine infusion would be only half the shift during CSF infusion. =
=
HIGH K+ OR VERATRIDINE INFUSION In order to demonstrate that
would actually detect a change in the entrained to the LD 12:12 schedule, and with assemblies filled with 20 mM K+ in artificial blinded, implanted cannula-pump CSF. Free-running circadian rhythms of drinking activity were then recorded during the infusions and for approximately 1 month after the infusions ended. During the infusions of high K+ into the SCN (n 10), drinking rhythmicity appeared disorganized, although periodic activity was transiently observed in some of the animals. In 3 of the 10 rats, drinking rhythms remained arrhythmic after the infusions ended, and histological examination disclosed >50% SCN destruction. In the other 7 rats (Fig. 3A), postinfusion drinking rhythms resumed with T’s unaltered from those expressed preinfusion (24.01 ± 0.02 hr preinfusion vs. 24.04 ± 0.04 hr postinfusion), but the phase of these restored rhythms was shifted. There was an apparent advance of the phase predicted by circadian
our
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rats were
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Department of Neurology, University of Massachusetts Medical School,
55 Lake Avenue,
North, Worcester, Massachusetts 01655 Abstract
We previously reported the results of an experimental paradigm in which tetrodotoxin (TTX) chronically infused by miniosmotic pump into the rat suprachiasmatic nuclei (SCN) (Schwartz et al., 1987). Although TTX reversibly blocked photic entrainment and overt expression of the circadian drinking rhythm, the circadian pacemaker in the SCN continued to oscillate unperturbed by the toxin, and we concluded that Na -dependent action potentials are not a part of the SCN pacemaker’s internal + timekeeping mechanism. In the research reported in the present paper, we used our paradigm to chronically infuse other agents, in order to evaluate the validity of this interpretation further. ) Infusion of 50% procaine into the SCN of blinded rats resulted in a disorganized circadian 1 ( drinking rhythm during the infusion, after which behavioral rhythmicity returned without apparent phase shift. In intact rats, procaine reduced the phase-resetting action of a reversed light-dark cycle + channel blockade by a local imposed during the infusion. Thus, the effects of voltage-dependent Na anesthetic resemble those produced by TTX. + or 100 μM veratridine into the SCN of blinded rats resulted in (2) Infusion of high (20 mM) K an apparent phase advance of the circadian drinking rhythm by over 4 hr. The phase-shifting effect of veratridine was blocked by simultaneous infusion of 1 μM TTX. Thus, membrane depolarization or direct activation of voltage-dependent Na + channels can affect the pacemaker’s oscillation. Our infusion paradigm can detect alterations of rhythm phase, and the lack of phase shift after TTX or procaine infusion is not an artifact of an insensitive method. was
There is considerable evidence to indicate that the suprachiasmatic nuclei (SCN) in the anterior hypothalamus are the site of an endogenous circadian pacemaker in mammals (see Ralph et al., 1990). Much current investigation is now directed to the cellular and molecular processes that might constitute the pacemaker’s oscillatory mechanism, and one of the obvious early objectives of this work will be a better understanding of the role played by SCN intercellular interactions. We recently presented evidence suggesting that Na+-dependent action potentials in the SCN are not a part of the internal timekeeping mechanism of the circadian pacemaker, although such spike activity is required for photic entrainment and overt expression of behavioral rhythmicity (Schwartz et al., 1987). Figure 1 outlines the results of our experimental paradigm, in which 1 pLM tetrodotoxin (TTX) or artificial cerebrospinal fluid (CSF) was chronically infused by miniosmotic pump into the SCN of unanesthetized, unrestrained albino rats while their free-running circadian rhythms of drinking activity were monitored. We first showed (Figs. lA and 1B) that TTX resulted in temporary behavioral arrhythmicity during its 14-day infusion, after which rhythmic drinking activity returned without apparent phase shift (i.e., the phase of the restored rhythm was that predicted by extrapolation of the free149 Downloaded from jbr.sagepub.com at NORTH CAROLINA STATE UNIV on May 1, 2015
FIGURE 1. (A and B) Actograms of two blinded rats with cannulae located in the SCN. Artificial CSF (A) or TTX (B) was continuously infused for the 14 days bracketed by arrows. (C and D) Actograms of two intact rats with cannulae located in the SCN and exposed to an LD 12:12 cycle progressively advanced by three successive 4-hr advances until a completely reversed cycle was attained. Artificial CSF (C) or TTX (D) was continuously infused for the 14 days bracketed by arrows.
running period [T] of the original [preinfusion] rhythm). We then showed (Figs. 1C and ID) that TTX prevented the phase-resetting action of a reversed light-dark (LD) cycle imposed during the infusion (i.e., the phase of the restored rhythm was what would have been expected had the rhythm continued its free run despite application of the light). Thus, the circadian pacemaker in the SCN continued to oscillate unperturbed by TTX treatment, although the 150 Downloaded from jbr.sagepub.com at NORTH CAROLINA STATE UNIV on May 1, 2015
reversibly inactivate the pacemaker’s input pathway for photic entrainment and its output pathway for overt expression of drinking rhythmicity. This paper reports the results of additional experiments using our infusion paradigm, in order to test the validity of this interpretation further. First, we infused 50% procaine to determine whether inhibition of SCN action potentials by another agent would reproduce the TTX results. Procaine blocks voltage-dependent Na+ channels and suppresses hypothalamic discharge activity (Harlan et al., 1983), but unlike TTX-which appears to bind to a receptor at the extracellular opening of the ion-conducting pore-the anesthetic is believed to inhibit action potential generation by binding to an internal receptor with differing affinities for the resting, active, and inactivated states of the channel (see Catterall, 1987). Second, we infused high (20 mM) K+ or 100 yvt veratridine to determine whether membrane depolarization would alter the pacemaker’s ongoing oscillation. Veratridine is an alkaloid that causes persistent activation of the Na+ channel by blocking its inactivation and by shifting the voltage dependence of its activation to more negative membrane potentials (see Catterall, toxin did
1987). z
MATERIALS AND METHODS
._
’
z
ANIMALS Adult male Sprague-Dawley rats were obtained from Charles River (Wilmington, MA) and housed individually in clear plastic cages contained within well-ventilated, light-proof end vironmental compartments as previously described (Schwartz et al., 1987). Light was provides by 15-W cool-white fluorescent tubes delivering an intensity of 600 lux at the midcage level. During darkness, 15-W safe lights with dark red (series 2) filters remained on in the facility to allow for routine care. Chow and water were freely available and replenished once every 6-7 days at irregular hours.
Some of the rats were blinded by bilateral orbital enucleation. Animals were fully anesthetized with ether; periorbital tissue was dissected carefully with iris scissors; optic nerves were cut; and eyes were removed. The entire procedure was accomplished in 50%) SCN damage. In the other 5 rats (Fig. 2A), postinfusion drinking rhythms resumed with T’s unaltered from those expressed preinfusion (24.13 ± 0.03 hr preinfusion vs. 24.16 ± 0.01 hr postinfusion). The phase of these restored rhythms was essentially that predicted by extrapolation of the T’s of the original (preinfusion) rhythms, with a phase shift of only 0.1± 0.5 hr for the population of animals. These results are similar to those previously reported with TTX (Schwartz et al., 1987). When
procaine
=
was
In order to test whether procaine treatment would uncouple the circadian pacemaker’s input pathway for photic entrainment, rats were first entrained to an LD 12:12 schedule for at least 2 weeks, after which cannula-pump assemblies were implanted. Rodents were maintained in constant environmental darkness for the first 8 days of the 14-day infusion period; during the last 6 days of the infusions, animals were exposed to an LD 12:12 cycle progressively delayed by three successive 4-hr delays until a completely reversed cycle was attained. At the end of the infusions, rats were placed in constant darkness, and free-running circadian rhythms of drinking activity were recorded for approximately 1 month. Drinking rhythms were successfully phase-shifted by the imposed LD cycle in rats with procaine infusions in the hypothalamus missing the SCN (n 5), to a degree (7.4 ± 0.6 hr) comparable to that previously reported in rats (Schwartz et al., 1987) with TTX infusions outside the SCN (6.2 ± 0.8 hr) or artificial CSF infusions into the SCN (6.6 ± 0.3 hr) (Fig. 2C). When procaine was infused into the SCN (n 5), a complete lesion of the nuclei by an abscess resulted in post infusion arrhythmicity in 1 animal. In the remaining 4 animals (Fig. 2B), procaine reduced the phase-resetting action of light, with an apparent phase shift of only 2.1 ± 0.5 hr for the population of animals-a value close to that previously reported with TTX (1.9 ± 0.7 hr) (Schwartz et al., 1987). This estimated 2.1-hr phase shift may be artifactually low, however, because procaine treatment resulted in a lengthened postinfusion T (24.30 ± 0.01 hr after procaine vs. 24.21 ± 0.01 hr after CSF). We calculate that this 0.09-hr T difference would lead to a 1.3-hr underestimate of the light-induced phase shift during procaine infusion; even with this corrected value, however, the phase shift during procaine infusion would be only half the shift during CSF infusion. =
=
HIGH K+ OR VERATRIDINE INFUSION In order to demonstrate that
would actually detect a change in the entrained to the LD 12:12 schedule, and with assemblies filled with 20 mM K+ in artificial blinded, implanted cannula-pump CSF. Free-running circadian rhythms of drinking activity were then recorded during the infusions and for approximately 1 month after the infusions ended. During the infusions of high K+ into the SCN (n 10), drinking rhythmicity appeared disorganized, although periodic activity was transiently observed in some of the animals. In 3 of the 10 rats, drinking rhythms remained arrhythmic after the infusions ended, and histological examination disclosed >50% SCN destruction. In the other 7 rats (Fig. 3A), postinfusion drinking rhythms resumed with T’s unaltered from those expressed preinfusion (24.01 ± 0.02 hr preinfusion vs. 24.04 ± 0.04 hr postinfusion), but the phase of these restored rhythms was shifted. There was an apparent advance of the phase predicted by circadian
our
infusion
paradigm
pacemaker’s ongoing oscillation,
rats were
=
153 Downloaded from jbr.sagepub.com at NORTH CAROLINA STATE UNIV on May 1, 2015
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