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Rhythmic Properties of the Hamster Suprachiasmatic Nucleus in Vivo

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  Rhythmic Properties of the Hamster SuprachiasmaticNucleus  In Vivo Shin Yamazaki, Marie C. Kerbeshian, Craig G. Hocker, Gene D. Block, and Michael Menaker National Science Foundation Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville,Virginia 22903 We recorded multiple unit neural activity [multiunit activity(MUA)] from inside and outside of the suprachiasmatic nucleus(SCN) in freely moving male golden hamsters housed inrunning-wheel cages under both light/dark cycles and constantdarkness. The circadian period of MUA in the SCN matched theperiod of locomotor activity; it was  24 hr in wild-type and 20hr in homozygous  tau  mutant hamsters. The peak of MUA in theSCN always occurred in the middle of the day or, in constantdarkness, the subjective day. There were circadian rhythms ofMUA outside of the SCN in the ventrolateral thalamic nucleus,the caudate putamen, the accumbens nucleus, the medialseptum, the lateral septum, the ventromedial hypothalamicnucleus, the medial preoptic region, and the stria medullaris.These rhythms were out-of-phase with the electrical rhythm inthe SCN but in-phase with the rhythm of locomotor activity,peaking during the night or subjective night. In addition tocircadian rhythms, there were significant ultradian rhythmspresent; one, with a period of   80 min, was in antiphasebetween the SCN and other brain areas, and another, with aperiod of   14 min, was in-phase between the SCN and otherbrain areas. The periods of these ultradian rhythms were notsignificantly different in wild-type and  tau  mutant hamsters. Ofparticular interest was the unique phase relationship betweenthe MUA of the bed nucleus of the stria terminalis (BNST) andthe SCN; in these two areas both circadian and ultradian com-ponents were always in-phase. This suggests that the BNST isstrongly coupled to the SCN and may be one of its major outputpathways. In addition to circadian and ultradian rhythms ofMUA, neural activity both within and outside the SCN wasacutely affected by locomotor activity. Whenever a hamster ranon its wheel, MUA in the SCN and the BNST was suppressed,and MUA in other areas was enhanced. Key words: circadian; ultradian; suprachiasmatic nucleus;  invivo  recording; hamster;  tau  mutant; locomotor activity; bed  nucleus of the stria terminalis; MUA Circadian locomotor activity rhythms in mammals are generatedby an endogenous pacemaker located in the suprachiasmaticnucleus (SCN) of the hypothalamus (for review, see Turek, 1985;Meijer and Rietveld, 1989; Klein et al., 1991). Lesions of the SCNcause arrhythmicity of locomotor activity (Moore and Eichler,1972; Stephan and Zucker, 1972; Rusak and Zucker, 1979), andtransplants of fetal SCN tissue restore circadian periodicities(Sawaki et al., 1984; Lehman et al., 1987; Ralph et al., 1990). TheSCN exhibits circadian rhythms in several  in vitro  preparations:the acute slice (Green and Gillette, 1982; Groos and Hendricks,1982; Shibata and Moore, 1988), slice culture (Bos and Mirmiran,1990; Herzog et al., 1997), and dispersed cell culture (Welsh etal., 1995; Liu et al., 1997). Both slice and dispersed cell cultures of SCN also display circadian rhythms of peptide release (Mu-rakami et al., 1991; Watanabe et al., 1993; Shinohara et al., 1995).In contrast the physiology of the SCN  in vivo  and its relationshipto circadian behavior in the intact animal have received littleexperimental attention.To understand how the circadian clock controls locomotorbehavior, we need to understand its connections to the motorcontrol system. Although output pathways from the SCN circa-dian pacemaker are not completely described, the motor controlsystem in mammals is relatively well characterized (Wichmann etal., 1995; Bergman et al., 1998). Because there are no knowndirect neural connections between the SCN and motor controlareas of the brain, it is likely that either humoral factors and/or“relay nuclei” serve to connect the SCN with the motor centers.Not only does the SCN regulate locomotor activity but there isreason to believe that locomotor activity feeds back on the SCN.The period of the free-running rhythm of rats housed in cages with a running wheel is different from that of rats housed in cages without a wheel (Yamada et al., 1988, 1990; Shioiri et al., 1990).Locking of the running wheel changes the free-running period inmice (Edgar et al., 1991). Access to running wheels induces phaseshifts of locomotor activity in hamsters (Mrosovsky, 1988; Reebsand Mrosovsky, 1989) as does injection of triazolam, which in-creases locomotor activity (van Reeth et al., 1987). In mice,forced treadmill running induces phase shifts of circadianrhythms and is able to entrain them (Marchant and Mistlberger,1996).The apparent complexity of the relationship between the SCNand locomotor centers, almost certainly involving reciprocal in- Received July 29, 1998; revised Sept. 28, 1998; accepted Oct. 1, 1998.This research was supported by the National Science Foundation (NSF) Scienceand Technology Center for Biological Timing along with Air Force Grants F49620-98-1-0174 to M.M. and F49620-97-1-0012 to G.D.B and M.M., by the NationalInstitutes of Health postdoctoral National Research Service Award NS09329 toC.G.H., and by an NSF postdoctoral fellowship in Biosciences Related to theEnvironment to M.C.K. We thank Dr. M. E. Geusz for computer programming forrecording neural activity (for spike discrimination) and Dr. M. Kawasaki for dis-cussing amplifier circuit design. Also our special thanks to Drs. M. Takahashi and M.Nishihara for general information on multiunit activity recording and to Dr. S.-I. T.Inouye for information on electrode design.We dedicate this work to Professor Hiroshi Kawamura on the occasion of his 70thbirthday (January 26, 1997).Correspondence should be addressed to Dr. Shin Yamazaki, National ScienceFoundation Center for Biological Timing, Department of Biology, Gilmer Hall,University of Virginia, Charlottesville, VA 22903.Copyright © 1998 Society for Neuroscience 0270-6474/98/1810709-15$05.00/0 The Journal of Neuroscience, December 15, 1998,  18 (24):10709–10723  teractions, and the fact that direct neuronal interconnectionsappear to be absent provide a strong rationale for exploring thefunctional relationships  in vivo . We have perfected a techniquethat allows us to record neuronal activity of several brain regionsin freely moving hamsters, enabling us to correlate electricalactivity within the SCN with activity in other brain regions and with locomotor activity. We have used this technique to describethe electrical characteristics of the SCN  in vivo , the differencesbetween the  tau  mutant and wild-type hamsters, the relationshipbetween the SCN and other regions of the brain, and the effect of the animal’s locomotor behavior on SCN activity. The resultsprovide a new framework for understanding the regulation of locomotor behavior by the circadian timing system. MATERIALS AND METHODS  Animals.  Three- to five-month-old golden hamsters (LVG wild type fromCharles River Laboratories, Wilmington, MA; LVG background  tau mutant animals from our colony) were used in this study. Animals wereentrained for at least 2 weeks to light/dark cycles (LDs) (14:10 hr LD for wild types; 11.7:8.3 hr LD for  tau  mutants; light intensity of   300 lux atcage level). We monitored wheel-running activity throughout the exper-iments and used only animals that showed clear locomotor rhythmicity.  Electrode implantation.  We implanted one or two bipolar electrodesconstructed from pairs of Teflon-coated stainless steel wires (bare diam-eter, 130   m; A-M Systems, Everett, WA; tip distance,   150   m forrecording from the SCN and 200–300  m for other brain regions) and anuncoated platinum–iridium wire (diameter, 130   m; A-M Systems) usedas a signal ground in the cortex. Wires were connected to an eight pin ICsocket wrapped in insulated copper tape. Distances between any twobipolar electrodes were determined according to the recording siteschosen.Electrode implantation was performed under pentobarbital anesthesia(90 mg/kg, i.p.). Animals were placed in a stereotaxic instrument with the nose bar set at  2 mm (David Kopf Instruments, Tujunga, CA).Four self-tapping screws (#0  1/8 inch; Small Parts, Miami Lakes, FL) were implanted into 1 mm holes in the skull made with a dental drill. Weused different stereotaxic coordinates for wild-type and  tau  mutanthamsters because the shape of bregma in  tau  mutants is different andmore variable than is that in wild types. Wild-type SCN coordinates were0.7 mm anterior to bregma, 0.2 mm lateral to the midsagittal, and 8.0–8.2mm below the dural surface.  Tau  mutant coordinates were 1.0–1.2 mmanterior to bregma, 0.2 mm lateral to the midsagittal, and 7.9–8.1 mmbelow the dural surface. The electrode was secured to the screws and theskull with dental cement.  Recording procedure.  One week after surgery, each hamster was trans-ferred to a 24 cm (width)  21 cm (length)  30 cm (height) cage witha running wheel 21 cm in diameter mounted on one side to allow thehamster equipped with wires access to the wheel. The electrodes wereconnected to head stage buffer amplifiers (J-FET input OP Amp; TL084)located on the hamster’s head. Buffer amplifiers were connected to a 12channel slip ring (Airflyte Electronics Company, Bayonne, NJ) thatallowed free movement for the animal. The wires between the head stageamplifiers and the slip ring were protected by a stainless steel spring.Output signals were processed by differential input integration amplifiers(INA 101 AM; Burr-Brown, Tucson, AZ; gain,   10) and then fed into AC amplifiers (OP Amp, 4558; bandpass, 500 Hz to 5 kHz; gain, 10,000).Spikes were discriminated by amplitude and counted in 1 min bins usinga computer-based window discrimination system (DAS-1801ST ADboard; Keithley Metrabyte, Taunton, MA). Wheel revolutions were re-corded using the Data Quest system (Data Science International, St.Paul, MN).  Reduction of recording noise.  In recording neural activity from freelymoving animals, the biggest problem is noise. We reduced microphonicnoise, which is caused by mechanical disturbances such as wire move-ments, by mounting the buffer amplifiers on the head (effectively decreas-ing the impedance). We directly coupled the output signals to theintegration amplifier (without any capacitors or resistors) to provide ahigh common mode rejection ratio that can reduce non-neuronal signalssuch as muscle potentials. We also used shielding material around theelectrode and its vicinity to reduce noise from the animal’s scratching.Because we could not entirely remove this source of noise, we used a  Figure 1.  Examples of MUA recorded for 10 d from the SCN of three wild-type hamsters. Animals were kept in light/dark cycles (14:10 hr LD) for4 d and then released into constant darkness (lighting condition indicated at the  bottom  of the figure). Neuronal spikes are plotted in 6 min bins.Wheel-running activity is plotted at the  bottom  of   A–C  as the number of revolutions per 6 min; the  y -axis for this portion of the figure shows 0–200revolutions per 6 min.  A , Recorded from the ventrolateral portion of the central region of the left SCN.  B , Recorded from the ventromedial portion of the right SCN at the center of the rostrocaudal axis.  C , Recorded from the ventromedial portion of the right SCN near the caudal end of the nucleus. 10710  J. Neurosci., December 15, 1998,  18 (24):10709–10723 Yamazaki et al.  ã  Circadian and Ultradian Rhythms in Hamster SCN  highly effective low-cut filter (500 Hz). Using these techniques, wereduced electrical noise generated by chewing or moving to undetectablelevels. Although scratching did generate detectable electrical noise, thisactivity was rare and did not create a problem in the analysis.  Identification of recording sites.  After the electrical recordings, eachhamster was anesthetized with halothane, the head amplifier was discon-nected, and a small positive current (50    A; 10 sec) was passed throughthe recording electrodes. The brain was removed and fixed in Zamboni’sfixative solution for a few days. Frozen sections (40   m thick) werestained with potassium ferrocyanide (5% potassium ferrocyanide in 10%HCl). Blue spots of deposited iron were used for identification of recording sites.  Data analysis.  The first report of SCN neuronal recordings from freelymoving rodents appeared 19 years ago and played a pivotal role inidentifying the SCN as the central mammalian circadian pacemaker(Inouye and Kawamura, 1979). Since then little use has been made of thistechnique. A primary reason for hesitation in the use of   in vivo  recordingtechniques is that changes in neuronal oscillations are oftentimes difficultto identify in the raw data, and thus robust time series statistical analysisis required. No single time series analysis tool can be applied in all cases.We applied a new method, singular-spectrum analysis (SSA), in combi-nation with older methods. Periodicities in multiunit activity (MUA)recorded  in vivo  were determined using SSA in combination with themultitaper method (MTM) approach to the fast Fourier transform(Thomson, 1982; Vautard et al., 1992).Singular-spectrum analysis or SSA is a linear, nonparametric methodbased on a principal component analysis in the vector space of the delaycoordinates for a times series (Elsner and Tsonis, 1996). In SSA, a singletime series is expanded into a set of multivariate time series of length  M  ,known as the “window length.”  M   determines what range of frequenciescan be resolved as a stationary signal in the calculated principal compo-nents. The principal component analysis orders the expanded time seriesas a new coordinate system with most information along the first coor-dinates. The principal components are processes of length  N     M     1that can be thought of as weighted moving averages of the time series in which each accounts for a certain percentage of the total variance. SSA allows optimal detrending, identification of the noise floor in spectralestimates, and identification of intermittent oscillatory components in thedata. In practice, we found that we could with reasonable success dividethe data into four parts (trend variance, circadian variance, ultradiancomponents variance, and noise variance) by use of two window lengthson the MUA data.  M   was set at 36 hr (  M     360 for 6 min bins) for thecircadian time scale and 5 hr for the ultradian time scale. SSA cannotresolve periods longer than  M   and treats them as trends. If   M   is muchgreater than the average lifetime of an episode of oscillation, SSA cannotresolve the intermittent oscillation.We used SSA for signal reconstruction from the noisy MUA data.Simple noise reduction by applying a fixed low-pass filter to the data isnot appropriate when the spectrum is not monotonic. Because steps weretaken to minimize instrument noise and a low-cut filter was used (500Hz) before binning the impulses in the MUA data, noise is representedhere mostly by random impulses from populations of neurons near theelectrode. Optimal filtering of signals that are not completely stablerequires methods such as Wiener filtering or SSA. Both provide optimalfilters in a least squares sense. However Weiner filters arbitrarily requireharmonic functions as a basis. SSA, in contrast, uses data-determinedgeneral functions that do not require any previous hypotheses about thenoise variance. Unlike SSA, the Wiener method requires smooth and very reliable estimates of the power spectrum that are impossible toobtain with very short data sets. “Noise-free” circadian or ultradian time  Figure 2.  Daily and circadian rhythms of neural activity inseveral regions of the brain. MUA and locomotor activityare plotted in 6 min bins as in Figure 1. Recordings weremade from the following:  A , right side of the ventrolateralthalamic nucleus ( VLT  );  B , right side of the medial septum(  MS );  C , right side of the stria medullaris (  sm );  D , right sideof the LS;  E , the optic chiasm (  oc ). Each plot represents adifferent wild-type hamster except for  A  (same animal as in  E ) and  C  (same animal as in Fig. 1 C ). Note (most clearly in  B ) that the peak of neural activity coincides with wheel-running activity. Yamazaki et al.  ã  Circadian and Ultradian Rhythms in Hamster SCN J. Neurosci., December 15, 1998,  18 (24):10709–10723  10711   Figure 3.  Wheel-running behavior and neural activity records from the SCN and LS of a wild-type hamster ( WT  ) in constant darkness. Electrodes werelocated in the left ventromedial region of the central SCN and in the right LS. The time scale shows hours after the hamster was released into constantdarkness.  A , MUA in the SCN in 6 min bins.  B , MUA in the LS in 6 min bins.  C , Wheel revolutions per 6 min. The data marked by the  double-headed arrows  are presented below (see Fig. 8).  Figure 4.  Mathematical analysis of the phase angle difference of the circadian rhythms recorded in the SCN and LS in a wild-type hamster. Originaldata are shown in Figure 3  A ,  B . Data reconstructed by SSA for the SCN (  A ) and for the LS (  B ).  C , Instantaneous phase angle difference between thecircadian rhythms shown in  A  and  B . The time series  A  and  B  were individually converted to their Hilbert transforms (the time series with a 90° phaseshift). The resultant time series are series of complex numbers representing the srcinal data (real part) and the Hilbert transform (imaginary part). Themagnitude of each of these complex numbers is an estimate at that time of the circadian rhythm amplitude (data not shown). The angle of each of thesecomplex numbers is an estimate at that time of the phase of the circadian rhythm relative to the beginning. The difference between the angles of   A  and  B  at each point is the instantaneous phase difference between the two circadian rhythms. 10712  J. Neurosci., December 15, 1998,  18 (24):10709–10723 Yamazaki et al.  ã  Circadian and Ultradian Rhythms in Hamster SCN
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