European Journal of Neuroscience, Vol.12, pp.3935±3943, 2000 ã Federation of European Neuroscience Societies

Changes in direct current (DC) potentials and infra-slow EEG oscillations at the onset of the luteinizing hormone (LH) pulse

Lisa Marshall,1 Matthias MoÈlle,1 Horst L. Fehm2 and Jan Born1 1Department of Clinical Neuroendocrinology and 2Department of Internal Medicine, Medical University of LuÈbeck, H. 23a, Ratzeburger Allee 160, D-23538 LuÈbeck, Germany

Keywords: human, hypothalamus, neocortex, pulse generator, sleep, slow potentials

Abstract An essential function of the neuroendocrine system lies in the coordination of hypothalamo-pituitary secretory activity with neocortical neuronal activity. Cortical direct current (DC) potential shifts and EEG were monitored in conjunction with the circulating concentration of luteinizing hormone (LH) in humans while asleep to assess a hypothalamic±neocortical interaction. The onset of an LH pulse was accompanied (i) at frontocortical locations by a transient positive DC potential shift of » 3 min duration and peak amplitude 50 mV; (ii) at frontal and central locations by an increase in power of infra-slow EEG oscillations for periodicities between 64 and 320 s. Results uniquely demonstrate a coupling of hypothalamo-pituitary activity with regulation of neocortical excitability.

Introduction A coupling of endocrine events to neocortical processing is a basic 1991; Pietrowsky et al., 1994). Also, during sleep pulsatile activity of requirement for integrating needs and behaviour. This coupling may this hypothalamo-pituitary axis is more regular, with initiation of LH reside in a hypothalamic±neocortical dialogue. Many neuroanatomic pulses predominantly occurring during NREM (nonrapid eye move- pathways relay a direct or indirect interaction between hypothalamus ment) sleep (Fehm et al., 1991; Rossmanith & Lauritzen, 1991). In and neocortex (Morecraft et al., 1992; Risold et al., 1997). addition, during the night hypothalamic multiunit activity associated Stimulation of the hypothalamus has been found to affect, for with the LHRH/LH pulse generator is increased (O'Byrne et al., instance, cortical slow potential activity and sleep-related thalamo± 1993). These characteristics led us to focus the investigation on a cortical spindle activity (Aladjalova, 1964; Rowland, 1968; Suntsova coupling between endocrine and cortical activity on LHRH/LH & Burikov, 1997), and electrical stimulation of the frontal neocortex activity generated during sleep. DC potentials and EEG activity were can exert an effect, albeit inhibitory, on luteinizing hormone (LH) measured continuously to determine changes in cortical activity secretion (Caceres & Taleisnik, 1980; Brutus et al., 1984). The during LHRH/LH secretory pulses for an interval from 15 min prior objective of the present study was to demonstrate a temporal link until 15 min after estimated onset of the LH pulse. between hypothalamus-dependent endocrine activity and neocortical activity in humans. For this purpose scalp-recorded direct current (DC) potentials and electroencephalogram (EEG) activity were Materials and methods recorded in conjunction with monitoring of LH secretory activity. Subjects and procedures LH secretion was chosen because it displays a distinct pulsatile pattern known to be closely linked to the hypothalamic release of Subjects were 11 healthy men (n = 7) and women (unmedicated, luteinizing hormone-releasing hormone (LHRH) into the pituitary nonsmokers) between 20 and 27 years, who judged themselves to be portal circulation. The neuronal system underlying the rhythmic good sleepers, had not been on shift work, and who suf®ciently activation of hypothalamic LHRH cells and the release of the ful®lled criteria of a preliminary screening test. For this, subjects were neuropeptide from their terminals has been described as the `pulse initially asked how well they judge themselves to sleep at home, in a generator', and has been localized to the arcuate nucleus within the new environment, and if they can sleep lying on their backs. Because mediobasal hypothalamus in the monkey (Knobil, 1989). Changes in restrained head movement is a necessary prerequisite for proper DC multiunit activity within the mediobasal hypothalamus parallel to potential recording, subjects were then asked to practice at home pulsatile LH secretion have been reported in males and females of sleeping throughout the night on their back wearing a cervical collar different mammalian species (Kawakami et al., 1982; Wilson et al., for 2±3 nights. Subjects documented how long they slept, if they 1984; Mori et al., 1991; Cardenas et al., 1993). In adult humans, LH remembered rolling over, and in what position they awoke. Volunteers levels and incremental LH pulse amplitude are on average larger able to sleep at least 5 h with the cervical collar, and without knowingly during the night-time sleep than during wakefulness (Fehm et al., changing their position more than about three times during the night, were subsequently recruited for an adaptation night. Normally cycling women were to participate on a night between days 4 and 11 of their Correspondence: Dr Lisa Marshall, as above. menstrual cycle (follicular stage). During the complete recording E-mail: [email protected], session subjects remained lying on their backs with their heads ®xed. Received 8 May 2000, revised 10 August 2000, accepted 31 August 2000 Blood was collected in an adjacent room through a long thin tube 3936 L. Marshall et al. connected to a forearm catheter. The studies took place in a sleep calculated for 30-sec epochs with the averaged values assigned to the laboratory at the Medical University of LuÈbeck, and the experimental centre time of the 30-sec epoch. The DC potential, integrated EMG protocol had been approved by the local ethics committee. activity and the polysomnographically determined sleep stages were averaged time-locked to the estimated onset of the LH pulse. These Recording averaged data segments covered an interval from 15 min before to For determination of LH pulses, blood was sampled every 7.5 min 15 min after the LH pulse onset. Data segments containing transitions between lights-off and lights-on from » 23 : 00 to 06 : 30 h. For to REM sleep, frequent transitions between stage 2 sleep and SWS, standard polysomnographic sleep recordings EEG (0.15±30 Hz), and periods of wakefulness within 615 min of estimated LH pulse electrooculogram (EOG; 0.045±30 Hz), and submental electromyo- onset were excluded. Exclusion of epochs containing transitions to gram (EMG; 1.5 Hz±1.5 kHz, with two electrodes placed horizontally REM sleep was based on the ®nding by Fehm et al. (1991) that LH over the M. submentalis) were ampli®ed by a Nicolet electroence- onset occurred predominantly during the mid portion of the NREM phalograph (Nicolet Biomedical Instruments, WI, USA). The sleep phase, and to exclude superimposition with the REM-transition ampli®ed EMG signal was sent further to analogue-to-digital positive shift as described previously (Marshall et al., 1998). Also, conversion (sampling rate 100 Hz). data segments with scores of `movement time' (but not of `movement DC potential and EEG signals were obtained from electrodes arousal', according to Rechtschaffen & Kales, 1968) occurring within located at F3, Fz, F4, C3, Cz, and C4 of the international 10±20 5 min of estimated LH pulse onset, and segments with changes in system. All electrodes were referenced to linked electrodes at the scalp temperature (> 0.5 °C) within 610 min of the estimated LH mastoids. DC potential, polysomnographic and temperature measure- pulse onset were eliminated, because artifacts in DC potential data ment were as described previously (Marshall et al., 1998). In brief, at could not be ruled out. Thus, of the original 30 data segments, 15 each electrode site a `clip-on' electrode socket was attached with segments remained for analyses. The ®rst 5 min of the 30-min data collodion and the scalp was punctured with a sterile hypodermic segment served as baseline. For a supplementary analysis the DC needle until minor bleeding occurred. Electrode gel (Electrode potential of the same selected 15 epochs was averaged within Electrolyte, TECA Corp., NY, USA) was applied to the socket, and 660 min around LH pulse onset. Prior to averaging DC potential the electrode, also ®lled with gel, was clipped on. Non-polarizable data, linear trends occurring across the 30-min and 120-min intervals, Ag/AgCl electrodes (8 mm diameter, ZAK GmbH, Simbach/Inn, respectively, were eliminated. Germany) were used. Air bubbles had been previously eliminated Differences in DC potential level as well as differences in DC from the electrode gel by centrifugation (1300 r.p.m. for 3 h at 10 °C) potential slope were assessed by analysis of covariance (ANCOVA) and care was taken not to produce any air bubbles when clipping with repeated measures. Factors were Electrode site and Time. electrodes onto their sockets. Electrode impedance was always Covariates, introduced to control for possible covariations of the DC <5 kW. A direct-current ampli®er (Toennies DC/AC ampli®er, Jaeger potential, were either sleep stage, movements (movement arousals/ GmbH and Co., KG, Germany; input resistance 2*100 MW) was used movement time), or integrated EMG activity. For the ANCOVA, sleep for recording and ampli®cation of DC potentials. Ampli®cation, stages were given the following values, as in a previous study 2000-fold; low pass ®lter, 30 Hz; threshold for automatic DC offset (Marshall et al., 1998): sleep stage 1, 1; stage 2, 2; stage 3, 3; stage 4, correction, 64 mV. With short-circuited input, the ampli®er drift, if 4; awake, ±1; REM sleep, 0. Degrees of freedom were corrected present, was < 3 mV/h. Analog DC/EEG signals were digitized at according to Greenhouse±Geisser (Greenhouse & Geisser, 1959). 100 Hz/channel (CED 1401, Cambridge Electronics, UK) and stored Post hoc contrasts were assessed only when respective main effects or on a PC together with a time marker (every 30 s) for off-line analysis. interactions reached statistical signi®cance at the P < 0.05 level. Skin temperature was measured near each mastoid reference and To characterize activity in the EEG frequency bands between on the scalp halfway between each frontolateral (F3, F4) and its 15 min before and after estimated LH pulse onset, a moving fast corresponding centrolateral (C3, C4) electrode (Mini Mitter, OR, Fourier transform (FFT) was calculated using 10.24-sec windows USA; 60.1 °C level of accuracy), and stored every 30 s. (1024 data points) shifted every 5 s (frequency resolution 0.0977 Hz). Data of each window had been multiplied by a raised cosine function Data processing and statistical analysis prior to FFT to taper the signal towards zero at the extremes of the Serum concentrations of LH were measured by a commercial data window, thus reducing errors induced by edge-effects. The immunoradiometric assay (Biermann, Bad Nauheim, Germany). moving FFT resulted in a new time series representing the change Sensitivity of the assay was 0.15 mIU/mL; intra-assay coef®cient of over time in power within ®ve selected frequency bands: delta (0.49± variation < 1.2% for concentrations < 16.4 mlU/mL, interassay 4 Hz), theta (4±8 Hz), alpha (8±12 Hz), beta (15±25 Hz) and sigma, coef®cient of variation < 2.2%, for concentrations < 16.8 mlU/mL. representing spindle activity (12±15 Hz). For each window of FFT Onsets of LH pulses in individual data were detected using the cluster analyses results were assigned to the corresponding centre time. The analysis as described (Veldhuis & Johnson, 1988).This method marks time series of EEG power were subsequently smoothed by applying a all signi®cant increases from nadir to peak clusters, all signi®cant 7-point (i.e. 30-s) moving average. decreases in the data series of known variance, and then designates a To prove statistical signi®cance of very slow periodic ¯uctuations peak as a region comprising an increase followed by a decrease. observed at visual inspection in the above-mentioned time series of Selected sample size of the nadir and peak clusters (two points each) power, autocorrelations were calculated for the intervals of all 15 data and the t-statistics for signi®cant increases and decreases resulted in a segments with SPSS statistical software (SPSS Inc., Chicago, USA). false positive rate < 1%. LH pulse onset was de®ned as the last nadir The cumulative distribution of the time-lags for the statistically sample point preceding a signi®cant peak. Sleep stages (1, 2, 3, 4 and signi®cant autocorrelations determined across the 15 data segments REM sleep), awake time and movement artifacts were scored off-line indicated considerable jitter in the period length of these oscillations, for 30-sec intervals (Rechtschaffen & Kales, 1968). Stage 3 and 4 with most of the `infra-slow' EEG oscillations occurring in a band correspond to slow-wave sleep (SWS). corresponding to period lengths between 64 and 320 s (Fig. 4). For To adapt electrophysiological data to a common time scale, this band, average power was calculated by FFT for evaluating the average values of DC potentials and integrated EMG activity were time course of infra-slow oscillations in relation to the LH pulse

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 DC shifts, infra-slow oscillations and LH 3937 onset. The average power for the 64±320 s band was calculated P < 0.003 for the Electrode±Time interaction). Mean slope values separately for the time series (temporal resolution 5 s) of delta, theta, for the rising and falling intervals of the large positive DC potential alpha, beta and sigma activity as well as for the time series of DC shift were, respectively, for F3 10.2 vs. ±13.2 mV/min (F(1,13) = 10.57, potential data, using again a moving FFT. Window length of these P < 0.01), for Fz 10.0 vs. ±9.5 mV/min (F(1,13) = 5.45, P < 0.05), and FFTs was 10.67 min (128 data points, resulting in a frequency for F4 13.0 vs. ±13.4 mV/min (F(1,13) = 15.73, P < 0.01). At » 8 min resolution of 0.00156 Hz), and windows were shifted every minute. after the LH pulse onset the DC potential at frontal locations Resulting values were assigned to the centre time of the FFT window; thus the ®rst window began 5 min before and the last window ended 5 min after the 30-min interval centred around the estimated LH pulse onset. Changes in EEG power and in infra-slow oscillatory power (corresponding to the 64±320 s range of period length) occurring before and after LH pulse onset were statistically evaluated using an ANCOVA model as described for DC potential data.

Results In general, sleep parameters were close to typical sleep under laboratory conditions with the somewhat increased time spent awake, and decreased time spent in stage 4 probably due to the restrained position of head and body during sleep. Mean 6 SEM total sleep time was 433.1 6 8.1 min, and percentages of time spent awake, in sleep stages 1±4 and in REM sleep were 9.7 6 0.02, 9.8 6 0.03, 48.7 6 0.03, 9.8 6 0.01, 6.2 6 0.01 and 15.6 6 0.01%, respectively. Mean LH concentration increased by 3.88 6 0.55 mIU/mL within 15 min after estimated pulse onset and reached a peak value of 7.19 6 0.84 mIU/mL. Thereafter LH concentration gradually declined with baseline levels not fully recovered by 60 min after estimated LH pulse onset (Fig. 2A).

DC potential changes at the onset of the LH pulse Close to the time of LH pulse onset (0 min) a prominent large positive shift occurred being most obvious in recordings over the frontal cortex (F3, Fz, F4) where peak amplitudes between 40 and 50 mVas compared to baseline were measured (Fig. 1B). ANCOVA con®rmed that the DC potential level between ±1 and +2 min (referenced to estimated LH pulse onset) was signi®cantly more positive than baseline over the frontal cortex (Fig. 1C). At central electrodes (C3, Cz, C4) a parallel increase in positivity of the DC potential remained nonsigni®cant compared with baseline (P > 0.2). Also, the covariate sleep stage remained nonsigni®cant (P > 0.4), indicating that the DC potential changes were not contaminated by sleep stage-related in¯uences (Fig. 1D). Likewise neither movement arousal (Fig. 1E) nor integrated EMG activity when used as covariate obtained any signi®cance (P > 0.2). The transient character of the large positive DC potential shift around the time of LH pulse onset is indicated by the clear change in direction of slope between the ±3 to +1 min interval and the +2 to

+6 min interval (F(1,13) = 10.50, P < 0.007; covariates sleep stage, movement arousal, integrated EMG: all P > 0.4; F(5,70) = 5.46,

FIG. 1. Time courses of LH concentration, DC potential, sleep stage and movements for the interval from 15 min before to 15 min after the estimated onset of the LH pulse. (A) Mean 6 SEM LH concentration; (B) DC potential collapsed across frontal electrode sites (F3, Fz, F4; heavy line) and central electrode sites (C3, Cz, C4; thin line). (The mean potential value of the ®rst 60 s was set to 0 mV). The hatched horizontal bar indicates the 5-min interval used as baseline (±15 to ±10 min); (C) P-values for the effect of Time (compared to baseline) in ANCOVA with sleep stage as covariate. P-values refer to changes in the DC potential at frontal sites. Changes at central sites remained statistically nonsigni®cant; (D) Averaged sleep stage (6 SEM); (E) Number of movements (movement time given the value 2; movement arousal given the value 1) summed across the 15 data segments.

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 3938 L. Marshall et al. recovered to baseline levels (Fig. 1). The small yet distinct negative dip in the DC potential » 3 min before estimated LH pulse onset was not statistically signi®cant, although clearly visible in several single data segments.

DC potential changes within 660 min of LH pulse onset A supplementary analysis of the DC potential 660 min around LH pulse onset examined whether the DC potential shift associated with the LH pulse onset bore any relationship to the NREM-REM sleep cycle and related DC potential changes (Hoffmann et al., 1996; Marshall et al., 1998). Figure 2 reveals that within a 660 min interval the immediate change in DC potential at LH onset was preceded by a pronounced DC potential shift toward negative values being steepest » 30 min prior to LH pulse onset. Apparently, this negative shift coincides with a shift in sleep stage from prevailing REM sleep to NREM sleep. Thereafter a more gradual DC potential shift in positive direction developed, onto which the immediate changes in DC potential at LH pulse onset were superimposed. This gradual shift was re¯ected in a signi®cantly more positive mean DC potential level between 25 and 30 min after LH pulse onset as compared to the potential level between 20 and 15 min before pulse onset (F(1,13) = 6.76, P < 0.05; P > 0.1 for covariate Sleep stage). Comparing the potential level within intervals even more distant from LH pulse onset yielded no more signi®cant changes. Both the strong negative shift in the DC potential seen in Fig. 2 and the subsequent gradual positive slope have been documented in pervious experiments (Marshall et al., 1998) where they were termed `NREM-transition negative shift' and `NREM-positive slope'. They indicate a close link between DC-potential changes and the NREM±REM sleep cycle, upon which potential changes related to LHRH/LH secretory activity appear to be superimposed during the NREM phase.

Delta, theta, alpha, beta and sigma power

Consistent with the criteria of sleep stage scoring, ANCOVA revealed that power within all EEG frequency bands correlated with the sleep stage covariate (P < 0.05). However, analyses with reference to the time course of the LH pulse did not reveal any signi®cant change in EEG power in the 0.5±25 Hz frequency range. Figure 3 reveals, for two individuals, LH concentration and the associated time series of EEG power, together with infra-slow EEG activity.

Infra-slow periodic EEG activity Inspection of individual records indicated that slow periodic ¯uctuations were superimposed on the time series of EEG power (Fig. 3). Autocorrelation functions for the intervals con®rmed the occurrence of signi®cant infra-slow oscillations in the time series of EEG power which were distributed in a rather broad range between 60 and 320 s (Fig. 4). A main periodicity appears to occur with a time lag of » 90 s, being most apparent in the time series of alpha and FIG. 2. Time courses of LH concentration, DC potential and sleep stage for sigma power. Given the wide range of signi®cant periodicities, the interval from 60 min before to 60 min after estimated LH pulse onset. average power was determined by FFT for a broad band covering the (A) Mean 6 SEM LH concentration; (B) DC potential collapsed across frontal periodicities between 64 and 320 s to further analyse the changes in electrode sites (F3, Fz, F4; black line) and central electrode sites (C3, Cz, C4; grey line). Open arrow indicates DC potential shift at LH onset. Prior to time of this infra-slow periodic activity time-locked to the LH peak averaging linear trends occurring across the 120-min interval were eliminated; onset. (In fact, when upper or lower subdivisions of this band were (C) Averaged sleep stages (6 SEM). For averaging, sleep stages were assigned separately evaluated, basically the same changes in time were the following values: sleep stage 1, 1; stage 2, 2; stage 3, 3; stage 4, 4; awake, revealed as those reported below for the broad 64±320 s band.) DC ±1; REM sleep, 0. The strong shift in the DC potential towards negativity » 30 min prior to estimated LH pulse onset and the subsequent gradual positive potential data were not included in this analysis, because respective shift represent, respectively, the `NREM-transition negative shift' and autocorrelation functions did not con®rm a suf®cient number of `NREM-positive slope' described previously (Marshall et al., 1998). Note signi®cant correlation coef®cients (< 45) for this range of infra-slow that the sleep scoring diagram indicates on average deepest NREM sleep in the oscillations (Fig. 4). middle of the 120-min interval.

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 DC shifts, infra-slow oscillations and LH 3939

FIG. 3. Time courses of LH concentration and power of EEG activity (left abscissa in log units) in delta, theta, alpha, beta and sigma frequency bands (thin lines) for two individual data segments (A,B). Note infra-slow periodic activity of delta, theta, alpha and beta power becoming prominent after estimated LH peak onset. The power of these infra-slow oscillations (with period lengths between 64 and 320 s) is additionally shown by thick lines referring to the right y-axis (in log power).

Compared to baseline, signi®cant enhancements in infra-slow the estimated LH pulse onset was preceded by a transient but oscillatory power were observed between 0 and 5 min after the statistically signi®cant suppression of infra-slow activity. estimated LH pulse onset for delta, theta, alpha and beta bands, but not in the sigma band (P < 0.05, for all ANOVA main effects of Time, Discussion Fig. 5). In addition, signi®cant (P < 0.05) Time±Electrode site interactions indicated differential topographical distributions in all Results indicate (i) that the onset of LH pulses is accompanied by a EEG bands except for theta. Accordingly, increases in infra-slow transient positive DC potential shift commencing shortly before the oscillatory activity around the time of LH pulse onset were most estimated LH pulse onset and ceasing » 8 min later; (ii) The positive distinct for delta at central sites (C3, Cz, C4), for alpha at frontal sites DC potential shift concentrated over frontal cortical areas; (iii) LH (F3, Fz, F4) and for beta activity at lateral sites (F3, F4, C3, C4). For pulse onset was associated with an increase in infra-slow oscillations the theta band, the increase in infra-slow oscillatory activity around in EEG activity occurring over a wide range of EEG frequencies.

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 3940 L. Marshall et al.

FIG. 4. (A) Autocorrelation functions for the time series of the DC potential and of delta, theta, alpha, beta and sigma power shown for two individual data segments (07, 09). Autocorrelation functions were calculated over intervals revealing slow periodic ¯uctuations. Vertical lines indicate the 5% level of signi®cance. Resolution, 5 s. (B) Cumulative distribution of statistically signi®cant positive time lags for the autocorrelation functions of all 15 data segments, for the time series of the DC potential as well as of delta, theta, alpha, beta and sigma power. Y-axis re¯ects the number of statistically signi®cant autocorrelations across the 15 data segments for a given time lag. For simplicity diagrams are only shown for positive time lags; correlation coef®cients with lag < 10 s were omitted. Note, while infra-slow periodic activity was poorly expressed in the time series of the DC potential, signi®cant infra-slow oscillations overlaying EEG activity considerably accumulated in a broad range of time lags (periodicities) between » 64 and 320 s.

Indications of a hypothalamic±neocortical interaction LH in the present study was used as an indicator of activation by the LHRH pulse generator because LHRH in portal blood in humans Whilst the data provide clear evidence for concurrent changes in cannot be measured directly. In studies where LHRH and LH have neocortical activity and endocrine activity of the LHRH/LH system, been measured simultaneously large-amplitude LH pulses have been the question arises what type of hypothalamic±neocortical interaction reported to accompany or be directly preceded by LHRH pulses, can be inferred. It can be excluded that the electrocortical changes in whereby the accompanying LH pulse, measured with 10-min the DC potential and infra-slow EEG oscillations were a consequence collection intervals, always occurred abruptly and peaked within 1± of the LH changes in peripheral blood. Signi®cant changes in cortical 2 collection intervals, similarly to the present study (Moenter et al., activity were well established before the LH plasma concentration 1992). The durations of the positive DC shift and of the increase in reached maximum levels. In addition, when the DC potential shift was infra-slow periodic EEG activity are similar to several temporal ceasing 8 min after estimated LH pulse onset mean LH concentration measures obtained for neuronal and neurosecretory activity asso- in blood was still enhanced and even rising (Figs 1 and 2). ciated with the LHRH pulse generating mechanism in mammals.

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 DC shifts, infra-slow oscillations and LH 3941

Enhanced multiunit activity of 1±5 min duration has been reported in conjunction with LHRH/LH neurosecretory activity (Kawakami et al., 1982; Williams et al., 1990; Goubillon et al., 1995), and release of LHRH into portal blood persisted for 2±8 min, subsequent to and preceding a rapid onset and cessation (Moenter et al., 1992). These temporal characteristics suggest a close linkage of the LHRH pulse generation to the positive DC potential shift starting shortly before the estimated LH pulse onset and lasting » 8 min, as well as to the increase in infra-slow oscillatory power commencing around the same time. A higher sampling rate of venous blood in the present study may have reduced some temporal jitter between LH concentration and electrophysiological measures. However, the inert release and distribution of LH into peripheral vessels contribute to the fact that plasma LH represents only an estimate of the actual timing of the LHRH pulse generator. Studies in rabbits and cats have shown that shifts in the cortical DC potential as well as infra-slow oscillatory activity in the neocortex can be readily induced by electrical stimulation of various medial hypothalamic sites (Aladjalova, 1964; Rowland, 1968). Moreover, Aladjalova measured infra-slow oscilla- tions simultaneously but with different period lengths in the hypothalamus and cerebral cortex, after hypothalamic stimulation. The latter studies imply that changes in neocortical electrical activity in association with pituitary LH secretion result from a direct or indirect collateral hypothalamic output to cortical structures. Although the temporal resolution of the present data do not allow exclusion of an inverse relationship, evidence that stimulation of LHRH/LH secretory activity could be a consequence of changes in activity at the cortical level is scarce (Caceres & Taleisnik, 1980; Brutus et al., 1984). As a further, though indirect, indication of a hypothalamic± neocortical interaction, the temporal association between LH pulse onset and previously reported phases of the DC potential change during the NREM-REM sleep cycle (i.e. the NREM-transition negative shift and the NREM-positive slope) could also be taken (Marshall et al., 1998). This data con®rms the strong link of LHRH/ LH secretory activity to the NREM phase of the NREM-REM sleep cycle observed in numerous foregoing studies (e.g. Fehm et al., 1991; Driver et al., 1996).

Cortical mechanisms of DC potentials and slow periodic EEG activity At present the exact sources of the large transient positive DC potential shift at frontal locations around the time of LH pulse onset are unclear. Slow cortical DC potential changes during wakefulness have been described as a measure re¯ecting a tonic tuning of excitability of cortical neuronal networks (Birbaumer et al., 1990; Elbert, 1993). It has become established to associate the occurrence of slow negative DC potential shifts during tasks with widespread synchronized membrane depolarizations of pyramidal apical den- drites and, accordingly, to associate DC potential shifts of positive polarity with reduced depolarization and hyperpolarization of apical dendrites. However, widespread hyperpolarizations in deeper cortical layers underlying slow oscillatory activity may also produce a FIG. 5. Mean log power of infra-slow EEG oscillations (between 64 and 320 s) super®cial potential of negative polarity (Steriade et al., 1993). in the time series of delta, theta, alpha, beta and sigma activity during the interval from 15 min before to 15 min after LH pulse onset. Time courses Further, it is of note that if activity is concentrated in the orbito- represent the average across electrode sites for theta. Because signi®cant frontal region it cannot be excluded that the direction of polarity at Electrode±Time interactions indicated speci®c topographies, for the other the frontal cortex is reversed due to the convexity of the orbito-frontal EEG bands averages were calculated across the electrode sites showing the region. most distinct changes in infra-slow oscillations, which were the central sites (C3, Cz, C4) for delta, the frontal sites (F3, Fz, F4) for alpha, and the lateral Regardless of the issue of exactly where the potential-generating sites (F3, F4, C3, C4) for beta activity. P-values indicate signi®cant changes in dipole is located in the neocortex, which would require more ®ne- infra-slow periodic EEG activity as compared to baseline (hatched bar). grained topographical analyses, our results indicate that modulations N = 15. in the DC potential and in infra-slow EEG oscillations are temporally

Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943 3942 L. Marshall et al. linked, with enhanced infra-slow periodic EEG activity and the pertains also to other hypothalamo-pituitary endocrine systems which positive DC potential shift obtaining signi®cance around the same would argue against a speci®c relevance within the reproductive axis. time. Yet, although the positive DC potential shift and the increase in Rather, such a link could serve a more general function, tuning infra-slow periodic EEG activity may share some of the generator activity between the endocrine and central nervous systems. On the mechanisms in cortical and subcortical tissues these phenomena do basis of the present data it may be speculated that neocortical not re¯ect identical processes because there are discrete distinctions excitability is down-regulated in temporal proximity to LH pulse in topography and time course. First, changes in infra-slow periodic onset, thus coinciding with the inhibitory effect of neocortical EEG activity in conjunction with LH release were observed at frontal, stimulation on LH secretion (Caceres & Taleisnik, 1980; Brutus et al., but also at central, electrode sites whereas the large transient positive 1984). Also, long-lasting effects on hippocampal excitability after in DC potential shift was clearly limited to frontal locations (Figs 1 and vitro LHRH administration have been observed (Chen et al., 1993). 5). Second, the DC potential level at the end of the 30-min interval Assuming that the association between LHRH/LH secretory activity did not differ from baseline level in contrast to the infra-slow and DC potential change disclosed here during night-time sleep can oscillations in delta, theta and alpha activity which at that time were be likewise demonstrated during the awake phase, the modulation in still enhanced. This suggests the shifts in the DC potential to be more neocortical excitability in conjunction with LH pulses may also closely linked to a temporally restricted hypothalamic±frontocortical acutely affect various aspects of ongoing behavioural regulation. interaction. Also, infra-slow oscillatory activity in the DC potential However, such actions remain to be speci®ed. was much weaker than that in the EEG bands. Interestingly, neuronal membrane potential level has been reported to in¯uence amplitude Acknowledgements and frequency of a very slow oscillatory activity in thalamic neurons (Leresche et al., 1991; Albrecht et al., 1998), and very slow periodic This work was supported by a DFG grant to J.B. and H.L.F. We gratefully excitability changes were also reported in neurons of the hippo- thank Ms J. Ries, Ms C. Beyer, Mr J. Czyborra and Mr J. Hoffmann for collecting data and for preliminary analyses, as well as Ms C. Zinke and campus (Penttonen et al., 1999). Ms A-K. JuÈrs for further technical assistance. An intriguing candidate mediating hypothalmic in¯uences on cortical excitability, DC potentials and infra-slow EEG activity is neuropeptide Y (NPY). Abundant NPY binding sites have been found Abbreviations in human frontal cortex (Statnick et al., 1997; Caberlotto et al., 1998), DC, direct current; EEG, electroencephalogram; EMG, electromyogram; FFT, and NPY release precedes LHRH by » 5 min (Woller et al., 1992). fast Fourier transform; LH, luteinizing hormone; LHRH, luteinizing hormone- NPY has been shown to produce shifts in EEG frequencies and releasing hormone; NPY, neuropeptide Y; NREM, nonrapid eye movement; REM, rapid eye movement. amplitude, particularly over the frontal cortex, and to in¯uence cortical excitability in vitro as well as after intracerebroventricular administration in rats (Zini et al., 1984; Ehlers et al., 1997; Klapstein References & Colmers, 1997; Woldbye et al., 1997). NPY closely interacts with Aladjalova, N.A. (1964) Slow Electric Processes in the Brain. Prog.Brain the noradrenergic system in LHRH pulse generation (Wuttke et al., Res. Vol. 7. Elsevier, Amsterdam. 1996; Terasawa, 1998), which points to a possible contribution of Albrecht, D., Royl, G. & Kaneoke, Y. (1998) Very slow oscillatory activities brainstem noradrenergic afferents in the joint modulation of in lateral geniculate neurons of freely moving and anesthetized rats. neocortical DC potential and infra-slow oscillatory activity Neurosci.Res. , 32, 209±220. Birbaumer, N., Elbert, T., Canavan, A.G. & Rockstroh, B. (1990) Slow (Terzano et al., 1985; Scheuler et al., 1990; Novak et al., 1992). potentials of the cerebral cortex and behavior. Physiol.Rev. , 70, 1±41. However, LHRH itself could also be involved in the transfer of action Brutus, M., Watson, R.E. Jr, Shaikh, M.B., Siegel, H.E., Weiner, S. & Siegel, to the neocortex. Receptor sites for LHRH have been identi®ed in the A. (1984) A [14C]2-deoxyglucose analysis of the functional neural pathways rat frontal cortex, limited to the rhinal sulcus (Jennes & Conn, 1994), of the limbic forebrain in the rat. IV. A pathway from the prefrontal cortical- which would correspond with the distinct frontal focus of the changes medial thalamic system to the hypothalamus. Brain Res., 310, 279±293. Caberlotto, L., Fuxe, K., Rimland, J.M., Sedvall, G. & Hurd, Y.L. (1998) in the DC potential observed in the present study. Regional distribution of neuropeptide Y Y2 receptor messenger RNA in the These neuropeptides and transmitters within the neocortex may human post mortem brain. Neuroscience, 86, 167±178. also affect glial cells which, due to their ability to spatially buffer K+, Caceres, A. & Taleisnik, S. (1980) Blockade of ovulation and release of LH in are supposed to participate in the generation of slow potentials (Lux the rat by electrochemical stimulation of the frontal lobe cortex. Brain Res., et al., 1986; Birbaumer et al., 1990). Glial cells have been shown to 188, 411±423. Cardenas, H., Ordog, T., O'Byrne, K.T. & Knobil, E. (1993) Single unit respond to electrical and various neurochemical stimuli, including components of the hypothalamic multiunit electrical activity associated with NPY, with an increase in intracellular Ca2+ concentration (Laming, the central signal generator that directs the pulsatile secretion of 1989; Gimpl et al., 1993; Pasti et al., 1997; Verkhratsky et al., 1998). gonadotropic hormones. Proc.Natl Acad.Sci.USA , 90, 9630±9634. In cultured astrocytes, oscillatory activity has been described with Chen, Y., Wong, M. & Moss, R.L. (1993) Effects of a biologically active LHRH fragment on CA1 hippocampal neurons. Peptides, 14, 1079±1081. periodicities up to the minute range (Pasti et al., 1997). Thus neural± Delamont, R.S., Julu, P.O. & Jamal, G.A. (1999) Periodicity of a noninvasive glial interactions may contribute to the enhancement of slow periodic measure of cardiac vagal tone during non-rapid eye movement sleep in non- EEG activity or DC potential positivity. sleep-deprived and sleep-deprived normal subjects. J.Clin.Neurophysiol. , The changes in the DC potential and infra-slow periodic EEG 16, 146±153. activity associated with LH pulse onset suggest a coordinated control Driver, H.S., Dijk, D.J., Werth, E., Biedermann, K. & Borbely, A.A. (1996) Sleep and the sleep electroencephalogram across the menstrual cycle in of neocortical activity in conjunction with hypothalamo-pituitary young healthy women. J.Clin.Endocrinol.Metab. , 81, 728±735. activation of LH release. The concordant increase in infra-slow EEG Ehlers, C.L., Somes, C., Lopez, A., Kirby, D. & Rivier, J.E. 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Ó 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 3935±3943