BRAIN RESEARCH

ELSEVIER Brain Research 718 (1996) 76-82

R e s e a r c h r e p o r t

Preoptic/anterior hypothalamic neurons: thermosensitivity in wakefulness and non rapid eye movement sleep

Md. N o o r A l a m a, D e n n i s M c G i n t y a,c, * , R o n a l d S z y m u s i a k b,c

a Department of Psychology, University of California, Los Angeles, CA, USA b Department of Medicine, University of California, Los Angeles, CA, USA

c Department of Neurophysiology Research (151A3), Veterans Affairs Medical Center, 16]11 Plummer Street, Sepulveda, CA 91344, USA

Accepted 27 December 1995

Abstract

Thermosensitive neurons of the preoptic/anterior hypothalamic area (POAH) have been implicated in the regulation of both body temperature and non rapid eye movement (NREM) sleep. During NREM sleep, a majority of POAH warm-sensitive neurons (WSN) exhibit increased discharge compared to wakefulness. Cold-sensitive neurons (CSN) exhibit reduced discharge in NREM sleep compared to wakefulness. To further study the mechanism underlying these processes, the present study compared discharge rate and thermosensi- tivity (discharge rate change/°C) of WSNs and CSNs in NREM sleep and wakefulness in freely moving adult cats. The thermosensitivity of 24 WSNs and 31 CSNs from the medial POAH was determined from responses to local POAH warming and cooling. WSNs with increased discharge in NREM sleep exhibited increased thermosensitivity during NREM sleep compared to wakefulness. CSNs with decreased discharge during NREM sleep exhibited decreased thermosensitivity in NREM sleep. The change in thermosensitivity from wakefulness to NREM sleep was correlated with the change in discharge rate in WSNs but not in CSNs. In addition, 9 of 47 neurons that were thermo-insensitive during wakefulness became warm-sensitive during NREM sleep. Changes in POAH neuronal thermosensitivity could be a component of the mechanism for stabilization of state after state transition.

Keywords: Preoptic area; Anterior hypothalamus; Thermosensitive neuron; Thermosensitivity; Wakefulness; Non-rapid eye movement sleep

1. Introduct ion

The preoptic/anterior hypothalamic area (POAH) is a critical thermoregulatory structure of the mammalian brain (see [2,12]). POAH warm-sensitive (WSN) and cold-sensi- tive neurons (CSN) have been found in several species both in vivo [1,10,23,34] and in vitro (e.g., [8]). A role for the POAH in the regulation of non rapid eye movement (NREM) sleep is also documented (see [30]). POAH le- sions cause long-lasting insomnia, and local chemical stim- ulation of the POAH promotes sleep [9,14,15,19,22,29,32].

The sleep-regulatory and thermoregulatory mechanisms of the POAH are closely integrated [17]. Local POAH warming promotes NREM sleep in addition to activating heat loss responses [2,24,25]. Within sustained NREM sleep, EEG slow wave activity is augmented by POAH warming [18]. These studies show that activation of POAH WSNs and/or inhibition of CSNs can induce EEG slow wave activity and NREM sleep. In a recent study, we

* Corresponding author at address 'c ' . Fax: (1) (818) 895-9575.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 0 3 5 - 2

found that the majority of POAH WSNs exhibited in- creased discharge at the onset of naturally-occurring NREM sleep. In contrast, a majority of CSNs exhibited decreased discharge during NREM sleep onset [1]. Since these changes in neuronal discharge are sufficient to induce NREM sleep and they occur spontaneously at natural sleep onset, we have hypothesized that these neural events play a role in initiating spontaneous sleep as well as sleep in- duced by POAH warming.

The mechanisms that may bring about changes in the discharge of POAH thermosensitive neurons during NREM sleep may include alteration in synaptic input, local effects of endogenous sleep factors and/or modulation of the intrinsic thermosensitivity. We have recently reported that WSNs with increased discharge in NREM sleep (sleep-re- lated WSNs) exhibit increased thermosensitivity in NREM sleep. CSNs with increased discharge in waking as com- pared to NREM sleep (wake-related CSNs) exhibit re- duced thermosensitivity in NREM sleep. However, several issues concerning these phenomena need further study. First, both warm- and cold-sensitive neuronal populations

Md.N. Alam et al. / Brain Research 718 (1996) 76-82 77

represent a mixture of subgroups including wake-related, sleep-related and state-indifferent neurons. The changes in the thermosensitivity in most of these subgroups have not been described. Second, ou~." preliminary analysis was car- fled out in WSN and CSN ,;ubgroups in which thermosen- sitivity changes were parallel with rate changes in NREM sleep. It is important to determine if changes in thermosen- sitivity in NREM sleep are closely correlated with changes in discharge rate in different thermosensitive neuronal subgroups. Third, since we found that thermosensitivity of WSNs may increase in NREM sleep, it is possible that some neurons that are therrno-insensitive in waking be- come thermosensitive in NREM sleep. The present study examined each of these problems.

2. Materials and methods

Experimental subjects were five adult cats. In brief, under Nembutal anesthesia (35 mg/kg , i.p.) and aseptic conditions, cats were surgically prepared for chronic recording of medial POAH neuronal activity and standard electrodes for monitoring sleep-wake states (see [1]). A three barrel mechanical microdrive (diameter, 0.055 ram) with 1.0 mm barrel spacing was implanted in the medial POAH. Four or five 25 tzm insulated stainless steel wires were inserted through each barrel into the medial POAH to record neuronal discharge. A stainless steel (24 gauge) water perfused thermode (diameter = 0.8 mm) was im- planted into medial POAH at a 20 degree angle to the microdrive with its tip centered 2.0 mm lateral to the plane of the recording wires. One microthermocouple was placed in the central barrel of the microdrive along with mi- crowires. Thus, we recorded local POAH temperature close to the neuronal discharge recording site. Another thermo- couple was positioned 2.0 mm lateral to the thermode. Electroencephalogram (EEG), Electrooculogram (EOG), electromyogram (EMG), and pontine-geniculate-occipital waves (PGO) electrodes were implanted for the quantifica- tion of sleep-wakefulness.

Experiments were started at least seven days after surgery. Cats were freely-moving within a 60 × 60 × 100 cm cubicle. The microdriw~ was advanced in 20-25 tzm steps, until stable and isolated single units (Signal/Noise > 3 /1 ) were found. Unit triggered pulses were recorded on the polygraph along with sleep-wake variables and POAH temperature (TpoAI~). EEG, EOG, EMG, TpOAH

and unit activity were also digitized (Cambridge Electronic Design 1401, London) and stored on disk.

Thermosensitivity was tested during wakefulness and NREM sleep. Each test included the recording of sponta- neous discharge rate at [1] baseline POAH temperature for 30-60 s, [2] after stabilization of POAH temperature mea- sured close to the recording site at about 1.5-2°C above or below baseline for 30-60 s, and [3] after withdrawal of thermal stimulus for 30-60 s. Changes in behavioral state

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Fig. 1. Polygraphic traces showing examples of wanning trials conducted for testing the thermosensitivity of a neuron during wakefulness (A) and NREM sleep (B). The upper panels show EEG, EOG, PGO, and EMG indicating that the animal was in the same state, i.e., either in wakefulness or NREM sleep throughout the trial. This neuron was cold-sensitive in waking as POAH warming induced a decrease in its spontaneous dis- charge rate (A). In NREM sleep, a decrease in the basal discharge rate and a smaller %change/°C in the discharge of this neuron in response to POAH warming can be seen in B. This shows that this neuron was wake-related and exhibited a decreased thermosensitivity in NREM sleep as compared to wakefulness. Those thermosensitivity tests during which the animal did not remain in the same state, i.e., either wakefulness or NREM sleep were not considered. EEG, electroencephalogram; EOG, electrooculogram; EMG, electromyogram; PGO, pontine-geniculate-oc- cipital spikes; SU, single units; IU, integrated units; TpOAH , preoptic/an- terior hypothalamic temperature; Nos, Discharge rate for the marked area.

could influence spontaneous neuronal firing rate, in turn influencing the quantification of thermosensitivity. There- fore, during tests, the sleep-wake state of the cat was carefully monitored both behaviorally and by reference to concurrent polygraphic recordings. Tests in which behav- ioral state was constant, i.e. either waking or NREM sleep, were used in analyses. Fig. 1 shows typical examples of warming trials conducted for testing neuronal thermosensi- fivity in wakefulness and NREM sleep. Most of the neu- rons were tested for sensitivity to both warming and cooling and by repeated tests. The average number of tests/neuron during waking was 2.8 and during NREM sleep it was 2.0. Neurons were considered as thermosensi- tive if a particular thermal stimulus, i.e. either warming or cooling, evoked a consistent pattern of responses in 2 of 3 trials and the average change/°C on all trials met the

78 Md.N. Alam et al. / Brain Research 718 (1996) 76-82

criteria for thermosensitivity. The mean thermosensitivity derived from the repeated tests was used to assess hypothe- ses. At the end of the recording session, under deep Nembutal anesthesia, microwire positions were marked by making microlesion and positions of the recorded neurons

were histologically traced. Thermosensitivity was quantified by three criteria used

in earlier studies, i.e. Ql0, i m p u l s e s / s / ° C , and %change/°C criteria (Table 1). For Q10 classification, neurons were considered as Warm-Sensitive Neurons

(WSN) if the Qm was > 2.0 and Cold-Sensitive Neurons

(CSN) if the Qm was < 0.5. For i m p u l s e s / s / ° C classifi- cation, neurons were defined as WSNs if they exhibited an

increase of > 0.8 i m p u l s e s / s / ° C and as CSNs if firing rate decreased by at least 0.6 i m p u l s e s / s / ° C . According to %change/°C criterion, neurons were classified as WSNs

if the %change/°C was _> 10% and CSNs if the %change/°C was _< - 1 0 % of the basal discharge rate. The basic hypothesis about the thermosensitivity during NREM sleep was tested by all the three criteria (Table 1). The three criteria defined slightly different populations of thermosensitive neurons. The Q10 criterion was liberal in classifying slow discharging neurons as thermosensitive. I m p u l s e s / s / ° C criterion was liberal for fast discharging neurons ( > l0 Hz) recognizing about twice as many fast discharging neurons compared with other criteria (see [1]). On the other hand, %change/°C was applicable to both

slow- and fast-discharging neurons and was used for de- tailed analyses [1]. Wakefulness and NREM sleep were identified on the basis of EEG, EOG, and EMG using standard criteria [33]. Neurons were considered to be wake-related if they had a N R E M / W a k e discharge ratio of < 0.8, NREM sleep-related if they had a N R E M / W a k e discharge ratio of > 1.2, and state-indifferent if they had a N R E M / W a k e discharge ratio of > 0.8 and < 1.2 [1].

The statistical reliability of the changes in thermosensi- tivity for individual subgroups of neurons during NREM sleep as compared to wakefulness was determined by the

Wilcoxon matched-pair signed rank test. The Mann-Whi t - ney test was used to determine statistical reliability of inter-group variabilities.

3. Results

The thermosensitivity of 62 medial POAH thermosensi- tive and 47 thermo-insensitive neurons was studied during wakefulness and NREM sleep. Thermosensitive neurons

constituted 21% of 308 POAH neurons whose thermosen- sitivity was tested. The discharge rate changes of these neurons with s leep-wake state have been reported earlier [1]. Most WSNs exhibited increased discharge and most CSNs exhibited reduced discharge in NREM sleep com-

pared to wakefulness. However, subgroups with other state-related discharge patterns such as wake-related WSNs and sleep-related CSNs could also be identified. These classifications are used below.

Among thermosensitive neuronal groups defined by any of three criteria adopted to quantify thermosensitivity, the subgroup of WSNs with sleep-related discharge exhibited a significant increase in thermosensitivity during NREM sleep (Table 1). The subgroup of CSNs with wake-related discharge showed a significant decrease in thermosensitiv- ity during NREM sleep as compared to wakefulness (Table 1). Other subgroups did not exhibit significant changes in thermosensitivity that were consistent across criteria for thermosensitivity (Table 1).

The %change/°C criterion was used in the detailed analyses (see Section 2). Fifty-five neurons qualified as thermosensitive during wakefulness using the %change/°C criterion including 24 WSNs and 31 CSNs.

3.1. Warm-sensitive neurons

The mean thermosensitivity (%change/°C _ S.E.M.) of

WSNs during NREM sleep, 2 4 . 1 3 _ 6.64, was slightly

Table 1 Mean ( _ S.E.M.) thermosensitivity in wakefulness and NREM sleep for different subgroups of warm-sensitive and cold-sensitive neurons as identified by different criteria

Group Subgroup Q10 Impulse/s/°C %change/°C

Wake Sleep Wake Sleep Wake Sleep

W WR 6.64 + 1.57 (5) 5.54 + 3.30 1.50 + 0.21 (5) 0.61 + 0.25 ÷ 23.87 + 0.04 (4) 6.26 + 6.79 S SR a 6.29 + 0.79 (16) 9.98 _ 1.83 + 1.64 + 0.50 (10) 4.32 + 2.62 ÷ 21.57 + 3.79 (15) 35.24 + 9.21 ÷ N SI 3.71 + 0.61 (8) 2.17 + 0.50 2.79 + 0.67 (7) 2.22 + 0.71 16.59 + 1.46 (5) 5.10 + 5.06

Average 5.64 + 0.59 (29) 7.06 + 1.33 1.97 + 0.34 (22) 2.81 + 1.25 20.92 + 2.46 (24) 24.13 + 6.64 C WR a 0.11+0.03(24) 0.45+0.13 ** -1.78+0.48(17) -0.34+0.13 ** -29.57+2.77(22) -18.63+3.49 ** S SR 0.17 + 0.04 (4) 0.69 + 0.32 -0.68 + 0.04 (3) -0.06 + 0.16 -22.03 + 2.58 (4) - 10.21 + 5.04 N SI 0.08 + 0.04 (5) 0.66 ___ 0.35 -2.65 + 0.76 (4) -0.39 + 0.11 -29.56 + 2.58 (5) - 12.04 + 5.53

Average 0.11 + 0.02 (33) 0.51 + 0.09 * * - 1.79 _ 0.38 (24) -0.32 _+ 0.07 * ~ -28.60 + 2.18 (31) - 16.48 + 2.78 * *

WSN, Warm-sensitive neuron; CSN, cold-sensitive neuron; WR, wake-related; SR, sleep-related; SI, state-indifferent. Numbers in parentheses represent number of neurons studied. a Data reported previously [1]. + P < 0.05 and * * P < 0.01 level of difference between thermosensitivity in wakefulness and NREM sleep (Wilcoxon matched-pair signed rank test).

Md.N. Alam et al./ Brain Research 718 (1996) 76-82 79

higher but insignificantly different from the mean ther- mosensit ivity during wakefulness, 20.92 _+ 2.46 (Table 1; Fig. 2A). The mean N R E M / W a k e discharge rate ratio of WSNs was 1.53 _+ 0.23. WSNs consisted of three sub- groups of neurons: wake-related, sleep-related, and state- indifferent neurons. There was no significant difference in thermosensitivity between these subgroups of neurons dur- ing wakefulness. The major subgroup (63% of WSNs) exhibited spontaneously increased discharge in NREM sleep. This subgroup of neurons displayed a significant increase in thermosensitivity during NREM sleep as com- pared to wakefulness (Table 1; Fig. 2A). The other sub- groups, i.e. wake-related WSNs and state-indifferent WSNs, exhibited decreased thermosensit ivity during NREM sleep, but these changes were not statistically significant (Fig. 2A).

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Fig. 3. The ratio of the %change/°C during NREM sleep and wakeful- ness as compared to the ratio of the spontaneous discharge rates during NREM sleep and wakefulness of WSNs (A) and CSNs (B). These two parameters exhibited a significant correlation in WSNs. In CSNs, the correlation between the above two parameters was not significant.

3.2. Cold-sensitive neurons

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Fig. 2. A: %change/°C (mean + S.E.M.) of wake-related, sleep-related and state-indifferent WSNs during wakefulness (blank bars) and NREM sleep (filled bars). A significant increase in %change/°C of sleep-related WSNs during NREM sleep can be seen in this figure. The %change/°C of other sub-groups during NREM sleep was not significantly different from wakefulness. ÷ P < 0.05 level of significant increase (Wilcoxon matched-pair signed rank test); WR, wake-related; SR, sleep-related; SI, state-indifferent; n, number of neurons. B: %change/°C (mean + S.E.M.) of wake-related, sleep-related, and state-indifferent CSNs during wakeful- ness (blank bars) and NREM sleep (filled bars). All three subgroups of CSNs showed reduced %change/°C during NREM sleep. However, among the subgroups, only the wake-related CSNs exhibited significant change. * * P < 0.01 level of significant decrease (Wilcoxon matched-pair signed rank test). Abbreviations are same as in A.

The mean thermosensitivity of CSNs during NREM sleep, - 16.48 _ 2.78, was significantly lower than the mean thermosensitivity during wakefulness, - 2 8 . 6 0 ___ 2.18 (Table 1, Fig. 2B). The N R E M / W a k e discharge rate ratio of CSNs was 0.73 + 0.14. CSNs also consisted of a mixture of wake-related, sleep-related and state-indifferent neurons. In case of the CSNs, the major subgroup, i.e. wake-related CSNs exhibited reduced thermosensitivity during NREM sleep. The other subgroups, i.e. sleep-re- lated CSNs and state-indifferent CSNs exhibited decreased thermosensitivity that was statistically insignificant (Table 1, Fig. 2B). There was no significant difference in the thermosensitivity between these subgroups of neurons dur- ing wakefulness.

3.3. Thermosensit ivi ty and discharge rate

We evaluated the correlation between changes in dis- charge rate and changes in thermosensitivity from wake to NREM sleep. Among WSNs, the change in thermosensi- tivity from wakefulness to NREM sleep was correlated significantly with the change in discharge rate (n = 24, r = 0.61, P = 0.002; Fig. 3A). However, among CSNs, no

80 Md.N. Alam et al. / Brain Research 718 (1996) 76-82

L) %

z

r j

40

30

20

10

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Fig. 4. %Change/°C in discharge rates of eleven thermo-insensitive neurons to POAH temperature changes during wakefulness (open circles) and NREM sleep (filled circles). These neurons did not meet the %change/°C criterion of thermosensitivity in wakefulness. During NREM sleep, these neurons became thermosensitive by exhibiting changes of at least 10%/°C in their discharge rate due to POAH temperature manipula- tion.

significant correlation between change in discharge rate and thermosensitivity was found (n = 31, r = 0.12, P = 0.52; Fig. 3B). The correlations between thermosensitivity and discharge rate within wakefulness or NREM sleep were not significant for either WSNs (wakefulness: r = 0.21, P = 0.47; NREM sleep: r = 0.18, P = 0.39) or CSNs (wakefulness: r = 0.22, P = 0.21; NREM sleep: r = 0.32, P = 0.07).

3.4. Thermo-insensitive neurons

The thermosensitivity of 47 randomly selected neurons found to be thermo-insensitive during wakefulness was retested during NREM sleep. Of these, 11 (23%) neurons became thermosensitive during NREM sleep. Nine became warm-sensitive and two became cold-sensitive during NREM sleep (Fig. 4). Seven of these were wake-related, two were sleep-related and the remaining two were state- indifferent.

3.5. Histology

The anatomical distribution of the above discussed neu- rons has been reported earlier [1]. These neurons were found in an area rostral to the anterior commissure, includ- ing the preoptic area, anterior hypothalamus and the hori- zontal limb of the nucleus of the diagonal band. There was no spatial distinction between WSNs and CSNs as well as WSNs showing increased and CSNs showing decreased thermosensitivity during NREM sleep. Both WSNs and CSNs were sometimes found in loose clusters. The thermo-insensitive neurons tested for their thermosensitiv- ity during NREM sleep were intermixed with WSNs and CSNs.

4. Discuss ion

In our previous study we found that most POAH WSNs exhibited increased discharge in NREM sleep compared to wakefulness (sleep-related WSNs) and that most CSNs showed decreased discharge in NREM sleep (wake-related CSNs). In the present study we found that among WSN subgroups, sleep-related WSNs also exhibited increased thermosensitivity in NREM sleep. In contrast, reduced thermosensitivity in NREM sleep was found in WSNs with reduced neuronal activity in NREM sleep and in WSNs showing no change in discharge with sleep-wake state, although the latter changes did not reach statistical signifi- cance. Some neurons identified as thermo-insensitive dur- ing wakefulness became predominantly WSNs in NREM sleep. Among CSNs, all subgroups showed reduced ther- mosensitivity in NREM sleep, but this change achieved statistical reliability only in the wake-related CSNs sub- group. There were few neurons in other subgroups, i.e. wake-related WSNs, sleep-related CSNs and state-indiffer- ent WSNs and CSNs. The failure to find statistical reliabil- ity in changes in the thermosensitivity of these subgroups might be due to small sample size. The change in ther- mosensitivity was correlated with discharge rate change in NREM sleep as compared to wakefulness in WSNs but not in CSNs.

The naturally-occurring increases in mean discharge rate of WSNs and decreases in discharge of CSNs at NREM sleep onset are equivalent to the neuronal re- sponses produced by local POAH warming by 2.0°C [1]. A 2.0°C local POAH warming during wakefulness will read- ily facilitate sleep. Thus, these changes are likely to exert powerful control over sleep-waking states. The changes in thermosensitivity we observed could facilitate and stabilize sleep-wake state changes as follows. The activity or out- put of a thermosensitive neuron at a given temperature is determined by both its thermosensitivity (rate vs. tempera- ture slope) and its 'set-point' ( 'y-intercept' of rate vs. temperature function, see [1]). But at a fixed set-point, increased thermosensitivity of a WSN should tend to in- crease its discharge rate, further increasing its putative hypnogenic and thermolytic effects. Similarly, decreased thermosensitivity in CSNs during NREM sleep should stabilize their lower discharge rates, reducing possible wake-promoting effects of these neurons. Thus, the ther- mosensitivity changes we observed should tend to amplify and stabilize state changes that are facilitated by these neurons. Such a mechanism could account for the relative stability or inertia of sleep and waking states, once they have begun.

Most WSNs and CSNs also exhibited baseline rate changes during NREM sleep compared to waking (see Fig. 1). However, it is unlikely that the changes in thermosensi- tivity we observed were due to baseline effects. The thermosensitivity measurements were based on the propor- tional change compared with baseline discharge rate in

Md.N. Alam et a l . / Brain Research 718 (1996) 76-82 81

each state. Thus, sleep-related WSNs whose baseline rate increased in NREM sleep had to display a greater numeri- cal rate change to achieve: the same thermosensitivity in NREM sleep as in waking. On the other hand, wake-re- lated CSNs whose discharge rate decreased in NREM sleep had to display a smaller numerical rate change to achieve the same thermosensitivity in NREM sleep. How- ever, sleep-related WSNs e, xhibited increased thermosensi- tivity, whereas, wake-related CSNs exhibited a decreased thermosensitivity in NREM sleep as compared to waking. Thus, change in thermosensitivity during NREM sleep was sufficient to overcome these baseline rate effect. More- over, the findings did not depend upon the criteria used to define thermosensitivity, as they were confirmed in neu- ronal populations defined by each of the criteria.

Our thermal response tests were conducted using mild degrees of warming and cooling. Thus, the cells we stud- ied were temperature-sensitive in the near neutral tempera- ture ranges that must be involved in sleep-wake regula- tion.

This study confirms previous findings that, in the unanesthetized animal, PC,AH neuronal thermosensitivity to local temperature changes is strongly modulated by shifts between wakefulness and NREM sleep [10,23]. Pro- gressive changes in thermosensitivity have also been rec- ognized in anesthetized animal [3]. Thermosensitivity can be confirmed in the isolated hypothalamic slice preparation under conditions of synaptic blockade or in dispersed POAH neurons, and can be intrinsic property of neurons. We cannot be certain that our sample included the intrinsi- cally thermosensitive neurons as identified in vitro [13]. However, some evidence favors the argument that our sample included such primary thermodetectors. Most of WSNs and CSNs in our study were responsive to both warming and cooling. That is, in the temperature range we studied, they did not exhihit the response thresholds that might be characteristic of thermoregulatory interneurons (see [2]). Since, in the intact animal, nearly all WSNs exhibited changes in thermosensitivity with state, and some of these are likely to be primary thermodetectors, we can suggest that either the primary mechanisms of thermosen- sitivity can be regulated by state variables, or that there are additive effects of primary mechanisms and state-related modulating factors in determining thermosensitivity.

We found that thermosensitivity was correlated with discharge rate changes in NREM sleep compared to wake- fulness in WSNs, but no1; in CSNs. This suggests that increased discharge in NREM sleep may be coupled to increased thermosensitivity in WSNs. There is some evi- dence that thermosensitivity can be altered by depolarizing processes. In a slice preparation, tonic depolarization by current injection of a temperature-insensitive neuron, lead- ing to an increased discharge rate induced warm-sensitivity [7]. In another study, Na+ /K + pump inhibition by ouabain increased discharge rate and induced warm-sensitivity in an initially thermo-insensifive neuron [6]. Therefore, it

seems reasonable to hypothesize that activation of WSNs during NREM sleep could contribute to increased ther- mosensitivity. The mechanisms underlying the coupling of thermosensitivity and discharge rate are not known.

A number of factors including steroid hormones, glu- cose level, osmotic pressure [4,31], prostaglandins [11,21], cytokines [27], and neuropeptides [26] can influence dis- charge of POAH thermosensitive neurons. Some of these variables exhibit slow cyclic changes in relation to the sleep-wake cycle. It is likely that circadian clock signals also directly modulate thermosensitive neurons. However, we have described discharge rate changes in thermosensi- tive neurons occurring during the waking-NREM sleep transition, in close relationship to the EEG-behavioral state transitions. These changes in thermosensitive neu- ronal activity are occurring within a time frame of several seconds to one min. Slow cyclic processes do not readily explain such relatively rapid events. Among more rapidly changing processes is brain temperature which falls before as well as after sleep onset [5]. It has been shown that cooling increases input resistance of POAH thermosensi- tive neurons [7]. Through this mechanism excitatory input to WSNs and inhibitory inputs to CSNs could have in- creased post-synaptic effects during NREM sleep. There are also dense serotonergic [28] and noradrenergic termi- nals [20] in the POAH. Changes in discharge/thermosensi- tivity of WSNs and CSNs could be secondary to decreased release of monoamines that would be expected at sleep onset [16]. Sleep regulation could involve the integration of two or more of these mechanisms. We can hypothesize that slower cyclic processes play a permissive role by altering thermosensitivity, and that changes in monoamin- ergic inputs to the POAH could have a final sleep-onset triggering role.

Acknowledgements

This research was supported by MH 47480 and the Veterans Administration. The authors are thankful to Ms. M. Mesbahi and L.T. Nguyen for their excellent assis- tance.

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