Thermosensitivity: An intrinsic property of hypothalamic neurons

Pergamon

O:~-4S6S(94)F_~O~

J. therm. Biol. Vol. 19, No. 4, pp. 219-236, 1994 Copyright © 1994 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0306-4565/94 $7.00 + 0.00

REVIEW THERMOSENSITIVITY: AN INTRINSIC PROPERTY HYPOTHALAMIC NEURONS

OF

V. Y. VASILENKO Institute of Physiology, Academy of Sciences of Belarus, Skarina str. 28, 220072 Minsk, Belarus (Received 8 May 1993; accepted in revised form 12 February 1994) Abstract--l. Marked temperature dependence of impulse activity is a property of mammalian neurons which are mainly localized in the hypothalamus and adjoining brain regions. 2. Data is presented on the similarity of thermal behaviour of neurons/n vivo and in vitro. It follows from the evidence on the synaptic blockade, postnatal development, and intracellular recordings that inherent thermosensitivity is a property primarily of warm-sensitive hypothalamic neurons. 3. The Na+/K + pump does not underlie hypothalamic neuron thermosensitivity. Warm-sensitive units possess highly thermosensitive non-inactivating inward sodium current. 4. Data is presented on thermo-induced membrane structural rearrangements based on changes in the physical State of lipids, changes in protein conformation and membrane skeleton activity. A hypothesis of the relation of threshold temperature responses of warm sensitive hypothalamic neurons with these membrane phenomena, as well as alternative hypotheses, are viewed. Key Word Index: Hypothalamus; brain slice; temperature regulation; thermosensitive neuron

THERMOSENSITIVE NEURONS

INTRODUCTION

Criteria f o r thermosensitivity

According to current views, the impulse activity of a nerve cell is determined primarily by its intrinsic properties. Most neurons of the CNS as a whole and the hypothalamic thermoregulatory center in particular are endogenously active, and stimuli only modulate their activity (Ling et aL, 1990; Milburn, 1990; Fidia, 1991). N o w there are reasons to believe that marked thermo-induced changes in the impulse activity of neurons of the thermoregulatory centers are determined by intrinsic neuronal properties as well (at least in cells with a definite type of thermal behaviour). Neural and humoral effects on the thermoregulatory centers are probably realized via synaptic and chemical action on the endogenous mechanism of neuronal thermosensitivity. The present review pays attention to data that directly or indirectly indicate this mechanism. Detailed evidence on nervous connections and functional significance of thermosensitive neurons, as well as on effects of different chemical agents with thermoregulatory action on them can be found in other reviews of the last decade (Nakayama, 1985; Simon et al., 1986; Hori et al., 1987; Boulant et al., 1989; Myers and Lee, 1989).

Thermosensitive neurons comprise cells with increasing firing rate (the number of action potentials per unit time) during increasing (warm-sensitive units) or decreasing (cold-sensitive units) brain tissue temperature. The slope of the thermoresponse curve is most widely used as a criterion of division of neurons to thermosensitive and temperature-insensitive because it can be applied both to the local (true) thermosensitivity of the neuron and its peripheral thermosensitivity due to afferent input from cutaneous thermoreceptors (Boulant et al., 1989). Using Ql0 of the firing rate is often difficult since, as is known, Ql0 expresses the ratio of the rate of a process at elevated temperature to that at initial temperature, but in experiments in vitro many neurons show no impulse activity at initial temperature. In various studies neurons are classified as warmor cold-sensitive if the slopes of their thermoresponse curves are at least from 0.5 to 0.8 or at least from - 0.3 to - 0.6 imp(impulses) s- l°C- l, respectively (Hori et al., 1980a; Kelso et al., 1982; Kobayashi, 1986; Watanabe et al., 1986; Nakashima et al., 1987). 219

220

V.Y. VASILENKO

of firing rates of such warm- and cold-sensitive neurons is considerably higher than 2.0 and lower than 0.5, respectively (Hori et al., 1980a; Nakayama, 1985; Baldino, 1986; Boulant et al., 1989). Some papers (Inenaga et al., 1987; Keenan and Chu, 1987) consider neurons with low thermosensitivity above 0.1 and below - 0 . 1 imp s-J°C -~ as warm- and cold-sensitive cells.

neurons makes about 30, 10 and 60%, respectively (Boulant et al., 1989), though in concrete studies with the same sample sizes and neuron division criteria this ratio strongly varies. Warm-sensitive neurons vary, e.g. from 17% (Hori et al., 1980a) to 63% (Watanabe et al., 1987) and even 80% (Ono et al., 1987). Several groups of thermosensitive neurons with different temperature-activity relationships were revealed in vivo in the PO/AH of endothermic animals (Helion, 1967; Guieu and Hardy, 1971; Boulant, 1974; Belyavsky, 1976). It was shown (Boulant, 1974; Boulant et al., 1989) that warm- and cold-sensitive neurons, the activity of which linearly and nonlinearly depends on a wide range of temperatures, receive inputs from peripheral thermoreceptors and, probably, control heat loss, heat production and heat retention by integrating information about skin and hypothalamic temperature. At the same time, a population of warm-sensitive neurons with the threshold temperature-activity dependence was found. These units which were excitable only during warming with hyperthermic temperatures (over 38°C, rabbit) receive virtually no inputs from peripheral thermoreceptors. It was suggested (Boulant, 1974) that a group of such PO/AH neurons may essentially contribute to the control of the heat loss responses by using information only about hypothalamic temperature. Evidently, the populations of threshold

Ql0

Types o f neurons

Initially thcrmoscnsitive neurons were revealed in the preoptic region and anterior hypothalamus (PO/AH), i.e. in an area belonging to the hypothalamic thermoregulatory center. Nakayama et al. (1961, 1963) in experiments in anesthetized cats found that local thermal stimulation of the hypothalamus not only triggered thermoregulatory responses, as it has been already known, but also changed the firing rates of PO/AH neurons (Fig. 1). Excitation of hypothalamic neurons preceded the occurrence of thermoregulatory responses. Subsequently the thermosensitive PO/AH neurons were studied both in vivo (mainly in rabbits) and in tissue slices (rats, guinea-pigs), tissue cultures and dissociated neurons (rats). Averaging of different data shows that both in vivo and in slice experiments the ratio of warm-sensitive, cold-sensitive and temperature-insensitive PO/AH Discharge frequency i m p ./s

Hypothalamic t e m p . °C

IIII~IUlIIlll I I I I1111 nl$1llllllllJllgl 8.g

14,3

..............

. .........

IIl.llll IlHllllltllllllllllllll ...............

38.0 a

Ii I Ildl

I1 _.dl

H$~$11111111illllllllhlllilill Illil/III 15.7

. . . . .

. . . . . . . . . . . . . . . . .

15 14 13

39,5 c

39.7

--

d

9

IlUlillillillllllilDlilid$39.1 f . . . . . . .

o'°~l

! 20

1o0

",..

f /

80

I'o.

A - D i s c h a r g e rate B - Respiration rate

7

IIIhlllillilllllill Ill tlllllllthlllilllllilllld 39.7 e

9.7 l l l l l l U l l l l h l

g

--7"'""_...-.-" \_ ....t

8

12.6

f

-

~, 12 "~ it -e~

Ililllll '

d

16

3S.7 b

I

. . . . . . . . . . . . . . . .

illHlllltilllltlllll/lllldllli 14.7

b

•~

Hypothalamic

temp.

r

38 8.3

II I1Ill LIII I111111 |111111 II Illllllllllll --'M t s b~---

R.F. Heating

[//l///////////~

38.4 g

L

I

I

I

I

0

1

2

3

4

Time (rain)

Fig. 1. The first evidence for the existence of thermosensitive neurons. Effect of local radio-frequency heating of the anterior hypothalamus on electric activity of single cell and respiration in cat. (From Nakayama et al., 1961.)

60

.-0=

Thermosensitivity warm-sensitive cells found in PO/AH slices (Hori et al., 1980a; Kelso et al., 1982; Kobayashi, 1986; Nakashima et al., 1987; Vasilenko and Gourine 1992b) belong to this group of neurons. Distribution in the brain

It is known that not only PO/AH, but many other diencephalic regions are implicated in thermoregulation. Utilizing horizontal tissue slices, Dean and Boulant (1989b) have recorded single-unit activity throughout the rat diencephalon. Warm- and cold-sensitive cells (thermosensitivities at least of 0.8 and - 0.6 imp s- J°C- l ) were identified in 18 nuclei. The authors have concluded that this wide distribution of thermosensitive cells suggests that many diencephalic areas, besides the PO/AH, are capable of thermoreception and thermointegration and that many of these thermosensitive cells may function in other systems (e.g. reproduction, feeding, and water-balance) which central and environmental temperatures are known to influence. These findings confirm the earlier in vivo and in vitro data on the presence of thermosensitive neurons in regions adjoining the PO/AH, i.e. the paraventricular nuclei, ventromedial and posterior hypothalamus, and septum (Edinger and Eisenman, 1970; Dymnikova, 1973; Zacharzhevskaya, 1974; Belyavsky, 1976; Kushakov, 1977; Inenaga et al., 1987; Imai-Matsumura et al., 1988). The experiments by Dean et al. (1992) show that there is a bi-directional rostral-caudal network linking thermosensitive neurons throughout the rat diencephalon. Some of the neurons displaying remote thermosensitivity (sensitivity to the changing temperature of the remote part of diencephalic slice) were recorded in this study at the lateral border of the diencephalic nuclei, near the median forebrain bundle. Virtually all of these neurons were warmsensitive to local temperature, but cold-sensitive to remote temperature. Thus, in a neuronal network, the synaptically derived thermosensitivity could sometimes compete with the local intrinsic thermosensitivity of the neuron. Besides the diencephalon, thermosensitive units are present in spinal and brainstem regions which are involved in processing information from peripheral thermoreceptors (Wfinnenberg and Briick, 1968; Simon and Iriki, 1971; Nakayama, 1985; Watanabe et al., 1986; Keenan and Chu, 1987). However, even the main thermosensitive area, PO/AH, contains about 60% of temperature-insensitive neurons (Boulant et al., 1989). Several studies were specially aimed at investigating the thermal behaviour of neurons in brain regions that do not affect thermoregulation. The data

221

obtained from cortical neurons are quite contradicting. Adey (1974) observed a decrease in the activity of cortical neurons in response to heating of a local cortical region, while Kozyreva (1972) observed an increase in the activity. Baker and Carpenter (1970) found thermosensitive neurons in the cat sensorimotor cortex, but the authors were changing the temperature of all of the brain supplied by the carotid and noted that alterations in the intensity of synaptic bombardment may contribute to the temperature dependency of spontaneous discharge. In our hippocampal slice study (Vasilenko et al., 1989a) we found neurons in the pyramidal layer, the firing rate of which depended not on the temperature level, but on the rate of temperature change. Later (Vasilenko, 1990) such neurons were found in PO/AH slices. The thermosensitivity of such PO/AH units was markedly higher than that of hippocampal units. In a study by Paton et al. (1991) in slices of the cardiorespiratory region of the medulla oblongata the firing rate of tonically rhythmic neurons linearly increased during warming from 32 to 39.5°C, the thermosensitivities averaged about 0.2imp s-~°C -~ (according to the data presented). Such neurons are considered temperature-insensitive. The above data result from few observations, and so it is not possible to state confidently either the presence or absence of thermosensitive neurons in brain regions not considered to be thermoregulatory centers. This issue, however, seems rather important for understanding the organization of brain thermosensory elements. Additional information

There is evidence that thermosensitivity is a permanent property of a neuron and is constant in different days of observation (20-days' testing) and in different time of a day (Reaves and Heath, 1983). However, according to other data (Parmeggiani et al., 1983; Glotzbach and Heller, 1984), neuronal thermosensitivity correlates with the sleep-awake cycle and decreases in sleep. Besides thermal stimuli, many warm- and coldsensitive neurons respond also to other stimulations, i.e. osmotic changes (Boulant and Silva, 1987; Hori et al., 1987), changes in the blood levels of glucose and steroids (Boulant and Silva, 1987), blood pressure and nonthermal emotional stimuli (Hori et al., 1987), mechanical influences on the skin and auditory stimulus (Dymnikova, 1987). This is suggestive of a possible involvement of PO/AH thermosensitive cells in the coordination of thermoregulation and nonthermal autonomic and behavioral responses controlled from the hypothalamus (Hori et al., 1987). However, there is no direct evidence for the

222

V.Y. VASILENKO

involvement of "multimodal" thermosensitive neurons in the functioning of non-thermal homeostatic systems. Morphological data (Ger6ks et al., 1986) show that the PO/AH of rats consists of medium-size neurons (14-22 micrometers in soma diameter) having 2-4, rarely more, main dendritic shafts. PO/AH neurons have fine axons originating either from one of the stem dendrites or from the pericarion. The maximal lengths of most dendrites are 200-600 micrometers from the cell body (Miilhouse, 1979; Griffin and Boulant, 1991). In studies in PO/AH tissue cultures of mice (Nakayama et al., 1978; Hori and Nakayama, 1982) it was found that cell bodies of warm-sensitive and other neurons had an egg-like shape and 10-17#m in diameter. To our knowledge, there is little convincing morphological evidence on the distinction between thermosensitive and temperatureinsensitive neurons. On the basis of electrophysiological parameters, it has been suggested (Boulant, 1980; Curras et al., 1991) that warm-sensitive neurons have smaller cell bodies compared to temperature-insensitive neurons. Recent morphological study (Griffin and Boulant, 1991) suggests that, unlike temperatureinsensitive neurons, anterior hypothalamic warmsensitive neurons have bipolar dendritic trees oriented medially and laterally, with the lateral dendrites projecting toward the median forebrain bundle. As to phylogenesis of neuronal thermosensitivity, there are data on the presence of thermosensitive nerve cells in the brain of lizard (Cabanac et al., 1967) and fish (Greer and Gardner, 1970; Nelson and Prosser, 1981a, b). Centrally located thermosensitive neurons are thought to be components of a thermoregulatory system in all vertebrates (Nelson and Prosser, 1981a). There is a review (Slonim, 1986) suggesting that thermosensitive brain neurons are phylogenetically more ancient thermoreceptors than peripheral warm and cold receptors. Ontogenetic data are included in the section "Postnatal development" of the next part of the review. EVIDENCE FOR INHERENT NEURONAL MECHANISM OF THERMOSENSITIVITY

The in vitro studies

From the first studies of neuronal thermosensitivity in PO/AH slices (Hori et al., 1980a; Kelso et al., 1982) and tissue cultures (Nakayama et al., 1978; Baldino and Geller, 1982; Hori and Nakayama, 1982) it became evident that warm and cold sensitivities of neurons are not underlain either by thermal and nonthermal nervous inputs to the hypothalamus or systemic humoral changes that are absent in these experimental conditions. In vitro, persistent neuronal thermosensitivity may be either a property of local

synaptic networks or intrinsic property of neurons. In both cases it may not be excluded that PO/AH neuron activity is modulated by thermo-induced release and uptake of various chemical agents by different cells in slices and tissue cultures. The firing rate of neurons in vitro as compared in vivo is decreased, which is characteristic of neurons of different brain regions. This may reflect a decrease in external neural inputs or some unknown deficit of neuronal metabolism which may occur in tissue slices (Hori et al., 1980a). However, both in vivo and in vitro, the same patterns of spontaneous activity in the hypothalamus were revealed. Different hypothalamic neurons in vivo displayed irregular single spikes and bursts (Kushakov, 1977; Paisley and Summerlee, 1985), groups and trains of impulses (Kushakov, 1977), and regular single spikes (Belyavsky, 1976). All these patterns were observed in our PO/AH slice experiments (Vasilenko et al., 1989a). Such patterns (regular and irregular single spikes and bursts) are inherent to different hypothalamic neurons in dissociated culture as well; moreover, as seen from synaptic block tests, spontaneous activity of neurons in dissociated hypothalamic culture is generated through an endogenous mechanism, independent of synaptic excitation (Ling et al., 1990). It follows from the collation of different papers and reviews (Hori et al., 1980a; Kelso et al., 1982; Nakayama, 1985; Kobayashi, 1986; Boulant et aL, 1989) that in vivo and in vitro the PO/AH shows the same quantitative proportions of warm-sensitive, cold-sensitive, and temperature-insensitive units, slope of neuronal thermoresponsive curves, linear, nonlinear, and threshold temperature-activity relationships in different neurons, threshold temperatures and ranges of cell thermosensitivity. Already the first PO/AH slice studies (Hori et al., 1980a; Kelso et al., 1982) found that more thermosensitive neurons with threshold thermosensitivity, i.e. cells set to temperature close to normal brain temperature, are observed in PO/AH slices as compared to intact animals. The authors suggested that this difference may be ascribed to the lack of extrahypothalamic synaptic inputs in slice preparations. If some thermosensitive cells acquire threshold thermosensitivity after elimination of extrahypothalamic inputs, the threshold temperature response is more likely due to the intrinsic neuronal mechanism than nonthreshold temperature responses. This is also seen from the synaptic block testing in slices (Kelso and Boulant, 1982) showing that in some warmsensitive neurons a decrease in the firing rate during blockade was accompanied by a shift in the range of maximum thermosensitivity toward the hyperthermic temperatures (Fig. 2A).

Thermosensitivity

10 - (A)

.

j

8--

i

x

2_ 0

.y¢y

I,,

.D.--,,---,,,lfr"~o

32

-

I

37 Tissue temperature (*C)

5t - (B) 4

x

I 42

oo

0

3

o

3

'

x x 0

2

xx

32

@'~ O"~O--

37 Tissue temperature (*C)

--

42

Fig. 2. The effect of synaptic blockade on the thermosensitivities of warm-sensitive (A) and cold-sensitive (B) preoptic neurons in rat brain slices. Thermoresponse curves marked: 1, O, are from initial control period; 2, ×, are during synaptic blockade; 3, (3, are after return to control medium. (From Kelso and Boulant, 1982.)

Effects o f synaptic blockade

The synaptic blockade in slices and tissue cultures was made using medium containing lowered (range for different studies 0-0.3 mM) calcium and elevated (range 6.5-12 mM) magnesium concentrations (Hori et al., 1980b; Baldino and Geller, 1982; Kelso and Boulant, 1982). In hippocampal slices which were placed in a recording chamber, along with hypothalamic slices, field potentials were completely blocked by 0.2 mM calcium and 11.4mM magnesium medium (Dean and Boulant, 1989a). Nakashima et al. (1987) showed that in hypothalamic slices use of a medium even with slightly higher Mg 2÷ (3.1 mM) with a complete absence of calcium abolished PO/AH neuron responses to electrical stimulation of the adjoining lateral preoptic area. According to a number of studies (Hori et al., 1980b; Baldino and Geller, 1982; Kelso and Boulant, 1982) nearly all warm-sensitive neurons in the PO/AH tend to retain their warm sensitivity during synaptic blockade (Fig. 2A). The decrease in the firing rate in most warm-sensitive neurons in the

223

blocking medium may indicate the effect of excitatory synaptic inputs from nearby warm-sensitive and temperature-insensitive neurons on these cells (Boulant et al., 1989). In our view, however, the decrease in neuronal activity and thermosensitivity in blocking medium is not necessarily underlain by the block of synaptic inputs to a cell. Replacement of calcium for magnesium in the neurons themselves may differently affect the excitability of these neurons (Vasilenko and Gourine, 1992a; Schmid and Pierau, 1993). In connection with this, Schmid and Pierau (1993) who also found the dependence of firing rate and thermosensitivity of PO/AH neurons in slices on calcium ion concentration in the medium even conclude that replacement of calcium for magnesium to distinguish between inherent and synpatically driven neuronal thermosensitivities is not applicable. Undoubtedly, this point of view must be taken into account in cases when blockade-induced changes in neuronal activity or thermosensitivity take place. While warm-sensitive PO/AH neurons, according to most studies, retain thermosensitivity during synaptic blockade the effects of the block on thermosensitivity of cold-sensitive neurons were different in different investigations. Studies using PO/AH slices (Hori et al., 1980b; Nakashima et al., 1987) and tissue cultures (Baldino and Geller, 1982) found the existence of neuronal cold sensitivity during synaptic blockade. In contrast to these results, synaptic blockade eliminated neuronal cold sensitivity in a work by Kelso and Boulant (1982) (Fig. 2B). In their discussion of possible causes of this discrepancy, Boulant et al. (1989) admit that the calcium-free medium used in PO/AH slice study of Hori et al. (1980b) may contribute to unstable or hyperexcitable neuronal membranes (Nakashima et al. (1987) also used such a medium) and that in 3-4-week cultures of preoptic explants from 1-dayold rats (Baldino and Geller, 1982) the types of neurons that develop and their synaptic connections are not representative of neurons recorded in slices. Boulant (1974) suggested that PO/AH coldsensitive neurons are merely interneurons whose thermosensitivity is derived totally from inhibitory synaptic input from nearby warm-sensitive neurons. The data of Kelso and Boulant (1982) suggest that cold-sensitive neurons are actually interneurons. Besides, the authors note that the fact that blockade usually decreased, rather than increased, spontaneous activity suggests that many of these interneurons are driven by excitatory inputs from temperature-insensitive or marginally warm-sensitive neurons. However, the alteration of neuronal excitability due to the replacement of calcium for magnesium may be an

224

V.Y. VASm~NKO

alternative explanation of this effect (Vasilenko and Gourine, 1992a; Schmid and Pierau, 1993). Studies using horizontal slices (Dean and Boulant, 1989a; Boulant et al., 1989) have revealed that, whereas PO/AH cold-sensitive neurons lost their thermosensitivities during blockade, the majority of septal and posterior hypothalamic cold-sensitive neurons retained their thermosensitivity during synaptic block. Thus, inherently cold-sensitive neurons may exist outside the PO/AH. Postnatal development

Along with different actions of synaptic blockade, warm- and cold-sensitive cells also differ ontogenetically. Warm-sensitive neurons are observable in the rat hypothalamus as early as at 1st postnatal day (Hori and Shinohara, 1979), i.e. before enhanced synaptogenesis and differentiation which in the PO/AH last for the first 3-5 postnatal weeks (Reier et al., 1977; Gerrcs et al., 1986). At the same time, cold-sensitive neurons were first visualized only at 8 days after birth (Hori and Shinohara, 1979), i.e. during formation of neuronal network. Warm-sensitive neurons are visible in preoptic tissue culture earlier than cold-sensitive cells as well (Hori and Nakayama, 1982). 1ntracellular recordings

Inherent thermosensitivity of warm-sensitive hypothalamic neurons is demonstrated in intracellular studies which are few due to, primarily, small cell bodies of PO/AH neurons. The first of these works (Nelson and Prosser, 1981a) performed in anesthetized fish revealed two groups of thermosensitive neurons in the preoptic area, i.e. units which appeared to be synaptically driven and endogenously active units. In warm- and cold-sensitive neurons of the 1st group the thermoinduced change in firing rate was underlain by a change in the frequency of postsynaptic potentials. The 2nd group consisted only of warm-sensitive cells which generated regular discharges (Fig. 3A). Local increase of brain temperature accelerated the decay of postspike hyperpolarization and, as a result, increased the firing rate of these cells. The authors concluded that only one type of true thermodetector cells (warm-sensitive neurons of the 2nd group) seems to exist in the preoptic region of the fish brain. Similar data were obtained recently in the rat PO/AH slices (Curras et al., 1991). In this study the thermo-induced increase in the firing rate of warmsensitive neurons was due to a faster rise of pacemaker-like depolarizing prepotentials (Fig. 3B,C) whereas the firing rate of cold-sensitive neurons

correlated with postsynaptic potentials whose frequency depended on temperature. In warm-sensitive cells the pacemaker-like depolarizing prepotential was not dependent on synaptic input, since excitatory postsynaptic potentials were not observed when spike activity was suppressed by hyperpolarizing current injection (Fig. 3B), suggesting that intrinsic mechanisms are responsible for neuronal warm sensitivity. An acceleration of the decay of postspike hyperpolarisation (Nelson and Prosser, 1981a) and faster rise of depolarizing prepotential (Curras et al., 1991) which underlie the excitation of warm-sensitive neurons during warming are probably the same process. Some authors who observed this process (Griffin et al., 1993) stressed that thermo-induced excitation of warm-sensitive hypothalamic neurons was not accompanied by a change in resting membrane potential. However, other data (Nakashima et aL, 1989; Kobayashi and Takahashi, 1993) showed depolarization in this case. In addition to the intrinsic mechanism for thermosensitivity, preoptic warm-sensitive neurons also displayed putative inhibitory postsynaptic potentials (Fig. 3B,C) whose frequency was not affected by temperature, suggesting that some inherently warmsensitive neurons receive synaptic inhibition from temperature-insensitive neurons that do not alter their thermosensitivity (Curras et al., 1991). Synaptically driven activity in different cold-sensitive neurons can be regulated by excitatory postsynaptic potentials from cold-sensitive or temperature-insensitive neurons and by inhibitory postsynaptic potentials from warm-sensitive or temperature-insensitive neurons (Curras et al., 1991). As in cold-sensitive neurons, action potentials of temperature insensitive cells studied by Curras et al. (1991) often were preceded by short duration, rapidly rising prepotentials, whose rates of rise were not affected by temperature. When depolarized, some temperature-insensitive neurons can display warmsensitive characteristics and show increased firing rates during warming (Boulant and Curras, 1989; Boulant, 1991; Curras et ol., 1991). A discussion of this phenomenon will be given in the section "Na+/K + pump". Input resistance is inversely related to temperature in warm-sensitive, cold-sensitive, and temperatureinsensitive neurons in PO/AH slices (Boulant and Curras, 1989; Curras et al., 1991). The above results of intracellular recordings, ontogenetic studies, and synaptic blockade testings show that warm-sensitive PO/AH neurons possess their inherent mechanism of thermosensitivity and do not answer whether cold-sensitive neurons are also inherently thermosensitive.

Thermosensitivity

(A)

225

(B)

(C) 32.6 *

C

36.6 °

C

~

~.~ 0

27

29

Temperature (°C)

41.0 ° C ~

20 m s

20 ms

~

~

20 mV [ 1 O0 m s

Fig. 3. Intracellular activity of three (A, B, C) warm-sensitive preoptic neurons (A--green sunfish, B, C--slices of rat brain). A--thermoresponse curve, intracellular traces; lower trace represents testing by injection of depolarizing current pulse that elicited a somewhat smaller action potential. B, C--intracellular traces; the action potentials are truncated; the arrows indicate inhibitory postsynaptic potentiallike activity; lower trace on B shows activity at 41°C during hyperpolarizing current injection that maintained the membrane potential at -98 inV. (A--from Nelson and Prosser, 1981a; B,C--from Curras et al., 1991.)

IONIC AND METABOLICBASESOF HYPOTHALAMIC NEURONALTHERMOSENSITIVITY N a +/K + p u m p

Carpenter in his review (1981) was inclined to suggest that thermosensitivity of mammalian neurons is not a unique property of neurons involved in thermal sensation or thermoregulation, but results from mechanisms common to all excitable cells. In Aplysia neurons the activity of an electrogenic sodium pump tends to membrane hyperpolarization and decrease the cell discharge with increasing temperature; at the same time, the temperature dependence of the passive permeabilities to sodium and potassium tends to depolarization and increase the cell discharge with increasing temperature as a result of higher Qt0 of Na ÷ permeability than that of K ÷ permeability. The discharge of pacemaker neurons in Aplysia, which are not dependent on the activity of other cells, can be adequately explained by interaction of these two functions (Willis et al., 1974). Recent experiments revealed peculiarities in the mechanisms of thermosensitivity in mammalian PO/AH neurons. PO/AH neuronal warm and cold sensitivities do not appear to rely on the Na+/K ÷ pump, since in

PO/AH slices ouabain (a Na+/K ÷ pump blocker) does not eliminate warm and cold unit sensitivities (Curras et al., 1986; Curras and Boulant, 1989), in contrast to cold sensitivity of Aplysia neurons (Willis et al., 1974; Carpenter, 1981) or mammalian skin cold receptors (Spray, 1974; Pierau et al., 1975). As to temperature-insensitive PO/AH neurons, about a half of these cells showed warm sensitivity when the Na+/K ÷ pump was blocked by ouabain (Curras et al., 1986; Curras and Boulant, 1989). But this ouabain enhancement of warm sensitivity did not occur in temperature-insensitive neurons exposed to elevated magnesium and reduced calcium concentrations, which distinguishes true warm sensitivity from ouabain-induced warm sensitivity. Curras et al. (1991) suggested that the Na÷/K + pump may be used to maintain a hyperpolarization necessary for temperature insensitivity and that blockade of the Na+/K ÷ pump may unmask a voltage-dependent depolarizing current that is thermosensitive, causing increased discharge of temperature-insensitive cells during warming. This suggestion is supported by the appearance of thermosensitive prepotential during warm-induced increasing of the firing rate of temperature-insensitive neurons in conditions of

226

V.Y. VASILENKO

artificial intracellular depolarization (Curras et al., 1991). Cessation of hyperpolarization maintained with the Na+/K + pump may account for a change of some temperature-insensitive neurons to warm-sensitive cells under the influence of the endogenous polypeptide bombesin in hypothalamic slices, which is found by Schmid et al. (1993). The same explanation may hold for the results of a study by Shibata and Blatteis (1991b), where some neurons in hypothalamic slices were temperature-insensitive in a medium aerated with 95% 02 and 5% CO2 but displayed warm sensitivity in a medium gassed with 21% 02, 5% CO2, and 74% N 2. Although the suggestion of the authors that oxygen toxicity due to free radical formation may account for the impairment of neuronal thermosensitivity in 95% O2-medium, may be the correct interpretation of the effect. Ionic permeability: cold-sensitive neurons

The inherent neuronal mechanism of cold transduction may be analogous to that described for spinal motoneurons. Like in motoneurons (Pierau et al., 1969, 1976), K + permeability in cold-sensitive PO/AH neurons may be more thermodependent than Na + permeability (Boulant et al., 1989; Hori, 1991). In this case cooling may significantly decrease K ÷ permeability, which would lead to depolarization and increased firing rate. This supposition has not received direct experimental substantiation. Indirectly, it is confirmed by a more negative reversal potential of warm-activated current in cold-sensitive neurons as' compared to warm-sensitive cells [clamp recordings in PO/AH slices (Kobayashi and Takahashi, 1993)]. The mean values of reversal potential in cold-sensitive neurons ( - 6 4 mV) and in warm-sensitive neurons ( - 4 3 mV) do not coincide with the equilibrium potential for Na ÷ , K + or CI-, suggesting that several different ion species may contribute to reversal potential (Kobayashi and Takahashi, 1993). Kobayashi and Takahashi (1993) showed that temperature changes altered the firing rates of coldsensitive neurons without significantly altering the membrane potential. This restricts a probability of the suggestion that in PO/AH cold-sensitive neurons a more thermodependent K + permeability as compared to Na + permeability leads to depolarization and thus to increased firing rate during cooling. lonic permeability: warm-sensitive neurons

Definite data exist on inherent neuronal mechanisms of warm transduction in the PO/AH. Carpenter (1981) suggests that inherent warm sensitivity of neurons may be underlain by warm-

induced passive depolarization associated with the effect of temperature on the ratio of Na ÷ and K ÷ permeabilities. In warm-sensitive neurons the Q~0 may be higher for Na ÷ permeability than for K + one. Therefore, at higher temperatures there would be relatively more depolarizing Na ÷ current than the hyperpolarizing K + current. We believe that Ca 2÷ current must be also taken into account. Intracellular analysis in slices of different brain regions of the guinea-pig has revealed that activation of a transient, low-threshold calcium current plays a pivotal role ir euronal rhythmogensis (Gutnick and Yarom, l ' ~ ) . In this study the membrane potential of each individual neuron may oscillate rhythmically as a result of interplay between calcium current and other inward and outward voltage- and calcium-dependent currents. Lowthreshold calcium channels were studied in detail in neurons freshly isolated from the hypothalamus of the rat (Akaike et al., 1989). Apparently, the main current with which Ca 2+ current may interplay resulting in rhythmical oscillations in inherently warmsensitive PO/AH neurons is inward sodium current. Recently clear evidence for the involvement of Na ÷ current to hypothalamic neuronal warm sensitivity was obtained in experiments with dissociated PO/AH neurons (Kiyohara et al., 1990). 24% of neurons s~ awed, by the voltage clamp recordings, a highly t. aperature sensitive (within 35-40°C, Qi0 about 6) inward sodium current underlain by the conductivity of tetrodotoxin-sensitive non-inactivating Na + channels (Fig. 4a,b). At lower temperatures the inward sodium current in these ceils was analogous to that recorded in the other cells which displayed a weak (Q~0 about 2) linear dependence in a wide temperature range (Fig. 4c). The similarities in the magnitudes of thermosensitivities in this study and in vivo or in vitro PO/AH neuronal thermosensitivity studies, as well as in the shapes of the thermal response curves and in the percentage of neuronal groups revealed suggest that the 76% neurons studied with linear temperature-current relationship and 24% cells with threshold temperature-current relationship (Fig. 4d) correspond to temperature-insensitive and warmsensitive neurons, respectively (Kiyohara et aL, 1990). This conclusion appears to be rather true although the study does not contain direct evidence that the two groups of neurons revealed are actually warm-sensitive and temperature-insensitive cells. These data also show that in temperature-insensitive neurons one of the main ionic currents underlying the generation of action potentials linearly depends, to a small extent, on a wide range of temperatures. This indicates that it is more reasonable to identify neurons as thermosensitive having

Thermosensitivity

227

(a)

o

34

~

28

I

36

\

32 18t-

t-

18 -

"~: . . . . . . . . . . . . .

~

~

~

_

~

•••f•a

0.1 nA

I nA

V hold: -80 mV

(b)

30 s

(d) 0.3

~

34

~

28 18

:::

]

8

_

~'-a~''a

0.1 nA V hold: -80 mV

1 min

0

I

I

I

1

I

I

30

32

34

36

38

40

T c (°C) Fig. 4. Changes in steady inward Na ÷ currents of two PO/AH neurons during changes in cellular temperature (T¢). (a,b) Changes in Na + current of a PO/AH neuron during continuous and step changes in T c. (c) Changes in Na ÷ current of the other neuron. (d) Thermal response curves of these two neurons showing changes in steady inward Na ÷ currents (AI) as a function of Tc. Data were taken from a, b and c. (From Kiyohara et al., 1990.)

not a low but high temperature dependence of firing rates, as well as neurons with threshold temperature responses. There are data on the effect of osmotic stimulation on sodium current in isolated neurons of anteroventral region of the third ventricle (Inenaga et al., 1989). The c o m m o n mechanism of sensitivity of Na ÷ channels to temperature and osmosis in some hypothalamic neurons may be quite a basis of interactions of the two homeostatic systems at the neuronal level. Recently in identified neurons of Aplysia it was shown (Sawada et al., 1991) that extracellular ejection of interleukin- 1~ induces outward current, sensitive to changes in extracellular concentrations of N a ÷ but not K ÷ , Ca 2+ , and C I - . The authors suggest that this current is associated with a decrease in sodium conductance and that interleukin-la may directly affect permeability of neuronal membrane. It is probably just this effect that is responsible for inhibition of P O / A H warm-sensitive neurons in response to different pyrogens (references see in: Nakayama, 1985; Hori et al., 1987), including recombinant interleukinla (Shibata and Blatteis, 1991a; Xin and Blatteis, 1992). Kiyohara et al. (1990) who discovered the highly thermosensitive non-inactivating sodium current in

dissociated preoptic neurons noted that these neurons may possess Na ÷ channels similar to threshold channels. C o m p a r e d with the " n o r m a l " N a ÷ channels, the threshold N a ÷ channels are activated at relatively negative voltages and close very slowly once opened (Gilly and Armstrong, 1984). Threshold Na ÷ channels are thought (Gilly and Armstrong, 1984) to be important in squid axon for phenomena like repetitive firing, and for pacemaking behaviour in autorhythmic cells if these channels are present in them. Probably, high thermosensitive non-inactivating Na + current in some preoptic neurons may also result from disordered inactivation.

PROSPECTS OF STUDIES O~ THE MECHANISMS OF HYPOTHALAM1C NEURONAL THERMOSENSITIVITY

"Working" idea o f the mechanism o f temperature transduction by hypothalamic neurons Undoubtedly, one should agree with Carpenter (1981) in that there are mechanisms of thermosensitivity c o m m o n for all excitable cells. Probably, the unique character of temperature transduction by hypothalamic thermosensitive neurons results from evolutionary development of these c o m m o n mechanisms.

228

V.Y. VASILENKO

The Na÷/K ÷ pump which hyperpolarizes and inhibits invertebrate neurons (Willis et al., 1974; Carpenter, 1981) and mammalian cold skin receptors (Spray, 1974; Pierau et al., 1975) during warming probably determines a difference between thermal behaviour of warm-sensitive and temperature-insensitive neurons in the vertebrate hypothalamus, maintaining hyperpolarization in the latter cells that counteracts the occurrence of warm sensitivity (Curras et al., 1991). Warm sensitivity of hypothalamic neurons is due to the presence of pacemaker-like depolarizing potentials in warm-sensitive units (Curras et al., 1991) which are similar to thermosensitive pacemaker potentials observed in a variety of excitable cells (Sperelakis, 1970; Willis et al., 1974). Differences in temperature dependence of membrane permeabilities for different ion species found in invertebrate neurons (Willis et al., 1974; Carpenter, 1981) and in mammalian motoneurons (Pierau et al., 1969, 1976) may underlie the warm and cold sensitivities of PO/AH neurons (Carpenter, 1981; Boulant et al., 1989; Kobayashi and Takahashi, 1993). In general, the mechanism of temperature transduction by a PO/AH warm-sensitive neuron may be described as follows. During warming of brain tissue when threshold temperature is reached membrane permeability for Na + of a warm-sensitive neuron increases (Kiyohara et al., 1990). This leads to accelerated decay of postspike hyperpolarization (Nelson and Prosser, 1981a) and accelerated rise of depolarizing prepotential (Curras et al., 1991) (perhaps it is the same process). According to Griffin et al. (1993), there is no depolarization in this case, but by other data (Nakashima et al., 1989; Kobayashi and Takahashi, 1993) there is a steady depolarization of neuronal membrane. In any case, the changes observed result in a sharp increase of neuronal firing rate in a suprathreshold temperature range. Cold-sensitive PO/AH neurons may not possess their inherent mechanism of cold transduction. They may be excited by possible abolition of inhibitory synaptic control from warm-sensitive neurons during cooling (Boulant, 1974) or by cold-induced increase in transmitter release (Curras et al., 1991). Combination of inherent and synaptically driven thermosensitivities is also possible, that is a higher input resistance of neuronal membrane during cooling leads to increased amplitude of excitatory postsynaptic potentials (according to Ohm's law) initiated by inputs from temperature-insensitive and other cold-sensitive neurons (Boulant et al., 1989; Curras et al., 1991). If hypothalamic coldsensitive neurons actually possess their inherent

mechanism of thermosensitivity the mechanism may be related to a prevalence of Ql0 permeability for K ÷ over Q~0 permeability for Na ÷ in coldsensitive cells. In this case, cooling would essentially decrease K ÷ permeability, which would lead to depolarization and excitation of neuron (Boulant et al., 1989). As it has been noted, some data show that (i) many of PO/AH warm- and cold-sensitive neurons receive inputs from peripheral thermoreceptors, (ii) some warm-sensitive units with linear temperature dependence in wide temperature range acquire threshold warm sensitivity after elimination of extrahypothalamic inputs in tissue slices, and (iii) a shift in the range of maximal thermosensitivity toward the hyperthermic temperatures during synaptic blockade in some warm-sensitive neurons in PO/AH slices is observed. All these findings indicate that interaction between inherent and synaptically driven thermosensitivities determines the thermoregulatory functioning of these neurons and that synaptic inputs often transform inherent threshold neuronal thermosensitivity to linear thermosensitivity. However, inherent thermosensitivity of PO/AH neurons is indispensible for their thermoregulatory behaviour [it was shown that PO/AH temperature-insensitive neurons receive virtually no inputs from skin thermoreceptors (Boulant and Hardy, 1974)]. There are reasons to believe that the inherent threshold thermosensitivity in many PO/AH warm-sensitive neurons, unlike above mentioned neurons, determines their thermal behaviour and is not essentially modulated by synaptic inputs. Experimental findings which support this conclusion are as follows: (a) the in vivo existence of warmsensitive hypothalamic neurons with threshold thermosensitivity (thermosensitivity only in hyperthermic temperature range) (Hellon, 1967; Guieu and Hardy, 1971; Boulant, 1974; Belyavsky, 1976; Kushakov,. 1977) including neurons precisely set to temperature close to normal brain temperature (Dymnikova and Chernova, 1990; Ivanov, 1990), (b) such warm-sensitive hypothalamic neurons do not receive inputs from skin thermoreceptors (Boulant, 1974), and (c) warm-sensitive neurons in PO/AH slices, unlike cold-sensitive cells, do not possess excitatory postsynaptic potentials and display pacemakerlike fluctuations of membrane potential (Curras et al., 1991). The next step of deepening our insight to the nature of neuronal thermosensitivity is the search for cellular mechanisms underlying the thermo-induced changes in ionic permeabilities of membranes of warmsensitive and, possibly, cold-sensitive hypothalamic neurons. At present we are able only to hypothesize these mechanisms.

Thermosensitivity M e m b r a n e structure rearrangements hypothesis

It is well known that phospholipid bilayers undergo a highly cooperative phase transition in a narrow temperature range, usually 1-2°C (for example, see: Lee and Chapman, 1987). Biological membranes, being composed of a mixture of lipid species, do not undergo a single sharp phase transition. However, on heating and cooling, phase separation can occur which result in the coexistence, over a particular temperature range, of distinct lipid phases of differing chemical composition (Lee and Chapman, 1987). The results of many studies and his own speculations have made Macdonald (1990) conclude that bilayer fluidity has a minor role in the electrophysiology and biochemistry of the Na + and other electrically-gated ion channels. At the same time, data of some papers indicate that such a conclusion can not be probably true for all cases. It is shown that a change in the composition of lipids and their components in membrane of different cells affect the kinetics of Na ÷ current (Spiegel et al., 1986; Hall et al., 1987; Carpenter et al., 1988; BiJsselberg et al., 1989). Evidence from mammalian diencephalic neurons is especially interesting in this respect. Kubarko and Tsaryuk (1986) revealed the effect of phosphatidylcholine liposome infusion to the rabbit hypothalamus on body temperature and impulse activity of hypothalamic neurons. According to Park and Ahmed (1992), the prevailing presence either of linolenic or linoleic acid in diencephalic neuron membranes of rats--both fatty acids are known to be taken in with food (for example, see: Bourre et al., 1990)--considerably changes the time of the increase of Na ÷ current and some other parameters of its kinetics. It is just time dependence of ionic conductance that is the most temperaturedependent parameter of Na ÷ current kinetics (see: Macdonald, 1990). The effect of a charge of headgroup on the Na ÷ channel conductivity and Na ÷ current kinetics is provided both by experimental (Cukierman et al., 1988) and theoretical (Krueger, 1989) investigations. Close examination of ionic interactions with the channel inactivation ball ("ball and chain" inactivating mechanism in K ÷ channel) shows that gating represents the physical plugging of the cytoplasmic face of the pore and that nonspecific electrostatic forces focus this positively charged domain into its pore-blocking site (reference see in: Miller, 1992). The above data are suggestive of a possible effect of membrane lipid composition and bilayer fluidity

229

largely determined by this composition on the permeability of the membrane ionic channels. The permeability of ionic channels is also regulated by cytoskeleton. Experimental evidence exists for Na ÷ channels (Vassilev and Kanazirska, 1990; Cantiello et al., 1991; Cook, 1991). The possibility of the involvement of actomyosin-like proteins in "membrane events" including sensory transduction was first proposed by C. J. Duncan in 1967 (reference see in: Bowler and Duncan, 1967). The cells possess the so-called membrane skeleton (references see in: Luna and Hitt, 1992) as well as subaxolemmal cytoskeleton probably involved not in axonal transport but in cell electrical excitability (Matsumoto et al., 1989). The existence of special stretch-activated ion channels whose functioning is probably associated with cytoskeletal structures is well known, too (see review: Sachs and Sokabe, 1990). A direct interaction between actin and membrane lipids is shown (St-Onge and Gicquaud, 1989). Membrane functions are regulated not only by lipid fluidity and membrane skeleton activity but also by membrane protein-lipid interactions (Cossins and Raynard, 1987; Bowler, 1987; Lee and Chapman, 1987; Yeagle, 1989; Eze, 1990; Behan-Martin et al., 1993) as well as protein-cytoskeletal interactions (Lee and Chapman, 1987). It is thought that membrane lipid-protein-cytoskeleton interactions may play an important role in mobilization and aggregation of erythrocytic intramembrane particles (Ferretti et al., 1990), in prevention of ishemia-induced death of brain cells through the effect on ionic conductivity (Kogure, 1988), and in thermosensitivity of mammalian cells (Konings and Ruifrok, 1985). Konev (1987) believe that the conformation-charge state of membrane elements, including lipids, can regulate gating of Na ÷ and other ionic channels, as well as change the size or number of spaces in the bilayer and at the lipid-protein interfaces. According to Konev (1987), the biological membrane is a continuous solid-resilient protein network filled with the lipid bilayer. This is a system of membrane proteins with spectrin-type protein associates and microfilaments and microtubules. Konev's view (Konev, 1987) holds that the membrane is in a tense state due to interaction between the cytoskeleton and the bilayer. Membrane tension is maintained by electrical and osmotic potentials which, in their turn, depend on membrane structural organization. The tensioned membrane can be considered to be in a local metastable minimum of potential energy, and so can easily "roll down" locally to a condition with a lowered reserve of free energy. Each sublevel of the metastable state has its own particular molecular packing in the lipid bilayer and configuration of

230

V.Y. VASILENKO

resilient-elastic protein cytoskeleton with degrees of freedom in placing elements (turns, straightening of folds, etc.). Switches between sublevels of the metastable state occur during critical changes in temperature, pH, and ligand concentrations. Thus, according to the concept of Konev, cellular functions are regulated by cooperative structural rearrangements of membranes, resulting in stepfunction changes in the activity of membrane and cell, as well as by other established processes, such as modulation of enzyme activity. The cooperative membrane rearrangements are most observable during temperature changes (Konev, 1987). These ideas of Konev are based on his own experimental material, indicating that cooperative structural rearrangements of cell membrane may participate in the aggregation of fibroblasts, proliferation and differentiation of cells, photobiological responses, and cell responses to action of various ligands. The data of other authors show that it is structural changes in membranes that finally lead to changes in ionic permeability of receptor cell membranes in visual, olfactory, and gustatory organs and to the appearance of electrical impulse [references in: Konev (1987)]. The involvement of specific change of lipid phasic state and protein conformation in thermoreception was suggested earlier (Gourine, 1980; Antonov, 1982; Konings and Ruifrok, 1985). It was shown (Tsaryuk and Kubarko, 1989) that temperature of endothermal peak of brain lipid phase transition is close to normal brain temperature and correlates with experimental body temperature changes, as well as with changes in temperature thresholds of excitation of hypothalamic neurons responding to general heating of the animal. In our PO/AH slice experiments (Vasilenko et al., 1989a, b; Vasilenko and Gourine, 1992a, b) threshold temperature responses of neurons were studied during temperature changes, analogous to natural slow low-amplitude brain temperature changes (Abrams and Hammel, 1964; Rawson et al., 1965; Meisenberg and Simmons, 1984; Atkins and Dinarello, 1985; Kruk et al., 1985). Threshold warmand cold-sensitive neurons (42% cells studied) had precise and stable threshold temperatures in the range about 36-39°C for different units (Fig. 5A,B) (Vasilenko and Gourine, 1992b). The difference between response thresholds in repeated temperature stimulations averaged 0.1°C. This is consistent with data obtained in experiments in unanesthetized animals, suggesting that some hypothalamic neurons are capable of responding to brain temperature changes of about 0.1-0.05°C (Ivanov, 1990). It was shown in unanesthetized rabbits (Dymnikova and Chernova, 1990) that there are neurons in the posterior

hypothalamus the firing rate of which significantly correlates with spontaneous changes in hypothalamic temperature within the range of about 0.15°C. The scatter in response thresholds for different neurons within 1.5-2°C observed in vivo (Hellon, 1967; Guieu and Hardy, 1971; Belyavsky, 1976) and in PO/AH slices (Kelso et al., 1982; Kobayashi, 1986; Vasilenko and Gourine, 1992b) may provide a gradual increase in total activity of warm- or cold-sensitive neurons during deviation of brain temperature from the normal level. Probably, it is PO/AH neurons precisely set to temperature with a small scatter of threshold temperatures in different units that maintain set point and provide narrow-band control of temperature. Our experiments in PO/AH slices of guinea-pig (Vasilenko and Gourine, 1992b) also found the stabilization of neuronal firing rate at a high level after exceeding the threshold, on average, only by 0.8°C (in some neurons only by 0.2°C). This new level of activity did not change further as temperature rose above the threshold (Fig. 5C). Nor did it do so during a prolonged maintenance of the suprathreshold temperature (Fig. 5D). Thermosensitivities in such responses in a narrow temperature range are several times higher than those established earlier for hypothalamic neurons in other papers which used only fast and high-amplitude temperature changes. The threshold character of the responses studied (excitation and inhibition of a cell at a fixed temperature) and the existence of two levels of firing rate with a transition process between them in a narrow suprathreshold temperature range are indicative of a possible association of these responses with structural rearrangements of neuronal membrane. The parameters of these responses of PO/AH neurons meet the experimentally revealed (Konev, 1987) criteria of membrane structure rearrangements (Vasilenko et al., 1989b; Vasilenko and Gourine, 1992a, b). These criteria are: the occurrence of step changes in the functional activity; sigmoid shape and narrowness of transitions in temperature-effect curves; the appearance of variations in the values of a structuralsensitive parameter at the point of semitransition (the latter criterion may be related to the appearance of trains or neuronal impulses between the absence and continuous generation of impulses in a close-tothreshold temperature range in most of neurons. (Fig. 5C,D). The virtually instant response to "temperature puncture" (CO2-1aser radiation) of neurons in PO/AH slices with a wide-range thermosensitivity and 15-30 s latency response to the same stimulus of threshold warm-sensitive cells (Fig. 5E) (Vasilenko and Gourine, 1992b) also indirectly show a more considerable degree of cooperativity of processes

Thermosensitivity underlying the threshold thermosensitivity of hypothalamic neurons. There is no direct evidence for the involvement of structure membrane rearrangements in the mechanism of threshold temperature response of hypotbalamic neuron. Besides, it must be noted that the idea of such rearrangements (Konev, 1987) follows from

data obtained in isolated membrane preparations, shadow red disks, artificial lipid bilayers. The absence o f intravital observations of such structural transformations in the membranes of intact cells has not permitted, so far, to consider the well-studied phase transitions and phase separations o f lipids and conformational changes of proteins as properties of

38.1

3,t

o~

(A)

36

38.1 °C

'

Hill

4 o

231

II

.... ~uillllll

I~

111111

fill II I I

11

I I

60 s (B)

38] P

36.5

36.7

36.5

36.7 36.7 °c 36.6

36

II 7

j

L

~'11 lull

i

3

O. ot

(C)

2.5]

36.2

37.0

J

37.3

38.9 oc ,

, I

%

Nil

[ ] i [] n i

Ilnlilllillliillllllliinilim

1o s (D) 37.5

37.7

38.0 °C

2.5] %

,

3 o

II BI

0

10

20

Min

38 I II II

4

~ o

I Ill ....

'

....

LIB

mi 9i

----._. 10 s

Fig. 5. Activity of five (A-E threshold thermosensitive neurons in PO/AH slices during thermal stimulations (B--cold-sensitive neuron, the remaining neurons are warm-sensitive; E--neuronal activity during CO2-1aser radiation). Temperature change, generation of impulses and change in integrated firing rate are shown. The trace of the temperature recordings was re-set at 37.0°C (C) and at 37.5°C (D) to keep the writing pen within the recording range. Dashed line (in E) notes temperature threshold. (From Vasilenko and Gourine, 1992b.)

232

V.Y. VASILENKO

assemblies of protein and lipid molecules functioning in the living membrane. No doubt, it restricts but is far from excluding a possible involvement of structural changes, comprising the whole membrane or its large areas and leading to a sharp increase in sodium permeability, in the threshold temperature response of hypothalamic warm-sensitive neuron. This suggestion (Vasilenko et al., 1989b; Vasilenko and Gourine, 1992a, b) relates primarily to warm-sensitive PO/AH cells. The same phenomenon may also occur with temperature-insensitive neurons but its influence on resting membrane potential and firing rate in these cells may be counterbalanced by Na+/K ÷ pump effect. Cold-sensitive PO/AH neurons, as noted above, may be controlled by warm-sensitive cells and are excitable in this case as a result of cold-induced abolition of inhibitory synaptic influences from warm-sensitive neurons. If the proposed hypothesis is valid, the thermoregulatory function of threshold hypothalamic neurons may be presented as follows (Vasilenko, 1991). Deviation of the hypothalamic temperature from normal level causes structural changes in membranes of warm-sensitive neurons, resulting in step increases in their firing rates (the firing rates of cold-sensitive neurons synaptically driven from these warm-sensitive neurons are changes oppositely). Total excitation of these thermosensitive neurons may be enough for rapid normalization of the temperature. If it is not enough and hyper- or hypothermia last quite long the hypothalamic tissue may undergo alterations affecting the structure of thermosensitive cell membranes (composition of synthesized lipids may change or a certain chemical agent may appear). In such modified membranes thermo-induced rearrangements would be caused at temperatures 1-2°C different from normal hypothalamic temperature, i.e. thresholds of excitation of thermosensitive neurons would change by 1-2°C. As a result, the temperature is set at a level of new thresholds, and hyper- or hypothermia develops. Alternative hypotheses

The sharp temperature response is known to be underlain by processes which require high energy of activation. As a rule, these are cooperative processes. However, cooperativity which a sharp thermoinduced change of firing rate of warm-sensitive PO/AH neuron is associated with may be realized not only at the level of aggregation of membrane molecules to large assemblies. Formation of small local assemblies of lipid or protein molecules which affect the conductivity of ionic channel or make conditions for formation of a membrane pore is also possible. Cooperativity may also manifest itself as aggregation

of warm-sensitive neurons to synchronously activating assemblies, which would be an additional factor promoting their sharp thermo-induced excitation. The threshold temperature response of warmsensitive neuron may not be necessarily underlain by structural changes in neuronal membrane. High energy of activation may be associated with the appearance in the neuron of a bioactive substance with an appreciable difference between the rates of its synthesis and destruction, as well as with the exit of bioactive substance hardly permeable through a membrane from the neuron. Oxidative phosphorylation can also participate in the mechanism of neuronal temperature response. It was shown (Kobayashi and Takahashi, 1993) that in PO/AH warm-sensitive neurons intracellular application of ATP noticeably shifts the reversal potential of warm-activated current in the negative direction. The authors suggest that phosphorylation of K + channels enhances their thermosensitivity, thus shifting the reversal potential to the negative direction and decreasing the response of warm-sensitive neuron to warming. Such a process is indirectly confirmed by a decrease in the thermosensitivity of warm-sensitive PO/AH neurons by cytokines and arachidonic acid, probably, via an intracellular modulation mechanism (Hori et al., 1988). Finally, the membrane of warm-sensitive PO/AH neuron may have specific ionic channels, e.g. lowthreshold sodium channels (Gilly and Armstrong, 1984), as it was suggested by Kiyohara et al. (1990), which are more sensitive to temperature change than the channels in membranes of temperature-insensitive neurons. Warm-sensitive neuron may also be specifically distinguished by a thermo-induced disorder of the ionic channel inactivation mechanism. CONCLUSION

The problem of mechanisms of temperature sensitivity of hypothalamic neurons is gradually growing beyond the field of thermophysiology. The property of thermo-induced conductivity of thermosensitive neuron membrane found recently may become both the subject and the tool of studying the regularities of ionic channel function. Perhaps, there is an urgent necessity for a search of approaches to coupling of investigations of thermo-induced changes in membrane structure and thermal behaviour of neurons. Thermosensitive PO/AH neuron may be used as an object of study of cell chemical cycles underlying a sharp change in the electrical state of biomembrane. The advance in studies of thermoregulatory functioning of warm- and cold-sensitive hypothalamic neurons is associated primarily with identification of

Thermosensitivity m e c h a n i s m s o f shift o f t e m p e r a t u r e thresholds o f excitation in P O / A H nerve cells precisely set to temperature. W e believe t h a t the hyper- a n d hypot h e r m i a d e v e l o p m e n t is underlain just by regulation o f these thresholds. Investigations in this direction c a n be assisted by the rich experimental material s u m m a r i z e d in a n u m b e r o f detailed reviews ( N a k a y a m a , 1985; S i m o n et al., 1986; Hori et al., 1987; M y e r s a n d Lee, 1989) which indicates the effects o f chemical substances with t h e r m o r e g u l a t o r y action o n w a r m - a n d cold-sensitive neurons.

Acknowledgements--The author is thankful to Professor V. N. Gourine and to Professor G. E. Dobretsov for the useful discussion of the problem reviewed. REFERENCES

Abrams R. and Hammel H. T. (1964) Hypothalamic temperature in unanesthetized albino rats during feeding and sleeping. Am. J. Physiol. 206, 641-646. Adey W. R. (1974) Biophysical and metabolic bases of cooling effects on cortical membrane potentials in the cat. Exp. Neurol. 42, 113-140. Akaike N., Kostyuk P. G. and Osipchuk Y. V. (1989) Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J. Physiol. 412, 181-195. Antonov V. F. (1982) Lipids and Ionic Permeability of Membranes, p. 151. Nauka, Moscow (in Russian). Atkins E. and Dinarello C. A. (1985) Reflections on the mechanism of the biphasic febrile response and febrile tolerance. In Physiol., Metab. and Immunol. Act. Interleukin-I Proc. Symp., pp. 97-106. New York. Baker J. L. and Carpenter D. O. (1970) Thermosensitivity of neurons in the sensorimotor cortex of the cat. Science 169, 597-598. Baldino F. (1986) Norepinephrine suppression of neuronal thermosensitivity. In Homeostasis and Ther. Adv. 6th Int. Symp. Pharmacol. Thermoregul., Jasper, Alta, Aug. 26-31, 1985, pp. 99-102. Karger, Basel. Baldino F. and Geller H. M. (1982) Electrophysiologlcal analysis of neuronal thermosensitivity in rat preoptic and hypothalamic tissue cultures. J. Physiol. 327, 173-184. Behan-Martin M. K., Jones G. R., Bowler K. and Cossins A. R. (1993) A near perfect temperature adaptation of bilayer order in vertebrate brain membranes. Biochim. Biophys. Acta 1151, 216-222. Belyavsky E. M. (1976) On neuronal organization of thermosensory area of the anterior hypothalamus. Fiziol. zhurn. SSSR 62, 175-181 (in Russian). Boulant J. A. (1974) The effect of firing rate on preoptic neuronal thermosensitivity. J. Physiol. 240, 661-669. Boulant J. A. (1980) Hypothalamic control of thermoregulation: neurophysiologlcal basis. In Handbook of the Hypothalamus (Edited by Morgane P. J. and Panksepp J.), Vol. 3, part A, pp. 1-82. Dekker, New York. Boulant J. A. (1991) Properties of hypothalamic temperature sensitive and insensitive neurons. In Regional Meeting of the International Union of Physiological Sciences: Abstr. (Edited by Turek S., Mare~ovfi D. and Stastn~' F.), p. 167. Prague. T B 19/4~B

233

Boulant J. A. and Curras M. C. (1989) Intracellular analysis of inherent and synaptic activity in hypothalamic thermosensitive neurons. In Thermal Physiological Symposium: Abstr. Satel. Symp. X X X I Int. Congr. Physiol. Sci., p. 4. Troms~. Boulant J. A., Curras M. C. and Dean J. B. (1989) Neurophysiologlcal aspects of thermoregulation. In Advances in Comparative and Environmental Physiology (Edited by Wang L. C. H.), VoL 4, pp. 117-160. SpringerVerlag, Berlin. Boulant J. A. and Hardy J. D. (1974) The effect of spinal and skin temperatures on the firing rate and thermosensitivity of preoptic neurones. J. Physiol. 240, 639-660. Boulant J. A. and Silva N. L. (1987) Interactions of reproductive steroids, osmotic pressure, and glucose on thermosensitive neurons in preoptic tissue slices. Can. J. Physiol. Pharmacol. 65, 1267-1273. Bourre J.-M., Dumont O., Piciotti M., Pascal G. and Durand G. (1990) Control of brain fatty acids. Upsala J. Med. Sci., Suppl. 48, 109-131. Bowler K. (1987) Cellular heat injury: are membranes involved? SEB Symposium 41, Animal Cells and Temperature (Edited by K. Bowler and B. J. Fuller), pp. 157-185. COB, Cambridge. Bowler K. and Duncan C. J. (1967) Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells. J. Cell. Physiol. 70, 121-125. Bfisselberg D., Evans M. L. and Carpenter D. O. (1989) Effects of exogenous ganglioside and cholersterol application on excitability of Aplysia neurons. Membrane Biochem. 8, 19-26. Cabanac M., Hammel H. T. and Hardy J. D. (1967) Temperature sensitive units in lizard brain. Science 158, 1050-1051. Cantiello H. F., Jennifer L. S., Adriana G. P. and Dennis A. A. (1991) Actin filaments regulate epithelial Na + channel activity. Am. J. Physiol. 261, C882-C888. Carpenter D. O. (1981) Ionic and metabolic bases of neuronal thermosensitivity. Fed. Proc. 40, 2808-2813. Carpenter D. O., Hall A. F. and Rahmann H. (1988) Exogenous ganglioside induce direct voltage and conductance changes on isolated neurons. Cell Mol. Neurobiol. 8, 245-250. Cook J. S. (1991) Regulation of epithelial Na + channels by the cytoskeleton. NIPS 6, 199. Cossins A. R. and Raynard R. S. (1987) Adaptive responses of animal cell membranes to temperature. SEB Symposium 41, Animals Cells and Temperature (Edited by K. Bowler and B. J. Fuller), pp. 95-111. COB, Cambridge. Cukierman S., Zinkand W. C., French R. J. and Krueger B. K. (1988) Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers. J. Gen. Physiol. 92, 431-447. Curras M. C. and Boulant J. A. (1989) Effects of ouabain on neuronal thermosensitivity in hypothalamic tissue slices. Am. J. Physiol. 257, R21-R28. Curras M. C., Dean J. B. and Boulant J. A. (1986) Effects of ouabain on neuronal thermosensitivity in hypothalamic tissue slices. Fed. Proc. 45, 406. Curras M. C., Kelso S. R. and Boulant J. A. (1991) Intracellular analysis of inherent and synaptic activity in hypothalamic thermosensitive neurones in the rat. J. Physiol. 440, 257-271. Dean J. B. and Boulant J. A. (1989a) Effects of synaptic blockade on thermosensitive neurons in rat diencephalon in vitro. Am. J. Physiol. 257, R65-R73.

234

V.Y. VASILENKO

Dean J. B. and Boulant J. A. (1989b) In vitro localization of thermosensitive neurons in the rat diencephalon. Am. J. PhysioL 257, R57-R64. Dean J. B., Kaple M. L. and Boulant J. A. (1992) Regional interactions between thermosensitive neurons in diencephalic slices. Am. J. Physiol. 263, R670-R678. Dymnikova L. P. (1973) Impulse activity of posterior hypothalamic neurons during changes in skin and brain temperature in unanesthetized rabbits. Neirofiziologiya 5, 490-496 (in Russian). Dymnikova L. P. (1987) Response of thermoregulatory center neurons to stimuli of different sensory modalities. Fiziol. zhurn. SSSR 73, 547-549 (in Russian). Dymnikova L. P. and Chernova M. D. (1990) Impulse activity of thermoregulatory center neurons in conditions of thermoneutral zone. Fiziol. zhurn. SSSR 76, 789-794 (in Russian). Edinger H. M. and Eisenman J. S. (1970). Thermosensitive neurons in tuberal and posterior hypothalamus of cats. Am. J. Physiol. 219, 1098-1103. Eze M. O. (1990) Consequences of the lipid bilayer to membrane-associated reactions. J. Chem. Educ. 67, 17-20. Ferretti G., Tangorra A. and Curatola G. (1990) Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects. Exper. Cell Res. 191, 14-21. Fidia research foundation neuroscience award lectures, Vol. 4, 1988-1989 (1991). Neurochem. Int. 19, 619-620. Ger6cs K., R6thelyi M. and Hahisz B. (1986) Quantitative analysis of dendritic protrusions in the medial preoptic area during postnatal development. Dev. Brain Res. 26, 49-57. Gilly W. F. and Armstrong C. M. (1984) Threshold channels--a novel type of sodium channel in squid giant axon. Nature 309, 448-450. Glotzbach S. F. and Heller H. C. (1984) Changes in the thermal characteristics of hypothalamic neurons during sleep and wakefulness. Brain Res. 309, 17-26. Gourine V. N. (1980) Central Mechanisms of Thermoregulation, p. 127. Belarus, Minsk (in Russian). Greer G. L. and Gardner D. R. (1970) Temperature sensitive neurons in the brain of brook trout. Science 169, 1220-1222. Griffin J. D. and Boulant J. A. (1991) Physiological and morphological characteristics of hypothalamic temperature sensitive neurons. FASEB J. 5, AI400. Griffin J. D., Burgoon P. W. and Boulant J. A. (1993) Neuronal thermosensitivity does not depend on thermal changes in resting membrane potential. FASEB J. 7, AI7. Guieu J. D. and Hardy J. D. (1971) Integrative activity of preoptic units: response to local and peripheral temperature changes. J. Physiol. (Paris) 63, 253-256. Gutnick M. J. and Yarom Y. (1989) Low threshold calcium spikes, intrinisic neuronal oscillation and rhythm generation in the CNS. J. Neurosci. Meth. 28, 93-99. Hall A. O., Carpenter D. O. and Rahmann H. (1987) Voltage clamp analysis of exogenous ganglioside (GMI) application on Aplysia neurons. In Gangliosides and modulation of neuronal functions: NA TO A SI Series, Cell Biol. 7, 547-548. Hellon R. (1967) Thermal stimulation of hypothalamic neurons in unanesthetized rabbits. J. Physiol. 193, 381-395. Hori T. (1991) Effects of temperature and neuroactive substances on hypothalamic neurons in vitro: possible

implications for the induction on fever. In Regional Meeting o f the International Union o f Physiological Sciences: Abstr. (Edited by Tu~ek S., Mare~ovfi D. and Stastn~ F.), p. 167. Prague. Hori T., Kiyohara T., Nakashima T., Shibata M. and Koga H. (1987) Muttimodal responses of preoptic and anterior hypothalamic neurons to thermal and nonthermal homeostatic parameters. Can. J. Physiol. Pharmacol. 65, 1290-1298. Hori T., Nakashima T., Hori N. and Kiyohara T. (1980a) Thermo-sensitive neurons in hypothalamic tissue slices in vitro. Brain Res. 186, 203-207. Hori T., Nakashima T., Kiyohara T., Shibata M. and Hori N. (1980b) Effect of calcium removal on thermosensitivity of preoptic neurons in hypothalamic slices. Neurosci. Lett. 20, 171-175. Hori T., Shibata M., Nakashima T., Yamasaki M., Asami A., Asami T. and Koga H. (1988) Effects of interleukin-1 and arachidonate on the preoptic and anterior hypothalamic neurons. Brain Res. Bull. 20, 75-82. Hori T. and Shinohara K. (1979) Hypothalamic thermoresponsive neurons in the newborn rat. J. Physiol. 294, 541-560. Hori Y. and Nakayama T. (1982) Temperature sensitivity of the preoptic and anterior hypothalamic neurons in organ culture. Tohoku J. Exp. Med. 136, 79-87. Imai-Matsumura K., Matsumura K., Tsai C.-L. and Nakayama T, (1988) Thermal responses of ventromedial hypothalamic neurons in vivo and in vitro. Brain Res. 445, 193-197. Inenaga k., Akaike N. and Yamashita H. (1989) Effect of osmotic stimulation on sodium current in isolated AV3V neurons. Neurosci. Res., Suppl. 9, 65. Inenaga K., Osaka T. and Yamashita H. (1987) Thermosensitivity of neurons in the paraventricular nucleus of the rat slice preparation. Brain Res. 424, 126-132. Ivanov K. P. (1990) The Principles of Energetics in Organism: Theoretical and Practical Aspects. Vol. 1. The General Energetics, Heatexchange, Thermoregulation, p. 307. Nauka, Leningrad (in Russian). Keenan C. L. and Chu N.-S. (1987) Thermosensitivity of dorsal raphe neurons in vitro. Brain Res. 410, 189-194. Kelso S. R. and Boulant J. A. (1982) Effect of synaptic blockade on thermosensitive neurons in hypothalamic tissue slices. Am. J. Physiol. 243, R480-R490. Kelso S. R., Perlmutter M. N. and Boulant J. A. (1982) Thermosensitive single-unit activity of in vitro hypothalamic slices. Am. J. Physiol. 242, R77-R84. Kiyohara T., Hirata M., Hori T. and Akaike N. (1990) Hypothalamic warm-sensitive neurons possess a tetrodotoxin-sensitive sodium channel with a high Qt0- Neurosci. Res. 8, 48-53. Kobayashi S. (1986) Warm- and cold-sensitive neurons inactive at normal core temperature in rat hypothalamic slices. Brain Res. 362, 132-139. Kobayashi S. and Takahashi T. (1993) Whole-cell properties of temperature-sensitive neurons in rat hypothalamic slices. Proc. R. Soc. Lond. B. 251, 89 94. Kogure K. (1988) Signal transducing system in postischemic death of selectively vulnerable brain cells. In Neurotransmitt.: Focus Excitatory Amino Acids: Proc. 5th Workshop Neurotrasmitt. and Diseases, Tokyo, June 18, 1988, pp. 95-114. Amsterdam. Konev S. V. (1987) Structural Lability of Biological Membranes and Regulatory Processes, p. 240. Nauka i technika, Minsk (in Russian).

Thermosensitivity Konings A. W. and Ruifrok A. C. C. (1985) Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells. Radiat. Res. 102, 86-98. Kozyreva T. V. (1972) Effect of local change in brain temperature on neuronal activity of sensorimotor cortex in rabbit. Fiziol. zhurn. SSSR 58, 1663-1668 (in Russian): Krueger B. K. (1989) Toward an understanding of structure and function of ion channels. FASEB J. 3, 1906-1914. Kruk B., Kaciuba-Uscilko H., Nazar K., Greenleaf J. E. and Kozlowcki S. (1985) Hypothalamic, rectal and muscle temperatures in exercising dogs: effect of cooling. J. Appl. Physiol. 58, 1444-1448. Kubarko A. I. and Tsaryuk V. V. (1986) Effect of phosphatidylcholine on body temperature and activity of posterior hypothalamic neurons in animals. Byull. Exper. Biol. i Med. 101, 652-654 (in Russian). Kushakov D. (1977) Characterization of thermosensitive neurons in the posterior hypothalamus. Fiziol. Zhurn. SSSR 63, 1261-1267 (in Russian). Lee D. C. and Chapman D. (1987) The effects of temperature on biological membranes and their models. SEB Symposium 41, Animal Cells and Temperature (Edited by K. Bowler and B. J. Fuller), pp. 35-52. COB, Cambridge. Ling D. S. F., Petroski R. E., Chou W. and Geller H. M. (1990) Development of spontaneous electrical activity by rat hypothalamic neurons in dissociated culture. Dev. Brain Res. 53, 276-282. Luna E. J. and Hitt A. L. (1992) Cytoskeleton-plasma membrane interactions. Science 258, 955-964. Macdonald A. G. (1990) The homeoviscous theory of adaptation applied to excitable membranes: A critical evaluation. Biochim. Biophys. Acta 1031, 291-310. Matsumoto G., Ichikawa M., Arai T. and Tsukita S. (1989) Subaxolemmal cytoskeleton of the squid giant axon and its possible relation to electrical excitability. Neurochem. Res. 14, 811. Meisenberg G. and Simmons W. H. (1984) Hypothermia induced by centrally administered vasopressin in rats. A structure-activity study. Neuropharmacology 23, 1195-1200. Milburn N. (1990) Falling stereotypes and new cell models in neurobiology. Amer. Zool. 30, 507-512. Miller C. (1992) Ion channel structure and function. Science 258, 240-241. Millhouse O. E. (1979) A Golgi anatomy of the rodent hypothalamus. In Handbook o f the Hypothalamus. Vol. 1., pp. 221-265. New York, Dekker. Myers R. D. and Lee T. F. (1989) Neurochemical aspects of thermoregulation. In Advances in Comparative and Environmental Physiology (Edited by Wang L. C. H.), Vol. 4, pp. 161-203. Springer-Verlag, Berlin. Nakashima T., Hori T. and Kiyohara T. (1989) lntracellular recording analysis of thermosensitive neurons in the rat hypothalamus and septum. In Thermal Physiology Symposium: Abstr. Satel. Symp. X X X I lnt. Congr. Physiol. Sci., p. 49, Tromso. Nakashima T., Pierau F.-K., Simon E. and Hori T. (1987) Comparison between hypothalamic thermoresponsive neurons from duck and rat slices. Pfliigers Arch. 409, 236-243. Nakayama T. (1985) Thermosensitive neurons in the brain. Jap. J. Physiol. 35, 375-389.

235

Nakayama T., Eisenman J. S. and Hardy J. D. (1961) Single unit activity of anterior hypothalamus during local heating. Science 134, 560-561. Nakayama T., Hammel H. T., Hardy J. D. and Eisenman J. S. (1963) Thermal stimulation of electrical activity of single units of the preoptic region. Am. J. Physiol. 204, i 122-1126. Nakayama T., Hory Y., Suzuki M., Yonezawa T. and Yamamoto K. (1978) Thermo-sensitive neurons in preoptic and anterior hypothalamic tissue cultures in vitro. Neurosci. Lett. 9, 23-26. Nelson D. O. and Prosser C. L. (1981a) Intracellular recordings from thermosensitive preoptic neurons. Science 213, 787-789. Nelson D. O. and Prosser C. L. (1981b) Temperature sensitive neurons in the preoptic regions of sunfish. Am. J. Physiol. 241, R259-R263. Ono T., Morimoto A., Watanabe T. and Murakami N. (1987) Effects of endogenous pyrogen and prostaglandin E 2 on hypothalamic neurons in guinea pig brain slices. J. Appl. Physiol 63, 175-180. Paisley A. C. and Summerlee A. J. S. (1985) Activity of preoptic neurons in conscious rabbits. J. Physiol. 364, 49. Park C. C. and Ahmed Z. (1992) Alterations of plasma membrane fatty acid composition modify the kinetics of Na + current in cultured rat diencephalic neurons. Brain Res. 570, 75-84. Parmeggiani P. L., Azzaroni A., Cevolani D. and Ferrari G. (1983) Responses of anterior hypothalamic-preoptic neurons to direct thermal stimulation during wakefulness and sleep. Brain Res. 269, 382-385. Paton J. F. R., Rogers W. T. and Schwaber J. S. (1991) Tonically rhythmic neurons within a cardiorespiratory region of the nucleus tractus solitarii of the rat. J. Neurophysiol. 66, 824-838. Pierau F.-K., Klee M. R. and Klussmann F. W. (1969) Effects of local hypo- and hyperthermia on mammalian spinal motoneurones. Fed. Proc. 28, 1006-1010. Pierau F.-K., Klee M. R. and Klussmann F. W. (1976) Effect of temperature on postsynaptic potentials of cat spinal motoneurones. Brain Res. 114, 21-34. Pierau F.-K., Torrey P. and Carpenter D. (1975) Effect of ouabain and potassium-free solution on mammalian thermosensitive afferents in vitro. Pfliigers Arch. 359, 349-356. Rawson R. O., Stolwijk J. A. J., Graichen H. and Abrams R. (1965) Continuous radio telemetry of hypothalamic temperatures from unrestrained animals. J. Appl. Physiol. 20, 321-325. Reaves T. A. Jr. and Heath J. E. (1983) Thermosensitive characteristics of a preoptic area neuron recorded over a 20 day period in the rabbit. Brain Res. Bull. 10, 39-41. Reier P. J., Cullen M. J., Froelich J. S. and Rothchild J. (1977) The ultrastructure of the developing medial preoptic nucleus in the postnatal rat. Brain Res. 122, 415-436. Sachs F. and Sokabe M. (1990) Stretch-activated ion channels and membrane mechanics. Neurosci. Res., Suppl. 12, SI-$4. Sawada M., Hara N. and Maeno T. (1991) Ionic mechanism of the outward current induced by extracellular ejection of interleukin-I onto identified neurons of Aplysia. Brain Res. 545, 248-256. Schmid H. A., Jansky L. and Pierau F.-K. (1993) Temperature sensitivity of neurons in slices of the rat PO/AH area:

236

V.Y. VASILENKO

effect of bombesin and substance P. Am J. Physiol. 264, R449-R455. Schmid H. A. and Pierau F.-K. (1993) Temperature sensitivity of neurons in slices of the rat PO/AH hypothalamic area: effect of calcium. Am. J. Physiol. 264, R440-R448. Shibata M. and Blatteis C. M. (1991a) Differential effects of cytokines on thermosensitive neurons in guinea pig preoptic area slices. Am. J. Physiol. 261, R1096-Rl103. Shibata M. and Blatteis C. M. (1991b) High perfusate PO2 impairs thermosensitivity of hypothalamic thermosensirive neurons in slice preparations. Brain Res. Bull. 26, 467-471. Simon E. and Iriki M. (1971) Ascending neurons highly sensitive to variations of spinal cord temperature. J. Physiol. (Paris) 63, 415-417. Simon E., Pierau F.-K. and Taylor D. C. (1986) Central and peripheral thermal control of effectors in homeothermic temperature regulation. Physiol. Rev. 66, 235-300. Slonim A. D. (1986) Evolution of Thermoregulation, p. 76. Nauka, Leningrad (in Russian). Sperelakis N. (1970) Effects of temperature on membrane potentials of excitable cells. In Physiological and Behavioral Temperature Regulation (Edited by Hardy J., Gagge A. P. and Stolwijk J. A. J.), pp. 408-441. Thomas, Springfield. Spiegel S., Handler J. S. and Fishman P. H. (1986) Gangliosides modulate sodium transport in cultured toad kidney epithelia. J. Biol. Chem. 261, 15755-15760. Spray D. C. (1974) Metabolic dependence of frog cold receptor sensitivity. Brain Res. 72, 354-359. St-Onge D. and Gicquaud C. (1989) Evidence of direct interaction between actin and membrane lipids. Cell Biol. 67, 297-300. Tsaryuk V. V. and Kubarko A. I. (1989) Study on central mechanisms of thermoregulation and their relations to brain lipid phase transitions. Fiziol. Zhurn. SSSR 75, 97-104 (in Russian). Vasilenko V. Y. (1990) The neurons responding to temperature changes in slices of hypothalamus. Fiziol. Zhurn. SSSR 76, 937-941 (in Russian). Vasilenko V. Y. (1991) On a putative neuronal mechanism of thermoregulation. In Temperature Monitoring of Functional State of Organism: Abstr. Sem.-Conf, p. 6. AlmaAta (in Russian).

Vasilenko V. Y., Belyavsky E. M. and Gourine V. N. (1989a) Temperature dependence of neuronal activity in guinea pig hypothalamic and hippocampal slices. Neirofiziologiya (Engl. Transl.)21, 259-265. Vasilenko V. Y., Belyavsky E. M. and Gourine V. N. (1989b) Threshold temperature responses of neurons in hypothalamic slices. Doklady Akademii Nauk BSSR 33, 947-950 (in Russian). Vasilenko V. Y. and Gourine V. N. (1992a) Effects of synaptic blockade on temperature responses of neurons in hypothalamic slices and putative membrane mechanisms of neuronal thermosensitivity. Biologicheskie Membrany 9, 1086-1089 (in Russian). Vasilenko V. Y. and Gourine V. N. (1992b) Threshold temperature responses of thermosensitive neurons in guinea-pig hypothalamic slices. J. Therm. Biol. 17, 367-374. Vassilev P. M. and Kanazirska M. P. (1990) Effect of antibodies directed against cytoskeletal proteins on single sodium channel activities in hippocampal neurons. Neurosei. Res. Commun. 7, 17-25. Watanabe T., Morimoto A. and Murakami N. (1986) Effect of amine on temperature-responsive neurons in slice preparation of rat brain stem. Am. J. Physiol. 250, R553-R559. Watababe T., Morimoto A. and Murakami N. (1987) Effect of PGE2 on preoptic and anterior hypothalamic neurons using brain slice preparation. J. Appl. Physiol. 63, 918-922. Willis J. A., Gaubatz G. L. and Carpenter D. O. (1974) The role of an electrogenic sodium pump in modulation of pacemaker discharge of Aplysia neurons. J. Cell. Physiol. 84, 463-472. W/innenberg W. and Brfick K. (1968) Single unit activity evoked by thermal stimulation of the cervical spinal cord in the guinea pig. Nature 218, 1268-1269. Xin L. and Blatteis C. M. (1992) Blockade by interleukin-I receptor antagonist of IL-l~-induced neuronal activity in guinea pig preoptic area slices. Brain Res. 569, 348-352. Yeagle P. L. (1989) Lipid regulation of cell membrane structure and function. FASEB J. 3, 1833-1842. Zacharzhevskaya N. P. (1974) Medial preoptic and septal neurons responsive to thermal stimulation of brain and skin. Fiziol. Zhurn. SSSR 60, 341-347 (in Russian).