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Journal of Physiology (1991), 440, pp. 257-271 With 8 figures Printed in Great Britain

INTRACELLULAR ANALYSIS OF INHERENT AND SYNAPTIC ACTIVITY IN HYPOTHALAMIC THERMOSENSITIVE NEURONES IN THE RAT

BY M. C. CURRAS, S. R. KELSO* AND J. A. BOULANTt From the Department of Physiology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA and the *Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680, USA

(Received 21 May 1990) SUMMARY

1. Intracellular neuronal activity was recorded in rat preoptic-anterior hypothalamic tissue slices. Thirty neurones were classified as warm sensitive, cold sensitive or temperature insensitive, based on their firing rate response to temperature changes. Seventy-seven per cent of the neurones were temperature insensitive, which included both spontaneously firing and silent neurones. Of all neurones, 10% were warm sensitive and 13% were cold sensitive. 2. Silent temperature-insensitive neurones had lower input resistances (126 + 21 MQ2) than thermosensitive neurones (179 + 24 MQ). Regardless of neuronal type, however, resistance was inversely related to temperature. 3. Warm-sensitive neurones were characterized by a slow, depolarizing prepotential, whose rate of rise was temperature dependent. This depolarizing potential disappeared during current-induced hyperpolarization, suggesting that intrinsic mechanisms are responsible for neuronal warm sensitivity. 4. Spike activity in cold-sensitive neurones correlated with putative excitatory and inhibitory postsynaptic potentials, whose frequency was thermosensitive. This suggests that cold sensitivity in these neurones depends on synaptic input from nearby neurones. 5. Like cold-sensitive neurones, action potentials of temperature-insensitive neurones often were preceded by short duration (< 20 ms), rapidly rising prepotentials, whose rates of rise were not affected by temperature. In some temperature-insensitive neurones, depolarizing current injection increased both firing rate (by 5-8 impulses s-1) and warm sensitivity, with pre-potentials having temperature-dependent rates of rise. We suggest that temperature-insensitive neurones employ two opposing, thermally dependent mechanisms: a voltagedependent depolarizing conductance and a hyperpolarizing sodium-potassium pump.

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S. R. KELSO AND J. A. BOULANT INTRODUCTION

The preoptic area and anterior hypothalamus are critical to the maintenance of body temperature. Certain preoptic neurones sense their own temperature, receive afferent information from peripheral thermoreceptors, and communicate with other brain stem areas to control thermoregulatory responses (Boulant, 1980). Some neuronal models suggest that preoptic warm-sensitive neurones control heat loss responses and cold-sensitive neurones control heat retention and heat production responses (Hammel, 1965; Boulant, 1980). These models also hypothesize that warm-sensitive neurones are intrinsically thermosensitive, while the thermosensitivity of cold-sensitive neurones is attributed to synaptic inhibition from nearby warm-sensitive neurones. The majority of preoptic neurones are classified as temperature insensitive, because they show little or no change in their firing rates during changes in temperature. It has been suggested that temperature-insensitive neurones serve as constant reference signals in synaptic networks that determine set point temperature (Hammel, 1965). While previous studies have revealed much about neuronal types and integration of thermal information, surprisingly little is known about the cellular mechanisms of temperature sensitivity and insensitivity in hypothalamic neurones (Boulant, Curras & Dean, 1989). One intracellular study in fish has investigated hypothalamic thermosensitive neurones (Nelson & Prosser, 1981); however, most intracellular studies of neuronal thermosensitivity have been confined to large neurones outside the hypothalamus (Pierau, Klee & Klussmann, 1976) or in invertebrates (Willis, Gaubatz & Carpenter, 1974). To understand the mechanisms of neuronal thermosensitivity in the mammalian hypothalamus, the present study recorded intracellular activity in rat preoptic and anterior hypothalamic tissue slices. The purpose of this study is to describe the effect of temperature on the intrinsic and synaptic activity of warm-sensitive, cold-sensitive and temperature-insensitive neurones. METHODS

Hypothalamic tissue slices were prepared from 120-250 g, male Sprague-Dawley rats. Following decapitation, a block containing the hypothalamus was cut, and 350-450 ,um thick frontal slices were sectioned rostral and caudal to the anterior commissure. Slices containing the preoptic area and anterior hypothalamic nucleus were incubated in an interphase perfusion chamber (Kelso, Nelson, Silva & Boulant, 1983) for 2 h prior to recording. In some experiments hippocampal slices were used to compare intracellular recordings in hypothalamic neurones with CAI hippocampal neurones. The slices were constantly perfused with a nutrient medium containing (in mM): 124 NaCl, 26 NaHC03, 10 glucose, 5 KCl, 3 CaCl2, 2MgSO4 and 1-24 KH2PO4. Both the chamber and the medium were bubbled with 95 % 02 and 5% CO2. Daily monitoring ensured that the medium had a pH of 7-4 and an osmolality of 300 mosmol kg-'. A micrometer in a microscope eyepiece allowed all recording sites to be mapped on frontal projections, using the dorsal edge of the optic chiasm and ventral edge of the anterior commissure as reference points. Controlled by a thermoelectric Peltier assembly (Kelso et al. 1983), the in-flowing medium was maintained at a constant neutral temperature between 36 and 38 'C. Periodically, temperature was changed (maximum range, 31-41 °C) to test neuronal thermosensitivity. Thermocouples measured the temperatures of the tissue slice and surrounding perfusion medium. The effect of temperature on neuronal firing rate was used to determine the thermosensitivity of each neurone. Firing rate is the frequency of action potentials, defined as impulses s-1. Thermoresponse curves of firing rate

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as a function of temperature were graphed, and each neurone's thermosensitivity was determined by the slope of the regression line over the 2-5 °C range in which the neurone was most temperature sensitive. To conform with previous studies, neurones were classified as warm sensitive if they had positive slopes of at least +0-8 impulses s-' 'C-1, or cold sensitive if they had negative slopes of at least -0-6 impulses s-' 'C-1 (Boulant, 1980; Boulant et al. 1989). All other neurones were classified as temperature insensitive. Intracellular recordings were obtained using fine-tipped microelectrodes filled with 3 Mpotassium acetate and having resistances of 80-160 MQ. Spontaneous potentials were recorded using a Dagan 8700 amplifier with bridge circuitry that allowed simultaneous current injection and voltage recording. Criteria for successful impalements were stable resting membrane potentials greater than -50 mV. Membrane potential, current and temperature were displayed on an oscilloscope, recorded on a VCR tape-recorder, and monitored on a chart-recorder along with integrated firing rate. Some of the recorded activity was photographed or analysed with a digital storage oscilloscope. Determinations were made of the effect of temperature on membrane potential and input resistance. Resting membrane potentials were measured at various temperatures and were corrected using control determinations of extracellular tip potentials for each electrode. Neuronal input resistance was determined by the slope of current-voltage plots obtained from electrotonic potentials during hyperpolarizing current injections (usually -0.1 to -0 4 nA). Current pulses of sufficient duration (50-80 ms) to fully charge the membrane capacitance were used, and only linear portions of the current-voltage relationship were used to calculate input resistance. Some neurones were injected periodically with constant hyperpolarizing current (1-5 min) to eliminate spike discharges and examine underlying synaptic potentials. Data recorded on VHS magnetic tape was analysed using computer software (Cambridge Electronic Design Ltd). At each temperature, spikes were averaged from at least fifteen representative action potentials superimposed on threshold. Computer averages of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were also generated. Putative postsynaptic potentials were characterized as brief, subthreshold changes in membrane potential with rapid rises to peak, 10-30 ms durations and > 2 mV amplitudes. At different temperatures, the frequencies of these putative EPSPs and IPSPs were counted from twenty, randomly selected, 1-3 s records. These frequencies were plotted as a function of temperature in order to characterize synaptic influences on neuronal thermosensitivity. Two-sample t tests were used to determine if temperature altered interspike intervals, rates of rise in depolarizing pre-potentials, and frequencies of postsynaptic potentials. Two-sample t tests were also used to compare mean firing rates, membrane potentials and input resistances between different neuronal populations. All statistical differences were considered significant if P < 0 05. The QIO or temperature quotient of the rate of change in membrane potential was determined by the equation: Qlo = (R2/R )°1OT2-TI, where R1 is the rate at the cooler temperature (Ti) and R2 is the rate at the warmer temperature (T2). RESULTS

Stable (20-120 min) resting membrane potentials -50 mV were obtained from forty-eight neurones in and near the preoptic region. Most neurones had spontaneous firing rates and displayed postsynaptic activity. At neutral temperature these neurones had a mean resting membrane potential of -55 + 1 mV (+ S.E.M., n = 48) and a mean input resistance of 152 + 10 MQ (n = 35). Similar input resistances have been reported for other hypothalamic neurones in paraventricular and supraoptic nuclei (Dudek & Andrew, 1985; Bourque, Randle & Renaud, 1986). Thirty neurones were recorded during at least one cyclic temperature change (normally lasting 8-10 min), permitting classification according to thermosensitivity. These neurones were located in the preoptic-anterior hypothalamus (74 %), in adjacent septal and hypothalamic areas (13%), and in the paraventricular nucleus (13 %). Ten per cent of the neurones were warm sensitive, 13 % were cold sensitive, and 77 % were temperature insensitive. Temperature-insensitive neurones included 9-2

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spontaneously firing neurones (40 % of all neurones), as well as silent neurones that only produced action potentials during depolarizing current injections. These silent neurones did not fire when the tissue temperature was changed at least 3 °C above and below 37 'C. They comprised 37 % of all neurones and 23 % of the preoptic A

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Neuronal input resistance Regardless of thermosensitivity, the input resistance of most neurones (i.e. 94 %) was inversely related to temperature. Figure 1 shows examples of the effect of temperature on warm-sensitive and cold-sensitive neurones, as well as on spontaneously firing and silent temperature-insensitive neurones. Each plot shows

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the relationship between injected hyperpolarizing current and the resulting membrane potential. Input resistance is the slope of the current-voltage plot. For each neurone, resistance either increased with cooling or decreased with warming. At neutral temperatures, the mean input resistance for the combined thermosensitive 35.8 OC

Fig. 2. Effect of temperature on the activity of a preoptic warm-sensitive neurone having a resting membrane potential of -67 mV. Arrows indicate two spontaneously occurring hyperpolarizing potentials.

neurones was 179 + 24 MQ2 (n = 6); and, when temperature was changed, the average thermally-induced resistance change was -17 MQIC-1. The mean resistance of temperature-insensitive neurones was 158+ 17 MQ (n = 8) for spontaneously firing neurones and 126 + 21 MQ (n -= 8) for silent neurones; and the thermally induced resistance changes were -11 and -12 MQ 0C-1, respectively. The mean input resistance of the silent temperature-insensitive neurones was significantly less than the resistance of the combined thermosensitive neurones. There were no other statistical differences between neuronal types, in terms of input resistance or thermally induced resistance changes.

Warm-sensitive neurones Figure 2 shows the activity of a warm-sensitive preoptic neurone at two different temperatures. Firing rate more than doubled during a 1-8 °C increase in temperature. At 35'8 °C, arrows indicate two spontaneously occurring hyperpolarizing potentials that were associated with prolonged interspike intervals. The primary determinant of spiking activity, however, was a slow, ramp-like depolarization that preceded each action potential. Figure 3A shows this slow depolarizing pre-potential in another warm-sensitive preoptic neurone, in this case, at three different temperatures. Warming increased the pre-potential's rate of rise, which decreased the interspike interval and increased firing rate. When compared from 10 ms to 40 ms before spike threshold, the rate of rise of the pre-potential at 41-0 °C was 0-25 + 0-01 mV ms-'. This was significantly greater (P = 0-004) than the rate of rise at 36-6 °C (i.e. 0 18+0-01 mV ms-1) or at

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32-6 °C (i.e. 0 17 + 002 mV ms-'). These values represent the averages of fifteen to twenty individually measured pre-potentials. Using the 41-0 and 32-6 °C values, the Q10 of the rate of rise was 1-6. A similar trend is shown in Fig. 4, which plots the computer-averaged pre-spike and post-spike activity superimposed on spike B

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Fig. 3. A, activity of a preoptic warm-sensitive neurones recorded at three different temperatures. These records are amplified to show pre-spike potentials, and the action potentials are truncated. Resting membrane potential is approximately -60 mV. The arrow indicates IPSP-like activity. B, shows activity recorded at the same three temperatures, but all of the interspike intervals are prolonged and contain IPSP-like activity (arrows). C, shows activity at 41 °C during hyperpolarizing current injection that maintained the membrane potential at -98 mV.

threshold. In this case, the rate of rise was greater at 41-0 °C (i.e. 0-183 mV ms-') than at 32-6 °C (i.e. 0 115 mV ms-1). Using these two values, the Q1o for the rate of rise of the slow pre-potential was 1-7. The nature of these pre-potentials was examined during injection of constant hyperpolarizing current to eliminate spiking activity. Figure 3A shows normal spiking activity at a resting membrane potential of approximately -60 mV. Figure 3C shows that spikes were eliminated by hyperpolarizing current injection that maintained the membrane potential at -98 mV. During this hyperpolarization, no postsynaptic activity was observed. Like the neurone in Fig. 2, the warm-sensitive neurone in Fig. 3B displayed spontaneous hyperpolarizing potentials associated with prolonged interspike intervals. At each of the three temperatures, average interspike intervals were obtained from either fifteen interspike intervals containing putative IPSPs or fifteen interspike intervals in which no putative IPSPs were observed. At 32-6 °C, the interspike interval was 189+12 ms (±S.E.M.) with IPSPs present, compared to 142 +6 ms

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without IPSPs; at 36-6 °C, this interval was 127 + 8 ms with IPSPs and 95+3 ms without IPSPs; and at 41-0 °C, the interval was 98 + 6 ms with IPSPs and 74 + 2 ms without IPSPs. At each temperature, average interspike intervals were significantly longer when putative IPSPs were present (P 0 003). While the frequency of these

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Fig. 4. Computer-averaged pre-spike and post-spike activity of the warm sensitive neurone in Fig. 3. For each temperature, the average of fifteen (truncated) action potentials are shown superimposed on the spike threshold. Cooling decreased the rate of rise in the averaged pre-spike potential.

putative IPSPs was not affected by temperature, the effectiveness of their inhibition increased with cooling, i.e. the differences between average interspike intervals (with and without putative IPSPs) increased with cooling. Using the average intervals mentioned above, the presence of IPSPs lengthened the interspike interval by 24 ms at 410 °C, 32 ms at 36-6 °C, and 47 ms at 32-6 °C. Therefore, cooling from 41 to 32-6 °C produced a 23 ms increase in the effectiveness of the IPSP to lengthen the interspike interval (i.e. 47-24 = 23 ms). This cooling-induced increase in IPSP effectiveness may be attributed to both presynaptic and postsynaptic thermal effects. Cooling increases spike amplitude and duration (Curras & Boulant, 1989b) which could produce greater presynaptic neurotransmitter release. In addition, changes in the resistance of the postsynaptic membrane may explain the increase in IPSP effectiveness during cooling. Coldinduced increases in resistance (Fig. 1) should increase IPSP amplitude; and the increased resistance should also increase the membrane's time constant, resulting in prolonged IPSPs. Figure 5 shows the same neurone's average IPSPs at different temperatures. These computer averages were generated from sixteen to nineteen putative IPSPs, superimposed at the point of deflection. As anticipated, IPSP amplitude and duration were inversely related to temperature. IPSP duration at 32-6 °C was 23 ms longer than the IPSP duration at 41-0 °C. This 23 ms increase is identical to the above-mentioned increase in interspike intervals with IPSPs. This increase in IPSP duration, therefore, could account for the cooling-induced increase in interspike interval when IPSPs are present.

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While cooling did increase the ability of IPSPs to lengthen the interspike interval, it should be noted that, at each temperature, the percentage increase (in interspike intervals with IPSPs) remained constant at approximately 33 %. At 41 0 °C, IPSPs lengthened the interspike interval by 24 ms, which represents a 32-4 % increase over the 74 ms interval without IPSPs; at 36-6 °C, the additional 32 ms represents a 33-7 % increase over the 95 ms interval without IPSPs; and at 32-6 °C, the additional 47 ms represents a 33-1 % increase over the 142 ms interval without IPSPs. A possible explanation for this constant percentage increase in the interspike interval may be seen in Fig. 5. During the IPSP recovery phase (as the membrane potential returns to resting level), temperature can affect the rate of decay of the returning potential. In Fig. 5, this rate of decay was 0 3 mV ms-1 at 41-0 °C, but slower, i.e.

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0-2 mV ms-1, at 32-6 'C. The Q1o of this rate of decay was 1P6, which was the same as the Qlo of the rate of rise in the slow depolarizing pre-potential. Since temperature has similar effects on the rate of rise of the pre-potential and the rate of IPSP decay, this may explain why cooling increased the duration of the interspike interval, while the percentage increase in this interval (with IPSPs) remained constant at 33%. A

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Temperature-insensitive neurones Temperature-insensitive neurones characteristically had low firing rates and showed little or no change in firing rate over a wide range of temperatures. Temperature-insensitive neurones often displayed putative excitatory and inhibitory C

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Fig. 8. Effect of temperature on the activity of a spontaneously firing, preoptic temperature-insensitive neurone. A, firing rate (impulses s-1) as a function of tissue temperature. B, computer-averaged pre-spike and post-spike activity of the neurone at two different temperatures. For each temperature, the average of 15 action potentials are shown superimposed on the spike threshold. While the pre-spike potential was unaffected, warming to 403 °C decreased the spike amplitude (arrow) and after-hyperpolarizing potential. C and D show the same neurone's activity during constant injection of 0-15 nA depolarizing current. C, depolarization increased both firing rate and the thermosensitivity of the firing rate response. D, depolarization also altered the effect of temperature on the rate of rise for the pre-spike potential.

postsynaptic activity with action potentials preceded by short duration, rapidly rising potentials. Even when slower pre-potentials were evident, the rate of rise of these depolarizations did not appear to be affected by temperature. As an example, Fig. 8A shows a preoptic temperature-insensitive neurone that fired about one action potential per second over a wide temperature range. Figure 8B presents the computer averages of this neurone's action potentials, showing that pre-spike activity was virtually identical at 35*8 and 40-3 'C. The only difference in these averaged action potentials was a decrease in spike amplitude and afterhyperpolarizing potential with warming.

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A recent study found that most preoptic temperature-insensitive neurones displayed warm sensitivity when the electrogenic sodium-potassium pump was blocked (Curras & Boulant, 1989a). Temperature-insensitive neurones may employ the thermally dependent hyperpolarizing pump to oppose a voltage-dependent depolarizing conductance that would normally contribute to warm sensitivity. To examine this possibility, the temperature-insensitive neurone in Fig. 8 was injected with constant depolarizing current. As shown in Fig. 8C-D, 0 15 nA depolarizing current not only increased spontaneous firing rate, but in addition, the neurone displayed increased thermosensitivity (i.e. +0-6 impulses s-1 'C-1) in the hyperthermic range. Much of this neurone's increased activity during depolarization was associated with bursts of two or three action potentials. These doublets occurred more often at hyperthermic temperatures. The second spike in each doublet usually had a smaller amplitude and was preceded by a slowly rising pre-potential. Figure 8D shows the averages of these second spikes at two temperatures. At 40 3 TC, there was an increased rate of rise in the depolarizing pre-potential. This response in a depolarized temperature-insensitive neurone is similar to the effect of temperature on the pre-potentials of warm-sensitive neurones (see Fig. 4), and its suggests that both types of neurones share similar mechanisms associated with warm sensitivity. DISCUSSION

This is the first intracellular study of neuronal thermosensitivity in the mammalian hypothalamus. Using the same criteria for thermosensitivity, several extracellular in vitro studies have found that approximately 30 % of the preoptic neurones are warm sensitive, 10 % are cold sensitive and 60 % are temperature insensitive (Boulant et al. 1989). These proportions are similar to those reported in extracellular in vivo studies (Boulant, 1980). In comparison, the present intracellular study found fewer warmsensitive neurones and more temperature-insensitive neurones: i.e. 10 % of the neurones were warm sensitive, 13 % were cold sensitive and 77 % were temperature insensitive. The only other intracellular study of thermosensitive hypothalamic neurones was conducted in sunfish (Nelson & Prosser, 1981). This previous study also reported a low incidence of thermosensitive neurones: i.e. 21 % of all neurones were temperature sensitive, and in most cases, the thermosensitivity was synaptically mediated. In this sunfish study, 3 % of the neurones were cold sensitive and only 3 % were intrinsically warm sensitive. The silent neurones in the present study account for part of the discrepancy with previous extracellular studies. Extracellular studies only detect spontaneously firing neurones and therefore cannot identify silent neurones. Of the spontaneously firing neurones in the present study, 16% were warm sensitive, 21 % were cold sensitive and 63 % were temperature insensitive. Thus, the proportions of spontaneously firing, temperature-insensitive neurones are similar in mammalian intracellular and extracellular studies. Another discrepancy with extracellular studies is the small proportion of warmsensitive neurones found in intracellular studies. It has been suggested that warmsensitive neurones have higher firing rates but smaller cell bodies compared to temperature-insensitive neurones (Boulant, 1980). Therefore, warm-sensitive

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neurones may be more difficult to impale, making their recordings less common in intracellular studies. In the present study, partial evidence for this may be the higher input resistance of the thermosensitive neurones. High resistance is associated with small cell size, and within a population, the smaller neurones are more excitable and have higher firing rates (Henneman, Somjen & Carpenter, 1965).

Neuronal input resistance Like a previous study of spinal motoneurones (Pierau, Klee, Faber & Klussmann, 1971), the present study found no consistent thermally induced changes in membrane potential or input resistance that could account for differences in neuronal thermosensitivity. Input resistance was inversely related to temperature in warmsensitive, cold-sensitive and temperature-insensitive hypothalamic neurones. Similar thermal changes in resistance were found in temperature-insensitive hippocampal neurones, both in the present study and in a previous study (Thompson, Masukawa & Prince, 1985). While temperature appeared to have the same effect on input resistance in each type of hypothalamic neurone, there may be more subtle differences between these neurones. For example, a few neurones, like the warm-sensitive neurone in Fig. 1, displayed anomalous rectification at neutral and cool temperatures, but not at warm temperatures. The physiological role of anomalous . rectification in neuronal thermosensitivity is not clear, since it is most prominent at very hyperpolarized potentials. In septal neurones, however, similar rectification can be blocked by caesium, which suggests that it involves changes in potassium conductance (Segal, 1986). Therefore, thermal differences in potassium conductance may provide a distinction between hypothalamic neuronal types.

Warm-sensitive neurones Warm-sensitive neurones displayed rhythmic firing with constant interspike intervals and action potentials preceded by slow ramp-like depolarizations. These depolarizing potentials were not dependent on synaptic input, since EPSPs were not observed when spike activity was suppressed by hyperpolarizing current injection. Instead, it appears that neuronal warm-sensitivity is associated with intrinsic depolarizing potentials that are similar to temperature-sensitive pacemaker potentials described in other cells. Large Q1o values, for example, have been reported for the rate of rise of pacemaker potentials in a variety of excitable cells (Sperelakis, 1970). In addition, warm sensitivity in Aplysia neurones has been associated with thermal effects on pacemaker potentials (Carpenter, 1970, 1973; Willis, Gaubatz & Carpenter, 1974), and similar depolarizing potentials have been observed in hypothalamic warm-sensitive neurones in the sunfish (Nelson & Prosser, 1981). The present study suggests that there are intrinsic thermosensitive mechanisms in mammalian warm-sensitive neurones. This is further supported by extracellular studies in rat tissue slices showing that neuronal warm-sensitivity is maintained during synaptic blockade with high-magnesium and low-calcium perfusions (Kelso & Boulant, 1982; Dean & Boulant, 1989). In addition to an intrinsic mechanism for thermosensitivity, preoptic warmsensitive neurones also displayed putative inhibitory postsynaptic potentials. The

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frequency of these IPSPs was not affected by temperature, suggesting that some inherently warm-sensitive neurones receive synaptic inhibition from temperatureinsensitive neurones. This input may serve to limit the firing rate of warm-sensitive neurones without altering their thermosensitivity. It is also possible that this input enhances neuronal warm sensitivity since cold-induced increases in resistance can enhance IPSP magnitude and duration and, therefore, accentuate a neurone's reduced firing rate during cooling. Cold-senrsitive neurones In the present study, neuronal cold-sensitivity appeared to be synaptically mediated. Previous extracellular studies indicate that, of all preoptic neurones, coldsensitive neurones receive the greatest synaptic input (Boulant, 1980). In sunfish, Nelson & Prosser (1981) found that the activity of cold-sensitive neurones depends on EPSPs and IPSPs, rather than on pacemaker potentials. Neuronal models propose that cold-sensitive neurones are inhibited by warm-sensitive neurones and, in some cases, excited by temperature-insensitive neurones (Hammel, 1965; Boulant, 1980). This type of synaptic integration is considered to be the primary determinant of preoptic neuronal cold sensitivity. The present intracellular study supports this conclusion. In general, action potentials of cold-sensitive neurones were preceded by apparent excitatory postsynaptic potentials, and the frequency of putative EPSP and IPSP activity correlated with the effect of temperature on firing rate. As shown in Fig. 7B, EPSP activity may remain constant, while IPSP frequency decreases with cooling. This supports the hypothesis that the neurones are inhibited by warmsensitive neurones and excited by temperature-insensitive neurones (Hammel, 1965). Other cold-sensitive neurons, however, may have different synaptic networks. As shown in Fig. 7A, IPSP activity can remain constant, while EPSP frequency increases with cooling. This would occur in neurones that are synaptically driven by other cold-sensitive neurones. Both types of activity support previous extracellular studies indicating that synaptic blockade abolishes cold sensitivity in preoptic neurones (Kelso & Boulant, 1982; Dean & Boulant, 1989). Pierau, Klee & Klussmann (1976) have suggested another synaptic mechanism for cold sensitivity in cat spinal motoneurones. These authors have proposed that motoneurones receive constant excitatory synaptic input and cooling-induced resistance increases cause increases in EPSP amplitude, which in turn, increase neuronal firing rate. A similar mechanism may also occur in some preoptic coldsensitive neurones. The present study has shown that cooling increases not only input resistance, but also the amplitude and duration of putative postsynaptic potentials. Therefore, even without synaptic inhibition from warm-sensitive neurones, a mechanism for cold sensitivity may exist in neurones that receive excitatory synaptic input. It is also possible that cooling increases presynaptic neurotransmitter release, causing increased firing rates in neurones receiving synaptic excitation. At the neuromusculature junction, for example, quantal content increases with cooling from 39 to 20 °C (Hubbard, Jones & Landau, 1971). Since cooling increases the amplitude and duration of action potentials (Curras & Boulant, 1989 b), this could cause greater Ca2' entry in presynaptic terminals and enhanced neurotransmitter release. Thus, thermal effects on presynaptic and postsynaptic

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events could account for cold sensitivity in neurones that only receive excitatory synaptic input. Temperature-insensitive neurones The primary difference between warm-sensitive neurones and temperaturesensitive neurones is illustrated in a comparison of Figs 4 and 8B. Action potentials of warm-sensitive neurones are preceded by slowly depolarizing pre-potentials, whose rates of rise are temperature sensitive. In temperature-insensitive neurones, pre-spike depolarizations have shorter durations and their rates of rise are not markedly affected by temperature. When depolarized, some temperature-insensitive neurones can display warmsensitive characteristics and show increased firing rates during warming. This increase in firing rate was associated with doublets or pairs of action potentials. Doublets have been reported in septal neurones exposed to depolarizing current (Segal, 1986); although in the septal neurones, the 5-10 ms interval between the two spikes is much shorter than the 60-120 ms intervals in the hypothalamic neurones in the present study. Figure 8D compares the second spike in the doublets and shows that warming increased the rate of rise of the pre-potential. This suggests that a component of the pre-potential includes a voltage-dependent depolarizing current that is thermosensitive. If the membrane potential is normally maintained at a more hyperpolarized level, this thermosensitive component may never be expressed. Our previous study suggested that the Na+-K+ pump may be used to maintain the hyperpolarization necessary for temperature insensitivity (Curras & Boulant, 1989 a). Like the neurone in Fig. 8 C-D, temperature-insensitive neurones increased their firing rates and warm sensitivity when the Na+-K+ pump was blocked with ouabain. Blockade of a temperature-sensitive Na+-K+ pump may unmask a voltage-dependent depolarizing current that is thermosensitive, causing increased discharge during warming. Despite the similarities between warm-sensitive and temperature-insensitive neurones, there remain important differences. Ouabain inhibition of the Na+-K+ pump does not alter the thermosensitivity of warm-sensitive neurones, but it does produce warm sensitivity in neurones that are normally temperature insensitive (Curras & Boulant, 1989a). In addition, this ouabain enhancement of warm sensitivity did not occur in temperature-insensitive neurones exposed to elevated magnesium and reduced calcium concentrations (Curras & Boulant, 1989a). Yet, numerous studies have shown that preoptic warm-sensitive neurones retain their thermosensitivity in these ionic concentrations (Kelso & Boulant, 1982; Dean & Boulant, 1989). It appears, therefore, that the thermosensitive slow pre-potential may be calcium-dependent in the depolarized temperature-insensitive neurones, but not in the warm-sensitive neurones. This research was supported in part by NIH grant NS 14644 from the National Institute for Neurological and Communicative Disorders and Stroke and by a grant from the American Heart Association, Ohio Affliate. J. A. Boulant is the Hitchcock Professor of Environmental Physiology. The authors express their gratitude to Lorry Kaple for her assistance in these experiments.

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