Temperature effects on neuronal membrane potentials and inward currents in rat hypothalamic tissue slices
1 Department of Physiology and Cell Biology, College of Medicine, Ohio State University, Columbus, OH 43210, USA
Abstract
Preoptic–anterior hypothalamic (PO/AH) neurones sense and regulate body temperature. Although controversial, it has been postulated that warm-induced depolarization determines neuronal thermosensitivity. Supporting this hypothesis, recent studies suggest that temperature-sensitive cationic channels (e.g. vanilloid receptor TRP channels) constitute the underlying mechanism of neuronal thermosensitivity. Moreover, earlier studies indicated that PO/AH neuronal warm sensitivity is due to depolarizing sodium currents that are sensitive to tetrodotoxin (TTX). To test these possibilities, intracellular recordings were made in rat hypothalamic tissue slices. Thermal effects on membrane potentials and currents were compared in PO/AH warm-sensitive, temperature-insensitive and silent neurones. All three types of neurones displayed slight depolarization during warming and hyperpolarization during cooling. There were no significant differences in membrane potential thermosensitivity for the different neuronal types. Voltage clamp recordings (at –92 mV) measured the thermal effects on persistent inward cationic currents. In all neurones, resting holding currents decreased during cooling and increased during warming, and there was no correlation between firing rate thermosensitivity and current thermosensitivity. To determine the thermosensitive contribution of persistent, TTX-sensitive currents, voltage clamp recordings were conducted in the presence of 0.5 μM TTX. TTX decreased the current thermosensitivity in most neurones, but there were no resulting differences between the different neuronal types. The present study found no evidence of a resting ionic current that is unique to warm-sensitive neurones. This supports studies suggesting that neuronal thermosensitivity is controlled, not by resting currents, but rather by currents that determine rapid changes in membrane potential between successive action potentials.
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Introduction
The preoptic–anterior hypothalamus (PO/AH) is important in sensing changes in core temperature and controlling both physiological and behavioural thermoregulatory responses (Hammel, 1965; Bligh, 1973; Boulant, 1980, 1996). About 20% of spontaneously firing PO/AH neurones are warm sensitive and increase their firing rates during temperature increases. Most of the remaining spontaneously firing neurones are considered to be temperature insensitive, showing little or no change in their firing rates during temperature changes (Boulant & Dean, 1986). PO/AH warm-sensitive neurones orientate their dendrites perpendicular to the midline third ventricle (Griffin et al. 2001), and these neurones receive much synaptic input from medial and lateral ascending pathways carrying afferent information from peripheral thermoreceptors (Boulant & Hardy, 1974). In contrast, PO/AH temperature-insensitive neurones orientate their dendrites parallel to the third ventricle and are predominantly unaffected by peripheral thermoreceptors. In addition to these two populations, intracellular studies have shown that another population of PO/AH neurones are silent and only fire action potentials when they are electrically stimulated (Griffin & Boulant, 1995; Griffin et al. 2001).
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The mechanisms of neuronal thermosensitivity remain unresolved. Conflicting studies point to either transient or persistent ionic currents as the basis of firing rate responses to temperature. Our previous studies suggest that thermosensitivity is due to transient currents that determine the pacemaker potentials or depolarizing prepotentials between successive action potentials (Curras et al. 1991; Griffin et al. 1996). Unlike temperature-insensitive neurones, PO/AH warm-sensitive neurones display depolarizing prepotentials that are affected by temperature; i.e. warming increases the prepotential's rate of depolarization which, in turn, shortens the interspike interval and increases firing rate. While different currents contribute to these prepotentials, one study (Griffin et al. 1996) has noted the importance of A-current inactivation. The A-current is a transient outward potassium current that hyperpolarizes the membrane for a brief time after each action potential. Since A-current inactivation is highly temperature dependent, warming will shorten the inactivation period and allow the prepotential to depolarize at a faster rate, thus increasing the firing rate.
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The mechanisms underlying neuronal thermosensitivity remain controversial, however. Recent studies propose that neuronal thermosensitivity is due to thermally induced changes in persistent, inward, cationic currents that determine the resting membrane potential. Heat-sensitive and vanilloid/capsaicin-sensitive transient receptor potential vanilloid (TRPV) channels have been identified in the dorsal root ganglion and are suggested to be transducers of hypothalamic temperature sensitivity (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003). These calcium and sodium TRP channels produce warm-induced depolarization which could produce increased firing rates. Studies by Kiyohara et al. (1990) and Kobayashi & Takahashi (1993) indicate that PO/AH neuronal thermosensitivity is due to a warm-induced membrane depolarization caused by a non-inactivating, inward sodium current. Kiyohara et al. (1990) suggest that warm-sensitive neurones possess a tetrodotoxin (TTX)-sensitive, persistent Na+ current that is highly thermosensitive in the hyperthermic range. In a small subpopulation of dissociated PO/AH neurones from newborn rats, TTX blocked this highly thermosensitive persistent Na+ current. Although this subpopulation did not fire spontaneous action potentials, the Kiyohara et al. (1990) study presumed that these cells were warm-sensitive neurones because their inward currents were more thermosensitive than the other neuronal population.
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The purpose of the present study was to test the hypothesis that thermosensitivity in PO/AH warm-sensitive neurones is due to persistent, depolarizing cationic currents that may or may not be TTX sensitive. Using whole-cell patch clamp recordings, these experiments sought to determine differences in the thermal properties of persistent currents of temperature-sensitive and -insensitive PO/AH neurones. These neurones were first characterized according to their firing rate thermosensitivities, and then the effects of temperature on their resting membrane potentials were determined. Finally, voltage clamp recordings of these neurones determined the effects of temperature on their inward cationic conductances, both before and during TTX application.
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Methods
Slice preparation
Hypothalamic tissue slices were prepared from 150–320 g, male Sprague-Dawley rats. Each rat was quickly decapitated with a guillotine according to procedures approved by the National Institutes of Health (USA) and the Ohio State University Laboratory Animal Care and Use Committee. Following removal of the brain, a hypothalamic tissue block was cut, mounted on a vibratome, and submerged in perfusion medium. Five or six 350 μm-thick horizontal slices were sectioned (see Dean & Boulant, 1988). One or two slices were transferred to a recording chamber where they incubated for 2 h before any recordings were made. The 7.4 pH, 300 mosmol l–1 control perfusion medium contained (mM): 124 NaCl, 26 NaHCO3, 10 glucose, 5 KCl, 2.4 CaCl2, 1.3 MgSO4 and 1.24 KH3PO4. This medium was oxygenated with 95% O2–5% CO2. In some experiments, the perfusion was also temporarily switched to a similar medium containing 500 nM TTX, in order to block voltage-gated Na+ channels. A thermocouple was placed beneath the slices to record tissue temperature. A thermoelectric Peltier assembly (Kelso et al. 1983) allowed the perfusion medium flowing to the chamber (1–2 ml min–1) to be heated to 36–37°C during the incubation. This assembly was also used to periodically warm and cool the tissue slices in order to determine neuronal thermosensitivity.
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Electrophysiological recordings
Whole-cell patch clamp recordings were made using 2 μm tip (3–5 M) glass microelectrodes filled with a 7.2–7.3 pH, 290–295 mosmol l–1 solution containing (mM): 140 potassium gluconate, 10 EGTA, 10 Hepes, 1 MgCl2, 1 CaCl2, 2 ATP and 5 NaCl. The Ag–AgCl earth electrode was maintained at a constant temperature in an outer bath connected by a filter paper bridge to the inner bath containing the tissue slice. The liquid junction potential (Barry & Lynch, 1991) was experimentally determined to be 12.0 mV (Griffin & Boulant, 1995), and this was subtracted from all recorded potentials.
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Initially for each whole-cell recording, a holding current (–5 to –20 pA) was applied to stabilize the membrane–electrode seal. This holding current was gradually removed over a 5–10 min period, and no holding current was applied during the periods that membrane potential data was collected. During voltage clamp recordings, each neurone was clamped at –92 mV to ensure that transient sodium channels were closed or inactive and to maximize the recorded persistent, inward cationic currents.
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Currents and voltages were recorded by an Axopatch 200A amplifier (Axon Instruments) and relayed via a TL-1 DMA interface to a computer. Acceptable recordings consisted of (1) action potential amplitudes equal to or greater than 55 mV, and (2) stable, non-fluctuating membrane potential recordings during the data collection periods. Action potentials and currents were displayed on an oscilloscope and stored on magnetic tape. A ratemeter measured action potential firing rate, which was recorded on a computer and polygraph along with tissue temperature, membrane potential and holding current. As previously described (Griffin & Boulant, 1995), a separate measurement of integrated membrane potential was made by filtering all rapidly changing potentials (including action potentials) using a Grass 7DA driver amplifier adjusted to 0.5 Hz half-amplitude response.
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After a control recording at 36–37°C, neuronal firing rate thermosensitivity was determined by changing the tissue temperature, usually between 32 and 39°C but not exceeding 30–40°C. In some experiments, a baffle was introduced in the perfusion line to remove bubbles from the bathing medium. When this baffle produced a delay between the actual tissue temperature and the temperature measured by a thermocouple, a correction factor was used to minimize hysteresis by synchronizing the firing rate and temperature peaks.
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Data analysis
The thermal coefficient m is the slope of the regression line produced when firing rate is plotted as a function of temperature (impulses s–1 °C–1). As in other studies (Kelso & Boulant 1982; Boulant & Dean, 1986; Griffin & Boulant, 1995), a neurone was classified as warm sensitive if it had a positive thermal coefficient that was at least +0.8 impulses s–1 °C–1, or a neurone was classified as cold sensitive if it had a negative thermal coefficient that was at least –0.6 impulses s–1 °C–1. All other neurones with spontaneous action potentials were classified as temperature insensitive. As in previous studies (Griffin & Boulant, 1995; Burgoon & Boulant, 2001), temperature-insensitive neurones were subdivided into two groups: moderate slope and low slope. Moderate slope temperature-insensitive neurones increased their firing rates slightly during increases in temperature; and their thermal coefficients were less than +0.8 impulses s–1 °C–1 but greater than +0.2 impulses s–1 °C–1. During temperature changes, the firing rates of low slope temperature-insensitive neurones were virtually unchanged, and the absolute values of their thermal coefficients were 0.2 impulses s–1 °C–1. If a neurone did not have spontaneous activity but could be stimulated by depolarizing current to produce action potentials, it was classified as a silent neurone.
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Software for data acquisition and analysis included pCLAMP, Axotape, AxoScope (Axon Instruments) and MiniAnalysis (Synaptosoft). Membrane potential (in current clamp mode) and resting current at –92 mV (in voltage clamp mode) were recorded continuously and plotted as functions of temperature. Responses of membrane potential or current to temperature were determined by linear regression, and this value defined the thermosensitivity of the response. If resting current in voltage clamp became larger than –300 pA, the recording was discounted because the membrane was considered to be unstable.
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Values are reported as means ± S.E.M. For different neuronal types, ANOVA and standard Student's t tests were used to determine the statistical differences in membrane potential and holding current. Differences were considered significant if P < 0.05.
Results
Effects of temperature on membrane potential
The examples in Fig. 1 show the effects of temperature on membrane potentials and action potentials in the three types of spontaneously firing PO/AH neurones. The warm-sensitive neurone in Fig. 1A showed a substantial warm-induced increase in firing rate, while the firing rate of the moderate slope temperature-insensitive neurone in Fig. 1B was only slightly affected by the temperature changes. In contrast, the low slope temperature-insensitive neurone in Fig. 1C showed no change in firing rate over the temperature range tested. The three examples in Fig. 1 displayed occasional inhibitory postsynaptic potentials, and each neurone had action potential amplitudes that decreased during warming and increased during cooling.
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A, warm-sensitive neurone having a thermosensitivity of 1.56 impulses s–1 °C–1. B, moderate slope temperature-insensitive neurone having a thermosensitivity of 0.56 impulses s–1 °C–1. C, low slope temperature-insensitive neurone having a thermosensitivity of –0.03 impulses s–1 °C–1. All three types of neurones displayed inhibitory postsynaptic potentials as indicated by arrow. In all neurones warming decreased and cooling increased the action potential amplitudes.
, 百拇医药 As shown in Table 1, firing rate thermosensitivity was determined in 117 neurones located in or near the PO/AH. This included 71 neurones in the medial preoptic nucleus, 37 neurones in the anterior hypothalamic nucleus, 3 neurones in the lateral preoptic area, 2 neurones in the bed nucleus of the stria terminalis, 2 neurones in medial septum, 1 neurone in the dorsal anterior nucleus, and 1 neurone in the dorsomedial hypothalamus. Of the total, 15 neurones were classified as silent, because they did not have spontaneous firing rates but only produced action potentials when stimulated electrically. Of the remaining 102 spontaneously firing neurones, 19 neurones were warm sensitive, 38 were moderate slope temperature insensitive, and 45 were low slope temperature insensitive. Warm-sensitive, moderate slope temperature-insensitive, and low slope temperature-insensitive neurones represented (respectively) 19%, 37% and 44% of the spontaneously firing neurones and 16%, 33% and 38% of the total population (that included silent neurones). In this study, no recorded neurones met the criterion (i.e. –0.6 impulses s–1 °C–1) for cold sensitivity.
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Figure 2 shows the effect of temperature on the firing rates and integrated membrane potentials for three different types of spontaneously firing neurones. For each neurone, the records in the left panels are the changes in firing rate (FR) and membrane potential (mV) during the changes in tissue temperature (°C). Figure 2 also plots the neuronal firing rates (centre panels) and membrane potentials (right panels, Vm) as a function of tissue temperature. The warm-sensitive neurone in Fig. 2A decreased its firing rate during cooling and increased its firing rate during warming; it had a thermal coefficient of +1.35 impulses s–1 °C–1. The moderate slope temperature-insensitive neurone in Fig. 2B showed more modest firing rate changes during cooling and warming; its thermal coefficient was +0.38 impulses s–1 °C–1. The low slope temperature-insensitive neurone in Fig. 2C showed very little change in its firing rate during the temperature change; its thermal coefficient was +0.14 impulses s–1 °C–1. The three graphs on the right of Fig. 2 show the plots of membrane potential as a function of temperature. Most neurones displayed subtle depolarization during tissue warming and hyperpolarization during tissue cooling; however, this occurred in both warm-sensitive and temperature-insensitive neurones. The three neurones in Fig. 2, for example, showed a wide range of firing rate thermosensitivities, and yet, their membrane potential thermosensitivities were all very small and virtually identical (i.e. 0.47, 0.48 and 0.47 mV °C–1).
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Left panels: time records of firing rate (FR) and membrane potential (Vm) during changes in tissue slice temperature (°C). Centre panels: plots of firing rate as a function of tissue temperature. Right panels: plots of membrane potential as a function of tissue temperature. m is the regression coefficient of the FR and Vm plots. A, warm-sensitive neurone with firing rate thermosensitivity of 1.35 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.47 mV °C–1. B, moderate slope temperature-insensitive neurone with firing rate thermosensitivity of 0.38 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.48 mV °C–1. C, low slope temperature-insensitive neurone with firing rate thermosensitivity of 0.14 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.47 mV °C. Temperature had similar effects on the membrane potential of all three neurones.
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The 15 silent neurones are aligned on the left and had a mean membrane potential (Vm) thermosensitivity of 0.50 ± 0.11 mV °C–1. The mean Vm thermosensitivity for all 117 neurones (including the silent neurones) was 0.53 ± 0.03 mV °C–1. For the spontaneously firing neurones (low slope temperature-insensitive neurones, moderate slope temperature-insensitive neurones, and warm-sensitive neurones), the extremely low regression coefficient (m) and correlation coefficient (r) indicate that there is no correlation between neuronal firing rate thermosensitivity and membrane potential thermosensitivity.
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Effects of temperature on persistent ionic currents
After each neurone was characterized according to its firing rate and membrane potential thermosensitivity, it was voltage clamped at –92 mV in order to determine the effect of temperature on persistent, inward, cationic currents. Figure 4 shows the thermally induced current changes in three different types of PO/AH neurones. Figure 4A and B shows the recordings from the same warm-sensitive and moderate slope temperature-insensitive neurones that were depicted in Fig. 2A and B; Fig. 4C is a low slope temperature-insensitive neurone having a firing rate thermal coefficient of 0.06 impulses s–1 °C–1. The centre panels of Fig. 4 show records of the holding currents during cyclic temperature changes. The holding current balances the inward resting currents and indicates the magnitude of the net resting current. In all three neurones, the resting ionic currents decreased during tissue cooling and increased during tissue warming. These ionic currents are plotted as a function of tissue temperature in the right-hand panels of Fig. 4, and the current thermosensitivities are the regression coefficients (pA °C–1) of these plots. A previous study by Kiyohara et al. (1990) indicated that warm-sensitive neurones display significantly higher current thermosensitivity in the hyperthermic range. Accordingly, Fig. 4 (right) provides separate current thermosensitivities for both the hypothermic range (m1, below 36°C) and hyperthermic range (m2, above 36°C).
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Left panels: firing rate (FR) responses to temperature during current clamp for warm-sensitive neurone (A), moderate slope temperature-insensitive neurone (B), and low slope temperature-insensitive neurone (C). Centre panels: during voltage clamp (–92 mV), time records of holding currents during changes in tissue temperature. Note that during warming, more negative current is required to maintain the voltage clamp at –92 mV, suggesting that there is a net increase in inward cationic current. Right panels: voltage clamp holding currents plotted as a function of temperature. Regression lines indicate the current thermosensitivities: m1 over temperatures below 36°C (), and m2 over temperatures above 36°C ().
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These examples suggest that current thermosensitivity is not a determinant of firing rate thermosensitivity. In fact, in the hyperthermic range the warm-sensitive neurone (Fig. 4A) had the lowest current thermosensitivity (–10.44 pA °C–1), while the low slope temperature-insensitive neurone (Fig. 4C) had the highest current thermosensivitivity (–21.30 pA °C–1). For the entire population, there were no significant differences between the average current thermosensitivities for low slope temperature-insensitive neurones (–9.61 ± 1.24 pA °C–1, n = 9), moderate slope temperature-insensitive neurones (–6.89 ± 0.75 pA °C–1, n = 9), and warm-sensitive neurones (–8.45 ± 1.39 pA °C–1, n = 7). The mean current thermosensitivity for the combined populations (n = 27, including 2 silent neurones) was –7.96 ± 0.66 pA °C–1.
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Figure 5 plots the current thermosensitivity of each neurone as a function of that neurone's firing rate thermosensitivity. This includes seven warm-sensitive neurones, nine moderate slope and nine low slope temperature-insensitive neurones and two silent neurones. The current thermosensitivities in Fig. 5 were determined over the hypothermic range (top panel), the hyperthermic range (middle panel), and the entire combined temperature range (bottom panel). In each case, the extremely low regression lines (m) and correlation coefficients (r) indicate that there is no relationship between current thermosensitivity and firing rate thermosensitivity. In addition to current thermosensitivity expressed as pA °C–1, the effect of temperature on ionic current can also be expressed as Q10, where:
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Thus, Q10 shows the effect of temperature on the relative change in a current compared to some basal current level. A previous study reported that the inward currents of warm-sensitive neurones have a much higher Q10 compared to temperature-insensitive neurones (Kiyohara et al. 1990). For the neurones in the present study, Fig. 6 plots each neurone's current Q10 as a function of its firing rate thermosensitivity. As in Fig. 5, the plots in Fig. 6 indicate that there is no relationship between neuronal current thermosensitivity and firing rate thermosensitivity.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The small regression coefficients (m) and correlation coefficients (r) suggest that thermally induced changes in resting currents do not determine neuronal thermosensitivity. n = 27 neurones.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The small regression coefficients (m) and correlation coefficients (r) suggest that thermally induced changes in resting currents do not determine neuronal thermosensitivity. n = 27 neurones.
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Effects of tetrodotoxin on resting current thermosensitivity
Kiyohara et al. (1990) have indicated that, in the hyperthermic range, the Na+ channel blocker tetrodotoxin (TTX) reduces the current thermosensitivity of warm-sensitive neurones, but has no effect on the current thermosensitivity of temperature-insensitive neurones. To test this hypothesis, neurones were first characterized by their firing rate thermosensitivity (current clamp mode). Following this, during voltage clamp, the same neurones were tested for their current thermosensitivity before and during perfusions with a medium containing 500 nM TTX.
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Figure 7 shows an example of a low slope temperature-insensitive neurone (0.06 impulses s–1 °C–1) and a record (top) of its thermally induced current changes during control medium perfusion and TTX medium perfusion. The current–temperature plots (bottom: centre and right panels) show current thermosensitivity both in the hypothermic range (m1) and hyperthermic range (m2). TTX reduced the neurone's hyperthermic current thermosensitivity by 6.46 pA °C–1; i.e. from –21.30 pA °C–1 (control) to –14.84 pA °C–1 (TTX). Figure 8 shows similar records for a warm-sensitive neurone (1.35 impulses s–1 °C–1) in which TTX reduced hyperthermic current thermosensitivity by 5.27 pA °C–1; i.e. from –10.44 pA °C–1 (control) to –5.17 pA °C–1 (TTX).
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Top record shows current changes before and during 500 nM TTX perfusion. Bottom left panel, neuronal firing rate (FR) plotted as a function of tissue temperature showing a thermosensitivity of 0.06 impulses s–1 °C–1. Bottom centre and right panels, resting current plotted as a function of tissue temperature. Current thermosensitivities are determined below 36°C (m1) and above 36°C (m2).
Top record shows current changes before and during 500 nM TTX perfusion. Bottom left panel, neuronal firing rate (FR) plotted as a function of tissue temperature showing a thermosensitivity of 1.35 impulses s–1 °C–1 in the hyperthermic range. Bottom centre and right panels, resting current plotted as a function of tissue temperature. Current thermosensitivities are determined below 36°C (m1) and above 36°C (m2). TTX decreased current thermosensitivity above 36°C from –10.44 to –5.17 pA °C–1.
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Figure 9 plots the current thermosensitivity as a function of firing rate thermosensitivity for neurones tested with control and TTX media. This included five warm-sensitive neurones, four moderate slope temperature-insensitive neurones, two low slope temperature-insensitive neurones and one silent neurone. The current thermosensitivities in Fig. 9 were determined over the hypothermic range (top panel), the hyperthermic range (middle panel), and the entire combined temperature range (bottom panel). In each case, the extremely low regression lines (m) and correlation coefficients (r) indicate that there is no relationship between current thermosensitivity and firing rate thermosensitivity during both control and TTX perfusions. Similarly, Fig. 10 plots the current Q10 as a function of firing rate thermosensitivity for these same 12 neurones. Once again, the low regression coefficients (m) and correlation coefficients (r) indicate that there is no relationship between the Q10 of the inward current and firing rate thermosensitivity.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The insets show the regression coefficients (m) and correlation coefficients (r) during control (Con) and tetrodotoxin (TTX) perfusions. TTX decreased the ionic currents and current thermosensitivities in all neurones. n = 12 neurones.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The insets show the regression coefficients (m) and correlation coefficients (r) during control (Con) and tetrodotoxin (TTX) perfusions. n = 12 neurones.
Figures 9 and 10 indicate that even though TTX blockade of Na+ currents often decreased current thermosensitivity, this decrease occurred in both temperature-sensitive and -insensitive neurones. In each figure, the regression lines before and during TTX perfusion are similar, relatively flat, and show no relationship between current and firing rate thermosensitivities. Accordingly, there is no evidence that a TTX-induced decrease in current thermosensitivity is different in warm-sensitive and temperature-insensitive neurones. Nor is there evidence that warm-sensitive neurones possess greater TTX-sensitive Na+ currents at hyperthermic temperatures.
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Discussion
There continues to be debate over the mechanisms that determine thermosensitivity in hypothalamic neurones. Early studies of molluscan neurones suggested that temperature had a greater effect on depolarizing Na+ currents (compared to hyperpolarizing K+ currents), consequently causing warm-induced membrane depolarization and increased firing rates (Gorman & Marmor, 1970; Carpenter, 1981). Recent reviews suggest that PO/AH neuronal thermosensitivity may be due to cationic channels that are part of the temperature-sensitive TRPV (i.e. transient receptor potential) family of vanilloid/capsaicin-sensitive channels (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003), although to date, studies of the thermosensitive properties of TRPV channels have not included hypothalamic neurones. Warming above a threshold temperature opens TRP channels and produces depolarization in dorsal root ganglion neurones. TRPV1 and TRPV2 channels have temperature thresholds greater than 43°C, therefore these channels are not likely explanations for the PO/AH warm-sensitive neurones that show proportional changes in their firing rates over the 30–40°C range. On the other hand, TRPV3 and TRPV4 have lower thresholds and appear to be sensitive over a range in which PO/AH neurones are thermosensitive. Hypothalamic warm-sensitive neurones, however, generally do not display either sensitization or desensitization during repeated temperature changes (Dean & Boulant, 1989; Boulant, 1996). Unfortunately, repetitive thermal stimulation strongly sensitizes TRPV3 channels and desensitizes TRPV4 channels (Benham et al. 2003). Accordingly, TRP channels do not offer a simple explanation for hypothalamic neuronal thermosensitivity. Perhaps more importantly, the mechanism of thermosensitivity for TRP channels is a thermally induced inward cationic (Ca2+ and Na+) current that depolarizes the membrane. While the present study shows that warming causes slight depolarization, this occurs in all neurones and is not restricted to warm-sensitive neurones.
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Some electrophysiological studies have suggested that warm-induced, voltage-gated Na+ currents are the mechanism for thermosensitivity in specific PO/AH neurones (Kiyohara et al. 1990). Using PO/AH tissue slices from 10- to 15-day-old rats, Kobayashi & Takahashi (1993) reported that one population of ‘warm responsive’ neurones showed warm-induced depolarizations leading to increased firing rates. Unfortunately, this previous study employed techniques that created technical problems. While the tissue slices were initially incubated for an hour at 37°C, during the intracellular recordings each neurone was maintained at 25°C, and it was thermally tested by warming to only 35°C. Therefore, the neurones were not maintained or tested over a normo-thermic range of temperatures. In addition, during current clamp recordings in this study, a holding current was continuously injected to maintain each neurone at a –68 mV membrane potential at 25°C. A more recent tissue slice study (Griffin & Boulant, 1995) has shown that such injections of hyperpolarizing holding currents introduce a temperature-dependent artifact that produces spurious membrane potential depolarizations during warming. Since neuronal membrane resistance decreases during warming, a constant negative current injection will produce an apparent decrease in the membrane potential (i.e. depolarization). If no holding current is injected, however, these depolarizations are not observed.
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Another intracellular recording study also claimed that PO/AH neuronal warm sensitivity is due to TTX-sensitive, non-inactivating, voltage-gated Na+ currents (Kiyohara et al. 1990). Again, however, there are methodological problems that would open this earlier study to reinterpretation. The electrode solution in the patch clamp microelectode did not contain ATP, and therefore the intracellular ATP of each recorded neurone would have been rapidly diluted. Also, the Kiyohara study used mechanically and enzymatically dissociated neurones from 1- to 4-week-old rats, and the morphology of these cells suggest that most neuronal processes were amputated or severely damaged. While some of these recorded neurones could be electrically stimulated to produce action potentials, the action potentials did not appear to be normal. For example, at 19°C the spikes did not pass through 0 mV, the spike durations were more than 20 ms, and there were no prominent after-hyperpolarizing potentials (AHPs). Most importantly, these neurones did not have spontaneous firing rates. Therefore, the authors could not classify neurones according to their firing rate thermosensitivities. Instead, the neurones were considered to be ‘warm sensitive’ if (during a –80 mV voltage clamp) they displayed a strong inward current that increased proportionally during warming over the hyperthermic range (i.e. approximately 35–40°C).
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The most obvious criticism of the 1990 Kiyohara et al. study is that the populations of warm-sensitive and temperature-insensitive neurones were not based on their firing rate thermosensitivities, but rather on current thermosensitivities. In the present study, the middle plot in Fig. 5 shows neuronal current thermosensitivities (pA °C–1) over the hyperthermic range (> 36°C). As an example, if one arbitrarily identified neurones as warm sensitive if their current thermosensitivities were greater than 11 pA °C–1, Fig. 5 (middle) shows 8 neurones with high current thermosensitivities. Unfortunately, 6 of these 8 neurones are temperature insensitive based on their firing rate thermosensitivities. Similarly, the middle plot of Fig. 6 shows the Q10 of each neurone's current in the hyperthermic range. Only 3 of 27 neurones had Q10 values greater than 2.5, and all three neurones were low slope temperature-insensitive neurones, based on their firing rate thermosensitivities. It appears, therefore, that there is little value in defining neuronal thermosensitivity based on current thermosensitivity, instead of firing rate thermosensitivity.
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The 1990 Kiyohara study also indicated that tetrodotoxin had a distinct and selective effect on the ‘warm-sensitive’ neurones. Specifically, 5 x 10–7 M TTX strongly reduced the current thermosensitivity of these neurones in the hyperthermic range. The present study found no evidence to support this contention. The middle sections of Figs 9 and 10 show the effects of TTX on the current thermosensitivities (pA °C–1) and current Q10 values in the hyperthermic range. Both figures show no indication that TTX has selective or predominant effects on the current thermosensitivity of warm-sensitive neurones.
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Recent studies offer some explanation for the differential effects of tetrodotoxin observed by Kiyohara et al. (1990), particularly if the mechanically and enzymatically dissociated neurones received varying degrees of injury during the dissociation process. Stretch injury to cultured neurones and their axons produces a Ca2+ influx that is mediated by a TTX-sensitive Na+ current (Wolf et al. 2001; Iwata et al. 2004). TTX blocks this trauma-induced Na+ current and the resulting Ca2+ influx. In the dissociated neurones of the Kiyohara study, it is possible that the most traumatized neurones displayed the strongest TTX-sensitive Na+ currents, and it is not unreasonable to suspect that these injury-induced currents were strongly affected by hyperthermic temperatures. Accordingly, if this current was not as pronounced in the less traumatized neurones, then these cells would constitute the second population of TTX-insensitive neurones.
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The electrode solution in the present study contained 10 mM EGTA (and 1 mM Ca2+) which should reduce intracellular free calcium concentrations (normally 50–100 nM) to less than 20 nM. If some membrane channels are gated by intracellular calcium, then these conductances may be reduced. On the other hand, like the present study, previous intracellular recording studies (Griffin & Boulant, 1995; Griffin et al. 2001; Burgoon & Boulant, 2001) have used this same electrode solution and found that the proportions of warm-sensitive and temperature-insensitive neurones are similar to the proportions found in extracellular recording studies (reviewed in Boulant & Dean, 1986). Accordingly, it does not appear that these reductions in intracellular free calcium significantly alter neuronal thermosensitivity.
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Apart from the tetrodotoxin experiments, the present study did not block synaptic activity, which could contribute to the measured resting potentials and currents. In PO/AH neurones, however, postsynaptic potentials have short durations (i.e. approximately 15 ms) and frequencies of less than 4 events s–1 (Griffin et al. 2001). Consequently, the contribution of postsynaptic activity to the measurements would be minimal. More importantly, there are no differences in the frequencies of postsynaptic potentials between warm-sensitive, temperature-insensitive and silent neurones, and temperature has no significant effect on these frequencies (Griffin et al. 2001). Therefore, it is unlikely that synaptic activity contributed to the results reported here.
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The present study found no evidence to suggest that neuronal thermosensitivity is due to temperature-induced changes in either the resting membrane potential or resting inward cationic currents. On the other hand, there is evidence that thermal effects on more transient currents play a role in neuronal thermosensitivity. Most PO/AH neurones display depolarizing prepotentials that produce action potentials. Figure 1A and B shows that depolarizing prepotentials exist in both warm-sensitive neurones and temperature- insensitive neurones. The rates of depolarization in the prepotentials determine the firing rates. Previous studies indicated that temperature has little or no effect on the prepotental depolarization rates in temperature-insensitive neurones; however, temperature has a pronounced effect on prepotential depolarization rates in warm-sensitive neurones (Curras et al. 1991; Griffin & Boulant, 1995). In warm-sensitive neurones, warming increases the prepotential's rate of depolarization, causing the membrane potential to reach threshold sooner. This, in turn, shortens the interspike interval and increases the firing rate.
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Either increasing depolarizing conductances (Na+ and/or Ca2+) or decreasing hyperpolarizing conductances (K+ and/or Cl–) can induce prepotentials. If the depolarizing prepotentials are caused by inward cationic currents, persistent Na+ currents might be possible candidates (French & Gage, 1985; Huang, 1993; Pennartz et al. 1997). Persistent Na+ currents activate around –60 to –50 mV (i.e. below the action potential thresholds) and can affect the resting membrane potential. This low-threshold Na+ current inactivates slowly, compared to the transient Na+ current associated with action potentials. The voltage dependence and kinetics of persistent Na+ currents suggest that they partially de-inactivate during the after-hyperpolarizing potential (AHP) and may contribute to the subsequent depolarizing prepotential (Pennartz et al. 1997).
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If the depolarizing prepotentials are primarily caused by increasing inward cationic currents, the membrane resistance might be expected to decrease as the prepotential depolarizes toward threshold. In warm-sensitive neurones, however, Griffin et al. (1996) found that the membrane resistance gradually increased as the prepotential depolarized toward threshold. This suggests that the thermosensitive prepotential of warm-sensitive neurones is primarily dependent upon a decreasing conductance, such as an outward (hyperpolaring) K+ current. In contrast, the membrane resistance of temperature-insensitive neurones remained constant during the interspike intervals, suggesting that the prepotentials of temperature-insensitive neurones are more dependent on a balance of both increased inward (e.g. Na+ and Ca2+) and decreased outward (e.g. K+) cationic currents.
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The previous study indicated that a transient K+ current, the potassium A-current (IA), is important in the prepotential of warm-sensitive neurones (Griffin et al. 1996). After each action potential and subsequent AHP, IA activation causes a transient outward potassium current that attenuates further depolarization, keeping the membrane potential at a hyperpolarized level. The IA then slowly inactivates, which allows the membrane potential to slowly depolarize. This could be a major component of the depolarizing prepotential in warm-sensitive neurones, since several studies have shown that IA inactivation rates are extremely temperature sensitive (Lee & Deutsch, 1990; Pahapill & Schlichter, 1990; Huguenard & Prince, 1991; Forsythe et al. 1992; Griffin et al. 1996). Warming increases IA inactivation, and this would increase the prepotential's depolarization rate and increase firing rate. Conversely, cooling slows IA inactivation which would decrease the prepotential's depolarization rate and decrease firing rate. For PO/AH neurones, Griffin et al. (1996) reported that the time constants for IA inactivation were about 78 ms at hyperthermic temperatures (38–40°C) and 156 ms at hypothermic temperatures (31–34°C). This thermally induced difference in IA inactivation rates could have a significant effect on faster firing, warm-sensitive neurones (with short interspike intervals) compared to slower firing temperature-insensitive neurones (with longer interspike intervals).
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The present experiments show that PO/AH neuronal thermosensitivity is not due to thermally induced changes in resting membrane potentials or persistent inward Na+/Ca2+ currents. These experiments therefore support previous studies (Griffin et al. 1996) which indicate that the warm sensitivity of hypothalamic neurones is due to the ionic currents (including IA inactivation) that determine the rate of rise of the depolarizing prepotentials occurring during the brief interval between one action potential and the next.
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While there is evidence that TRPV channels underlie transduction mechanisms in peripheral thermal receptors (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003), it is possible that central hypothalamic thermosensitivity evolved from completely different mechanisms. Little is known about the transduction mechanisms of peripheral warm receptors (Pierau, 1996), but it is generally assumed that the basis lies in temperature-induced changes in the resting membrane potential. One importance of peripheral receptors is their response to rapid changes in the external environment. In contrast, central hypothalamic receptors may have evolved to monitor slower changes in the internal environment (e.g. core temperature, plasma glucose and osmolality). In addition to acting as sensors, these same central neurones can also act as effector neurones, controlling various homeostatic responses. Accordingly, it may be important that these central neurones have relatively constant, spontaneous firing rates that can be slowly modified by the internal environment. In these central neurones, the sensing mechanisms probably became linked with the interspike interval that determines spontaneous firing rates. Various ionic conductances determine the interspike interval, and temperature affects most of these conductances. Therefore, it seems reasonable that different sensing strategies could have evolved under these two very different conditions.
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Abstract
Preoptic–anterior hypothalamic (PO/AH) neurones sense and regulate body temperature. Although controversial, it has been postulated that warm-induced depolarization determines neuronal thermosensitivity. Supporting this hypothesis, recent studies suggest that temperature-sensitive cationic channels (e.g. vanilloid receptor TRP channels) constitute the underlying mechanism of neuronal thermosensitivity. Moreover, earlier studies indicated that PO/AH neuronal warm sensitivity is due to depolarizing sodium currents that are sensitive to tetrodotoxin (TTX). To test these possibilities, intracellular recordings were made in rat hypothalamic tissue slices. Thermal effects on membrane potentials and currents were compared in PO/AH warm-sensitive, temperature-insensitive and silent neurones. All three types of neurones displayed slight depolarization during warming and hyperpolarization during cooling. There were no significant differences in membrane potential thermosensitivity for the different neuronal types. Voltage clamp recordings (at –92 mV) measured the thermal effects on persistent inward cationic currents. In all neurones, resting holding currents decreased during cooling and increased during warming, and there was no correlation between firing rate thermosensitivity and current thermosensitivity. To determine the thermosensitive contribution of persistent, TTX-sensitive currents, voltage clamp recordings were conducted in the presence of 0.5 μM TTX. TTX decreased the current thermosensitivity in most neurones, but there were no resulting differences between the different neuronal types. The present study found no evidence of a resting ionic current that is unique to warm-sensitive neurones. This supports studies suggesting that neuronal thermosensitivity is controlled, not by resting currents, but rather by currents that determine rapid changes in membrane potential between successive action potentials.
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Introduction
The preoptic–anterior hypothalamus (PO/AH) is important in sensing changes in core temperature and controlling both physiological and behavioural thermoregulatory responses (Hammel, 1965; Bligh, 1973; Boulant, 1980, 1996). About 20% of spontaneously firing PO/AH neurones are warm sensitive and increase their firing rates during temperature increases. Most of the remaining spontaneously firing neurones are considered to be temperature insensitive, showing little or no change in their firing rates during temperature changes (Boulant & Dean, 1986). PO/AH warm-sensitive neurones orientate their dendrites perpendicular to the midline third ventricle (Griffin et al. 2001), and these neurones receive much synaptic input from medial and lateral ascending pathways carrying afferent information from peripheral thermoreceptors (Boulant & Hardy, 1974). In contrast, PO/AH temperature-insensitive neurones orientate their dendrites parallel to the third ventricle and are predominantly unaffected by peripheral thermoreceptors. In addition to these two populations, intracellular studies have shown that another population of PO/AH neurones are silent and only fire action potentials when they are electrically stimulated (Griffin & Boulant, 1995; Griffin et al. 2001).
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The mechanisms of neuronal thermosensitivity remain unresolved. Conflicting studies point to either transient or persistent ionic currents as the basis of firing rate responses to temperature. Our previous studies suggest that thermosensitivity is due to transient currents that determine the pacemaker potentials or depolarizing prepotentials between successive action potentials (Curras et al. 1991; Griffin et al. 1996). Unlike temperature-insensitive neurones, PO/AH warm-sensitive neurones display depolarizing prepotentials that are affected by temperature; i.e. warming increases the prepotential's rate of depolarization which, in turn, shortens the interspike interval and increases firing rate. While different currents contribute to these prepotentials, one study (Griffin et al. 1996) has noted the importance of A-current inactivation. The A-current is a transient outward potassium current that hyperpolarizes the membrane for a brief time after each action potential. Since A-current inactivation is highly temperature dependent, warming will shorten the inactivation period and allow the prepotential to depolarize at a faster rate, thus increasing the firing rate.
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The mechanisms underlying neuronal thermosensitivity remain controversial, however. Recent studies propose that neuronal thermosensitivity is due to thermally induced changes in persistent, inward, cationic currents that determine the resting membrane potential. Heat-sensitive and vanilloid/capsaicin-sensitive transient receptor potential vanilloid (TRPV) channels have been identified in the dorsal root ganglion and are suggested to be transducers of hypothalamic temperature sensitivity (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003). These calcium and sodium TRP channels produce warm-induced depolarization which could produce increased firing rates. Studies by Kiyohara et al. (1990) and Kobayashi & Takahashi (1993) indicate that PO/AH neuronal thermosensitivity is due to a warm-induced membrane depolarization caused by a non-inactivating, inward sodium current. Kiyohara et al. (1990) suggest that warm-sensitive neurones possess a tetrodotoxin (TTX)-sensitive, persistent Na+ current that is highly thermosensitive in the hyperthermic range. In a small subpopulation of dissociated PO/AH neurones from newborn rats, TTX blocked this highly thermosensitive persistent Na+ current. Although this subpopulation did not fire spontaneous action potentials, the Kiyohara et al. (1990) study presumed that these cells were warm-sensitive neurones because their inward currents were more thermosensitive than the other neuronal population.
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The purpose of the present study was to test the hypothesis that thermosensitivity in PO/AH warm-sensitive neurones is due to persistent, depolarizing cationic currents that may or may not be TTX sensitive. Using whole-cell patch clamp recordings, these experiments sought to determine differences in the thermal properties of persistent currents of temperature-sensitive and -insensitive PO/AH neurones. These neurones were first characterized according to their firing rate thermosensitivities, and then the effects of temperature on their resting membrane potentials were determined. Finally, voltage clamp recordings of these neurones determined the effects of temperature on their inward cationic conductances, both before and during TTX application.
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Methods
Slice preparation
Hypothalamic tissue slices were prepared from 150–320 g, male Sprague-Dawley rats. Each rat was quickly decapitated with a guillotine according to procedures approved by the National Institutes of Health (USA) and the Ohio State University Laboratory Animal Care and Use Committee. Following removal of the brain, a hypothalamic tissue block was cut, mounted on a vibratome, and submerged in perfusion medium. Five or six 350 μm-thick horizontal slices were sectioned (see Dean & Boulant, 1988). One or two slices were transferred to a recording chamber where they incubated for 2 h before any recordings were made. The 7.4 pH, 300 mosmol l–1 control perfusion medium contained (mM): 124 NaCl, 26 NaHCO3, 10 glucose, 5 KCl, 2.4 CaCl2, 1.3 MgSO4 and 1.24 KH3PO4. This medium was oxygenated with 95% O2–5% CO2. In some experiments, the perfusion was also temporarily switched to a similar medium containing 500 nM TTX, in order to block voltage-gated Na+ channels. A thermocouple was placed beneath the slices to record tissue temperature. A thermoelectric Peltier assembly (Kelso et al. 1983) allowed the perfusion medium flowing to the chamber (1–2 ml min–1) to be heated to 36–37°C during the incubation. This assembly was also used to periodically warm and cool the tissue slices in order to determine neuronal thermosensitivity.
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Electrophysiological recordings
Whole-cell patch clamp recordings were made using 2 μm tip (3–5 M) glass microelectrodes filled with a 7.2–7.3 pH, 290–295 mosmol l–1 solution containing (mM): 140 potassium gluconate, 10 EGTA, 10 Hepes, 1 MgCl2, 1 CaCl2, 2 ATP and 5 NaCl. The Ag–AgCl earth electrode was maintained at a constant temperature in an outer bath connected by a filter paper bridge to the inner bath containing the tissue slice. The liquid junction potential (Barry & Lynch, 1991) was experimentally determined to be 12.0 mV (Griffin & Boulant, 1995), and this was subtracted from all recorded potentials.
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Initially for each whole-cell recording, a holding current (–5 to –20 pA) was applied to stabilize the membrane–electrode seal. This holding current was gradually removed over a 5–10 min period, and no holding current was applied during the periods that membrane potential data was collected. During voltage clamp recordings, each neurone was clamped at –92 mV to ensure that transient sodium channels were closed or inactive and to maximize the recorded persistent, inward cationic currents.
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Currents and voltages were recorded by an Axopatch 200A amplifier (Axon Instruments) and relayed via a TL-1 DMA interface to a computer. Acceptable recordings consisted of (1) action potential amplitudes equal to or greater than 55 mV, and (2) stable, non-fluctuating membrane potential recordings during the data collection periods. Action potentials and currents were displayed on an oscilloscope and stored on magnetic tape. A ratemeter measured action potential firing rate, which was recorded on a computer and polygraph along with tissue temperature, membrane potential and holding current. As previously described (Griffin & Boulant, 1995), a separate measurement of integrated membrane potential was made by filtering all rapidly changing potentials (including action potentials) using a Grass 7DA driver amplifier adjusted to 0.5 Hz half-amplitude response.
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After a control recording at 36–37°C, neuronal firing rate thermosensitivity was determined by changing the tissue temperature, usually between 32 and 39°C but not exceeding 30–40°C. In some experiments, a baffle was introduced in the perfusion line to remove bubbles from the bathing medium. When this baffle produced a delay between the actual tissue temperature and the temperature measured by a thermocouple, a correction factor was used to minimize hysteresis by synchronizing the firing rate and temperature peaks.
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Data analysis
The thermal coefficient m is the slope of the regression line produced when firing rate is plotted as a function of temperature (impulses s–1 °C–1). As in other studies (Kelso & Boulant 1982; Boulant & Dean, 1986; Griffin & Boulant, 1995), a neurone was classified as warm sensitive if it had a positive thermal coefficient that was at least +0.8 impulses s–1 °C–1, or a neurone was classified as cold sensitive if it had a negative thermal coefficient that was at least –0.6 impulses s–1 °C–1. All other neurones with spontaneous action potentials were classified as temperature insensitive. As in previous studies (Griffin & Boulant, 1995; Burgoon & Boulant, 2001), temperature-insensitive neurones were subdivided into two groups: moderate slope and low slope. Moderate slope temperature-insensitive neurones increased their firing rates slightly during increases in temperature; and their thermal coefficients were less than +0.8 impulses s–1 °C–1 but greater than +0.2 impulses s–1 °C–1. During temperature changes, the firing rates of low slope temperature-insensitive neurones were virtually unchanged, and the absolute values of their thermal coefficients were 0.2 impulses s–1 °C–1. If a neurone did not have spontaneous activity but could be stimulated by depolarizing current to produce action potentials, it was classified as a silent neurone.
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Software for data acquisition and analysis included pCLAMP, Axotape, AxoScope (Axon Instruments) and MiniAnalysis (Synaptosoft). Membrane potential (in current clamp mode) and resting current at –92 mV (in voltage clamp mode) were recorded continuously and plotted as functions of temperature. Responses of membrane potential or current to temperature were determined by linear regression, and this value defined the thermosensitivity of the response. If resting current in voltage clamp became larger than –300 pA, the recording was discounted because the membrane was considered to be unstable.
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Values are reported as means ± S.E.M. For different neuronal types, ANOVA and standard Student's t tests were used to determine the statistical differences in membrane potential and holding current. Differences were considered significant if P < 0.05.
Results
Effects of temperature on membrane potential
The examples in Fig. 1 show the effects of temperature on membrane potentials and action potentials in the three types of spontaneously firing PO/AH neurones. The warm-sensitive neurone in Fig. 1A showed a substantial warm-induced increase in firing rate, while the firing rate of the moderate slope temperature-insensitive neurone in Fig. 1B was only slightly affected by the temperature changes. In contrast, the low slope temperature-insensitive neurone in Fig. 1C showed no change in firing rate over the temperature range tested. The three examples in Fig. 1 displayed occasional inhibitory postsynaptic potentials, and each neurone had action potential amplitudes that decreased during warming and increased during cooling.
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A, warm-sensitive neurone having a thermosensitivity of 1.56 impulses s–1 °C–1. B, moderate slope temperature-insensitive neurone having a thermosensitivity of 0.56 impulses s–1 °C–1. C, low slope temperature-insensitive neurone having a thermosensitivity of –0.03 impulses s–1 °C–1. All three types of neurones displayed inhibitory postsynaptic potentials as indicated by arrow. In all neurones warming decreased and cooling increased the action potential amplitudes.
, 百拇医药 As shown in Table 1, firing rate thermosensitivity was determined in 117 neurones located in or near the PO/AH. This included 71 neurones in the medial preoptic nucleus, 37 neurones in the anterior hypothalamic nucleus, 3 neurones in the lateral preoptic area, 2 neurones in the bed nucleus of the stria terminalis, 2 neurones in medial septum, 1 neurone in the dorsal anterior nucleus, and 1 neurone in the dorsomedial hypothalamus. Of the total, 15 neurones were classified as silent, because they did not have spontaneous firing rates but only produced action potentials when stimulated electrically. Of the remaining 102 spontaneously firing neurones, 19 neurones were warm sensitive, 38 were moderate slope temperature insensitive, and 45 were low slope temperature insensitive. Warm-sensitive, moderate slope temperature-insensitive, and low slope temperature-insensitive neurones represented (respectively) 19%, 37% and 44% of the spontaneously firing neurones and 16%, 33% and 38% of the total population (that included silent neurones). In this study, no recorded neurones met the criterion (i.e. –0.6 impulses s–1 °C–1) for cold sensitivity.
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Figure 2 shows the effect of temperature on the firing rates and integrated membrane potentials for three different types of spontaneously firing neurones. For each neurone, the records in the left panels are the changes in firing rate (FR) and membrane potential (mV) during the changes in tissue temperature (°C). Figure 2 also plots the neuronal firing rates (centre panels) and membrane potentials (right panels, Vm) as a function of tissue temperature. The warm-sensitive neurone in Fig. 2A decreased its firing rate during cooling and increased its firing rate during warming; it had a thermal coefficient of +1.35 impulses s–1 °C–1. The moderate slope temperature-insensitive neurone in Fig. 2B showed more modest firing rate changes during cooling and warming; its thermal coefficient was +0.38 impulses s–1 °C–1. The low slope temperature-insensitive neurone in Fig. 2C showed very little change in its firing rate during the temperature change; its thermal coefficient was +0.14 impulses s–1 °C–1. The three graphs on the right of Fig. 2 show the plots of membrane potential as a function of temperature. Most neurones displayed subtle depolarization during tissue warming and hyperpolarization during tissue cooling; however, this occurred in both warm-sensitive and temperature-insensitive neurones. The three neurones in Fig. 2, for example, showed a wide range of firing rate thermosensitivities, and yet, their membrane potential thermosensitivities were all very small and virtually identical (i.e. 0.47, 0.48 and 0.47 mV °C–1).
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Left panels: time records of firing rate (FR) and membrane potential (Vm) during changes in tissue slice temperature (°C). Centre panels: plots of firing rate as a function of tissue temperature. Right panels: plots of membrane potential as a function of tissue temperature. m is the regression coefficient of the FR and Vm plots. A, warm-sensitive neurone with firing rate thermosensitivity of 1.35 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.47 mV °C–1. B, moderate slope temperature-insensitive neurone with firing rate thermosensitivity of 0.38 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.48 mV °C–1. C, low slope temperature-insensitive neurone with firing rate thermosensitivity of 0.14 impulses s–1 °C–1 and membrane potential thermosensitivity of 0.47 mV °C. Temperature had similar effects on the membrane potential of all three neurones.
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The 15 silent neurones are aligned on the left and had a mean membrane potential (Vm) thermosensitivity of 0.50 ± 0.11 mV °C–1. The mean Vm thermosensitivity for all 117 neurones (including the silent neurones) was 0.53 ± 0.03 mV °C–1. For the spontaneously firing neurones (low slope temperature-insensitive neurones, moderate slope temperature-insensitive neurones, and warm-sensitive neurones), the extremely low regression coefficient (m) and correlation coefficient (r) indicate that there is no correlation between neuronal firing rate thermosensitivity and membrane potential thermosensitivity.
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Effects of temperature on persistent ionic currents
After each neurone was characterized according to its firing rate and membrane potential thermosensitivity, it was voltage clamped at –92 mV in order to determine the effect of temperature on persistent, inward, cationic currents. Figure 4 shows the thermally induced current changes in three different types of PO/AH neurones. Figure 4A and B shows the recordings from the same warm-sensitive and moderate slope temperature-insensitive neurones that were depicted in Fig. 2A and B; Fig. 4C is a low slope temperature-insensitive neurone having a firing rate thermal coefficient of 0.06 impulses s–1 °C–1. The centre panels of Fig. 4 show records of the holding currents during cyclic temperature changes. The holding current balances the inward resting currents and indicates the magnitude of the net resting current. In all three neurones, the resting ionic currents decreased during tissue cooling and increased during tissue warming. These ionic currents are plotted as a function of tissue temperature in the right-hand panels of Fig. 4, and the current thermosensitivities are the regression coefficients (pA °C–1) of these plots. A previous study by Kiyohara et al. (1990) indicated that warm-sensitive neurones display significantly higher current thermosensitivity in the hyperthermic range. Accordingly, Fig. 4 (right) provides separate current thermosensitivities for both the hypothermic range (m1, below 36°C) and hyperthermic range (m2, above 36°C).
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Left panels: firing rate (FR) responses to temperature during current clamp for warm-sensitive neurone (A), moderate slope temperature-insensitive neurone (B), and low slope temperature-insensitive neurone (C). Centre panels: during voltage clamp (–92 mV), time records of holding currents during changes in tissue temperature. Note that during warming, more negative current is required to maintain the voltage clamp at –92 mV, suggesting that there is a net increase in inward cationic current. Right panels: voltage clamp holding currents plotted as a function of temperature. Regression lines indicate the current thermosensitivities: m1 over temperatures below 36°C (), and m2 over temperatures above 36°C ().
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These examples suggest that current thermosensitivity is not a determinant of firing rate thermosensitivity. In fact, in the hyperthermic range the warm-sensitive neurone (Fig. 4A) had the lowest current thermosensitivity (–10.44 pA °C–1), while the low slope temperature-insensitive neurone (Fig. 4C) had the highest current thermosensivitivity (–21.30 pA °C–1). For the entire population, there were no significant differences between the average current thermosensitivities for low slope temperature-insensitive neurones (–9.61 ± 1.24 pA °C–1, n = 9), moderate slope temperature-insensitive neurones (–6.89 ± 0.75 pA °C–1, n = 9), and warm-sensitive neurones (–8.45 ± 1.39 pA °C–1, n = 7). The mean current thermosensitivity for the combined populations (n = 27, including 2 silent neurones) was –7.96 ± 0.66 pA °C–1.
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Figure 5 plots the current thermosensitivity of each neurone as a function of that neurone's firing rate thermosensitivity. This includes seven warm-sensitive neurones, nine moderate slope and nine low slope temperature-insensitive neurones and two silent neurones. The current thermosensitivities in Fig. 5 were determined over the hypothermic range (top panel), the hyperthermic range (middle panel), and the entire combined temperature range (bottom panel). In each case, the extremely low regression lines (m) and correlation coefficients (r) indicate that there is no relationship between current thermosensitivity and firing rate thermosensitivity. In addition to current thermosensitivity expressed as pA °C–1, the effect of temperature on ionic current can also be expressed as Q10, where:
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Thus, Q10 shows the effect of temperature on the relative change in a current compared to some basal current level. A previous study reported that the inward currents of warm-sensitive neurones have a much higher Q10 compared to temperature-insensitive neurones (Kiyohara et al. 1990). For the neurones in the present study, Fig. 6 plots each neurone's current Q10 as a function of its firing rate thermosensitivity. As in Fig. 5, the plots in Fig. 6 indicate that there is no relationship between neuronal current thermosensitivity and firing rate thermosensitivity.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The small regression coefficients (m) and correlation coefficients (r) suggest that thermally induced changes in resting currents do not determine neuronal thermosensitivity. n = 27 neurones.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The small regression coefficients (m) and correlation coefficients (r) suggest that thermally induced changes in resting currents do not determine neuronal thermosensitivity. n = 27 neurones.
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Effects of tetrodotoxin on resting current thermosensitivity
Kiyohara et al. (1990) have indicated that, in the hyperthermic range, the Na+ channel blocker tetrodotoxin (TTX) reduces the current thermosensitivity of warm-sensitive neurones, but has no effect on the current thermosensitivity of temperature-insensitive neurones. To test this hypothesis, neurones were first characterized by their firing rate thermosensitivity (current clamp mode). Following this, during voltage clamp, the same neurones were tested for their current thermosensitivity before and during perfusions with a medium containing 500 nM TTX.
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Figure 7 shows an example of a low slope temperature-insensitive neurone (0.06 impulses s–1 °C–1) and a record (top) of its thermally induced current changes during control medium perfusion and TTX medium perfusion. The current–temperature plots (bottom: centre and right panels) show current thermosensitivity both in the hypothermic range (m1) and hyperthermic range (m2). TTX reduced the neurone's hyperthermic current thermosensitivity by 6.46 pA °C–1; i.e. from –21.30 pA °C–1 (control) to –14.84 pA °C–1 (TTX). Figure 8 shows similar records for a warm-sensitive neurone (1.35 impulses s–1 °C–1) in which TTX reduced hyperthermic current thermosensitivity by 5.27 pA °C–1; i.e. from –10.44 pA °C–1 (control) to –5.17 pA °C–1 (TTX).
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Top record shows current changes before and during 500 nM TTX perfusion. Bottom left panel, neuronal firing rate (FR) plotted as a function of tissue temperature showing a thermosensitivity of 0.06 impulses s–1 °C–1. Bottom centre and right panels, resting current plotted as a function of tissue temperature. Current thermosensitivities are determined below 36°C (m1) and above 36°C (m2).
Top record shows current changes before and during 500 nM TTX perfusion. Bottom left panel, neuronal firing rate (FR) plotted as a function of tissue temperature showing a thermosensitivity of 1.35 impulses s–1 °C–1 in the hyperthermic range. Bottom centre and right panels, resting current plotted as a function of tissue temperature. Current thermosensitivities are determined below 36°C (m1) and above 36°C (m2). TTX decreased current thermosensitivity above 36°C from –10.44 to –5.17 pA °C–1.
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Figure 9 plots the current thermosensitivity as a function of firing rate thermosensitivity for neurones tested with control and TTX media. This included five warm-sensitive neurones, four moderate slope temperature-insensitive neurones, two low slope temperature-insensitive neurones and one silent neurone. The current thermosensitivities in Fig. 9 were determined over the hypothermic range (top panel), the hyperthermic range (middle panel), and the entire combined temperature range (bottom panel). In each case, the extremely low regression lines (m) and correlation coefficients (r) indicate that there is no relationship between current thermosensitivity and firing rate thermosensitivity during both control and TTX perfusions. Similarly, Fig. 10 plots the current Q10 as a function of firing rate thermosensitivity for these same 12 neurones. Once again, the low regression coefficients (m) and correlation coefficients (r) indicate that there is no relationship between the Q10 of the inward current and firing rate thermosensitivity.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The insets show the regression coefficients (m) and correlation coefficients (r) during control (Con) and tetrodotoxin (TTX) perfusions. TTX decreased the ionic currents and current thermosensitivities in all neurones. n = 12 neurones.
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Current thermosensitivities were determined over the hypothermic range < 36°C (top panel), hyperthermic range > 36°C (middle panel), and entire range, ‘Total’ (bottom panel). The vertical dotted line shows 0.8 impulses s–1 °C–1, the minimal criterion for neuronal warm sensitivity. The insets show the regression coefficients (m) and correlation coefficients (r) during control (Con) and tetrodotoxin (TTX) perfusions. n = 12 neurones.
Figures 9 and 10 indicate that even though TTX blockade of Na+ currents often decreased current thermosensitivity, this decrease occurred in both temperature-sensitive and -insensitive neurones. In each figure, the regression lines before and during TTX perfusion are similar, relatively flat, and show no relationship between current and firing rate thermosensitivities. Accordingly, there is no evidence that a TTX-induced decrease in current thermosensitivity is different in warm-sensitive and temperature-insensitive neurones. Nor is there evidence that warm-sensitive neurones possess greater TTX-sensitive Na+ currents at hyperthermic temperatures.
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Discussion
There continues to be debate over the mechanisms that determine thermosensitivity in hypothalamic neurones. Early studies of molluscan neurones suggested that temperature had a greater effect on depolarizing Na+ currents (compared to hyperpolarizing K+ currents), consequently causing warm-induced membrane depolarization and increased firing rates (Gorman & Marmor, 1970; Carpenter, 1981). Recent reviews suggest that PO/AH neuronal thermosensitivity may be due to cationic channels that are part of the temperature-sensitive TRPV (i.e. transient receptor potential) family of vanilloid/capsaicin-sensitive channels (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003), although to date, studies of the thermosensitive properties of TRPV channels have not included hypothalamic neurones. Warming above a threshold temperature opens TRP channels and produces depolarization in dorsal root ganglion neurones. TRPV1 and TRPV2 channels have temperature thresholds greater than 43°C, therefore these channels are not likely explanations for the PO/AH warm-sensitive neurones that show proportional changes in their firing rates over the 30–40°C range. On the other hand, TRPV3 and TRPV4 have lower thresholds and appear to be sensitive over a range in which PO/AH neurones are thermosensitive. Hypothalamic warm-sensitive neurones, however, generally do not display either sensitization or desensitization during repeated temperature changes (Dean & Boulant, 1989; Boulant, 1996). Unfortunately, repetitive thermal stimulation strongly sensitizes TRPV3 channels and desensitizes TRPV4 channels (Benham et al. 2003). Accordingly, TRP channels do not offer a simple explanation for hypothalamic neuronal thermosensitivity. Perhaps more importantly, the mechanism of thermosensitivity for TRP channels is a thermally induced inward cationic (Ca2+ and Na+) current that depolarizes the membrane. While the present study shows that warming causes slight depolarization, this occurs in all neurones and is not restricted to warm-sensitive neurones.
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Some electrophysiological studies have suggested that warm-induced, voltage-gated Na+ currents are the mechanism for thermosensitivity in specific PO/AH neurones (Kiyohara et al. 1990). Using PO/AH tissue slices from 10- to 15-day-old rats, Kobayashi & Takahashi (1993) reported that one population of ‘warm responsive’ neurones showed warm-induced depolarizations leading to increased firing rates. Unfortunately, this previous study employed techniques that created technical problems. While the tissue slices were initially incubated for an hour at 37°C, during the intracellular recordings each neurone was maintained at 25°C, and it was thermally tested by warming to only 35°C. Therefore, the neurones were not maintained or tested over a normo-thermic range of temperatures. In addition, during current clamp recordings in this study, a holding current was continuously injected to maintain each neurone at a –68 mV membrane potential at 25°C. A more recent tissue slice study (Griffin & Boulant, 1995) has shown that such injections of hyperpolarizing holding currents introduce a temperature-dependent artifact that produces spurious membrane potential depolarizations during warming. Since neuronal membrane resistance decreases during warming, a constant negative current injection will produce an apparent decrease in the membrane potential (i.e. depolarization). If no holding current is injected, however, these depolarizations are not observed.
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Another intracellular recording study also claimed that PO/AH neuronal warm sensitivity is due to TTX-sensitive, non-inactivating, voltage-gated Na+ currents (Kiyohara et al. 1990). Again, however, there are methodological problems that would open this earlier study to reinterpretation. The electrode solution in the patch clamp microelectode did not contain ATP, and therefore the intracellular ATP of each recorded neurone would have been rapidly diluted. Also, the Kiyohara study used mechanically and enzymatically dissociated neurones from 1- to 4-week-old rats, and the morphology of these cells suggest that most neuronal processes were amputated or severely damaged. While some of these recorded neurones could be electrically stimulated to produce action potentials, the action potentials did not appear to be normal. For example, at 19°C the spikes did not pass through 0 mV, the spike durations were more than 20 ms, and there were no prominent after-hyperpolarizing potentials (AHPs). Most importantly, these neurones did not have spontaneous firing rates. Therefore, the authors could not classify neurones according to their firing rate thermosensitivities. Instead, the neurones were considered to be ‘warm sensitive’ if (during a –80 mV voltage clamp) they displayed a strong inward current that increased proportionally during warming over the hyperthermic range (i.e. approximately 35–40°C).
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The most obvious criticism of the 1990 Kiyohara et al. study is that the populations of warm-sensitive and temperature-insensitive neurones were not based on their firing rate thermosensitivities, but rather on current thermosensitivities. In the present study, the middle plot in Fig. 5 shows neuronal current thermosensitivities (pA °C–1) over the hyperthermic range (> 36°C). As an example, if one arbitrarily identified neurones as warm sensitive if their current thermosensitivities were greater than 11 pA °C–1, Fig. 5 (middle) shows 8 neurones with high current thermosensitivities. Unfortunately, 6 of these 8 neurones are temperature insensitive based on their firing rate thermosensitivities. Similarly, the middle plot of Fig. 6 shows the Q10 of each neurone's current in the hyperthermic range. Only 3 of 27 neurones had Q10 values greater than 2.5, and all three neurones were low slope temperature-insensitive neurones, based on their firing rate thermosensitivities. It appears, therefore, that there is little value in defining neuronal thermosensitivity based on current thermosensitivity, instead of firing rate thermosensitivity.
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The 1990 Kiyohara study also indicated that tetrodotoxin had a distinct and selective effect on the ‘warm-sensitive’ neurones. Specifically, 5 x 10–7 M TTX strongly reduced the current thermosensitivity of these neurones in the hyperthermic range. The present study found no evidence to support this contention. The middle sections of Figs 9 and 10 show the effects of TTX on the current thermosensitivities (pA °C–1) and current Q10 values in the hyperthermic range. Both figures show no indication that TTX has selective or predominant effects on the current thermosensitivity of warm-sensitive neurones.
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Recent studies offer some explanation for the differential effects of tetrodotoxin observed by Kiyohara et al. (1990), particularly if the mechanically and enzymatically dissociated neurones received varying degrees of injury during the dissociation process. Stretch injury to cultured neurones and their axons produces a Ca2+ influx that is mediated by a TTX-sensitive Na+ current (Wolf et al. 2001; Iwata et al. 2004). TTX blocks this trauma-induced Na+ current and the resulting Ca2+ influx. In the dissociated neurones of the Kiyohara study, it is possible that the most traumatized neurones displayed the strongest TTX-sensitive Na+ currents, and it is not unreasonable to suspect that these injury-induced currents were strongly affected by hyperthermic temperatures. Accordingly, if this current was not as pronounced in the less traumatized neurones, then these cells would constitute the second population of TTX-insensitive neurones.
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The electrode solution in the present study contained 10 mM EGTA (and 1 mM Ca2+) which should reduce intracellular free calcium concentrations (normally 50–100 nM) to less than 20 nM. If some membrane channels are gated by intracellular calcium, then these conductances may be reduced. On the other hand, like the present study, previous intracellular recording studies (Griffin & Boulant, 1995; Griffin et al. 2001; Burgoon & Boulant, 2001) have used this same electrode solution and found that the proportions of warm-sensitive and temperature-insensitive neurones are similar to the proportions found in extracellular recording studies (reviewed in Boulant & Dean, 1986). Accordingly, it does not appear that these reductions in intracellular free calcium significantly alter neuronal thermosensitivity.
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Apart from the tetrodotoxin experiments, the present study did not block synaptic activity, which could contribute to the measured resting potentials and currents. In PO/AH neurones, however, postsynaptic potentials have short durations (i.e. approximately 15 ms) and frequencies of less than 4 events s–1 (Griffin et al. 2001). Consequently, the contribution of postsynaptic activity to the measurements would be minimal. More importantly, there are no differences in the frequencies of postsynaptic potentials between warm-sensitive, temperature-insensitive and silent neurones, and temperature has no significant effect on these frequencies (Griffin et al. 2001). Therefore, it is unlikely that synaptic activity contributed to the results reported here.
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The present study found no evidence to suggest that neuronal thermosensitivity is due to temperature-induced changes in either the resting membrane potential or resting inward cationic currents. On the other hand, there is evidence that thermal effects on more transient currents play a role in neuronal thermosensitivity. Most PO/AH neurones display depolarizing prepotentials that produce action potentials. Figure 1A and B shows that depolarizing prepotentials exist in both warm-sensitive neurones and temperature- insensitive neurones. The rates of depolarization in the prepotentials determine the firing rates. Previous studies indicated that temperature has little or no effect on the prepotental depolarization rates in temperature-insensitive neurones; however, temperature has a pronounced effect on prepotential depolarization rates in warm-sensitive neurones (Curras et al. 1991; Griffin & Boulant, 1995). In warm-sensitive neurones, warming increases the prepotential's rate of depolarization, causing the membrane potential to reach threshold sooner. This, in turn, shortens the interspike interval and increases the firing rate.
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Either increasing depolarizing conductances (Na+ and/or Ca2+) or decreasing hyperpolarizing conductances (K+ and/or Cl–) can induce prepotentials. If the depolarizing prepotentials are caused by inward cationic currents, persistent Na+ currents might be possible candidates (French & Gage, 1985; Huang, 1993; Pennartz et al. 1997). Persistent Na+ currents activate around –60 to –50 mV (i.e. below the action potential thresholds) and can affect the resting membrane potential. This low-threshold Na+ current inactivates slowly, compared to the transient Na+ current associated with action potentials. The voltage dependence and kinetics of persistent Na+ currents suggest that they partially de-inactivate during the after-hyperpolarizing potential (AHP) and may contribute to the subsequent depolarizing prepotential (Pennartz et al. 1997).
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If the depolarizing prepotentials are primarily caused by increasing inward cationic currents, the membrane resistance might be expected to decrease as the prepotential depolarizes toward threshold. In warm-sensitive neurones, however, Griffin et al. (1996) found that the membrane resistance gradually increased as the prepotential depolarized toward threshold. This suggests that the thermosensitive prepotential of warm-sensitive neurones is primarily dependent upon a decreasing conductance, such as an outward (hyperpolaring) K+ current. In contrast, the membrane resistance of temperature-insensitive neurones remained constant during the interspike intervals, suggesting that the prepotentials of temperature-insensitive neurones are more dependent on a balance of both increased inward (e.g. Na+ and Ca2+) and decreased outward (e.g. K+) cationic currents.
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The previous study indicated that a transient K+ current, the potassium A-current (IA), is important in the prepotential of warm-sensitive neurones (Griffin et al. 1996). After each action potential and subsequent AHP, IA activation causes a transient outward potassium current that attenuates further depolarization, keeping the membrane potential at a hyperpolarized level. The IA then slowly inactivates, which allows the membrane potential to slowly depolarize. This could be a major component of the depolarizing prepotential in warm-sensitive neurones, since several studies have shown that IA inactivation rates are extremely temperature sensitive (Lee & Deutsch, 1990; Pahapill & Schlichter, 1990; Huguenard & Prince, 1991; Forsythe et al. 1992; Griffin et al. 1996). Warming increases IA inactivation, and this would increase the prepotential's depolarization rate and increase firing rate. Conversely, cooling slows IA inactivation which would decrease the prepotential's depolarization rate and decrease firing rate. For PO/AH neurones, Griffin et al. (1996) reported that the time constants for IA inactivation were about 78 ms at hyperthermic temperatures (38–40°C) and 156 ms at hypothermic temperatures (31–34°C). This thermally induced difference in IA inactivation rates could have a significant effect on faster firing, warm-sensitive neurones (with short interspike intervals) compared to slower firing temperature-insensitive neurones (with longer interspike intervals).
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The present experiments show that PO/AH neuronal thermosensitivity is not due to thermally induced changes in resting membrane potentials or persistent inward Na+/Ca2+ currents. These experiments therefore support previous studies (Griffin et al. 1996) which indicate that the warm sensitivity of hypothalamic neurones is due to the ionic currents (including IA inactivation) that determine the rate of rise of the depolarizing prepotentials occurring during the brief interval between one action potential and the next.
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While there is evidence that TRPV channels underlie transduction mechanisms in peripheral thermal receptors (Caterina et al. 1997; Guler et al. 2002; Benham et al. 2003; Patapoutian et al. 2003), it is possible that central hypothalamic thermosensitivity evolved from completely different mechanisms. Little is known about the transduction mechanisms of peripheral warm receptors (Pierau, 1996), but it is generally assumed that the basis lies in temperature-induced changes in the resting membrane potential. One importance of peripheral receptors is their response to rapid changes in the external environment. In contrast, central hypothalamic receptors may have evolved to monitor slower changes in the internal environment (e.g. core temperature, plasma glucose and osmolality). In addition to acting as sensors, these same central neurones can also act as effector neurones, controlling various homeostatic responses. Accordingly, it may be important that these central neurones have relatively constant, spontaneous firing rates that can be slowly modified by the internal environment. In these central neurones, the sensing mechanisms probably became linked with the interspike interval that determines spontaneous firing rates. Various ionic conductances determine the interspike interval, and temperature affects most of these conductances. Therefore, it seems reasonable that different sensing strategies could have evolved under these two very different conditions.
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