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Physiology and Pharmacology of Temperature Regulation
Intelligence Science and Technology
Graduate School of Informatics
Kyoto University, Kyoto, Japan
POINT: HEAT-INDUCED MEMBRANE DEPOLARIZATION OF HYPOTHALAMIC NEURONS: A PUTATIVE MECHANISM OF CENTRAL THERMOSENSITIVITY
HEATING AND COOLING VARIOUS sites of the preoptic area and anterior hypothalamus (PO/AH) of mammals with fine thermodes reflexively evoke thermoregulatory responses, indicating that many central thermostats regulate temperature (T) of the brain (16). In PO/AH, heating- and cooling-activated neurons have been recorded in vivo (5, 14) and in vitro (1, 2, 6–8, 10, 12, 20). This suggests that some PO/AH neurons have primary T receptors similar to peripheral T receptors, and that these PO/AH neurons themselves act as the central thermostats (9–11). However, it has been controversial whether primary PO/AH neurons show heating-induced depolarization (6, 12) or not (1, 2, 20). Here, we state that primary PO/AH neurons show heating-induced depolarization based on our study (6) and thermo transient receptor potential (TRP) channels (4, 13, 18, 19).
As reported by Hori et al. (7) and Kelso and Boulant (8), T-sensitive neurons in PO/AH slices have been classified into two types. One is primary T-sensitive neurons, which retain thermal sensitivity after synaptic blockade. The other is secondary T-sensitive neurons whose T sensitivities disappear after synaptic blockade. Therefore, we analyzed the ionic basis of primary heating-activated neurons visualized in PO/AH slices with the patch-clamp method (6, 12).
To inhibit evoked release of synaptic vesicles, solution of Ca2+-free/high Mg2+ was prepared from normal Krebs solution by replacing Ca2+ with Mg2+ ions (7, 8). However, this cannot eliminate miniature release of synaptic vesicles (17) that strongly affect postsynaptic neurons in the brain. In the brain, the main excitatory transmitter is glutamate affecting non-N-methyl-D-aspartate (non-NMDA) receptors and the main inhibitory transmitter is GABA affecting GABAA receptors. Therefore, blockers (CNQX) for non-NMDA receptors and blockers (bicuculline methiodide) for GABAA receptors were added to the above Ca2+-free/high-Mg2+ solution. This was finally used as a test solution for synaptic blockade. In this solution, evoked or miniature postsynaptic currents disappeared in postsynaptic neurons (17), indicating that this procedure enabled synaptic blockade.
Secondary T-Sensitive Neurons Do Not Show Heating-Induced Depolarization
In this study, T sensitivity was tested at 32–40°C (6). In normal Krebs solution, we easily found heating-sensitive neurons. In whole cell current-clamp recordings, these neurons evoked action potentials (impulses) spontaneously with regular intervals. When heated, frequency of action potentials increased substantially, but heating-induced depolarization from resting potential did not appear. Thus, these neurons behaved as if they were heating-sensitive neurons without depolarization. However, when normal Krebs solution was replaced by the test solution for synaptic blockade, all of these neurons stopped impulse generation. Therefore, we identified them as secondary T-sensitive neurons, which received inputs from presynaptic T-sensitive cells in the same slice.
These secondary T-sensitive neurons may correspond to the T-sensitive neurons that Boulant and his colleagues have repeatedly recorded in rat PO/AH slices in normal Krebs solution without synaptic blockade. Their heating-activated neurons have not shown membrane depolarization (1, 2, 20).
Primary T-Sensitive Neurons Show Heating-Induced Depolarization
Throughout the following experiments, we recorded neurons in the test solution for synaptic blockade (6). With loose-seal cell-attached patch recordings of impulses, we extracellularly searched for primary T-sensitive neurons. Under synaptic blockade, the proportion of primary T-sensitive neurons was very low. When found, we made whole cell configuration with a new patch pipette. In current-clamp recordings (Fig. 1A), when T increased from 32 to 40°C, membrane potential started to depolarize from resting potential at threshold T (35.5 ± 2.1°C, n = 9). The heating-induced depolarization (receptor potential) increased with T. When the depolarization increased above threshold potential of excitation, these neurons generated impulses repetitively. The discharge frequency increased with depolarization. Thus, primary T-sensitive neurons showed heating-induced depolarization, which evoked impulses repetitively. We have proposed that this neuron acts as a comparator that compares T with its threshold T, as expressed by a symbol of a bimetal switch (Fig. 1B).
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These results implied that there were a few primary T-sensitive neurons in a slice, which sent extensive synaptic outputs to secondary T-sensitive neurons in the same slice.
If there are efferent paths from primary heating-activated neurons in PO/AH to heat-loss effectors in vivo, these comparator neurons act as the central thermostats (Fig. 1B) to regulate the brain T against heat with coolers (9, 11). This assumption is reasonable, because local heating of PO/AH evokes various heat-loss responses (16).
There may be afferent paths from peripheral heating-activated receptors to primary heating-activated neurons in PO/AH. Then, primary PO/AH neurons play two roles. One is to work as the central thermostats against heat. The other is to work as relays (interneurons) on a path from peripheral thermostats (receptors) (9, 11) to heat-loss effectors.
Recently-cloned T receptors (4, 13, 18, 19) exist in the TRP-nonselective cation channel family and are expressed in peripheral sensory neurons. TRPM8 (melastatin) channels are receptors, causing depolarization to 0 mV when T is below threshold (
28°C). TRPV1 (vanilloid), TRPV2, TRPV3, and TRPV4 are receptors, also causing depolarization to 0 mV when T is above their respective thresholds. These thermo TRP channels may be comparators with different active ranges, working based on phase transition (15).
Active T ranges (near-normal core T) of TRPV3 (19) and TRPV4 (4) are similar to those of primary PO/AH neurons. TRPV3 (3) and TRPV4 (4) have been reported to be present in the brain. Therefore, TRPV3 (3) or TRPV4 (4) might be expressed in primary PO/AH neurons. However, we must test this hypothesis by future studies. From these results, we conclude that heating-induced depolarization of primary PO/AH neurons is a putative mechanism of central thermosensitivity.
FOOTNOTES
* Faculty of Information Science and Technology
Osaka Institute of Technology
Hirakata, Osaka, Japan E-mail: skoba{at}i.kyoto-u.ac.jp 
REFERENCES
Intelligence Science and Technology
Graduate School of Informatics
Kyoto University, Kyoto, Japan
Department of Physiology and Cell Biology
College of Medicine
Ohio State University
Columbus, Ohio
E-mail: boulant.1@osu.edu
THE HYPOTHALAMIC PREOPTIC region contains thermosensitive neurons that are important in the regulation of body temperature. Mechanisms for preoptic neuronal thermosensitivity remain controversial, and some laboratories continue to believe that the primary mechanism is a heat-induced depolarization of the resting membrane potential. It is my contention that this is not true. As explained below, my reasons are based on the following lines of evidence. First, preoptic neuronal warm sensitivity is due primarily to the effects of temperature on brief depolarizing prepotentials or pacemaker potentials that determine the intervals between successive action potentials. Second, there is no correlation between firing rate thermosensitivity and the thermosensitivity of resting membrane potentials or resting currents. Third, in some laboratories, experimentally-induced conditions (especially the use of injected holding currents and traumatized neurons) can artificially produce neurons having heat-induced membrane depolarization.
As illustrated in Fig. 1, electrophysiological studies have identified two predominant types of preoptic neurons in both intact animals and hypothalamic tissue slices. The majority of preoptic neurons are classified as "temperature insensitive" (Fig. 1A) and show little or no change in their firing rates during a change in preoptic temperature. In contrast, about 20% of preoptic neurons are classified as "warm sensitive" (Fig. 1B) because their firing rates increase significantly with an increase in temperature (4).
When comparing warm-sensitive and temperature-insensitive neurons, it should be noted that different laboratories employ different criteria for neuronal thermosensitivity, which is defined by the linear regression of the action potential firing rate (i.e., impulses per second) plotted as a function of hypothalamic temperature. Studies by Kobayashi and colleagues (11, 13, 14) have used 0.5 impulses·s–1·°C–1 as the criterion to distinguish between temperature-insensitive and warm-sensitive neurons. For many years, however, our laboratory has used 0.8 impulses·s–1·°C–1 as the minimal criterion for warm sensitivity (2, 3). Both physiological and morphological neuronal differences provide compelling reasons to use this later criterion. If the 0.8 impulses·s–1·°C–1 criterion is used, most preoptic warm-sensitive neurons (unlike temperature insensitive neurons) can be shown to receive afferent information from skin and spinal thermoreceptors (5). This indicates that the warm-sensitive neurons are capable of integrating central and peripheral thermal information, suggesting a functional role in thermoregulatory responses. Similarly, there are morphological differences between neurons having thermosensitivities greater than 0.8 impulses·s–1·°C–1 compared with neurons having lesser thermosensitivities (9). Preoptic temperature-insensitive neurons do not receive peripheral thermal information and often orient their dendrites parallel to the midline third ventricle. In contrast, warm-sensitive neurons tend to orient their dendrites perpendicular to the midline third ventricle, presumably to receive synaptic input from peripheral thermoreceptor pathways that ascend both medially and laterally through the hypothalamus (9). Again, this suggests that that warm-sensitive neurons may integrate central and peripheral thermal information to serve a functional role in thermoregulation.
There continues to be debate over the mechanisms of preoptic neuronal thermosensitivity. Kobayashi and colleagues (11, 14) and Kiyohara et al. (12) suggest that neuronal warm sensitivity is due to heat-induced, inward, cationic currents causing a slow depolarization of a neuron's resting membrane potential. Our own studies, however, indicate that temperature has very little influence on resting potentials, and the small thermal effects on resting membrane potentials are essentially identical for both warm-sensitive and temperature-insensitive neurons (6, 7, 18). Therefore, heat-induced depolarization cannot be the explanation for warm sensitivity.
Warm Sensitivity Is Due To Depolarizing Prepotentials
Our studies indicate that the basis of neuronal thermosensitivity lies in the brief, transient currents that determine the timing between one action potential and the next. As illustrated in Fig. 1, C and D, the primary distinction between temperature-sensitive and -insensitive neurons is the effect of temperature on the pacemaker potential or depolarizing prepotential. These depolarizing prepotentials are not dependent on synaptic inputs and occur in both warm-sensitive neurons and temperature-insensitive neurons. In warm-sensitive neurons, however, temperature strongly affects the prepotential's rate of depolarization. Figure 1D shows that warming increases the depolarizing prepotential's rate of rise. This allows threshold to be reached sooner, causing an increase in firing rate. As illustrated in Fig. 1, it is this prepotential (not the resting membrane potential) that is the primary mechanism of warm sensitivity.
Although various inward cationic currents contribute to the depolarizing prepotential, one study emphasizes the importance of a transient, outward hyperpolarizing K+ current, i.e., the A-type potassium current (IA) (8). Unlike temperature-insensitive neurons, warm-sensitive neurons display a gradual decrease in their net ionic conductance as the prepotential depolarizes toward threshold. This suggests that, in warm-sensitive neurons, much of the depolarizing prepotential is due to a gradually decreasing outward potassium current. The IA is important because temperature strongly affects its inactivation phase (8, 15, 16). Immediately following an action potential, when the membrane begins to depolarize, IA activates and the outward K+ current briefly contributes to membrane hyperpolarization. After a short time, however, this outward K+ current inactivates, which allows the membrane to depolarize. Warming increases the rate of IA inactivation, and in turn, this allows the prepotential to depolarize at a faster rate, as shown for 39°C in Fig. 1D. Also shown in Fig. 1D, is that it is important to note that at all three temperatures, the average membrane potential (i.e., the voltage halfway between each interspike interval) remains unchanged. Therefore, the neuron's overall resting membrane potential is not significantly affected by temperature.
No Correlation Between Firing Rate And Membrane Potential Thermosensitivities
Perhaps the best evidence that neuronal thermosensitivity is not due to heat-induced depolarization comes from a recent study comparing the thermosensitivities of resting membrane potentials and resting currents in all types of preoptic neurons (18). As examples, the effects of temperature on firing rate and resting membrane potential are shown in Fig. 2A for a warm-sensitive neuron (1.35 impulses·s–1·°C–1) and in Fig. 2B for a temperature-insensitive neuron (0.14 impulses·s–1·°C–1). Even though these two neurons have widely differing firing rate thermosensitivities, their membrane potential thermosensitivites are identical (i.e., 0.47 mV/°C). For all 117 neurons recorded in this study, Fig. 2C plots the resting membrane potential thermosensitivity as a function of each neuron's firing rate thermosensitivity. This includes 19 warm-sensitive neurons, 83 temperature-insensitive neurons, and 15 silent neurons. As indicated by the low regression and correlation coefficients in Fig. 2C, there is no correlation between resting membrane thermosensitivity and firing rate thermosensitivity (i.e., r = 0.05). This same study also conducted –92 mV voltage-clamp recordings on these neurons. This was done to maximize inward cationic currents, because other studies suggest that neuronal warm sensitivity is due to persistent, thermally-dependent Na+ or Ca2+ currents (1, 10, 12, 17). Once again, when all of these neuronal current thermosensitivities are plotted as a function of firing rate thermosensitivities, there is no correlation (i.e., r = 0.01). These are strong reasons to reject the hypothesis that neuronal warm sensitivity is due to heat-induced depolarization.
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Why then do some studies report that certain preoptic neurons display heat-induced depolarization? Our previous publications list several possible reasons stemming from experimentally-induced conditions (7, 8, 18). These include grounding problems where the temperature of the indifferent electrode is allowed to change. Also, in some studies (11, 14), the stability of the recording is suspect, particularly when the records are very short (i.e., <2 min) or when the tissue is maintained for long periods at hypothermic temperatures, with brief periods of rapid warming lasting only a minute. Other studies (12) use mechanically- and enzymatically-dissociated neurons from neonatal animals; and because of this, neuronal trauma should be considered a reason for abnormal membrane responses to temperature (reviewed in Ref. 18). Usually, the instability of neuronal recordings is apparent from the amplitude and shape of recorded action potentials and the absence of after-hyperpolarizing potentials following each action potential.
Another problem concerns sample sizes where comparisions are made between a small number of temperature-sensitive and -insensitive neurons. Consider, for example, the data shown in Fig. 2C which plots membrane potential thermosensitivity as a function of firing rate thermosensitivity for 117 preoptic neurons. Because of the variability in membrane potential thermosensitivity, this plot shows that there is no correlation between heat-induced membrane depolarization and firing rate thermosensitivity. On the other hand, within this sample it would certainly be possible to find some warm-sensitive neurons with relatively high membrane potential thermosensitivities and some temperature-insensitive neurons with low membrane potential thermosensitivities.
Finally, an example of experimentally induced artifact comes in studies in which temperature is changed while a neuron is injected with a negative holding current to maintain the membrane potential at a hyperpolarized level. All neurons show thermally induced changes in their input resistance, where warming decreases resistance and cooling increases resistance. By simply applying Ohm's law (V = IR), we see that the recorded voltage (V) is equal to the membrane resistance (R) times the current (I). If a study applies constant negative current injection to artificially "hold" a neuron at a hyperpolarized membrane potential, then a warm-induced decrease in resistance will decrease this "experimentally-imposed" hyperpolarization. In other words, during warming the neuron will appear to depolarize, but this is totally artificial. Our earlier study (7) has shown that if no holding current is applied, temperature has little or no effect on a preoptic neuron's membrane potential. On the other hand, the application of a hyperpolarizing holding current causes a dramatic increase in the same neuron's membrane potential thermosensitivity. These conditions appear to be similar to intracellular recordings conducted by Kobayashi and colleagues (11, 14) in which heat-induced depolarization was observed in some preoptic neurons. One of these studies reported that a hyperpolarizing current was constantly injected to hold the membrane potential at –68 mV (14). Whether negative currents are injected intentionally or unintentionally, these conditions should not be considered physiological and could easily lead to mistaken conclusions regarding the effect of temperature on membrane potentials.
In conclusion, our studies of preoptic neurons find no correlation between the temperature sensitivity of resting membrane potentials and firing rate thermosensitivity. These studies do not support the hypothesis that neuronal warm sensitivity is due to heat-induced membrane depolarization. Instead, the mechanisms for warm sensitivity reside in the brief ionic currents of the prepotential that determines the timing between successive action potentials.
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This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-14644 and NS-045758, the Hitchcock Professorship in Environmental Physiology, and funds provided by Dr. Owen C. Peck.
REFERENCES
Intelligence Science and Technology
Graduate School of Informatics
Kyoto University, Kyoto, Japan
Department of Physiology and Cell Biology
College of Medicine
Ohio State University
Columbus, Ohio
E-mail: boulant.1@osu.edu
We analyzed the ionic basis of primary heating-activated neurons under synaptic blockade. In voltage-clamp recordings, heating activated whole cell conductance with nonselective cation channel properties, similar to those of TRP. This conductance would cause heating-induced depolarization to generate impulses (see our Fig. 1). We conclude that heating-induced depolarization explains thermosensitivity of primary heating-activated neurons in PO/AH.
FOOTNOTES
* Faculty of Information Science and Technology
Osaka Institute of Technology
Hirakata, Osaka, Japan E-mail: skoba{at}i.kyoto-u.ac.jp
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0.8 impulses·s–1·°C–1. B: temperature-insensitive neuron with firing rate thermosensitivity of 0.14 impulses·s–1·°C–1 and membrane potential thermosensitivity of 0.47 mV/°C. A and B, left: time records of firing rate (FR) and averaged membrane potential (MP) during changes in tissue slice temperature (°C). Averaged membrane potential was recorded by filtering out rapid changes in membrane potentials, including action potentials. Middle: firing rate is plotted as a function of tissue temperature. Right: membrane potential is plotted as a function of tissue temperature. m, Regression coefficient of FR and MP plots. Temperature had similar effects on the membrane potential thermosensitivity of both neurons. C: relationship between firing rate thermosensitivity and membrane potential thermosensitivity for 117 neurons recorded in rat hypothalamic tissue slices. This includes 15 silent neurons (plotted, left) that did not have spontaneous activity. For the remaining 102 spontaneously firing neurons, 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. Vertical dashed line indicates the criterion for neuronal warm-sensitivity, 0.8 impulses·s–1·°C–1 (Reproduced with permission from Ref.