Jack A. Boulant

Role of the Preoptic-Anterior Hypothalamus in Thermoregulation and Fever

Lesion and thermal stimulation studies suggest that temperature regulation is controlled by a hierarchy of neural structures. Effector areas for specific thermoregulatory responses are located throughout the brain stem and spinal cord. The preoptic region, in and near the rostral hypothalamus, acts as a coordinating center and strongly influences each of the lower effector areas. The preoptic area contains neurons that are sensitive to subtle changes in hypothalamic or core temperature. Preoptic thermosensitive neurons also receive a wealth of somatosensory input from skin and spinal thermoreceptors. In this way, preoptic neurons compare and integrate central and peripheral thermal information. As a result of this sensory integration and its control over lower effector areas, the preoptic region elicits the thermoregulatory responses that are the most appropriate for both internal and environmental thermal conditions. Thermosensitive preoptic neurons are also affected by endogenous substances, such as pyrogens. By reducing the activity of warm-sensitive neurons and increasing the activity of cold-sensitive neurons, pyrogens cause fever, a state in which all thermoregulatory responses have elevated set-point temperatures.

Intracellular analysis of inherent and synaptic activity in hypothalamic thermosensitive neurones in the rat.

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

Cellular mechanisms for neuronal thermosensitivity in the rat hypothalamus.

1. To study the basic mechanisms of neuronal thermosensitivity, rat hypothalamic tissue slices were used to record and compare intracellular activity of temperature‐sensitive and ‐insensitive neurones. This study tested the hypothesis that different neuronal types have thermally dependent differences in the transient potentials that determine the interspike interval. 2. Most spontaneously firing neurones displayed depolarizing prepotentials that preceded each action potential. In warm‐sensitive neurones, warming increased the rate of rise of the depolarizing prepotential which, in turn, decreased the interspike interval and increased the firing rate. In contrast, temperature had little or no effect on the rate of rise in prepotentials of temperature‐insensitive neurones. 3. Prepotential depolarization can be due to increasing depolarizing conductances or decreasing hyperpolarizing conductances. These are differences in the ionic conductances responsible for prepotentials in temperature‐sensitive and ‐insensitive neurones. In warm‐sensitive neurones, the net ionic conductance decreased as the prepotential depolarized towards threshold, suggesting that the prepotential is primarily determined by a decrease in outward potassium conductances. In contrast, in low‐slope temperature‐insensitive neurones, the net conductance remained constant during the interspike interval, suggesting a more balanced combination of both depolarizing and hyperpolarizing conductances. 4. Transient outward potassium currents, including A‐currents, are important determinants of neuronal firing rates. These currents were identified in all warm‐sensitive neurones tested, as well as in many temperature‐insensitive and silent neurones. Since warming increased the rates of inactivation of these currents, transient K+ currents may contribute to the temperature‐dependent prepotentials of some hypothalamic neurones.

Synaptic and morphological characteristics of temperature‐sensitive and ‐insensitive rat hypothalamic neurones

1 Intracellular recordings were made from neurones in rat hypothalamic tissue slices, primarily in the preoptic area and anterior hypothalamus, a thermoregulatory region that integrates central and peripheral thermal information. The present study compared morphologies and local synaptic inputs of warm‐sensitive and temperature‐insensitive neurones. 2 Warm‐sensitive neurones oriented their dendrites perpendicular to the third ventricle, with medial dendrites directed toward the periventricular region and lateral dendrites directed toward the medial forebrain bundle. In contrast, temperature‐insensitive neurones generally oriented their dendrites parallel to the third ventricle. 3 Both warm‐sensitive and temperature‐insensitive neurones displayed excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs). In most cases, EPSP and IPSP frequencies were not affected by temperature changes, suggesting that temperature‐insensitive neurones are responsible for most local synapses within this hypothalamic network. 4 Two additional neuronal groups were identified: silent neurones having no spontaneous firing rates and EPSP‐driven neurones having action potentials that are primarily dependent on excitatory synaptic input from nearby neurones. Silent neurones had the most extensive dendritic trees, and these branched in all directions. In contrast, EPSP‐driven neurones had the fewest dendrites, and usually the dendrites were oriented in only one direction (either medially or laterally), suggesting that these neurones receive more selective synaptic input.

Temperature effects on membrane potential and input resistance in rat hypothalamic neurones.

1. Whole‐cell recordings were conducted in rat hypothalamic tissue slices to test the hypothesis that thermal changes in membrane potential contribute to neuronal thermosensitivity. Intracellular recordings of membrane potential and input resistance were made in eighty‐two neurones, including twenty‐four silent neurones and fifty‐eight spontaneously firing neurones (22 warm‐sensitive neurones and 36 temperature‐insensitive neurones). Fifty‐seven of the neurones were recorded in the preoptic and anterior hypothalamus. 2. Warm‐sensitive neurones increased their firing rates during increases in temperature (1.07 +/‐ 0.06 impulses s‐1 degree C‐1), but their resting membrane potentials were not affected by temperature (0.06 +/‐ 0.06 mV degree C‐1). Similarly, temperature did not affect the membrane potentials of temperature‐insensitive neurones or silent neurones. 3. Silent neurones had significantly lower input resistances (256.9 +/‐ 20.0 M omega), compared with temperature‐insensitive (362.6 +/‐ 57.2 M omega) and warm‐sensitive neurones (392.2 +/‐ 50.0 M omega). Temperature had the same effect on all three types of neurones, such that resistance increased during cooling and decreased during warming. 4. If hyperpolarizing or depolarizing holding currents were applied to neurones, temperature caused changes in the membrane potentials. This spurious effect can be explained by thermally induced changes in the input resistance. 5. Measurements of electrode tip potentials indicated that artificial changes in membrane potential may also be recorded if grounding electrodes are not isolated from the changes in temperature. 6. These results suggest that physiological changes in resting membrane potentials do not determine neuronal warm sensitivity, and thermal changes in input resistance do not determine the primary differences between warm‐sensitive and temperature‐insensitive hypothalamic neurones.

Altered CNS neuroanatomical organization of spontaneously hypertensive (SHR) rats.

Compared to Wistar-Kyoto (WKY) normotensive control rats, spontaneously hypertensive (SHR) rats have significantly reduced brain weights (-10.6%) and brain volumes (-11.8%). Computerized morphometric analysis of soma cross-sectional areas of single neurons in 12 selected hypothalamic regions revealed significant differences between SHR and WKY animals. Neurons from the periventricular, medial and lateral preoptic nuclei and ventromedial hypothalamus show significantly increased soma cross-sectional areas in SHR animals when compared to normotensive controls. Cells located in the two circumventricular organs, organ vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), also showed significantly greater cross-sectional areas in the SHR. In contrast, neurons in the paraventricular and arcuate nuclei and dorsomedial hypothalamus were significantly smaller in spontaneously hypertensive rats when compared to normotensive controls. Only neurons in supraoptic nucleus, lateral and anterior hypothalamus have equivalent cross-sectional areas in WKY and SHR animals. Differences also exist in the number of cells in certain nuclei in SHR animals. Cell densities in periventricular preoptic nucleus, paraventricular nucleus, arcuate nucleus, ventromedial and anterior hypothalamus, organ vasculosum lamina terminalis and subfornical organ were reduced in SHR animals compared to WKY controls. Because of decreased brain weight and volume along with observed morphometric differences in individual neuronal soma size and cell densities, it is suggested that the SHR brain differs significantly from normotensive control rats. The differences may underlie some of the abnormalities in cardiovascular and endocrine regulation associated with neurogenic hypertension.

Dopamine effects on thermosensitive neurons in hypothalamic tissue slices

To understand the role of hypothalamic dopamine in thermoregulation, single unit activity was recorded in vitro, from constantly perfused tissue slices of rat preoptic area and anterior hypothalamus, PO/AH. The firing rate and thermosensitivity of individual PO/AH neurons were determined before, during and after tissue perfusion with media containing dopamine. Dopamine excited 41% of the warm-sensitive neurons, inhibited 100% of the cold-sensitive neurons, and had no effect on 83% of the temperature-insensitive neurons. In addition, dopamine decreased the local thermosensitivity of most cold-sensitive neurons. There were no major differences between these neuronal types in terms of the time course or latency of dopamines effect. These results are consistent with the hypothesis that dopamine is involved in hypothalamic synapses controlling thermoregulatory responses which oppose increases in body temperature.

Neurophysiological Aspects of Thermoregulation

The ability to react to thermal challenges and regulate body temperature depends on the ability of the nervous system to sense temperature both in the environment and deep within the body core. The basis of the neural control of thermoregulation is the synaptic communication between peripheral and central thermal receptors. Peripheral cutaneous thermoreceptors relay ambient temperature (Ta) information over afferent pathways to central neurons in the lower brain stem and hypothalamus. Many of these central neurons are, themselves, thermosensitive, sensing changes in core temperature that occur, for example, during exercise or under drastic environmental conditions. Perhaps most important is the ability of these neurons to integrate this central thermal information with afferent peripheral thermal information. As a result of this integration, efferent signals are produced that select the most appropriate responses to maintain a constant central temperature.

Temperature effects on neuronal membrane potentials and inward currents in rat hypothalamic tissue slices

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.

A slice chamber for intracellular and extracellular recording during continuous perfusion.

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.