Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation

Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these "functional" in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of -60 mV evoked in control neurons potassium currents with a peak current of 10.0 +/- 2.5 nA and a large steady state current of 8.5 +/- 2.6 nA, which was still activated from a holding potential of -40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 +/- 2.4 nA; steady state: 3.6 +/- 2.0 nA). This current was inactivated at a holding potential of -40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42 degrees C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices.     

The effect of firing rate on preoptic neuronal thermosensitivity

1. In anaesthetized rabbits, preoptic single units were recorded having positive or negative thermal coefficients (impulses/sec. degrees C) for changes in preoptic temperature.2. A population of forty-two positive coefficient units was divided into four groups based on their level of firing rate at 38 degrees C. In each group, the average thermoresponse curve was determined by averaging the firing rates of the units at 1 degrees C intervals over the 33-43 degrees C range of preoptic temperatures.3. A population of twenty-six negative coefficient units was divided into three groups based on their firing rates at 38 degrees C. Similar average thermoresponse curves were determined for each group.4. As the level of firing rate increased in the positive coefficient units, the preoptic thermosensitivity progressively decreased at temperatures above 39 degrees C, but generally increased at temperatures below 39 degrees C.5. In the negative coefficient units, preoptic thermosensitivity generally increased (especially above 39 degrees C) as the firing rate at 38 degrees C increased.6. The results indicate that in positive coefficient units the level of firing rate determines the temperature range in which units are most thermosensitive. This range reflects whether a neurone is more likely to function in heat-loss or heat-production responses.7. Since peripheral thermal input affects the level of firing rate of positive coefficient units, a neuronal model is suggested to explain the role of peripheral and central thermal signals in temperature regulation.     

The effect of spinal and skin temperatures on the firing rate and thermosensitivity of preoptic neurones

1. In anaesthetized rabbits, preoptic single unit activity was recorded while preoptic, spinal cord and skin temperatures were independently manipulated.2. The units that were insensitive to preoptic temperature were characterized by low firing rates and also by a very low incidence of extrahypothalamic thermosensitivity.3. Thirty-seven units having positive coefficients to preoptic temperature were tested for their response to spinal or skin temperature. Of these, twenty-two units responded to extrahypothalamic temperature, seventeen with positive thermal coefficients. In addition, the incidence of extrahypothalamic thermosensitivity generally increased among the higher firing units.4. Twenty-two units had negative coefficients for preoptic temperature and were tested for their extrahypothalamic thermosensitivities. Of these, sixteen units had dual thermosensitivities, ten with negative coefficients for the extrahypothalamic temperatures. In addition, there was no correlation between the incidence of extrahypothalamic thermosensitivity and the level of firing rate.5. In the units having positive coefficients for preoptic temperature, an increased firing rate, due to extrahypothalamic temperature, generally resulted in a decreased preoptic thermosensitivity. Conversely, a decreased firing rate usually resulted in an increased preoptic thermosensitivity.6. In the units having negative coefficients for preoptic temperature, an increased firing rate, due to extrahypothalamic temperature, usually increased the preoptic thermosensitivity; while a decreased firing rate tended to decrease the sensitivity to preoptic temperature.     

A looming-sensitive pathway responds to changes in the trajectory of object motion

Two identified locust neurons, the lobula giant movement detector (LGMD) and its postsynaptic partner, the descending contralateral movement detector (DCMD), constitute one motion-sensitive pathway in the visual system that responds preferentially to objects that approach on a direct collision course and are implicated in collision-avoidance behavior. Previously described responses to the approach of paired objects and approaches at different time intervals (Guest BB, Gray JR. J Neurophysiol 95: 1428-1441, 2006) suggest that this pathway may also be affected by more complicated movements in the locust's visual environment. To test this possibility we presented stationary locusts with disks traveling along combinations of colliding (looming), noncolliding (translatory), and near-miss trajectories. Distinctly different responses to different trajectories and trajectory changes demonstrate that DCMD responds to complex aspects of local visual motion. DCMD peak firing rates associated with the time of collision remained relatively invariant after a trajectory change from translation to looming. Translatory motion initiated in the frontal visual field generated a larger peak firing rate relative to object motion initiated in the posterior visual field, and the peak varied with simulated distance from the eye. Transition from translation to looming produced a transient decrease in the firing rate, whereas transition away from looming produced a transient increase. The change in firing rate at the time of transition was strongly correlated with unique expansion parameters described by the instantaneous angular acceleration of the leading edge and subtense angle of the disk. However, response time remained invariant. While these results may reflect low spatial resolution of the compound eye, they also suggest that this motion-sensitive pathway may be capable of monitoring dynamic expansion properties of objects that change the trajectory of motion.     

The effect of rate of cooling, time of day and light regime on the shivering response in rabbits.

In man hypothermia exists when core temperature (Tc) falls below 35 degrees C. The onset of hypothermia is often insidious in that it can occur without any particular symptoms of serious discomfort. There is evidence that this decrease in thermosensitivity is most likely to occur when the rate of body cooling is slow. In addition there is some evidence that the susceptibility to become hypothermic varies with the circadian rhythm of Tc, particularly in animals maintained under constant light conditions. A systematic investigation has been carried out to determine whether the thermoregulatory response to body core cooling is affected by the rate of change of body temperature, the time of day at which the cooling takes place and/or by the light regime under which the animals are maintained. The investigation was made in rabbits maintained either under a 12-h light/dark (LD) cycle or under conditions of continuous light (LL). Thermosensitivity (relationship between falling Tc and the induced increase in metabolic heat production) was determined at two different rates of body cooling (1 degrees C decrease in core temperature in either 30 or 160 min.) and at different times of the day. A chronically implanted intravascular heat exchanger was used to extract heat from the animals. The results indicate that neither the rate of body cooling nor the time of day at which the cooling took place had any clear effect on the shivering response. Likewise there was no clear difference in the shivering response of the animals maintained under (LD) conditions as compared to those maintained under (LL) conditions.     

Brief heat shock treatment induces a long-lasting alteration in the glycolipid receptor binding specificity and growth rate of Haemophilus influenzae.

After brief heat shock treatment, clinical strains of nontypeable Haemophilus influenzae show a long-lasting change in the binding specificity for glycolipids and a markedly increased growth rate in vitro. Non-heat-shocked H. influenzae specifically binds to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and gangliotriosylceramide (Gg3) and binds minimally to sulfatoxygalactosylceramide (SGC; also called sulfatide). After a 5-min heat shock at 42 degrees C, strains of H. influenzae showed a marked increase in binding to SGC and acquired the ability to bind to sulfatoxygalactosylglycerol (SGG) in thin-layer chromatography overlays. Additionally, heat-shocked H. influenzae cells showed an increased growth rate (twofold). Increased sulfatide binding and growth rate were retained for approximately 60 generations, after which the heat-shocked organisms reverted to their original glycolipid binding pattern (i.e., PE, Gg3, and Gg4) and growth rate. Such organisms could then be reexposed to heat, and the heat shock phenotype would be reestablished. After exposure of the organisms to brief heat shock, Western blotting of a surface extract of H. influenzae with anti-bovine-brain hsp-70 monoclonal antibody showed an increase in two protein bands at 82 and 60 kDa. This antibody was a potent inhibitor of the binding of heat-shocked H. influenzae to SGC and SGG but had no effect on PE, Gg3, or Gg4 binding in vitro. In contrast, an antibody against an H. influenzae PE-Gg3-Gg4-binding adhesin that was recently identified (J. Busse, E. Hartmann, and C. A. Lingwood, J. Infect. Dis. 175:77-83, 1996) selectively inhibited the organism's binding to PE and Gg3. This indicates that cell surface hsp-70-related heat shock proteins can mediate H. influenzae attachment to sulfoglycolipids following heat shock. We suggest that such increased binding to sulfated glycolipids may be a response to fever following H. influenzae infection in humans.     

Responses of a looming-sensitive neuron to compound and paired object approaches

The lobula giant movement detector (LGMD) and its target neuron, the descending contralateral movement detector (DCMD), constitute a motion-sensitive pathway in the locust visual system that responds preferentially to objects approaching on a collision course. LGMD receptive field properties, anisotropic distribution of local retinotopic inputs across the visual field, and localized habituation to repeated stimuli suggest that this pathway should be sensitive to approaches of individual objects within a complex visual scene. We presented locusts with compound looming objects while recording from the DCMD to test the effects of nonuniform edge expansion on looming responses. We also presented paired objects approaching from different regions of the visual field at nonoverlapping, closely timed and simultaneous approach intervals to study DCMD responses to multiple looming stimuli. We found that looming compound objects evoked characteristic responses in the DCMD and that the time of peak firing was consistent with predicted values based on a weighted ratio of the half size of each distinct object edge and the absolute approach velocity. We also found that the azimuthal position and interval of paired approaches affected DCMD firing properties and that DCMDs responded to individual objects approaching within 106 ms of each other. Moreover, comparisons between individual and paired approaches revealed that overlapping approaches are processed in a strongly sublinear manner. These findings are consistent with biophysical mechanisms that produce nonlinear integration of excitatory and feed-forward inhibitory inputs onto the LGMD that have been shown to underlie responses to looming stimuli.     

Effect of Heat Shock on Susceptibility of Normal Lymphoblasts and of a Heat Shock Protein 70-Defective Tumour Cell Line to Cytotoxic T Lymphocytes In Vitro

The effect of heat shock pretreatment of target cells on their lysability by cytotoxic T lymphocytes was analysed. Killing of Concanavalin A-stimulated normal lymphocytes by minor or major histocompatibi-lity antigen-specific cytotoxic T lymphocytes is unchanged or even slightly enhanced after heat shock, whereas cells of the myeloma line Y3, which is derived from one of the lymphocyte donor strains, become nearly resistant to killing after the same pretreatment. Cold target inhibition experiments show that heat-shocked cells are recognized specifically and that untreated and heat-shocked target cells possess similar inhibitory potential. Y3 cells are unable to express the strongly heat-inducible heat shock protein of 70 kDa (hsp70) after heat shock; the acquired resistance is thus independent of hsp70 induction. Possible mechanisms of the different lysability seen in lymphoblasts and tumour cells after heat shock are discussed.     

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.     

Spontaneous activity and properties of two types of principal neurons from the ventral tegmental area of rat

We investigated the spontaneous activity and properties of freshly isolated ventral tegmental area (VTA) principal neurons by whole cell recording and single-cell RT-PCR. The VTA principal neurons, which were tyrosine hydroxylase-positive and glutamic acid decarboxylase (GAD67)-negative, exhibited low firing frequency and a long action potential (AP) duration. The VTA principal neurons exhibited a calretinin-positive and parvalbumin-negative Ca2+-binding protein mRNA expression pattern. The VTA principal neurons were classified into two subpopulations based on their firing frequency coefficient of variation (CV) at room temperature (21-23 degrees C): irregular-type neurons with a large CV and tonic-type neurons with a small CV. These two firing patterns were also recorded at the temperature of 34 degrees C and in nystatin-perforated patch recording. In VTA principal neurons, the AP afterhyperpolarization (AHP) amplitude contributed to the firing regularity and AHP decay slope contributed to the firing frequency. The AHP amplitude in the irregular-type VTA principal neurons was smaller than that in the tonic-type VTA principal neurons. There was no significant difference in the AHP decay slope between the two-types of VTA principal neurons. Apamin-sensitive small-conductance Ca2+-activated K+ (SK) channels contributed to the AHP and the regular firing of the tonic-type neurons but contributed little to the AHP and firing of the irregular-type neurons. In voltage-clamp tail-current analysis, in both conventional and nystatin-perforated whole cell recording, the apamin-sensitive AHP current density of the tonic-type neurons was significantly larger than that of the irregular-type neurons. We suggest that apamin-sensitive SK current contributes to intrinsic firing differences between the two subpopulations of VTA principal neurons.     

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.     

Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells

Intestinal epithelial cells secrete the chemokine interleukin (IL)-8 in the course of inflammation. Because heat shock proteins (Hsps) and butyrate confer protection to enterocytes, we investigated whether they modulate Salmonella enterica serovar Enteritidis (S. serovar Enteritidis)-induced secretion of IL-8 in enterocyte-like Caco-2 cells. Caco-2 cells incubated with or without butyrate (0-20 m M, 48 h) were infected with S. serovar Enteritidis after (1 h at 42 degrees C, 6 h at 37 degrees C) or without prior heat shock (37 degrees C). Levels of Hsp70 production and IL-8 secretion were analysed using immunostaining of Western blots and enzyme-linked immunosorbent assay (ELISA), respectively. The cells secreted IL-8 in response to S. serovar Enteritidis and produced Hsp70 after heat shock or incubation with butyrate. The IL-8 secretion was inhibited by heat shock and butyrate concentrations as low as 0.2 m M for crypt-like and 1 m M for villous-like cells. In a dose-dependent manner, higher butyrate concentrations enhanced IL-8 secretion to maximal levels followed by a gradual but stable decline. This decline was associated with increasing production of Hsp70 and was more vivid in crypt-like cells. In addition, the higher concentrations abolished the heat shock inhibitory effect. Instead, they promoted the IL-8 production in heat-shocked cells even in the absence of S. serovar Enteritidis. We conclude that heat shock and low concentrations of butyrate inhibit IL-8 production by Caco-2 cells exposed to S. serovar Enteritidis. Higher butyrate concentrations stimulate the chemokine production and override the inhibitory effect of the heat shock. The IL-8 down-regulation could in part be mediated via production of Hsp70.     

Intracellular recordings from medial septal neurons during hippocampal theta rhythm

 The electrophysiological properties of neurons of the medial septal nucleus and the nucleus of the diagnonal band of Broca (MS/DB) were studied using intracellular methods in urethane-anesthetized rats. Three types of rhythmically bursting neurons were identified in vivo on the basis of their action potential shapes and durations, afterhyperpolarizations (AHPs), membrane characteristics, firing rates and sensitivities to the action of muscarinic antagonist: (1) Cells with short-duration action potentials and no AHPs (2 of 34 rhythmic cells, 6%) had high firing rates and extremely reliable bursts with 6–16 spikes per theta cycle, which were highly resistant to scopolamine action. (2) Cells with short-duration action potentials and short-duration AHPs (8 of 34 rhythmic cells, 24%) also had high firing rates and reliable bursts with 4–13 spikes per theta cycle, phase-locked to the negative peak of the dentate theta wave. Hyperpolarizing current injection revealed a brief membrane time constant, time-dependent membrane rectification and a burst of firing at the break. Depolarizing current steps produced high-frequency repetitive trains of action potentials without spike frequency adaptation. The action potential and membrane and characteristics of this cell type are consistent with those described for GABAergic septal neurons. Many of these neurons retained their theta-bursting pattern in the presence of muscarinic antagonist. (3) Cells with long-duration action potentials and long-duration AHPs (24 of 34 rhythmic cells, 70%) had low firing rates, and usually only 1–3 spikes per theta cycle, locked mainly to the positive peak of the dentate theta rhythm. Hyperpolarizing current injection revealed a long membrane time constant and a break potential; a depolarizing pulse caused a train of action potentials with pronounced spike frequency adaptation. The action potential and membrane properties of this cell type are consistent with those reported for cholinergic septal neurons. The theta-related rhythmicity of this cell type was abolished by muscarinic antagonists. The phasic inhibition of ”cholinergic” MS/DB neurons by ”GABAergic” MS/DB neurons, followed by a rebound of their firing, is proposed as a mechanism contributing to recruitment of the whole MS/DB neuronal population into the synchronized rhythmic bursting pattern of activity that underlies the occurrence of the hippocampal theta rhythm. Received: 5 February 1996 / Accepted: 6 November 1996     

Changes in protein turnover after heat shock are related to accumulation of abnormal proteins in aging Drosophila melanogaster

Adult Drosophila melanogaster kept at 24 degrees C show a progressive decline in the synthesis and degradation of proteins with age. After exposure of young, 7-10 days old flies to 20 min of heat shock at 37 degrees C, the incorporation of [35S]-methionine into trichloroacetic acid precipitable proteins decreases to more than 60% of that observed in non-stressed flies. This decrease is also accompanied by a lower protein degradation rate. In contrast, the same stress in old, 49 days old insects results in a 3-fold increase in protein synthesis as compared to either non-heat shocked senescent flies or to young heat-shocked flies. The older flies also have faster protein turnover than unshocked controls. An effect similar to that observed in senescent Drosophila also occurs in young flies that have been fed canavanine, an arginine analogue, before and during heat shock. These results suggest that an age dependent accumulation of abnormal proteins may be responsible for the changes in protein turnover observed in the heat-shocked old flies.     

Orthopteran DCMD neuron: a reevaluation of responses to moving objects. I. Selective responses to approaching objects.

1. The "descending contralateral movement detector" (DCMD) neuron in the locust has been challenged with a variety of moving stimuli, including scenes from a film (Star Wars), moving disks, and images generated by computer. The neuron responds well to any rapid movement. For a dark object moving along a straight path at a uniform velocity, the DCMD gives the strongest response when the object travels directly toward the eye, and the weakest when the object travels away from the eye. Instead of expressing selectivity for movements of small rather than large objects, the DCMD responds preferentially to approaching objects. 2. The neuron shows a clear selectivity for approach over recession for a variety of sizes and velocities of movement both of real objects and in simulated movements. When a disk that subtends > or = 5 degrees at the eye approaches the eye, there are two peaks in spike rate: one immediately after the start of movement; and a second that builds up during the approach. When a disk recedes from the eye, there is a single peak in response as the movement starts. There is a good correlation between spike rate and angular acceleration of the edges of the image over the eye. 3. When an object approaches from a distance sufficient for it to subtend less than one interommatidial angle at the start of its approach, there is a single peak in response. The DCMD tracks the approach, and, if the object moves at 1 m/s or faster, the spike rate increases throughout the duration of object movement. The size of the response depends on the speed of approach. 4. It is unlikely that the DCMD encodes the time to collision accurately, because the response depends on the size as well as the velocity of an approaching object. 5. Wide-field movements suppress the response to an approaching object. The suppression varies with the temporal frequency of the background pattern. 6. Over a wide range of contrasts of object against background, the DCMD gives a stronger response to approaching than to receding objects. For low contrasts, the selectivity is greater for objects that are darker than the background than for objects that are lighter.     

Resilient RTN fast spiking in Kv3.1 null mice suggests redundancy in the action potential repolarization mechanism

Fast spiking (FS), GABAergic neurons of the reticular thalamic nucleus (RTN) are capable of firing high-frequency trains of brief action potentials, with little adaptation. Studies in recombinant systems have shown that high-voltage-activated K(+) channels containing the Kv3.1 and/or Kv3.2 subunits display biophysical properties that may contribute to the FS phenotype. Given that RTN expresses high levels of Kv3.1, with little or no Kv3.2, we tested whether this subunit was required for the fast action potential repolarization mechanism essential to the FS phenotype. Single- and multiple-action potentials were recorded using whole-cell current clamp in RTN neurons from brain slices of wild-type and Kv3.1-deficient mice. At 23 degrees C, action potentials recorded from homozygous Kv3.1 deficient mice (Kv3.1(-/-)) compared with their wild-type (Kv3.1(+/+)) counterparts had reduced amplitudes (-6%) and fast after-hyperpolarizations (-16%). At 34 degrees C, action potentials in Kv3.1(-/-) mice had increased duration (21%) due to a reduced rate of repolarization (-30%) when compared with wild-type controls. Action potential trains in Kv3.1(-/-) were associated with a significantly greater spike decrement and broadening and a diminished firing frequency versus injected current relationship (F/I) at 34 degrees C. There was no change in either spike count or maximum instantaneous frequency during low-threshold Ca(2+) bursts in Kv3.1(-/-) RTN neurons at either temperature tested. Our findings show that Kv3.1 is not solely responsible for fast spikes or high-frequency firing in RTN neurons. This suggests genetic redundancy in the system, possibly in the form of other Kv3 members, which may suffice to maintain the FS phenotype in RTN neurons in the absence of Kv3.1.     

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 μ m 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.     

Electrophysiological properties and thermosensitivity of mouse preoptic and anterior hypothalamic neurons in culture

Responses of mouse preoptic and anterior hypothalamic neurons to variations of temperature are key elements in regulating the setpoint of homeotherms. The goal of the present work was to assess the relevance of culture preparations for investigating the cellular mechanisms underlying thermosensitivity in hypothalamic cells. Our working hypothesis was that some of the main properties of preoptic/anterior hypothalamic neurons in culture are similar to those reported by other authors in slice preparations. Indeed, cultured preoptic/anterior hypothalamic neurons share many of the physiological and morphological properties of neurons in hypothalamic slices. They display heterogenous dendritic arbors and somatic shapes. Most of them are GABAergic and their activity is synaptically driven by the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors. Active membrane properties include a depolarizing "sag" in response to hyperpolarization, and a low threshold spike, which is present in a majority of cells and is generated by T-type Ca2+ channels. In a fraction of the cells, the low threshold spike repeats rhythmically, either spontaneously, or in response to depolarization. The background synaptic noise in cultured neurons is characterized by the presence of numerous postsynaptic potentials which can be easily distinguished from the baseline, thus providing an opportunity for assessing their possible roles in thermosensitivity. An unexpected finding was that GABA-A receptors can generate both hyper- and depolarizing postsynaptic potentials in the same neuron. About 20% of the spontaneously firing preoptic/anterior hypothalamic neurons are warm-sensitive. Warming (32-41 degrees C) depolarizes some cells, a phenomenon which is Na+-dependent and tetrodotoxin-insensitive. The increased firing rate of warm-sensitive cells in response to warming can be prepotential and/or synaptically driven. Overall, our data suggest that a warm-sensitive phenotype is already developed in cultured cells. Therefore, and despite obvious differences in their networks, cultured and slice preparations of hypothalamic neurons can complement each other for further studies of warm-sensitivity at the cellular and molecular level.     

Modulation of pristane-induced arthritis by mycobacterial antigens.

Several prominent mycobacterial protein antigens involved in antibody and T cell responses have been identified as members of highly conserved heat shock protein families. In particular, immune responses to the mycobacterial 65 kD heat shock protein (hsp65) have been implicated in the pathogenesis of autoimmune diseases both in experimental animal models and in man. Additionally, hsp65 has been shown to modulate the course of autoimmune disease in such experimental animal systems. In this report, we have examined the synthesis of heat shock proteins by a fast growing mycobacterial strain, M. vaccae, in heat stressed cultures and used the pristane induced arthritis model to investigate the immunoprophylactic and immunotherapeutic potential of heat killed M. vaccae. Heat shock of M. vaccae cultures at 48 degrees C demonstrated a 43-fold increase in hsp65 over that expressed at 37 degrees C. It is therefore suggested that heat killed M. vaccae contains sufficient hsp that can be presented in the context of appropriate adjuvant properties for use as an effective immunomodulatory agent. Immunisation experiments with M. vaccae revealed that protection or exacerbation of pristane induced arthritis was dependent on the dose (given in an oil or aqueous suspension), route and time of immunisation. In addition, it was demonstrated that the development of arthritis correlated with high levels of agalactosyl IgG and that "protected" animals had significantly depressed levels.