An inexpensive programmable stimulator for the study of thermal sensation

This report describes a simple thermal stimulator that uses a single-layer Peltier device to produce heating or cooling of the skin in the range 0-50 degrees C. The output temperature of the stimulator is determined by an external command voltage. The device incorporates a precision thermometer, and a novel feedback controller which employs out of phase gains to minimize oscillation of the stimulus temperature about the command value. Command waveforms are followed to within 0.05 degrees C for rates of up to 2 degrees C/s.     

Cooling/heating module for tissue chambers and solutions: theoretical considerations and practical design.

We provide a theoretical framework for the estimation of the performance of a modular cooling/heating device for tissue baths. The framework can be adapted to other designs using Peltier elements for cooling and heating. The design employs a Peltier as a heat pump and a flat heat pipe to transport heat to or from a 'remote' site. In the cooling mode heat from the hot side of the Peltier is removed by a heat sink cooled by a fan. The small cross section of the heat pipe permits cooling/heating of tissue chambers on microscope stages or in locations where it would be impractical to mount a Peltier element. The faces of the heat pipe can be used to pre-cool/heat solutions using a simple capillary heat exchanger.     

Rapid cooling aborts seizure-like activity in rodent hippocampal-entorhinal slices

PURPOSE: As a preliminary step in the development of an implantable Peltier device to abort focal neocortical seizures in vivo, we have examined the effect of rapid cooling on seizures in rodent hippocampal-entorhinal slices. METHODS: Seizure-like discharges were induced by exposing the slices to extracellular saline containing 4-aminopyridine (50 micromol/L). RESULTS: When we manually activated a Peltier device that was in direct contact with the slice, seizures terminated within seconds of the onset of cooling, sometimes preceding a detectable decrease in temperature measured near the top of the slice. However, activation of the Peltier device did not stop seizures when slices were no longer in direct physical contact with the device, indicating that this was not a field effect. When cooling was shut off and temperature returned to 33 degrees C, bursting sometimes returned, but a longer-term suppressive effect on seizure activity could be observed. In two of our experiments, a custom computer program automatically detected seizure discharges and triggered a transistor-transistor logic pulse to activate the Peltier device. In these experiments, the Peltier device automatically terminated the slice bursting in less than 4 seconds. When the Peltier device was placed in contact with the normal, exposed cortex of a newborn pig, we found that the cortical temperature decreased rapidly from 36 degrees C to as low as 26 degrees C at a depth of 1.7 mm below the cooling unit. CONCLUSIONS: These experiments show that local cooling may rapidly terminate focal paroxysmal discharges and might be adapted for clinical practice.     

Neocortical seizure termination by focal cooling: temperature dependence and automated seizure detection

PURPOSE: The therapy for focal neocortical epilepsy remains suboptimal. We have, therefore, worked to develop techniques to cool small regions of the neocortical surface for seizure mapping and, ultimately, for long-term suppression of focal seizures. METHODS: We induced focal neocortical seizures in halothane-anesthetized rats by the microinjection of 4-aminopyridine (4-AP) into the motor cortex. The dura over the injection site was cooled with a Peltier device, and the temperature at the interface between dura and Peltier was measured with a thermocouple. In some experiments, seizures were automatically detected by a computer program that activated the Peltier device. RESULTS: Monopolar EEG indicated that our seizures were focal and suppressed when cooling was applied directly over the injection site. The threshold temperature required to observe any reduction in seizure duration was 24 degrees C. The temperature gradient across the cooled neocortex was sharp, with the temperature increasing to 31 degrees C at 4 mm below the Peltier, which was cooled to 20 degrees C. Automatic seizure detection reduced the total seizure duration from 43.4 +/- 33.6 s to 5.6 +/- 5.3 s. CONCLUSIONS: Cooling terminates neocortical seizures when applied very close to the epileptogenic focus. The threshold for seizure termination (24 degrees C) may be lower than the threshold for termination of normal cortical activity, suggesting that this technique will not dissociate the anticonvulsant effect of cooling from the disruption of normal behavior. However, when coupled with automatic seizure detection, focal cooling remains an attractive option for development as a treatment for focal epilepsy.     

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.     

Focal cooling rapidly terminates experimental neocortical seizures.

The efficacy of surgical resection for epilepsy is considerably lower for neocortical epilepsy than for temporal lobe epilepsy. We have explored focal cooling with a thermoelectric (Peltier) device as a potential therapy for neocortical epilepsy. After creating a cranial window in anesthetized rats, we induced seizures by injecting artificial cerebrospinal fluid containing 4-aminopyridine (4-AP), a potassium channel blocker. Within 30 minutes of 4-AP injection, animals developed recurrent seizures (duration 85.7 +/- 26.2 seconds; n = 10 rats) that persisted for 2 hours. When a small Peltier device cooled the exposed cortical surface to 20-25 degrees C at seizure onset, the seizure duration was reduced to 8.4 +/- 5.0 seconds (n = 10 rats; p < 0.001). When the Peltier device was placed close to the cortical surface, but not allowed to make physical contact, there was no effect on seizure duration (104.3 +/- 20.7 seconds; p > 0.05 compared to control). Interestingly, the duration of uncooled seizures was reduced after we allowed the cortex to rewarm from prior cooling. Histological examination of the cortex after cooling has shown no evidence of acute or delayed neuronal injury, and blood pressure and temperature remained stable. It may be possible to use Peltier devices for cortical mapping or, when seizure detection algorithms improve, for chronic seizure control.     

Noxious heat-induced CGRP release from rat sciatic nerve axons in vitro

Noxious heat may act as an endogenous activator of the ionotropic capsaicin receptor (VR1) and of its recently found homologue VRL1, expressed in rat dorsal root ganglion cells and present along their nerve fibres. We have previously reported that capsaicin induces receptor-mediated and Ca++-dependent calcitonin gene-related peptide (CGRP) release from axons of the isolated rat sciatic nerve. Here we extended the investigation to noxious heat stimulation and the transduction mechanisms involved. Heat stimulation augmented the CGRP release from desheathed sciatic nerves in a log-linear manner with a Q10 of approximately 15 and a threshold between 40 and 42 degrees C. The increases were 1.75-fold at 42 degrees C, 3.8-fold at 45 degrees C and 29.1-fold at 52 degrees C; in Ca++-free solution these heat responses were abolished or reduced by 71 and 92%, respectively. Capsazepine (10 microm) and Ruthenium Red (1 microm) used as capsaicin receptor/channel antagonists did not significantly inhibit the heat-induced release. Pretreatment of the nerves with capsaicin (100 microm for 30 min) caused complete desensitization to 1 microm capsaicin, but a significant heat response remained, indicating that heat sensitivity is not restricted to capsaicin-sensitive fibres. The sciatic nerve axons responded to heat, potassium and capsaicin stimulation with a Ca++-dependent CGRP release. Blockade of the capsaicin receptor/channels had little effect on the heat-induced neuropeptide release. We conclude therefore that other heat-activated ion channels than VR1 and VRL1 in capsaicin-sensitive and -insensitive nerve fibres may cause excitation, axonal Ca++ influx and subsequent CGRP release.     

A fast solution switching system with temperature control for single cell measurements

This article describes a perfusion system for biophysical single cell experiments at the physiological temperature. Our system regulates temperature of test solutions using a small heat exchanger that includes several capillaries. Water circulating inside the heat exchanger warms or cools test solutions flowing inside the capillaries. Temperature-controlled solutions are delivered directly to a single cell(s) through a multibarreled manifold that switches solutions bathing a cell in less than 1s. This solution exchange is optimal for patch clamp, single-cell microamperometry, and microfluorometry experiments. Using this system, we demonstrate that exocytosis from pancreatic β cells and activation of TRPV1 channels are temperature sensitive. We also discuss how to measure local temperature near a single cell under investigation.     

A cooling/heating system for use with in vitro preparations: study of temperature effects on newborn rat rhythmic activities.

We have developed a universal stage that is particularly suitable for use with vertebrate and invertebrate in vitro preparations. Based on the Peltier effect, the same compact apparatus can be used to cool or heat within a wide temperature range (from 5 to 50 degrees C). Due to the use of interchangeable rings, the stage is compatible with a wide variety of dishes. We used this system to analyze the effects of temperature on the spinal networks that generate fictive locomotion in newborn rats. The results showed that these spinal networks can be active in a wide temperature range, producing an organized pattern (bursts alternating between the right and left sides) even at very low temperatures (15 degrees C). From 15 to 20 degrees C the period decreased before reaching a plateau (between 20 to 30 degrees C).     

A proportional controlled thermoregulator circuit of simple design.

This paper describes a proportional controlled thermoregulator circuit. The instrument controls D.C. current in the heating device, precluding the introduction of radiated interference by the heater. This circuit is capable of regulating the temperature of a typical decerebrated cat to within 0.1 degrees C. A dual channel temperature regulator can be constructed for about $ 300.     

An instrument using a multiple layer peltier device to change skin temperature rapidly

A thermal stimulator that uses multiple layer Peltier devices produces rapid heating or cooling of the skin of experimental animals. Rate of change of temperature can be controlled from 1°C/sec to 5°/sec (cooling) or to 10°/sec (heating). Rapid thermal transients are not accompanied by thermal overshoot. Maintenance of and return to a preset temperature are within ± 0.05°.     

Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain

Protons cause a sustained depolarization of human dorsal root ganglion (DRG) neurons [Baumann et al. (1996) Pain, 65, 31-38]. In the present study we sought to determine which ion channels are expressed in human DRG neurons that could mediate the sustained responses observed in the patch-clamp recordings. RT-PCR of material from the DRG tissue revealed the presence of mRNAs for a nonselective cation channel that is activated by protons (TRPV1) and background potassium channels that are blocked by protons (TASK-1, TASK-3 and Kir2.3). Highly acidic solution (pH 5.4) applied to cultured DRG neurons evoked prolonged currents that were associated with a net increase in membrane conductance. Consistent with the involvement of TRPV1, these proton-evoked currents were blocked by capsazepine and were only found in neurons that responded to capsaicin with an increase in membrane conductance. Less acidic extracellular solution (pH 6.0) evoked such currents only rarely, but was able to strongly enhance the currents evoked by capsaicin. Capsazepine (1 microm) blocked the currents evoked by capsaicin at pH 7.35, as well as the potentiated responses to capsaicin at pH 6.0. In neurons that were not excited by capsaicin, moderate extracellular acidification (pH 6.0) caused a sustained decrease in resting membrane conductance. The decrease in membrane conductance by protons was associated with inhibition of background potassium channels. This excitatory effect of protons was not blocked by capsazepine. We conclude that in most neurons the sustained depolarization in response to moderately acidic solutions is the result of blocked background potassium channels. In a subset of neurons, TRPV1 also contributes.     

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices

During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.     

GABA- and glycine-induced Cl channels in cultured mouse spinal neurons require the same energy to close

Fluctuation analysis has been used to study the influence of temperature on the conductance and mean open time of Cl- permeable ion channels induced by gamma-aminobutyric acid (GABA) and glycine in the membrane of tissue cultured mouse spinal neurons. The conductance of GABA- and glycine-activated channels increased slightly at higher temperatures (Q10 approximately 1.3). The mean open times of these channels decreased at higher temperatures (Q10 approximately 3). Arrhenius plots for the open times of GABA- and glycine-induced channels were parallel and showed no obvious transition behavior over the temperature range 16-40 degrees C.     

Relation between temperature and processes of spontaneous quantum and non-quantum mediator release from motor nerve endings of the mouse

The spontaneous quantal and nonquantal acetylcholine release was investigated at the temperature range from 10 to 35 degrees C in white mouse semidiaphragm. The quantal release was evaluated by calculation of miniature end-plate potentials frequency, while the nonquantal one--from the H-effect value. The spontaneous quantal release increased exponentially with the temperature growth. The temperature dependence of the nonquantal release showed two relative maxima: at 20 degrees and 35 degrees C. At 10 degrees C the nonquantal release was absent. The value of calculated effective energy of activation of the quantal release was 57.0 kJ/mol in the investigated temperature range. The effective energy of activation for the nonquantal release process in intervals 15-20 degrees C and 25-35 degrees C was 45.5 and 38.2 kJ/mol, relatively. It is suggested that the nonquantal release is rather due to active transport processes than to simple diffusion of acetylcholine molecules.     

Single-channel characteristics of the large-conductance anion channel in rat cortical astrocytes in primary culture

Cultured rat cortical astrocytes, in addition to a variety of voltage-sensitive potassium channels, also express anion channels. However, the behavior and regulation of these anion channels have been far less studied. This paper describes a patch-clamp study on a voltage-sensitive 200–300 pS high-conductance single-channel anion current, which seems to possess at least five different open sublevels or, alternatively, be formed froin five or more small-conductance ion channels linked together. This channel is voltage dependent, showing a bell-shaped open probability curve with highest open probability close to the reversal potential (zero-current). Although potassium channels are commonly detected in astrocytes in cell-attached and excised patches with both normal osmolarity and hypoosmotic solutions, the occurrence of the anion channel is clearly increased in isolated patches when hypoosmotic bath solutions are used. Also, cell aging in culture and the preparation of secondary cell cultures by trypsinization seem to increase the rate of occurrence of the anion channel. Though this channel is more routinely seen when a membrane patch is excised from the cell, occasionally cell-attached configurations with instant channel activity can be formed. While the modulation of this anion channel was being studied, it was found to be blocked by an anion transport inhibitor, L-644,711, reported to affect cell volume regulation in astrocytes. © 1993 Wiley-Liss, Inc.     

首次发现大鼠海马CA1区神经元的TRP样通道：脑片膜片钳记录及2-氨乙氧二苯基硼酸酯和镧验证

TRP通道最初从果蝇中发现,目前已发展成为具有分子同源性的一个阳离子通道超家族。哺乳动物TRP通道蛋白是具有6个跨膜域和胞浆N、C末端的阳离子通透性通道,并按照其序列相似性,分为TRPC、TRPV、TRPM、TRPA1等亚家族。与TRP通道功能相关的特性还仍然未知,但TRP通道有可能介导阳离子因循其电化学梯度的跨膜流,进而增加细胞内的Ca2+、Na+浓度,并使细胞在多型激活过程中去极化。目前认为,TRP通道与大脑神经元功能的多种特性相关联。我们采用全细胞膜片钳方法,在急性分离的大鼠脑片中,首次记录到了海马CA1神经元的特征性TRP样通道,并且发现2-APB 或镧灌流能够增强该通道电流。     

Dorsal horn neuron response patterns to graded heat stimuli in the rat

Sensory input from various receptors in the periphery first becomes integrated in the spinal cord dorsal horn. The response of the spinal cord dorsal horn neurons to mechanical stimuli are classified as low threshold, high threshold, and wide dynamic range neurons. However, the response pattern of deep dorsal horn cells to heat has not been well described. In this study, the response of the spinal cord dorsal horn neurons to graded heat stimuli were characterized in 147 neurons in rats by extracellular single cell recording. After a differentiable cell was identified, the Peltier heat stimulator was applied to the receptive field and the base temperature was set at 30 degrees C. The heat stimulus was delivered for 10 s from 37-51 degrees C in 2 degrees C increments, with an inter-stimulus interval of 30 s. Out of the 147 neurons, five statistically distinguishable response patterns were identified by latent class cluster analysis. It is concluded that variation of temperature may account for the observed results and indicate functionally different subsets of heat-responsive cells in the deep dorsal horn.     

Thermal and nociceptive sensations from menthol and their suppression by dynamic contact

It was recently found that cooling the skin to temperatures as mild as 25-30 degrees C can induce nociceptive sensations (burning, stinging or pricking) that are strongly suppressed by dynamic contact between the thermode and skin (contact suppression). Here we investigated whether nociceptive sensations produced by menthol can be similarly suppressed. In the first experiment subjects rated the intensity of cold and burning/stinging/pricking sensations before and after application of 10% l-menthol to the forearm. Ratings were compared at resting skin temperature ( approximately 33 degrees C) and at 28, 24, or 20 degrees C during static or dynamic contact cooling via a Peltier thermode. At resting skin temperature, menthol produced cold and nociceptive sensations, both of which were suppressed by dynamic contact. When the skin was cooled during static contact, menthol increased nociceptive sensations but not cold sensations; when the skin was cooled during dynamic contact, cold sensations were again unchanged while nociceptive sensations were suppressed. A second experiment tested whether contact suppression of menthol's cold and nociceptive sensations at resting skin temperature was caused by slight deviations of thermode temperature above skin temperature. The results showed that suppression occurred even when the thermode was slightly cooler (-0.5 degrees C) than the skin. These findings support other evidence that the menthol-sensitive channel, TRPM8, plays a role in cold nociception, and raise new questions about how dynamic tactile stimulation may modify perception of nonpainful cold stimulation.