Short-term treatment with the GABAA receptor antagonist pentylenetetrazole produces a sustained pro-cognitive benefit in a mouse model of Down's syndrome.

BACKGROUND AND PURPOSE: Down's syndrome is a common genetic cause of intellectual disability, for which there are no drug therapies. Mechanistic studies in a model of Down's syndrome [Ts65Dn (TS) mice] demonstrated that impaired cognitive function was due to excessive neuronal inhibitory tone. These deficits were normalized by low doses of GABAA receptor antagonists in adult animals. In this study, we explore the therapeutic potential of pentylenetetrazole, a GABAA receptor antagonist with a history of safe use in humans. EXPERIMENTAL APPROACH: Long-term memory was assessed by the novel object recognition test in different cohorts of TS mice after a delay following a short-term chronic treatment with pentylenetetrazole. Seizure susceptibility, an index of treatment safety, was studied by means of EEG, behaviour and hippocampus morphology. EEG spectral analysis was used as a bio-marker of the treatment. KEY RESULTS: PTZ has a wide therapeutic window (0.03-3 mg·kg(-1)) that is >10-1000-fold below its seizure threshold and chronic pentylenetetrazole treatment did not lower the seizure threshold. Short-term, low, chronic dose regimens of pentylenetetrazole elicited long-lasting (>1 week) normalization of cognitive function in young and aged mice. Pentylenetetrazole effectiveness was dependent on the time of treatment; cognitive performance improved after treatment during the light (inactive) phase, but not during the dark (active) phase. Chronic pentylenetetrazole treatment normalized EEG power spectra in TS mice. CONCLUSIONS AND IMPLICATIONS: Low doses of pentylenetetrazole were safe, produced long-lasting cognitive improvements and have the potential of fulfilling an unmet therapeutic need in Down's syndrome.

Heat loss through the glabrous skin surfaces of heavily insulated, heat-stressed individuals.

Insulation reduces heat exchange between a body and the environment. Glabrous (nonhairy) skin surfaces (palms of the hands, soles of the feet, face, and ears) constitute a small percentage of total body surface area but contain specialized vascular structures that facilitate heat loss. We have previously reported that cooling the glabrous skin surfaces is effective in alleviating heat stress and that the application of local subatmospheric pressure enhances the effect. In this paper, we compare the effects of cooling multiple glabrous skin surfaces with and without vacuum on thermal recovery in heavily insulated heat-stressed individuals. Esophageal temperatures (T(es)) and heart rates were monitored throughout the trials. Water loss was determined from pre- and post-trial nude weights. Treadmill exercise (5.6 km/h, 9-16% slope, and 25-45 min duration) in a hot environment (41.5 degrees C, 20-30% relative humidity) while wearing insulating pants and jackets was used to induce heat stress (T(es)>or=39 degrees C). For postexercise recovery, the subjects donned additional insulation (a balaclava, winter gloves, and impermeable boot covers) and rested in the hot environment for 60 min. Postexercise cooling treatments included control (no cooling) or the application of a 10 degrees C closed water circulating system to (a) the hand(s) with or without application of a local subatmospheric pressure, (b) the face, (c) the feet, or (d) multiple glabrous skin regions. Following exercise induction of heat stress in heavily insulated subjects, the rate of recovery of T(es) was 0.4+/-0.2 degrees C/h(n=12), but with application of cooling to one hand, the rate was 0.8+/-0.3 degrees C/h(n=12), and with one hand cooling with subatmospheric pressure, the rate was 1.0+/-0.2 degrees C/h(n=12). Cooling alone yielded two responses, one resembling that of cooling with subatmospheric pressure (n=8) and one resembling that of no cooling (n=4). The effect of treating multiple surfaces was additive (no cooling, DeltaT(es)=-0.4+/-0.2 degrees C; one hand, -0.9+/-0.3 degrees C; face, -1.0+/-0.3 degrees C; two hands, -1.3+/-0.1 degrees C; two feet, -1.3+/-0.3 degrees C; and face, feet, and hands, -1.6+/-0.2 degrees C). Cooling treatments had a similar effect on water loss and final resting heart rate. In heat-stressed resting subjects, cooling the glabrous skin regions was effective in lowering T(es). Under this protocol, the application of local subatmospheric pressure did not significantly increase heat transfer per se but, presumably, increased the likelihood of an effect.

Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain.

Previous studies have documented changes in expression of the immediate early gene (IEG) c-fos and Fos protein in the brain between sleep and wakefulness. Such expression differences implicate changes in transcriptional regulation across behavioral states and suggest that other transcription factors may also be affected. In the current study, we examined the expression of seven fos/jun family member mRNAs (c-fos, fosB, fos related antigen (fra)1, fra-2, junB, c-jun, and junD) and three other IEG mRNAs (egr-1, egr-3, and nur77) in mouse brain following short-term (6 h) sleep deprivation (SD) and 4 h recovery sleep (RS) after SD. Gene expression was quantified in seven brain regions by real-time reverse transcription-polymerase chain reaction (RT-PCR). Multivariate analysis of variance revealed statistically significant variation in cerebral cortex, basal forebrain, thalamus and cerebellum. Levels of c-fos and fosB mRNA were elevated during SD in all four of these brain regions. In the cerebral cortex, junB mRNA was also elevated during SD whereas, in the basal forebrain, fra-1 and fra-2 mRNA levels increased in this condition. During RS, the only IEG mRNA to undergo significant increase was fra-2 in the cortex. C-jun and junD mRNAs were invariant across experimental conditions. These results indicate that the expression of fos/jun family members is diverse during SD. Among other IEGs, nur77 mRNA expression across conditions was similar to c-fos and fosB, egr-1 mRNA was elevated during SD in the cortex and basal forebrain, and egr-3 mRNA was elevated in the cortex during both SD and RS. The similarity of fosB and nur77 expression to c-fos expression indicates that these genes might also be useful markers of functional activity. Along with our previous results, the increased levels of fra-2 and egr-3 mRNAs during RS reported here suggest that increased mRNA expression during sleep is rare and may be anatomically restricted.

Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep.

Although sleep is thought to be restorative from prior wakeful activities, it is not clear what is being restored. To determine whether the synthesis of macromolecules is increased in the cerebral cortex during sleep, we subjected C57BL/6 mice to 6 hours of sleep deprivation and then screened the expression of 1176 genes of known function by using cDNA arrays. The expression of the heat shock proteins (HSP), endoplasmic reticulum protein (ERp72) and glucose-regulated protein (GRp78), was among the genes whose expression was significantly elevated in the cortex during sleep deprivation, whereas GRp78 and GRp94 mRNAs were elevated in the cortex during recovery sleep after sleep deprivation, as confirmed by conventional and quantitative real-time polymerase chain reaction and/or Northern analyses. A systematic evaluation of the expression of six heat shock protein family members (ERP72, GRp78, GRp94, HSP27, HSP70-1, and HSP84) in seven brain regions revealed increased mRNA levels in cortex, basal forebrain, hypothalamus, cerebellum and medulla during sleep deprivation, whereas increased mRNA levels during recovery sleep were limited to the cortex and medulla. Immunohistochemical studies identified increased numbers of GRp78-, GRp94-, and ERp72-immunoreactive cells in the dorsal and lateral cortex during sleep deprivation but, during recovery sleep, elevated numbers of these cells were found only in the lateral cortex. In the medulla, increased numbers of GRp94-immunoreactive cells were observed in nucleus tractus solitarius, dorsal motor nucleus of the vagus and the rostroventrolateral medulla during recovery sleep. The widespread increase of heat shock protein family mRNAs in brain during sleep deprivation may be a neuroprotective response to prolonged wakefulness. In contrast, the relatively limited heat shock protein family mRNA expression during recovery sleep may be related to the role of heat shock proteins in protein biogenesis and thus to the restorative function of sleep.

Sleep deprivation elevates plasma corticosterone levels in neonatal rats.

Plasma corticosterone (CORT) levels were measured after short periods of sleep deprivation in rats at postnatal days 12, 16, 20, and 24. There was an age-dependent increase in basal CORT levels and sleep deprivation significantly elevated CORT at all ages compared to non-sleep deprived controls. The levels of CORT after sleep deprivation in P16, P20 and P24 animals were similar, resulting in an age-dependent decrease of the magnitude of the response. Sleep deprived P12 animals had lower levels of CORT. However, the observed response to sleep deprivation suggests that sleep loss is a significant stressor at this age. These observations suggest that younger animals are more sensitive to the effects of mild sleep deprivation than older ones.

Prenatal nicotine alters vigilance states and AchR gene expression in the neonatal rat: implications for SIDS.

Maternal smoking is a major risk factor for sudden infant death syndrome (SIDS). The mechanisms by which cigarette smoke predisposes infants to SIDS are not known. We examined the effects of prenatal nicotine exposure on sleep/wake ontogenesis and central cholinergic receptor gene expression in the neonatal rat. Prenatal nicotine exposure transiently increased sleep continuity and accelerated sleep/wake ontogeny in the neonatal rat. Prenatal nicotine also upregulated nicotinic and muscarinic cholinergic receptor mRNAs in brain regions involved in regulating vigilance states. These findings suggest that the nicotine contained in cigarette smoke may predispose human infants to SIDS by interfering with the normal maturation of sleep and wake.

Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice.

The hypocretin/orexin ligand-receptor system has recently been implicated in the sleep disorder narcolepsy. During the dark (active) period, null mutants of the prepro-orexin (prepro-hypocretin) gene have cataplectic attacks and increased levels of both rapid eye movement (REM) and non-REM (NREM) sleep. Intracerebroventricular injection of one of the encoded neuropeptides, orexin-A, early in the light period increases wakefulness and reduces REM sleep in the rat, suggesting that this system may be involved in the normal regulation of sleep and wakefulness. To further test this hypothesis, we measured hypocretin (hcrt) mRNA levels by both Northern hybridization and Taqman analysis in mouse and rat hypothalamus after short-term (6 h) sleep deprivation (SD) and 2-4 hours after recovery from SD. Although our SD procedures effectively induced a sleep debt and increased c-fos mRNA expression in the cortex and hypothalamus as described by other investigators, we found that hcrt mRNA levels were not significantly changed in either species either after SD or after recovery from SD. If the hcrt system is involved in normal regulation of sleep and wakefulness, longer periods of SD may be necessary to affect hcrt mRNA levels or changes may occur at the protein rather than mRNA level. Alternatively, this system may also be involved in another function that counterbalances any SD-induced changes in hcrt mRNA levels.

Siberian hamsters that fail to reentrain to the photocycle have suppressed melatonin levels.

Siberian hamsters readily reentrain to a 3-h phase delay of the photocycle (16 h light/day) but fail to reentrain to a 5-h phase delay. This study tested whether melatonin production was suppressed in animals that failed to reentrain. Melatonin was measured on the day before, day of, or several days after each phase shift. Melatonin levels measured 4 h after dark onset were approximately 83 microg/ml on the day before each phase delay and undetectable (<6 microg/ml) during the light phase on the day of the phase shift. Activity onsets regained their prior phase relationship to the photocycle 4 (3 h) or 5 (5 h) days after the phase shift; on that day, melatonin levels were measured 4 h after dark onset. Melatonin levels were unaffected by the 3-h phase delay (>57.6 microg/ml) but were undetectable after a 5-h phase delay (<8 microg/ml). Thus melatonin remained suppressed only after the phase delay to which hamsters also fail to reentrain. This relationship suggests that the propensity for reentrainment may be influenced by changes in melatonin production following a phase shift of the photocycle.

HSP70 expression is increased during the day in a diurnal animal, the golden-mantled ground squirrel Spermophilus lateralis.

Heat shock protein 70 (HSP70) gene expression was studied in a seasonal hibernator, the diurnal ground squirrel, Spermophilus lateralis. RNA transcripts of 2.7 and 2.9 kb hybridizing to an HSP70 cDNA were expressed in both brain and peripheral tissues of pre-hibernation euthermic animals; higher levels of expression were observed during the day than during nighttime samples. A decline in the expression of both transcripts occurred in all tissues examined during hibernation that remained low throughout the hibernation season, including the interbout euthermic periods and regardless of time of day. Quantitative comparisons showed pre-hibernation nighttime HSP70 expression to be as low as that observed during hibernation, despite the drastic increase in metabolic state and nearly 30 degrees C difference in body temperature. In contrast to HSP70, some mRNAs, such as beta-actin and HSP60, remained relatively constant, while others, such as glyceraldehyde 3-phosphate dehydrogenase, increased in specific tissues during the hibernation season. These results indicate that the expression of a highly conserved gene involved in protection from cellular stress, HSP70, can vary with an animal's arousal state.

Circadian rhythms in the suprachiasmatic nucleus are temperature-compensated and phase-shifted by heat pulses in vitro.

Temperature compensation and the effects of heat pulses on rhythm phase were assessed in the suprachiasmatic nucleus (SCN). Circadian neuronal rhythms were recorded from the rat SCN at 37 and 31 degrees C in vitro. Rhythm period was 23.9 +/- 0.1 and 23.7 +/- 0.1 hr at 37 and 31 degrees C, respectively; the Q(10) for tau was 0.99. Heat pulses were administered at various circadian times (CTs) by increasing SCN temperature from 34 to 37 degrees C for 2 hr. Phase delays and advances were observed during early and late subjective night, respectively, and no phase shifts were obtained during midsubjective day. Maximum phase delays of 2.2 +/- 0.3 hr were obtained at CT 14, and maximum phase advances of 3.5 +/- 0.2 hr were obtained at CT 20. Phase delays were not blocked by a combination of NMDA [AP-5 (100 microM)] and non-NMDA [CNQX (10 microM)] receptor antagonists or by tetrodotoxin (TTX) at concentrations of 1 or 3 microM. The phase response curve for heat pulses is similar to ones obtained with light pulses for behavioral rhythms. These data demonstrate that circadian pacemaker period in the rat SCN is temperature-compensated over a physiological range of temperatures. Phase delays were not caused by activation of ionotropic glutamate receptors, release of other neurotransmitters, or temperature-dependent increases in metabolism associated with action potentials. Heat pulses may have phase-shifted rhythms by directly altering transcriptional or translational events in SCN pacemaker cells.

8-OH-DPAT-sensitive neurons in the nucleus raphe magnus modulate thermoregulatory output in rats.

The nucleus raphe magnus (NRM) is purported to be a relay through which peripheral thermoafferent information is transmitted to thermointegrative centers located in the preoptic/anterior hypothalamus (POAH). Therefore, suppression of neural activity in the NRM should reduce thermoregulatory responses to peripheral thermal challenges, but not affect responses elicited by manipulation of POAH temperature. At low ambient temperatures lidocaine injections into the NRM of nonanesthetized rats resulted in decreases in POAH temperature, oxygen consumption, and electromyographic activity. At a warm ambient temperature, lidocaine injections into the NRM decreased the elevations in oxygen consumption and electromyographic activity elicited by cooling the POAH. The effects of lidocaine injections were duplicated by injection of a 5-HT(1A) agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) into the NRM. The effect of 8-OH-DPAT was eliminated by pre-treatment with a selective autoreceptor antagonist. These results suggest that NRM 5-HT neurons are modulating the relationship between output of thermointegrative centers and thermoregulatory effector responses rather than processing thermoafferent information.

Gene expression in the brain across the hibernation cycle.

The purpose of this study was to characterize changes in gene expression in the brain of a seasonal hibernator, the golden-mantled ground squirrel, Spermophilus lateralis, during the hibernation season. Very little information is available on molecular changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern analysis. Although some small differences were observed across hibernation states, none of the 29 had significant changes. However, a more direct approach, investigating expression of putative hibernation-responsive genes by Northern analysis, revealed an increase in expression of transcription factors c-fos, junB, and c-Jun, but not junD, commencing during late torpor and peaking during the arousal phase of individual hibernation bouts. In contrast, prostaglandin D2 synthase declined during late torpor and arousal but returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine decarboxylase, and a sleep-related transcript SD464 did not change significantly during individual hibernation bouts. We also observed no decline in total RNA or total mRNA during torpor; such a decline had been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that accompany hibernation involve only modest reprogramming of gene expression or steady-state mRNA levels.

Developmental changes in nicotinic receptor mRNAs and responses to nicotine in the suprachiasmatic nucleus and other brain regions.

Our previous studies demonstrated that nicotine induces c-fos expression in the suprachiasmatic nucleus (SCN) of the rat during a narrow developmental window occurring in the perinatal period. We have extended these observations by showing that c-fos cannot be induced in the adult SCN by nicotine even during the subjective night, when phase shifts do occur. In contrast to the SCN, significant induction of c-fos and NGFI-A was observed in the medial habenula and paraventricular nucleus at all circadian times. In the fetal rat SCN we show that NGFI-A and junB are also induced by nicotine, but not c-jun. To investigate whether changes in nicotinic acetylcholine receptor (nAChR) expression in the SCN may underlie this change in sensitivity during the perinatal period, we examined nAChR mRNAs across this developmental period. By Northern analyses, alpha2, alpha3 and alpha4 subunit mRNAs are relatively abundant in the fetal SCN but decline substantially in the adult. alpha7 mRNA increases substantially while beta2 mRNA is relatively abundant throughout development. We also examine expression in the whole mouse brain beginning at embryonic day 11. Many mRNA sizes for nAChR subunits in both the rat and mouse are characterized here for the first time by Northern analyses and some show very large changes in expression across development. In particular, a small 1.4 kb alpha2-related mRNA is highly expressed during early development, perhaps indicating an important novel function for this subunit.

Sleep after arousal from hibernation is not homeostatically regulated.

Electroencephalographic slow-wave activity (SWA) in non-rapid eye movement (NREM) sleep is directly related to prior sleep/wake history, with high levels of SWA following extended periods of wake. Therefore, SWA has been thought to reflect the level of accumulated sleep need. The discovery that euthermic intervals between hibernation bouts are spent primarily in sleep and that this sleep is characterized by high and monotonically declining SWA has led to speculation that sleep homeostasis may play a fundamental role in the regulation of the timing of bouts of hibernation and periodic arousals to euthermia. It was proposed that because the SWA profile seen after arousal from hibernation is strikingly similar to what is seen in nonhibernating mammals after extended periods of wakefulness, that hibernating mammals may arouse from hibernation with significant accumulated sleep need. This sleep need may accumulate during hibernation because the low brain temperatures during hibernation may not be compatible with sleep restorative processes. In the present study, golden-mantled ground squirrels were sleep deprived during the first 4 h of interbout euthermia by injection of caffeine (20 mg/kg ip). We predicted that if the SWA peaks after bouts of hibernation reflected a homeostatic response to an accumulated sleep need, sleep deprivation should simply have displaced and possibly augmented the SWA to subsequent recovery sleep. Instead we found that after caffeine-induced sleep deprivation of animals just aroused from hibernation, the anticipated high SWA typical of recovery sleep did not occur. Similar results were found in a study that induced sleep deprivation by gentle handling (19). These findings indicate that the SWA peak immediately after hibernation does not represent homeostatic regulation of NREM sleep, as it normally does after prolonged wakefulness during euthermia, but instead may reflect some other neurological process in the recovery of brain function from an extended period at low temperature.

The glutamate induced phase shift in the SCN slice: a two pulse study.

The short-term dynamics of resetting the circadian 'clock' was assessed by a double-pulse paradigm in vitro. On day 1, single and double 1 h 'pulses' of 1 mM l-glutamate were applied to the rat suprachiamastic nuclei (SCN). On days 2 and 3, single unit activity (SUA) was recorded and time-of-peak SUA was used as a phase marker of the circadian rhythm. The time-of-peak in untreated slices at 'Zeitgeber' time (ZT; hours after lights-on) 6, was used to evaluate effects of glutamate on phase. In accordance with published data, a single glutamate pulse at ZT 14 resulted in a 3 h delay of peak SUA on days 2 and 3. A 2nd pulse, given 3 h after a 1st pulse, resulted in two distinct peaks on day 2: a 1st at ZT 7 and a 2nd at ZT 12, i. e., a 6 h phase delay and hence twice the delay obtained after a single pulse. On day 3, no peak in SUA was observed which indicates that a new steady state was not reached on day 2. The bimodal distribution of SUA on day 2 corroborates other findings which suggest that the SCN comprises two distinct neuronal populations with circadian firing patterns that are normally coupled but, possibly due to different sensitivities to glutamate, can desynchronize. The additive phase-shifting effect of two consecutive glutamate pulses suggests that, at least for one sub-population of SCN neurons, the phase shift is completed within 3 h.

Phase shift magnitude and direction determine whether Siberian hamsters reentrain to the photocycle.

Body temperature (Tb) or activity rhythms were monitored in male Siberian hamsters (Phodopus sungorus) housed in an LD cycle of 16 h light/day from birth. At 3 months of age, rhythms were monitored for 14 days, and then the LD cycle was phase delayed by 1, 3, or 5 h or phase advanced by 5 h in four separate groups of animals. Phase delays were accomplished via a 1- or 3-h extension of the light phase or via a 5-h extension of the dark phase. The phase advance was accomplished via a 5-h shortening of the light phase. After 2 to 3 weeks, hamsters that were phase delayed by 1 or 3 h were then phase advanced by 1 or 3 h, respectively, via a shortening of the light phase. All of the animals reentrained to phase delays of 1 or 3 h and to a 1-h phase advance; 79% reentrained to a 3-h phase advance. In contrast, only 13% of the animals reentrained to the 5-h phase advance, 13% became arrhythmic, and 74% free ran for several weeks. After the 5-h phase delay, however, reentrainment was observed in 50% of the animals although half of them required more than 21 days to reentrain. The response to a phase shift could not be predicted by any parameter of circadian rhythm organization assessed prior to the phase shift. These data demonstrate that a phase shift of the LD cycle can permanently disrupt entrainment mechanisms and eliminate circadian Tb and activity rhythms. Magnitude and direction of a phase shift of the LD cycle determine not only the rate but also the probability of reentrainment. Furthermore, the phase of the LD cycle at which the phase shift is made has a marked effect on the proportion of animals that reentrain. Light exposure during mid-subjective night combined with daily light exposure during the active phase may explain these phenomena.

Neurons containing hypocretin (orexin) project to multiple neuronal systems.

The novel neuropeptides called hypocretins (orexins) have recently been identified as being localized exclusively in cell bodies in a subregion of the tuberal part of the hypothalamus. The structure of the hypocretins, their accumulation in vesicles of axon terminals, and their excitatory effect on cultured hypothalamic neurons suggest that the hypocretins function in intercellular communication. To characterize these peptides further and to help understand what physiological functions they may serve, we undertook an immunohistochemical study to examine the distribution of preprohypocretin-immunoreactive neurons and fibers in the rat brain. Preprohypocretin-positive neurons were found in the perifornical nucleus and in the dorsal and lateral hypothalamic areas. These cells were distinct from those that express melanin-concentrating hormone. Although they represent a restricted group of cells, their projections were widely distributed in the brain. We observed labeled fibers throughout the hypothalamus. The densest extrahypothalamic projection was found in the locus coeruleus. Fibers were also seen in the septal nuclei, the bed nucleus of the stria terminalis, the paraventricular and reuniens nuclei of the thalamus, the zona incerta, the subthalamic nucleus, the central gray, the substantia nigra, the raphe nuclei, the parabrachial area, the medullary reticular formation, and the nucleus of the solitary tract. Less prominent projections were found in cortical regions, central and anterior amygdaloid nuclei, and the olfactory bulb. These results suggest that hypocretins are likely to have a role in physiological functions in addition to food intake such as regulation of blood pressure, the neuroendocrine system, body temperature, and the sleep-waking cycle.

Recovery from mild hypothermia can be accelerated by mechanically distending blood vessels in the hand.

Peripheral vasoconstriction decreases thermal conductance of hypothermic individuals, making it difficult to transfer externally applied heat to the body core. We hypothesized that increasing blood flow to the skin of a hypothermic individual would enhance the transfer of exogenous heat to the body core, thereby increasing the rate of rewarming. External auditory meatus temperature (TEAM) was monitored in hypothermic subjects during recovery from general anesthesia. In 10 subjects, heat (45-46 degreesC, water-perfused blanket) was applied to a single forearm and hand that had been placed in a subatmospheric pressure environment (-30 to -40 mmHg) to distend the blood vessels. Heat alone was applied to control subjects (n = 6). The application of subatmospheric pressure resulted in a 10-fold increase in rewarming rates as determined by changes in TEAM [13.6 +/- 2.1 (SE) degreesC/h in the experimental group vs. 1.4 +/- 0.1 degreesC/h in the control group; P < 0.001]. In the experimental subjects, the rate of change of TEAM decreased sharply as TEAM neared the normothermic range.

Early synaptogenesis in vitro: role of axon target distance.

In contrast to some previous reports suggesting a delay in synapse formation in vitro, we found that under ideal conditions, most hippocampal and hypothalamic rat neurons were synaptically coupled after 3 or 4 days in vitro. Synaptophysin immunocytochemistry revealed strongly stained presynaptic boutons by 3 days in vitro. Studies with time-lapse laser confocal imaging of FM1-43 revealed that axonal boutons were recycling their synaptic vesicles, an indication of synapse formation, as early as 3 days after plating. To test the hypothesis that neurite outgrowth was enhanced in high-density cultures, thereby increasing the probability of synapse formation, neurons were transfected with the jellyfish green fluorescent protein (GFP) gene. After 2 days in high-density cultures, green fluorescent neurites were about three times longer than in sister neurons plated in low-density cultures. Even in single dishes, GFP-transfected cells in contact with other neurons had neurites that were at least three times longer and grew faster than more isolated cells. Neurons grew longer neurites (+51%) when growing on surface membranes of heat-killed neurons than on polylysine, underlining the importance of plasma membrane contact. Calcium imaging with fura-2 and whole cell recording showed that both GABA and glutamate presynaptic release occurred after 3 or 4 days in vitro in high-density cultures but was absent in low-density cultures at this time. Together, these morphological, cytochemical, and physiological data suggest that the distance an axon must grow to find a postsynaptic partner plays a substantial role in the timing of synapse formation. Although other factors in vitro may also play a role, the distance to a postsynaptic target, which defines the interval during which an axon grows to its target, can probably account for much of the difference in timing of synapse formation previously reported in vitro. A short intercell distance may increase the concentration of limited amounts of trophic factors available to a nearby cell, and once contact is made, a neuronal membrane provides a superior substrate for neuritic elongation.