Vinh H. Cao

Embryonic hypothalamic expression of functional glutamate receptors

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamates critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS)

Nicotine and nicotinic receptors in the circadian system

Considerable data support a role for cholinergic influences on the circadian system. The extent to which these influences are mediated by nicotinic acetylcholine receptors (nAChRs) has been controversial, as have the specific actions of nicotine and acetylcholine in the suprachiasmatic nucleus (SCN) of the hypothalamus. In this article we review the existing literature and present new data supporting an important role for nAChRs in both the developing and adult SCN. Specifically, we present data showing that nicotine is capable of causing phase shifts in the circadian rhythms of rats. Like light and carbachol, nicotine appears to cause phase delays in the early subjective night and phase advances in the late subjective night. In the isolated SCN slice, however, only phase advances are seen, and, surprisingly, nicotine appears to cause the inhibition rather than the excitation of neurons. Among nAChR subunit mRNAs, alpha 7 appears to be the most abundant subunit in the adult SCN, whereas in the perinatal period, the more typical nAChRs with higher affinity for nicotine predominate in the SCN. This developmental change in subunit expression may explain the dramatic sensitivity of the perinatal SCN to nicotine that we have previously observed. The effects of nicotine on the SCN may contribute to alterations caused by nicotine in other physiological systems. These effects might also contribute to the dependence properties of nicotine through influences on arousal.

Acute Light Exposure Suppresses Circadian Rhythms in Clock Gene Expression

Light can induce arrhythmia in circadian systems by several weeks of constant light or by a brief light stimulus given at the transition point of the phase response curve. In the present study, a novel light treatment consisting of phase advance and phase delay photic stimuli given on 2 successive nights was used to induce circadian arrhythmia in the Siberian hamster ( Phodopus sungorus). We therefore investigated whether loss of rhythms in behavior was due to arrhythmia within the suprachiasmatic nucleus (SCN). SCN tissue samples were obtained at 6 time points across 24 h in constant darkness from entrained and arrhythmic hamsters, and per1, per2 , bmal1, and cry1 mRNA were measured by quantitative RT-PCR. The light treatment eliminated circadian expression of clock genes within the SCN, and the overall expression of these genes was reduced by 18% to 40% of entrained values. Arrhythmia in per1, per2, and bmal1 was due to reductions in the amplitudes of their oscillations. We suggest that these data are compatible with an amplitude suppression model in which light induces singularity in the molecular circadian pacemaker.

Circadian system of mice integrates brief light stimuli

Light is the primary sensory stimulus that synchronizes or entrains the internal circadian rhythms of animals to the solar day. In mammals photic entrainment of the circadian pacemaker residing in the suprachiasmatic nuclei is due to the fact that light at certain times of day can phase shift the pacemaker. In this study we show that the circadian system of mice can integrate extremely brief, repeated photic stimuli to produce large phase shifts. A train of 2-ms light pulses delivered as one pulse every 5 or 60 s, with a total light duration of 120 ms, can cause phase shifts of several hours that endure for weeks. Single 2-ms pulses of light were ineffective. Thus these data reveal a property of the mammalian circadian clock: it can integrate and store latent sensory information in such a way that a series of extremely brief photic stimuli, each too small to cause a phase shift individually, together can cause a large and long-lasting change in behavior.

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.

Work volume and strength training responses to resistive exercise improve with periodic heat extraction from the palm.

Abstract Grahn, DA, Cao, VH, Nguyen, CM, Liu, MT, and Heller, HC. Work volume and strength training responses to resistive exercise improve with periodic heat extraction from the palm. J Strength Cond Res 26(9): 2558–2569, 2012—Body core cooling via the palm of a hand increases work volume during resistive exercise. We asked: (a) “Is there a correlation between elevated core temperatures and fatigue onset during resistive exercise?” and (b) “Does palm cooling between sets of resistive exercise affect strength and work volume training responses?” Core temperature was manipulated by 30–45 minutes of fixed load and duration treadmill exercise in the heat with or without palm cooling. Work volume was then assessed by 4 sets of fixed load bench press exercises. Core temperatures were reduced and work volumes increased after palm cooling (Control: Tes = 39.0 ± 0.1° C, 36 ± 7 reps vs. Cooling: Tes = 38.4 ± 0.2° C, 42 ± 7 reps, mean ± SD, n = 8, p < 0.001). In separate experiments, the impact of palm cooling on work volume and strength training responses were assessed. The participants completed biweekly bench press or pull-up exercises for multiple successive weeks. Palm cooling was applied for 3 minutes between sets of exercise. Over 3 weeks of bench press training, palm cooling increased work volume by 40% (vs. 13% with no treatment; n = 8, p < 0.05). Over 6 weeks of pull-up training, palm cooling increased work volume by 144% in pull-up experienced subjects (vs. 5% over 2 weeks with no treatment; n = 7, p < 0.001) and by 80% in pull-up naïve subjects (vs. 20% with no treatment; n = 11, p < 0.01). Strength (1 repetition maximum) increased 22% over 10 weeks of pyramid bench press training (4 weeks with no treatment followed by 6 weeks with palm cooling; n = 10, p < 0.001). These results verify previous observations about the effects of palm cooling on work volume, demonstrate a link between core temperature and fatigue onset during resistive exercise, and suggest a novel means for improving strength and work volume training responses.

Light Pulses Do Not Induce C-Fos or Per1 in the SCN of Hamsters That Fail to Reentrain to the Photocycle

Circadian activity rhythms of most Siberian hamsters (Phodopus sungorus sungorus) fail to reentrain to a 5-h phase shift of the light-dark (LD) cycle. Instead, their rhythms free-run at periods close to 25 h despite the continued presence of the LD cycle. This lack of behavioral reentrainment necessarily means that molecular oscillators in the master circadian pacemaker, the SCN, were unable to reentrain as well. The authors tested the hypothesis that a phase shift of the LD cycle rendered the SCN incapable of responding to photic input. Animals were exposed to a 5-h phase delay of the photocycle, and activity rhythms were monitored until a lack of reentrainment was confirmed. Hamsters were then housed in constant darkness for 24 h and administered a 30-min light pulse 2 circadian hours after activity onset. Brains were then removed, and tissue sections containing the SCN were processed for in situ hybridization. Sections were probed with Siberian hamster c-fosand per1mRNA probes because light rapidly induces these 2 genes in the SCN during subjective night but not at other circadian phases. Light pulses induced robust expression of both genes in all animals that reentrained to theLDcycle, but no expression was observed in any animal that failed to reentrain. None of the animals exhibited an intermediate response. This finding is the first report of acute shift in a photocycle eliminating photosensitivity in the SCN and suggests that a specific pattern of light exposure may desensitize the SCN to subsequent photic input.

A Chemical Heat Pack-Based Method For Consistent Heating of Intravenous Fluids

BACKGROUND Transfusion of cold intravenous fluids (IVF) can exacerbate hypothermia. Civilian and military guidelines recommend heated IVF for hypothermic patients; however, there is currently no ideal IVF heating system for use in resource-limited settings. OBJECTIVE Development of a system that uses flameless ration heaters (FRH) and an insulated sleeve for the consistent delivery of IVF at physiologically appropriate temperatures (40°-42°C) over the range of ambient conditions typical of the prehospital and wilderness environments. METHODS The temperatures of 0.9% normal saline (NS) 1-L bags were measured under 3 ambient conditions: 3°C, 10°C, and 20°C. The IVF was placed in an insulated pouch along with a predetermined number of activated FRH (5 FRH for 3°C, 4 FRH for 10°C, and 3 FRH for 20°C) for 10 minutes before removing the FRHs. The insulated IVF bag was drained through 280 cm of intravenous tubing at a flow rate of 77 mL/min. Raw temperature data for internal and delivery temperatures were collected and analyzed. RESULTS The temperature of the IVF throughout the delivery of 1 L of NS under the 3 ambient conditions was as follows (mean ± SD): at 3°C ambient, 47° ± 2.1°C internal and 42.6°C ± 1.4°C at delivery; at 10°C ambient, 52.3° ± 2.7°C and 45.2° ± 1.6°C; and at 20°C ambient, 45.5° ± 1°C and 39.7° ± 0.7°C. CONCLUSIONS The IVF heating system described here reliably delivered physiologically appropriate temperature intravenous fluids in 2 of the 3 ambient treatment conditions. With the appropriate number of FRH for the ambient conditions, this system enables the delivery of warmed IVF to provide active warming, which may be clinically beneficial in the prevention and treatment of hypothermia.

Novel Application of Chemical Cold Packs for Treatment of Exercise-Induced Hyperthermia: A Randomized Controlled Trial

OBJECTIVE Heat-related illness is a common disease with significant morbidity and mortality. Despite no proven efficacy, application of chemical cold packs (CCP) to the skin overlying the large vessels of the neck, groin, and axillae is a traditional recommended cooling modality. The study objective was to compare the cooling rates of CCP applied to these traditional areas vs the glabrous skin surfaces of the cheeks, palms, and soles in exercise-induced hyperthermia. METHODS Ten healthy adult male volunteers walked on a treadmill in a heated room (40°±0.5°C) while wearing insulated military overgarments until their esophageal temperatures (Tes) reached 39.2°C. Each participant had three heat stress trials on separate days: no treatment followed by randomly ordered traditional (neck, groin, and axillae) cooling and glabrous skin cooling. RESULTS With no treatment, Tes remained stable after the first 5 minutes of the heat trial (ΔTes=0.12°±0.07°C/10 min). Traditional cooling followed a linear decline (ΔTes=0.17°±0.04°C/10 min; P<.001). Glabrous cooling enhanced the treatment effect by a steeper decline (ΔTes=0.30°±0.06°C/10 min; P<.001), significantly different from traditional cooling by 2-way analysis of variance (P<.001). CONCLUSIONS Application of CCP to glabrous skin surfaces was more effective for treating exercise-induced heat stress than the traditional CCP cooling intervention. This novel cooling technique may be beneficial as an adjunctive treatment for heat-related illness in the prehospital environment.