Jean-Marc Pequignot

Intermittent hypoxia during development induces long-term alterations in spatial working memory, monoamines, and dendritic branching in rat frontal cortex.

Exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing, is associated with increased apoptosis in vulnerable brain regions as well as with spatial reference memory deficits in adult and developing rats. The latter are more susceptible to IH, suggesting that early exposure to IH may have long-term consequences. Rats were exposed to 14 d of room air (RA) or IH starting at postnatal d 10. Working memory was then assessed in the water maze at 4 mo of age using a delayed matching to place task in which the rats were required to locate a submerged platform hidden in a novel location on the first trial (T1 or acquisition trial), and then remember that position after a delay (T2 or test trial). Mean escape latencies and swim distances were derived and the savings (T1–T2) were used as a measure of working memory. Male but not female rats exposed to IH showed working memory deficits at both a 10- and 120-min delay (for both latency and pathlength). Additionally, Sholl analysis of Golgi-stained neurons revealed decreased dendritic branching in the frontal cortex, but not the hippocampus, of male rats exposed to IH. Norepinephrine concentrations, dopamine turnover, and tyrosine hydroxylase activity were increased similarly in males and females. However, increased dopamine concentrations were present only in the frontal cortex of female rats. In conclusion, exposure to IH during a critical developmental period is associated with long-term alterations in frontal cortical dopaminergic pathways that may underlie gender differences in neurobehavioral deficits.

Prenatal hypoxia impairs the postnatal development of neural and functional chemoafferent pathway in rat

1 To define the effects of prenatal hypoxia on the postnatal development of the chemoafferent pathway, ventilation and metabolism, pregnant rats were exposed to normobaric hypoxia (10 % oxygen) from embryonic day 5 to embryonic day 20. Offspring were studied at 1, 3 and 9 weeks of age in three separate protocols. 2 Prenatal hypoxia decreased the dopamine content in the carotid bodies at all ages, and decreased the utilisation rate of noradrenaline in the caudal part of the A2 (A2c), A1 and A5 noradrenergic brainstem cell groups at 3 weeks after birth. At 9 weeks of age, the level of dopamine in the carotid bodies was still reduced but the utilisation rate of noradrenaline was enhanced in A1. 3 Rats from dams subjected to hypoxia during pregnancy hyperventilated until 3 weeks after birth. In these rats, the biphasic hypoxic ventilatory response was absent at 1 week and the increase in minute ventilation was amplified at 3 weeks. 4 Prenatal hypoxia disturbed the metabolism of offspring until 3 weeks after birth. A weak or absent hypometabolism in response to hypoxia was observed in these rats in contrast to control animals. 5 Prenatal hypoxia impairs the postnatal development of the chemoafferent pathway, as well as the ventilatory and metabolic responses to hypoxia. These alterations were mostly evident until 3 weeks after birth.

Effects of gestational hypoxia on mRNA levels of Glut3 and Glut4 transporters, hypoxia inducible factor-1 and thyroid hormone receptors in developing rat brain.

Alterations of brain development result from noxious intrauterine signals, as oxygen deprivation, which decrease glucose energetic yield. To verify the hypothesis that a defect of brain energetic adaptation is responsible for these alterations, we have studied the effects of gestational hypoxia (10% oxygen during the last 2 weeks of fetal life) on cerebral ontogenesis of glucose transporters which control the limiting step of glucose utilization by neurons. This study is realised in rats by quantification of whole brain Glut3 and Glut4 mRNA in 14- and 19-day-old embryos (E14, E19), newborn (P0) and 7 postnatal-day-old rats (P7) by using reverse transcription-polymerase chain reaction (RT-PCR) method. We have associated our study with the analysis of a transcriptional factor, the hypoxia inducible factor-1alpha (HIF-1alpha), known to control the expression of glucose transporter, and with a family of transcriptional factors, the thyroid hormone receptors (TR), regulating specific genes involved in brain development. The data show (1) for the first time the Glut4 and HIF-1alpha gene expression in fetal rat brain which are detected as soon as E14, (2) that gestational hypoxia induces an increase of mRNA transcript levels of Glut3, Glut4, TRalpha2, TRbeta1 and HIF-1alpha genes mainly or exclusively at E14, and (3) that the absence of response of Glut3 and HIF-1alpha at E19 in hypoxic vs. normoxic group could indicate an insufficient energetic adaptation at this period of development which could lead to the neural alterations observed postnatally.

Plasma free and sulphated catecholamines after ultra-long exercise and recovery

SummaryWe investigated the early and late effects of two types of ultra-long exercise on sympatho-adrenal and dopaminergic activity. With this aim both free and sulphoconjugated plasma catecholamines (CA), noradrenaline (NA), adrenaline (A), and dopamine (DA) were determined in two groups of athletes immediately after completion of 24-h running or a 10-h triathlon and on recovery during the next 1–3 days. Both races stimulated the sympathetic activity, but differences were observed in the CA pattern: the 24-h run induced a marked elevation of free and sulphoconjugated NA (+175% and +180%, respectively) but failed to alter significantly A and DA levels. The triathlon challenge increased the three conjugated CA (NA sulphate +350%; A sulphate + 110%; DA sulphate +270%) and to a lesser extent free CA (NA +45%; A +30%). On the first post-exercise morning, a sustained intense noradrenergic activity was still present in the 24h-runners, as evidenced by the large increase in free and sulphated NA levels (+ 140% and + 100%, respectively). Such a prolonged activity was also indicated after completion of the triathlon, by the increase of NA sulphate (+ 140%) observed on the 1st recovery day. However, after the triathlon there was a decreased release of A from the adrenal medulla for several days. These data show that both types of ultralong exercise are able to induce for several hours a sustained sympathetic activation during the test and in the recovery period. Furthermore, the study shows that plasma conjugated CA may provide delayed and cumulative indexes of sympathetic activation, complementary to the instantaneous markers such as free CA.

Dopamine and norepinephrine dynamics in rat carotid body during long-term hypoxia

The contents of dopamine (DA), norepinephrine (NE) and 3,4-dihydroxyphenylacetic acid (DOPAC), and the utilization rates (secretion plus breakdown) of DA and NE were measured in carotid bodies of rats exposed, to normobaric hypoxia (10% O2 + 90% N2) for 0, 2, 7, 14 or 28 days. Long-term hypoxia elicited gradual increase in DA, NE and DOPAC contents, which after 28 days were increased 27, 51 and 4.6 fold, respectively. The DA utilization rate estimated after blockade of biosynthesis by alpha-methyl-p-tyrosine gradually increased from two days to the end of hypoxic exposure. The utilization rate of NE was unaltered within the first 7 days but was greatly increased after 14 and 28 days. It was concluded that the utilization rates of both DA and NE were increased by long-term hypoxia but these increases had different time courses. Dopamine, whose utilization increased in the early stage of hypoxia, might exert neurochemical effects on the chemoreceptors throughout the exposure, whereas NE, whose utilization was stimulated after two weeks of hypoxia, might play a significant role only after a delay.

Optimal neuroprotection by erythropoietin requires elevated expression of its receptor in neurons

Erythropoietin receptor (EpoR) binding mediates neuroprotection by endogenous Epo or by exogenous recombinant human (rh)Epo. The level of EpoR gene expression may determine tissue responsiveness to Epo. Thus, harnessing the neuroprotective power of Epo requires an understanding of the Epo–EpoR system and its regulation. We tested the hypothesis that neuronal expression of EpoR is required to achieve optimal neuroprotection by Epo. The ventral limbic region (VLR) in the rat brain was used because we determined that its neurons express minimal EpoR under basal conditions, and they are highly sensitive to excitotoxic damage, such as occurs with pilocarpine-induced status epilepticus (Pilo-SE). We report that (i) EpoR expression is significantly elevated in nearly all VLR neurons when rats are subjected to 3 moderate hypoxic exposures, with each separated by a 4-day interval; (ii) synergistic induction of EpoR expression is achieved in the dorsal hippocampus and neocortex by the combination of hypoxia and exposure to an enriched environment, with minimal increased expression by either treatment alone; and (iii) rhEpo administered after Pilo-SE cannot rescue neurons in the VLR, unless neuronal induction of EpoR is elicited by hypoxia before Pilo-SE. This study thus demonstrates using environmental manipulations in normal rodents, the strict requirement for induction of EpoR expression in brain neurons to achieve optimal neuroprotection. Our results indicate that regulation of EpoR gene expression may facilitate the neuroprotective potential of rhEpo.

Sex differences in the sympatho-adrenal response to isometric exercise.

SummaryThe effects on heart rate, blood pressure, and plasma catecholamines (epinephrine, norepinephrine, dopamine) of sustained isometric contraction (SIC) were studied in six women and nine men. Each subject held a tension equivalent to 30% of maximal handgrip strength until exhaustion. There were no significant differences between women and men in the duration of handgrip. Rise of heart rate and blood pressure were similar for women and men. Considering the absolute plasma levels of each catecholamine, no sex differences was observed at rest and at any time during SIC, except for epinephrine whose concentration was higher in men at first min of SIC. On the other hand, women and men exhibited different adrenergic patterns in response to SIC: in the first min of exercise the plasma level of the three catecholamines increased in men whereas for women plasma catecholamines levels were essentially unaffected. Thus, epinephrine seems to play a minor role in the regulation of heart rate and blood pressure during SIC for women. Another interesting result of our study is that SIC is able to induce an increase in dopamine plasma level for women as well as for men.

Monoamines (norepinephrine, dopamine, serotonin) in the rat medial vestibular nucleus: endogenous levels and turnover

SummaryMonoamine (norepinephrine, dopamine, serotonin) and metabolite endogenous levels were determined in the rat medial vestibular nucleus (MVN) using HPLC with electrochemical detection. As a comparison, the locus cœruleus (LC) and dorsal raphe nucleus (RD) which contain the cell bodies of MVN noradrenergic and serotoninergic neurons respectively were also analyzed. Norepinephrine (NE) and serotonin (5-HT) basal levels of MVN were high (33.8 and 39.2pmol/mg protein respectively) but lesser than in LC or RD. Great amounts of MHPG and 5-HIAA were also present in the MVN. The turnover of NE assessed both from the ratio MHPG/NE and by the decrease in the NE content after treatment with α-methylparatyrosine was faster in the MVN (half-life∶ 1.5h) than in LC (half-life∶ 3.6h). On the other hand, the ratio 5-HIAA/5-HT was lower in the MVN (0.58) than in the RD (0.85) indicating a smaller 5-HT turnover in the MVN. In addition, like LC and RD, the MVN contained meaningful amounts of dopamine (DA) and DOPAC. The high ratio DA/NE (0.27) suggests the presence of non precursor specific dopaminergic pools. However, individualized dopaminergic neurons have not yet been demonstrated. The data are discussed in line with the possible neurotransmitter function of monoamines in the MVN.

Ventilatory and central neurochemical reorganisation of O2 chemoreflex after carotid sinus nerve transection in rat

The first step of this study was to determine the early time course and pattern of hypoxic ventilatory response (HVR) recovery following irreversible bilateral carotid sinus nerve transection (CSNT). The second step was to find out if HVR recovery was associated with changes in the neurochemical activity of the medullary catecholaminergic cell groups involved in the O2 chemoreflex pathway. The breathing response to acute hypoxia (10% O2) was measured in awake rats 2, 6, 10, 45 and 90 days after CSNT. In a control group of sham‐operated rats, the ventilatory response to hypoxia was principally due to increased respiratory frequency. There was a large reduction in HVR in the CSNT compared to the sham‐operated rats (−65%, 2 days after surgery). Within the weeks following denervation, the CSNT rats progressively recovered a HVR level similar to the sham‐operated rats (‐37% at 6 days, −27% at 10 days, and no difference at 45 or 90 days). After recovery, the CSNT rats exhibited a higher tidal volume (+38%) than the sham‐operated rats in response to hypoxia, but not a complete recovery of respiratory frequency. Fifteen days after CSNT, in vivo tyrosine hydroxylase (TH) activity had decreased in caudal A2C2 (−35%) and A6 cells (−35%). After 90 days, the CSNT rats displayed higher TH activity than the sham‐operated animals in caudal A1C1 (+51%), caudal A2C2 (+129%), A5 (+216%) and A6 cells (+79%). It is concluded that HVR following CSNT is associated with a profound functional reorganisation of the central O2 chemoreflex pathway, including changes in ventilatory pattern and medullary catecholaminergic activity.

Sympathetic stimulation induced by hand cooling alters cold-induced vasodilatation in humans.

Abstract Hand cooling is a cold pressor test, which induces general sympathetic stimulation. This cooling procedure is often performed to investigate cold induced vasodilatation (CIVD) in one finger. To investigate the effects of this sympathetic stimulation on local CIVD, 12 subjects immersed either the right index finger (T1), right hand (T2) or left hand and right index finger (T3) for 30 min in water at 5°C followed by 15-min recovery. Skin temperature and skin blood flow (Q˙sk) measured by laser Doppler flowmetry on the right index finger, as well as heart rate (fc) and mean arterial blood pressure (), were continuously monitored during the three tests. Cutaneous vascular conductance was calculated as Q˙sk/. Concentrations of plasma noradrenaline (NA) and adrenaline (AD) were measured at different times during the tests. The results showed no cardiovascular change in T1, whereas fc and increased significantly at the beginning of both T2 and T3. Similarly, sympathetic stimulation was reflected in the NA concentrations, which increased significantly (P < 0.01) during T2 and T3 after 5 min of immersion, and remained elevated until the recovery period. The AD concentration did not change during the three tests. During T2, the CIVD appeared later and slower in comparison with T1 [CIVD onset: 12.81 (SEM 2.30) min in T2 and 5.62 (SEM 0.33) min in T1] . During T3, the CIVD onset was not delayed compared to T1 [6.38 (SEM 0.67) min], but the rewarming was lower [+5.40 (SEM 0.86)°C in T3 and +9.10 (SEM 1.31)°C in T1]. These results showed that CIVD could be altered by sympathetic stimulation but it also appeared that the onset of CIVD could be influenced by local cooling, independently of the general sympathetic stimulation.