Specific changes in cerebral second messenger accumulation underline REM sleep inhibition induced by the exposure to low ambient temperature

Brain Research 1022 (2004) 62 – 70 www.elsevier.com/locate/brainres

Research report

Specific changes in cerebral second messenger accumulation underline REM sleep inhibition induced by the exposure to low ambient temperature Giovanni Zamboni*, Christine Ann Jones, Rosa Domeniconi, Roberto Amici, Emanuele Perez, Marco Luppi, Matteo Cerri, Pier Luigi Parmeggiani Dipartimento di Fisiologia umana e generale, Universita` di Bologna, Bologna, Italy Accepted 6 July 2004 Available online 11 August 2004

Abstract In the rat the exposure to an ambient temperature (Ta) of 10 8C induces an almost total REM sleep deprivation that results in a proportional rebound in the following recovery at normal laboratory Ta when the exposure lasts for 24 h, but in a rebound much lower than expected when the exposure lasts 48 h. The possibility that this may be related to plastic changes in the nervous structures involved in the control of thermoregulation and REM sleep has been investigated by measuring changes in the concentration of adenosine 3V:5V-cyclic monophosphate (cAMP) and d-myo-inositol 1,4,5-trisphosphate (IP3) in the preoptic-anterior hypothalamic area (PO-AH), the ventromedial hypothalamic nucleus (VMH) and, as a control, the cerebral cortex (CC). Second messenger concentration was determined in animals either stimulated by being exposed to hypoxia, a depolarizing condition that induces maximal second messenger accumulation or unstimulated, at the end of a 24-h and a 48-h exposure to 10 8C and also between 4 h 15 min and 4 h 30 min into recovery (early recovery). At the end of both exposure conditions, cAMP concentration significantly decreased in PO-AH-VMH, but did not change in CC, whilst changes in IP3 concentration were similar in all these regions. The low cAMP concentration in PO-AH-VMH was concomitant with a significantly low accumulation in hypoxia. The normal capacity of cAMP accumulation was only restored in the early recovery following 24 h of exposure, but not following 48 h of exposure, suggesting that this may be a biochemical equivalent of the REM sleep inhibition observed during 48 h of exposure and which is carried over to the recovery. D 2004 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Osmotic and thermal regulation Keywords: Preoptic-anterior hypothalamic area; Adenosine cyclic monophosphate; Inositol trisphosphate; Low ambient temperature; REM sleep

1. Introduction

Abbreviations: cAMP, adenosine 3V:5V-cyclic monophosphate; CC, cerebral cortex; IP3, d-myo-inositol 1,4,5-trisphosphate; LC, locus coeruleus; PO-AH, preoptic-anterior hypothalamic area; Ta, ambient temperature; VMH, ventromedial hypothalamic nucleus * Corresponding author. Dipartimento di Fisiologia umana e generale Piazza di Porta San Donato 2, 40127 Bologna BO, Italy. Tel.: +39 051 2091742; fax: +39 051 251731. E-mail address: [email protected] (G. Zamboni). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.07.002

The exposure to low ambient temperature (Ta) represents a physiological procedure for sleep deprivation which was first applied to the cat and was shown to affect REM sleep more selectively than NREM sleep [32]. In the rat, it has been observed that the exposure to low Ta induces a REM sleep loss and an immediate rebound which is proportional to the thermal load of exposure (Ta level by duration of exposure) when animals were returned to recover at normal laboratory Ta [2,3,4,15]. However, it has been observed that

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this relationship is lost following a 48-h exposure to Ta 10 8C since the amount of REM sleep produced in the subsequent recovery is significantly lower than that found following a 24-h exposure to the same Ta [3], in spite of the fact that a 48-h exposure causes a much larger sleep deprivation. The close relationship between the inhibition of REM sleep occurrence and the thermal load is a result of the suspension of thermoregulation observed in mammals during REM sleep [33]. Thus, in a laboratory condition which does not allow any other appropriate behavior [43] such as burrowing, huddling or, eventually, migrating, the inhibition of REM sleep occurrence would represent the main form of behavioral thermoregulation. This mechanism has a manifest adaptational role which, however, is counteracted by the build up of a sleep debt which becomes greater with time and, for this reason, requires a proportional increase in the inhibition of REM sleep to maintain the same level of thermoregulatory efficiency [2,3]. The reduction of REM sleep occurrence during the recovery following 48-h exposure to Ta 10 8C may therefore be interpreted as an effect of the active inhibition of REM sleep increased to such a level that the mechanisms involved in lifting this inhibition cannot be induced rapidly enough on return to normal laboratory Ta. The possibility that this may be due to plastic changes induced by the exposure to low Ta in structures controlling both thermoregulation and the rate of occurrence of REM sleep episodes [16,33,34,51] is suggested by two findings: firstly, that the concentration of adenosine 3V:5V-cyclic monophosphate (cAMP) is significantly reduced in the preoptic-anterior hypothalamic area, but not in the cerebral cortex, at the end of a 48-h exposure to Ta 10 8C [36] and, secondly, that this is caused by a reduction in the capacity to accumulate cAMP which is carried over to the first hours of the recovery period [57]. This work has been carried out to further clarify the specificity of second messenger accumulation during the exposure to Ta 10 8C and the following recovery. To this end, the capacity to accumulate both cAMP and d-myoinositol 1,4,5-trisphosphate (IP3) was determined in the preoptic-anterior hypothalamic area (PO-AH), in the ventromedial hypothalamic nucleus (VMH) and in the cerebral cortex (CC). These brain regions were selected for the following reasons: (i) it is well known that the PO-AH is the highest-order thermoregulatory structure [9] and is involved in the control of the wake–sleep cycle [33,44]; (ii) VMH is involved in the control of brown adipose tissue thermogenesis [38,55]; (iii) it is the cortical bioelectrical activity which is used to classify behavioral states.

of 23F0.5 8C (normal laboratory Ta) and to a 12 h:12 h light–dark (LD) cycle (L: 09.00–21.00; 100–150 lx at cage level); food and water were ad libitum. The experiments have been approved by the Ethical Committee of the University of Bologna under the supervision of the Health Authority (Ministero della Sanita`) in accordance with the EEC Directive (86/609). Animal care was under the direct control of the University Veterinary Service. The experiment consisted in the determination of the cerebral concentration of cAMP and IP3 under five different ambient conditions. Animals were placed either at normal laboratory Ta (23F0.5 8C; n-lab) or low Ta ( 10F1 8C; exposure) as follows: (i) n-lab; (ii) 24-h exposure; (iii) 48-h exposure; (iv) recovery (4 h 15 min to 4 h 30 min) under the n-lab condition following 24-h exposure; (v) recovery (4 h 15 min to 4 h 30 min) under the n-lab condition following 48-h exposure. All changes in the ambient conditions were made at the onset of the L period of the LD cycle. Second messenger concentration was measured following an unspecific stimulation (hypoxia) or without stimulation (normoxic control). Hypoxia was induced by placing animals under a bell jar in which the oxygen concentration was quickly reduced by forcedly flushing with 100% nitrogen. Two different schedules of hypoxia were used: fixed time of hypoxia, in which animals were exposed to the condition for 75 s, and a time course of hypoxia, in which animals were exposed to the condition for times ranging from 30 to 120 s. Normoxic control animals were kept at normal oxygen concentration. Animals were sacrificed by immersion in liquid nitrogen and stored at 80 8C for the subsequent biochemical determinations.

2. Materials and methods

(i)

2.1. Animals and general outline of the experiment Male Sprague–Dawley rats (200–250 g; Harlan) were used. Animals were adapted to an ambient temperature (Ta)

2.2. Hypoxia It has been observed that hypoxia induces different transients of increase in the accumulation of both cAMP and IP3 in the mouse [17,23] and in the rat [22,56,57], that are thought to be determined by a generalized release of neurotransmitters triggered by the increase in cytosolic calcium concentration in nerve endings [35]. Because of this pattern of release and its short duration, the measurement of second messenger concentration in hypoxia may be considered as a way to assess the maximal capacity of the accumulation determined by the relationship between the processes of synthesis and degradation activated by the occupancy of G-protein linked receptors. This condition shows some features and limitations that may be considered as follows: The limiting step in the enzymatic formation of cAMP is the stoichiometric availability of adenylyl cyclase (AC) [18], whilst the stimulated rise in the nucleotide concentration is rapidly attenuated by phosphodiesterase (PDE) activity, which is at least 10 times that of AC [11].

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(ii)

IP3 is released from the membrane pool by phospholipase C (PLC) and recycled by means of successive dephosphorylation/phosphorylation steps that require several ATP molecules [50,52]. (iii) The kinetics of the accumulation of cAMP during hypoxia show that, in rodent brain, the concentration increases to a relatively higher level and declines more slowly than that of IP3 [23,57]. (iv) Since cAMP accumulation appears more stoichiometrically favoured than IP3 accumulation, the changes in the kinetics of IP3 accumulation may be interpreted as the result of the fall in brain ATP concentration that declines rapidly over approximately 120 s [23,24]; however, more complex mechanisms may be involved in the explanation of these differences as suggested by the observation that an increase in cAMP accumulation can exert, in platelets, a direct inhibitory effect on phosphoinositol kinase activity [42]. 2.3. Experimental plan and statistical analysis of the results The study has been performed in three distinct experiments, each organized according to the results of the previous: (a)

Second messenger concentration at a fixed time of hypoxia at the end of a 24-h or 48-h exposure to low Ta. This experiment was carried out as a randomized block design incorporated within a factorial design. Factors were: blocks (six), ambient condition (levels: n-lab, 24-h exposure and 48-h exposure) and hypoxia (levels: 0 s (normoxic control) and 75 s). Each block consisted of eighteen animals (three for each combination of ambient condition and hypoxia factor). (b) Second messenger concentration at different times of hypoxia at the end of a 48-h exposure to low Ta. The experimental design was factorial and factors were: ambient condition (levels: n-lab and 48-h exposure) and hypoxia (levels: 0 s (normoxic control), 30 s, 60 s, 75 s, 90 s, 120 s, for cAMP; levels: 0 s (normoxic control), 30 s, 75 s, 120 s, for IP3). Animals were randomly assigned to each of the combination of levels (for cAMP, eight animals for each of 12 combinations; for IP3, twenty-two animals for each of 8 combinations). (c) Second messenger concentration at a fixed time of hypoxia in the early recovery period following 24-h or 48-h of exposure to low Ta. In this experiment a randomized block design was applied. Animals were exposed to 75 s of hypoxia in the interval between 4 h 15 min and 4 h 30 min from the start of the following three experimental conditions: n-lab, recovery under n-lab conditions following 24-h exposure, recovery under n-lab conditions following 48-h exposure. The experiment was performed in three

blocks of fifteen animals (five for each experimental condition). Since the standard deviation and the mean of the samples were varying in direct proportion, results were evaluated after the logarithmic (ln) transformation of the data [48] by using the analysis of variance (ANOVA); significance levels were pre-set at Pb0.05 and Pb0.01. Individual comparisons were evaluated by means of the modified t-statistics corrected for the significance levels according to the Bonferroni’s method [54]. 2.4. Biochemical determinations Samples from the cerebral cortex and from both the preoptic anterior-hypothalamic area and the ventromedial hypothalamus were removed from frozen brains, kept on dry ice, using stainless steel needles of 0.8 mm i.d. cAMP and IP3 concentrations were determined in the same animal for experiment (a), in different animals for experiment (b), whilst only cAMP concentration was determined in experiment (c). Samples from PO-AH and VMH were pooled (PO-AH-VMH) in experiments (b) and (c). Samples were sonicated in either 150 Al (experiments (b) and (c) or 300 Al (experiment a)) of ice-cold 5% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000g at 4 8C for 10 min. Following centrifugation, an aliquot of the supernatant (140 or 280 Al, respectively) was mixed with an equal volume of 1 M HCl and, following the extraction of TCA with water-saturated diethyl ether, lyophilized and stored at 80 8C. cAMP content was determined by means of a radiobinding assay using [3H]cAMP (New England Nuclear), as the radiolabelled ligand, cAMP free acid as the standard (Sigma) and a commercial cAMP dependent protein kinase from bovine heart (Sigma), as the binding protein. Incubations were carried out overnight at 4 8C in 50 mM tris(hydroxymethyl)aminomethane buffer containing 4 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5 (TrisEDTA buffer), in which lyophilized samples were reconstituted. The final incubation volume was 200 Al. Separation of bound and free ligands was obtained by adding bovine gamma globulins as carrier (50 Al of a 10 mg/ml solution), 150 Al of 45% (w/v) polyethylene glycol (PEG, mw 8000; Sigma) in Tris-EDTA buffer and centrifuging at 10,000g for 15 min at 4 8C. Pellets were washed with 500 Al of 20% PEG in Tris-EDTA buffer and recentrifuged. IP3 levels were assessed by means of a modified radioreceptor binding assay which has previously been described [10]. Briefly, the IP3 binding protein was prepared from bovine adrenal cortices, taken from glands obtained on the same day from the slaughter house. Glands were kept in oxygenated, ice-cold Krebs-Henseleit bicarbonate buffer (118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM

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glucose, pH 7.4) and the dissected cortices homogenized in 20 mM NaHCO3, 1 mM dithiothreitol (DTT), 4 mM (EDTA) buffer pH 8.0 (NaHCO3/DTT/EDTA). Two supernatant fractions were collected following two centrifugations (5000g for 15 min, at 4 8C), separated by a rehomogenization of the pellet. The pooled supernatant fractions were centrifuged at 38,000g (20 min at 4 8C), the pellet was resuspended in 0.14 M H3BO4–Na2B4O7 buffer pH 9.0 containing 4 mM EDTA and 1 mM DTT (borate buffer) and centrifuged (38,000g for 20 min at 4 8C). The pellet was then resuspended in borate buffer at a concentration of approximately 10 mg protein/ml, as determined by the spectrophotometric assay described below, divided into aliquots and stored at 20 8C. The binding characteristics of the IP3 receptor protein were determined by assessing in triplicates: (1) the degree of competition for [3H]IP3 (New England Nuclear) binding by: (i) d-myo-inositol 1,4-bisphosphate (I(1,4)P2, Sigma); (ii) d-myo-inositol 1,3,4-trisphosphate (I(1,3,4)P3, Sigma); (iii) d-myo-inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4, Calbiochem); (iv) d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (I(1,2,3,4,5,6)P6, Calbiochem); (v) heparin (mw 5000, from porcine intestinal mucosa, Calbiochem); (vi) ATP (Sigma); these determinations were made as specified below for assay conditions; (2) Kd and Bmax of the receptor preparation were determined by utilizing progressively increasing concentrations (0.3–85 nM) of [3H]IP3 and 625 Ag of heparin used for non specific binding; values were determined by plotting data according to the Scatchard equation [7]. The results are shown in Table 1. Before each assay of the experiment, a separate assay to confirm the percentage cross-reactivity between I(1,3,4)P3 and I(1,3, 4,5)P4 was performed to ensure the binding protein’s stability. The assay was performed by using [3H]IP3 as the radiolabelled ligand and IP3 trilithium salt as the standard.

Table 1 Assessment of the binding characteristics of IP3 receptor preparation I(1,4)P2 I(1,3,4)P3 I(1,4,5)P3 I(1,3,4,5)P4 I(1,2,3,4,5,6)P6 ATP Heparin

I(1,4,5)P3

IC50(meanFSEM)

% Cross-reactivty

127.10F3.29 AM (3) 15.30F0.30 AM (2) 10.32F0.73 nM (20) 80.40F13.83 nM (17) 23.31F0.36 AM (3) 750.0F0.15 AM (3) 7.35F0.12 AM (3)

0.008 0.07 100 12.84 0.04 0.001 0.14

Kd

Bmax

10.14 nM

2.68 pmol/mg protein

Determinations were made in triplicate; values are the meanFS.E.M. with the number of experiments given in parenthesis. IC50, concentration of the different ligands inhibiting 50% of maximum [3H]-I(1,4,5)P3 binding; % cross-reactivity, ratio (100) of the IC50 for a ligand to that of I(1,4,5)P3; Kd, equilibrium dissociation constant; Bmax, apparent maximum number of binding sites. Kd and Bmax were calculated by means of the Scatchard equation.

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The incubation was carried out for 60 min at 4 8C in borate buffer pH 9.0 in a total volume of 300 Al. Nonspecific binding was determined in the presence of 5 AM IP3 standard and separation of bound and free ligands was obtained by centrifugation at 14,000g for 10 min at 4 8C. For both cAMP and IP3 assays, the pellets were solubilized in 50 Al of 5% (w/v) sodium dodecyl sulfate (SDS), followed by the addition of 6.0 ml of scintillation liquid (Ultima Gold, Packard) and the radioactivity determined in a Beckman LS 1800 scintillation counter. The concentration of cAMP and IP3 in each sample was referred to the protein content. Pellets from the original samples were resuspended in 70 Al of 0.5M NaOH and brought to a volume of 500 Al with 0.4 M H3BO4. Protein was reacted with 4% (w/v) CuSO4, the cuprous proteinates with bicinchoninic acid (Pierce) and the resulting chromogen read at 565 nm in 96 well polycarbonate plates with an ELISA spectrophotometer (Titertek).

3. Results Table 2 shows the changes in second messenger concentration observed after 75 s of hypoxia at the end of a 24-h or 48-h exposure to low Ta (experiment a). Results are shown separately for each second messenger and brain region. It can be seen that the overall response to hypoxia is different with respect to each second messenger system and that, whilst there is a consistent pattern of change concerning cAMP, such a pattern is absent for IP3. With respect to cAMP, the main effects of the ambient condition (df 2, 97) are significant in PO-AH ( F=17.07, Pb0.01) and VMH ( F=10.22, Pb0.01), but not in CC, whilst those of hypoxia are significant in all the brain regions (PO-AH: F=143.44, Pb0.01; VMH: F=327.09, Pb0.01; CC: 611.77, Pb0.01). Moreover, in PO-AH the interaction between the ambient condition and hypoxia is significant ( F=8.05, Pb0.01). The individual comparisons of the results show that hypoxic stimulation is effective in increasing cAMP accumulation in all the conditions and brain regions studied. However, the exposure to low Ta significantly ( Pb0.01) reduces the capacity to accumulate cAMP under hypoxic stimulation in both PO-AH and VMH. It should be noted that the lowest hypoxic values found at the end of the 48-h exposure to Ta 10 8C, are not significantly different from those found following the 24-h exposure. The picture that emerges from the analysis of the results concerning IP3 is quite different and somehow simpler, since the only significant results concern the main effect of both the ambient condition in VMH [ F(2,97) 4.02, Pb0.05] and hypoxia in PO-AH [ F(1,97) 13.65, Pb0.01]. Figs. 1 and 2, respectively, show changes in cAMP and IP3 concentration at different times of hypoxia in animals kept for either 48 h at Ta 10 8C or under the n-lab

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Table 2 cAMP and IP3 concentration (pmol/mg protein) induced in different brain regions by a fixed time of hypoxia (75 s) at the end of the exposure to Ta PO-AH(a,b,c)**

10 8C

cAMP

CCb,**

VMH(a,b)**

Normoxic Control

Hypoxia

Normoxic control

Hypoxia

Normoxic control

Hypoxia

n-lab E24 E48

13.77F0.71d,** 14.17F0.82d,** 14.66F0.77d,**

39.13F1.86 39.76F2.30 40.10F2.55

21.39F1.10d,** 19.50F1.18d,** 18.58F0.87d,**

66.37F5.74 37.85F4.32e,** 32.83F2.72e,**

19.54F1.00d,** 17.44F0.91d,** 16.24F0.81d,**

55.55F3.72 42.63F4.07e,** 38.97F2.38e,**

IP3

CC Normoxic Control

Hypoxia

Normoxic Control

Hypoxia

Normoxic Control

Hypoxia

n-lab E24 E48

60.36F5.37 62.02F8.17 59.59F7.42

42.35F3.13 54.19F5.96 61.11F5.84

39.73F3.71 36.02F3.12 41.00F3.24

29.58F2.18 27.83F2.24 33.20F1.71

49.71F2.72 42.15F3.39 44.66F2.39

46.90F3.45 41.00F2.54 45.62F1.44

PO-AHb,**

VMHa,*

CC, cerebral cortex; PO-AH, preoptic-anterior hypothalamic area; VMH, ventromedial hypothalamic nucleus; n-lab, normal laboratory Ta; E24, E48, 24-h and 48-h of exposure to Ta 10 8C, respectively; values are expressed as meanFS.E.M. of 18 different determinations. Significant results of the statistical analysis (*Pb0.05; **Pb0.01): (i) ANOVA: amain effect of ambient condition factor; bmain effect of factor for hypoxia; csignificant interaction between ambient condition and factor for hypoxia; (ii) modified t-statistics: dcomparison with the correspondent value in hypoxia; ecomparison with the correspondent n-lab value.

condition (experiment b). The time of exposure and the decision to pool samples from PO-AH and VMH were made after considering the results given in Table 2. The time of exposure (48 h) had led to the largest variation in second messenger concentration and the pooling of samples from PO-AH and VMH, which showed a similar pattern of response, allowed more tissue for replicate determinations. The results depicted in Fig. 1 concern the time course of cAMP accumulation. The statistical analysis shows that for PO-AH-VMH the main effect of hypoxia [ F(5,84) 26.53, Pb0.01] and ambient condition [ F(1,84) 56.02, Pb0.01] and the interaction between factors [ F(5,84), 4.36, Pb0.01] are significant. Moreover, the individual comparisons, following the 48-h exposure to Ta 10 8C, show that the values observed from 75 s of hypoxia onwards are significantly lower ( Pb0.05) than those under the n-lab condition. This difference in the accumulation kinetics would explain why the interaction between the two experimental factors becomes significant. In contrast to PO-AH-VMH, only the main effect of hypoxia is statistically significant [ F(5,84) 95.20, Pb0.01] for CC. Changes in IP3 concentration (Fig. 2) are different from those observed for cAMP. Firstly, the time course of IP3 accumulation in PO-AH-VMH is very similar to that of CC and, secondly, the exposure to low Ta increases the overall accumulation rate with respect to that observed under the nlab condition. For PO-AH-VMH the main effect of hypoxia [ F(3,168) 93.21, Pb0.01] and ambient condition [ F(1,168) 18.94, Pb0.01] and the interaction between factors [ F(3,168), 8.06, Pb0.01] are significant, whilst the main effect of hypoxia [ F(3,168) 56.11, Pb0.01] and the interaction between factors (F(3,168) 4.233, Pb0.01) were significant for CC. The individual comparisons of the results show that the values observed following 48-h exposure to Ta 10 8C, are significantly higher (respec-

tively Pb0.01 and Pb0.05) than those under the n-lab condition, in PO-AH-VMH from 75 s of hypoxia onwards and at 75 s in CC. As for the kinetics of cAMP accumulation, these differences would explain the significance of the interaction between the two experimental in both POAH-VMH and CC. In Fig. 3 the concentration of cAMP observed at 0 and 75 s of hypoxia given in the early recovery (4 h 15 min–4 h 30 min) from either a 24-h or 48-h exposure to Ta 10 8C (experiment c), is compared to that of the corresponding nlab and exposure conditions taken from Table 2. Since cAMP concentration in the cerebral cortex has been shown to be stable, the values have been expressed as the ratio of the cAMP concentration in PO-AH-VMH to that in CC (i.e. relative hypothalamic cAMP concentration). The individual comparisons clearly show that both the 24-h and 48-h exposure to Ta 10 8C significantly decrease ( Pb0.01) the relative hypothalamic cAMP concentration to approximately the same level. However, it appears that only during the early recovery that follows the 24-h exposure, does the capacity to accumulate cAMP attain control levels. In fact, the relative hypothalamic cAMP concentration at the end of the early recovery period following the 48-h exposure, although increased with respect to that observed at the end of exposure, still remains significantly ( Pb0.05) below the control level. 3.1. Discussion This study demonstrates that hypoxia may be considered a useful method to determine the capacity of second messenger accumulation in the brain. The results following 75 s of hypoxia show that in both PO-AH and VMH, but not in CC, the hypoxia-stimulated changes in cAMP concentration at the end of the 24 and 48 h periods of exposure to Ta 10 8C are significantly lower than those under n-lab conditions, whilst those concerning IP3 do not show

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reduced, at the PO-AH-VMH level, whereas no significant change occurs in the cerebral cortex. Moreover, the kinetics of IP3 accumulation shows the same pattern of variation in both regions: an initial peak, irrespective of the experimental condition, followed by a decline which appears significantly slower following the exposure to low Ta than under the n-lab condition. Thus, it may be assumed that following either a 48-h or a 24-h exposure to Ta 10 8C, the

Fig. 1. Second messenger accumulation at different times of hypoxia. cAMP concentration (pmol/mg protein; values are the mean of eight different determinations; bars represent S.E.M.) at different times (s) of hypoxia. Top: preoptic-anterior hypothalamic area-ventromedial hypothalamic nucleus (pooled samples; PO-AH-VMH). Bottom: cerebral cortex (CC). Empty circles: normal laboratory Ta (n-lab); filled circles: end of the 48 h exposure to Ta 10 8C (E48). Time 0 is equivalent to normal oxygen concentration (normoxic control). Significant results from the statistical analysis: (i) PO-AH-VMH; ANOVA: main effect of ambient condition factor ( Pb0.01) and factor for hypoxia ( Pb0.01); interaction between ambient condition factor and factor for hypoxia ( Pb0.01); modified tstatistics: comparison with the correspondent control value (*Pb0.05); (ii) CC; ANOVA: main effect of factor for hypoxia ( Pb0.01).

significant variations with respect to either exposure or brain region. Moreover, the concentration of cAMP in the preoptic-hypothalamic region at the end of a 48-h exposure is lower, although not significantly, than that observed at the end of a 24-h exposure. Such a structural and biochemical specificity was further supported by the study on the kinetics of second messenger accumulation carried out on samples from CC and on pooled samples from PO-AH and VMH during hypoxia at the end of the 48-h exposure. With respect to the n-lab condition the kinetics of cAMP accumulation is greatly

Fig. 2. Second messenger accumulation at different times of hypoxia. IP3 concentration (pmol/mg protein; values are the mean of twenty-two different determinations; bars represent S.E.M.) at different times (s) of hypoxia. Top: preoptic-anterior hypothalamic area-ventromedial hypothalamic nucleus (pooled samples; PO-AH-VMH). Bottom: cerebral cortex (CC). Empty circles: normal laboratory Ta (n-lab); filled circles: end of the 48 h exposure to Ta 10 8C (E48). Time 0 is equivalent to normal oxygen concentration (normoxic control). Significant results from the statistical analysis: (i) PO-AH-VMH; ANOVA: main effect of ambient condition factor ( Pb0.01) and factor for hypoxia ( Pb0.01); interaction between ambient condition factor and factor for hypoxia ( Pb0.01); modified tstatistics: comparison with the correspondent control value (**Pb0.01); (ii) CC; ANOVA: main effect of factor for hypoxia ( Pb0.01); interaction between ambient condition factor and factor for hypoxia ( Pb0.01); modified t-statistics: comparison with the correspondent control value (*Pb0.05).

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Fig. 3. Ratio of PO-AH-VMH to CC cAMP concentration (pmol/mg protein; bars represent S.E.M.) in the same animal, at a fixed time of hypoxia, at: (i) normal laboratory Ta (n-lab); (ii) end of the 24-h and 48-h exposure to Ta 10 8C (E24, E48, respectively); (iii) following the correspondent recovery period (R24, R48, respectively). PO-AH-VMH, preoptic-anterior hypothalamic area-ventromedial hypothalamic nucleus; CC, cerebral cortex. Empty circles, data from exposure; filled circles, data from recovery. Dashed lines mark the range of control values. Significant results from the comparison with the correspondent control value by means of the modified t-statistics analysis: *Pb0.05; **Pb0.01.

mechanism of cAMP accumulation is desensitised in POAH-VMH but it is working normally in CC. In contrast, as far as IP3 is concerned, it appears that the mechanism of accumulation is sensitised in both regions studied. The finding that the increase in cAMP concentration induced by hypoxic stimulation is totally prevented by the administration of dl-propranolol [56] suggests that betanoradrenergic transmission should play a major part in explaining the changes observed when the nucleotide accumulation is maximally stimulated by hypoxia. This viewpoint may be further supported by the observation that, whilst dl-propranolol also affects a subtype of serotonergic receptors (5HT1), these are known to inhibit cAMP production [39]. Thus, the observed changes in cAMP accumulation in PO-AH-VMH may be related to a change in noradrenergic (NA) transmission induced by the 48-h exposure to Ta 10 8C and/or to the concomitant REM sleep inhibition. Since during this exposure there is an almost total inhibition of REM sleep [3], whilst NREM sleep is much less affected both in the cat [32] and in the rat (unpublished observations), it appears that REM sleep deprivation induced by an exposure to a very low Ta is comparable to the selective deprivation induced by classical techniques such as the water platform [53]. The role of NA activity in sleep has been mainly investigated in locus coeruleus (LC) neurons that have been shown to be active during wakefulness, but decrease their discharge during NREM sleep and become silent during REM sleep [25]. Thus, it may be expected that the intensification of wakefulness induced by sleep deprivation increases the activity of NA neurons located in the LC

inducing, since they innervate the cerebral cortex [31,46,47], an effect on cortical beta-receptors. Results from selective REM sleep deprivation studies, carried out in the rat by means of the water-platform method, showed a significant downregulation of beta-noradrenergic receptors in the cerebral cortex after either 72 h [30] or 96 h of deprivation [19]. In keeping with these findings, it has been observed that at least 72 h of selective REM sleep deprivation using the same technique were required to induce a significant increase in tyrosine hydroxylase mRNA [6,40] in LC neurons, but that, once attained, both mRNA [6] and protein [26] remain at significantly high levels for longer deprivation periods. However, under the same experimental conditions, neither beta-receptor number nor noradrenaline content changed in the anterior hypothalamus [19,40] and, following REM sleep deprivation obtained by means of cold exposure (48 h at Ta 10 8C), the kinetic parameters (Km, Vmax) of tyrosine hydroxylase in the POAH do not change with respect to the control [37]. Noradrenergic activity, in response to an exposure to low ambient temperature, has been examined in several studies which have mainly concerned LC neurons, but, with respect to our approach, following exposures to less severe ambient temperatures and, above all, without any reference to sleep [13,20,21,27,28,29,41]. On the overall, the results suggest that the exposure to cold elicits immediate cellular activation followed by a regional difference in the level of adaptation that may depend on either the brainstem NA field or local regulatory factors in the target neurons [14,49]. Although the NA innervation of the cerebral cortex and the hypothalamus originates from different brainstem neurons [31,46,47], it is not known to which extent this would affect cAMP accumulation in these structures following the exposure to Ta 10 8C. Moreover, this activity may be further influenced by the increase in IP3 accumulation induced by cold exposure in both cerebral structures studied, since a postreceptorial response, affecting adenylyl cyclase activity irrespective of beta-receptor regulation, may arise from a cross-talk between these different signal transduction pathways [1,8,12,18,45]. These studies show that, when viewed separately, the effects of sleep deprivation and exposure to a low Ta on beta-noradrenergic transmission are different from those observed when the processes of deprivation and exposure are acting together. Although the reduction in cAMP accumulation at the hypothalamic level following cold exposure would depend on a mechanism which is as yet unknown, we would expect that it evokes a downstream response such as a reduction in cAMP-dependent protein phosphorylation [5]. This reduction in cAMP accumulation is manifest following either 24 h or 48 h at Ta 0 8C, but, whilst the capacity of accumulating cAMP is fully restored following 24 h of exposure, this does not occur after an exposure lasting for 48 h. Thus, it appears that the reduction in cAMP-dependent protein phosphorylation in a brain region involved in the

G. Zamboni et al. / Brain Research 1022 (2004) 62–70

control of vegetative activity and sleep is concomitant: firstly, with the inhibition of REM sleep occurrence during the exposure to low Ta and, secondly, with an impairment of REM sleep occurrence during the early phase of the recovery following 48 h of exposure. Interestingly, this impairment has been shown to affect the type of REM sleep characterized by episodes separated by short intervals (sequential REM sleep episodes) since in the first 5 h of the recovery period the amount of REM sleep in the form of sequential episodes was higher (1693 s above base line) following 24 h of exposure than after an exposure of 48 h (595 s above base line) [3]. It is well known that while REM sleep is characterized by vegetative regulations that are not fully operant, these regulations are fully operant during the interval between each REM sleep episode [34]. Since, by definition, sequential REM sleep episodes entail the frequent switching between these two states, it may be hypothesized that the persistent reduction of cAMP-dependent phosphorylation during the early recovery may keep hypothalamic vegetative control close to the level of that causing REM sleep inhibition (i.e. continued thermoregulation) impeding the high level of sequential REM sleep episodes needed for the recovery from a strong deprivation.

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Acknowledgements Preliminary results of this work have been presented at the 14th Congress of the European Sleep Research Society (Madrid, 1998). This work has been supported by grants from both the Ministero dell’Universita` e della Ricerca Scientifica, Italy and the University of Bologna. The authors would like to thank: Mr. G. Mancinelli and Mr. L. Sabattini for the wiring and the mechanical work needed in the adaptation of both the recording apparatus and room.

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