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• W1628011621 abstract "What is the central question of this study? Early life exposure to stress augments the hypoxic chemoreflex of adult male rats. This effect predisposes to respiratory instability during sleep. We used pharmacological and neuroanatomical approaches to determine whether a change in glutamatergic neurotransmission contributes to this abnormal respiratory phenotype. What is the main finding and its importance? We initially showed that neonatal stress augments sensitivity to an AMPA/kainate receptor antagonist. Results from autoradiography experiments support this observation because they demonstrate that neonatal stress increases expression of AMPA receptors in key regions that regulate breathing. Region-specific changes in brain-derived neurotrophic factor expression may contribute to these changes in glutamatergic neurotransmission. These results bring new insight into the pathophysiology of sleep-disordered breathing. Neonatal stress disrupts the developmental trajectory of homeostatic systems. Adult (8- to 10-week-old) male rats exposed to maternal separation (a form of neonatal stress) display several traits reported in patients suffering from sleep-disordered breathing, including an augmented hypoxic chemoreflex. To understand the mechanisms behind this effect, we tested the hypothesis that neonatal stress augments glutamatergic neurotransmission in three regions involved in respiratory regulation, namely the nucleus of the solitary tract, the paraventricular nucleus of the hypothalamus and the phrenic motor nucleus. Maternal separation was performed for 3 h day−1 from postnatal day 3 to 12. Control pups were undisturbed. Adult rats were instrumented for intracerebroventricular injection of the AMPA/kainate receptor antagonist CNQX (0–4.3 μm). Using plethysmography, ventilatory activity was measured at rest in awake animals during normoxia (fractional inspired O2= 0.21) and during acute hypoxia (fractional inspired O2= 0.12; 20 min). Following vehicle injection, the hypoxic ventilatory response of stressed rats was 35% greater than that of controls. Microinjection of CNQX attenuated the hypoxic ventilatory response, but the effect observed in stressed rats was greater than that in control animals. Autoradiography experiments showed that neonatal stress augments expression of AMPA receptors within the paraventricular nucleus of the hypothalamus and the phrenic motor nucleus. Quantification of brain-derived neurotrophic factor showed that neonatal stress augments brain-derived neurotrophic factor expression only within the paraventricular nucleus. We conclude that neonatal stress augments the hypoxic chemoreflex by increasing the efficacy of glutamatergic synaptic inputs projecting onto key respiratory structures, especially the paraventricular nucleus of the hypothalamus. These data provide new insight into the aetiology of sleep-disordered breathing. The chemosensory, integrative and motor components of the respiratory control system regulate ventilatory activity to maintain homeostasis of arterial blood gases. When facing environmental challenges or changes in metabolic demands, the ability of the system to generate ventilatory responses that are in line with the gas exchange requirements of the organism is a hallmark of a ‘healthy’ control system. In contrast, excessive or insufficient ventilatory responses to O2- and/or CO2-related challenges contribute to respiratory disorders or indicate a poor prognosis for patients with congestive heart failure (Kara et al. 2003). In humans, an augmented hypoxic ventilatory response contributes to sleep-disordered breathing and has been associated with hypertension (Dempsey et al. 2010). Sleep-disordered breathing is a complex, multifaceted disease, and its aetiology remains poorly understood. In rats, exposure to stress during early life alters the developmental trajectory of the respiratory control system (Bavis & Mitchell, 2008; Cayetanot et al. 2009). In adulthood, the respiratory phenotype of adult male rats previously subjected to neonatal maternal separation (NMS) shares several traits reported in sleep-disordered breathing patients; by comparison with control animals, these rats are hypertensive, produce a greater hypoxic ventilatory response (HVR) and show more respiratory instability during sleep (Genest et al. 2004; Kinkead et al. 2009). Interestingly, these effects are also sex specific (males but not females). Neonatal maternal separation also interferes with early life programming of the hypothalamic–pituitary–adrenal axis. In adulthood, male (but not female) rats previously subjected to NMS are more responsive to stress and can show higher than normal corticosterone levels (Genest et al. 2004; Lippmann et al. 2007). Within the CNS, changes in the efficacy of synaptic inputs converging onto neuronal structures influence behaviour and function. Given that stress and glucocorticoids influence glutamatergic transmission by regulating AMPA receptors (Shank & Scheuer, 2003; Krugers et al. 2010), we proposed that NMS augments AMPA-mediated glutamatergic neurotransmission within the respiratory control system. To test this hypothesis, we first determined whether NMS alters the effect of intracerebroventricular (i.c.v.) injection of the AMPA receptor antagonist CNQX on ventilatory activity at rest and during acute exposure to moderate hypoxia [fractional inspired O2 (Fi) = 0.12; 20 min]. We then used autoradiography to compare AMPA receptor binding sites between NMS and control rats in the following three areas involved in respiratory regulation: (i) the paraventricular nucleus of the hypothalamus (PVH) which, in addition to orchestrating the neuroendocrine response to stress, has direct projections to key respiratory control areas, including the pre-Bötzinger complex, the nucleus tractus solitarii (NTS) and phrenic motoneurons (Kc & Dick, 2010; Behan & Kinkead, 2011); (ii) the NTS, a key integrative area, which is the primary projection site for peripheral O2 chemosensors (Finley & Katz, 1992); and (iii) the inspiratory (phrenic) motoneurons located in the cervical spinal cord. Brain-derived neurotrophic factor (BDNF) plays an important role in mediating synaptic plasticity. One of the main mechanisms by which BDNF influences synaptic function is by modulating glutamatergic neurotransmission, because BDNF can augment AMPA receptor expression and trafficking (Caldeira et al. 2007). In the context of respiratory control, it is also noteworthy that BDNF exerts a tonic action within the NTS to regulate autonomic function (via glutamatergic receptors; Clark et al. 2011). Given that neonatal stress has persistent effects on basal expression of BDNF (Cirulli et al. 2009) and our results from CNQX icv injection indicate that an augmented glutamatergic neurotransmission contributes to NMS-related augmentation of the hypoxic ventilatory response, we therefore proposed that BDNF is a good candidate for transducing adverse early life events into an abnormally high HVR during adulthood. To address this issue, we used immunohistochemistry and enzyme-lined immunosorbent assays (ELISAs) on isolated tissue to compare basal BDNF expression between NMS and control rats in the NTS and other relevant respiratory regions. Experiments were performed on NMS and control (undisturbed) male Sprague–Dawley rats (Charles River Canada, St Constant, Québec, Canada). All animals were born and raised in our animal care facilities. Rats were supplied with food and water ad libitum and maintained in standard laboratory conditions (21°C, 12 h–12–h dark–light cycle, with lights on at 06.00 h and off at 18.00 h). Université Laval Animal Care Committee approved all the experimental procedures described in this report, and the protocols were in accordance with the guidelines detailed by the Canadian Council on Animal Care. Virgin females were mated and delivered 10–15 pups. One day after delivery, litters were culled to 12 pups, when necessary, with a roughly equal number of males and females. The NMS protocol was identical to the one used in our previous studies (Genest et al. 2004; Kinkead et al. 2009). Briefly, the entire litter was separated daily from their mother for 3 h (from 09.00 to 12.00 h) from day 3 to 12. Separated pups were placed in a temperature- (35°C) and humidity (45%)-controlled incubator and isolated from each other by a Plexiglass partition. On day 21, rats were weaned and housed in standard animal care conditions until adulthood (8–10 weeks old), at which time experimental procedures were performed. The respiratory and neuroanatomical data obtained from this experimental group were then compared with those of animals not subjected to NMS and continuously maintained in standard animal care procedures. Note that for each group, rats originated from at least three different litters to ensure that treatment-related differences were not due to a litter-specific effect. The first series of experiments used whole-body plethysmography to measure ventilatory activity in quiet, freely moving animals while proceeding with i.c.v. microinjections of a glutamatergic antagonist. Measurements were performed at rest in awake animals during normoxia (Fi= 0.21) and during moderate hypoxia (Fi= 0.12; 20 min); data were compared between groups (NMS versus control animals). Surgical procedures Rats (NMS, n= 19 and control, n= 20) were placed in an isoflurane-saturated induction chamber. After losing consciousness, the rat was positioned in a stereotaxic holder (David Kopf Instruments, Tujunga, CA, USA) equipped with a nose cone to maintain anaesthesia with isoflurane (2.0–2.5% delivered in 30% O2; balance N2). A craniotomy was made above the right lateral ventricle, and the ventricle was reached with a single guide cannula (C315/6 mm; Plastic One, Roanoke, VA, USA). The coordinates from bregma were as follows: anteroposterior, −1.2 mm; lateral, +1.6 mm; and dorsoventral, −3.6 mm (Paxinos & Watson, 1998). Once in place, the guide was secured with screws and cranioplastic cement (Dentsply, Woodbridge, ON, Canada). A telemetry temperature probe (Minimitter, Bend, OR, USA) was surgically inserted into the peritoneal cavity and fixed to the abdominal muscle for body temperature monitoring during ventilatory measurements according to our standard procedures (Montandon et al. 2006). Post-surgical care consisted of three subcutaneous injections of a non-steroidal anti-inflammatory (ketoprophene; 2 mg kg−1) mixed in lactated Ringer solution (5 ml) immediately after the surgery and 24 and 48 h post-operatively. Microinjections and respiratory measurements Seven days after the surgery, a 33 gauge injection cannula (Plastic One) was inserted into the guide cannula and positioned in the lateral ventricle. The animal was placed in the plethysmographic chamber, and the cannula was connected to a 25 μl Hamilton glass syringe with a long polyethylene catheter, which swivelled at the top of the chamber to allow free and unrestricted movement of the animal. The cannula and catheter were filled with the drug or PBS (sham treatment) prior to the insertion. The rat acclimated to the chamber for roughly 1 h, and baseline (normoxic) measurements were made when the animal was quiet but appeared awake and the ventilatory variables were stable. Then, measurements of minute ventilation (), breathing frequency (f) and tidal volume (Vt) in awake and unrestrained rats were obtained by whole-body flow-through plethysmography as described in our previous studies (Genest et al. 2007a,b). Briefly, the system consisted of a 4.5 l Plexiglass experimental chamber. The flow of air or hypoxic gas mixture delivered to the chamber was kept constant and ranged between 2.0 and 2.5 l min−1. Barometric pressure, chamber temperature and humidity were also measured to express Vt in millilitres (body temperature and pressure when saturated with water vapour; BTPS) per 100 g. After baseline values had been obtained, the injection began using a ‘Pico pump’ (Harvard Apparatus, Holliston, MA, USA). All rats received a total volume of 3 μl; all drug concentrations were infused at a constant rate of 1 μl min−1 for 3 min. AMPA receptor antagonist 6-Cyano-7-nitroquinoxa-line-2,3-dione (CNQX; a potent, competitive AMPA/kainate ionotropic receptor antagonist; Sigma-Aldrich, Oakville, ON, Canada) was first dissolved in DMSO and then in Dulbecco's PBS (Fisher Scientific, Ottawa, ON, Canada). Two CNQX concentrations (2.2 and 4.3 μm) were used based on the literature (Boivin & Beninger, 2008) and results from preliminary experiments measuring the effects of drug injection on basal respiratory activity and hypoxic ventilatory response. Physiological parameters changed minimally during microinjection of the vehicle (Table 1). Ten minutes after the injection, a hypoxic gas mixture (Fi= 0.12) was delivered to the chamber for 20 min. All measurements were performed between 9.00 and 13.00 h to minimize variability associated with the circadian rhythm. Note that each rat received only one drug concentration (or vehicle) and was subjected to the protocol once, because multiple exposures to hypoxia can elicit persistent changes in respiratory control. At the end of the experiment, rats were deeply anaesthetized with a ketamine (80 mg kg−1) and xylazine (10 mg kg−1) solution administered intraperitoneally. Brains were harvested and frozen. Tissue was cut into 30-μm-thick coronal sections, and the placement of the cannula was verified visually (Nissl staining). Animals with a misplaced cannula were excluded from the analysis. Note that the numbers of animals reported previously do not include the two rats with a misplaced cannula. Receptor autoradiography The AMPA receptors are responsible for the primary depolarization in glutamate-mediated neurotransmission (Carvalho et al. 2008). In a distinct series of experiments, 28 rats (NMS, n= 14 and control, n= 14) were used to measure 3H-AMPA receptor binding in selected structures involved in respiratory regulation. The animals were placed in an isoflurane-saturated induction chamber and decapitated after they had lost consciousness. The brains were immediately removed and frozen with dry ice. Using a cryostat, coronal sections (12 μm thick) were cut through the paraventricular nucleus of the hypothalamus (PVH; bregma −1.8 to −2.12 mm), the caudal region of the nucleus tractus solitarius (cNTS; bregma −13.68 to −14.3 mm) and the cervical spinal cord (C3–C5). Sections were mounted onto gelatin-coated microscope slides and stored at −80°C. The sections were thawed, dried, and then pre-incubated for 30 min in Tris–acetate buffer (100 mm, pH 7.4) at 37°C containing 100 mm potassium thiocyanate and 100 μm EGTA. Brain sections were incubated with 3H-AMPA (Perkin-Elmer, Woodbrige, ON, Canada) for 45 min at 4°C in the same buffer. The 3H-AMPA concentration added to the solution varied for each region (PVH, 70 nm; cNTS, 90 nm; and C3–C5, 200 nm). These concentrations are based on estimates of the maximum receptor binding (Bmax) obtained by obtaining preliminary saturation curves for each brain region from control tissue (concentration range 0–250 nm). In these experiments, the relationships of bound ligand versus concentration (saturation curves) were analysed using a non-linear regression analysis of total and non-specific binding (GraphPad Prism 3; GraphPad software, Inc. La Jolla, CA, USA). For each region, non-specific binding was assessed by adding glutamate (500 nm; Tocris Bioscience, Ellisville, MO, USA). After incubation, the sections were rinsed and dried rapidly and exposed to 3H-sensitive film (Sigma Aldrich, Oakville, ON, Canada) with 3H-standards (GE Healthcare, Mississauga, ON, Canada) for 21 days at −80°C. Films were then processed using Kodak GBX developer (Sigma Aldrich). The relative expression of precursor (pro-BDNF) and mature forms of BDNF can differ between conditions and structures. To reduce the impact of this factor in data interpretation, we used an ELISA to measure the mature form and immunohistochemistry to measure both forms (pro- and mature BDNF) in discrete brain regions. Brain-derived neurotrophic factor ELISA in discrete brain regions These experiments were performed on brain tissue harvested from 164 rats (85 control and 79 NMS animals) that were anaesthetized using isoflurane and then decapitated. Following dissection, brains were immediately frozen on dry ice and stored at −80°C. Tissue from discrete brain regions was collected in a cryostat (−18°C) using the ‘Palkovits Punch’ technique (needle diameter 0.3 mm; Zivic Instruments, Pittsburgh, PA, USA). Tissue samples were collected from the PVH, cNTS and cervical spinal cord at the same level as in series II. For each region, the tissues were pooled (10–12 animals per pool), homogenized in 50 μl of cold lysis buffer (50 mm Tris pH 8.4, 150 mm NaCl, 1% Tergitol™ type NP-40, 10% glycerol, 10 mm phenylmethanesulfonyl fluoride, 1 μg ml−1 aprotinin, 10 μg ml−1 leupeptin and 5 mm sodium orthovanate; Sigma Aldrich), using a pellet mixer (VWR International, Mont-Royal, QC, Canada). The homogenate was then incubated for 30 min on ice, sonicated (two times for 5 s), centrifuged (16,000g at 4°C for 30 min). The supernatant was stored at −80°C until use. Total protein concentrations were determined with Micro BSA™ protein assay kit (Fisher Scientific). The free (mature) BDNF levels in tissue homogenate were measured with the BDNF Emax ImmunoAssay System (Promega, Madison, WI, USA) following the manufacturer's protocol. Briefly, 96-well flat-bottommed immunoplates (Nunc-Immuno™ Modules; Fischer Scientific) were coated overnight at 4°C with monoclonal anti-BDNF antibody. After rinsing once, the wells were blocked for 1 h with the blocking buffer. The plates were rinsed again and incubated with samples or BDNF standards for 2 h. The antigen was incubated with polyclonal anti-human BDNF antibody for 2 h, and the secondary antibody was detected by an additional incubation with anti-IgY horseradish peroxidase conjugate for 1 h. After a final wash, tetramethylbenzidine substrate was added into the wells and incubated for 10 min. Colour development was stopped by the addition of 1 n HCl, after which the absorbance at 450 nm of each well content was measured using a Microplate Reader (μ-Quant; BioTek, Winooski, VT, USA). All incubation periods were carried out at room temperature with shaking (400 r.p.m.), and each wash procedure consisted of five individual washes of the plates unless otherwise stated. Samples were assayed in duplicate, and BDNF concentration was normalized per milligram of total protein. Brain-derived neurotrophic factor immunohistochemistry To corroborate the ELISA results, immunohistochemistry was performed to visualize and quantify changes in BDNF (both precursor and mature)-expressing perikarya within the PVH, the cNTS and the cervical spinal cord. These experiments were performed on tissue obtained from a distinct group of rats (four control and four NMS animals) that were deeply anaesthetized (ketamine and xylazine, i.p., 80 mg kg−1 and 10 mg kg−1, respectively), and perfused with cold saline (0.9%) followed by cold 4% paraformaldehyde in 0.1 m sodium tetraborate buffer (pH 9.5). The brain and cervical spinal cord were removed, postfixed for 24 h in 4% paraformaldehyde and borax (4°C), and then placed in 20% sucrose, 4% paraformaldehyde and borax for 48 h at 4°C. Frozen brains were mounted on a microtome, and the PVH, cNTS and cervical spinal cord were cut into 30-μm-thick coronal sections. The section levels used correspond to those described in the previous series of experiments. Slices were collected in a cold cryoprotectant solution (0.05 m sodium phosphate buffer, 30% ethylene glycol and 20% glycerol) and stored at −20°C until processing. Tissue was first washed with 0.1 m Tris-buffered saline pH 7.4 (TBS) before being treated with fresh 0.1% H2O2 in TBS. After washing with TBS, non-specific binding sites were blocked with 0.5% bovine serum albumin in TBS. After washing with 0.1% Triton X-100 in TBS, staining was performed by incubating the sections with BDNF (N-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in 0.1% Triton X-100 in TBS (1:500) overnight at 4°C. Sections were washed and incubated with a biotinylated secondary antibody (1:1000) diluted in 0.1% Triton X-100 in TBS for 1 h at room temperature. After washing, the sections were incubated with an avidin–biotin–peroxidase reagent (ABC Elite kit; Vector laboratories, Burlington, ON, Canada) for 1 h. Sections were washed and then reacted with a solution containing 0.04% diaminobenzidine tetrahydrochloride, 0.01% hydrogen peroxide and 0.14% nickel chloride in PBS. Negative controls without primary antibody were performed and confirmed the specificity of the labelling. Respiratory measurements: effects of vehicle and CNQX injection on basal breathing Baseline measurements of ventilatory variables were obtained by averaging the last 10 min of stable recording before drug injection (t= 0 min). The effect of drug injection on basal (normoxic) f, VT, and O2 consumption were assessed by calculating the percentage change between the ventilatory variables at the end of the injection procedure (t=+10 min) and the baseline value. Absolute values are reported in Table 1. At the end of the hypoxic exposure, measurements of ventilatory variables were obtained by averaging the last 5 min of recording. The response to hypoxia was then assessed by expressing the values obtained at the end of the protocol as a percentage change from the post-injection value (t=+10 min). For each drug, the effect of protocol (pre- and post-injection + hypoxia), dose effect and stress (control versus NMS) were analysed using three-way ANOVA with protocol as a repeated measure (Statview 5.0; SAS Institute, Cary, NC, USA). For each ventilatory variable, the hypoxic responses were first analysed with a three-way ANOVA on absolute values (hypoxia × dose × stress). This analysis was then validated by performing a two-way ANOVA (dose × stress) on normalized results expressed as a percentage change from the normoxic value measured after the injection. Blind quantitative analysis was performed on the digitized film (AGFA Duoscan T1200, Mortsel, Belgium). The signal was analysed with NIH Image software (version 1.61; W. Rasband, National Institutes of Health, Bethesda, MD, USA). Densitometric analysis was performed by comparison with a standard 3H-microscale. Specific binding was calculated by subtracting ‘background’ signal determined from autoradiographs of sections with non-specific binding. Results were first transformed into tissue radioactivity (in nanocuries per milligram of tissue) and then converted to femtomoles per milligram of tissue using the specific activity of the tritiated ligand (Genest et al. 2007a). These results were then analysed using a two-way ANOVA (region × stress). Structures were identified using the atlas of Paxinos & Watson (1998). For each region of interest (PVH, NTS and cervical spinal cord), the levels analysed were the same as those used for AMPA receptor autoradiography (series II). Sections for NMS and control animals were matched for rostro-caudal level. Within each structure, the number of BDNF-positive perikarya observed within a circle (diameter = 300 μm) that corresponded to the area sampled for ELISA were counted bilaterally and averaged to produce a mean value for each section (see Fig. 3). Three sections were obtained per rat, and sections originating from the same animal were then averaged before producing a group mean for the specific brain region. These results were analysed using a two-way ANOVA (region × stress). Immunohistochemistry and ELISA results were analysed independently. All ANOVAs were followed by a post hoc Fisher's least significant difference test when appropriate. The ANOVA values are reported in the text, while results of post hoc tests are reported in the figures with symbols. Differences were considered significant if P < 0.05. All values are expressed as means ± SEM. In normoxic conditions, measurement of several ventilatory variables and indicators of metabolism showed that the effects of NMS and the microinjection protocol were limited to body temperature, which was lower in rats subjected to NMS than in control animals (Table 1; stress, P= 0.02). Body temperature decreased slightly over the course of the injection procedure (P < 0.0001); however, the change was not dose dependent (dose, P= 0.37). Tidal volumes of rats used for CNQX injection were lower than those assigned to the PBS group (dose effect, P= 0.03); this difference was significant in NMS rats (Table 1). As we discuss below in the section titled `Neonatal stress, drug injection, and ventilatory measurements' the factors explaining the elevated VT measurements observed in PBS-treated NMS rats are unknown. Consistent with previous findings (Genest et al. 2004; Kinkead et al. 2005, 2009), results obtained in PBS-injected rats showed that the HVR of NMS rats was 35% greater than that of control rats (Fig. 1; stress, P= 0.04) owing mainly to a larger tidal volume response (stress effect, P= 0.04). Analysis of absolute (non-normalized) results confirmed these observations (hypoxia × stress, P= 0.04 and 0.03, respectively; data not shown). Analysis of variance on absolute data indicated that CNQX microinjection attenuated the HVR (hypoxia × dose, P= 0.0006) and that this effect was greater in NMS rats than control animals (hypoxia × dose × stress, P= 0.02). Figure 1A reports these data expressed as a percentage change from baseline. Note that performing statistical analyses on normalized data yielded similar results (stress × dose, P= 0.01). Comparison of the effects of CNQX intracerebroventricular injection on the hypoxic ventilatory response of adult male rats subjected to neonatal maternal separation (NMS; filled bars) or undisturbed [open bars; control (Ctrl)]. A, minute ventilation response following intracerebroventricular injection of vehicle (PBS) or CNQX (2.2 and 4.3 μm). B, representative plethysmography recordings illustrating ventilatory activity in each group of rats (NMS versus control) during normoxia (baseline) and near the end of hypoxia (fractional inspired O2= 0.12; 20 min). The top traces were obtained following vehicle injection and the bottom traces following CNQX injection. The frequency and tidal volume components of the minute ventilation response are presented in C and D, respectively. Data are shown as means ± SEM. The number of animals used in each group is detailed in Table 1. A three-way ANOVA with repeated measures was first performed to assess the effects of hypoxia, stress and CNQX concentration on absolute data (minute ventilation, frequency and tidal volume). A two-way ANOVA was then used to confirm the effects of stress and CNQX concentration on the hypoxic responses. *Different from corresponding PBS value at P < 0.05. †Different from corresponding control value at P < 0.05. While CNQX attenuated both the f and VT components of the hyperventilatory response (hypoxia × dose, P= 0.0014 and 0.0013, respectively), VT was the only variable for which the effect of CNQX was influenced by NMS (stress × dose × hypoxia, P= 0.06 and 0.04 for f and VT, respectively). Figure 1C and D presents the f and VT components of the hypoxic responses expressed as a percentage change from baseline. Analysis of the normalized results confirmed these observations (dose × stress, P= 0.09 and 0.05 for f and VT, respectively). Quantification of the 3H-AMPA autoradiographic signal indicated that neonatal stress augmented the number of AMPA binding sites in a region-specific manner (Fig. 2; region × stress, P= 0.02). Specifically, the number of AMPA binding sites in the PVH of NMS rats was 32% greater than that in control rats. In the cervical spinal cord, the bound ligand value measured in tissue from NMS rats was 49% greater than that in control animals. However, 3H-AMPA binding measured in the NTS did not differ between groups. Comparison of 3H-AMPA binding signal between rats previously subjected to NMS (filled bars, n= 14) and control animals (open bars, n= 14) Quantification of the 3H-AMPA binding sites was performed in the nucleus tractus solitarii, the paraventricular nucleus of the hypothalamus and the phrenic motor nucleus located in the ventral horn of the cervical spinal cord (C3–C5). For each region, representative autoradiographs of 3H-AMPA are presented for each group; the non-specific binding is also illustrated. The area analysed is indicated in red in the cross-section diagram of each specific region. The diagrams are adapted from Paxinos & Watson (1998). Data are shown as means + SEM. The effects of stress and region were analysed using a two-way ANOVA. †Different from corresponding control value at P < 0.05. Neonatal stress elicits region-specific changes in BDNF levels The BDNF levels were influenced by NMS, but the effect varied significantly between structures (Fig. 3; region × stress, P= 0.05). Neonatal stress augmented BDNF levels within the PVH. In the cervical spinal cord, however, BDNF levels of rats subjected to NMS were lower than those of control rats. Within the NTS, BDNF levels did not differ between groups. Quantification of BDNF-positive perikarya within these regions was consistent with the ELISA results (Fig. 3; region × stress, P= 0.0008). Comparison of expression level of brain-derived neurotrophic factor (BDNF) between rats previously subjected to NMS (filled bars) and control animals (open bars) Using the micropunch technique for tissue harvesting, quantification of the mature form of BDNF was performed with ELISA in specific brain regions, namely" @default.
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• W1628011621 date "2013-05-15" @default.
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• W1628011621 title "Neonatal stress augments the hypoxic chemoreflex of adult male rats by increasing AMPA receptor-mediated modulation" @default.
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