Conditioned breathing depression during neonatal life as a function of associating ethanol odor and the drug's intoxicating effects.

Fetal and neonatal ethanol-related alterations upon the respiratory system have been described in different mammals. Studies also indicate that perinates learn about the sensory attributes of ethanol and associate them with diverse physiological effects of the state of intoxication. The present study was conducted in rat neonates during a developmental stage equivalent to the third human gestational trimester. The major goal was to analyze the consequences of ethanol odor exposure, the state of intoxication, or the temporal contiguity between these factors upon breathing patterns. The main findings were as follows: (a) a conditioned breathing depression was observed following few trials defined by the association between ethanol odor and the state of intoxication and (b) sequential exposure to ethanol sensitizes the organism to the drug's respiratory depressant effects without affecting ethanol metabolism. These results indicate that early breathing disruptions caused by ethanol can be determined or modulated via learning processes. © 2016 Wiley Periodicals, Inc. Dev Psychobiol 58:670-686, 2016.

Comparison of dipsogenic responses of adult rat offspring as a function of different perinatal programming models.

The perinatal environment interacts with the genotype of the developing organism resulting in a unique phenotype through a developmental or perinatal programming phenomenon. However, it remains unclear how this phenomenon differentially affects particular targets expressing specific drinking responses depending on the perinatal conditions. The main goal of the present study was to compare the dipsogenic responses induced by different thirst models as a function of two perinatal manipulation models, defined by the maternal free access to hypertonic sodium solution and a partial aortic ligation (PAL-W/Na) or a sham-ligation (Sham-W/Na). The programmed adult offspring of both perinatal manipulated models responded similarly when was challenged by overnight water dehydration or after a sodium depletion showing a reduced water intake in comparison to the non-programmed animals. However, when animals were evaluated after a body sodium overload, only adult Sham-W/Na offspring showed drinking differences compared to PAL and control offspring. By analyzing the central neurobiological substrates involved, a significant increase in the number of Fos + cells was found after sodium depletion in the subfornical organ of both programmed groups and an increase in the number of Fos + cells in the dorsal raphe nucleus was only observed in adult depleted PAL-W/Na. Our results suggest that perinatal programming is a phenomenon that differentially affects particular targets which induce specific dipsogenic responses depending on matching between perinatal programming conditions and the osmotic challenge in the latter environment. Probably, each programmed-drinking phenotype has a particular set point to elicit specific repertoires of mechanisms to reestablish fluid balance.

Early ethanol pre-exposure alters breathing patterns by disruptions in the central respiratory network and serotonergic balance in neonate rats.

Early ethanol exposure alters neonatal breathing plasticity. Respiratory EtOH's effects are attributed to central respiratory network disruptions, particularly in the medullary serotonin (5HT) system. In this study we evaluated the effects of neonatal pre-exposure to low/moderate doses upon breathing rates, activation patterns of brainstem's nuclei and expression of 5HT 2A and 2C receptors. At PD9, breathing frequencies, tidal volumes and apneas were examined in pups pre-exposed to vehicle or ethanol (2.0 g/kg) at PDs 3, 5 and 7. This developmental stage is equivalent to the 3rd human gestational trimester, characterized by increased levels of synaptogenesis. Pups were tested under sobriety or under the state of ethanol intoxication and when subjected to normoxia or hypoxia. Number of c-Fos and 5HT immunolabelled cells and relative mRNA expression of 5HT 2A and 2C receptors were quantified in the brainstem. Under normoxia, ethanol pre-exposed pups exhibited breathing depressions and a high number of apneas. An opposite phenomenon was found in ethanol pre-treated pups tested under hypoxia where an exacerbated hypoxic ventilatory response (HVR) was observed. The breathing depression was associated with an increase in the neural activation levels of the raphe obscurus (ROb) and a high mRNA expression of the 5HT 2A receptor in the brainstem while desactivation of the ROb and high activation levels in the solitary tract nucleus and area postrema were associated to the exacerbated HVR. In summary, early ethanol experience induces respiratory disruptions indicative of sensitization processes. Neuroadaptive changes in central respiratory areas under consideration appear to be strongly associated with changes in their respiratory plasticity.

Co-existence of ethanol-related respiratory and motivational learning processes based on a tactile discrimination procedure in neonatal rats.

In rats, high ethanol doses during early postnatal life exert deleterious effects upon brain development that impact diverse social and cognitive abilities. This stage in development partially overlaps with the third human gestational trimester, commonly referred to as the brain growth spurt period. At this stage in development, human fetuses and rat neonates (postnatal days [PD] 3-9) exhibit relatively high respiratory rates that are affected by subteratogenic ethanol doses. Recent studies suggest conditioned breathing responses in the developing organism, given that there are explicit associations between exteroceptive stimuli and the state of ethanol intoxication. Furthermore, studies performed with near-term rat fetuses suggest heightened sensitivity to ethanol's motivational effects. The present study was meant to analyze the unconditioned effects of ethanol intoxication and the possible co-occurrence of learning mechanisms that can impact respiratory plasticity, and to analyze the preference for cues that signal the state of intoxication as well as the effects of the drug, related with motor stimulation. Neonatal rats were subjected to differential experiences with salient tactile cues explicitly paired or not paired with the effects of vehicle or ethanol (2.0 g/kg). A tactile discrimination procedure applied during PDs 3, 5, 7, and 9 allowed the identification of the emergence of ethanol-derived non-associative and associative learning processes that affect breathing plasticity, particularly when considering apneic disruptions. Ethanol was found to partially inhibit the disruptions that appeared to be intimately related with stressful circumstances defined by the experimental procedure. Tactile cues paired with the drug's effects were also observed to exert an inhibitory effect upon these breathing disruptions. The level of contingency between a given tactile cue and ethanol intoxication also resulted in significant changes in the probability of seeking this cue in a tactile preference test. In addition, the state of intoxication exerted motor-stimulating effects. When contrasting the data obtained via the analysis of the different dependent variables, it appears that most ethanol-derived changes are modulated by positive and/or negative (anti-anxiety) reinforcing effects of the drug. As a whole, the study indicates co-existence of ethanol-related functional changes in the developing organism that simultaneously affect respiratory plasticity and preference patterns elicited by stimuli that signal ethanol's motivational effects. These results emphasize the need to consider significant alterations due to minimal ethanol experiences that argue against "safe" levels of exposure in a critical stage in brain development.

Brief ethanol exposure and stress-related factors disorganize neonatal breathing plasticity during the brain growth spurt period in the rat.

RATIONALE: The effects of early ethanol exposure upon neonatal respiratory plasticity have received progressive attention given a multifactorial perspective related with sudden infant death syndrome or hypoxia-associated syndromes. The present preclinical study was performed in 3-9-day-old pups, a stage in development characterized by a brain growth spurt that partially overlaps with the 3rd human gestational trimester. METHODS: Breathing frequencies and apneas were examined in pups receiving vehicle or a relatively moderate ethanol dose (2.0 g/kg) utilizing a whole body plethysmograph. The experimental design also considered possible associations between drug administration stress and exteroceptive cues (plethysmographic context or an artificial odor). Ethanol exposure progressively exerted a detrimental effect upon breathing frequencies. A test conducted at PD9 when pups were under the state of sobriety confirmed ethanol's detrimental effects upon respiratory plasticity (breathing depression). RESULTS: Pre-exposure to the drug also resulted in a highly disorganized respiratory response following a hypoxic event, i.e., heightened apneic episodes. Associative processes involving drug administration procedures and placement in the plethysmographic context also affected respiratory plasticity. Pups that experienced intragastric administrations in close temporal contiguity with such a context showed diminished hyperventilation during hypoxia. In a 2nd test conducted at PD9 while pups were intoxicated and undergoing hypoxia, an attenuated hyperventilatory response was observed. In this test, there were also indications that prior ethanol exposure depressed breathing frequencies during hypoxia and a recovery normoxia phase. CONCLUSION: As a whole, the results demonstrated that brief ethanol experience and stress-related factors significantly disorganize respiratory patterns as well as arousal responses linked to hypoxia in neonatal rats.

Pup circadian rhythm entrainment--effect of maternal ganglionectomy or pinealectomy.

In rodents, during late embryonic and early neonatal development, circadian rhythms develop in synchrony with those of their mothers, which in turn are synchronized with the environmental photoperiod. This paper examines the effect of maternal ganglionectomy (pineal gland sympathetic denervation) or extirpation of the pineal gland on pups' drinking rhythms, a behavior that is continuously monitored in individual animals starting after weaning and studied up to 3 weeks later. Maternal ganglionectomy or pinealectomy performed on the 7th day of gestation significantly disrupts rat pups' drinking behavior, within and among litters. In both treatments, circadian rhythm characteristics of the free-running period (tau), phase, amplitude and alpha were significantly altered compared to those of the control pups born from sham-operated mothers. With the exception of the alpha component, both maternal treatments have similar effects. When melatonin was given to the mothers instead of the endogenous pineal secretory activity for 5 days during the late period of gestation, this treatment reversed the effects of maternal ganglionectomy and pinealectomy. These observations, together with previous studies of our group, indicate that the maternal superior cervical ganglia and pineal gland are necessary components of the mechanism for maternal synchronization, and that maternal melatonin may, directly or indirectly, affect the performance of the pups' central oscillator during early pup rat development.

Early free access to hypertonic NaCl solution induces a long-term effect on drinking, brain cell activity and gene expression of adult rat offspring.

Exposure to an altered osmotic environment during a pre/postnatal period can differentially program the fluid intake and excretion pattern profile in a way that persists until adulthood. However, knowledge about the programming effects on the underlying brain neurochemical circuits of thirst and hydroelectrolyte balance, and its relation with behavioral outputs, is limited. We evaluated whether early voluntary intake of hypertonic NaCl solution may program adult offspring fluid balance, plasma vasopressin, neural activity, and brain vasopressin and angiotensinergic receptor type 1a (AT1a)-receptor gene expression. The manipulation (M) period covered dams from 1 week before conception until offspring turned 1-month-old. The experimental groups were (i) Free access to hypertonic NaCl solution (0.45 M NaCl), food (0.18% NaCl) and water [M-Na]; and (ii) Free access to food and water only [M-Ctrol]. Male offspring (2-month-old) were subjected to iv infusion (0.15 ml/min) of hypertonic (1.5M NaCl), isotonic (0.15M NaCl) or sham infusion during 20 min. Cumulative water intake (140 min) and drinking latency to the first lick were recorded from the start of the infusion. Our results indicate that, after systemic sodium overload, the M-Na group had increased water intake, and diminished neuronal activity (Fos-immunoreactivity) in the subfornical organ (SFO) and nucleus of the solitary tract. They also showed reduced relative vasopressin (AVP)-mRNA and AT1a-mRNA expression at the supraoptic nucleus and SFO, respectively. The data indicate that the availability of a rich source of sodium during the pre/postnatal period induces a long-term effect on drinking, neural activity, and brain gene expression implicated in the control of hydroelectrolyte balance.

Temporal dissociation between sodium depletion and sodium appetite appearance: Involvement of inhibitory and stimulatory signals.

Our aim was to analyze the participation of inhibitory and stimulatory signals in the temporal dissociation between sodium depletion (SD) induced by peritoneal dialysis (PD) and the appearance of sodium appetite (SA), particularly 2h after PD, when the rats are hypovolemic/natremic but SA is not evident. We investigated the effects of bilateral injections of the serotonin (5-HT) receptor antagonist, methysergide, into the lateral parabrachial nucleus (LPBN) on hypertonic NaCl and water intake 2h vs. 24h after PD. We also studied plasma renin activity (PRA) and aldosterone (ALDO) concentration 2h vs. 24h after PD. Additionally, we combined the analysis of brain Fos immunoreactivity (Fos-ir) with the detection of double immunoreactivity in 5HT and oxytocinergic (OT) cells 2h after PD. Bilateral LPBN injections of methysergide (4µg/200nl at each site) increased NaCl intake when tested 2h after PD compared to controls. We found a significant increase in PRA and ALDO concentration after PD but no differences between 2 and 24h after PD. We also found for the first time a significant increase 2h after PD in the number of Fos-ir neurons in the brainstem nuclei that have been shown to be involved in the inhibition of SA. In summary, the results show that 5HT-mechanisms in the LPBN modulate sodium intake during the delay of SA when the renin angiotensin aldosterone system (RAAS) is increased. In addition, the activation of brainstem areas previously associated with the satiety phase of SA is in part responsible for the temporal dissociation between SD and behavioral arousal.

Availability of a rich source of sodium during the perinatal period programs the fluid balance restoration pattern in adult offspring.

Osmoregulatory mechanisms can be vulnerable to electrolyte and/or endocrine environmental changes during the perinatal period, differentially programming the developing offspring and affecting them even in adulthood. The aim of this study was to evaluate whether availability of hypertonic sodium solution during the perinatal period may induce a differential programming in adult offspring osmoregulatory mechanisms. With this aim, we studied water and sodium intake after Furosemide-sodium depletion in adult offspring exposed to hypertonic sodium solution from 1 week before mating until postnatal day 28 of the offspring, used as a perinatal manipulation model [PM-Na group]. In these animals, we also identified the cell population groups in brain nuclei activated by Furosemide-sodium depletion treatment, analyzing the spatial patterns of Fos and Fos-vasopressin immunoreactivity. In sodium depleted rats, sodium and water intake were significantly lower in the PM-Na group vs. animals without access to hypertonic sodium solution [PM-Ctrol group]. Interestingly, when comparing the volumes consumed of both solutions in each PM group, our data show the expected significant differences between both solutions ingested in the PM-Ctrol group, which makes an isotonic cocktail; however, in the PM-Na group there were no significant differences in the volumes of both solutions consumed after Furosemide-sodium depletion, and therefore the sodium concentration of total fluid ingested by this group was significantly higher than that in the PM-Ctrol group. With regard to brain Fos immunoreactivity, we observed that Furosemide-sodium depletion in the PM-Na group induced a higher number of activated cells in the subfornical organ, ventral subdivision of the paraventricular nucleus and vasopressinergic neurons of the supraoptic nucleus than in the PM-Ctrol animals. Moreover, along the brainstem, we found a decreased number of sodium depletion-activated cells within the nucleus of the solitary tract of the PM-Na group. Our data indicate that early sodium availability induces a long-term effect on fluid drinking and on the cell activity of brain nuclei involved in the control of hydromineral balance. These results also suggest that availability of a rich source of sodium during the perinatal period may provoke a larger anticipatory response in the offspring, activating the vasopressinergic system and reducing thirst after water and sodium depletion, as a result of central osmosensitive mechanism alterations.