Nicotine-exposed Pups Research Articles

Investigating the Modulation of the VTA Neurons in Nicotine-Exposed Pups during Early Maturation Using Optogenetics.

Advancing the understanding of the relationship between perinatal nicotine addiction and the reward mechanism of the brain is crucial for uncovering and implementing new treatments for addiction control and prevention. The mesolimbic pathway of the brain, also known as the reward pathway, consists of two main areas that regulate dopamine (DA) and addiction-related behaviors. The ventral tegmental area (VTA) releases DA when stimulated, causing the propagation of neuronal firing along the pathway. This ends in the release of DA into the extracellular space of the nucleus accumbens (NAc), which is directly modulated by the uptake of DA. Much research has been conducted on the effects of nicotine addiction, but little research has been conducted concerning nicotine addiction and the mesolimbic pathway regarding maturation due to the small brain size. In this study, we apply our novel microstimulation experimental system to rat pups that have been perinatally exposed to nicotine. By using our self-fabricated photo-stimulation (PS) device, we can stimulate the VTA and collect dialysate, which is then used to estimate DA released into the NAc. The proposed platform has demonstrated the potential to monitor neural pathways as the pups mature.

Perinatal nicotine exposure alters development of cardiac performance

Perinatal nicotine exposure alters development of brainstem neurons, including those involved in the control of breathing and cardiovascular function. Whether or not perinatal nicotine exposure alters development of cardiac performance is unknown. Pregnant rats were exposed to nicotine plus saccharin in drinking water beginning on the 4thday of gestation. Developing fetuses were exposed via the placental circulation, and exposure continued via breast milk after birth, up until the day of study. Plasma cotinine levels in the nicotine‐exposed pups ranged from 50‐100 ng/ml. Control neonates drank water with saccharin, but no nicotine. Animals aged postnatal day 10‐12 (P10‐P12) were lightly anesthetized with isoflurane, and cardiac function was assessed non‐invasively with echocardiography. Compared to controls (N=13), nicotine‐exposed pups (N=13) had reduced body weight (28.4 ± 1 vs. 21 ± 1 g, P=0.00032), significant reductions in stroke volume (39.1 ± 1 vs. 33.7 ± 1.7 μL/beat, P=0.027) and cardiac output (14.0 ± 0.8 vs. 12.4 ± 1 ml/min, P=0.011), and had significantly slower isovolumetric contraction (15 ± 1 vs. 22 ± 2 ms, P=0.005) and relaxation times (21 ± 1 vs. 33 ± 3 ms, P=0.04). There were also trends toward a lower heart rate (P=0.055), ejection fraction (P=0.065) and left ventricular wall thickness (P=0.059) in the nicotine exposed animals. These are the first data showing that perinatal nicotine exposure alters development of the heart’s pumping capacity.

Prenatal nicotine exposure enhances the trigeminocardiac reflex via serotonin receptor facilitation in brainstem pathways

In this study we used a rat model for prenatal nicotine exposure to test whether clinically relevant concentrations of brain nicotine and cotinine are passed from dams exposed to nicotine to her pups, whether this changes the trigeminocardiac reflex (TCR), and whether serotonergic function in the TCR brainstem circuitry is altered. Pregnant Sprague-Dawley dams were exposed to 6 mg·kg(-1)·day(-1) of nicotine via osmotic minipumps for the duration of pregnancy. Following birth dams and pups were killed, blood was collected, and brain nicotine and cotinine levels were measured. A separate group of prenatal nicotine-exposed pups was used for electrophysiological recordings. A horizontal brainstem slice was obtained by carefully preserving the trigeminal nerve with fluorescent identification of cardiac vagal neurons (CVNs) in the nucleus ambiguus. Stimulation of the trigeminal nerve evoked excitatory postsynaptic current in CVNs. Our data demonstrate that prenatal nicotine exposure significantly exaggerates both the TCR-evoked changes in heart rate in conscious unrestrained pups, and the excitatory neurotransmission to CVNs upon trigeminal afferent nerve stimulation within this brainstem reflex circuit. Application of the 5-HT1A receptor antagonist WAY 100635 (100 μM) and 5-HT2A/C receptor antagonist ketanserin (10 μM)significantly decreased neurotransmission, indicating an increased facilitation of 5-HT function in prenatal nicotine-exposed animals. Prenatal nicotine exposure enhances activation of 5-HT receptors and exaggerates the trigeminocardiac reflex.

Postnatal effects of maternal nicotine exposure on the striatal dopaminergic system in rats

The biochemical effects of continuous prenatal exposure to nicotine (1.5 mg kg-1 day-1) have been examined in 14 day old male and female rat pups. The ability of striatal tissue slices from the nicotine-exposed pups to synthesize [3H]dopamine from [3H]tyrosine was enhanced. However, the ability of (+)-amphetamine to stimulate formation and release of [3H]dopamine from [3H]tyrosine was not affected. Furthermore, prenatal exposure to nicotine increased the rate of striatal dopamine turnover in the male, but not the female nicotine-exposed pups.

Postnatal behavioural effects of maternal nicotine exposure in rats.

The effects of nicotine on locomotor activity have been studied in neonate rats exposed to nicotine (1.5 mg kg-1 day-1) throughout the gestational period. Both 14 day old male and female offspring demonstrated an increase in spontaneous locomotor activity when compared with saline-exposed controls. However, systemic administration of (+)-amphetamine was effective in attenuating the hyperactivity of these nicotine-exposed pups.

Chronic Nicotine Blunts Hypoxic Sensitivity in Perinatal Rat Adrenal Chromaffin Cells via Upregulation of KATPChannels: Role of α7 Nicotinic Acetylcholine Receptor and Hypoxia-Inducible Factor-2α

Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K(ATP) channels via stimulation of alpha7 nicotinic ACh receptors (AChRs). These K(ATP) channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K(+) current, membrane depolarization, rise in cytosolic Ca(2+), and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K(ATP) channels, was present. Both K(ATP) current density and K(ATP) channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), suggesting involvement of the HIF pathway. The therapeutic potential of K(ATP) channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

Prenatal nicotine exposure impairs postnatal sensitivity to hypoxia and hypercapnia

Data on the influence of prenatal nicotine exposure on the postnatal development of hypoxic and hypercapnic sensitivity are equivocal. We previously presented preliminary data showing some abnormalities in the response to hypercapnia and hypoxia in nicotine-exposed neonatal rats. Here, we report the responses to hypoxia (10% inspired O2), hyperoxia (100% O2), hyperoxic hypercapnia (9% CO2, 50% O2, balance N2) and asphyxia (12% O2, 5% CO2, balance N2) in 26 neonatal rats; 13 underwent in utero saline exposure, and 13 in utero nicotine exposure. Each animal was studied on postnatal days 1,3,6,9,12 and 18 with head-out body plethysmography. Ventilatory responses were analyzed with 3-factor ANOVA, with treatment, age and test gas the main factors. Nicotine-exposed pups showed blunted ventilatory responses to hypoxia on days 3 and 9, but a larger response on day 18. The drop in ventilation in response to hyperoxia was blunted on days 6 and 9 in nicotine-exposed pups, but they showed a larger drop on day 18. Nicotine blunted the ventilatory response to hypercapnia on days 6 and 12, while the change in ventilation with asphyxia was significantly blunted only on day 1. Prenatal nicotine exposure impairs the development of chemoreceptor reflexes, with manifestation of the developmental abnormalities dependent on both age and stimulus modality.

Effects of maternal intravenous nicotine administration on locomotor behavior in pre-weanling rats

Maternal tobacco use is associated with adverse developmental outcomes in offspring, including hyperactivity. Animal studies attempting to model this phenomenon have primarily used continuous s.c. nicotine infusion as the method of nicotine administration, which does not model the intermittent bolus delivery of nicotine associated with smoking in humans. The purpose of the present experiment was to examine the locomotor activity of pre-weanling offspring of pregnant rats exposed to an i.v. nicotine dosing protocol that approximates the pattern of nicotine exposure in moderate to heavy smokers. Pregnant rats were administered an i.v. bolus of 0.03 mg/kg nicotine ( N = 13) or saline ( N = 10) every 14 min for 16 h/day, resulting in a total daily dose of 2 mg/kg (base), from gestational day 4 to delivery. Pups from each litter were tested for spontaneous locomotor activity on postnatal days (PND) 19–21 and nicotine-induced locomotor activity on PND 22. Mean birth weight was significantly lower in nicotine-exposed pups compared to controls, but body weights were equivalent between groups by the time of behavioral testing. Mean total distance traveled, vertical counts, and stereotypy counts were lower on PND 19 in nicotine-exposed pups compared to controls, but only the difference in mean stereotypy counts was statistically significant. Within-session analysis revealed that both distance traveled and stereotypy were significantly decreased in nicotine-exposed pups in the first 5 min of the session on PND 19. Total time spent in the center of the field was also lower in nicotine-exposed pups. Nicotine-induced increases in activity on PND 22 did not differ according to gestational exposure. These findings demonstrate that prenatal nicotine exposure in a model that mimics the pattern of nicotine exposure from cigarette smoking in humans results in offspring that exhibit low birth weight and hypoactivity in a novel environment.

Cholinergic Modulation of Appetite-Related Synapses in Mouse Lateral Hypothalamic Slice

Nicotine administration reduces appetite and alters feeding patterns; a major deterrent to smoking cessation is hyperphagia and resultant weight gain. We demonstrate here that lateral hypothalamic (LH) circuits involving melanin-concentrating hormone (MCH) neurons are subject to cholinergic modulation that may be related to the effects of nicotine on appetite control. Cholinergic input to the perifornical LH area of the mouse is confirmed by examination of immunostaining for vesicular acetylcholine (ACh) transporter (VAT) in conjunction with antibodies to MCH and the vesicular GABA transporter (vGABAT). vAChT-positive neurons border the LH, and VAT-positive projections are detected throughout the perifornical area. MCH-positive dendrites appear studded with vGABAT-positive contacts, consistent with recordings of GABAergic inputs to LH/MCH neurons identified by their location, morphology, electrophysiological profile, and MCH expression. Activation of presynaptic nicotinic ACh receptors (nAChRs) enhances GABAergic transmission. GABAergic transmission is potentiated by (1) direct nicotine application, (2) increasing local ACh concentration, and (3) stimulation of cholinergic projections. Based on pharmacological studies and comparisons of wild-type versus alpha7 nAChR subunit mutant mice, we propose that alpha7*-nAChRs are required for the modulation of GABAergic inputs in LH. Prenatal exposure to nicotine elicits a persistent elevation of GABAergic transmission in the LH of postnatal pups. Furthermore, GABAergic inputs to LH of prenatal nicotine-exposed pups are insensitive to subsequent nicotine challenge. Our studies support the hypothesis that nicotine administration or elevated cholinergic tone enhance inhibition of perifonical LH/MCH neurons via activation of presynaptic alpha7*-nAChRs.

Influence of prenatal nicotine exposure on postnatal development of breathing pattern.

To determine if prenatal nicotine exposure alters the postnatal development of the ventilatory pattern and the frequency and duration of apneas, we recorded respiratory airflow with head-out body plethysmography in awake neonates on postnatal days 1, 2, 6, 10, 14, and 18. Data from 12 nicotine-exposed animals were compared with data from 12 saline-exposed animals. Nicotine (6 mg/kg of nicotine tartrate per day) or saline exposure was induced by osmotic minipumps that were implanted subdermally on the fifth day of gestation in Sprague-Dawley Dams. Although both saline- and nicotine-exposed pups gained weight at the same rate throughout the studies, there were subtle differences in ventilatory indices between the two groups. Nicotine-exposed animals had a significantly higher breathing frequency on day 10, and a lower tidal volume on days 14 and 18. Although ventilation tended to be lower in the nicotine-exposed animals, the difference was not significant. There was a significantly higher frequency of apneas in the nicotine-exposed compared with the saline-exposed animals on postnatal days 1 and 2, but the apnea duration did not differ between the groups. No apneas were observed in any of the animals after the sixth postnatal day. Prenatal nicotine exposure is associated with a greater incidence of apneas on the first two postnatal days, and then an altered breathing pattern that manifests at a later stage of development.

Changes in the glutamate release and uptake of cerebellar cells in perinatally nicotine-exposed rat pups.

Cerebellar granule and glial cells were cultured from 7 day-old rat pups after pre- and post-natal nicotine treatment. Ten days later, the basal release of glutamate in the granule cells prepared from the pre- and post-natally nicotine-exposed pups was higher and lower than the controls, respectively. The N-methyl-D-aspartate-induced release of glutamate was higher in the granule cells of post-natal nicotine exposed rats. However, the nicotine-induced glutamate release was either unchanged or was lower in the granule cells of all nicotine-treated pups. The basal glutamate uptake was higher in the glial cells from those exposed pre-natally and lower in the continuously nicotine-exposed pups. The sensitivities of L-trans-pyrrolidine-2,4-dicarboxylic acid on glutamate uptake were higher in all nicotine treated groups. There was a higher number of specific [3H]dizocilpine binding sites in the pre- or continuously nicotine-exposed group. These results suggest that the cerebellar cell properties are altered after perinatal nicotine exposure and that the development of an excitatory amino acid system might be affected differently depending on the nicotine exposure time.

Effects of gestational nicotine exposure on hippocampal morphology.

Morphology of the dentate gyrus and Ammon's horn of the hippocampus was observed postnatally in Wistar rats exposed to prenatal nicotine. Pregnant Wistar rats were injected with nicotine (2.5 mg/kg/ day) from gestational day (GD) 6 to term and pups were delivered at term normally. The brains of nicotine-exposed pups at postnatal day (PND) 40 were dissected and processed for araldite embedding. Semithin (0.5 microm) toluidine blue-stained sections of the dentate gyrus, CA3, and CA1 regions of the hippocampus were studied for morphological details. Morphometric analysis showed a significant reduction in the neuronal area of nicotine-exposed brains in all three regions studied. Distal dendritic arbor was significantly less and spine density was more in the granule cells, terminal and basal dendrites of the pyramidal neurons of CA3 and CA1 regions of the hippocampus in the experimental group. CA3 pyramidal neurons showed significantly less apical dendritic thorny excrescence after gestational nicotine exposure. Ultrastructurally the granule cells and the pyramidal neurons of the CA3 and CA1 regions showed increase in free ribosomes and dilatation of rough endoplasmic reticulum (RER) and Golgi apparatus cisternae in the nicotine-treated group. The morphological changes in the hippocampus following prenatal nicotine exposure, as observed in the present study, may contribute to the behavioural abnormalities.