Prenatal nicotine exposure increases apnoea and reduces nicotinic potentiation of hypoglossal inspiratory output in mice

We examined the effects of in utero nicotine exposure on postnatal development of breathing pattern and ventilatory responses to hypoxia (7.4 % O₂) using whole-body plethysmography in mice at postnatal day 0 (P0), P3, P9, P19 and P42. Nicotine delayed early postnatal changes in breathing pattern. During normoxia, control and nicotine-exposed P0 mice exhibited a high frequency of apnoea ( f _A) which declined by P3 in control animals (from 6.7 ± 0.7 to 2.2 ± 0.7 min⁻¹) but persisted in P3 nicotine-exposed animals (5.4 ± 1.3 min⁻¹). Hypoxia induced a rapid and sustained reduction in f _A except in P0 nicotine-exposed animals where it fell initially and then increased throughout the hypoxic period. During recovery, f _A increased above control levels in both groups at P0. By P3 this increase was reduced in control but persisted in nicotine-exposed animals. To examine the origin of differences in respiratory behaviour, we compared the activity of hypoglossal (XII) nerves and motoneurons in medullary slice preparations. The frequency and variability of the respiratory rhythm and the envelope of inspiratory activity in XII nerves and motoneurons were indistinguishable between control and nicotine-exposed animals. Activation of postsynaptic nicotine receptors caused an inward current in XII motoneurons that potentiated XII nerve burst amplitude by 25 ± 5 % in control but only 14 ± 3 % in nicotine-exposed animals. Increased apnoea following nicotine exposure does not appear to reflect changes in basal activity of rhythm or pattern-generating networks, but may result, in part, from reduced nicotinic modulation of XII motoneurons.     

Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.     

Effects of chronic neonatal nicotine exposure on nicotinic acetylcholine receptor binding, cell death and morphology in hippocampus and cerebellum

Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric alpha7 nAChRs, neuroprotection to heteromeric alpha4beta2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for alpha4, alpha7 and beta2 mRNA was determined by in situ hybridization; nAChR binding sites by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl Violet-stained sections. In control cerebellum, strong expression of alpha4, beta2 mRNA and heteromeric nAChRs labeled with [125I]-epibatidine was found in granule cells, and alpha7 mRNA and homomeric nAChRs labeled with [125I]-alpha-bungarotoxin were in the external germinal layer. In control hippocampus, low expression of alpha4 mRNA and heteromeric nAChRs and high expression of alpha7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.     

Effect of repeated nicotine exposure on high-affinity nicotinic acetylcholine receptor density in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHRs) are often used as a model of attention deficit hyperactivity disorder (ADHD) and to investigate the effects of hypertension on cognitive function. Further, they appear to have reduced numbers of central nicotinic acetylcholine receptors (nAChRs) and, therefore, may be useful to model certain aspects of Alzheimer's disease (AD) and other forms of dementia given that a decrease in nAChRs is thought to contribute to cognitive decline in these disorders. In the present study, based on reports that chronic nicotine exposure increases nAChRs in several mammalian models, we tested the hypothesis that repeated exposures to a relatively low dose of the alkaloid would ameliorate the receptor deficits in SHR. Thus, young-adult SHRs and age-matched Wistar-Kyoto (WKY) control rats were treated with either saline or nicotine twice a day for 14 days (total daily dose = 0.7 mg/kg nicotine base) and then sacrificed. Quantitative receptor autoradiography with [125I]-IPH, an epibatidine analog, revealed: (1) that high-affinity nAChRs were higher in saline-treated WKY (control) rats compared to saline-treated SHRs in 18 of the 19 brain region measured, although statistically different only in the mediodorsal thalamic nuclei, (2) that nicotine significantly increased nAChR binding in WKY rats in six brain areas including cortical regions and the anterior thalamic nucleus, (3) that there were no cases where nicotine significantly increased nAChR binding in SHRs. These results indicate that subjects deficient in nAChRs may be less sensitive to nAChR upregulation with nicotine than normal subjects and require higher doses or longer periods of exposure.     

Prenatal nicotine exposure alters the types of nicotinic receptors that facilitate excitatory inputs to cardiac vagal neurons

Nicotinic receptors play an important role in modulating the activity of parasympathetic cardiac vagal neurons in the medulla. Previous work has shown nicotine acts via at least three mechanisms to excite brain stem premotor cardiac vagal neurons. Nicotine evokes a direct increase in holding current and facilitates both the frequency and amplitude of glutamatergic neurotransmission to cardiac vagal neurons. This study tests whether these nicotinic receptor-mediated responses are endogenously active, whether alpha4beta2 and alpha7 nicotinic receptors are involved, and whether prenatal exposure to nicotine alters the magnitude of these responses and the types of nicotinic receptors involved. Application of neostigmine (10 microM) significantly increased the holding current, amplitude, and frequency of miniature excitatory postsynaptic current (mEPSC) glutamatergic events in cardiac vagal neurons. In unexposed animals, the nicotine-evoked facilitation of mEPSC frequency, but not mEPSC amplitude or holding current, was blocked by alpha-bungarotoxin (100 nM). Prenatal nicotine exposure significantly exaggerated and altered the types of nicotinic receptors involved in these responses. In prenatal nicotine-exposed animals, alpha-bungarotoxin only partially reduced the increase in mEPSC frequency. In addition, in prenatal nicotine-exposed animals, the increase in holding current was partially dependent on alpha-7 subunit-containing nicotinic receptors, in contrast to unexposed animals in which alpha-bungarotoxin had no effect. These results indicate prenatal nicotine exposure, one of the highest risk factors for sudden infant death syndrome (SIDS), exaggerates the responses and changes the types of nicotinic receptors involved in exciting premotor cardiac vagal neurons. These alterations could be responsible for the pronounced bradycardia that occurs during apnea in SIDS victims.     

Disconnection between activation and desensitization of autonomic nicotinic receptors by nicotine and cotinine

Cotinine is the major metabolite of nicotine in humans, and the substance greatly outlasts the presence of nicotine in the body. Recently, cotinine has been shown to exert pharmacological properties of its own that include potential cognition enhancement, anti-psychotic activity, and cytoprotection. Since the metabolite is generally less potent than nicotine in vivo, we considered whether part of cotinine's efficacy could be related to a reduced ability to desensitize nicotinic receptors as compared with nicotine. Rats freely moving in their home cages were instrumented to allow ongoing measurement of mean arterial blood pressure (MAP). The ganglionic stimulant dimethylphenylpiperazinium (DMPP) maximally increased MAP by 25mmHg. Slow (20min) i.v. infusion of nicotine (0.25-1micromol) produced no change in resting MAP, but the pressor response to subsequent injection of DMPP was significantly attenuated in a dose-dependent manner by up to 51%. Pre-infusion of equivalent doses of cotinine produced the same maximal degree of inhibition of the response to DMPP. Discrete i.v. injections of nicotine also produced a dose dependent increase in MAP of up to 43mmHg after the highest tolerated dose. In contrast, injection of cotinine produced no significant change in MAP up to 13 times the highest dose of nicotine. These results illustrate the disconnection between nicotinic receptor activation and receptor desensitization, and they suggest that cotinine's pharmacological actions are either mediated through partial desensitization, or through non-ganglionic subtypes of nicotinic receptors.     

Sex differences in anxiety-like behavior and locomotor activity following chronic nicotine exposure in mice

Smoking appears to increase overall levels of stress, despite self-reports that men and women smoke to control symptoms of anxiety. The overall incidence of anxiety disorders is also significantly higher in women. This study examined whether behavioral sensitivity to chronic nicotine varies across sexes in mice. Male and female C57BL/6J mice were exposed chronically to nicotine in the drinking water (50, 100, or 200 microg/ml) and tested for locomotor activation and anxiety-like behavior in the elevated plus maze (EPM). Female mice were less sensitive to the locomotor activating effects of chronic nicotine. Whereas both males and females showed increases in locomotor activity at the highest (200 microg/ml) concentration of nicotine, only males showed locomotor activation at the middle (100 microg/ml) concentration. The decreased sensitivity in females could not be explained by reduced nicotine intake compared to males. In the EPM, nicotine produced an anxiogenic-like response in females, but had no effect in males. Treatment with the high (200 microg/ml) dose of nicotine reduced the amount of time spent in the open arms of the EPM in female, but not male mice. No differences in the anxiogenic-like response to chronic nicotine was observed between beta2-subunit knockout and wildtype mice, suggesting that beta2-subunit containing nicotinic receptors do not mediate the anxiogenic-like response to chronic nicotine in females. This shows that female mice have an anxiogenic-like response to chronic nicotine, but are less sensitive to nicotine's psychostimulant properties, which may be related to the increased relapse to smoking following abstinence in women.     

Regulation of nicotinic acetylcholine receptor desensitization by Ca2+

The relationship between the concentration of intracellular Ca2+ ([Ca2+](i)) and recovery from desensitization of nicotinic acetylcholine receptors (nAChRs) in rat medial habenula (MHb) neurons was investigated using the whole cell patch-clamp techniques in combination with microfluorescent [Ca2+](i) measurements. Recovery from desensitization was assessed with a paired-pulse agonist application protocol. Application of 100 microM nicotine (5 s) caused pronounced desensitization of nAChRs, after which recovery proceeded with two components. The relative weight of the two phases of recovery was sensitive to the nature of the intracellular Ca2+ chelator, with a greater fraction of channels recovering during the fast phase in the presence of BAPTA than EGTA. Recovery was affected by differential Ca2+ buffering only when Ca2+ was present in the extracellular solution, implying that Ca2+ influx through nAChRs was responsible for slowing the recovery. Simultaneous [Ca2+](i) measurements showed that recovery from desensitization was inversely correlated with the instantaneous [Ca2+](i), further supporting the suggestion that elevation of [Ca2+](i) limits the return of nAChRs to the resting state. In a separate set of experiments, activation of voltage-gated Ca2+ channels during the recovery phase produced a sufficiently large increase in [Ca2+](i) to reduce recovery from desensitization even in the absence of Ca2+ influx through nAChRs. Overall, it is suggested that Ca2+ entry through both nAChRs and voltage-gated Ca2+ channels exerts a negative feedback on nAChR activity through stabilization of desensitized states. The interaction of these two Ca2+ sources could form the basis of a coincidence detector under specific circumstances.     

A neuronal nicotinic acetylcholine receptor in crayfish neurons

In warm-blooded vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) are distinguished from muscle endplate receptors by their ligand affinities and sensitivity to several toxins. In the crayfish optic lobe, synaptic and acetylcholine (ACh)-elicited responses are blocked by toxins (F-toxin and neosurugatoxin) selective for neuronal nAChRs and are insensitive to the alpha-neurotoxins selective for endplate nAChRs.     

Herpes simplex virus infection of motor neurons: hypoglossal model.

Herpes simplex virus type 1 (HSV) was inoculated into the tongue muscle of A/J mice. Typical HSV vesicles developed on the tongue surface 4 days after HSV inoculation. Virus was isolated from hypoglossal nerve explants, and inflammatory cells appeared where the hypoglossal nerve exists from the ventral medulla. HSV viral capsids were present in astroglial cells near the point of nerve exit. A focal encephalitis ensued with immunoperoxidase staining of HSV antigens in neurons of the hypoglossal nucleus. These findings indicate that HSV can penetrate the neuromuscular junction, travel in a pure motor nerve, and produce a focal encephalitis in the corresponding central nervous system motor nucleus.     

Increased responsiveness of rat dorsal horn neurons in vivo following prolonged intrathecal exposure to interferon-gamma

Prolonged increases in the level of the pro-inflammatory cytokine interferon-gamma occur in the CNS during some disease states associated with persistent pain. Administration of interferon-gamma to both humans and rodents has produced pain or pain-related behavior but the underlying mechanisms are unknown. The present study examined the effects of repeated intrathecal administration of interferon-gamma on dorsal horn neuronal responses under in vivo conditions. In addition, behavioral effects of interferon-gamma treatment were studied. Intrathecal cannulae were implanted into anesthetized rats. Animals then received either 1000 U of recombinant rat interferon-gamma in 10 microl buffer intrathecally, repeated four times over 8 days, or similarly administered buffer (controls). Interferon-gamma-treated animals showed a significant reduction in paw withdrawal threshold to mechanical stimulation of the hind paw. Electrophysiological experiments were performed under halothane anesthesia. Extracellular recordings of spontaneous and evoked responses were obtained from dorsal horn neurons (n=64) in the lumbar spinal cord. There was a significantly higher proportion of spontaneously active neurons in the interferon-gamma-treated animals (50%) when compared with controls (19%). A significantly increased proportion of neurons from interferon-gamma-treated animals displayed afterdischarges following both innocuous and noxious mechanical stimulation of the receptive field (brush: 21% in interferon-gamma-treated, 3% in controls; pinch: 97% in interferon-gamma-treated, 50% in controls). Neurons from interferon-gamma-treated animals also showed significantly increased wind-up of action potentials in response to repeated electrical stimulation of the sciatic nerve at C-fiber strength at both 0.5 and 1 Hz. Paired-pulse inhibition, evoked through electrical stimulation of the cutaneous receptive field, was significantly decreased in neurons from interferon-gamma-treated animals at 50 and 100 ms inter-stimulus intervals. We propose that this demonstrated reduction in inhibition may underlie the enhanced excitatory responses. Such interferon-gamma-induced changes in evoked responses may contribute to persistent pain following damage or disease states in the nervous system.     

Increased expression of VMAT2 in dopaminergic neurons during nicotine withdrawal

Evidence suggests that the vesicular monoamine transporter-2 (VMAT2) is regulated in striatum and dopamine (DA) may play a role in its regulation. DA is an important mediator of the behavioral actions of nicotine, and dopaminergic neurotransmission is altered following nicotine administration. We investigated the effect of nicotine withdrawal on the expression of VMAT2 in the midbrain DA neurons in animals dependent to nicotine. Mice were injected with nicotine free base 2 mg/kg, sc, four times daily for 14 days and killed 12–72 h after drug discontinuation. VMAT2 protein was increased in the striatum of nicotine-treated mice in a time-dependent fashion at all times studied. Furthermore, in situ hybridization studies demonstrated that VMAT2 mRNA was elevated in the substantia nigra pars compacta and ventral tegmental area, indicating enhanced gene expression and subsequent protein synthesis. Tissue DA content and synthesis were unaltered in the striatum of nicotine-treated mice at the times studied. However, basal DA release was decreased at 12 and 24 h after nicotine discontinuation which coincided with the elevated levels of VMAT2 protein. VMAT2 up-regulation might be a compensatory mechanism to restore and maintain synaptic transmission in dopaminergic midbrain neurons during nicotine withdrawal.     

Regulation of ciliary neurotrophic factor receptor alpha in sciatic motor neurons following axotomy.

Spinal motor neurons are one of the few classes of neurons capable of regenerating axons following axotomy. Injury-induced expression of neurotrophic factors and corresponding receptors may play an important role in this rare ability. A wide variety of indirect data suggests that ciliary neurotrophic factor receptor alpha may critically contribute to the regeneration of injured spinal motor neurons. We used immunohistochemistry, in situ hybridization and retrograde tracing techniques to study the regulation of ciliary neurotrophic factor receptor alpha in axotomized sciatic motor neurons. Ciliary neurotrophic factor receptor alpha immunoreactivity, detected with two independent antisera, is increased in a subpopulation of caudal sciatic motor neuron soma one, two and six weeks after sciatic nerve transection and reattachment, while no changes are detected at one day and 15 weeks post-lesion. Ciliary neurotrophic factor receptor alpha messenger RNA levels are augmented in the same classes of neurons following an identical lesion, suggesting that increased synthesis contributes, at least in part, to the additional ciliary neurotrophic factor receptor alpha protein. Separating the proximal and distal nerve stumps with a plastic barrier does not noticeably affect the injury-induced change in ciliary neurotrophic factor receptor alpha regulation, thereby indicating that this injury response is not dependent on signals distal to the lesion traveling retrogradely through the nerve or signals generated by axonal growth through the distal nerve. The prolonged increases in ciliary neurotrophic factor receptor alpha protein and messenger RNA found in regenerating sciatic motor neurons contrast with the responses of non-regenerating central neurons, which are reported to display, at most, a short-lived increase in ciliary neurotrophic factor receptor alpha messenger RNA expression following injury. The present data are the first to demonstrate, in vivo, neuronal regulation of ciliary neurotrophic factor receptor alpha protein in response to injury. Moreover, they suggest that the ability of a subpopulation of spinal motor neurons to regulate ciliary neurotrophic factor receptor alpha levels in response to injury may play a role in their survival and axonal regeneration. Consistent with such a role, we also find relatively high, and probably elevated, levels of ciliary neurotrophic factor receptor alpha immunoreactivity in regenerating axons.     

Size-dependent dendritic maladaptations of hypoglossal motor neurons in SOD1G93A mice

The total motor neuron (MN) somato-dendritic surface area is correlated with motor unit type. MNs with smaller surface areas innervate slow (S) and fast fatigue-resistant (FR) motor units, while MNs with larger surface areas innervate fast fatigue-intermediate (FInt) and fast fatigable (FF) motor units. Differences in MN surface area (equivalent to membrane capacitance) underpin the intrinsic excitability of MNs and are consistent with the orderly recruitment of motor units (S > FR > FInt > FF) via the Size Principle. In amyotrophic lateral sclerosis (ALS), large MNs controlling FInt and FF motor units exhibit earlier denervation and death, compared to smaller and more resilient MNs of type S and FR motor units that are spared until late in ALS. Abnormal dendritic morphologies in MNs precede neuronal death in human ALS and in rodent models. We employed Golgi-Cox methods to investigate somal size-dependent changes in the dendritic morphology of hypoglossal MNs in wildtype and SOD1^G93A mice (a model of ALS), at postnatal (P) day ~30 (pre-symptomatic), ~P60 (onset), and ~P120 (mid-disease) stages. In wildtype hypoglossal MNs, increased MN somal size correlated with increased dendritic length and spines in a linear fashion. By contrast, in SOD1^G93A mice, significant deviations from this linear correlation were restricted to the larger vulnerable MNs at pre-symptomatic (maladaptive) and mid-disease (degenerative) stages. These findings are consistent with excitability changes observed in ALS patients and in rodent models. Our results suggest that intrinsic or synaptic increases in MN excitability are likely to contribute to ALS pathogenesis, not compensate for it.     

Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons.

The actions of the trivalent cation Gd(3+) on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd(3+) (0.1-10 microM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 microM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd(3+) also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd(3+) reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd(3+) inhibited both peak and steady-state currents, indicating that Gd(3+) both reduces desensitization and inhibits these currents. Gd(3+) reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd(3+). The Gd(3+)-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd(3+) reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd(3+) potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.     

Chick embryonic development following exposure to caffeine and nicotine

Because cigarette smoking is considered to be deleterious to the fetus and caffeine is held in suspect as a potential human teratogen, the combined effects of caffeine and nicotine on early chick embryos were investigated. Treatment of the embryos at 48 h incubation with both caffeine (1 mg) and nicotine (1 mg) resulted in a high incidence of embryonic death and developmental defects. At 72 h incubation teratogenicity was potentiated following the same treatment. Embryonic growth was not affected. Embryotoxic interactions of this nature might account for congenital anomalies of doubtful etiology.