Gestational nicotine exposure regulates expression of AMPA and NMDA receptors and their signaling apparatus in developing and adult rat hippocampus.

Untimely activation of nicotinic acetylcholine receptors (nAChRs) by nicotine results in short- and long-term consequences on learning and behavior. In this study, the aim was to determine how prenatal nicotine exposure affects components of glutamatergic signaling in the hippocampus during postnatal development. We investigated regulation of both nAChRs and glutamate receptors for AMPA and N-methyl-D-aspartate (NMDA), from postnatal day 1 (P1) to P63 after a temporally restricted exposure to saline or nicotine for 14 days in utero. We analyzed postsynaptic density components associated with AMPA receptor (AMPAR) and NMDA receptor (NMDAR) signaling: calmodulin (CaM), CaM Kinase II alpha (CaMKIIα), and postsynaptic density-95 (PSD95), as well as presynaptically localized synaptosomal-associated protein 25 (SNAP25). At P1, there was significantly heightened expression of AMPAR subunit GluR1 but not GluR2, and of NMDAR subunits NR1, NR2a, and NR2d but not NR2b. NR2c was not detectable. CaM, CaMKIIα, and PSD95 were also significantly upregulated at P1, together with presynaptic SNAP25. This enhanced expression of glutamate receptors and signaling proteins was concomitant with elevated levels of [³H]epibatidine (³H]EB) binding in prenatal nicotine-exposed hippocampus, indicating that α4ß2 nAChR may influence glutamatergic function in the hippocampus at P1. By P14, neither [³H]EB binding nor the expression levels of subunits GluR1, GluR2, NR1, NR2a, NR2b, NR2c, or NR2d seemed changed with prenatal nicotine. However, CaMKIIα was significantly upregulated with nicotine treatment while CaM showed downregulation at P14. The effects of nicotine persisted in P63 young adult brains which exhibited significantly downregulated GluR2, NR1, and NR2c expression levels in hippocampal homogenates and a considerably muted overall distribution of [³H]AMPA binding in areas CA1, CA2 and CA3, and the dentate gyrus. Our results suggest that prenatal nicotine exposure can regulate the glutamatergic signaling system throughout postnatal development by enhancing or inhibiting availability of AMPAR and NMDAR or their signaling components. The persistent depression, in adults, of the requisite NR1 subunit for NMDAR assembly, and of GluR2, important for assembly, trafficking, and biophysical properties of AMPAR, indicates that nicotine may alter ionotropic glutamate receptor stoichiometry and functional properties in adults after prenatally restricted nicotine exposure.

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression.

A synthetic peptide corresponding to the C-terminus of the alpha 3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal alpha 3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the alpha 3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of alpha 3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the alpha 3 subunit in the SCG was also determined. The alpha 3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the alpha 3 subunit participate in important roles in specific regions of the rat brain and the SCG.

Evidence of a functional alpha7-neuronal nicotinic receptor subtype located on motoneurons of the dorsal motor nucleus of the vagus.

In vitro autoradiography using 125I-alpha-bungarotoxin (alpha-BGTx) and anti-alpha7 immunohistochemistry were performed on the dorsal motor nucleus of the vagus (DMV) of sham and chronically vagotomized rats to determine whether the alpha7-nicotinic acetylcholine receptor (nAChR) is located postsynaptically on DMV neurons whose axons contribute to the vagus nerve. Intense bilateral 125I-alpha-BGTx binding and anti-alpha7 immunostaining were observed in coronal brain sections containing the DMV of sham-vagotomized animals. Unilateral cervical vagotomy resulted in ipsilateral losses of 125I-alpha-BGTx binding and anti-alpha7 immunostaining from the DMV. Simultaneous staining of rat brainstem sections with anti-alpha7 and anti-choline acetyltransferase (ChAT) antibodies (to identify cholinergic DMV neurons that project into the vagus nerve) revealed that every DMV neuron that was stained for ChAT showed alpha7-staining as well. In vagotomized animals, no ChAT-positive neurons expressing alpha7-nAChRs remained in the ipsilateral DMV. We conclude that the alpha7-nAChR subtype is located postsynaptically on DMV neurons. To test whether the alpha7-nAChR is similar to the alpha7-homomeric nAChR, experiments were performed in anesthetized rats, and compounds were microinjected into the DMV while monitoring intragastric pressure (IGP). alpha-BGTx and strychnine antagonized nicotine-induced increases in IGP; no antagonism was observed with methyllycaconitine, a compound known to block the homomeric alpha7-nAChR subtype. Recovery from alpha-BGTx-induced antagonism of the nicotine response was observed. We conclude that there is a nAChR containing the alpha7-subunit in the DMV that is different from the homomeric alpha7-nAChR subtype.

Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies.

Chronic administration of nicotine increases the density of neuronal cholinergic nicotinic receptors in cells and in rodent brain, and similar increases have been reported in brains from human smokers. To further examine this phenomenon, we measured nicotinic receptor binding sites in brain regions from matched populations of smokers and nonsmokers. We first measured binding of [3H](+/-)epibatidine ([3H]EB) and [3H]cytisine in homogenate preparations from samples of prefrontal and temporal cerebral cortex. Binding of each radioligand was significantly higher (250-300%) in both cortical regions from brains of smokers. Frozen sections from each of the cerebral cortical regions and the hippocampus were used for autoradiographic analysis of [3H]EB binding. In cerebral cortex, binding was most dense in layer VI in the prefrontal cortex and layers IV and VI in the temporal cortex. Densitometric analysis of [3H]EB binding sites revealed marked increases of 300 to 400% of control in all cortical regions examined from smokers' brains. Binding in the hippocampal formation was heterogeneously distributed, with dense areas of binding sites seen in the parasubiculum, subiculum, and molecular layer of the dentate gyrus, and the lacunosum-moleculare layer of the CA1/2. Binding of [3H]EB was significantly higher in all six regions of the hippocampus examined from brains of smokers compared with nonsmokers. These increases ranged from 160% of control in parasubiculum to 290% in the molecular layer of the dentate gyrus. The increase in nicotinic receptors in the cerebral cortex and hippocampus of smokers may modify the central nervous system effects of nicotine and contribute to an altered response of smokers to nicotine.

Nicotinic receptor binding sites in rat primary neuronal cells in culture: characterization and their regulation by chronic nicotine.

We have characterized high affinity neuronal nicotinic acetylcholine receptors labeled by [3H]cytisine in primary neuronal cell cultures from fetal rat brains. After 15 days in culture, the highest density of [3H]cytisine binding sites (Bmax approximately 57 fmol/mg protein) was found in cells from the brainstem, which includes the following subcortical brain areas: the septum, thalamus, hypothalamus, midbrain, pons and medulla. A lower density of sites was found in cells from the cerebral cortex, hippocampus, and caudate nucleus. [3H]Cytisine binds to receptors in primary cells from the brainstem and cerebral cortex with a Kd of approximately 0. 5 nM, and the binding is inhibited by the agonists nicotine, acetylcholine, and epibatidine with IC50 values of 1 to 20 nM, and by carbachol and the antagonist dihydro-beta-erythroidine with IC50 values of 0.5 to 1.5 microM. Chronic treatment of neuronal cultures with nicotine for 7 days differentially affected the number of nicotinic receptors in cells from different brain areas; it significantly increased the number of nicotinic binding sites in cells from the cerebral cortex, hippocampus, and caudate, but not in cells from the brainstem. The nicotine-induced increase of receptors in cerebral cortical cultures was not blocked by either mecamylamine or dihydro-beta-erythroidine. These results indicate that primary cultures of rat neuronal cells provide a good model system in which to study and compare the properties and regulation of native neuronal nicotinic acetylcholine receptors.

Pharmacology of neuronal nicotinic acetylcholine recceptors: effects of acute and chronic nicotine.

Neuronal nicotinic acetylcholine receptors mediate nicotine's diverse effects on the brain, spinal cord and autonomic nervous system. These receptors are composed of alpha and beta subunits. Eight different alpha and three different beta subunits have been identified in vertebrate nervous systems, giving rise to the possibility of multiple subtypes of nicotinic receptors, defined by their constituent subunits. The pharmacological and channel conductance properties of the recombinant receptor subtypes studied in cellular expression systems differ from one another. In addition, the regulation of the receptor density and function during and after acute and chronic exposure to nicotine appears to differ among the subtypes. The predominant receptor subtypes in specific brain regions and peripheral neurons are beginning to be identified and their characteristics studied using new ligands and methods. As more is learned of the differences among the receptor subtypes, it should be possible to identify which specific subtype mediates a specific function within the nervous system and which subtypes are associated with the reinforcing and addictive actions of nicotine.

[125I]IPH, an epibatidine analog, binds with high affinity to neuronal nicotinic cholinergic receptors.

An analog of epibatidine (EB) was synthesized with an iodine atom in the 2 position of the pyridyl ring. This analog, (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane (IPH), as well as its two stereoisomers, displayed high affinity for neuronal nicotinic receptors; therefore, radioiodinated IPH, [125I]IPH, was synthesized with specific radioactivities consistently > 1000 Ci/mmol, and its properties as a radioligand for neuronal nicotinic receptors were evaluated. The characteristics of [125I]IPH binding in tissue homogenates appeared to be virtually identical to those reported for [3H]epibatidine binding; but the high specific radioactivity of [125I]IPH greatly facilitated measurements of nicotinic receptors in tissues with relatively low receptor densities and/or where tissues are in limited supply. Autoradiography with [125I]IPH provided clear localization of nicotinic receptors in brain and adrenal gland after film exposure times of < or = 2 days. We conclude that [125I]IPH will be a very useful radioligand for the study of neuronal nicotinic receptors in brain and in peripheral ganglia.

Differential regulation of neuronal nicotinic receptor binding sites following chronic nicotine administration.

Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up-regulated sites labeled by [3H]cytisine in cerebral cortex appear to be composed exclusively of alpha4 and beta2 subunits. It is unknown whether receptor subtypes that do not bind [3H]cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [3H]cytisine with that of [3H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [3H]cytisine and [3H]epibatidine. In contrast, no change in [3H]epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high-affinity [3H]cytisine binding. These data indicate that, under the conditions used here, nicotine up-regulates the alpha4beta2 nicotinic receptor subtype, which can be labeled by [3H]cystisine and [3H]epibatidine, but not non-alpha4beta2 subtypes, which can be labeled by [3H]epibatidine.

Characterization of (+/-)(-)[3H]epibatidine binding to nicotinic cholinergic receptors in rat and human brain.

Epibatidine is an alkaloid that was first isolated from the skin of the Ecuadoran frog Epipedobates tricolor by Daly et al. [J. Am. Chem. Soc. 102:803-836 (1980)] and was found to have very high affinity for neuronal nicotinic receptors, where it acts as a potent agonist. Here we have measured and characterized the binding of (+/-)(-)[3H]epibatidine to nicotinic receptors in rat brain. In rat forebrain homogenates, (+/-)(-)[3H]epibatidine binds to two sites, with apparent affinities of 15 pM and 360 pM. Both of these binding sites have pharmacological profiles consistent with neuronal nicotinic receptors and a similar brain regional distribution. (+/-)(-)[3H]Epibatidine also binds to sites in rat adrenal gland, suggesting that it can label a subtype of nicotinic receptor found in peripheral ganglia as well as the subtype that predominates in brain. In human cerebral cortex as well, (+/-)(-)[3H]epibatidine binds two sites, one of which appears to have an affinity of < 1 pM. We conclude that (+/-)(-)[3H]epibatidine should be a very useful new tool for characterizing the properties and regulation of neuronal nicotinic receptors, including those not easily measurable with other radioligands.

Neuropeptides as positive or negative neuronal growth regulatory factors: effects of ACTH and leu-enkephalin on cultured serotonergic neurons.

In summary, we have presented evidence that neuropeptides can function as either positive or negative growth regulatory factors during development. The ACTH family of peptides appear to act predominantly as a positive growth regulatory factor-enhancing neurite outgrowth, cell survival, biochemical maturation and behavioral expression. These effects of ACTH are most pronounced prior to the time the afferent cell has reached its target. Thus, ACTH may act as a low level general neurotrophic growth regulatory factor. The opioids have the opposite effect. These neuropeptides inhibit neurite extension, cell survival, and biochemical maturation. The effects of these negative growth regulatory factors are observed even when the afferents have reached their targets. The action of the opioids is thought to occur through specific receptors and known second messenger systems. Thus, CNS neuropeptide levels can have important actions in regulating the development of a variety of CNS systems, and permanently influencing the structure and function of the brain.

Effects of acute and chronic administration of Leu-enkephalin on cultured serotonergic neurons: evidence for opioids as inhibitory neuronal growth factors.

Leu-enkephalin, at concentrations between 18 microM and 1.8 pM, was administered in a single or daily dose to dissociated mesencephalic raphe cell cultures maintained for 3 or 5 days. Daily administration of Leu-enkephalin produced an inhibition of high affinity uptake of [3H]5-HT, a measure of serotonergic process outgrowth in cultures of fetal neurons. This inhibition was maximal at a dose of 18 nM in both 3 (59%, P less than 0.05)- and 5 (38%, P less than 0.05)-day cultures. The expression of uptake was consistently lower in 5-day cultures than in 3-day cultures at all concentrations tested. In marked contrast, a single dose of Leu-enkephalin at the time of plating stimulated uptake in 3- and 5-day cultures. Maximal stimulation was observed at 180 nM for both 3 (191%, P less than 0.05)- and 5 (140%, P less than 0.05)-day cultures. The results obtained after a single dose of the opioid may reflect a paradoxical stimulation probably due to a rebound mechanism of receptors since co-administration of bacitracin (0.5 mg/ml), an aminopeptidase inhibitor, resulted in inhibition of the uptake expression. Together these results indicate that Leu-enkephalin can function as an inhibitory regulatory growth factor for neuronal cultures when constant exposure to this opioid is maintained over time.