Expression of alpha2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.     

Chronic nicotine induces growth retardation in neonatal rat pups

In the United State, 20% of pregnant women smoke. One of the most consistent adverse outcomes is reduced birth weight in the off-spring. Animal studies using chronic nicotine, the major psychoactive tobacco ingredient, have shown conflicting results, questioning the role of nicotine in growth retardation. To evaluate the direct effects of nicotine during a period equivalent to the human third trimester, we developed an oral gastric intubation model using neonatal rat pups. Nicotine (6 mg/kg/day) was dissolve in milk-formula and delivered during three feedings daily from postnatal day (P)1 to P7. Nicotine immediately and significantly [P<0.05] decreased weight gain per day (WGD) by 13.5% (+/-) 1 day after onset of treatment in both genders and throughout the treatment period. This resulted in significantly lower body weight at P4 and P5 in male and female pups, respectively. After nicotine withdrawal, WGD returned to control level within 1 day, whereas total body weight recovered by P18. There were no long-term consequences on body weight or growth pattern in either gender. The nicotinic acetylcholine receptor (nAChR) antagonist dihydro-beta-erythroidine (DHbetaE) reversed nicotine's effects on WGD suggesting an involvement of heteromeric alpha4beta2, whereas methyllycaconitine (MLA) an antagonist for the homomeric alpha7-type receptor was ineffective. The immediate decrease of growth in neonatal pups suggests that nicotine's effect on birth weight results from direct anorexic rather then indirect effects due to placental dysfunction or increased fetal hypoxia. The postnatal oral gastric intubation model seems to accurately reflect the direct effects of nicotine in neonates.     

Glial Regulation of α7-Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7-type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum-containing medium without mitotic inhibitors, specific ¹²⁵I-α-bungarotoxin binding to α7-type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5'-fluorodeoxyuridine (80 µ M ) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased ¹²⁵I-α-bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment-induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5'-Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N -[³H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased ¹²⁵I-α-bungarotoxin binding in both control and 5'-fluorodeoxyuridine-treated cultures, suggesting the release of a soluble factor that inhibits α7-type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µ M ) increased ¹²⁵I-α-bungarotoxin binding in astrocyte-poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7-type nicotinic receptors in developing brain.     

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.     

Varenicline and nicotine enhance GABAergic synaptic transmission in rat CA1 hippocampal and medial septum/diagonal band neurons

AimsThe FDA approved smoking cessation aid varenicline can effectively attenuate nicotine-stimulated dopamine release. Varenicline may also exert important actions on other transmitter systems that also influence nicotine reinforcement or contribute to the drug's cognitive and affective side effects. In this study, we determined if varenicline, like nicotine, can stimulate presynaptic GABA release.Main methodsUsing whole-cell patch-clamp techniques, we measured GABA_AR-mediated asynchronous, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in acute brain slices from two brain regions important for learning and memory, the hippocampus and basal forebrain.Key findingsBoth varenicline (10 μM) and nicotine (10 μM) applications alone resulted in small but significant increases in amplitude, as well as robustly enhanced frequency of mIPSCs in hippocampal CA1 pyramidal neurons and medial septum/diagonal band (MS/DB) neurons. A unique subpopulation of MS/DB neurons showed decreases in frequency. In the presence of nicotine, varenicline effectively attenuated the expected enhancement of hippocampal mIPSC frequency like a competitive antagonist. However, in the MS/DB, varenicline only partially attenuated nicotine's effects. Reversing the order of drug application by adding nicotine to varenicline-exposed slices had little effect.SignificanceVarenicline, like nicotine, stimulates presynaptic GABA release, and also exerts a partial agonist action by attenuating nicotine-stimulated release in both the hippocampus and basal forebrain. These effects could potentially affect cognitive functions.