Katumi Sumikawa

Acute and chronic nicotine exposure differentially facilitate the induction of LTP.

We report here that acute and chronic nicotine exposure facilitated the induction of long-term potentiation (LTP), a leading candidate for a cellular mechanism underlying learning and memory, in the hippocampus. Furthermore, acute application of nicotine in chronic nicotine-treated hippocampus further facilitated the induction of LTP, suggesting that acute and chronic nicotine effects on LTP induction are mediated by different mechanisms. These findings not only provide evidence for chronic nicotine-induced synaptic changes in the hippocampus, but also an explanation of the cellular basis of nicotine-induced cognitive enhancement.

Inactivation of α7 ACh receptors and activation of non-α7 ACh receptors both contribute to long term potentiation induction in the hippocampal CA1 region

Acute and chronic nicotine exposure differentially facilitate the induction of long-term potentiation (LTP), a synaptic model of learning and memory, in the hippocampal CA1 region. The mechanisms underlying these effects of nicotine, however, are unknown. In the present study, both nicotinic acetylcholine receptor (nAChR) agonists and an alpha7 nAChR antagonist facilitated the induction LTP in the hippocampal CA1 region of naive rat. Furthermore, chronic nicotine treatment lowered the threshold for induction of LTP, and acute application of nicotinic agonists, but not an alpha7 antagonist, further facilitated LTP induction in the chronic-nicotine-treated hippocampus. These results suggest not only that both activation of non-alpha7 nAChRs and inactivation of alpha7 nAChRs contribute to LTP induction, but also that chronic-nicotine-mediated facilitation of LTP induction is due to chronic-nicotine-induced desensitization of alpha7 nAChRs.

Nicotine reverses GABAergic inhibition of long-term potentiation induction in the hippocampal CA1 region.

Nicotine is known to enhance cognitive function but the mechanism is unknown. The present study examined the modulatory effect of nicotine on the induction of long-term potentiation (LTP), a synaptic model of learning and memory. A weak tetanic stimulation consisting of 20 pulses at 100 Hz induced stable LTP in the hippocampal CA1. The induction of LTP was completely blocked if the tetanus was delivered in the presence of muscimol (2.5 microM), a gamma-aminobutyric acid (GABA) receptor agonist. This inhibition was sensitive to, and reversed by, not only nicotinic acetylcholine receptor (nAChR) agonists (nicotine and epibatidine), but also the alpha7 nAChR-selective antagonist methyllycaconitine (MLA). Furthermore, ACh-puff activation of alpha7 nAChRs on feedforward interneurons induced inhibitory postsynaptic currents in pyramidal cells that were blocked by nicotine or MLA. In addition, nicotine reduced field monosynaptic inhibitory postsynaptic potentials in the presence of MLA. These results suggest not only two pathways of nicotine-induced disinhibition of pyramidal cells, one involving desensitization of alpha7 nAChRs and the other involving non-alpha7 nAChRs, but also two potential mechanisms underlying the modulatory effect of nicotine on LTP induction, both reducing GABAergic inhibition, thereby indirectly increasing the excitability of pyramidal cells.

Properties of acetylcholine receptors translated by cat muscle mRNA in Xenopus oocytes.

Poly(A)+ mRNA, extracted from denervated skeletal muscles of the cat, directs the synthesis of acetylcholine receptors in Xenopus laevis oocytes. The receptors are inserted in the oocyte membrane where they form acetylcholine receptor‐channel complexes which have properties like those of the native receptors in the muscle membrane.

Assembly and N-glycosylation of all ACh receptor subunits are required for their efficient insertion into plasma membranes

Various combinations of synthetic acetylcholine receptor (AChR) subunit mRNAs were injected into Xenopus oocytes, and assembly of incomplete AChRs and their insertion into the plasma membrane was studied. Assembly of incomplete AChRs is not greatly affected by the absence of one or two of the other subunits. In contrast, the membrane insertion of incomplete AChRs is profoundly reduced as compared with complete AChRs. The role of N-glycosylation on the assembly of AChR subunits, and on their insertion into plasma membranes, was also studied by using the Xenopus oocyte expression system and tunicamycin. Assembly of non-N-glycosylated AChR subunits occurs in tunicamycin-treated oocytes, but these subunits remain in intracellular compartments, suggesting that N-glycosylation of AChR subunits is not a prerequisite for receptor assembly, but is required for their efficient insertion into the plasma membrane.

Nicotine gates long-term potentiation in the hippocampal CA1 region via the activation of α2* nicotinic ACh receptors

Hippocampal CA1 pyramidal cells receive two major excitatory synaptic inputs via the Schaffer collateral (SC) and temporoammonic (TA) pathways. Nicotine promotes induction of long‐term potentiation (LTP) in the SC path; however, it is not known whether the modulatory effect of nicotine on LTP induction is pathway‐specific. Here we show that nicotine suppresses LTP induction in the TA path. Interestingly, these opposing effects of nicotine were absent or greatly reduced in α2 nicotinic acetylcholine receptor (nAChR)‐knockout (KO) mice, suggesting that an α2‐containing nAChR subtype mediates these effects. Optical imaging with a voltage‐sensitive dye revealed significantly stronger membrane depolarization in the presence of nicotine in the SC path, facilitating spread of excitatory neural activity along both the somatodendritic and the CA1 proximodistal axes. These effects of nicotine were also absent in α2 nAChR‐KO mice, suggesting that the enhanced optical signal is related to the nicotine‐induced facilitation of LTP induction. In contrast, in the TA path nicotine terminated depolarization more quickly and increased the delayed hyperpolarization in the termination zone of the TA path input. These inhibitory effects of nicotine were absent in α2 nAChR‐KO mice and, thus, most probably underlie the nicotine‐induced suppression of LTP induction. Our results suggest that nicotine influences the local balance between excitation and inhibition, gates LTP, and directs information flow through the hippocampal circuits via the activation of α2* nAChRs. These effects of nicotine may represent the cellular basis of nicotine‐mediated cognitive enhancement.

Site-directed mutagenesis of the conserved N-glycosylation site on the nicotinic acetylcholine receptor subunits

The role of the conserved N-glycosylation site on each subunit of the Torpedo acetylcholine receptor (AChR) in the biogenesis and function of the receptor was examined by expressing site-directed mutant subunits in Xenopus oocytes. Different mutant subunits caused different effects. The most striking effect was seen with the mutant gamma subunit which, when co-expressed with alpha, beta, and delta subunits, caused degradation of all the subunits. N-Glycosylation of the other subunits appears to contribute to stability of the subunits and/or efficient insertion of the receptor into the plasma membrane, but is not required for assembly. The AChRs containing the mutant alpha subunit formed functional ion channels in the plasma membrane and their activity could be blocked by alpha-bungarotoxin (alpha-BuTX). Thus, attachment of a carbohydrate moiety at the conserved N-glycosylation site is not an absolute requirement for the formation of ACh and alpha-BuTX binding sites.

Comparison of α2 nicotinic acetylcholine receptor subunit mRNA expression in the central nervous system of rats and mice

The nicotinic acetylcholine receptor (nAChR) α2 subunit was the first neuronal nAChR to be cloned. However, data for the distribution of α2 mRNA in the rodent exists in only a few studies. Therefore, we investigated the expression of α2 mRNA in the rat and mouse central nervous systems using nonradioactive in situ hybridization histochemistry. We detected strong hybridization signals in cell bodies located in the internal plexiform layer of the olfactory bulb, the interpeduncular nucleus of the midbrain, the ventral and dorsal tegmental nuclei, the median raphe nucleus of the pons, the ventral part of the medullary reticular nucleus, the ventral horn in the spinal cord of both rats and mice, and in a few Purkinje cells of rats, but not of mice. Cells that moderately express α2 mRNA were localized to the cerebral cortex layers V and VI, the subiculum, the oriens layer of CA1, the medial septum, the diagonal band complex, the substantia innominata, and the amygdala of both animals. They were also located in a few midbrain nuclei of rats, whereas in mice they were either few or absent in these areas. However, in the upper medulla oblongata α2 mRNA was expressed in several large neurons of the gigantocellular reticular nucleus and the raphe magnus nucleus of mice, but not of rats. The data obtained show that a similar pattern of α2 mRNA expression exists in both rats and mice, with the exception of a few regions, and provide the basis for cellular level analysis. J. Comp. Neurol. 493:241–260, 2005.

N-Glycosylation at the conserved sites ensures the expression of properly folded functional ACh receptors

The role of the conserved carbohydrate moiety in the expression of complete acetylcholine receptor (AChR), alpha2 beta gamma delta was re-investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subunits were stably expressed and appeared to associate with other normal subunits; however, removal of carbohydrate on the alpha subunit inhibited the formation of 125I-alpha-bungarotoxin (alpha-BuTX) binding sites and functional ACh-gated ion channels. 125I-alpha-BuTX binding to AChRs was also significantly reduced by removal of the conserved carbohydrate on the gamma or delta subunits. Immunoprecipitation with monoclonal antibodies that recognize the two distinct alpha-BuTX sites on the AChR indicated that the mutant gamma subunit did not interfere with efficient formation of the alpha-BuTX binding site at the alpha/delta interface, but loss of the carbohydrate did interfere with formation of the alpha-BuTX binding site at the alpha/mutant gamma interface. A similar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated efficiently into the mature (correct tertiary conformation capable of alpha-BuTX binding) alpha beta delta or alpha beta gamma complexes, respectively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these results suggest that the formation of the two alpha-BuTX binding sites requires correct folding of the alpha gamma and alpha delta complexes, which is aided by the conserved carbohydrate on the gamma and delta subunits. Electrophysiological experiments demonstrated that functional receptors containing mutant subunits were produced, but the functional properties of the mutant receptors were differentially altered, depending on the subunit mutated. Together, our results suggest that N-glycosylation of AChR subunits ensures the correct folding of important functional domains and expression of proper functional receptors in the plasma membrane.

Nicotine‐induced switch in the nicotinic cholinergic mechanisms of facilitation of long‐term potentiation induction

Nicotine facilitates the induction of long‐term potentiation (LTP) in the hippocampal CA1 region. The present study reveals the potential mechanisms underlying this effect of nicotine. Timed ACh‐mediated activation of α7 nicotinic acetylcholine receptors (nAChRs) on pyramidal cells is known to promote LTP induction. Nicotine could suppress this timing‐dependent mechanism by desensitizing nAChRs. Timed ACh‐mediated activation of α7 nAChRs on feedforward interneurons can prevent LTP induction by inhibiting pyramidal cells. Nicotine diminished this ACh‐mediated inhibition by desensitizing α7 nAChRs, thereby reducing the inhibitory influence on pyramidal cells. In addition to these desensitizing effects, nicotine activated presynaptic non‐α7 nAChRs on feedforward interneurons to decrease the evoked release of γ‐aminobutyric acid (GABA) onto pyramidal cells. Furthermore, nicotine increased the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in pyramidal cells, and concomitantly caused a reduction in the size of responses to focal GABA application onto the dendrites of pyramidal cells, suggesting that the nicotine‐induced increase in interneuronal activity leads ultimately to a use‐dependent depression of evoked IPSCs in pyramidal cells. These nicotine‐induced suppressions of inhibition of pyramidal cells were accompanied by enhanced N‐methyl‐d‐aspartate (NMDA) responses in pyramidal cells. Thus, our results suggest that nicotine promotes the induction of LTP by diminishing inhibitory influences on NMDA responses while suppressing the ACh‐mediated mechanisms. These ACh‐independent mechanisms probably contribute to the nicotine‐induced cognitive enhancement observed in the presence of cholinergic deficits, such as those in Alzheimers disease patients.