Robin A. J. Lester

Quisqualate receptor-mediated depression of calcium currents in hippocampal neurons.

The modulation of Ca2+ currents by the excitatory neurotransmitter glutamate and its analogs was investigated in hippocampal neurons in culture. In the presence of glutamate receptor-gated ion channel antagonists, all of the analogs tested caused either a small reversible depression or had no effect on the Ca2+ current. However, in neurons dialyzed with GTP gamma S, quisqualate and glutamate but not NMDA, kainate, AMPA, or L-APB caused marked and irreversible depressions of the Ca2+ current. This inhibition was only observed if Ca2+ was present in either the internal or external medium. Intracellular H-7, staurosporine, IP3, cAMP, cGMP, or calmodulin inhibitors failed to prevent the quisqualate-induced Ca2+ current inhibition. These observations are consistent with an interaction between a G protein-coupled glutamate receptor and Ca2+ channels.

Interactions between the glycine and glutamate binding sites of the NMDA receptor

The interactions between the glycine and glutamate binding sites of the NMDA receptor have been studied in outside-out patches and synapses from hippocampal neurons in culture using rapid drug application techniques. Desensitization of NMDA receptor-mediated currents elicited by glutamate in newly excised outside-out patches was reduced in the presence of saturating concentrations of glycine. This suggests that the glutamate and glycine binding sites of the NMDA receptor are allosterically coupled as has been reported in whole-cell preparations. A glycine-insensitive form of desensitization increased rapidly over the first few minutes of recording and largely occluded the glycine concentration-sensitive desensitization in outside-out patches. However, even in old patches that displayed no glycine-sensitive desensitization, the unbinding rate of glycine was increased fourfold by the presence of glutamate, suggesting that the two binding sites were still allosterically coupled. These data suggest the existence of two forms of NMDA receptor desensitization in outside-out patches, only one of which is dependent on the concentration of glycine. In the presence of saturating levels of glycine, activation of NMDA receptors by synaptic stimulation or by exogenous glutamate resulted in currents that relaxed biexponentially. Addition of the partial glycine-site agonist 1-hydroxy-3-aminopyrrolid-2-one (HA-966) increased the rate of decay of both synaptic and patch currents. This suggests that HA-966 increases the dissociation rate of glutamate from NMDA receptors. These results support the hypothesis that the glutamate and glycine binding sites of NMDA receptors interact allosterically; ligand binding at both types of sites can affect the affinity of the other type for its agonist.

Simvastatin enhances learning and memory independent of amyloid load in mice

Normal aging is often associated with a decline in learning and memory functions. This decline is manifested to a much greater extent in Alzheimers disease. Recent studies have indicated statins, a class of cholesterol‐lowering drugs, as a potential therapy for Alzheimers disease. Our objective was to determine whether administering a statin drug (simvastatin) would protect against the development of behavioral deficits in an established mouse model of Alzheimers disease.

Rapid Upregulation of α7 Nicotinic Acetylcholine Receptors by Tyrosine Dephosphorylation

α7 nicotinic acetylcholine receptors (nAChRs) modulate network activity in the CNS. Thus, functional regulation of α7 nAChRs could influence the flow of information through various brain nuclei. It is hypothesized here that these receptors are amenable to modulation by tyrosine phosphorylation. In both Xenopus oocytes and rat hippocampal interneurons, brief exposure to a broad-spectrum protein tyrosine kinase inhibitor, genistein, specifically and reversibly potentiated α7 nAChR-mediated responses, whereas a protein tyrosine phosphatase inhibitor, pervanadate, caused depression. Potentiation was associated with an increased expression of surface α7 subunits and was not accompanied by detectable changes in receptor open probability, implying that the increased function results from an increased number of α7 nAChRs. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis was shown to be a plausible mechanism for the rapid delivery of additional α7 nAChRs to the plasma membrane. Direct phosphorylation/dephosphorylation of α7 subunits was unlikely because mutation of all three cytoplasmic tyrosine residues did not prevent the genistein-mediated facilitation. Overall, these data are consistent with the hypothesis that the number of functional cell surface α7 nAChRs is controlled indirectly via processes involving tyrosine phosphorylation.

Nicotinic acetylcholine receptor subunit mRNA expression and channel function in medial habenula neurons.

Relationships between nicotinic acetylcholine receptor (nAChR) channel function and nAChR subunit mRNA expression were explored in acutely isolated rat medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT-PCR techniques. Following amplification using subunit-specific primers, subunits could be categorized in one of three ways: (i) present in 95-100% cells: alpha3, alpha4, alpha5, beta2 and beta4; (ii) never present: alpha2; and (iii) sometimes present ( approximately 40% cells): alpha6, alpha7 and beta3. These data imply that alpha2 subunits do not participate in nAChRs on MHb cells, that alpha6, alpha7 and beta3 subunits are not necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of alpha3, alpha4, alpha5, beta2 and beta4 subunits. Little difference in the patterns of subunit expression between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may be an insufficient determinant of nAChR channel function. Normalization of nAChR subunit levels to the amount of actin mRNA, however, revealed that cells with functional channels were associated with high levels (>0.78 relative to actin; 11/12 cells) of all of the category (i) subunits: alpha3, alpha4, alpha5, beta2 and beta4. Conversely, one or more of these subunits was always low (<0.40 relative to actin) in all cells with no detectable response to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs.

Temporal appearance of the presynaptic cytomatrix protein bassoon during synaptogenesis.

Bassoon is a 420-kDa presynaptic cytomatrix protein potentially involved in the structural organization of neurotransmitter release sites. In this study, we have investigated a possible role for Bassoon in synaptogenesis and in defining synaptic vesicle recycling sites. We find that it is expressed at early stages of neuronal differentiation in which it is selectively sorted into axons. As synaptogenesis begins, Bassoon clusters appear along dendritic profiles simultaneously with synaptotagmin I, sites of synaptic vesicle recycling, and the acquisition of functional excitatory and inhibitory synapses. A role for Bassoon in the assembly of excitatory and inhibitory synapses is supported by the colocalization of Bassoon clusters with clusters of GKAP and AMPA receptors as well as GABA(A) receptors. These data indicate that the recruitment of Bassoon is an early step in the formation of synaptic junctions.

Desensitization of Nicotinic Receptors in the Central Nervous System

hronic exposure to nicotine renders some CNS neuronal nicotinic acetylcholine receptors (nAChRs) “permanently” desensitized. These long-lived inactive receptor states are likely to underlie the development of tolerance to nicotine and may be responsible for some of the more long-term addictive properties of this drug. An understanding of nicotine dependency would benefit from knowledge of the neuronal factors that control desensitization of nAChRs. During recordings of nAChR channel activity in excised outside-out patches from medial habenula (MHb) neurons, there is a time-dependent increase in the rate of onset of receptor desensitization, assessed from the decay of macroscopic currents produced by brief pulses of nicotine. This alteration in desensitization precedes a complete rundown in channel activity. FIGURE 1A shows that after rundown of channel activity in an outsideout patch, a partial recovery of channel function is possible, provided that nAChRs are not exposed to nicotine for a prolonged period. These data are consistent with the idea that rundown of channel activity results from a build-up of nAChRs in a nonfunctional desensitized state; this may occur because recovery from desensitization is slowed due to the loss of certain factors upon patch formation. Knowledge of the factors that control how nAChRs exit from the desensitized states of the receptor should lead to an understanding of how neurons control the level of nAChR activity. Because receptors at synapses are only likely to be exposed to neurotransmitter very transiently, the physiological importance of these slowly reached desensitized states is unknown. However, because desensitized receptor conformations have high affinities for agonist, these states will become occupied during the continuous presence of drug, for example, as occurs during smoking. Furthermore, because certain nAChRs appear to become “permanently” inactivated during chronic exposure to nicotine, we hypothesize that nAChRs become trapped in a desensitized state(s) due to a long-term biochemical alteration. Chronic nicotine, in addition to causing a long-term inactivation of nAChRs, causes an up-regulation in the number of high-affinity [H]nicotine (α4β2 nAChR subunit-containing) binding sites. In accordance with these observations, we have shown that tobacco-related levels of nicotine (≈30–300 nM) will preferentially interact with the desensitized state of α4containing nAChRs: in Xenopus oocytes expressing rat α4β2 nAChRs, nicotine opens channels with an EC50 of ≈10 μM, whereas the IC50 for inducing receptor desensitization is ≈60 nM. After a 30 min exposure to 300 nM nicotine, receptors recover from desensitization with a relatively slow time course. Consistent with the idea that nAChRs could become trapped in the desensitized state, the rate of recovery from desensitization is strongly influenced by

Time-dependent changes in central nicotinic acetylcholine channel kinetics in excised patches.

The behavior of nicotinic acetylcholine receptor (nAChR) channels in acutely isolated habenula neurons was examined by rapidly applying nicotinic agonists to outside-out membrane patches. At negative membrane potentials, applications of 100 microM nicotine routinely produced macroscopic currents due to the opening of a large number of channels. During the continuous application of the agonist, the number of open nAChR channels decreased exponentially, i.e. receptor desensitization. A progressive loss in the number of channels contributing to the peak current was observed with time following outside-out patch excision, i.e. receptor rundown. In addition to rundown there was a time-dependent increase in the rate of desensitization and a concomitant slowing in the rate of recovery from desensitization. The extent of rundown and the changes in desensitization were coupled to the time after patch excision and were not dependent on ligand activation of nicotinic channels.

Synaptic excitation mediated by glutamate-gated ion channels

Excitatory synaptic transmission in the central nervous system relies predominantly on stimulation of L-glutamate-gated ion channels in postsynaptic membranes. Activation of these channels not only mediates millisecond to millisecond signalling but can also have long term influences on synaptogenesis and synaptic plasticity. Recent work has resolved some longstanding problems involving the identity of the transmitter, the postsynaptic localization of the receptor subtypes, and the time course of the transmitter in the synaptic cleft.

Interactions of atropine with heterologously expressed and native α3 subunit-containing nicotinic acetylcholine receptors

Atropine, a classical muscarinic antagonist, has been reported previously to inhibit neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, the action of atropine has been examined on α3β4 receptors expressed heterologously in Xenopus oocytes and native nAChRs in medial habenula neurons. At concentrations of atropine often used to inhibit muscarinic receptors (1 μM), responses induced by near‐maximal nicotine concentrations (100 μM) at negative holding potentials (−65 mV) are inhibited (14–30%) in a reversible manner in both α4 and α3 subunit‐containing heteromeric nAChRs. Half‐maximal effective concentrations (IC50 values) for atropine inhibition are similar for the four classes of heteromeric receptors studied (4–13 μM). For α3β4 nAChRs in oocytes, inhibition by atropine (10 μM) is not overcome at higher concentrations of agonist, and is increased with membrane hyperpolarization. These results are consistent with non‐competitive antagonism – possibly ion channel block. At low concentrations of both nicotine (10 μM) and atropine (<10 μM), potentiation (∼25%) of α3β4 nAChR responses in oocytes is observed. The relative balance between potentiation and inhibition is dependent upon membrane potential. In rat medial habenula (MHb) neurons, atropine (0.3–3.0 μM) inhibited nicotine‐induced responses in both a concentration and membrane potential‐dependent manner (at −40 mV, IC50=4 μM), similar to the effects on α3β4–nAChRs in oocytes. However, unlike heterologously expressed receptors, potentiation was barely detectable at depolarized membrane potentials using low concentrations of nicotine (3–10 μM). Conversely, the weak agonist, choline (1–3 mM) was observed to augment responses of MHb nAChRs.