Lincoln H. Wilkins

Alcohol‐Induced Neurodegeneration: When, Where and Why?

This manuscript reviews the proceedings of a symposium organized by Drs. Antonio Noronha and Fulton Crews presented at the 2003 Research Society on Alcoholism meeting. The purpose of the symposium was to examine recent findings on when alcohol induced brain damage occurs, e.g., during intoxication and/or during alcohol withdrawal. Further studies investigate specific brain regions (where) and the mechanisms (why) of alcoholic neurodegeneration. The presentations were (1) Characterization of Synaptic Loss in Cerebella of Mature and Senescent Rats after Lengthy Chronic Ethanol Consumption, (2) Ethanol Withdrawal Both Causes Neurotoxicity and Inhibits Neuronal Recovery Processes in Rat Organotypic Hippocampal Cultures, (3) Binge Drinking-Induced Brain Damage: Genetic and Age Related Effects, (4) Binge Ethanol-Induced Brain Damage: Involvement of Edema, Arachidonic Acid and Tissue Necrosis Factor alpha (TNFalpha), and (5) Cyclic AMP Cascade, Stem Cells and Ethanol. Taken together these studies suggest that alcoholic neurodegeneration occurs through multiple mechanisms and in multiple brain regions both during intoxication and withdrawal.

Neurotoxic effects of the human immunodeficiency virus type-1 transcription factor Tat require function of a polyamine sensitive-site on the N-methyl-d-aspartate receptor

Human immunodeficiency virus type-I (HIV-1) infection is often associated with neuronal loss in cortical and subcortical regions that may manifest as motor dysfunction and dementia. The function of the HIV-1 transcription protein Tat and subsequent activation of N-methyl-D-aspartate receptors (NMDAr) have been implicated in this form of neurodegeneration. However, it is unclear if Tat interacts directly with the NMDAr and the role of specific NMDAr subunit composition in mediating effects of Tat is also unclear. The present studies examined the ability of HIV-1 Tat1-72 protein (10 pM-1.0 microM) to displace [3H]MK-801 binding and to attenuate spermidine-induced potentiation of this binding in rat brain homogenate comprised of cerebellum, hippocampus, and cerebral cortex. The role of NMDAr polyamine-site function in the neurotoxic effects of Tat was determined using organotypic hippocampal slice cultures. Binding of [3H]MK-801 in adult rat brain homogenate was not reduced by Tat at concentrations below 1 microM. Tat potently inhibited the potentiation of [3H]MK-801 binding produced by co-exposure of membranes to the NMDAr co-agonist spermidine (IC(50)=3.74 nM). In hippocampal explants, Tat produced neurotoxicity in the CA3 and CA1 pyramidal cell layers, as well as in the dentate gyrus, that was significantly reduced by co-exposure to MK-801 (20 microM) and the NMDAr polyamine-site antagonist arcaine (10 microM). Exposure to the HIV-1 Tat deletion mutant (Tatdelta31-61) did not produce neurotoxicity in hippocampal explants. These data suggest that the neurotoxic effects of HIV-1 Tat are mediated, in part, by direct interactions with a polyamine-sensitive site on the NMDAr that positively modulates the function of this receptor.

N-n-Alkylpyridinium Analogs, a Novel Class of Nicotinic Receptor Antagonists: Selective Inhibition of Nicotine-Evoked [3H]Dopamine Overflow from Superfused Rat Striatal Slices

Structural simplification of N-n-alkylnicotinium analogs, antagonists at neuronal nicotinic acetylcholine receptors (nAChRs), was achieved by removal of the N-methylpyrrolidino moiety affording N-n-alkylpyridinium analogs with carbon chain lengths of C1 to C20. N-n-Alkylpyridinium analog inhibition of [3H]nicotine and [3H]methyllycaconitine binding to rat brain membranes assessed interaction with α4β2* and α7* nAChRs, respectively, whereas inhibition of nicotine-evoked 3H overflow from [3H]dopamine ([3H]DA)-preloaded rat striatal slices assessed antagonist action at nAChR subtypes mediating nicotine-evoked DA release. No inhibition of [3H]methyllycaconitine binding was observed, although N-n-alkylpyridinium analogs had low affinity for [3H]nicotine binding sites, i.e., 1 to 3 orders of magnitude lower than that of the respective N-n-alkylnicotinium analogs. These results indicate that the N-methylpyrrolidino moiety in the N-n-alkylnicotinium analogs is a structural requirement for potent inhibition of α4β2* nAChRs. Importantly, N-n-alkylpyridinium analogs with n-alkyl chains < C10 did not inhibit nicotine-evoked [3H]DA overflow, whereas analogs with n-alkyl chains ranging from C10 to C20 potently and completely inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.12-0.49 μM), with the exceptions of N-n-pentadecylpyridinium bromide (C15) and N-n-eicosylpyridinium bromide (C20), which exhibited maximal inhibition of ∼50%. The mechanism of inhibition of a representative analog of this structural series, N-n-dodecylpyridinium iodide, was determined by Schild analysis. Linear Schild regression with slope not different from unity indicated competitive antagonism at nAChRs mediating nicotine-evoked [3H]DA overflow and a KB value of 0.17 μM. Thus, the simplified N-n-alkylpyridinium analogs are potent, selective, and competitive antagonists of nAChRs mediating nicotine-evoked [3H]DA overflow, indicating that the N-methylpyrrolidino moiety is not a structural requirement for interaction with nAChR subtypes mediating nicotine-evoked DA release.

Development of a Novel Class of Subtype-Selective Nicotinic Receptor Antagonist: Pyridine-N-Substituted Nicotine Analogs

n spite of the extensive diversity in neuronal nicotinic receptors, only a limited number of tools with receptor subtype selectivity are available to study the pharmacology of native receptors. Primarily, efforts have been directed toward development of novel nicotinic receptor agonists as targets for drug discovery (see Ref. 1). A limited number of nicotinic receptor antagonists are available, but most of these are noncompetitive open-channel blockers (e.g., mecamylamine). Dihydro-β-erthyroidine (DHβE) is a commonly used competitive antagonist that blocks the agonist binding site of neuronal nicotinic receptors, but has no inherent receptor subtype-selectivity. Neuronal-bungarotoxin (n-BTX) may be a selective, competitive antagonist at α3β2 receptors as evidenced by potent antagonism of nicotinic responses in oocytes expressing these receptors; however, while n-BTX is inactive at α3β4 and α2β2 receptors, it has an intermediate potency at α4β2 receptors. More recently, α3β2 receptor subtype selectivity has been reported for the competitive antagonist, α-conatoxin MII. However, limited availability and high cost of these toxins limit their usefulness. The focus of the present work is the development of subtype-selective nicotinic receptor antagonists via facile pyridine-N alkylation of the nicotine (NIC) molecule, which will provide useful tools for elucidating structural/functional diversity of native nicotinic receptors in brain. A series of quaternary, pyridine-N-n-alkyl nicotinium analogs with alkyl substituents ranging from C1 to C12 were synthesized and tested for their ability to inhibit NIC-evoked [H]dopamine ([H]DA) release from [H]DA-preloaded striatal slices using the methods of Dwoskin and Zahniser, to inhibit [H]NIC binding to rat striatal membranes using the methods of Crooks et al., and to inhibit NIC-evoked Rb efflux from rat striatal synaptosomes using the methods of Marks et al. Results indicate that increasing the alkyl chain length from C1 to C9 directly correlated with increasing potency to inhibit NIC-evoked [H]dopamine release, purportedly mediated by the α3β2 receptor subtype. The C10 analog, N-n-decylnicotinium iodide (NDNI), did not inhibit NIC-evoked [H]DA release. Schild analysis with the C8 analog, N-n-octylnicotinium iodide (NONI) indicated a competitive interaction with this nicotinic receptor site and the potential involvement of more than one subtype. Assays for [H]NIC binding to striatal membranes and Rb efflux from striatal synaptosomes were used to assess interaction of these analogs with the α4β2 nicotinic receptor subtype. All analogs displaced [H]NIC binding; however, no correlation was found between alkyl chain length and binding affinity. Both NDNI and NONI competitively displaced [H]NIC binding with Ki values of 60 nM and 20 μM, respec-

Pharmacology, Distribution and Development of Muscarinic Acetylcholine Receptor Subtypes in the Optic Tectum of Rana Pipiens

Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5-9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes.These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for excitatory subtypes.

Introduction of unsaturation into theN-n-alkyl chain of the nicotinic receptor antagonists, NONI and NDNI: Effect on affinity and selectivity

N-n-Octylnicotinium iodide (NONI) andN-n-decylnicotinium iodide (NDNI) are selective nicotinic receptor (nAChR) antagonists mediating nicotine-evoked striatal dopamine (DA) release, and inhibiting [3H]nicotine binding, respectively. This study evaluated effects of introducing unsaturation into theN-n-alkyl chains of NONI and NDNI on inhibition of [3H]nicotine and [3H]methyllycaconitine binding (α4β2* and α7* nAChRs, respectively),86Rb+ efflux and [3H]DA release (agonist or antagonist effects at α4β2* and α6β2*-containing nAChRs, respectively). In the NONI series, introduction of a C3-cis-(NONB3c), C3-trans-(NONB3t), C7-double-bond (NONB7e), or C3-triple-bond (NONB3y) afforded a 4-fold to 250-fold increased affinity for [3H]nicotine binding sites compared with NONI. NONB7e and NONB3y inhibited nicotine-evoked86Rb+ efflux, indicating α4β2* antagonism. NONI analogs exhibited a 3-fold to 8-fold greater potency inhibiting nicotine-evoked [3H]DA overflow compared with NONI (IC50=0.62 μM; Imax=89%), with no change in Imax, except for NONB3y (Imax=50%). In the NDNI series, introduction of a C4-cis-(NDNB4c), C4-trans-double-bond (NDNB4t), or C3-triple-bond (NDNB3y) afforded a 4-fold to 80-fold decreased affinity for [3H]nicotine binding sites compared with NDNI, whereas introduction of a C9-double-bond (NDNB9e) did not alter affinity. NDNB3y and NDNB4t inhibited nicotine-evoked86Rb+ efflux, indicating anatogonism at α4β2* nAChRs. Although NDNI had no effect, NDNB4t and NDNB9e potently inhibited nicotine-evoked [3H]DA overflow (IC50=0.02–0.14μM, Imax=90%), as did NDNB4c (IC50=0.08 μM; Imax=50%), whereas NDNB3y showed no inhibition. None of the analogs had significant affinity for α7* nAChRs. Thus, unsaturated NONI analogs had enhanced affinity at α4β2*-and α6β2*-containing nAChRs, however a general reduction of affinity at α4β2* and an uncovering of antagonist effects at α6β2*-containing nAChRs were observed with unsaturated NDNI analogs.

N-n-alkylnicotinium analogs, a novel class of antagonists at α4β2* Nicotinic acetylcholine receptors: Inhibition of S(-)-nicotine-evoked 86Rb+Efflux from rat thalamic synaptosomes

PyridineN-n-alkylation of S(-)-nicotine (NIC) affordsN-n-alkylnicotinium analogs, previously shown to competitively inhibit [3H]NIC binding and interact with α4β2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked86Rb+ efflux from rat thalamic synaptosomes to assess functional interaction with α4β2* nAChRs. In a concentration-dependent manner, NIC evoked86Rb+ efflux (EC50=170 nmol/L). Analoginduced inhibition of NIC-evoked86Rb+ efflux varied over a ≈450-fold range. Analogs with longn-alkyl chain lengths (C9−C12) inhibited efflux in the low nmol/L range (IC50=9–20 nmol/L), similar to dihydro-β-erythroidine (IC50=19 nmol/L). Compounds with shortern-alkyl chain lengths (C1−C8) produced inhibition in the low μmol/L range (IC50 =3–12 μmol/L). C10 and C12 analogs completely inhibited NIC-evoked efflux, whereas C1–9 analogs produced maximal inhibition of only 10% to 60%. While the C10 analogN-n-decylnicotiniumiodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [3H]NIC binding sites (Ki=90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked86Rb+ efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at α4β2* nAChRs.