Thalamic Synaptosomes Research Articles

Perinatal hypoxia and thalamus brain region: increased efficiency of antiepileptic drug levetiracetam to inhibit GABA release from nerve terminals

Levetiracetam (LV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an antiepileptic drug. The exact mechanisms of anticonvulsant effects of LV remain unclear. In this study, rats (Wistar strain) underwent hypoxia and seizures at the age of 10–12 postnatal days (pd). [3H]GABA release was analysed in isolated from thalamus nerve terminals (synaptosomes) during development at the age of pd 17–19 and pd 24–26 (infantile stage), pd 38–40 (puberty) and pd 66–73 (young adults) in control and after perinatal hypoxia. The extracellular level of [3H]GABA in the preparation of thalamic synaptosomes increased during development at the age of pd 38–40 and pd 66–73 as compared to earlier ones. LV did not influence the extracellular level of [3H]GABA in control and after perinatal hypoxia at all studied ages. Exocytotic [3H]GABA release in control increased at the age of pd 24–26 as compared to pd 17–19. After hypoxia, exocytotic [3H]GABA release from synaptosomes also increased during development. LV elevated [3H]GABA release from thalamic synaptosomes at the age of pd 66–73 after hypoxia and during blockage of GABA uptake by NO-711 only. LV realizes its antiepileptic effects at the presynaptic site through an increase in exocytotic release of [3H]GABA in thalamic synaptosomes after perinatal hypoxia at pd 66–73. LV exhibited a more significant effect in thalamic synaptosomes after perinatal hypoxia than in control ones. The action of LV is age-dependent, and the drug was inert at the infantile stage that can be useful for an LV application strategy in child epilepsy therapy. Keywords: brain development, exocytosis, GABA, levetiracetam, perinatal hypoxia, thalamic synaptosomes

Pharmacological activation of mGlu5 receptors with the positive allosteric modulator VU0360172, modulates thalamic GABAergic transmission

Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABAA receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 h and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Changes in VGLUT2 expression and function in pain-related supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model

Vesicular glutamate transporters (VGLUTs) control the storage and release of glutamate, which plays a critical role in pain processing. The VGLUT2 isoform has been found to be densely distributed in the nociceptive pathways in supraspinal regions, and VGLUT2-deficient mice exhibit an attenuation of neuropathic pain; these results suggest a possible involvement of VGLUT2 in neuropathic pain. To further examine this, we investigated the temporal changes in VGLUT2 expression in different brain regions as well as changes in glutamate release from thalamic synaptosomes in spared nerve injury (SNI) mice. We also investigated the effects of a VGLUT inhibitor, Chicago Sky Blue 6B (CSB6B), on pain behavior, c-Fos expression, and depolarization-evoked glutamate release in SNI mice. Our results showed a significant elevation of VGLUT2 expression up to postoperative day 1 in the thalamus, periaqueductal gray, and amygdala, followed by a return to control levels. Consistent with the changes in VGLUT2 expression, SNI enhanced depolarization-induced glutamate release from thalamic synaptosomes, while CSB6B treatment produced a concentration-dependent inhibition of glutamate release. Moreover, intracerebroventricular administration of CSB6B, at a dose that did not affect motor function, attenuated mechanical allodynia and c-Fos up-regulation in pain-related brain areas during the early stages of neuropathic pain development. These results demonstrate that changes in the expression of supraspinal VGLUT2 may be a new mechanism relevant to the induction of neuropathic pain after nerve injury that acts through an aggravation of glutamate imbalance.

Perinatal hypoxia: different effects of the inhibitors of GABA transporters GAT1 and GAT3 on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals.

AimTo analyze the effects of highly selective blocker GAT1, NO-711, and substrate inhibitor GAT3, β-alanine, on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals (synaptosomes) after perinatal hypoxia.MethodsAnimals were divided into two groups: control (n = 17) and hypoxia (n = 12). Rats in the hypoxia group underwent hypoxia and seizures (airtight chamber, 4% O2 and 96% N2) at the age of 10-12 postnatal days and were used in the experiments 8-9 weeks after hypoxia.ResultsIn cortical synaptosomes, the effects of NO-711 (30 μΜ) and β-alanine (100 μΜ) on [3H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [3H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of β-alanine was increased after hypoxia from 14.4% to 22.1%. In thalamic synaptosomes, the effect of NO-711 was decreased by 79.6% in controls and by 70.9% in hypoxia group, whereas the effect of β-alanine was increased after hypoxia from 20.2% to 30.2%.ConclusionsThe effectiveness of β-alanine to influence GABA uptake was increased in hippocampal and thalamic nerve terminals as a result of perinatal hypoxia and the effectiveness of NO-711 in thalamic nerve terminals was decreased. These results may indicate changes in the ratio of active GAT1/GAT3 expressed in the plasma membrane of nerve terminals after perinatal hypoxia. We showed a possibility to modulate non-GAT1 GABA transporter activity in different brain regions by exogenous and endogenous β-alanine.

86Rb + efflux mediated by α4β2*-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization

The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration–effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine-stimulated 86Rb + efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC 50 values for each agonist for desensitization of 86Rb + efflux both with high (EC 50 ≈ 3 μM) and low (EC 50 ≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective K D values for receptor binding. Even though the two components of α4β2*-nAChR-mediated 86Rb + efflux from mouse brain differ markedly in EC 50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/h and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high-sensitivity and low-sensitivity acetylcholine-stimulated 86Rb + efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86Rb + efflux from thalamus, IC 50 values in both brain regions were unaffected by chronic nicotine treatment.

Trace amine associated receptor 1 mediates methamphetamine effects on monoamine transporter function

Trace amine associated receptor 1 (TAAR1) is activated by a spectrum of biogenic amines, amphetamine‐like compounds and thyronamines. It is present in brain monoaminergic regions and co‐localized with the dopamine transporter (DAT) in some neurons. Here, we determined whether TAAR1 signaling in response to methamphetamine (METH) is involved in non‐competitive effects of METH on DAT, norepinephrine transporter (NET) and serotonin transporter (SERT) regulation. Uptake and efflux assays were performed in monoamine transporter/TAAR1 co‐transfected cell lines and in rhesus monkey, wild‐type (WT) and TAAR1 knockout mouse (KO) striatal ([3H]DA) and [3H]5‐HT) and thalamic ([3H]NE) synaptosomes. METH activation of TAAR1 resulted in enhancement of uptake inhibition and promotion of efflux of [3H]monoamine by DAT, NET or SERT in vitro and ex vivo, across high and low loading concentrations of [3H]monoamine. At low loading concentrations (10–20 nM [3H]monoamine), METH action on uptake and efflux was entirely TAAR1‐ and PKA‐/PKC‐dependent. Further experiments revealed that 1 μM METH‐induced DAT internalization was TAAR1‐ and PKC‐dependent in vitro and ex vivo. Accordingly, METH targets and interacts with TAAR1 to initiate cellular phosphorylation cascades that regulate monoamine transporter function and trafficking. Support: NIH DA022323, DA016606, DA025697, DA025802 and RR00168

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at alpha4beta2 * nicotinic acetylcholine receptors, has moderate affinity (K(i)=5.46microM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2-20 of lobeline were synthesized and evaluated for interaction with alpha4beta2 * and alpha7 * neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at alpha7 * nAChRs. Similar to lobeline (K(i)=4nM), sulfonic acid esters had high affinity at alpha4beta2 * (K(i)=5-17nM). Aromatic carboxylic acid ester analogs of lobeline (2-4) were 100-1000-fold less potent than lobeline at alpha4beta2 * nAChRs, whereas aliphatic carboxylic acid ester analogs were 10-100-fold less potent than lobeline at alpha4beta2 *. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the (36)Rb(+) efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC(50) values of 0.85microM and 1.60microM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13-45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K(i)=1.98-10.8microM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at alpha4beta2 * nAChRs (K(i)=19.3microM) and was equipotent with lobeline, at VMAT2 (K(i)=2.98microM), exhibiting a 6.5-fold selectivity for VMAT2 over alpha4beta2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.

Acetylcholine-Stimulated [3H]GABA Release from Mouse Brain Synaptosomes is Modulated by α4β2 and α4α5β2 Nicotinic Receptor Subtypes

Nicotinic acetylcholine receptor (nAChR) agonists stimulate the release of GABA from GABAergic nerve terminals, but the nAChR subtypes that mediate this effect have not been elucidated. The studies reported here used synaptosomes derived from the cortex, hippocampus, striatum, and thalamus of wild-type and alpha4-, alpha5-, alpha7-, beta2-, and beta4-null mutant mice to identify nAChR subtypes involved in acetylcholine (ACh)-evoked GABA release. Null mutation of genes encoding the alpha4 or beta2 subunits resulted in complete loss of ACh-stimulated [(3)H]GABA release in all four brain regions. In contrast, alpha5 gene deletion exerted a small but significant decrease in maximal ACh-evoked [(3)H]GABA release in hippocampus and striatum, with a more profound effect in cortex. Acetylcholine-stimulated [(3)H]GABA release from thalamic synaptosomes was not significantly affected by alpha5 gene deletion. No effect was detected in the four brain regions examined in alpha7- or beta4-null mutant mice. Further analysis of ACh-evoked [(3)H]GABA release revealed biphasic concentration-response relationships in the four brain regions examined from all wild-type animals and in alpha5 null mutant mice. Moreover, a selective reduction in the maximum response of the high-affinity component was apparent in alpha5-null mutant mice. The results demonstrate that alpha4beta2-type nAChRs are critical for ACh-stimulated [(3)H]GABA release from all four brain regions examined. In addition, the results suggest that alpha5-containing receptors on GABAergic nerve terminals comprise a fraction of the high ACh-sensitivity component of the concentration-response curve and contribute directly to the ability of nicotinic agonists to evoke GABA release in these regions.

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the 86Rb+ efflux assay in thalamic synaptosomes. AEA reversibly inhibited 86Rb+ efflux induced by 300 microM ACh with an IC50 value of 0.9 +/- 2 microM. Pre-treatment with the cannabinoid (CB1) receptor antagonist SR141716A (1 microM), the CB2 receptor antagonist SR144528 (1 microM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of 86Rb+ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [3H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 microM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of 86Rb+ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.

β-Phenylethylamine Alters Monoamine Transporter Function via Trace Amine-Associated Receptor 1: Implication for Modulatory Roles of Trace Amines in Brain

Brain monoamines include common biogenic amines (dopamine, norepinephrine, and serotonin) and trace amines [beta-phenylethylamine (beta-PEA), tyramine, tryptamine, and octopamine]. Common biogenic amines are well established as neurotransmitters, but the roles and functional importance of trace amines remain elusive. Here, we re-evaluated the interaction of trace amines with trace amine-associated receptor 1 (TAAR1) and investigated effects of beta-PEA on monoamine transporter function and influence of monoamine autoreceptors on TAAR1 signaling. We confirmed that TAAR1 was activated by trace amines and demonstrated that TAAR1 activation by beta-PEA significantly inhibited uptake and induced efflux of [3H]dopamine, [3H]norepinephrine, and [3H]serotonin in transfected cells. In brain synaptosomes, beta-PEA significantly inhibited uptake and induced efflux of [3H]dopamine and [3H]serotonin in striatal and [3H]norepinephrine in thalamic synaptosomes of rhesus monkeys and wild-type mice, but it lacked the same effects in synaptosomes of TAAR1 knockout mice. The effect of beta-PEA on efflux was blocked by transporter inhibitors in either the transfected cells or wild-type mouse synaptosomes. We also demonstrated that TAAR1 signaling was not affected by monoamine autoreceptors at exposure to trace amines that we show to have poor binding affinity for the autoreceptors relative to common biogenic amines. These results reveal that beta-PEA alters monoamine transporter function via interacting with TAAR1 but not monoamine autoreceptors. The functional profile of beta-PEA may reveal a common mechanism by which trace amines exert modulatory effects on monoamine transporters in brain.

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

Pyridine N-n-alkylation of S(-)-nicotine (NIC) affords N-n-alkylnicotinium analogs, previously shown to competitively inhibit [(3)H]NIC binding and interact with alpha4beta2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked (86)Rb(+) efflux from rat thalamic synaptosomes to assess functional interaction with alpha4beta2* nAChRs. In a concentration-dependent manner, NIC evoked (86)Rb(+) efflux (EC(50) = 170 nmol/L). Analog-induced inhibition of NIC-evoked (86)Rb(+) efflux varied over a approximately 450-fold range. Analogs with long n-alkyl chain lengths (C(9)-C(12)) inhibited efflux in the low nmol/L range (IC(50) = 9-20 nmol/L), similar to dihydro-beta-erythroidine (IC(50) = 19 nmol/L). Compounds with shorter n-alkyl chain lengths (C(1)-C(8)) produced inhibition in the low micromol/L range (IC(50) = 3-12 micromol/L). C(10) and C(12) analogs completely inhibited NIC-evoked efflux, whereas C(1-9) analogs produced maximal inhibition of only 10% to 60%. While the C(10) analog N-n-decylnicotinium iodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [(3)H]NIC binding sites (K(i) = 90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked (86)Rb(+) efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at alpha4beta2* nAChRs.

Bis-Azaaromatic quaternary ammonium analogues: ligands for α4β2* and α7* subtypes of neuronal nicotinic receptors

A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N, N′-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [ 3H]nicotine binding sites ( K i=330 nM), but did not inhibit [ 3H]methyllycaconitine binding ( K i >100 μM), indicative of an interaction with α4β2*, but not α7* receptor subtypes, respectively. Also, bNDI inhibited (IC 50=3.76 μM) nicotine-evoked 86Rb + efflux from rat thalamic synaptosomes, indicating antagonist activity at α4β2* nAChRs. N, N′-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [ 3H]methyllycaconitine binding sites ( K i=1.61 μM), but did not inhibit [ 3H]nicotine binding ( K i>100 μM), demonstrating an interaction with α7*, but not α4β2* nAChRs. Thus, variation of N- n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the α4β2* nAChR subtype, as well as ligands with selectivity at α7* nAChRs.

Lobeline inhibits nicotine-evoked [ 3H]dopamine overflow from rat striatal slices and nicotine-evoked 86Rb + efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [ 3H] overflow from [ 3H]dopamine ([ 3H]DA)-preloaded rat striatal slices and 86Rb + efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(−)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(−)-nicotine was determined. Both S(−)-nicotine (0.1–1 μM) and lobeline (>1.0 μM) evoked [ 3H] overflow from superfused [ 3H]DA-preloaded striatal slices. However, lobeline-evoked [ 3H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 μM) which did not evoke [ 3H] overflow, lobeline inhibited S(−)-nicotine (0.1–10 μM)-evoked [ 3H] overflow, shifting the S(−)-nicotine concentration–response curve to the right. S(−)-Nicotine (30 nM–300 μM) increased (EC 50 value=0.2 μM) 86Rb + efflux from thalamic synaptosomes. In contrast, lobeline (1 nM–10 μM) did not evoke 86Rb + efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC 50 value=0.7 μM) 86Rb + efflux evoked by 1 μM S(−)-nicotine, a concentration which maximally stimulated 86Rb + efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(−)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.

TC-2559: A novel orally active ligand selective at neuronal acetylcholine receptors

TC-2559 [( E)- N-Methyl-4-[3-(5-ethoxypyridin)yl]-3-buten-1-amine] is a novel nicotinic agonist markedly more selective than recently reported novel nicotinic receptor ligands (selectivity ratio for central nervous system (CNS) to peripheral nervous system (PNS)>4000). TC-2559 competes effectively with [ 3H]-nicotine binding ( K i=5 nM) but not with [ 125I]-bungarotoxin (>50,000 nM). Dopamine release from striatal synaptosomes and ion flux from thalamic synaptosomes indicate that TC-2559 is potent and efficacious in the activation of CNS receptors and significantly reduced glutamate-induced neurotoxicity in vitro. TC-2559 has no detectable effects on muscle and ganglion-type nicotinic acetylcholine receptors at concentrations up to 1 mM. TC-2559 significantly attenuates scopolamine-induced cognitive deficits in a step-through passive avoidance task. Acute and repeated oral dosing of TC-2559 enhances performance in a radial arm maze task. In contrast to the effects of equimolar concentrations of (−) nicotine, TC-2559 does not induce hypothermia and locomotor activity is not enhanced following repeated daily administration of 14 days. TC-2559 has a markedly enhanced CNS-PNS selectivity ratio and an intra-CNS selectivity as evidenced by the improved cognition without increased locomotor activity. The in vitro and in vivo studies in the present study suggest that TC-2559 has the desired profile to be further evaluated as a potential therapeutic agent for neurodegenerative diseases.

UB-165: A Novel Nicotinic Agonist with Subtype Selectivity Implicates the α4β2* Subtype in the Modulation of Dopamine Release from Rat Striatal Synaptosomes

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

GABA release and uptake measured in crude synaptosomes from Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

GABA release and uptake were examined in Genetic Absence Epilepsy Rats from Strasbourg and in non-epileptic control animals, using crude synaptosomes prepared from the cerebral cortex and thalamus. Uptake of [ 3H]GABA over time was reduced in thalamic synaptosomes from epileptic rats, compared to controls. The affinity of the uptake process in thalamic synaptosomes was lower in epileptic animals. NNC-711, a ligand for the GAT-1 uptake protein, reduced synaptosomal uptake by more than 95%; β-alanine, an inhibitor selective for the uptake proteins GAT-2 and -3, did not significantly reduce synaptosomal uptake. Autoradiography studies using [ 3H]tiagabine, a ligand selective for GAT-1, revealed no differences between the strains in either affinity or levels of binding. Ethanolamine O-sulphate (100 μM), a selective inhibitor of GABA-transaminase, did not affect uptake levels. Aminooxyacetic acid (10–100 μM), an inhibitor of GABA-transaminase and, to a lesser extent, glutamate decarboxylase, caused an increase in measured uptake in both thalamic and cortical synaptosomes, in both strains. We found no difference in in vitro basal or KCl-stimulated endogenous GABA release between epileptic and control rats. These results indicate that GABA uptake in the thalamus of Genetic Absence Epilepsy Rats from Strasbourg was reduced, compared to control animals. The lower uptake affinity in the epileptic animals probably contributed to the reduction in uptake over time. Uptake appeared to be mediated primarily by the ‘neuronal’ transporter GAT-1. Autoradiography studies revealed no differences in the number or affinity of this uptake protein. It is therefore possible that altered functional modulation of GAT-1 caused the decrease in uptake shown in the epileptic animals. Inhibition of GABA-transaminase activity had no effect on measured GABA uptake, whereas a reduction in glutamate decarboxylase activity may have affected measured uptake levels.

Metanicotine: A nicotinic agonist with central nervous system selectivity?in vitro and in vivo characterization

A growing body of evidence suggests that disruption of nicotinic cholinergic systems may be an important factor in the etiology of a number of different diseases, ranging from neurodegenerative diseases, such as Alzheimer's and Parkinson's, to ulcerative colitis. The mechanistic basis for such diverse nicotinic effects is likely to lie in the ever growing number of potential receptor subtypes. Therefore, the development of receptor subtype-selective probes is essential to understand the emerging complexity of nicotinic cholinergic systems and the mechanisms underlying diseases that may involve these systems. Toward this end, we have evaluated the nicotinic agonist metanicotine, (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine, using the following in vitro and in vivo methods: 1) receptor binding and up-regulation, 2) neurotransmitter release and ion flux in synaptosomes/cells, 3) in vivo microdialysis in rats, 4) reversal of scopolamine-induced amnesia in a step-through passive-avoidance paradigm, 5) water maze performance in mice, 6) radial-arm maze performance in brain-lesioned rats, 7) changes in heart rate and blood pressure, and 8) physiological depression of body temperature, locomotor activity, acoustic startle, and respiration rate. Our in vitro results indicate that metanicotine binds with high affinity to the major receptor subtype in brain (α4β2), evokes dopamine release from striatal synaptosomes and Rb+ efflux from thalamic synaptosomes, but does not activate ganglionic, muscle, or other peripheral type nicotinic receptors. These results suggest that metanicotine is selective for α4-containing central nervous system (CNS) nicotinic receptors and has reduced selectivity for peripheral nervous system (PNS) receptor subtypes. These conclusions are further supported by in vivo studies with metanicotine showing enhanced cognitive effects and significantly lower peripheral effects. Our in vivo results indicate that metanicotine increases the release of acetylcholine, norepinephrine, dopamine, and serotonin in cortex and is equal to or better than nicotine on measures of cognitive enhancement. By comparison, metanicotine is significantly less potent than nicotine in increasing heart rate and blood pressure and in causing physiological depression. These results are consistent with in vitro data indicating metanicotine's CNS receptor selectivity, and they suggest that this ligand may be a suitable tool for probing the relationships that underlie the complex central and peripheral pharmacology of nicotinic cholinergic systems. Furthermore, metanicotine may be a good lead candidate for developing nicotinic agonists as CNS therapeutics with reduced peripheral side effects. Drug Dev. Res. 38:169–176 © 1996 Wiley-Liss, Inc.

GABAB Receptor‐Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Previously, we have shown a significant increase in number of GABAB receptor binding sites in neocortex and thalamus of lethargic (lh/lh) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABAB receptors would inhibit [3H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [3H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [3H]GABA release evoked by 12 mM KCl, an effect mediated by GABAB receptors. The maximal inhibition (Imax) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC50 (3 microM) was unchanged. In the thalamic preparation, the effect of baclofen (50 microM) was 58% less robust in lh/lh mice. Other effects mediated by GABAB receptors (inhibitions in Ca2+ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABAB receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABAB receptors or GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.