MMP-1 overexpression selectively alters inhibition in D1 spiny projection neurons in the mouse nucleus accumbens core.

Protease activated receptor-1 (PAR-1) and its ligand, matrix metalloproteinase-1 (MMP-1), are altered in several neurodegenerative diseases. PAR-1/MMP-1 signaling impacts neuronal activity in various brain regions, but their role in regulating synaptic physiology in the ventral striatum, which is implicated in motor function, is unknown. The ventral striatum contains two populations of GABAergic spiny projection neurons, D1 and D2 SPNs, which differ with respect to both synaptic inputs and projection targets. To evaluate the role of MMP-1/PAR-1 signaling in the regulation of ventral striatal synaptic function, we performed whole-cell recordings (WCR) from D1 and D2 SPNs in control mice, mice that overexpress MMP-1 (MMP-1OE), and MMP-1OE mice lacking PAR-1 (MMP-1OE/PAR-1KO). WCRs from MMP1-OE mice revealed an increase in spontaneous inhibitory post-synaptic current (sIPSC), miniature IPSC, and miniature excitatory PSC frequency in D1 SPNs but not D2 SPNs. This alteration may be partially PAR-1 dependent, as it was not present in MMP-1OE/PAR-1KO mice. Morphological reconstruction of D1 SPNs revealed increased dendritic complexity in the MMP-1OE, but not MMP-1OE/PAR-1KO mice. Moreover, MMP-1OE mice exhibited blunted locomotor responses to amphetamine, a phenotype also observed in MMP-1OE/PAR-1KO mice. Our data suggest PAR-1 dependent and independent MMP-1 signaling may lead to alterations in striatal neuronal function.

Differential electrophysiological properties of D1 and D2 spiny projection neurons in the mouse nucleus accumbens core.

The striatum consists of the dorsal (caudate/putamen) and the ventral (nucleus accumbens) regions. The nucleus accumbens is further divided into a core and shell. Both the dorsal and ventral striatum contain populations of spiny projection neurons, which make up 95% of the neurons within the striatum. SPNs are canonically categorized into those that express the D1-type dopamine receptor (D1 SPNs) and those that express the D2-type dopamine receptor (D2 SPNs). D1 and D2 SPNs differ with respect to both synaptic inputs and projection targets. In the dorsal striatum, it is well established that these populations of SPNs differ in terms of their electrophysiological and morphological properties. However, there remains a gap in our knowledge of the electrophysiological properties of SPNs in the nucleus accumbens core. To evaluate the differential properties of these SPNs, we performed whole-cell recordings from D1 and D2 SPNs in BAC transgenic mice in which D1 SPNs fluoresce red and D2 SPNs fluoresce green. The two SPN subtypes did not differ in terms of their time constant, capacitance, resting membrane potential, or tonic current. However, D2 SPNs displayed heightened inhibitory postsynaptic current (IPSC) and miniature excitatory PSC frequency as compared with D1 SPNs. Furthermore, D2 SPNs displayed decreased rheobase, increased excitability as measured by firing rates to depolarizing current injections, increased inward rectification, increased input resistance, and decreased dendritic complexity compared to D1 SPNs. Our results demonstrate a dichotomy in the electrophysiological properties of D1 and D2 SPNs in the nucleus accumbens core, which contributes to our knowledge of ventral striatal circuitry.

Absolute Configuration and Pharmacology of the Poison Frog Alkaloid Phantasmidine.

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.

Neonatal phenobarbital exposure disrupts GABAergic synaptic maturation in rat CA1 neurons.

OBJECTIVE: Phenobarbital is the most commonly utilized drug for the treatment of neonatal seizures. The use of phenobarbital continues despite growing evidence that it exerts suboptimal seizure control and is associated with long-term alterations in brain structure, function, and behavior. Alterations following neonatal phenobarbital exposure include acute induction of neuronal apoptosis, disruption of synaptic development in the striatum, and a host of behavioral deficits. These behavioral deficits include those in learning and memory mediated by the hippocampus. However, the synaptic changes caused by acute exposure to phenobarbital that lead to lasting effects on brain function and behavior remain understudied. METHODS: Postnatal day (P)7 rat pups were treated with phenobarbital (75 mg/kg) or saline. On P13-14 or P29-37, acute slices were prepared and whole-cell patch-clamp recordings were made from CA1 pyramidal neurons. RESULTS: At P14 we found an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in the phenobarbital-exposed as compared to the saline-exposed group. In addition to this change in mIPSC frequency, the phenobarbital group displayed larger bicuculline-sensitive tonic currents, decreased capacitance and membrane time constant, and a surprising persistence of giant depolarizing potentials. At P29+, the frequency of mIPSCs in the saline-exposed group had increased significantly from the frequency at P14, typical of normal synaptic development; at this age the phenobarbital-exposed group displayed a lower mIPSC frequency than did the control group. Spontaneous inhibitory postsynaptic current (sIPSC) frequency was unaffected at either P14 or P29+. SIGNIFICANCE: These neurophysiological alterations following phenobarbital exposure provide a potential mechanism by which acute phenobarbital exposure can have a long-lasting impact on brain development and behavior.

AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Rat α3ß4 Nicotinic Cholinergic Receptors.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3ß4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3ß4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3ß4 and α4ß2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3ß4 receptors over α4ß2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3ß4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3ß4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat α3ß4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4ß2 nAChRs. Both α3ß4 and α4ß2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3ß4 receptors than rat α3ß4 and human α4ß2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3ß4 nAChR and shortest for the human α4ß2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

APOE Genotype Alters Immunoglobulin Subtypes in Knock-In Mice.

Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer's disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen, and plasma. Levels of total IgG in brain and spleen were highest in APOE-ɛ3 mice, significantly higher than in APOE-ɛ2 and APOE-ɛ4 mice; no differences were observed for levels of total IgG in plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-ɛ3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-ɛ4 mice compared to APOE-ɛ2 and APOE-ɛ3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-ɛ4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-ɛ2 mice. In total, murine IgG2a and IgM were highest in APOE-ɛ4 mice, while total IgG and Ig2b were highest in APOE-ɛ3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes.

Synthesis and pharmacological characterization of new neuronal nicotinic acetylcholine receptor ligands derived from Sazetidine-A.

The enantiomers of two analogs of Sazetidine-A as well as several other novel biosteric analogues were synthesized. Their binding affinities at three major nAChRs subtypes and selectivity profiles were determined. Though many (S)-enantiomers of Sazetidine-A analogs have high binding affinities and good subtype selectivities, it is not a general rule that (S)-enantiomers are better than their (R) counterparts. Compound 11, of which the ethynyl group was replaced by its' bioisostere-the triazole via click chemistry, showed a high binding affinity to α4ß2 subtype (Ki=1.3 nM) and better selectivity to the α4ß2 subtype over α3ß4 subtype with that of Sazetidine-A. The azide compound 15, a potential photoaffinity label, showed improved high selectivity and similar binding property profile with that of Sazetidine-A. The biaryl analog 17 exhibited a much lower affinity as compared to Sazetidine-A indicating the importance of a 'long tail' side chain for α4ß2 nAChR binding.

Design, synthesis and discovery of picomolar selective α4ß2 nicotinic acetylcholine receptor ligands.

Developing novel and selective compounds that desensitize α4ß2 nicotinic acetylcholine receptors (nAChRs) could provide new effective treatments for nicotine addiction, as well as other disorders. Here we report a new class of nAChR ligands that display high selectivity and picomolar binding affinity for α4ß2 nicotinic receptors. The novel compounds have Ki values in the range of 0.031-0.26 nM and properties that should make them good candidates as drugs acting in the CNS. The selected lead compound 1 (VMY-2-95) binds with high affinity and potently desensitizes α4ß2 nAChRs. At a dose of 3 mg/kg, compound 1 significantly reduced rat nicotine self-administration. The overall results support further characterizations of compound 1 and its analogues in preclinical models of nicotine addiction and perhaps other disorders involving nAChRs.

Chemistry and pharmacological studies of 3-alkoxy-2,5-disubstituted-pyridinyl compounds as novel selective α4ß2 nicotinic acetylcholine receptor ligands that reduce alcohol intake in rats.

Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4ß2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogues containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed overall pharmacological property profiles similar to that of sazetidine-A but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In rat studies, compound (S)-9 significantly reduced alcohol uptake. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.

Consequences of linker length alteration of the α7 nicotinic acetylcholine receptor (nAChR) agonist, SEN12333.

A series of ligands based on SEN12333, containing either contracted or elongated alkyl chains, were synthesized and evaluated in molecular docking studies against a homology model of the α7 nicotinic acetylcholine receptor (nAChR) subtype. The predicted binding of all ligands was highly similar, with the exception of the analog containing a 5 methylene unit spacer. However, in vitro competition binding assays revealed that the ligands possessed dissimilar binding affinities, with a K(i) range of more than an order of magnitude (K(i)=0.50 to >10 µM), and only SEN12333 itself exhibited functional activity at the α7 nAChR.