Comparing tonic and phasic dendritic calcium in cholinergic pedunculopontine neurons and dopaminergic substantia nigra neurons.

Several brainstem nuclei degenerate in Parkinson's disease (PD). In addition to the well-characterized dopaminergic neurons of the substantia nigra pars compacta (SNc), the cholinergic neurons of the pedunculopontine nucleus (PPN) also degenerate in PD. One leading hypothesis of selective vulnerability is that pacemaking activity and the activation of low-threshold L-type calcium current are major contributors to tonic calcium load and cellular stress in SNc dopaminergic neurons. However, it is not yet clear whether the vulnerable PPN cholinergic neurons share this property. Therefore, we used two-photon dendritic calcium imaging and whole-cell electrophysiology to evaluate the role of L-type calcium channels in tonic and phasic dendritic calcium signals in PPN and SNc neurons. In addition, we investigated N- and P/Q-type calcium channel regulation of firing properties and dendritic calcium in PPN neurons. We found that blocking L-type channels reduces tonic firing rate and dendritic calcium levels in SNc neurons. By contrast, the tonic calcium load in PPN neurons did not depend on L-, N- or P/Q-type channels. However, we found that blocking either L-type (with nifedipine) or N- and P/Q-type (with omega-conotoxin MVIIC) channels reduces phasic calcium influx in PPN dendrites. Together, these findings show that L-type calcium channels play different roles in the activity of SNc and PPN neurons, and suggest that low-threshold L-type channels are not responsible for tonic calcium levels in PPN cholinergic neurons and are therefore not likely to be a source of selective vulnerability in these cells.

Comparing tonic and phasic calcium in the dendrites of vulnerable midbrain neurons.

Several midbrain nuclei degenerate in Parkinson's Disease (PD). Many of these nuclei share the common characteristics that are thought to contribute to their selective vulnerability, including pacemaking activity and high levels of calcium influx. In addition to the well-characterized dopaminergic neurons of the substantia nigra pars compacta (SNc), the cholinergic neurons of the pedunculopontine nucleus (PPN) also degenerate in PD. It is well established that the low-threshold L-type calcium current is a main contributor to tonic calcium in SNc dopaminergic neurons and is hypothesized to contribute to their selective vulnerability. However, it is not yet clear whether the vulnerable PPN cholinergic neurons share this property. Therefore, we used two-photon dendritic calcium imaging and whole-cell electrophysiology to evaluate the role of L-type calcium channels in the tonic and phasic activity of PPN neurons and the corresponding dendritic calcium signal and directly compare these characteristics to SNc neurons. We found that blocking L-type channels reduces tonic firing rate and dendritic calcium levels in SNc neurons. By contrast, the calcium load in PPN neurons during pacemaking did not depend on L-type channels. However, we find that blocking L-type channels reduces phasic calcium influx in PPN dendrites. Together, these findings show that L-type calcium channels play different roles in the activity of SNc and PPN neurons, and suggest that low-threshold L-type channels are not responsible for tonic calcium levels in PPN cholinergic neurons and are therefore not likely to be a source of selective vulnerability in these cells.

Functional characterization of N-octyl 4-methylamphetamine variants and related bivalent compounds at the dopamine and serotonin transporters using Ca2+ channels as sensors.

The early characterization of ligands at the dopamine and serotonin transporters, DAT and SERT, respectively, is important for drug discovery, forensic sciences, and drug abuse research. 4-Methyl amphetamine (4-MA) is a good example of an abused drug whose overdose can be fatal. It is a potent substrate at DAT and SERT where its simplest secondary amine (N-methyl 4-MA) retains substrate activity at them. In contrast, N-n-butyl 4-MA is very weak, therefore it was categorized as inactive at these transporters. Here, N-octyl 4-MA and other related compounds were synthesized, and their activities were evaluated at DAT and SERT. To expedite this endeavor, cells expressing DAT or SERT were co-transfected with a voltage-gated Ca2+ channel and, the genetically-encoded Ca2+ sensor, GCaMP6s. Control compounds and the newly synthesized molecules were tested on these cells using an automated multi-well fluorescence plate reader; substrates and inhibitors were identified successfully at DAT and SERT. N-Octyl 4-MA and three bivalent compounds were inhibitors at these transporters. These findings were validated by measuring Ca2+-mobilization using quantitative fluorescence microscopy. The bivalent molecules were the most potent of the series and were further characterized in an uptake-inhibition assay. Compared to cocaine, they showed comparable potency inhibiting uptake at DAT and higher potency at SERT. These observations support a previous hypothesis that amphetamine-related (and, here, N-extended alkyl and) bivalent arylalkylamine molecules are active at monoamine transporters, showing potent activity as reuptake inhibitors, and implicate the involvement of a distant auxiliary binding feature to account for their actions at DAT and SERT.

Impact of menthol on nicotine intake and preference in mice: Concentration, sex, and age differences.

Menthol has been shown to contribute to the appeal of tobacco products in humans. However, factors such as sex, age and menthol concentration remain unclear in the interaction between menthol and nicotine. To understand these factors, we utilized a mouse model to determine the impact of menthol on oral nicotine consumption. A range of menthol concentrations (oral and systemic) were tested with or without oral nicotine using the two-bottle choice paradigm in adolescent and adult female and male C57BL/6J mice. Moreover, genetically modified mice were used to investigate the role of α7 nicotinic acetylcholine receptors (nAChRs) on the effects of menthol. Menthol addition to nicotine solution increased oral nicotine consumption in C57BL/6J mice in a sex- and menthol concentration-dependent manner. At lower menthol concentrations, female mice demonstrated an enhancement of nicotine consumption and male mice showed a similar behavior at higher menthol concentrations. Menthol drinking alone was only significantly different by sex at 60 µg/ml menthol concentration where female mice had higher menthol intake than males. Menthol administered both orally and systemically (intraperitoneal) increased oral nicotine consumption. Adolescent female mice had a higher nicotine intake at lower menthol concentrations compared to their adult counterparts. While α7 nAChR wild type mice consumed more mentholated nicotine solution than nicotine only solution, this effect was abolished in KO mice. Effects of menthol are concentration-, sex-, age-, and α7 nAChR-dependent. Oral and intraperitoneal menthol increases nicotine intake, suggesting that sensory, peripheral, and/or central mechanisms are involved in effects of menthol on oral nicotine consumption.

Spreading Depression Promotes Astrocytic Calcium Oscillations and Enhances Gliotransmission to Hippocampal Neurons.

Spreading depression (SD) is a pathophysiological phenomenon characterized by propagating waves of profound neuronal and glial depolarization in central nervous system gray matter. Although SD is primarily mediated by neurons with a subsequent astrocytic response, it remains unclear how astrocytic activity is modulated after SD and how altered astrocyte signaling contribute to neuronal excitability. Here, we report that after the concurrent Ca2+ wave, SD enhanced astrocytic activity by promoting a secondary period of Ca2+ oscillations. SD-induced Ca2+ oscillations did not require the activation of metabotropic glutamate receptors or purinergic receptors; instead, they were mediated by the activation of GABAB receptors and 1,4,5-trisphosphate (IP3) receptors. Furthermore, SD increased the number of NMDA receptor-mediated slow inward currents (SICs) in CA1 pyramidal neurons. The frequency of SD-induced SICs was reduced by blockade of GABAB receptors or by limiting Ca2+ efflux from the ER. Selective inhibition of astrocytic Ca2+ signals by dialysis of BAPTA into astrocytes or by knocking out the astrocytic type of IP3 receptors suppressed SICs after SD. These results demonstrated a causative link between the SD-induced Ca2+ oscillations and the enhanced glutamatergic astrocyte-neuron signaling. Therefore, we conclude that SD enhances the astrocyte Ca2+ signals and further promotes gliotransmission and neuronal excitability.

Luteolin inhibits GABAA receptors in HEK cells and brain slices.

Modulation of the A type γ-aminobutyric acid receptors (GABAAR) is one of the major drug targets for neurological and psychological diseases. The natural flavonoid compound luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone) has been reported to have antidepressant, antinociceptive, and anxiolytic-like effects, which possibly involve the mechanisms of modulating GABA signaling. However, as yet detailed studies of the pharmacological effects of luteolin are still lacking, we investigated the effects of luteolin on recombinant and endogenous GABAAR-mediated current responses by electrophysiological approaches. Our results showed that luteolin inhibited GABA-mediated currents and slowed the activation kinetics of recombinant α1ß2, α1ß2γ2, α5ß2, and α5ß2γ2 receptors with different degrees of potency and efficacy. The modulatory effect of luteolin was likely dependent on the subunit composition of the receptor complex: the αß receptors were more sensitive than the αßγ receptors. In hippocampal pyramidal neurons, luteolin significantly reduced the amplitude and slowed the rise time of miniature inhibitory postsynaptic currents (mIPSCs). However, GABAAR-mediated tonic currents were not significantly influenced by luteolin. These data suggested that luteolin has negative modulatory effects on both recombinant and endogenous GABAARs and inhibits phasic rather than tonic inhibition in hippocampus.