Comparative Neuropharmacology and Pharmacokinetics of Methamphetamine and Its Thiophene Analog Methiopropamine in Rodents

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (Emax 12.5 mg/kg; i.p.) compared to methamphetamine (Emax 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

Autoradiographic localization of [3H]-Nisoxetine binding sites in the CNS of male and female Japanese quail

Background In the central nervous system of mammals, transporters localized on the presynaptic nerve terminals regulate the reuptake of neurotransmitters. These transporters are selective for a specific neurotransmitter such as dopamine (DA) and norepinephrine (NE). Specifically in the synapse, the dopamine transporter (DAT) reuptakes DA and the norepinephrine transporter (NET) reuptakes NE. However previous research has found that avian species do not have a gene for DAT, and therefore, birds may be using the NET to clear both NE and DA from the synapse. The current study aimed to extend this finding by localizing NET expression in male and female Japanese quail (Coturnix japonica) brains using [3H]Nisoxetine, a selective NET blocker. Results High densities of binding sites were observed in the olfactory tubercle (OTu), the medial striatum (MSt), and the lateral striatum (LSt). Lower densities of binding sites were detected in the amygdala (AMY) and hypothalamus (Hyp), and low binding was found in the medial preoptic area (mPOA) and the pallium. Conclusion The areas with the highest densities of NET are also areas that previous research has shown to have high levels of DA activity but low levels of NE innervation (e.g. striatum). The distribution of this reuptake transporter is consistent with the theory that NET acts to clear both DA and NE from the synapse.

Neuroprotective Role of the B Vitamins in the Modulation of the Central Glutamatergic Neurotransmission

Background: Regulation of glutamate release is crucial for maintaining normal brain function, but excess glutamate release is implicated in many neuropathological conditions. Therefore, the minimum glutamate release from presynaptic nerve terminals is an important neuroprotective mechanism. Objective: In this mini-review, we analyze the three B vitamins, namely vitamin B2 (riboflavin), vitamin B6 (pyridoxine), and vitamin B12 (cyanocobalamin), that affect the 4-aminopyridine (4-AP)-evoked glutamate release from presynaptic nerve terminal in rat and discuss their neuroprotective role. Methods: In this study, the measurements include glutamate release, DiSC3(5), and Fura-2. Results: The riboflavin, pyridoxine, and cyanocobalamin produced significant inhibitory effects on 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes) in a dose-dependent relationship. These presynaptic inhibitory actions of glutamate release are attributed to inhibition of physiologic Ca2+-dependent vesicular exocytosis but not Ca2+-independent nonvesicular release. These effects also did not affect membrane excitability, while diminished cytosolic [Ca2+]c through a reduction of direct Ca2+ influx via Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, rather than through indirect Ca2+ induced Ca2+ release from ryanodine-sensitive intracellular stores. Furthermore, their effects were attenuated by GF109203X and Ro318220, two protein kinase C (PKC) inhibitors, suggesting suppression of PKC activity. Taken together, these results suggest that riboflavin, pyridoxine, and cyanocobalamin inhibit presynaptic vesicular glutamate release from rat cerebrocortical synaptosomes, through the depression Ca2+ influx via voltage-dependent Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels, and PKC signaling cascade. Conclusion: Therefore, these B vitamins may reduce the strength of glutamatergic synaptic transmission and is of considerable importance as potential targets for therapeutic agents in glutamate-induced excitation-related diseases.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Intact synapse structure and function after combined knockout of PTPδ, PTPσ and LAR

It has long been proposed that Leukocyte common Antigen-Related Receptor Protein Tyrosine Phosphatases (LAR-RPTPs) are cell-adhesion proteins for the control of synapse assembly. Their synaptic nanoscale localization, however, has not been established, and the fine structure of synapses after knockout of the three vertebrate genes for LAR-RPTPs (PTPδ, PTPσ and LAR) has not been tested. Here, we find that PTPδ is precisely apposed to postsynaptic scaffolds at excitatory and inhibitory synapses using superresolution microscopy. We generated triple-conditional knockout mice for PTPδ, PTPσ and LAR to test whether they are essential for synapse structure. While mild effects on synaptic vesicle clustering and active zone architecture were detected, synapse numbers and their overall structure were unaffected, membrane anchoring of the active zone persisted, and vesicle docking and release were normal. We conclude that LAR-RPTPs, despite their localization at synaptic appositions, are dispensable for the organization and function of presynaptic nerve terminals.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed “dendrons,” and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

TMEM16B regulates anxiety-related behavior and GABAergic neuronal signaling in the central lateral amygdala

TMEM16B (ANO2) is the Ca2+-activated chloride channel expressed in multiple brain regions, including the amygdala. Here we report that Ano2 knockout mice exhibit impaired anxiety-related behaviors and context-independent fear memory, thus implicating TMEM16B in anxiety modulation. We found that TMEM16B is expressed in somatostatin-positive (SOM+) GABAergic neurons of the central lateral amygdala (CeL), and its activity modulates action potential duration and inhibitory postsynaptic current (IPSC). We further provide evidence for TMEM16B actions not only in the soma but also in the presynaptic nerve terminals of GABAergic neurons. Our study reveals an intriguing role for TMEM16B in context-independent but not context-dependent fear memory, and supports the notion that dysfunction of the amygdala contributes to anxiety-related behaviors.

Noncompetitive antagonists induce cooperative AMPA receptor channel gating

Glutamate is released from presynaptic nerve terminals in the central nervous system (CNS) and spreads excitation by binding to and activating postsynaptic iGluRs. Of the potential glutamate targets, tetrameric AMPA receptors mediate fast, transient CNS signaling. Each of the four AMPA subunits in the receptor channel complex is capable of binding glutamate at its ligand-binding domains and transmitting the energy of activation to the pore domain. Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Here, we investigate how two AMPA receptor-selective noncompetitive antagonists, GYKI-52466 and GYKI-53655, disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors using single-channel recordings from cell-attached patches. Interactions of these 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, we provide insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary. Our results suggest that this region may be a tunable locus for AMPA receptor channel gating.