Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles

Abstract

Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H + -ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca 2+ -dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP + ), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H + countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs.M ost antipsychotic drugs (APDs) are competitive dopamine (DA) D2 receptor (D2R) antagonists (1, 2). The standard view is that APDs, by being weak bases that are in equilibrium with an uncharged form, cross the blood-brain barrier to access extracellular receptor binding sites from the circulation. However, because APDs are weak bases, they have been proposed to accumulate also in acidic intracellular organelles by acidic trapping (i.e., the less abundant uncharged basic hydrophobic form enters organelles passively and then is protonated to become membrane-impermeant). This hypothesis was first explored by examining a D2 antagonist with a covalently added fluorophore and APD displacement of acridine orange (3). More recently, APDs were found to be released from brain tissue by depolarization, which is expected for any positively charged drug. However, follow-up experiments in hippocampal neuron cultures showed that APDs displace the acidophilic dye lysotracker red, implying there is overlap in intracellular distribution between APDs and lysotracker red. Furthermore, lysotracker red was found to accumulate in hippocampal neuron synaptic vesicles by acidic trapping without displacing native neurotransmitter and to be released in response to electrical stimulation. Thus, b...

Striatal dopamine neurotransmission: Regulation of release and uptake

“…New work confirms that while amphetamines indeed collapse acidic gradients in DA synaptic vesicles in mammalian striatum and in Drosophila brain, the net reverse transport of protons through VMAT during vesicular amphetamine uptake plays a larger role in the collapse than does a “weak base” action, in which protons are buffered by intravesicular amphetamine [85,96]. In striatal brain slices, 20 µM para-chloroamphetamine can release a fluorescent antipsychotic compound from synaptic vesicles; however, this effect is blocked by a VMAT2 inhibitor, reserpine, indicating a requirement for VMAT mediated-proton efflux.…”

“…In striatal brain slices, 20 µM para-chloroamphetamine can release a fluorescent antipsychotic compound from synaptic vesicles; however, this effect is blocked by a VMAT2 inhibitor, reserpine, indicating a requirement for VMAT mediated-proton efflux. Reserpine does not block such redistribution by a higher concentration of para-chloroamphetamine (100 µM), however, indicating that amphetamine concentration plays a role in these mechanisms [96]. In Drosophila, reserpine prevents methamphetamine-induced loss of protons, while chloroquine, a non-VMAT substrate weak base, is not prevented by reserpine.…”

Addictive neurons

“…The antipsychotic drug cyamemazine at 5 µM started to accumulate into puncta after 10 min of exposure in rat SNc as revealed by two-photon microscopy in vitro [39] .…”

“…The phenotype of sag in neurons differs depending on region were they reside. In SNc DA neurons of male P14−28 rats the time course of the sag is fast and the steady-state is reached within ~1 sec [39] . The 1 st rebound AP lack AHP in contrast to subsequent two spikes.…”

“…The AHP is either masked by the depolarizing phase of the RTP or it is a double spiking as observed in LS neurons [20] . Spontaneous spikes in SNc DA neurons occur in a pacemaker manner and possess AHP [39] . The GABAergic cells of SNc trigger 'a pure sodium spike, while dopamine neurons have a major contribution of calcium current to their spikes.…”