Special Delivery: Antipsychotic Drugs Released From Nerve Terminals
15 June 2012. In an interesting twist on antipsychotic drug (APD) action, a study published June 7 in Neuron reports that APDs are taken up into synaptic vesicles, then released via neural activity at concentrations sufficient to inhibit further exocytosis. Led by Teja Groemer of Friedrich-Alexander University of Erlangen-Nürnberg, Germany, the study suggests that the time it takes APDs to build up within these vesicles might help explain why they can take weeks to reach their full therapeutic effectiveness in treating symptoms of schizophrenia.
This prolonged time course for relieving some symptoms of schizophrenia has been ascribed to the accumulation of APDs or protein intermediaries in the brain (e.g., Rayport and Sulzer, 1995; Kornhuber et al., 1995; Kuhar and Joyce, 2001), and the new study supports and extends this idea by highlighting known, but disregarded, actions of APDs. Though APD efficacy is mainly attributed to their blockade of the D2 subtype of dopamine receptors, previous research has shown that antipsychotics such as haloperidol or chlorpromazine can inhibit voltage-gated sodium, calcium, and potassium channels (Ogata et al., 1989). These seemingly off-target effects occurred at micromolar concentrations of APDs, however, which are not thought to occur at therapeutic doses of APDs estimated to be in the nanomolar range (Baumann et al., 2004). The new research suggests otherwise, finding evidence for highly localized micromolar concentrations of weakly basic APDs (both typical and atypical) in the acidic interiors of synaptic vesicles. There, APDs were treated like the usual neurotransmitter cargo, and released as a consequence of neural activity. Once expelled into the synaptic cleft, the APDs exerted functional effects, acting on presynaptic sodium channels to dampen further release.
“The most important aspect of the work was the demonstration of the functional consequences of vesicular delivery of APDs on neurotransmitter release,” write Andrew Morton and Michael Cousin of the University of Edinburgh, U.K., in a preview of the paper. This raises the possibility of harnessing this ready-made delivery system inside the presynaptic terminal to precisely administer other kinds of weakly basic drugs to the brain, Groemer told SRF by e-mail.
First authors Carsten Tischbirek, Eva Wenzel, and Fang Zheng began by examining the buildup of a proxy fluorescent molecule similar to APDs called lysotracker red (LTR) in living hippocampal neurons from rats. Like APDs, LTR is a weak base, and live cell microscopy found that incubating cultured neurons with LTR led to its enrichment in acidic organelles, including synaptic vesicles stained by pH-dependent antibodies. LTR reached micromolar concentrations similar to those calculated for APDs in a cell model. Bathing the neurons with four different APDs (haloperidol, chlorpromazine, clozapine, or risperidone) decreased the LTR fluorescence signal, suggesting that the APDs displaced LTR molecules from the vesicles, but the researchers stopped short of directly confirming accumulation of APDs themselves inside the vesicles.
The researchers also found that electrical stimulation of these cultured neurons led to a loss of LTR signal at synaptic sites marked with a synaptic vesicle antibody. This LTR signal loss was calcium dependent and increased with stimulus strength, suggesting that LTR was shuttling through the usual exocytosis process governing neurotransmitter release.
To look for an in-vivo equivalent of this type of release, the researchers turned to microdialysis in freely behaving rats. The researchers implanted three probes to sample chemicals in the extracellular space of three different brain regions: prefrontal cortex, dorsal striatum, and nucleus accumbens. In rats treated with haloperidol for two weeks, the researchers found that local stimulation of these brain regions led to increases in haloperidol levels, consistent with APD accumulation and release. In the prefrontal cortex, the stimulation-induced haloperidol levels were nearly twice that measured in the dorsal striatum, which suggests that some regions are more prone to accumulation and/or release of APDs. These fluctuations contrast with a picture of steady-state APD concentrations in the brain.
The researchers then looked downstream to see what the effects of synaptically released APDs might be, and found that APDs interfere with voltage-sensitive triggers for exocytosis. Using a fluorescent dye to mark synaptic vesicles in cultured hippocampal neurons, they found that, while electrical stimulation evoked increases in the fluorescent signal (indicative of exocytosis), bathing the neurons in APDs decreased the exocytosis signal in a dose-dependent manner. This seemed to stem from decreases in the calcium trigger: APDs decreased the size of the calcium influx measured by a calcium indicator dye in presynaptic terminals evoked by electrical stimulation. Across the four different APDs tested, the amplitude of the exocytosis signals correlated with the amplitude of the calcium signals (R = 0.86, p <0.001).
This APD-induced inhibition could be attributed to interference with the triggers for exocytosis, rather than with the release machinery itself. Sodium channels in the presynaptic terminal are normally responsible for boosting membrane voltage high enough to pen calcium channels, which then sets off the cascade of events leading to synaptic vesicle release. In the presence of haloperidol, however, the researchers found sluggish sodium channels: voltage-clamp recordings revealed that haloperidol limited sodium channel activation, particularly once the channels entered the inactivated state. This would limit release triggered by a subsequent action potential, and raised the idea that haloperidol’s effect might depend on a neuron’s past firing history.
Similarly, the researchers found that the APD-induced inhibition was use dependent, with increasing stimulation strength boosting the braking power of haloperidol on exocytosis—even a 0.5 μM dose that had a negligible effect on exocytosis evoked by 60 action potentials showed substantial inhibition after 180 action potentials. This suggests an auto-inhibitory dosing mechanism—the more active a terminal is, the more strongly it would be inhibited by the APD. A similar activity-dependent effect on excitatory postsynaptic currents (EPSCs) was found in the more preserved neural networks of brain slices made from hippocampus and nucleus accumbens. Notably, the haloperidol-induced inhibition of the EPSCs was particularly strong in nucleus accumbens compared to hippocampus.
Finally, the researchers found that disrupting the accumulation of APDs inside synaptic vesicles limited their inhibitory ability. In the presence of folimycin, which neutralizes the acidic interior of synaptic vesicles and thus would be expected to decrease APD buildup inside, haloperidol had a reduced inhibitory effect on exocytosis or calcium transients. This partial reversal argues that haloperidol accumulation inside synaptic vesicles is key to its release-dampening powers.
Further research will have to piece together whether these APD effects actually translate into therapeutic consequences for the people taking these drugs. In the meantime, the novel drug-delivery mechanism outlined in the study suggests that the synaptic release machinery is willing and able to accommodate APDs, and potentially other drugs. Whether there are synapse-specific differences in the ability to take on unconventional cargo remains to be seen, but the pharmacopeia for brain disorders may eventually include drugs tailor made for “on demand” synaptic vesicle delivery.—Michele Solis.
Tischbirek CH, Wenzel EM, Zheng F, Huth T, Amato D, Trapp S, Denker A, Welzel O, Lueke K, Svetlitchny A, Rauh M, Deusser J, Schwab A, Rizzoli SO, Henkel AW, Müller CP, Alzheimer C, Kornhuber J, Groemer TW. Use-Dependent Inhibition of Synaptic Transmission by the Secretion of Intravesicularly Accumulated Antipsychotic Drugs. Neuron. 2012 June 7; 74: 830-844. Abstract
Morton A, Cousin MA. The Best Things Come in Small Packages—Vesicular Delivery of Weak Base Antipsychotics. Neuron. 2012 June 7; 74: 765-767. Abstract