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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.

Accumulating evidence
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.

Downstream doses
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.

Unconventional cargo
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

Comments on News and Primary Papers

Primary Papers: Use-dependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs.

Comment by:  Philip Seeman (Disclosure)
Submitted 15 June 2012
Posted 15 June 2012

Regrettably, despite the elegant methods, the antipsychotic concentrations applied were not clinically relevant and would all be toxic or lethal to humans if found at those concentrations in the human plasma water or in the spinal fluid.

For example, haloperidol was used at 500 to 5,000 nanomolar. However, the therapeutic concentration of haloperidol in the spinal fluid is between 1 and 5 nanomolar. The toxic concentration is about 10 times higher, and the comatose-lethal concentration is approximately 20 times higher (see Regenthal et al., 1999), all these concentrations being far lower than those used by Tischbirek et al.

In addition, chlorpromazine was used at 500 to 5,000 nanomolar. But the therapeutic concentration of chlorpromazine is between 3 and 10 nanomolar, the toxic concentration is about 10 times higher, and the comatose-lethal concentration is about 100 times higher (Regenthal et al., 1999), all these concentrations being far lower than those used by Tischbirek et al.

All the concentrations mentioned here are in the spinal fluid or in the plasma water, where allowance has already been made for binding to plasma proteins.

Finally, it should be noted that the toxic concentrations used in vitro in this study are known to be membrane-lytic.

Considering the toxic and lethal concentrations used by Tischbirek et al., their proposed mechanism of antipsychotic drug action is not clinically relevant.


Regenthal R, Krueger M, Koeppel C, Preiss R. Drug levels: therapeutic and toxic serum/plasma concentrations of common drugs. J Clin Monit Comput . 1999 Dec ; 15(7-8):529-44. Abstract

View all comments by Philip Seeman

Primary Papers: Use-dependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs.

Comment by:  Herbert Meltzer (Disclosure)
Submitted 15 June 2012
Posted 15 June 2012

Round and Round They Go

Tischbirek and colleagues are to be congratulated on completing a complicated, elegant in-vitro as well as in-vivo microdialysis study of the accumulation of a variety of antipsychotic drugs in synaptic vesicles in hippocampal tissue, their release upon neuronal depolarization, and their re-accumulation in neuronal terminals, where they appear to inhibit the further release of vesicles, leading to what they call use-dependent auto-inhibitory effects on synaptic transmission. The latter is the least convincing part of the study because they did not compare the concentrations of drugs in vesicles or in dialysates over periods that might parallel clinical treatment periods in patients, for example, weeks and months. Follow-up longitudinal studies of treatment effects on the levels of drugs within and releasable from vesicles will be of interest.

Tischbirek et al. claim their results are consistent with the delayed onset of clinical response to antipsychotic drugs, while others, for example, Kane and Kapur, are pressing the view that clinical response, when it does occur, does so rapidly. I would agree with Tischbirek on the issue of the slower response rate. The suggestion that this mechanism may explain why ECT, which would be expected to lead to vesicular emptying, is effective, as augmentation to antipsychotic drug treatment is intriguing and imaginative. Some specific tests of this in rodents would be welcome.

To the extent that accumulation of drugs into synaptic vesicles and their release into the synapse, leading to higher local concentrations of drugs than has been the common expectation does occur, polypharmacy would have to lead to complex mixtures which might be highly variable over time, although some steady state might eventually be reached.

View all comments by Herbert Meltzer