An FSCV Exploration of Serotonin`s In Vivo Mechanisms

An FSCV Exploration of Serotonin’s In Vivo Mechanisms
Lama, R.1, Wood, K.1, Takmakov, P.2, Wightman, R. M.2, Hashemi, P.*1
1
Department of Chemistry, Wayne State University, Cass Avenue, Detroit, MI
2
Department of Chemistry, UNC at Chapel Hill, Chapel Hill, NC
*[email protected]
Introduction
Serotonin is a neuromodulator that has gained notoriety in recent times due its involvement in
mood disorders. This is primarily because mood stabilizing therapeutic agents, such as
antidepressants, commonly target the serotonergic system. There is great interest surrounding
serotonin, its receptors, its transporter and its interaction with other molecules such as dopamine,
however, its precise mechanisms as a neuromodulators remain a matter of dispute.
We have recently described a fast-scan cyclic voltammetry (FSCV) technique that can measure
serotonin release and uptake in anesthetized rodents [1]. We now apply this method to some
fundamental aspects of serotonin transmission. Firstly, we pharmacologically compare serotonin
and dopamine synthesis, packaging, release, reuptake and metabolism. We find fundamental
differences in the regulation of dopamine and serotonin neurotransmission. Notably while
dopamine transmission is a synthesis/packaging driven system, serotonin transmission is an
uptake/metabolism driven system. This is interesting since the serotonin transporter (SERT) is
the target of the most common antidepressants. We next compare the effects of two common
antidepressants, fluoxetine and escitalopram, commercially known as prozac and lexapro,
respectively. Escitalopram is known to be one of most clinically efficacious antidepressants with
minimal side-effects while fluoxetine is known to be less efficacious with more prevalent sideeffects. We find clear differences in their actions on serotonin release and uptake in vivo.
Specifically escitalopram induces large increases in the magnitude of stimulated serotonin
release while fluoxetine’s effects are primarily on serotonin reuptake. These in vivo studies will be
a back-bone on which to base a fundamental understanding of serotonin’s chemical actions in the
synapse. It is already clear that mechanisms at this synaptic level correspond to clinical
behavioral findings.
Methods
Sprague Dawley rats (male, 8-12 weeks, 250-350g) and C57BL6J mice (male, 4-7 weeks, 2025g) were purchased from Charles River Labs (Raleigh, NC, USA) and The Jackson Laboratory
(Bar Harbor, Maine, USA). The Institutional Animal Care and Use Committees of Wayne State
University approved the protocols for animal care, handling and experimentation. Rats and mice
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were anesthetized with urethane (15 g kg rat weight and 1.5 g kg mouse weight). A stereotaxic
frame (David Kopf Instruments, Tujunga, CA, USA) was utilized for all brain surgeries. On rats,
surgeries were performed as previously described [2], on mice, the surgeries were slightly
modified to accommodate the smaller mouse brain. In rats, Nafion-coated carbon fiber
microelectrodes were implanted into the substantia nigra, pars reticulata (SNr) and the nucleus
accumbens core (NAc) while a bi-polar stainless steel stimulating electrode (0.2 mm diameter,
Plastics One, Roanoke, VA, USA) was implanted into the medial forebrain bundle (MFB) as
previously described [2]. In the mouse, the SNR coordinates used were AP -3.2 to -3.4 mm; ML
+1.5 mm; DV -4.5 to -4.7 mm) and the MFB coordinates were AP -1.2 mm; ML +1.0 mm; DV -5.0
mm). The rat and mouse brain atlases were used for reference from Bregma [3, 4]
Electrode construction and modification has previously been described [1]. Concisely, a glass
capillary (A-M Systems, Inc., Sequim, WA) was filled with a carbon fiber (T-650, Thornel, Amoco
Co.). A seal between the carbon fiber and the glass was established with a micropipette puller
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(Narishige, Tokyo, Japan). The exposed length of fiber was 100 -150 µm in length. Nafion
modification was as previously described [1]. Readings were referenced to Ag/Ag Cl. Waveform
generation and data acquisitions were performed by UEI potentiostats and quad potentiostats
(University of North Carolina Department of Chemistry Electronics Shop). Serotonin and
dopamine specific waveforms were used at previously described [2].
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Drugs were from Sigma-Aldrich (St. Louis, MO, USA). Fluoxetine hydrochloride (10mg kg ) and
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escitalopram oxalate (10mg kg ) were dissolved in sterile saline and injected in the peritoneum
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as previously described for rats [2] and for mice, at a volume of 0.3 ml kg .
Results and Discussion
We found that a common stimulation of the MFB elicits dopamine in the NAc and serotonin in the
SNr, however, the magnitude of dopamine release is three hundred fold greater than the
magnitude of serotonin release. We found that this was not a consequence of the axon’s reaction
to the stimulation (by varying stimulation parameters), nor a consequence of limited serotonin
stores (it is not possible to deplete serotonin in the time frame of our experiments).
Pharmacologically, we found that dopamine transmission was highly disrupted by synthesis and
packaging inhibition while serotonin transmission remained relatively unchanged, indicating an
abundance of intra-cellular serotonin stores. Conversely, serotonin transmission was highly
sensitive to disruption of reuptake and metabolism while dopamine transmission is less sensitive
to reuptake inhibition and almost unchanged by metabolism inhibition. These data draw important
distinctions between dopamine and serotonin transmission. Mainly that serotonin inactivation is a
critical part of its in vivo mechanisms. On this note, we explored different selective serotonin
reuptake inhibitors, SSRIs.
Our data indicates that there are important differences in the mechanisms that common SSRIs
exert on the serotonin system. For example, in mice we observe large increases in the amplitude
of serotonin release after escitalopram administration, while fluoxetine does not significantly
increase serotonin stimulated release amplitude. The kinetics of action of these two SSRIs are
significantly different. It takes 7 minutes for escitalopram to exert its maximal effect while it takes
around 15 minutes for the maximal effects of fluoxetine. Because of the difference in the t1/2 of the
two agents, they are cleared from the mouse system, as evidenced by the cessation of their
effects on the FSCV signal, at different rates. For escitalopram, this is around 60 minutes and for
fluoxetine, it is 90-120 minutes.
This study highlights important differences between the serotonergic and dopamine transmission
systems, shedding light on the importance of serotonin inactivation in its in vivo mechanisms.
Furthermore, serotonin inactivation through SERTs is modulated differently by antidepressants
with different clinical behavioral effects. FSCV studies of serotonin therefore are an important
foundation for linking in vivo mechanisms to behavioral mechanisms.
References
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Hashemi, P., et al., Voltammetric detection of 5-hydroxytryptamine release in the rat
brain. Anal Chem, 2009. 81(22): p. 9462-71.
Hashemi, P., et al., Brain Dopamine and Serotonin Differ in Regulation and its
Consequences. Proc Natl Acad Sci U S A, 2012. In Press.
Franklin, K., Paxinos, G. , The Mouse Brain in Stereotaxic Coordinates 2007: Academic
Publisher. 360.
Paxinos, G. and C. Watson, The Rat Brain in Stereotaxic Coordinates. 6th ed. 2007:
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pharmacokinetic/pharmacodynamic models of 5-hydroxytryptamine reuptake inhibitors in
mice. Br J Pharmacol, 2008. 155(2): p. 276-84.
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