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Vol. 294, Issue 2, 571-579, August 2000
Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, Piscataway, New Jersey
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Systemic administration of selective serotonin reuptake inhibitors (SSRIs) elicits larger increases in serotonin (5-HT) in raphe than in forebrain sites. Because serotonergic neuronal activity is suppressed, the mechanism underlying SSRI-induced increases in extracellular 5-HT is unclear. This study determined whether local infusion of SSRIs also elicited regionally selective increases in extracellular 5-HT, and whether changes depended on serotonergic neuronal depolarization. Conventional microdialysis methods were used to measure 5-HT in dorsal raphe (DRN), median raphe, nucleus accumbens (NAcc), and frontal cortex of unanesthetized rats. During infusion of SSRIs into each site, the maximum response was an ~6- to 7-fold increase in 5-HT in NAcc and frontal cortex, and an ~20-fold increase in DRN and median raphe. The larger increase in 5-HT in raphe was confirmed using zero-net-flux microdialysis. In NAcc, baseline 5-HT was 0.7 nM, and levels increased to a maximum of 3.1 nM during infusion of the SSRI citalopram. Baseline 5-HT in DRN was greater, 1.3 nM, and increased to 12.4 nM in response to citalopram. Consistent with evidence that autoreceptor activation inhibits serotonergic neuronal discharge, SSRI infusion into DRN produced a moderate decrease in 5-HT in NAcc. However, increases in 5-HT in DRN elicited by SSRI infusion were attenuated by 8-hydroxydipropylaminotetralin and tetrodotoxin. These data indicate that depolarization-dependent 5-HT release was not fully inhibited during SSRI infusion into DRN. In summary, SSRIs produce larger increases in extracellular 5-HT in raphe than in forebrain sites. Increases depend in part on depolarization-induced release, which may be greater in raphe than in forebrain.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Serotonin
(5-HT) in extracellular space is inactivated primarily by high-affinity
reuptake. Hence, drugs that block this process, such as the selective
serotonin reuptake inhibitors (SSRIs), can produce increases in
extracellular 5-HT (Invernizzi et al., 1992
; Perry and Fuller, 1992)
and have been used in treatment of depression (Blier and de Montigny,
1994). However, the efficacy of SSRIs is limited by an autoinhibitory
mechanism. Serotonergic neuronal discharge (Chaput et al., 1986;
Gartside et al., 1995) and 5-HT release in forebrain sites (Rutter et
al., 1995) is inhibited after systemic administration of SSRIs. This is
attributable in part to elevation of extracellular 5-HT in the raphe
and the consequent activation of somatodendritic autoreceptors (Adell
and Artigas, 1991; Invernizzi et al., 1992). Nerve terminal
autoreceptors also contribute to inhibition of 5-HT release and
restrain the increase in extracellular levels after SSRI administration
(Hjorth, 1993). The clinical efficacy of SSRIs may depend in part on
autoreceptor desensitization during prolonged inhibition of reuptake
(Blier and de Montigny, 1994). Thus, the interaction between reuptake and autoreceptors in regulation of serotonergic neurotransmission has
been studied intensively.
Reuptake inhibitors may have a differential effect on extracellular
5-HT in the raphe and forebrain (for review, see Gardier et al., 1996).
For example, at a dose just sufficient for suppressing serotonergic
neuronal activity, the SSRI paroxetine elicited about a 2-fold increase
in extracellular 5-HT in the raphe, with no change in extracellular
levels in the frontal cortex (FCx) of rats (Gartside et al., 1995).
Paradoxically, at a dose supramaximal for suppressing neuronal
discharge, paroxetine produced about a 4-fold increase in extracellular
5-HT in the raphe and 2-fold increase in the FCx. Thus, despite
sustained and apparently complete inhibition of serotonergic neuronal
discharge, synaptic 5-HT in the forebrain may be enhanced in response
to acute administration of an SSRI (Gartside et al., 1995). This might
indicate that increases in extracellular 5-HT produced by SSRIs do not
depend on depolarization-induced release. However, tetrodotoxin (TTX)
and autoreceptor agonists can attenuate increases in forebrain 5-HT
elicited by systemic administration of reuptake inhibitors (Carboni and
DiChiari, 1989; Perry and Fuller, 1992; Rutter and Auerbach, 1993).
Furthermore, the possibility that SSRI-elicited increases in 5-HT in
the raphe may be sustained by depolarization-independent release has
not been directly tested. Thus, the mechanism underlying the ability of
SSRIs to preferentially increase 5-HT in the raphe is unclear.
The major aims of this study were to compare the effect of local SSRI
infusion into the raphe and forebrain and to directly test the
hypothesis that SSRI-induced increases in extracellular 5-HT do not
depend on depolarization-induced release. For this purpose, we used
both conventional in vivo microdialysis to measure the percentage of
change in extracellular 5-HT elicited by SSRIs and zero-net-flux
microdialysis for determining actual concentrations of 5-HT. Changes in
5-HT were measured in response to reverse dialysis infusion of SSRIs
into the dorsal raphe nucleus (DRN), median raphe nucleus (MRN),
nucleus accumbens (NAcc), and FCx of unanesthetized rats. The DRN and
MRN contain almost all of the serotonergic cell bodies with projections
to the forebrain. The FCx is of particular interest because 5-HT
release is more tightly regulated here than in other forebrain sites
(for review, see Gardier et al., 1996). The NAcc is preferentially
innervated by DRN serotonergic neurons (Azmitia and Segal, 1978). Thus,
the role of somatodendritic autoreceptors in regulation of forebrain release could be characterized by infusion of citalopram into DRN while
measuring 5-HT in the NAcc. Dependence of 5-HT release on
depolarization was further tested by administration of the 5-HT1A receptor agonist
8-hydroxydipropylaminotetralin (8-OH-DPAT) to stimulate somatodendritic
autoreceptors and TTX to block action potential conduction. The results
provide evidence of differential regulation of extracellular 5-HT in
the raphe compared with forebrain sites. However, even when reuptake
was maximally inhibited, increases in extracellular 5-HT remained
partly dependent on depolarization-induced release.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Animal Preparation. Male Sprague-Dawley rats purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN) were individually housed with food and water available ad libitum. The animals were kept at least 2 weeks on a reversed light/dark cycle (lights off from 9:30 AM to 9:30 PM) and were briefly handled three to four times a week.
All animal-use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Rutgers University Institutional Review Board. Rats weighing 300 to 350 g were anesthetized with a combination of xylazine (4 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.) and then mounted in a Kopf stereotaxic frame in the flat skull position. Guide cannulas (22-gauge stainless steel tubing) were implanted above the dura (0.9 mm ventral to the skull surface). According to a rat brain atlas (Paxinos and Watson, 1986), the coordinates for the guide cannulas relative to
interaural zero were DRN, AP 1.2, ML 4.0, at a 32° angle
lateral to midline; MRN, AP 1.2, ML 4.0, at an angle of 26° lateral
to midline; NAcc, AP 10.7, ML 1.4; and FCx, AP 12.2, ML 3.2. Rats were
allowed at least 1 week for recovery from surgery.
Microdialysis Procedures.
Microdialysis was performed with
an I-shaped probe constructed from 26-gauge stainless steel tubing and
glass silica as previously described in detail (Auerbach et al., 1989).
The dialysis tubing was hollow nitrocellulose fiber (0.2 mm o.d., 6000 mol. wt. cut-off; Spectrum Medical Industries, Los Angeles, CA). The
length of the steel shaft was adjusted to place a 1.0-mm-long segment
of dialysis tubing in the DRN (DV 5.5-6.4, 32° angle) or MRN
(DV 7.7-8.6, 26° angle). Similarly, the length of the probe was
adjusted to place 2.5-mm-long segments of dialysis tubing in the NAcc
(DV 6.0-8.5) or FCx (DV 2.0-4.5).
). The HPLC-EC mobile phase composition
was 0.12 M NaOH, 0.18 mM EDTA, 0.15 M monochloroacetic acid, 1.0 mM
sodium octane sulfonic acid, and 56 ml/l acetonitrile, pH 3.4, and was
pumped at a rate of 0.90 ml/min. Detection limit of the assay was 0.3 pg per sample.
Experimental Protocol.
Drugs were administered to rats after
5-HT levels in three or four successive samples were stable (less than
±10% fluctuation of baseline. In some experiments, to study the
effect of somatodendritic autoreceptor activation on 5-HT release in
the forebrain, 8-OH-DPAT or citalopram was administered by reverse
dialysis in the DRN while 5-HT was measured by a second dialysis probe
in the NAcc. Other experiments involved drug administration by reverse
dialysis and measurement of 5-HT in the same site. These conventional
microdialysis experiments provided data concerning changes relative to
baseline 5-HT during local infusion of an SSRI. In addition, we used
the zero-net-flux method to estimate actual concentrations of 5-HT in
extracellular space. Details of this method have been described previously by others (Lönnroth et al., 1987; Justice, 1993). In
brief, 5-HT was added to the aCSF at concentrations above and below the
expected concentration in extracellular space (0-30 nM), and 5-HT was
measured in the aCSF effluent from the brain. To minimize loss of 5-HT
due to decomposition across time at neutral pH, paired samples were
collected simultaneously from the inlet and outlet lines and analyzed
within 30 min.
Data Analysis.
Data from the zero-net-flux experiments were
plotted as gain or loss of 5-HT in the dialysis probe effluent against
the concentration of exogenous 5-HT infused through the probe inlet.
Linear regression was used to interpolate the point of no net flux.
This provides an estimate of the concentration of 5-HT in extracellular
space (Lönnroth et al., 1987). For all other experiments, the
mean of three or four successive samples before drug administration was
taken as the baseline level and reported in the figure legends as
picograms per sample, uncorrected for probe recovery. Also, the data
were normalized and presented in figures as mean ± S.E. percentage of change from the averaged baseline measurements. Significance (P < .05) was determined using
repeated-measures ANOVA followed by Scheffé's test.
Materials.
All chemicals were reagent grade or better.
Citalopram hydrobromide was provided courtesy of Lundbeck A/S
(Copenhagen-Valby). Fluoxetine was a gift of Lilly Research
Laboratories (Indianapolis, IN) and (
)-penbutolol was generously
provided by Hoechst-Roussel (Somerville, NJ). (±)-8-OH-DPAT
hydrobromide was purchased from Research Biochemicals International
(Natick, MA), and TTX was obtained from Calbiochem-Novabiochem Corp.
(La Jolla, CA). Drugs were dissolved in aCSF to produce specific
concentrations for local infusion by reverse microdialysis. For
systemic administration, 8-OH-DPAT was dissolved in water and injected
in a volume of 1 ml/kg. The systemic dose of 8-OH-DPAT refers to the
salt form.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Microdialysis probes with a 1-mm-long exchange surface were implanted in the DRN and MRN. Probes with a 2.5-mm-long exchange surface were implanted in the NAcc and FCx. After overnight perfusion with aCSF containing no SSRI, baseline 5-HT levels in these sites were low but usually above the detection limit of our HPLC-EC assay. Averaged across experiments, basal 5-HT (pg/sample; uncorrected for probe recovery) in the absence of reuptake inhibitor was 0.7 ± 0.1 in the DRN (n = 61), 0.8 ± 0.1 in the MRN (n = 34), 0.9 ± 0.1 in the NAcc (n = 45), and 0.6 ± 0.1 in the FCx (n = 14).
Effect of Local SSRI Infusion on 5-HT in the Raphe and Forebrain
Sites.
SSRIs were infused locally by reverse dialysis into the
area of serotonergic cell bodies in the DRN and MRN and into two
forebrain sites, NAcc or FCx. As shown in Fig.
1, SSRI infusion produced larger
increases in the raphe than in forebrain sites. Reverse dialysis
infusion of citalopram (0.3-1000 µM in aCSF) or fluoxetine (10-1000
µM in aCSF) into the DRN produced similar dose-dependent increases in
5-HT in the DRN (Fig. 1, a and b). The maximum effect was an ~20-fold
increase. The effect of infusing citalopram into the MRN is shown in
Fig. 1c. Similar to the DRN, 5-HT was dose dependently elevated with a
maximal increase of nearly 20-fold.
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)-penbutolol into the NAcc to block terminal 5-HT autoreceptors (Hjorth and Sharp, 1993).
As shown in Fig. 3, ()-penbutolol (300 µM in aCSF) alone did not produce a significant increase in 5-HT in
the NAcc. However, ()-penbutolol significantly enhanced the effect of
infusing citalopram (1000 µM) into the NAcc on 5-HT in this site.
Thus, 5-HT in the NAcc was increased ~6-fold with citalopram alone,
significantly less (P < .05) than the ~12-fold
increase with ()-penbutolol pretreatment. Presumably, ()-penbutolol
infusion at a concentration of 300 µM produced a maximal blockade of
terminal autoreceptors. This is based on evidence that the effect of
SSRI infusion on extracellular 5-HT was similarly enhanced using a
30-fold lower concentration of ()-penbutolol (Rutter et al., 1995).
Nevertheless, despite terminal autoreceptor blockade, infusing
citalopram into the forebrain had a significantly smaller effect than
in the raphe [F(1,12) = 7.764, P = .0165].
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Zero-Net-Flux Method for Estimating the Concentration of 5-HT in
Extracellular Fluid.
Conventional microdialysis provided evidence
of differences in the effect of infusing uptake blockers into the raphe
compared with forebrain. However, differences in dialysis probe
recovery of neurotransmitter in these sites may have been a factor in
the apparently larger effect of blocking 5-HT reuptake in the raphe. To
examine this possibility, we used zero-net-flux microdialysis to
estimate the concentration of 5-HT in extracellular space. Varying
concentrations of 5-HT were added to the aCSF, and the amount of 5-HT
gained or lost during dialysis in the DRN or NAcc was measured. Net
gain or loss of 5-HT in the aCSF effluent is plotted against the
concentration of exogenous 5-HT added to the aCSF (Fig.
4). The interpolated point of no net flux
is an estimate of the actual concentration of 5-HT in extracellular
fluid that does not depend on probe length or other factors that can
affect in vivo recovery of neurotransmitters (Justice, 1993). We
estimated basal 5-HT and the increase in response to 1 and 300 µM
citalopram. These concentrations were chosen because they produced,
respectively, submaximal and maximal increases in extracellular 5-HT as
determined by conventional microdialysis (see Fig. 1).
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Influence of Direct and Indirect Stimulation of Somatodendritic
Autoreceptors in the DRN on 5-HT Release in the NAcc.
By eliciting
increased extracellular 5-HT, reuptake inhibitors indirectly activate
somatodendritic autoreceptors and thus inhibit serotonergic neuronal
discharge (Sheard et al., 1972). Hence, we did not anticipate the very
large increases in extracellular 5-HT during infusion of citalopram and
fluoxetine into the raphe. To evaluate the influence of somatodendritic
autoreceptors on 5-HT release, we infused 8-OH-DPAT, a
5-HT1A receptor agonist (Sharp et al., 1989), or
citalopram into the DRN while measuring extracellular 5-HT with a
second dialysis probe in the NAcc. As shown in Fig.
5a, infusion of 8-OH-DPAT (100 µM in
aCSF) produced a significant, ~40%, decrease in 5-HT in the NAcc.
Similarly, infusion of citalopram (1-1000 µM in aCSF) into the DRN
caused a dose-dependent decrease in extracellular 5-HT in the NAcc
(Fig. 5b). Figure 5b (inset) shows the dose-response curve for the
inhibitory effect of citalopram infusion. There was no significant
effect at the two lowest doses, 1 and 10 µM citalopram. However, 100, 300, and 1000 µM citalopram produced significant reductions of ~15,
~20, and ~30%, respectively. As calculated from these data, the
EC50 value for the inhibitory effect of
citalopram in the DRN on 5-HT in the NAcc was ~100 µM in the
perfusion medium. Presumably, citalopram infusion into the DRN
indirectly activates somatodendritic autoreceptors, but decreases in
extracellular 5-HT in the NAcc were significant only at relatively high
perfusate concentrations.
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Interaction between Citalopram and 8-OH-DPAT in the DRN: To What
Extent Are Somatodendritic Autoreceptors Activated by Local Infusion of
an SSRI into the Raphe?
Citalopram infusion into the DRN produced
relatively small decreases in 5-HT in the NAcc. Thus, it is possible
that 5-HT1A receptors in the DRN were not maximally
activated by endogenous 5-HT even during local infusion of high
concentrations of citalopram. In contrast, the direct acting
5-HT1A receptor agonist 8-OH-DPAT might produce a more
complete suppression of serotonergic neuronal activity. To test these
inferences, we measured the effect of citalopram followed by 8-OH-DPAT
infusion on 5-HT in the DRN. Figure 6
shows that, in the presence of 1 µM citalopram, 8-OH-DPAT decreased
extracellular 5-HT. At concentrations of 10 and 100 µM in the aCSF,
8-OH-DPAT infusion into the DRN produced similar, ~40%, reductions
in 5-HT in DRN (Fig. 6a). As shown in Fig. 6b, during infusion of 1000 µM citalopram into the DRN, higher doses of 8-OH-DPAT were necessary
for reducing 5-HT in the DRN. With the SSRI present at a high
concentration in the DRN, the maximal reduction induced by 100 and 1000 µM 8-OH-DPAT was ~25 and ~40%, respectively. Thus, the potency
but not the efficacy of 8-OH-DPAT infused into the DRN was attenuated
during infusion of citalopram at a high concentration.
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Interaction between Citalopram and TTX in DRN and NAcc: Is
Depolarization-Dependent Release Necessary for SSRI-Elicited Increases
in Extracellular 5-HT?
Even during citalopram infusion at a high
concentration, 8-OH-DPAT produced a decrease in extracellular 5-HT.
Presumably, 8-OH-DPAT, via direct stimulation of somatodendritic
autoreceptors, inhibited serotonergic neuronal activity. This result
provides evidence that citalopram alone did not completely inhibit
serotonergic neuronal activity and that the SSRI-induced increases in
5-HT were dependent at least in part on depolarization-induced release. To further test this inference, we infused TTX to block the generation and conduction of action potentials. Two sets of experiments were carried out. In the first, citalopram (1 or 1000 µM in aCSF) was infused into the DRN followed by TTX (1 µM in combination with citalopram). As shown in Fig. 7a, during
infusion of 1 µM citalopram, TTX elicited an ~60% reduction in
5-HT in the DRN, and during infusion of 1000 µM citalopram, TTX
elicited an ~40% reduction. Although the maximal effect of TTX was
apparently smaller during infusion of 1000 than of 1 µM citalopram,
this difference was not significant (P > .05). In
the second set of experiments, citalopram (1 or 1000 µM) and TTX were
infused into the NAcc. As shown in Fig. 7b, TTX infused into the NAcc
elicited similar, ~60%, maximal reductions in 5-HT in NAcc during
infusion of 1 and 1000 µM citalopram.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Consistent with the effect of systemic administration of reuptake
inhibitors (Adell and Artigas, 1991; Invernizzi et al., 1992), local
SSRI infusion produced greater increases in extracellular 5-HT in the
raphe than in forebrain. Thus, citalopram infusion produced a 3-fold
greater increase relative to baseline levels in the raphe compared with
forebrain (Fig. 1). Also, we determined absolute 5-HT concentrations
using zero-net-flux microdialysis, and these results support the
conclusion that reuptake inhibition has an ~3-fold greater effect on
5-HT in raphe (Fig. 4). Moreover, the predrug concentration of 5-HT in
DRN, ~1.3 nM, was about twice the level in NAcc at ~0.7 nM. Our
estimate of basal 5-HT in NAcc is consistent with the concentration
determined using a similar approach (Smith and Weiss, 1999). However,
as far as we are aware, there are no previous studies comparing
absolute concentrations of 5-HT in raphe with forebrain.
The EC50 value for SSRI-elicited increases in
extracellular 5-HT in raphe was high compared with forebrain (Fig. 2).
A semi-independent measure, the concentration of citalopram in the DRN
necessary for inhibition of 5-HT release in the NAcc, provides a
similar estimate for SSRI affinity to transporters in the raphe (Fig. 5b). Because the dialysis membrane prevents free diffusion, drug concentrations in extracellular fluid are low compared with amounts added to the dialysis solution (Dykstra et al., 1992). Nevertheless, the very high concentration of citalopram necessary for producing a
maximal increase in 5-HT in DRN and inhibition of 5-HT release in
forebrain was unexpected. We obtained similar results with citalopram
in MRN and fluoxetine in DRN, suggesting that this is a robust finding.
Several factors may be involved in the high EC50
values in the raphe. 1) Compared with forebrain, the higher extracellular concentration of 5-HT in raphe would provide more competition for SSRI binding to the 5-HT reuptake transporter (SERT).
Furthermore, the density of SERT in raphe is high compared with
forebrain (Hensler et al., 1994). Thus, binding to the transporter may
effectively lower the concentration of SSRIs in the raphe. Both of
these factors might cause an increase in the apparent affinity of an
SSRI. 2) Another possibility is that the SSRIs at high concentrations
in the raphe acted as 5-HT releasers. However, increases in
extracellular 5-HT produced by releasing drugs are unaffected by
inhibitors of serotonergic neuronal discharge and nerve terminal
depolarization (Carboni and DiChiara, 1989; Gundlah et al., 1997).
Thus, the decrease in 5-HT during infusion of 8-OH-DPAT or TTX (Figs. 6
and 7) suggests that the SSRIs were not acting as 5-HT-releasing
agents. 3) SSRIs have measurable, albeit remarkably low, affinity for
some neurotransmitter receptors (for review, see Sanchez and Hyttel,
1999). Thus, it is conceivable that citalopram and fluoxetine at high
enough doses bind to, for example,
1-adrenergic receptors in the raphe and thus
might stimulate 5-HT release. Finally, the high
EC50 value and biphasic response to SSRIs infused into the DRN and MRN may indicate that there is a low-affinity, high-capacity mechanism for 5-HT uptake in the raphe. For example, multiple mRNA species resulting from alternative splicing of the SERT
gene have been detected (Bengel et al., 1997). Thus, there could be
heterogeneous SERT proteins with different affinities for 5-HT. Indeed,
some evidence from in vitro binding studies supports the presence of
low- and high-affinity binding sites on 5-HT transporter proteins (Sur
et al., 1998), and there may be regional differences in the
distribution of the two sites (Rothman et al., 1994). However, in vitro
experiments to directly test for low-affinity uptake of 5-HT would be
difficult because of the small amount of tissue obtainable from rat raphe.
The balance between the rates of release and clearance determines
steady-state extracellular neurotransmitter levels. If a low-affinity
form of SERT regulated 5-HT clearance in the raphe, this could be one
explanation for higher basal levels in DRN compared with NAcc. However,
assuming that SERT is the predominant mechanism for clearance, greater
release may be the major factor in higher 5-HT in raphe compared with
forebrain when reuptake was fully blocked by SSRIs. Nerve terminal
autoreceptors might inhibit forebrain 5-HT release during local SSRI
infusion. However, even during infusion of ()-penbutolol to block
terminal autoreceptors, the increase in 5-HT in forebrain during
citalopram infusion was still less than in raphe (Fig. 3). These
observations are consistent with other evidence (Héry and
Ternaux, 1981) that 5-HT release is greater in raphe than in forebrain.
Increased Extracellular 5-HT Elicited by SSRIs Is Dependent in Part
on Depolarization.
SSRIs produce sustained increases in
extracellular 5-HT at doses that suppress serotonergic neuronal
discharge (Gartside et al., 1995). This suggests that SSRI-induced
increases in extracellular 5-HT are mainly independent of
depolarization-induced release. However, the direct somatodendritic
autoreceptor agonist 8-OH-DPAT produced decreases in 5-HT during
citalopram infusion into the DRN (Figs. 5 and 6). This agrees with
reports that 8-OH-DPAT attenuated the increase in 5-HT evoked by
systemic SSRI administration (Rutter and Auerbach, 1993). TTX infusion
also attenuated the increases in extracellular 5-HT produced by
citalopram infusion into the raphe or NAcc (Fig. 7). This is consistent
with reports that TTX reversed the effect of systemic administration of
reuptake inhibitors (Kalén et al., 1988; Carboni and DiChiara,
1989; Perry and Fuller, 1992). Thus, microdialysis results indicate
that neuronal impulse activity is partly responsible for increased
extracellular 5-HT elicited by reuptake inhibitors. With respect to
this hypothesis, it is important to note that in contrast to the
inhibitory effects of direct autoreceptor agonists such as 8-OH-DPAT,
the decrease in serotonergic neuronal activity in response to SSRIs
depends on the evoked increase in extracellular 5-HT. Moreover, because the elevation in extracellular 5-HT remains dependent on
depolarization, our results suggest that the inhibitory effect of SSRI
administration is self-limiting and in principle should not produce a
total suppression of serotonergic discharge.
;
Perry and Fuller 1992), these results suggest that SSRI-induced
increases in 5-HT are partly independent of impulse-induced release. A
possible source of the depolarization-independent fraction of 5-HT in
extracellular fluid is spontaneous vesicular release of
neurotransmitter, a physiological process that has been demonstrated,
for example, in recordings of miniature end-plate potentials at the
neuromuscular junction. When depolarization is inhibited, the
releasable pool of 5-HT is increased (Auerbach and Lipton, 1985). Thus,
during SSRI infusion into the raphe and, consequently,
autoreceptor-mediated attenuation of depolarization-induced exocytosis,
there could be an increase in the amount of spontaneously released
5-HT.
Other Methodological Considerations and Significance.
Maximal
increases in 5-HT in raphe and forebrain were greater during SSRI
infusion than those observed after systemic administration. Systemic
SSRIs produce 2- to 4-fold increases in 5-HT (for review, see Fuller,
1994). In contrast, local infusion induced an ~6-fold increase in
forebrain and an ~20-fold increase in midbrain levels (Fig. 1).
Presumably, limited autoreceptor activation during local infusion is
the explanation for this difference. Thus, during local SSRI infusion,
increased 5-HT is confined to a small area around the microdialysis
probe. In contrast, cell bodies that release 5-HT in the area of the
probe may be located far away. Furthermore, some evidence suggests that
part of the inhibitory effect of systemic SSRI administration is
mediated by long-loop feedback (Ceci et al., 1994; Bosker et al., 1997;
Hajós et al., 1998). Consistent with these inferences, local
infusion of 8-OH-DPAT or citalopram into the DRN produced relatively
small decreases in NAcc 5-HT (Fig. 5). In summary, the inhibitory
effect of local SSRI or 8-OH-DPAT infusion may be limited because this
route of administration would not cause widespread activation of
somatodendritic autoreceptors or activate long-loop feedback inhibition
of serotonergic neuronal discharge.
). Furthermore,
because the dialysis probe membrane hinders diffusion of substances
into the dialysate, 5-HT recovery is only a fraction of the amount in
extracellular space. Thus, baseline levels may be near the sensitivity
limit of detection methods. To increase recovery, a reuptake inhibitor
is often added to the dialysis perfusion medium. For example, SSRI
infusion was necessary for detecting changes in forebrain 5-HT during
electrical stimulation in raphe (Brodin et al., 1990; Sharp et al.,
1990). Our results suggest that at low concentrations of SSRI in the
dialysate, 5-HT release remains predominantly dependent on
depolarization. However, it is important to carry out appropriate
control experiments and to be cautious in interpretation of
microdialysis data when a reuptake inhibitor is used to enhance
neurotransmitter recovery (for further discussion, see DeBoer and
Abercrombie, 1996).
In summary, our results provide evidence that 5-HT release capacity is
greater and suggest that SERT affinity is lower in the raphe compared
with forebrain projection sites. Thus, physiological activation of
serotonergic neuronal discharge would result in relatively large
increases in extracellular 5-HT in the raphe, stimulation of
somatodendritic autoreceptors, and consequently, restrained increases
in extracellular 5-HT in the forebrain. These inferences are in line
with evidence that serotonergic neuronal discharge and 5-HT release in
forebrain are confined to a remarkably narrow range even during periods
of strong behavioral activation in response to physiological challenges
(for review, see Rueter et al., 1997). However, our results do not
support the hypothesis that reuptake blocker-induced increases in
forebrain extracellular 5-HT are completely independent of neuronal
activity. Increases in 5-HT in the forebrain were smaller than in the
raphe, but they remained largely dependent on depolarization during
SSRI treatment.
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Acknowledgments |
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We are grateful to Lundbeck, Eli Lilly, and Hoechst-Roussel for generous gifts of drugs.
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Footnotes |
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Accepted for publication April 17, 2000.
Received for publication January 7, 2000.
1
This work was supported by U.S. Public Health Service
Grant MH51080. Some of these results were presented in preliminary form to the Society for Neuroscience (Tao et al., 1997).
Send reprint requests to: Sidney B. Auerbach, Department of Cell Biology and Neuroscience, Rutgers University Nelson Biological Laboratories, 604 Allison Rd., Piscataway, NJ 08854-8082. E-mail: auerbach{at}biology.rutgers.edu
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Abbreviations |
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5-HT, 5-hydroxytryptamine, serotonin; SSRI, selective serotonin reuptake inhibitor; SERT, 5-HT reuptake transporter; TTX, tetrodotoxin; 8-OH-DPAT, 8-hydroxydipropylaminotetralin; DRN, dorsal raphe nucleus; MRN, median raphe nucleus; NAcc, nucleus accumbens; FCx, frontal cortex; aCSF, artificial cerebrospinal fluid; HPLC-EC, HPLC with electrochemical detection.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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an in vivo brain microdialysis study.
Naunyn-Schmiedeberg's Arch Pharmacol
343:
237-244[Medline].a microdialysis study.
J Neurochem
60:
776-779[Medline].)penbutolol.
J Pharmacol Exp Ther
265:
707-712sensitivity to tetrodotoxin, calcium and calcium antagonists.
Neuroscience
39:
629-637[Medline].This article has been cited by other articles:
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, DRN
citalopram; 
, DRN fluoxetine; 
, MRN citalopram; 
, NAcc
citalopram.
, (
