Introduction

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Alterations in the 5-hydroxytryptamine (serotonin) (5-HT) system are associated with the pathology of a variety of mood disorders (Maes and Meltzer, 1995), including depression and anxiety. Many drugs used to treat psychiatric disorders, such as selective serotonin reuptake inhibitors (SSRIs), alter levels of extracellular 5-HT. SSRIs produce their behavioral and clinical therapeutic effects by blocking the reuptake of 5-HT at the 5-HT transporter, thereby increasing extracellular levels of 5-HT in limbic regions mediating affective behavior (Wong et al., 1995; Delgado et al., 1999). The substantial serotonergic innervation of the forebrain is derived directly from two distinct midbrain regions: the dorsal raphe nucleus (DR) and the median raphe nucleus (MR). The DR innervates brain regions such as the striatum, frontal cortex, and lateral septum, whereas the MR innervates brain regions such as the dorsal and ventral hippocampus, the hypothalamus, and the medial septum (Vertes, 1991; Vertes et al., 1999).

5-HT transmission in the brain and the effects of drugs that alter levels of 5-HT are critically regulated by 5-HT autoreceptors (Fuller, 1994). These receptors on 5-HT neurons are sensitive to the endogenous neurotransmitter and supply negative feedback by inhibiting 5-HT release, synthesis, or discharge. The role of 5-HT autoreceptors is to confine increases in extracellular levels of 5-HT produced by SSRIs or other physiological stimuli within a limited range (Bel and Artigas, 1992). Two types of 5-HT autoreceptors have been identified with different neuronal locations (for review, see Hen, 1992). 5-HT_1A autoreceptors are located on serotonergic cell bodies and dendrites, such as those located in the dorsal and median raphe (Weissmann-Nanopoulos et al., 1985), whereas 5-HT_1B autoreceptors are located on axon terminals in brain regions, such as the cerebellum, basal ganglia, and hippocampus (Boschert et al., 1994). Activation of 5-HT_1A and 5-HT_1B receptors results in corresponding decreases in the release of 5-HT from forebrain terminals (Hjorth and Sharp, 1991; Kreiss and Lucki, 1994; Knobelman et al., 2000). 5-HT autoreceptors are especially important in the clinical effects of SSRIs because the "lag time" between the onset of drug treatment and therapeutic efficacy may be associated with the progressive desensitization of one or more 5-HT autoreceptors (Blier and de Montigny, 1994). Blockade of 5-HT autoreceptors in combination with acute administration of an SSRI augments the increase in extracellular 5-HT induced by the SSRIs (Pineyro and Blier, 1999). This physiological effect provides the basis for suggesting that the therapeutic effects of SSRIs can be augmented by coadministration of 5-HT autoreceptor antagonists such as pindolol (Artigas et al., 1996).

The individual contributions of 5-HT_1A or 5-HT_1B autoreceptors to the overall regulation of 5-HT release in discrete brain regions is under current investigation. Some evidence from pharmacological studies in rats and guinea pigs suggests that important regional differences in the effects of SSRIs may be related to the neural origin of the 5-HT afferents (for review, see Hjorth et al., 2000). 5-HT_1A autoreceptors in the DR and MR differ in their regulation of extracellular 5-HT in different brain regions (Kreiss and Lucki, 1994, 1997). Furthermore, augmentation of the effects of SSRIs by the coadministration of selective 5-HT_1A or 5-HT_1B/1D autoreceptor antagonists have been shown to vary between brain regions (Invernizzi et al., 1997; Sharp et al., 1997; Hervas and Artigas, 1998). Regional differences in the regulation of 5-HT release may also underlie the consequences of genetic mutation involving 5-HT_1A and 5-HT_1B receptors (Zhuang et al., 1999; Veenstra-VanderWeele et al., 2000).

Because 5-HT_1A and 5-HT_1B receptors exist as separate autoreceptors, the serotonergic system provides a unique opportunity to examine the role of different types of autoreceptors in the topographic organization of the regulation of extracellular 5-HT. The present study used genetic and pharmacological methods to assess the regional selectivity of different autoreceptors regulating extracellular 5-HT. The contributions of individual autoreceptors to the regulation of 5-HT release were determined by comparing the effects of the SSRI fluoxetine in 5-HT_1A (Ramboz et al., 1998) and 5-HT_1B receptor knockout mice (Saudou et al., 1994) measured in two different brain regions, the striatum and ventral hippocampus. The ability of selective 5-HT receptor antagonists given with fluoxetine to produce a similar pattern of effects in wild-type mice was also examined. The results of our studies showed that topographical variations in the regulation of extracellular 5-HT suggested by pharmacological methods were reproduced by genetic variation. Specifically, the 5-HT_1A autoreceptor plays a larger role in regulating 5-HT release in the striatum, whereas the role of the 5-HT_1B autoreceptor is relatively greater in regulating the release of 5-HT in the ventral hippocampus.

	Materials and Methods

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Subjects. Male 5-HT_1A receptor knockout, 5-HT_1B receptor knockout, and wild-type mice with a 129/Sv background were bred and housed in a colony at the University of Pennsylvania (Philadelphia, PA). Breeding founders were obtained from established colonies derived originally from the same 129/SV background (Saudou et al., 1994; Ramboz et al., 1998) by Dr. René Hen, Columbia University (New York, NY) (for more details about the genetic background, see Phillips et al., 1999). Mice were generated by breeding homozygote mutant or wild-type mice. After weaning, mice were housed four per cage, given free access to standard rodent chow and water, and maintained on a 12-h light/dark schedule, with lights on at 7:00 AM. They were 8 to 12 weeks of age when used in these studies. All studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals by the U.S. National Institutes of Health and were reviewed by the University of Pennsylvania Institutional Animal Care and Use Committee.

Surgery. Mice were anesthetized with chloral hydrate (400 mg/kg i.p.) and positioned in a mouse stereotaxic instrument (Kopf Instruments, Tujunga, CA). Mice were implanted with a probe at the following coordinates (in millimeters) taken from bregma according to the atlas of Franklin and Paxinos (1997): striatum, +0.6 AP, +1.7 ML, and 4.5 DV; ventral hippocampus, 2.8 AP, 3.5 ML, and 5.0 DV. A drop of cyanoacrylate was spread thinly over the exposed skull, and the probe was then cemented in place. Following surgery, the mice were placed into a 21.5-cm-high, clear polycarbonate cylindrical in vivo microdialysis apparatus with a counterbalance arm holding a liquid swivel (Instech Laboratories, Plymouth Meeting, PA) and allowed to recover overnight.

Dialysis Procedure. Microdialysis procedures were performed with custom made microdialysis probes, as previously described (Knobelman et al., 2000). The probes, constructed from 26-gauge stainless steel tubing, were continuously perfused with filtered artificial cerebrospinal fluid (147 mM NaCl, 1.7 mM CaCl₂, 0.9 mM MgCl₂, and 4 mM KCl, pH 6.3-6.5) at a rate of 0.8 µl/min using a Harvard Apparatus syringe pump (Instech Laboratories). Dialysate samples were collected into polypropylene microcentrifuge vials 17 to 20 h after surgery at 20-min intervals for 2 h before and for 3 h after drug injections.

Histology. At the completion of the experiment, brains were removed and frozen at 80°C. The brains were then sectioned with a refrigerated cryostat, stained with Neutral Red, and the tissue examined for the location of the dialysis probe. Data from animals with probes located outside of the target regions were not used. This procedure was not followed in all cases because of experimental error. Histology was examined in 42 of the 77 animals with placements in the striatum (59% wild type, 20% 5-HT_1A/, and 72% 5-HT_1B/), and 51 of the 83 animals with placements in the ventral hippocampus (44% wild type, 63% 5-HT_1A/ and 86% 5-HT_1B/). Of the 93 animals in which placements were verified, it was only necessary to exclude one animal, whose target region was in the striatum, due to probe placement outside of this region.

Data Analysis. The first four samples were averaged to derive the baseline value and were corrected for individual probe recovery to compare against archived values from this laboratory. Probe recovery in vitro was measured with a standard solution of artificial cerebrospinal fluid containing 5-HT (10 nM) at room temperature. Drug effects were then expressed as a percentage of baseline values. The overall effect of treatments with fluoxetine on extracellular 5-HT levels was determined by two-factor ANOVA, with genotype and repeated measures on time the two main effects. The results for main effects and interaction terms of all ANOVA comparisons are shown in Table 1. Subsequent analysis of simple main effects were determined using Student-Newman-Keuls post hoc test. For studies examining the combination of 5-HT receptor antagonists and fluoxetine, comparisons were based on area under the curve (AUC) values using time points after administration of the antagonist until the end of the experiment (80-180 min following fluoxetine). Comparisons between experimental groups were made using ANOVA (Table 1) followed by the Student-Newman-Keuls post hoc test.

Drugs. Fluoxetine hydrochloride (Eli Lilly, Indianapolis, IN), WAY 100635 maleate (Wyeth-Ayerst Laboratories, Princeton, NJ), and GR 127935 hydrochloride (GlaxoSmithKline, Uxbridge, Middlesex, UK) were dissolved in deionized water and administered in a volume of 8 ml/kg i.p. Drug doses were calculated as the weight of the base.

	Results

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Basal 5-HT Levels. The basal dialysate levels for 5-HT in the striatum and ventral hippocampus of the 5-HT_1A receptor knockout, the 5-HT_1B receptor knockout, and wild-type mice are indicated in Table 2. There was no significant effect of genotype on basal 5-HT levels in either the striatum (F_2,74 = 0.1; P = 0.90) or the ventral hippocampus (F_2,80 = 1.73; P = 0.18).

Effects of Fluoxetine in 5-HT_1A and 5-HT_1B Receptor Knockout Mice and Wild-Type Mice. The acute administration of fluoxetine (2.5 mg/kg) evoked a significant increase of 5-HT from basal levels in the striatum of wild-type mice, to a maximum of 167 ± 13% at peak, as shown in Fig. 1A. The response to fluoxetine was significantly greater in the 5-HT_1A receptor knockout mice (P < 0.01), to 269 ± 25% at peak, but was not significantly changed in the 5-HT_1B receptor knockout mice (P > 0.05; 179 ± 18%). Acute administration of a higher dose of fluoxetine (20 mg/kg) evoked a larger increase of striatal 5-HT in the wild-type mice, to a maximum of 349 ± 46% at peak. The response to the higher dose of fluoxetine was also significantly greater in the 5-HT_1A receptor knockout mice (P < 0.01), rising to 877 ± 103% at peak, but was not significantly changed in the 5-HT_1B receptor knockout mice at 319 ± 43% (P > 0.05).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of fluoxetine on extracellular 5-HT levels in the 5-HT1A receptor knockout mouse, 5-HT1B receptor knockout mouse, and wild-type mouse. Values represent mean changes in 5-HT content, expressed as percentage of baseline values. Vertical lines indicate 1 S.E.M. The top graph in A shows the effects of systemic administration of 2.5 mg/kg fluoxetine in wild-type mice (; N = 7; baseline = 8.00 ± 1.14 fmol/10 µl), 5-HT1A receptor knockout mice (; N = 7; baseline = 8.58 ± 1.11 fmol/10 µl), and 5-HT1B receptor knockout mice (; N = 6; baseline = 6.75 ± 0.42 fmol/10 µl) on extracellular 5-HT in the striatum. The bottom graph in A shows the effects of systemic administration of 20 mg/kg fluoxetine in wild-type mice (N = 7; baseline = 8.41 ± 1.06 fmol/10 µl), 5-HT1A receptor knockout mice (N = 6; baseline = 8.56 ± 1.21 fmol/10 µl), and 5-HT1B receptor knockout mice (N = 6; baseline = 9.22 ± 0.80 fmol/10 µl) in the striatum. The top graph in B shows the effects of systemic administration of 2.5 mg/kg fluoxetine in wild-type mice (N = 8; baseline = 7.27 ± 0.75 fmol/10 µl), 5-HT1A receptor knockout mice (N = 7; baseline = 9.17 ± 0.83 fmol/10 µl), and 5-HT1B receptor knockout mice (N = 9; baseline = 6.56 ± 0.85 fmol/10 µl) on extracellular 5-HT in the ventral hippocampus. The bottom graph in B shows the effects of systemic administration of 20 mg/kg fluoxetine in wild-type mice (N = 7; baseline = 6.69 ± 0.64 fmol/10 µl), 5-HT1A receptor knockout mice (N = 6; baseline = 7.51 ± 1.34 fmol/10 µl), and 5-HT1B receptor knockout mice (N = 7; baseline = 4.78 ± 0.36 fmol/10 µl) on extracellular 5-HT in the ventral hippocampus.

Effects of WAY 100635 and Fluoxetine in 5-HT_1A Receptor Knockout Mice and Wild-Type Mice. The effects of the 5-HT_1A receptor antagonist WAY 100635 (0.1 mg/kg) on the increase in extracellular 5-HT elicited by 2.5 mg/kg fluoxetine in the striatum of 5-HT_1A receptor knockout and wild-type mice is shown in Fig. 2A. WAY 100635, given 80 min after fluoxetine, increased extracellular 5-HT levels to a maximum of 239 ± 26% above baseline at peak in the wild-type mice. In the 5-HT_1A receptor knockout mice, WAY 100635 and fluoxetine together elicited an increase of 273 ± 26% above basal extracellular 5-HT values. The AUC values in Fig. 2B show the amount of change produced by the WAY 100635 treatment. As expected, the augmentation was significantly larger in wild-type mice than in 5-HT_1A receptor knockout mice (P < 0.05). In contrast, the administration of WAY 100635 (0.1 mg/kg) did not evoke a significant augmentation of the effects of fluoxetine (2.5 mg/kg) on extracellular 5-HT in the ventral hippocampus (Fig. 2, C and D).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Effect of fluoxetine (2.5 mg/kg) with and without concurrent administration of WAY 100635 (0.1 mg/kg) on extracellular 5-HT in wild-type and 5-HT1A receptor knockout mice. A, effects of systemic administration of either 2.5 mg/kg fluoxetine alone in the wild-type mice (; N = 7; baseline = 8.00 ± 1.14 fmol/10 µl; same as Fig. 1) and 5-HT1A receptor knockout mice (; N = 7; baseline = 8.58 ± 1.11 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.1 mg/kg WAY 100635 in the wild-type mice (; N = 7; baseline = 7.64 ± 0.97 fmol/10 µl) and 5-HT1A receptor knockout mice (; N = 6; baseline = 6.76 ± 0.72 fmol/10 µl) on extracellular 5-HT levels in the striatum. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. The bars in B represent mean AUC summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of WAY 100635, as indicated by the arrow in A. Vertical lines indicate 1 S.E.M. C, effects of systemic administration of either 2.5 mg/kg fluoxetine alone in the wild-type mice (; N = 8; baseline = 7.27 ± 0.75 fmol/10 µl; same as Fig. 1) and 5-HT1A receptor knockout mice (; N = 7; baseline = 9.17 ± 0.83 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.1 mg/kg WAY 100635 in the wild-type mice (; N = 6; baseline = 5.42 ± 0.67 fmol/10 µl) and 5-HT1A receptor knockout mice (; N = 7; baseline = 8.16 ± 0.93 fmol/10 µl) on extracellular 5-HT levels in the ventral hippocampus. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. Values of the bars shown in D represent mean AUC summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of WAY 100635, as indicated by the arrow in C. Vertical lines indicate 1 S.E.M.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Effect of fluoxetine (20 mg/kg) with and without concurrent administration of WAY 100635 (0.1 mg/kg) on extracellular 5-HT in wild-type and 5-HT1A receptor knockout mice. A, effects of systemic administration of either 20 mg/kg fluoxetine alone in the wild-type mice (; N = 7; baseline = 8.41 ± 1.06 fmol/10 µl; same as Fig. 1) and 5-HT1A receptor knockout mice (; N = 6; baseline = 8.56 ± 1.21 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.1 mg/kg WAY 100635 in the wild-type mice (; N = 6; baseline = 6.67 ± 1.27 fmol/10 µl) and 5-HT1A receptor knockout mice (; N = 6; baseline = 6.32 ± 1.11 fmol/10 µl) on extracellular 5-HT levels in the striatum. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. The bars shown in B represent mean AUC summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of WAY 100635, as indicated by the arrow in A. Vertical lines indicate 1 S.E.M. C, effects of systemic administration of either 20 mg/kg fluoxetine alone in the wild-type mice (N = 7; baseline = 6.69 ± 0.64 fmol/10 µl; same as Fig. 1) and 5-HT1A receptor knockout mice (N = 6; baseline = 7.51 ± 1.34 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.1 mg/kg WAY 100635 in the wild-type mice (N = 7; baseline = 8.33 ± 1.24 fmol/10 µl) and 5-HT1A receptor knockout mice (N = 7; baseline = 5.52 ± 0.29 fmol/10 µl) on extracellular 5-HT levels in the ventral hippocampus. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. The bars shown in D represent mean AUC values summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of WAY 100635, as indicated by the arrow in C. Vertical lines indicate 1 S.E.M.

Effect of GR 127935 and Fluoxetine in 5-HT_1B Receptor Knockout Mice and Wild-Type Mice. The administration of GR 127935 (0.056 mg/kg) failed to alter the effect of 20 mg/kg fluoxetine in the striatum of either wild-type or 5-HT_1B receptor knockout mice, as shown in Fig. 4, A and B. In the ventral hippocampus, however, GR 127935 (0.056 mg/kg) augmented the effect of 20 mg/kg fluoxetine in wild-type mice as shown in Fig. 4, C and D. Follow-up tests showed that the AUC values for GR 127935 plus fluoxetine were significantly greater than fluoxetine alone in the wild-type mice (P < 0.05), but not for 5-HT_1B receptor knockout mice. The effects of GR 127935 plus fluoxetine in 5-HT_1A receptor knockout mice appear in a companion manuscript.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Effect of fluoxetine (20 mg/kg) with and without concurrent administration of GR 127935 (0.056 mg/kg) on extracellular 5-HT in wild-type and 5-HT1B receptor knockout mice. A, effects of systemic administration of either 20 mg/kg fluoxetine alone in the wild-type mice (; N = 7; baseline = 8.41 ± 1.06 fmol/10 µl; same as Fig. 1) and 5-HT1B receptor knockout mice (; N = 6; baseline = 9.22 ± 0.80 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.056 mg/kg GR 127935 in the wild-type mice (; N = 7; baseline = 8.51 ± 0.56 fmol/10 µl) and 5-HT1B receptor knockout mice (; N = 6; baseline = 7.78 ± 1.89 fmol/10 µl) on extracellular 5-HT levels in the striatum. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. The bars shown in B represent mean AUC values summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of GR 127935, as indicated by the arrow in A. Vertical lines indicate 1 S.E.M. C, effects of systemic administration of either 20 mg/kg fluoxetine alone in the wild-type mice (N = 7; baseline = 6.69 ± 0.64 fmol/10 µl; same as Fig. 1) and 5-HT1B receptor knockout mice (N = 7; baseline = 4.78 ± 0.36 fmol/10 µl; same as Fig. 1) or followed 80 min later by systemic administration of 0.056 mg/kg GR 127935 in the wild-type mice (N = 6; baseline = 9.42 ± 0.65 fmol/10 µl) and 5-HT1B receptor knockout mice (N = 6; baseline = 7.97 ± 0.67 fmol/10 µl) on extracellular 5-HT levels in the ventral hippocampus. Values represent mean changes in 5-HT content, expressed as a percentage of baseline values. Vertical lines indicate 1 S.E.M. Values of the bars shown in D represent mean AUC values summed from the effects measured 80 to 200 min after administration of fluoxetine and immediately following acute systemic administration of GR 127935, as indicated by the arrow in C. Vertical lines indicate 1 S.E.M.

	Discussion

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Regional patterns in the regulation of 5-HT release by 5-HT autoreceptors were examined by measuring extracellular levels of 5-HT in the striatum and ventral hippocampus of wild-type, 5-HT_1A receptor knockout, and 5-HT_1B receptor knockout mice. There were no differences in basal 5-HT levels measured in mice lacking 5-HT_1A or 5-HT_1B receptors under these experimental conditions. Although this was somewhat surprising given the absence of somatodendritic or terminal autoreceptors in the 5-HT receptor mutants, the issue of autoregulation on serotonergic function is a topic of ongoing debate. The absence of changes in basal dialysate 5-HT levels is consistent with evidence that 5-HT autoreceptors are not tonically activated, and many microdialysis studies using pharmacological antagonists fail to increase extracellular 5-HT levels (Hjorth et al., 2000). Other studies found no change in baseline 5-HT levels in the cortex and hippocampus of 5-HT_1B receptor knockout mice (Trillat et al., 1997; Malagie et al., 2001), but higher baseline levels were reported in 5-HT_1A receptor knockout mice (Parsons et al., 2001). Variant background strains (129 versus C57BL/6) and ages of mice (2-3 versus 8-10 months) could underlie these different results. More sensitive quantitative microdialysis methods are necessary to address issues regarding phenotypic differences in basal extracellular 5-HT levels in 5-HT receptor knockout mice (Parsons and Justice, 1994).

5-HT receptor mutant mice demonstrated different patterns of regulation in the striatum and hippocampus in response to challenge with the SSRI fluoxetine. At the low dose of fluoxetine (2.5 mg/kg), 5-HT_1A receptor knockout mice demonstrated greater increases of 5-HT than wild-type mice in the striatum but not the hippocampus, whereas 5-HT_1B receptor knockout mice showed the converse. At the higher dose of fluoxetine (20 mg/kg), the significantly greater response of 5-HT_1B receptor knockout in the ventral hippocampus was selectively maintained. However, 5-HT_1A receptor knockout mice showed a significantly greater response to the higher dose of fluoxetine (20 mg/kg) in both the striatum and ventral hippocampus, perhaps indicating more generalized functional regulation of 5-HT release by the 5-HT_1A receptor, depending on the tone of the system. These results agree with other recent microdialysis studies in mutant mice. Fluoxetine (15 mg/kg i.p.) produced a larger increase of 5-HT in the frontal cortex than the ventral hippocampus of 5-HT_1A receptor knockout mice (Parsons et al., 2001). In 5-HT_1B receptor knockout mice, paroxetine (1 and 5 mg/kg i.p.) caused larger increases of 5-HT in the ventral hippocampus, although paroxetine was potentiated in the frontal cortex at the low dose (Malagie et al., 2001).

The effects of fluoxetine were likely augmented in the 5-HT receptor mutants by the genetic deletion of autoreceptors that ordinarily inhibit 5-HT release. Somatodendritic 5-HT_1A autoreceptors located in the DR and MR inhibit 5-HT neuronal discharge, synthesis, and release in the striatum and hippocampus, respectively, and blockade of 5-HT_1A receptors in the DR and MR can increase the impact of SSRIs in forebrain regions (Kreiss and Lucki, 1994; Hjorth et al., 1997; Romero and Artigas, 1997). However, postsynaptic 5-HT_1A receptors in the frontal cortex or the amygdala may also limit the ability of endogenous 5-HT to activate inhibitory feedback loops traversing back to 5-HT cell bodies in the brain stem (Bosker et al., 1997; Casanovas et al., 1999). 5-HT_1B autoreceptors are located at presynaptic nerve terminals, although 5-HT_1B autoreceptors also regulate 5-HT transmission by modulating the efflux of 5-HT from recurrent collaterals onto 5-HT cell bodies or by interacting with other neurotransmitters (Boschert et al., 1994). Because the present studies involved systemic administration, they did not determine the location of the autoreceptors responsible for augmenting the effects of fluoxetine in 5-HT receptor mutants.

The present studies systematically combined pharmacological and genetic approaches to support the existence of topographical differences in the regulation of 5-HT release in the mouse. In wild-type mice, systemic administration of fluoxetine produced substantially larger increases of extracellular 5-HT in the ventral hippocampus than in the striatum. The 5-HT_1A receptor antagonist WAY 100635 augmented the effects of the low dose of fluoxetine in the striatum and not the hippocampus but augmented the effects of the high dose of fluoxetine in both regions, mimicking the topographical pattern of the fluoxetine response in 5-HT_1A receptor mutants. The 5-HT_1B/1D receptor antagonist GR 127935 and fluoxetine augmented the effects of fluoxetine in the ventral hippocampus but not the striatum, a regional pattern shown by 5-HT_1B receptor mutants to fluoxetine. The limited (but significant) response to GR 127935 in the present study, substantially less than that of 5-HT_1B receptor knockout mice, may be due to its poor selectivity (versus 5-HT_1D receptors), partial efficacy at 5-HT_1B receptors, or the need to block receptors for longer periods of time. It is also possible that reduced sensitivity of 5-HT_1A receptors (Knobelman et al., 2001) contributed to the augmented effects of fluoxetine in the hippocampus of 5-HT_1B receptor knockout mice. Newer compounds that discriminate the effects of 5-HT_1B and 5-HT_1D receptors may be more effective in future studies (Price et al., 1997). Furthermore, pharmacological antagonists failed to augment the effects of fluoxetine after genetic deletion of the corresponding receptor, confirming the absence of corresponding functional 5-HT receptors in these mice. We and others have shown the absence of effects by 5-HT_1A or 5-HT_1B receptor agonists on extracellular 5-HT levels in mice with corresponding genetic deletions (Trillat et al., 1997; Knobelman et al., 2001).

As in the mouse, previous pharmacological studies in rats and guinea pigs have shown important regional differences in the net impact of SSRIs, but several ideas have been proposed as to the exact reason. One idea suggests that regional differences are related to the origin of the 5-HT afferents projecting to individual terminal areas (for review, see Hjorth et al., 2000). Thus, 5-HT_1A receptor antagonists appear to augment the effects of SSRIs in areas with predominate DR innervation, such as the frontal cortex and striatum, but are less effective (or not at all) in the MR-innervated dorsal hippocampus (Malagie et al., 1996; Invernizzi et al., 1997; Romero and Artigas, 1997; Sharp et al., 1997; Hervas and Artigas, 1998; Hjorth et al., 2000). However, the dose of SSRI is also a critical treatment variable because GR 127935 potentiated citalopram's effects on extracellular 5-HT in the rat ventral hippocampus at lower treatment doses than were sensitive to WAY 100635 (Cremers et al., 2000), and this response pattern was also measured in the present study in the mouse hippocampus. A second idea suggests that regional variations in the strength of 5-HT_1B autoreceptor feedback, such as between the frontal cortex and hippocampus, determine regional sensitivity to SSRIs (Hjorth et al., 2000). 5-HT_1B and 5-HT_1B/1D receptor antagonists may increase extracellular 5-HT in MR-innervated areas, such as the dentate gyrus or dorsal hippocampus, in rats and guinea pigs (Roberts et al., 1998; but see Hervas and Artigas, 1998; Hervas et al., 2000). A third idea is that regions demonstrate differential sensitivity to autoreceptor antagonists depending on factors regulating the balance between release and autoreceptor inhibition (Hervas et al., 2000). For example, greater effects of reuptake blockade are offset by greater autoreceptor-mediated inhibition in the frontal cortex resulting in greater sensitivity to autoreceptor antagonists, The higher density of 5-HT transporters and 5-HT_1A receptors in the DR than the MR (Hensler et al., 1994) supports this idea. Differential afferent input and heteroreceptor regulation could also contribute to regional differences in regulating the effects of SSRIs.

The similar topographical patterns in the regulation of 5-HT transmission between 5-HT receptor mutant mice and pharmacological challenges likely reflect endogenous regional patterns in the physiological regulation of extracellular 5-HT under genetic control by different types of 5-HT autoreceptors and not the development of compensatory mechanisms. Taking the pharmacological and genetic evidence together, the 5-HT_1A autoreceptor appears to play a larger role in regulating extracellular 5-HT in the striatum, and possibly other brain regions preferentially innervated by the DR, such as the frontal cortex, whereas the role of the 5-HT_1B autoreceptor was relatively greater in the ventral hippocampus, and potentially other MR-innervated brain regions. However, as described above, the precise mechanisms determining the topographical patterns of 5-HT release remain to be determined and are likely to arise from distributed local densities of transporters and autoreceptors, the recurrent collaterals supplying 5-HT innervation to cell body regions, differential afferent innervation, and endogenous tone at both the terminal and corresponding cell body regions.

The different regional patterns of 5-HT release attendant with 5-HT receptor mutants could contribute to varying expression of behavioral phenotypes (Zhuang et al., 1999) or, in the case of humans with relevant polymorphisms, to distinct behavioral traits, vulnerabilities, or drug interactions (Veenstra-VanderWeele et al., 2000). For example, enhanced sensitivity of 5-HT_1B receptor mutants to the antidepressant-like response of fluoxetine in the tail suspension test could be mediated by increased release of 5-HT in the hippocampus (Mayorga et al., 2001). 5-HT_1A receptor knockout mice demonstrated increased release of 5-HT in the frontal cortex but not the hippocampus after exposure to an open field (Parsons et al., 2001). Finally, since the 5-HT_1A and 5-HT_1B receptor antagonist pindolol is used to augment the clinical effects of fluoxetine, regional variations in the regulation of extracellular 5-HT may be pertinent to understanding its mechanism of action (Artigas et al., 1996).