Dopamine release induced by atypical antipsychotics in prefrontal cortex requires 5-HT(1A) receptors but not 5-HT(2A) receptors

Abstract

Atypical antipsychotic drugs (APDs) increase dopamine (DA) release in prefrontal cortex (PFC), an effect probably mediated by the direct or indirect activation of the 5-HT(1A) receptor (5-HT(1A)R). Given the very low in-vitro affinity of most APDs for 5-HT(1A)Rs and the large co-expression of 5-HT(1A)Rs and 5-HT(2A) receptors (5-HT(2A)Rs) in the PFC, this effect might result from the imbalance of 5-HT(1A)R and 5-HT(2A)R activation after blockade of these receptors by APDs, for which they show high affinity. Here we tested this hypothesis by examining the dependence of the APD-induced DA release in medial PFC (mPFC) on each receptor by using in-vivo microdialysis in wild-type (WT) and 5-HT(1A)R and 5-HT(2A)R knockout (KO) mice. Local APDs (clozapine, olanzapine, risperidone) administered by reverse dialysis induced a dose-dependent increase in mPFC DA output equally in WT and 5-HT(2A)R KO mice whereas the DA increase was absent in 5-HT(1A)R KO mice. To examine the relative contribution of both receptors to the clozapine-induced DA release in rat mPFC, we silenced G-protein-coupled receptors (GPCRs) in vivo with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) while 5-HT(1A)Rs or 5-HT(2A)/2CRs in the mPFC were selectively protected with the respective antagonists WAY-100635 or ritanserin. The inactivation of GPCRs while preserving ∼70% of 5-HT(2A)/(2C)Rs prevented the clozapine-induced DA rise in mPFC. In contrast, clozapine increased DA in mPFC of EEDQ-treated rats whose 5-HT(1A)Rs were protected (∼50% of control rats). These results indicate that (1) 5-HT(1A)Rs are necessary for the APDs-induced elevation in cortical DA transmission, and (2) this effect does not require 5-HT(2A)R blockade by APDs.

Fig. 1

Representative autoradiograms of [³H]8-OH-DPAT binding in the brains of wild-type (WT) (A1–A3, upper panels) and 5-HT_1AR knockout (KO) mice (A4–A6, lower panels). In WT mice, note the high density for 5-HT_1ARs in (A1) prefrontal cortex [cingulated (Cg1), prelimbic (PrL), infralimbic (IL); AP: 1.78 mm), (A2) hippocampus (AP: −2.06 mm) and (A3) dorsal raphe nuclei (DR) and entorhinal cortex (Ent) (AP: −4.36 mm). Parallel sections of the corresponding null mutant mice (KO) show a lack of [³H]8-OH-DPAT binding (A4–A6). Scale bar, 1 mm.

Fig. 2

Representative autoradiograms of [³H]mesulergine (a, b) and [³H]8-OH-DPAT (c) binding in the brains of wild-type (WT) (upper panels) and 5-HT_2AR knockout (KO) mice (lower panels). In WT mice, note the expected high density for 5-HT_2ARs as visualized with [³H]mesulergine plus 10⁻⁷ m SB242084 in (A1) prefrontal cortex [cingulated (Cg1). Motor (M1, M2), AP: 2.10 mm] and, (A2) frontal cortex (FCx) and claustrum (CL) (AP: 0.74 mm). Parallel sections of the corresponding null mutant mice (KO) show a conspicuous lack of binding (A3-A4). No differences between genotypes were noted for 5-HT_2CRs as visualized with [³H]mesulergine plus 10⁻⁷ m spiperone in the choroid plexus (ChP) (B1-B2) nor for 5-HT_1ARs, as visualized with [³H]8-OH-DPAT in prefrontal cortex (C1–C2) in WT and 5-HT_2AR KO mice, respectively. Scale bar, 1 mm.

Fig. 3

Local effect of the 5-HT_2A/2CR agonist DOI (100–300 μm) on the output of 5-HT (c) and DA (d) in the mPFC of wild-type (WT) and 5-HT_2AR knockout (KO) mice. The perfusion of DOI increased 5-HT and DA levels in mPFC of WT mice (n = 10). Both effects were absent in 5-HT_2AR KO mice (n = 7–10). The administration of aCSF did not alter prefrontal 5-HT (a) and DA (b) in either genotype (n = 5–6). Data are expressed as mean±s.e.m. See Results section for statistical analysis.

Fig. 4

The local administration of (a) clozapine (300 μm, n = 9), (b) olanzapine (100 μm, n = 6–7) and (c) risperidone (100 μm, n = 5) increased similarly DA levels in mPFC of wild-type (WT) and 5-HT_2AR knockout (KO) mice. This effect was not observed when APDs were infused in the mPFC of 5-HT_1AR KO mice (n = 4–6, a–c). Data are expressed as mean±s.e.m. See Results section for statistical analysis.

Fig. 5

Local administration of (a) clozapine and (b) olanzapine at increasing concentrations (30–100–300 μm) raised a similar DA output in mPFC of wild-type (WT) and 5-HT_2AR KO mice. This effect was not observed when APDs were perfused in the mPFC of 5-HT_1AR KO mice. (c) Local effect of clozapine (300 μm) and olanzapine (100 μm) on 5-HT output in mPFC of WT, 5-HT_2AR KO and 5-HT_1AR KO mice. Data are AUCs (fractions 6–16) expressed as percentage of baseline. N = 4–9 mice for all groups, except for 300 μm olanzapine in mPFC of 5-HT_2AR KO mice, where n = 3. See Results section for statistical analysis. * p <0.05, ** p <0.01 vs. WT and 5-HT_2AR KO mice.

Fig. 6

(a) The perfusion of 300 μm clozapine in mPFC of control rats increased local DA output (n = 10). This effect was absent in the mPFC of rats whose GPCRs were silenced by a prior EEDQ injection (6 mg/kg i.p.) and their prefrontal 5-HT_2A/2CRs had been protected by ritanserin (300 μm) administration through the microdialysis probe (n = 5) (see Methods section). (b) Conversely, clozapine administration (300 μm) elicited a significant DA elevation in mPFC of rats treated with EEDQ whose 5-HT_1ARs were preserved by prior WAY-100635 (300 μm) administration (n = 6). This effect was significantly greater than in control rats (n = 10). Data are expressed as mean ± s.e.m. See Results section for statistical analysis.

Fig. 7

(a) The perfusion of 300 μm DOI (5-HT_2A/2C agonist) increased DA output in the mPFC of control rats and of rats treated with EEDQ and whose mPFC 5-HT_2A/2CRs were unilaterally preserved by prior ritanserin administration. The perfusion of clozapine (300 μm) reversed the effect of DOI only in control rats (n = 6–8) but not in those with protected 5-HT_2A/2CR. (b) In control rats, the local perfusion of BAY × 3702 (30 μm) antagonized the increase of DA output in mPFC induced by the local administration of DOI (300 μm) (n = 5). Data are expressed as mean ± s.e.m. See Results section for statistical analysis.

Fig. 8

(a) Representative autoradiograms showing the density of 5-HT_2A/2CRs labelled with [³H]mesulergine in coronal sections of PFC (AP in mm: 3.70–3.20) from different pretreatment groups: (A1) controls (rats received the EEDQ vehicle i.p. and aCSF through the dialysis probe), (A2) GPCR-silenced rats (injected with 6 mg/kg i.p EEDQ and perfused with aCSF through the dialysis probe), (A3) GPCR-silenced+5-HT_1AR-protected rats (treated with 6 mg/kg i.p. EEDQ while 300 μm WAY-100635 was perfused through the dialysis probe), and (A4) GPCR-silenced+5-HT_2A/2CR-protected rats (treated with 6 mg/kg i.p. EEDQ while 300 μm ritanserin was perfused through the dialysis probe). A5 shows non-specific binding. Panels A1a–A4a are photomicrographs showing enlargements of the marked area in panels A1–A4. Note the higher 5-HT_2A/2CR binding in ipsilateral (protected) mPFC with respect to the contralateral (unprotected) side of panel A4 and the very low occupancy for 5-HT_2A/2CRs in both hemispheres of panels A2 and A3. Scale bars, 2 mm (A1–A4) and 500 μm (A1a–A4a). (b) Densitometric quantification of 5-HT_2A/2CR binding in mPFC including cingulate, prelimbic and infralimbic cortices of the different group of rats (B1–B4). Bars represent mean 5-HT_2A/2CR fmol/mg tissue ±s.e.m. of 4–8 observations (two or four observations at left and right hemispheres of two consecutive sections per animal and two to four animals per group). ** p<0.001 significantly different from corresponding contralateral (C) and ipsilateral (I) mPFC of control rats, ⁺⁺p<0.001 significantly different from contralateral mPFC of GPCR-silenced+5-HT_2A/2CR-protected rats, using one-way ANOVA followed by Newman–Keuls post-hoc test.

Fig. 9

(a) Representative autoradiograms showing the density of 5-HT_1ARs labelled with [³H]8-OH-DPAT in coronal sections of PFC (AP in mm: 3.70–3.20) from different pretreatment groups: (A1) controls (rats received the EEDQ vehicle i.p. and aCSF through the dialysis probe), (A2) GPCR-silenced rats (injected with 6 mg/kg i.p. EEDQ and perfused with aCSF through the dialysis probe), (A3) GPCR-silenced + 5-HT_2A/2CR-protected rats (injected with 6 mg/kg i.p. EEDQ while 300 μm ritanserin was perfused through the dialysis probe), and (A4) GPCR-silenced + 5-HT_1AR-protected rats (injected with 6 mg/kg i.p. EEDQ while 300 μm WAY-100635 was perfused through the dialysis probe). A5 shows non-specific binding. Panels A1a–A4a are photomicrographs showing enlargements of the marked area in panels A1–A4. Note the higher 5-HT_1AR binding in ipsilateral (protected) mPFC with respect to the contralateral (unprotected) side of A4 and the very low occupancy for 5-HT_1ARs in both hemispheres of panels A2 and A3. Scale bars, 2 mm (A1–A4) and 500 μm (A1a–A4a). (b) Densitometric quantification of 5-HT_1AR binding in mPFC including cingulate, prelimbic and infralimbic cortices of the different group of rats (B1–B4). Bars represent mean 5-HT_1AR fmol/mg tissue ±s.e.m. of 4–8 observations (two or four observations at left and right hemispheres of two consecutive sections per animal and two to four animals per group). ** p<0.001 significantly different from corresponding contralateral (C) and ipsilateral (I) mPFC of control rats, ⁺⁺p<0.001 significantly different from contralateral mPFC of GPCR-silenced + 5-HT_1AR-protected rats, using one-way ANOVA followed by Newman–Keuls post-hoc test.