In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism

Abstract

Background and purpose: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503.

Experimental approach: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907.

Key results: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity.

Conclusions and implications: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.

Figure 1

Spike train (upper trace) and rate-meter recordings showing the inhibitory effect of the putative 5-HT_2C receptor agonist WAY 161503 on the firing rate of individual DRN 5-HT neurones in the anaesthetized rat. WAY 161503 produced a dose-related inhibition of 5-HT cell firing (a), which was attenuated by pretreatment with the 5-HT_2C receptor antagonist SB 242084 (b, c). The effect of WAY 161503 was reversed by both SB 242084 (a) and the 5-HT₂ receptor antagonist ritanserin (a, b), but not by the 5-HT_1A receptor antagonist WAY 100635 (c). Note also the characteristic inhibitory response of 5-HT neurones to the 5-HT_1A receptor agonist 8-OH-DPAT, which was reversed by the 5-HT_1A receptor antagonist WAY 100635 (a, b). Drug administration (mg kg⁻¹ i.v.) as indicated by arrows. Abbreviations: 8-OH-DPAT (DPAT), SB 242084 (SB), ritanserin (Rit).

Figure 2

Inhibition of 5-HT cell firing by WAY 161503 and its blockade by the 5-HT_2C receptor antagonist SB 242084 (1.0 mg kg⁻¹). Control animals received WAY 161503 alone and showed a dose-related inhibition of cell firing. Note that pre-treatment with SB 242084 caused a significant shift to the right of the dose response to WAY 161503. Data are mean±s.e.m. of n observations at agonist doses of 0.125, 0.25, 0.5 mg kg⁻¹, respectively: control n=8,8,7; SB 242084 n=6,6,5. Effect of WAY 161503 alone, P<0.0001, one-way ANOVA; P<0.01 at 0.25 and 0.5 mg kg⁻¹, Dunnett's post hoc test. Effect of SB 242084 pre-treatment, P<0.01, two-way ANOVA; P<0.05 at 0.25 mg kg⁻¹ WAY 161503, Bonferroni post hoc test.

Figure 3

Binding of the radioligand [¹¹C]MDL 100907 in rat brain determined by (a) PET and (b) ex vivo methods. Rats received [¹¹C]MDL 100907 alone (open columns, n=5), 5 min after SB 242084 1 mg kg⁻¹ i.v. (filled columns, n=4), or 5 min after non-radioactive MDL 100907 (0.2–0.4 mg kg⁻¹ i.v.) (striped columns, n=4). Brain regions: a, olfactory bulbs; b, hypothamus; c, striatum; d, frontal with cingulate cortex; e, hippocampus, f, superior colliculi; g, medulla. Data are mean±s.d. values. Note that SB 242084 did not affect [¹¹C]MDL 100907 binding in any region whereas unlabelled MDL 100907 reduced [¹¹C]MDL 100907 binding to unity (P<0.05: Student's t-test).

Figure 4

Spike train (upper trace) and rate-meter recording illustrating a partial reversal of the inhibitory effect of WAY 161503 on 5-HT cell firing by administration of the GABA_A receptor antagonist picrotoxin. Drug administration (mg kg⁻¹ i.v.) as indicated by arrows.

Figure 5

Effect of WAY 161503 on the expression of Fos in GAD-positive DRN neurones. (a) Drug treatments were vehicle-vehicle, vehicle-WAY 161503 (3 mg kg⁻¹), SB 242084 (1.0 mg kg⁻¹)-vehicle and SB 242084 (1.0 mg kg⁻¹)-WAY 161503 (3 mg kg⁻¹). Data (n=6) are mean±s.e.m. Note that WAY 161503 increased the number of Fos/GAD double-labelled cells and that this effect was reduced by pre-treatment with SB 242084. ^**P<0.001 versus vehicle–vehicle (one way ANOVA with Dunnett's post hoc test). ^#P<0.05 versus vehicle–WAY 161503 (one-way ANOVA with Bonferroni's post hoc test). (b) Photomicrographs showing cells double labelled (arrows) with Fos and GAD immunoreactivity in the DRN of rats administered one the following drug treatments: vehicle–vehicle, SB 242084 (1.0 mg kg⁻¹)–vehicle, SB 242084 (1.0 mg kg⁻¹)–WAY 161503 (3 mg kg⁻¹), vehicle–WAY 161503 (3 mg kg⁻¹). Images at × 40 magnification.

Figure 6

Photomicrographs of the rat DRN showing fluorescent images of 5-HT_2C receptor immunoreactivity with either GAD_65/67 immunoreactivity (left) or Fos immunoreactivity induced by WAY 161503 (3 mg kg⁻¹ i.p.) (right). Arrows indicate co-localized neurones as seen in the merged images. Note the presence of GAD positive cells double-labelled with 5-HT_2C receptor immunoreactivity. Also Fos positive cells were double-labelled with 5-HT_2C receptor immunoreactivity after WAY 161503 administration.