5-hydroxytryptamine (5-HT)1A autoreceptor adaptive changes in substance P (neurokinin 1) receptor knock-out mice mimic antidepressant-induced desensitization

Abstract

Antagonists at substance P receptors of the neurokinin 1 (NK1) type have been shown to represent a novel class of antidepressant drugs, with comparable clinical efficacy to the selective serotonin (5-HT) reuptake inhibitors (SSRIs). Because 5-HT(1A) receptors may be critically involved in the mechanisms of action of SSRIs, we examined whether these receptors could also be affected in a model of whole-life blockade of NK1 receptors, i.e. knock-out mice lacking the latter receptors (NK1-/-). 5-HT(1A) receptor labeling by the selective antagonist radioligand [(3)H]N-[2-[4-(2-methoxyphenyl)1-piperazinyl]-ethyl]-N-(2-pyridinyl)-cyclohexanecarboxamide (WAY 100635) and 5-HT(1A)-dependent [(35)S]GTP-gamma-S binding at the level of the dorsal raphe nucleus (DRN) in brain sections, as well as the concentration of 5-HT(1A) mRNA in the anterior raphe area were significantly reduced (-19 to -46%) in NK1-/- compared with NK1+/+ mice. Furthermore, a approximately 10-fold decrease in the potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit the discharge of serotoninergic neurons in the dorsal raphe nucleus within brainstem slices, and reduced hypothermic response to 8-OH-DPAT, were noted in NK1-/- versus NK1+/+ mice. On the other hand, cortical 5-HT overflow caused by systemic injection of the SSRI paroxetine was four- to sixfold higher in freely moving NK1-/- mutants than in wild-type NK1+/+ mice. Accordingly, the constitutive lack of NK1 receptors appears to be associated with a downregulation/functional desensitization of 5-HT(1A) autoreceptors resembling that induced by chronic treatment with SSRI antidepressants. Double immunocytochemical labeling experiments suggest that such a heteroregulation of 5-HT(1A) autoreceptors in NK1-/- mutants does not reflect the existence of direct NK1-5-HT(1A) receptor interactions in normal mice.

Fig. 1.

Representative autoradiographic labeling of 5-HT_1A receptors by [³H] WAY 100635 in brain sections from NK1−/− mutants compared with NK1+/+ wild-type mice. Coronal sections (20 μm) at the level of the DRN and the hippocampus were labeled with 1 nm [³H]WAY 100635. Similar autoradiograms were obtained from three to five mice per group. Cx, Cerebral cortex; DRN, dorsal raphe nucleus; Ent. Cx, entorhinal cortex;Hip, hippocampus; MRN, median raphe nucleus; Sup. Coll., superior colliculi.

Fig. 2.

Representative autoradiograms of 5-HT_1A receptor-mediated increase in [³⁵S]GTP-γ-S binding to brain sections from NK1−/− mutants compared with NK1+/+ wild-type mice. Coronal sections (20 μm) were labeled by [³⁵S]GTP-γ-S (50 pm) without (BASAL) or with 10 μm 5-CT at the level of the DRN and the hippocampus. Nonspecific (NS) labeling was obtained from adjacent sections exposed to 10 μm 5-CT plus 10 μmWAY 100635 (5-HT_1A receptor antagonist). Similar autoradiograms were obtained from six mice per group.Cx, Cerebral cortex; DRN, dorsal raphe nucleus; Ent. Cx, entorhinal cortex; Hip, hippocampus; MRN, median raphe nucleus; Sup. Coll., superior colliculi.

Fig. 3.

Immunohistochemical labeling of serotonin-positive neurons and NK1 receptors in the dorsal raphe nucleus.A, A′, Bright-field photomicrographs of close serial transverse sections through the DRN illustrating the distribution of serotoninergic neurons in A and NK1 receptors in A′. Scale bar (shown in A′ for A and A′), 100 μm. Bshows dual antigen immunocytochemical labeling (DAB technique), with serotonin-positive elements in purple and NK1 receptor-like immunoreactivity in brown. Serotonin antibody labels cell bodies and primary dendrites, whereas NK1 receptor antibody mainly labels dendrites and neuropil. Most serotonin neurons are surrounded by NK1-positive dendrites and neuropil. Scale bar, 20 μm. C shows a confocal photomicrograph of dual antigen immunocytochemistry developed with fluorescence secondary antibodies. Sections were stained for serotonin in green (examples at arrows) and for NK1 receptors in red(examples at arrowheads). Serotonin and NK1 receptors do not colocalize in the same neurons, but NK1-positive neuropil inred is intermingled with serotonin-positive cell bodies and primary dendrites. Scale bar, 50 μm.

Fig. 4.

Effect of ipsapirone on the firing of DRN 5-HT neurons in NK1−/− mutants compared with NK1+/+ wild-type mice.A, Ipsapirone-induced inhibition and prevention by WAY 100635. Integrated firing rate histograms (in spikes per 10 sec) show the effect of increasing concentrations of the 5-HT_1Areceptor agonist ipsapirone on the electrical activity of a DRN 5-HT neuron in a NK1−/− mutant (right) compared with a paired NK1+/+ control (left). In both strains, ipsapirone-induced inhibition was markedly reduced by WAY 100635 (1 nm), a selective 5-HT_1A receptor antagonist (Fletcher et al., 1996). B, Concentration–response curves of ipsapirone-induced inhibition of the firing of DRN 5-HT neurons in brainstem slices from NK1−/− compared with NK1+/+ mice. Ipsapirone-induced inhibition is expressed as percentage of the baseline firing rate. Each point is the mean ± SEM of data obtained from 10–13 individual cells. The dotted linesillustrate the EC₅₀ values of ipsapirone (abscissa) in NK1−/− mutants and NK1+/+ wild-type mice. **p < 0.01, ***p < 0.001 as compared with the respective inhibition in wild-type mice.

Fig. 5.

Effects of paroxetine with or without the selective 5-HT_1A receptor antagonist WAY 100635 on 5-HT outflow in the frontal cortex of NK1−/− mutants compared with NK1+/+ wild-type mice. Data are the means ± SEM of extracellular 5-HT levels expressed as percentages of baseline in NK1+/+ wild-type (open symbols) and NK1−/− knock-out (closed symbols) mice after exposure to saline (0.9% NaCl) (A), paroxetine (Prx) (B), and WAY 100635 given 15 min before paroxetine (C). The first arrowrepresents injection of saline (A, B) or WAY 100635 (0.5 mg/kg, s.c.) (C), and thesecond arrow represents injection of saline (A) or paroxetine (1 mg/kg, i.p.) (B, C). Basal [5-HT]_extvalues (in femtomoles per 20 μl) were 2.20 ± 0.20 and 2.34 ± 0.25 in the frontal cortex of NK1+/+ and NK1−/− mice, respectively. D, AUC (mean ± SEM) values calculated for the amount of 5-HT collected during the 0–180 min period after treatment are expressed as percentages of basal values. Each value is the mean ± SEM of at least 12 independent determinations. **p < 0.01 and ***p < 0.001 compared with the corresponding control group (treated with NaCl/NaCl);^†††p < 0.001 relative to wild-type;^##p < 0.01 compared with wild-type treated with NaCl/paroxetine.

Fig. 6.

Time course of 8-OH-DPAT-induced hypothermia in NK1−/− mutants compared with NK1+/+ wild-type mice. Wild-type (A) and knock-out (B) mice received a subcutaneous injection of vehicle (▵, 10 ml of 0.9% NaCl/kg) or 8-OH-DPAT (○, 0.25 mg/kg; ●, 0.5 mg/kg), and body temperature was measured at 5 min intervals for up to 60 min. Change (in °C) in body temperature relative to baseline values (immediately before treatment, 0 on abscissa) is the mean ± SEM of determinations in six mice in each group. At both doses tested, body temperature was significantly (p < 0.05; Student's Newman–Keuls multiple t test) less in 8-OH-DPAT-treated than in vehicle-treated mice for the 10–60 min interval after injection in NK1+/+ as well as NK1−/− mice.