Neurokinin-1 receptor-expressing neurons in the amygdala modulate morphine reward and anxiety behaviors in the mouse

Abstract

Mice lacking the neurokinin-1 (NK1) receptor, the preferred receptor for the neuropeptide substance P (SP), do not show many of the behaviors associated with morphine reward. To identify the areas of the brain that might contribute to this effect, we assessed the behavioral effects of ablation of neurons expressing the NK1 receptor in specific regions of the mouse brain using the neurotoxin substance P-saporin. In a preliminary investigation, bilateral ablation of these neurons from the amygdala, but not the nucleus accumbens and dorsomedial caudate putamen, brought about reductions in morphine reward behavior. Subsequently, the effect of ablation of these neurons in the amygdala on anxiety behavior was assessed using the elevated plus maze (EPM), before conditioned place preference (CPP), and locomotor responses to morphine were measured. Loss of NK1 receptor-expressing neurons in the amygdala caused an increase in anxiety-like behavior on the EPM. It also brought about a reduction in morphine CPP scores and the stimulant effect of acute morphine administration relative to saline controls, without affecting CPP to cocaine. NK1 receptor-expressing neurons in the mouse amygdala therefore modulate morphine reward behaviors. These observations mirror those observed in NK1 receptor knock-out (NK1-/-) mice and suggest that the amygdala is an important area for the effects of SP and the NK1 receptor in the motivational properties of opiates, as well as the control of behaviors related to anxiety.

Figure 1.

Effects of SP-SAP in the NAcc. A, Sections from the NAcc of a naive and SP-SAP-injected mouse stained immunohistochemically for the NK₁ receptor. After 5 weeks, SP-SAP caused a clearance of NK₁ receptor immunoreactivity around the injection site, in both the core (AcbC) and shell (AcbSh) regions. Scale bar, 1 mm. B, Injection of SP-SAP into the NAcc caused the additional loss of neurons from the dorsomedial edge of the CPu, adjacent to the lateral ventricle (LV). Scale bar, 1 mm. C, Sections from the NAcc core stained immunohistochemically for the NK₁ receptor, NeuN, or GFAP. Loss of NK₁ receptor immunoreactivity at the injection site was not accompanied by nonspecific loss of neurons, as assessed by IHC for NeuN, or gliosis, as assessed by IHC for GFAP. Scale bar, 50 μm. ac, Anterior commissure.

Figure 2.

Effects of SP-SAP in the amygdala. A, Sections from the amygdala of a naive and SP-SAP-injected mouse stained immunohistochemically for the NK₁ receptor. After 5 weeks, SP-SAP caused a reduction in NK₁ receptor immunoreactivity around the injection site, particularly in the medial (MeA), basomedial (BMA), and central (CeA) nuclei. Scale bar, 1 mm. B, Sections from the medial nucleus stained immunohistochemically for NeuN or GFAP. Loss of NK₁ receptor immunoreactivity was not accompanied by loss of NeuN immunoreactivity or increases in GFAP immunoreactivity. Injection of SAP alone also failed to bring about loss of neurons or gliosis at the injection site. Sections from naive mice and SP-injected mice (data not shown) were similar to those injected with SAP. Scale bar, 250 μm. BLA, Basolateral nucleus of the amygdala.

Figure 3.

Localization of ablated cells in the amygdala. A, Sections of the amygdala of an NK₁^-/- mouse stained with X-Gal and counterstained with neutral red. βGal-positive cells are visible throughout the amygdala, notably in the medial nucleus. Right, Medial nucleus. Arrows indicate βGal-positive nuclei. Scale bars: left, 1 mm; right, 250 μm. B, Sections from an NK₁^+/- mouse 5 weeks after unilateral injection of SP-SAP into the amygdala. On the uninjected (Naive) side of the brain, βGal-positive nuclei are present though the central, basolateral, and medial nuclei of the amygdala, but these are absent on the side of the brain injected with SP-SAP. Right, Medial nucleus. Scale bars: left, 1 mm; right, 250 μm. BLA, Basolateral nucleus of the amygdala; MeA, medial nucleus of the amygdala; Pir, piriform cortex.

Figure 4.

Preliminary findings: effects of NK₁ receptor-expressing neuron ablation in the NAcc and dorsomedial CPu or amygdala on morphine reward behavior. Mean ± SEM. CPP scores after conditioning with 3.0 mg/kg morphine in naive mice and those with ablation in the NAcc and dorsomedial CPu (NAcc) or in the amygdala. Mice with ablation of NK₁ receptor-expressing neurons in the amygdala, but not the NAcc and dorsomedial CPu, exhibited reductions in CPP scores. ^*p < 0.05 versus naive (one-way ANOVA).

Figure 5.

Effects of NK₁ receptor-expressing neuron ablation in the amygdala on anxiety and morphine reward and stimulation. A, Mean ± SEM time spent in the open arms of the EPM and mean ± SEM number of entries into the open arms of the EPM in mice with ablation in the amygdala and mice receiving control injections of SP or SAP alone. Cell ablation caused decreases in the amount of time spent in the open arms of the maze and in the number of entries made into them relative to control groups. ^*p < 0.05; ^**p < 0.01 versus SP-SAP (post hoc Tukey comparisons). B, Mean ± SEM. CPP scores in mice conditioned with saline or morphine (3.0 mg/kg). Mice injected with SP or SAP showed a significant CPP to morphine, whereas those with ablation of NK₁ receptor-expressing neurons did not. ^*p < 0.05 versus saline. C, Mean ± SEM number of beam crosses in 10 min, beginning 30 min after an injection of saline or morphine (10.0 mg/kg). Mice injected with SP or SAP into the amygdala showed a stimulant effect of morphine, but this was absent in mice with ablation of NK₁ receptor-expressing neurons. ^**p < 0.01; ^***p < 0.001.

Figure 6.

Effects of NK₁ receptor-expressing neuron ablation in the amygdala on cocaine reward. Mean ± SEM. CPP scores in naive mice and those with ablation of NK₁ receptor-expressing neurons in the amygdala, after conditioning with saline or cocaine (5.0 mg/kg). Ablation did not affect cocaine reward.