Mice Lacking Serotonin 2C Receptors Have increased Affective Responses to Aversive Stimuli

Abstract

Although central serotonergic systems are known to influence responses to noxious stimuli, mechanisms underlying serotonergic modulation of pain responses are unclear. We proposed that serotonin 2C receptors (5-HT2CRs), which are expressed within brain regions implicated in sensory and affective responses to pain, contribute to the serotonergic modulation of pain responses. In mice constitutively lacking 5-HT2CRs (2CKO mice) we found normal baseline sensory responses to noxious thermal, mechanical and chemical stimuli. In contrast, 2CKO mice exhibited a selective enhancement of affect-related ultrasonic afterdischarge vocalizations in response to footshock. Enhanced affect-related responses to noxious stimuli were also exhibited by 2CKO mice in a fear-sensitized startle assay. The extent to which a brief series of unconditioned footshocks produced enhancement of acoustic startle responses was markedly increased in 2CKO mice. As mesolimbic dopamine pathways influence affective responses to noxious stimuli, and these pathways are disinhibited in 2CKO mice, we examined the sensitivity of footshock-induced enhancement of startle to dopamine receptor blockade. Systemic administration of the dopamine D2/D3 receptor antagonist raclopride selectively reduced footshock-induced enhancement of startle without influencing baseline acoustic startle responses. We propose that 5-HT2CRs regulate affective behavioral responses to unconditioned aversive stimuli through mechanisms involving the disinhibition of ascending dopaminergic pathways.

Fig 1. Heat-induced nociceptive responses are similar in 2CKO and wildtype mice.

A. No difference between wildtype (open bar) and mutant (filled bar) paw withdrawal latencies in Hargreave’s assay. Data analyzed by Student’s t-test, bars are ± 1 standard error. B. No difference between wildtype (open bar) and mutant (filled bar) latencies to move paws from hot plate at 48, 52.5, and 55°C. C. No acute difference in the temporal pattern of paw licking following hindpaw formalin injection. Data analyzed by ANOVA, bars are ± 1 standard error. D. Twenty-four hours after formalin injection, paw withdrawal thresholds significantly decreased to a similar extent in 2CKO and wildtype mice. * = p<0.001, Students’s t-test, bars are ± 1 standard error.

Fig 2. 2CKO mice display ultrasonic vocalizations to footshock indicative of enhanced affective reactivity.

A. Representative sound spectrogram from a wildtype mouse following a 0.4 mA footshock stimulus administered 2.7 sec into recording trial. Two vocalization-during-shock (VDSs, two short noise spikes) followed by vocalization-after-discharge (VAD, long noise burst with visible “chatter” lines) vocalization patterns can be observed. B. Representative sound spectrogram from a 2CKO mouse following a 0.4 mA footshock stimulus. A single VDS vocalization followed by a prolonged VAD (with prominent chatter) is observed. C. Spectral power (above ambient noise) of VDS vocalizations. Open bars depict wildtype responses, and filled bars depict 2CKO responses. Bars are ± 1 standard error. D. Spectral power (above ambient noise) of VAD vocalizations. Open bars depict wildtype responses, and filled bars depict 2CKO responses. * p<0.001, Student’s t-test, bars are ± 1 standard error. Since our background noise measurements averaged around 70 dB, the background ultrasound power for these figures shows up in red on the colormap, with higher powers depicted as darker colors.

Fig 3. 2CKO mice display enhanced fear-sensitized startle responses after unsignaled footshock.

Open bars depict wildtype, and filled bars depict 2CKO data. All footshock stimuli were 0.4 mA in amplitude. The primary factors of genotype, startle stimulus intensity, and condition (pre vs. post shock) were all significant at p<0.001, ANOVA, bars are ± 1 standard error.

Fig 4. Wildtype mice require higher shock intensities to exhibit levels of startle potentiation equivalent to those of 2CKO mice.

Responses of 2CKO mice to footshocks of 0.15 mA intensity (black bars) are similar to those of wildtype mice receiving 0.8 mA shocks (filled bars). ANOVA, bars are ± 1 standard error.

Fig 5. Raclopride blocks expression of fear-sensitized startle responses in 2CKO mice without affecting baseline startle responses.

Open bars depict preshock responses, black bars depict postshock responses. Bars surrounded by no background derive from wildtype mice; bars surrounded by grey background derive from 2CKO mutant mice. Raclopride dosages given on x axis. A. Responses to 90 decibel (dB) startle stimuli. B. Responses to 105 dB startle stimuli. C. Responses to 120 dB startle stimuli. Whereas the footshock-induced enhancement of startle responses in 2CKO mice is blocked by raclopride, baseline startle responses are unaffected by the drug. ANOVA, bars are ± 1 standard error.