Simon Beckett

Central C-Fos Expression Following 20kHz/Ultrasound Induced Defence Behaviour in the Rat

Exposure of rats to aversive stimuli produces specific defence behaviour including the emission of 20-27kHz ultrasonic calls. Recent studies in this laboratory have shown that rats exposed to a 20kHz ultrasound tone display flight behaviour similar to that seen naturally, or following stimulation of brain regions associated with anxiety and defence. The present study examines the effect of ultrasound exposure on the central expression of the immediate early gene c-fos in the rat, in order to examine the brain structures activated by such behaviour. Ultrasound presentation produced rapid locomotor activity characteristic of defence behaviour, including brisk running and jumping behaviour. Animals showed dense c-fos like immunoreactivity in the dorsal periaqueductal grey matter, basolateral, medial, central amygdala, paraventricular thalamic nuclei and the dorsomedial nuclei of the hypothalamus, which was significantly greater than in either home-cage or arena control rats. These results suggest that exposure to artificially generated ultrasound can induce defence behaviour which is associated with activity in brain regions important in mediating aversion. This technique offers the potential of generating unconditioned aversive behaviour in rats in a non invasive way.

Evidence for differential modulation of conditioned aversion and fear-conditioned analgesia by CB1 receptors

Fear‐conditioned analgesia is an important survival response mediated by substrates controlling nociception and aversion. Cannabinoid1 (CB1) receptors play an important role in nociception and aversion. However, their role in fear‐conditioned analgesia has not been investigated. This study investigated the effects of systemic administration of the CB1 receptor antagonist, SR141716A (1 mg/kg, ip), on fear‐conditioned analgesia and conditioned aversion in rats. Twenty‐four hours after receiving footshock, rats exhibited reduced formalin‐evoked nociceptive behaviour, increased freezing and increased defecation when tested in the footshock apparatus, compared with non‐footshocked formalin‐injected rats. SR141716A attenuated fear‐conditioned analgesia, freezing and defecation. Importantly, SR141716A had no effect on formalin‐evoked nociceptive behaviour over an equivalent time period in rats not receiving footshock. SR141716A had no effect on contextually induced freezing during the first half of the test trial in rats receiving intra‐plantar injection of saline. Administration of SR1417176A did, however, attenuate short‐term extinction of contextually induced freezing and ultrasound emission in rats receiving intra‐plantar saline, compared with vehicle‐treated saline controls. These data suggest an important role for the CB1 receptor in mediating fear‐conditioned analgesia and provide evidence for differential modulation of conditioned aversive behaviour by CB1 receptors during tonic, persistent pain.

Effects of coadministration of cannabinoids and morphine on nociceptive behaviour, brain monoamines and HPA axis activity in a rat model of persistent pain

The antinociceptive effects of Δ9‐tetrahydrocannabinol (Δ9‐THC) have been widely described; however, its therapeutic potential may be limited by secondary effects. We investigated whether coadministration of low doses of cannabinoids or cannabinoids and morphine produced antinociception in the absence of side‐effects. Effects of preadministration (i.p.) of Δ9‐THC (1 or 2.5 mg/kg), cannabidiol (5 mg/kg), morphine (2 mg/kg), Δ9‐THC + morphine, Δ9‐THC + cannabidiol or vehicle on formalin‐evoked nociceptive behaviour were studied over 60 min. Trunk blood and brains were collected 60 min after formalin injection and assayed for corticosterone and tissue levels of monoamines and metabolites, respectively. Drug effects on locomotor activity, core body temperature and grooming were assessed. Δ9‐THC reduced both phases of formalin‐evoked nociceptive behaviour, enhanced the formalin‐evoked corticosterone response and increased the 4‐hydroxy‐3‐methoxyphenylglycol : noradrenaline ratio in the hypothalamus. Cannabidiol alone had no effect on these indices and did not modulate the effects of Δ9‐THC. Morphine reduced both phases of formalin‐evoked nociceptive behaviour. Coadministration of Δ9‐THC and morphine reduced the second phase of formalin‐evoked nociceptive behaviour to a greater extent than either drug alone, and increased levels of thalamic 5‐hydroxytryptamine. While the antinociceptive effects of Δ9‐THC and morphine alone occurred at doses devoid of effects on locomotor activity, coadministration of Δ9‐THC and morphine inhibited locomotor activity. In conclusion, coadministration of a low dose of morphine, but not cannabidiol, with Δ9‐THC, increased antinociception and 5‐hydroxytryptamine levels in the thalamus in a model of persistent nociception. Nevertheless, these enhanced antinociceptive effects were associated with increased secondary effects on locomotor activity.

Pharmacological manipulation of ultrasound induced defence behaviour in the rat

Abstract Rats exposed to aversive stimuli display species specific defence behaviour as part of their natural survival strategy. One component of this behaviour is the production of ultrasonic calls in the 20 to 27-kHz range, which are thought to serve a communicative role. The present study has examined the behavioural effects of exposing rats to artificially generated ultrasound and the ability of three distinct pharmacological agents to modify this response. Single tone 20 kHz ultrasound exposure for 1 min produced intensity-related locomotor behaviour, characteristic of defence behaviour, which could be measured using a computer tracking system. This was significantly reduced by peripheral pretreatment with the benzodiazepine, diazepam (0.3 and 3.0 mg/kg IP). Pretreatment with the 5-HT agonist 1-(3-chlorophenyl) piperazine (mCPP) (0.5–2.0 mg/kg IP) produced a dose-related reduction in the ultrasound-induced response. The α2 adrenoceptor antagonist, yohimbine (0.5–5.0 mg/kg IP), caused an increase in the response at the lower dose (0.5 mg/kg) and a decrease at the two higher doses (2.0 and 5.0 mg/kg). The present findings suggest that defence behaviour in the rat can be artificially produced by 20 kHz ultrasound; this is sensitive to pharmacological manipulation and may offer a novel animal model of aversive behaviours that are associated with human panic.

Effects of lesioning noradrenergic neurones in the locus coeruleus on conditioned and unconditioned aversive behaviour in the rat

1. The brain noradrenergic system may have a role in anxiety disorder. This study has examined the effect of bilateral 6-hydroxydopamine lesions of the noradrenergic neurones in the locus coeruleus (LC) of male Lister hooded rats on behaviour produced by unconditioned and conditioned aversive stimuli. 2. The 6-hydroxydopamine (4 microg) lesions markedly reduced the noradrenaline content of the locus coeruleus hypothalamus, frontal cortex and the periaqueductal grey area without altering the levels of either dopamine or 5-hydroxytryptamine measured 14 days after administration. 3. Exposure to ultrasound (20 kHz at 98 dB for 60 sec), an unconditioned aversive stimulus, induced a defence response in the rats characterised by an increase in activity (running and jumping) followed by a period of inactivity (freezing). 4. Lesioning of the LC significantly attenuated the duration of freezing but was without effect on the active phase of the response. A similar reduction in freezing behaviour was seen with LC lesions when rats were exposed (3 hours after the acquisition) to the contextual cue of the conditioned emotion response paradigm. 5. These findings confirm that the locus coeruleus is involved in the regulation of fear-related behaviour in the rat both in an unconditioned and a conditioned model. Furthermore the results indicate that noradrenaline modifies defence behaviour rather than being the principle activating mechanism.

Behavioral, central monoaminergic and hypothalamo–pituitary–adrenal axis correlates of fear-conditioned analgesia in rats

Fear-conditioned analgesia is an important survival response which is expressed upon re-exposure to a context previously paired with a noxious stimulus. The aim of the present study was to characterize further the behavioral, monoaminergic and hypothalamo-pituitary-adrenal axis alterations associated with expression of fear-conditioned analgesia. Rats which had received footshock conditioning 24 h earlier, exhibited reduced formalin-evoked nociceptive behavior upon re-exposure to the footshock chamber, compared with non-footshocked formalin-treated rats. Intra-plantar injection of formalin reduced the duration of contextually-induced freezing and 20-40 kHz ultrasound emission. Intra-plantar injection of formalin to non-footshocked, non-conditioned rats did not induce ultrasonic vocalizations. Intra-plantar injection of formalin to footshock-conditioned rats, significantly increased tissue levels of 3,4-dihydroxyphenylacetic acid and the 3,4-dihydroxyphenylacetic acid:dopamine ratio in the periaqueductal gray and reduced levels of dopamine in the thalamus, compared with saline-treated footshocked controls. Non-footshocked, non-conditioned rats were capable of mounting a robust formalin-evoked increase in plasma corticosterone levels. Moreover, plasma corticosterone levels were significantly higher in saline-treated, footshock conditioned rats compared with saline-treated non-footshocked rats and levels did not differ between saline- and formalin-treated footshock conditioned rats. Assessment of the effects of the intra-plantar injection procedure revealed an attenuation of short-term extinction of contextually-induced freezing in rats anesthetized for intra-plantar injection of saline compared with non-anesthetized, non-injected rats as well as discrete effects on monoamines, their metabolites and plasma corticosterone levels. These data extend behavioral characterization of the phenomenon of fear-conditioned analgesia and suggest that measurement of ultrasound emission may be used as an ethologically relevant index of the defense response during fear-conditioned analgesia. Ultrasonic vocalization may also be a useful behavioral output to aid separation of nociception and aversion. The data provide evidence for discrete alterations in dopaminergic activity in the periaqueductal gray and thalamus and for altered hypothalamo-pituitary-adrenal axis activity following expression of defensive behavior.

Strain differences to the effects of aversive frequency ultrasound on behaviour and brain topography of c-fos expression in the rat

Previous studies have shown that ultrasound at 20 kHz produces an escape (defence) response in the hooded Lister rat. This study compares the ultrasound-induced behavioural response in the hooded Lister and albino Wistar rat. Ultrasound (continuous tone, square wave, 20 kHz) produced an initial characteristic startle response (brisk running) in the hooded Lister rat that was followed immediately after cessation of the ultrasound by a period of freezing behaviour. In contrast, Wistar rats showed no initial escape response but a prolonged period of freezing that started during the ultrasound and continued for a period after the end of the ultrasound. Immunohistochemical assessment of c-fos expression also showed a difference between the two strains with preferential expression in the dorsal region of the rostral and caudal periaqueductal grey (PAG) in the hooded Lister rat, while the expression occurred in the ventral PAG in the Wistar rats. In summary, the two strains exhibit distinct defensive behaviours and patterns of neuronal activation in response to the same aversive signal. It remains to be determined whether these differences relate to neuronal circuitry or perception of the signal, but analysis of the mechanisms involved may help our understanding of the heterogeneity of anxiety disorders.

Acoustically elicited behaviours in Lister hooded and Wistar rats.

It has been reported previously that experimenter-presented 20-kHz tones at low intensities produce bursts of locomotor running in Lister hooded rats, but reduced locomotion (freezing) in Wistar rats. Because rats emit 20-kHz tones when stressed, it was proposed that this ultrasound-elicited running and freezing behaviour in Lister hooded and Wistar rats, respectively, represents a model for qualitative strain differences in fear behaviour. The present studies examined the acoustic specificity of acoustically elicited locomotor behaviours in Lister hooded and Wistar rats. In Experiment 1, it was found that brief exposure (i.e., 15 s) of Lister hooded rats to tones at frequencies of 7, 12, or 20 kHz and intensities of 85-95 dB SPL, elicited running behaviour characterised by brief bursts of locomotion followed by periods of quiescence. Somewhat surprisingly, the 7- and 12-kHz tones elicited running behaviour at lower intensities than did the 20-kHz tones. In Experiment 2, it was found that exposure of Lister hooded rats to the 20-kHz acoustic stimulus (91-101 dB, SPL) for a much longer duration, up to 9 min, resulted in episodic bursts of locomotion and convulsions in a significant proportion of subjects. Both the maximal velocity of locomotion and the likelihood of occurrence of convulsions was related to the intensity of the acoustic stimulus. Exposure of Lister hooded rats to white noise for up to 9 min also elicited episodic bursts of locomotion and convulsions in an intensity-dependent manner. The white noise stimulus was found to be a more effective stimulus than the 20-kHz stimulus in this regard. In Experiment 3, it was found that Lister hooded rats exhibited reduced locomotion when they were exposed to a low-intensity 20-kHz acoustic stimulus (e.g., 81 dB, SPL). In Experiment 4, it was found that Wistar rats did not exhibit locomotor bursts or convulsions when presented with 20-kHz tones using stimulus parameters equal to and even greater than those that had been shown to be effective in producing locomotor bursts in Lister hooded rats. Rather, Wistar rats exhibited only reduced locomotion. The present data indicate that (1) running behaviour in Lister hooded rats is not specific for the 20-kHz stimulus. Moreover, (2) when compared to Lister hooded rats, Wistar rats are relatively insensitive to the running and convulsions elicited by acoustic stimuli. Finally, (3) both Lister hooded and Wistar rats exhibited reduced locomotion when presented with the 20-kHz tones, although the range of stimulus intensities that produces freezing behaviour is much more limited in Lister hooded rats because of their propensity to exhibit locomotor bursting and convulsions. Thus, it appears that the difference between the two strains with respect to their unconditioned locomotor responses to novel acoustic stimuli relates to the fact that Lister hooded rats are uniquely susceptible to acoustically elicited locomotor bursts and/or convulsions.

The role of the central nucleus of the amygdala in nociception and aversion.

&NA; The role of the central nucleus of the amygdala in nociception ‐ conditioned and unconditioned aversion ‐ was studied. Rats received microinjection of vehicle or the N‐type Ca2+ channel blocker Ω‐conotoxin GVIA (0.2 &mgr;g/250 nl) into the central amygdaloid nucleus prior to intra‐plantar injection of formalin, ultrasound exposure or immediately prior to the acquisition phase of an aversive conditioning trial. Intra‐amygdala Ω‐conotoxin GVIA resulted in an earlier onset of nociceptive response to formalin and increased nociceptive behaviour during the first 5min. Ω‐Conotoxin GVIA significantly reduced conditioned freezing behaviour with no effect on ultrasound‐induced unconditioned aversive behaviour. These data indicate that N‐type Ca2+ channels in the central amygdaloid nucleus play a role in mediating behavioural responses to nociceptive and conditioned aversive stimuli. NeuroReport 14:981–984

Effects of chronic infusion of neurotensin and a neurotensin NT1 selective analogue PD149163 on amphetamine-induced hyperlocomotion

Neurotensin (NT) has been proposed as an endogenous antipsychotic based in part on the similarity in behavioural effects to antipsychotic drugs, for example, attenuation of both amphetamine-induced hyperlocomotion (AH) and amphetamine disrupted pre-pulse inhibition in the rat. However, there is some evidence that repeated administration of NT or an analogue produces behavioural tolerance to such effects. The present experiments sought to confirm and extend these findings by testing the effects on AH of 7 days central administration of NT and the NT 1 selective analogue PD 149163 and the effects of 21 days central administration of NT. NT and PD149163 continuously administered for 7 days produced no effect on AH (in contrast to attenuation with a single injection here and previously reported), whereas 21 days of NT administration potentiated AH. Together, these studies report that the effects of NT or a NT analogue on AH depends on the duration of administration of peptide. The results are discussed in comparison with the reported antipsychotic properties of acute administration of NT and possible mechanisms involving NT1 receptors.