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

doi:10.1016/S0361-9230(96)00332-2

Brain Research Bulletin

Volume 42, Issue 6, 1997, Pages 421-426

https://doi.org/10.1016/S0361-9230(96)00332-2 Get rights and content

Abstract

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.

Introduction

Immunohistological detection of Fos, the protein product of the immediate early gene c-fos, has been used extensively to monitor the activity of central neurones following various kinds of stimuli, and although the significance of c-fos expression in neurones remains incompletely understood, it is known to be a reproducible marker of neuronal activation (for review see [16]). In rats, the production of Fos-like immunoreactivity (FLI) increases in specific brain regions following relatively mild stressors, such as tactile stimulation of the whiskers [24] and brief restraint [15], and also following more severe conditioned stimuli, such as foot shock 13, 26, 3. Unconditioned stressors, such as pain and immobilisation stress, induce FLI in a variety of brain nuclei [32], an effect also seen in rats subjected to swim stress [17]. In the present study, the central activation of c-fos, assessed by measurement of FLI in the forebrain and midbrain, has been used as an indication of those areas involved in the expression of a novel model of defensive behaviour in rats.

It is well documented that rats emit ultrasonic calls as part of their natural behavioural repertoire. The frequency and duration of this vocalisation is determined by specific environmental stimuli and is thought to have an important communicative role [11]. Aversive stimuli such as conspecific encounters cause the production of short pulse 50kHz and long pulse 25kHz calls [29], whereas predator exposure results in 18–27kHz calls [9]. Such antipredator vocalisations have been shown to elicit components of defensive behaviour and vocalisations in other members of the same colony [7]. As the production of these calls is enhanced by the presence of familiar conspecifics [8], it has been suggested that they may alter the behaviour of other rats not directly exposed to a predator [10]. This may be due to the inherent aversive nature of the calls.

Recent studies in this laboratory have demonstrated that predator naive rats, exposed to artificially generated continuous tone 20kHz ultrasound, display species specific defence-like behaviour which is related to the intensity of stimulation [6]. This aversive behaviour, characterised by a combination of freezing and rapid locomotion to “escape” the stimulus, is extremely similar to that seen following electrical or chemical stimulation of brain regions associated with anxiety and defence 21, 27, 4.

The aims of the present study were to determine which brain regions participate in the defence response elicited by exposure to 20kHz ultrasound in an open-field arena. Preliminary reports of part of this work have been presented previously [2].

Section snippets

Animals

Experiments were performed on 12 male hooded Lister rats (250–300g, Biomedical Services Unit, Queen's Medical Centre, Nottingham). Animals were group housed with a 12 h light/dark cycle (lights on 7.00 am). Food (Pilsbury animal diet) and water were available ad libitum.

Ultrasound Production

Ultrasound (continuous tone, square wave) was produced using a multifunction signal generator (Jupiter 500, Black Star, UK) at a sound intensity of 75dB. This was delivered to the testing arena via a piezoelectric speaker

Ultrasound Generated Behaviour

Arena-only animals when placed in the testing arena displayed normal exploratory locomotor behaviour, characterised by stable values of maximum speed, average speed and distance travelled, which were unchanged throughout the total 5 min test period (Fig. 2). Ultrasound stimulated animals placed in the testing arena also showed the normal pattern of exploratory behaviour for the initial 2 min prior to ultrasound stimulation. On exposure to the ultrasound tone, they displayed an initial freezing

Discussion

The results of the present experiment indicate that while exposure to an open-field arena causes FLI expression in various discreet brain nuclei, further expression in areas known to be involved in the mediation of defence behaviour can be elicited on presentation of a 20kHz ultrasound tone in the same arena. Furthermore, exposure to 20kHz ultrasound causes escape behaviour characterised by rapid locomotor activity which ceases when the sound is removed. The overt behavioural response to the

Acknowledgements

This work was supported by the MRC.

References (34)

F. Anton et al.
C-fos like immunoreactivity in the rat brainstem neurones following noxious stimulation of the nasal mucosa
Neurosci.
(1991)
S.R.G. Beckett et al.
Computer analysis and quantification of periaqueductal grey-induced defence behaviour
J. Neurosci. Meths.
(1995)
R.J. Blanchard et al.
The characterisation and modelling of antipredator defence behaviour
Neurosci. Biobehav. Rev.
(1990)
R.J. Blanchard et al.
Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in a visible burrow system
Physiol. Behav.
(1991)
S.M. Brudzynski et al.
Behavioural responses of laboratory rats to playback of 22 kHz ultrasonic calls
Physiology and Behavior
(1995)
S. Campeau et al.
Induction of the c-fos proto oncogene in rat amygdala during unconditioned and conditioned fear
Brain Research
(1991)
N. Chastrette et al.
Effects of daytime and nighttime stress of Fos-like immunoreactivity in the paraventricular nucleus of the hypothalamus, the habenula, and the posterior paraventricular nucleus of the thalamus
Brain Res.
(1991)
M. Dragunow et al.
The use of c-fos as a metabolis marker in neuronal pathway tracing
J. Neurosci. Meths.
(1989)
G.E. Duncan et al.
Neuroanatomical characterization of Fos induction in rat behavioral models of anxiety
Brain Research
(1996)
G. Le Gal La Salle et al.
Audiogenic siezures evoked in DBA/2 mice induce c-fos oncogene expression into subcortical auditory nuclei
Brain. Res.
(1990)

R.A. Leslie et al.

Serotonin(2/1C) receptor activation causes a localized expression of the immediate-early gene c fos in rat brain: Evidence for involvement of dorsal raphe nucleus projection fibres

Neuroscience

(1993)

K.J. Mack et al.

Induction of transcription factors in the somatosensory cortex after tactile stimulation

Molec. Brain Res.

(1992)

M.A. Pezzone et al.

Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli

Brain Res.

(1992)

G.D. Sales

Ultrasound and aggressive behaviour in rats and other small mammals

Anim. Behav.

(1972)

G. Sandner et al.

What brain structures are active during emotions? Effects of brain stimulation elicited aversion on c-fos immunoreactivity and behaviour

Behav. Brain Res.

(1993)

E. Senba et al.

Stress-induced c-fos expression in the rat brain: Activation mechanism of sympathetic pathway

Brain. Res. Bull.

(1993)

M.A. Smith et al.

Induction of c-fos mRNA in rat brain by conditioned and unconditioned stressors

Brain Research

(1992)

Cited by (90)

The effect of playback of 22-kHz and 50-kHz ultrasonic vocalizations on rat behaviors assessed with a modified open-field test
2021, Physiology and Behavior
Juvenile and adult rats emit two affectively different types of ultrasonic vocalizations (USVs), namely aversive 22-kHz and appetitive 50-kHz USVs. Aversive 22-kHz USVs are considered to be alarm calls that communicate negative affective states to conspecific receivers. Although the alarming effects of playback of 22-kHz USVs were reported recently, behavioral data showing those effects are still not abundant. Appetitive 50-kHz USVs are considered to communicate positive affective states to conspecific receivers, to pace and coordinate social behavior. In line with this, playback of 50-kHz USVs has been found to initiate behavioral activation and induce approach behavior in receiver rats. However, most of these playback studies have used male 50-kHz USVs; thus, it seems to remain unclear whether female 50-kHz USVs exert a similar social attractant effect on male rats. To investigate these issues, we performed modified open-field tests, during which USVs were continuously presented for 15 min to male receivers. In these tests, if negative affective changes are evoked in subject rats, the time spent in the open arena decreases, while the time spent on defensive behaviors increases. In contrast, when positive affective changes are evoked, the opposite phenomenon is observed. Playback of male aversive 22-kHz USVs induced anxiety-related defensive responses in receivers. However, playback of female appetitive frequency-modulated (FM) 50-kHz USVs increased opposite, appetitive pattern of exploratory behavior with increased exploration. The results indicate that playback of male aversive 22-kHz and female appetitive 50-kHz USVs might induce behavioral responses probably associated with negative and positive affective states in male rats, respectively, suggesting the validity of rat USVs as an animal model of vocal communication of emotion.
Playback of Alarm and Appetitive Calls Differentially Impacts Vocal, Heart-Rate, and Motor Response in Rats
2020, iScience
Our rudimentary knowledge about rat intraspecific vocal system of information exchange is limited by experimental models of communication. Rats emit 50-kHz ultrasonic vocalizations in appetitive states and 22-kHz ones in aversive states. Both affective states influence heart rate. We propose a behavioral model employing exposure to pre-recorded playbacks in home-cage-like conditions. Fifty-kHz playbacks elicited the most vocalizations (>60 calls per minute, mostly of 50-kHz type), increased heart rate, and locomotor activity. In contrast, 22-kHz playback led to abrupt decrease in heart rate and locomotor activity. Observed effects were more pronounced in singly housed rats compared with the paired housed group; they were stronger when evoked by natural playback than by corresponding artificial tones. Finally, we also observed correlations between the number of vocalizations, heart rate levels, and locomotor activity. The correlations were especially strong in response to 50-kHz playback.
Fear paradigms: The times they are a-changin’
2018, Current Opinion in Behavioral Sciences
Fear is considered an integral part of the brain's defensive mechanism that evolved to protect animals and humans from predation and other ecological threats. Hence, it is logical to study fear from the perspective of antipredator-survival behaviors and circuits by sampling a range of threatening situations that organisms are likely to encounter in the wild. In the past several decades, however, mainstream fear research has focused on the importance of associative learning; that is, how animals become frightened of innocuous cues as consequences of their contingent pairing with aversive events. While significant discoveries have been made, contemporary fear models derived from learning studies are likely to provide only a partial picture of the brain's fear system because they cannot simulate the dynamic range of risky situations in nature that require various adaptive actions and decisions. This review considers two different approaches to study fear, grounded on behaviorism and ethology and examines their contributions in revealing the naturalistic workings of fear in guiding and shaping behavior as animals make real-world choices.
Playback of Ultrasonic Vocalizations to Juvenile and Adult Rats: Behavioral and Neuronal Effects
2018, Handbook of Behavioral Neuroscience
Citation Excerpt :
When looking in detail, however, the specificity and strength of the behavioral response to playback becomes questionable. Often, effects were only clearly evident with loud and artificial continuous sine-wave tones (Beckett et al., 1996, 1997; Commissaris et al., 1998, 2000; Neophytou et al., 2000; Nicolas et al., 2007; Nobre & Brandão, 2004; Voits et al., 1999), whereas behavioral responses were rather weak or absent in the case of natural stimuli (Brudzynski & Chiu, 1995; Burman et al., 2007; Endres et al., 2007; Parsana, Li, & Brown, 2012; Parsana, Moran, & Brown, 2012; Sales, 1991; Wöhr & Schwarting, 2007), indicating that signal features such as loudness have to be controlled carefully. In the first study using natural 22-kHz USVs as playback stimuli, Sales (1991) showed that these natural signals led to slight locomotor inhibition, that is, around 20% reduction compared to noise controls.
Ultrasonic vocalizations in rats serve as important social signals, thereby receiving substantial scientific attention in behavioral and neurobiological research. To study their effects on call recipients, playback techniques were developed with the use of natural or artificial auditory signals that can be tested in terms of their effects on behavior and brain functions. The approach is also used to study the outcomes of psychopharmacological manipulations and their applicability in preclinical models of human disorders where social communicatory deficits are known to play a role. The present review summarizes the current scientific evidence for roles and effects of 22-kHz and 50-kHz ultrasonic vocalizations on juvenile and adult rats. These vocalizations represent two functionally opposite signal classes that serve aversive/defensive and affiliative/appetitive functions, respectively, and which are related to specific and partly distinct brain regions.
Inhibition of substance P-induced defensive behavior via neurokinin-1 receptor antagonism in the central and medial but not basolateral nuclei of the amygdala in male Wistar rats
2017, Progress in Neuro-Psychopharmacology and Biological Psychiatry
The production of unconditioned defensive behaviors has been related to the amygdala, a key component of the encephalic aversion system. Microinjection of the neuropeptide substance P (SP) in the amygdala elicits defensive behaviors via the activation of type 1 neurokinin (NK-1) receptors. However, no studies have investigated whether intra-amygdala SP/NK-1 mechanisms can elicit other types of defensive responses, such as antinociception and ultrasonic vocalizations (USVs).
The present study investigated the effects of SP-induced activation of the neurokininergic system in three main nuclei of the amygdala—basolateral (BLA), central (CeA), and medial (MeA) nuclei—in rats that were subjected to the elevated plus maze (EPM), tail-flick test, and USV recording. The effects of SP in these amygdaloid nuclei were challenged with combined injections of the NK-1 receptor antagonist spantide.
The present study showed that SP injections in the CeA and MeA but not BLA exerted anxiogenic-like effects. In contrast to the CeA, the anxiogenic-like effects of SP in the MeA were not dependent on NK-1 mechanisms. In the tail-flick test, SP microinjections produced antinociceptive effects only in the MeA through NK-1 receptor activation. No USV emissions were detected after the SP microinjections.
The present study showed that NK-1 receptors in the CeA and MeA but not BLA are involved in defensive reactions to conditions of fear. The present results may provide a better understanding of the neurochemical mediation of fear states.
Effects of chemical stimulation of the lateral wings of the dorsal raphe nucleus on panic-like defensive behaviors and Fos protein expression in rats
2017, Behavioural Brain Research
Citation Excerpt :
Treatment with kainic acid did not change Fos protein expression in the three amygdala nuclei investigated, i.e. the BLA, MeA and Cea. As observed here with lwDR stimulation, exposure of rats to aversive stimuli/situations that evoke escape such as confrontation with predators [16–18], 20–22 kHz ultrasound [19,20] or open-arm experience in the elevated T-maze [3,21], also increases Fos expression in the dPAG/lPAG and/or mH. However, differently from our results, in all these situations Fos protein expression was also up- regulated in some or all of the amygdala nuclei investigated by us, notably in the MeA.
The lateral wings subnucleus of the dorsal raphe nucleus (lwDR) has been implicated in the modulation of panic-like behaviors, such as escape. Infusion of non- excitotoxic doses of the excitatory amino acid kainic acid into this subnucleus promptly evokes a vigorous escape response. In addition, rats exposed to panic-inducing situations show an increase in Fos protein expression in neurons within the lwDR. In the present study, we first investigated whether key structures associated with the mediation of escape behavior are recruited after chemical stimulation of the lwDR with kainic acid. We next investigated whether the infusion of the GABA_A receptor antagonist bicuculline into the lwDR also evoked escape responses measured both in a circular arena and in the rat elevated T-maze. The effects of bicuculline in the circular arena were compared to those caused by the infusion of this antagonist into the ventrolateral periaqueductal gray (vlPAG), an area in close vicinity to the lwDR. The results showed that kainic acid infusion into the lwDR increased Fos protein immunostaining in brain structures deeply involved in panic-like defensive behaviors, such as the periaqueductal gray and hypothalamus, but not the amygdala. As observed with kainic acid, bicuculline evoked a pronounced escape response in the circular arena when microinjected in the lwDR, but not in the vlPAG. The escape-promoting effect of bicuculline in the lwDR was also evidenced in the elevated T-maze. These findings strength the view that dysfunction in mechanisms controlling escape in the lwDR is critically implicated in the pathophysiology of panic disorder.

View all citing articles on Scopus

View full text

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

Abstract

Introduction

Section snippets

Animals

Ultrasound Production

Ultrasound Generated Behaviour

Discussion

Acknowledgements

Neurosci.

J. Neurosci. Meths.

Neurosci. Biobehav. Rev.

Physiol. Behav.

Physiology and Behavior

Brain Research

Brain Res.

J. Neurosci. Meths.

Brain Research

Brain. Res.

Neuroscience

Molec. Brain Res.

Brain Res.

Anim. Behav.

Behav. Brain Res.

Brain. Res. Bull.

Brain Research