Elsevier

Brain Research

Volume 1128, 12 January 2007, Pages 107-119
Brain Research

Research Report
Neurotoxic lesions centered on the perifornical hypothalamus abolish the cardiovascular and behavioral responses of conditioned fear to context but not of restraint

https://doi.org/10.1016/j.brainres.2006.10.058Get rights and content

Abstract

The aim of this study was to test the role of the perifornical hypothalamus and adjacent areas in the behavioral and cardiovascular responses to two forms of stress, conditioned fear to context and restraint. Of particular interest was the role of the hypocretin (orexin) containing neurons in these responses. Rats implanted with radio-telemetric probes and fear conditioned to a context received bilateral injections of the neurotoxin hypocretin-2-saporin centered on the perifornical area. One week later, the animals were tested for conditioned fear to context and restraint while recording freezing, 22 kHz ultrasonic vocalizations, activity, mean arterial pressure and heart rate. Histological verification revealed that the lesions were not specific since virtually all the neurons within the injection area were lost. Nevertheless, these lesions, which were centered on the perifornical area, markedly reduced all recorded components of the contextual fear response (by 70%) but had no effect on the response to restraint. The lesions also caused a reduction in body weight and reduced the circadian rhythm of the recorded parameters. The results show (i) that hypocretin-2-saporin was not specific enough to produce lesions restricted to the hypocretin system, (ii) that neurons of the perifornical area are necessary for the expression of both the cardiovascular and behavioral components of conditioned fear to context, and (iii) that the same neurons are not necessary for the cardiovascular response to restraint. Thus, the perifornical hypothalamus is critical for some forms of stress but not others. We propose that it is a necessary relay for emotional responses in which the psychological component is stronger than the sensory component.

Introduction

Conditioned fear is a well-established model of psychological stress. Fear conditioning consists of pairing a once neutral stimulus, such as a tone, with an innately aversive stimulus, such as an electric footshock. As a result, the tone becomes capable of eliciting a stress response when presented alone because it predicts the occurrence of the footshock (i.e., the tone becomes a conditioned stimulus) (Fanselow, 1984, Davis, 1992, Fendt and Fanselow, 1999, Maren, 2001, LeDoux, 2003). In the absence of tone or other discrete stimuli, the conditioned stimulus can be the context or environment (i.e., the footshock chamber). This form of conditioning is known as fear conditioning to context (Fendt and Fanselow, 1999, LeDoux, 2003). Conditioned fear to a tone and to a context differ in the duration of the response. With conditioned fear to a tone, the response lasts for between 10 s and 2 min (Iwata et al., 1986, LeDoux et al., 1988), whereas with contextual fear, the reaction can last for up to 30–40 min (Carrive, 2002, Carrive, 2006).

Previous work by Iwata et al., 1986, LeDoux et al., 1988 has shown that conditioned fear to a tone is associated with a characteristic freezing immobility and an increase in arterial blood pressure of approximately + 15 mm Hg. These authors also showed that these two components of the response are mediated by neurons located in different parts of the brain. More specifically, they demonstrated that excitotoxic lesions of the lateral hypothalamus (LH) abolish the pressor response but not the freezing response (Iwata et al., 1986, LeDoux et al., 1988), whereas excitotoxic lesions of the caudal periaqueductal gray (PAG) abolish the freezing response but not the pressor response (LeDoux et al., 1988). More recent work has extended some of these findings to contextual fear, using new approaches and tools such as radio-telemetric recording, which allows simultaneous recording of behavioral and cardiovascular changes. Thus, work from our laboratory and others has shown that contextual fear is associated not only with freezing and a rise in arterial pressure (+ 25 mm Hg) but also with a rise in heart rate (+ 100 bpm) and the emission of 22 kHz ultrasonic vocalizations (Nijsen et al., 1998, Antoniadis and McDonald, 1999, Carrive, 2002, Vianna and Carrive, 2005, Carrive, 2006). Further, we have shown that temporary blockade of the caudal ventrolateral PAG abolishes freezing and ultrasonic vocalizations, but does not reduce the pressor and tachycardic responses (Walker and Carrive, 2003), which is consistent with LeDoux et al. (1988) findings. However, the role of the hypothalamus in these 4 responses to contextual fear has not yet been tested. In particular, it is not known if the pressor and tachycardic responses to contextual fear are mediated by the hypothalamus.

As described above, the hypothalamic lesions that reduced the pressor response to the conditioned tone in the LeDoux et al. (1988) study were centered on the LH. In most cases these lesions extended medially to the fornix, to include the lateral part of the perifornical (PeF) area. The PeF area, which also corresponds to the classic hypothalamic defense area (Abrahams et al., 1964, Smith et al., 1980, Hilton, 1982), is currently receiving much attention because it is the principle location of neurons containing hypocretin (Hcrt), a neuropeptide also known as orexin. The neurons that produce Hcrt form a restricted group centered on the PeF, but they also extend laterally into LH and medially into the dorsomedial hypothalamic nucleus (DMH) (Sakurai et al., 1998, Chen et al., 1999, Sutcliffe and de Lecea, 2002). Hcrt, which comes in two forms (Hcrt-1 and Hcrt-2), is thought to play an important role in maintaining arousal and wakefulness. Thus activity of Hcrt neurons is higher when animals are awake or active than when asleep or inactive (Estabrooke et al., 2001, Fujiki et al., 2001, Yoshida et al., 2001, Kiyashchenko et al., 2002, Martinez et al., 2002, Wu et al., 2002, Torterolo et al., 2003, Kodama et al., 2005, Mileykovskiy et al., 2005). Hcrt has also been shown to be involved in feeding, motor activity and autonomic regulation, which are well-established functions of the PeF (Kilduff and Peyron, 2000, Sutcliffe and de Lecea, 2002, Sakurai, 2003, Taylor and Samson, 2003, Berridge and Espana, 2005). Finally, there is growing evidence that Hcrt may play a role in stress (Zhang et al., 2006). In particular, it has been shown that Hcrt knock-out mice have a markedly attenuated cardiovascular response to stimulation of the hypothalamic defense area as well as to psychosocial stress (resident–intruder model) compared to wild-type mice (Kayaba et al., 2003). Thus, Hcrt neurons could be involved in psychological stress, and perhaps also in the conditioned fear response, since the lesions in the study of LeDoux et al. (1988) would have included a large portion of Hcrt neurons.

There were two aims in this study. The first aim was to test the role of the PeF area or hypothalamic defense region in the response to contextual fear, not just its pressor and freezing components but also its cardiac and ultrasonic vocalization components. The second aim was to test the role of the Hcrt system in this response. The two aims were combined by testing contextual fear after PeF lesions with the neurotoxin hypocretin-2-saporin (Hcrt-saporin). This toxin has been shown to destroy Hcrt neurons, as well as neurons harbouring Hcrt receptors (Gerashchenko et al., 2001, Gerashchenko et al., 2003). In addition, to find out if this could be generalized to other forms of stress we also tested the response to restraint, a commonly used stressor.

Section snippets

Histology and grouping

The specificity and extent of the Hcrt-saporin lesions were assessed with Hcrt, melanin concentrating hormone (MCH) and Neuronal nuclei (NeuN) immunostaining (Fig. 1). The lesions resulted in loss of Hcrt neurons and in some cases the loss was complete (Figs. 1A, B). However, Hcrt neurons were not the only neurons destroyed. MCH neurons, for example, which have a wider distribution, were also lesioned (Figs. 1C, D). In fact NeuN immunostaining (which was confirmed by Nissl staining, not shown)

Discussion

There were two aims in this study. The first aim was to test the role of the PeF area in the expression of the pressor, cardiac, freezing and ultrasonic responses of contextual fear and of the pressor and cardiac responses of restraint. The second aim, more specific, was to test the role of Hcrt neurons in these two responses. Unfortunately, because the Hcrt-saporin toxin produced lesions that were not restricted to the Hcrt system, it was not possible to determine the role of Hcrt neurons in

Animals

Thirty four male Wistar rats (400–500 g) obtained from the colony of specific pathogen-free rats maintained by the University of New South Wales were used. Rats were individually housed in plastic boxes with metal lids (65 × 40 × 22 cm) on a 12:12 light–dark cycle (lights on 8 am) with ad lib access to food and water. The procedures were approved by the animal ethics committee of the University of New South Wales and conformed to the rules and guidelines on animal experiments in Australia.

Experimental design

The rats

Acknowledgment

This work was supported by the National Heart Foundation of Australia.

References (64)

  • M. Fendt et al.

    The neuroanatomical and neurochemical basis of conditioned fear

    Neurosci. Biochem. Rev.

    (1999)
  • D. Gerashchenko et al.

    Effects of lateral hypothalamic lesion with the neurotoxin hypocretin-2-saporin on sleep in Long-Evans rats

    Neuroscience

    (2003)
  • J. Hara et al.

    Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity

    Neuron

    (2001)
  • G.J. Hervieu et al.

    Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord

    Neuroscience

    (2001)
  • J. Iwata et al.

    Destruction of intrinsic neurons in the lateral hypothalamus disrupts the classical conditioning of autonomic but not behavioral emotional responses in the rat

    Brain Res.

    (1986)
  • T.S. Kilduff et al.

    The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders

    Trends Neurosci.

    (2000)
  • T. Kodama et al.

    High Fos expression during the active phase in orexin neurons of a diurnal rodent, Tamias sibiricus barberi

    Peptides

    (2005)
  • G.S. Martinez et al.

    Diurnal and nocturnal rodents show rhythms in orexinergic neurons

    Brain Res.

    (2002)
  • B.Y. Mileykovskiy et al.

    Behavioral correlates of activity in identified hypocretin/orexin neurons

    Neuron

    (2005)
  • M.J. Nijsen et al.

    Conditioned fear-induced tachycardia in the rat: vagal involvement

    Eur. J. Neurosci.

    (1998)
  • T. Sakurai et al.

    Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior

    Cell

    (1998)
  • C.B. Saper et al.

    The hypothalamic integrator for circadian rhythms

    Trends Neurosci.

    (2005)
  • A. Shekhar et al.

    Defense reaction elicited by injection of GABA antagonists and synthesis inhibitors into the posterior hypothalamus in rats

    Neuropharmacology

    (1987)
  • P. Trivedi et al.

    Distribution of orexin receptor mRNA in the rat brain

    FEBS Lett.

    (1998)
  • P. Walker et al.

    Role of ventrolateral periaqueductal gray neurons in the behavioral and cardiovascular responses to contextual conditioned fear and poststress recovery

    Neuroscience

    (2003)
  • W. Zhang et al.

    Multiple components of the defense response depend on orexin: evidence from orexin knockout mice and orexin neuron-ablated mice

    Auton. Neurosci.

    (2006)
  • V.C. Abrahams et al.

    The role of active muscle vasodilatation in the early stage of the defence reaction

    J. Physiol. (Lond.)

    (1964)
  • M. Backberg et al.

    Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake

    Eur. J. Neurosci.

    (2002)
  • C.T. Beuckmann et al.

    Orexins: from neuropeptides to energy homeostasis and sleep/wake regulation

    J. Mol. Med.

    (2002)
  • P. Carrive

    Dual activation of cardiac sympathetic and parasympathetic components during conditioned fear to context in the rat

    Clin. Exp. Pharmacol. Physiol.

    (2006)
  • T.C. Chou et al.

    Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral rhythms

    J. Neurosci.

    (2003)
  • M. Davis

    The role of the amygdala in fear and anxiety

    Annu. Rev. Neurosci.

    (1992)
  • Cited by (55)

    • Dopamine D<inf>4</inf> receptors in the lateral habenula regulate depression-related behaviors via a pre-synaptic mechanism in experimental Parkinson's disease

      2020, Neurochemistry International
      Citation Excerpt :

      To determine the extent of dopaminergic neuron degeneration in the SNc and VTA of rats with unilateral sham lesions and 6-OHDA lesions of the SNc, the sections were examined for immunohistochemical staining of tyrosine hydroxylase (TH) as previously described (n = 30–32 rats/group; Wang et al., 2009). The extent of ibotenic acid lesions in the RMTg was determined by immunohistochemistry of neuronal nuclei (NeuN; n = 32 rats/group) according to the method of Furlong and Carrive (2007). The stained sections were examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DP71, Olympus) interfaced to a computer with Image-Pro Plus (version 5.1; Media Cybernetics, Inc., MD, USA).

    View all citing articles on Scopus
    View full text