Research ReportNeurotoxic lesions centered on the perifornical hypothalamus abolish the cardiovascular and behavioral responses of conditioned fear to context but not of restraint
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.
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2020, Neurochemistry InternationalCitation 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).