The Britannica Encyclopaedia defines pain as - A complex experience consisting of a physiological (bodily) response to a noxious stimulus followed by an affective (emotional) response to that event. Pain is a warning mechanism that helps to protect an organism by influencing it to withdraw from harmful stimuli. It is primarily associated with injury or the threat of injury, to bodily tissues. Thus pain processing is a multi-facetted experience that integrates the functioning of several limbic and cortical structures to coordinate the behavioural response to nociceptive stimulation. There are two major types of pain. Acute pain is of sudden onset, lasting for hours to days and disappears once the underlying cause is treated. Acute pain is due to peripheral or visceral noxious stimuli trigger a cascade of physiological events that propagate neural signals to the brain, which are then integrated and processed by limbic and cortical structures to manifest as a painful sensation. Acute pain signals that there is something wrong and motivates the person to get help. Chronic pain begins as acute pain and continues beyond the normal time expected for resolution of the problem or persists or recurs for various other reasons and is no longer therapeutically beneficial to the patient. In the treatment of acute pain, attention is focused to treat the cause of pain, whereas in chronic pain the emphasis is laid upon reducing the pain to give relief, limit disability and improve function. Chronic pain can be further divided into neuropathic and nociceptive pain. Neuropathic pain is caused by damage to nerve tissue. It is often felt as a burning, crawling, stabbing or shocking pain. Nociceptive pain is caused by an injury or disease outside the nervous system. It is often an ongoing dull ache or pressure, rather than the sharper, trauma-like pain that is characteristic of neuropathic pain. The annual cost of treatment of chronic pain in the United States, is estimated to be $100 billion annually, (Holden and Pizzi et al., 2003). More than half of all hospitalized patients experienced pain in the last days of their lives and although therapies are present to alleviate most pain for those with terminal illnesses, research shows that 50-75% of patients die in moderate to severe pain, (Weiss et al., 2001). In Europe, one in five people suffer from chronic pain of moderate-to-severe intensity (Holden and Pizzi, 2003; Breivik et al., 2006).
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Pain - 76.2 million people, National Centres for Health Statistics
Diabetes - 20.8 million people (diagnosed and estimated undiagnosed), American Diabetes Association
Coronary Heart Disease (including heart attack and chest pain) and Stroke - 18.7 million people, American Heart Association
Cancer - 1.4 million people, American Cancer Society
Figure 1: National Center for Health Statistics Report: Health, United States, 2006, Special Feature on Pain.
1.1 The Pain Pathway
The sensation of pain usually depends on the activation of a set of neurons that includes primary afferent nociceptors, interneurons in the spinal cord, cells of the ascending tracts, thalamic neurons and neurons of the cerebral cortex. Hence, the pain system involves a set of ascending pathways that convey nociceptive information from peripheral nociceptors to higher levels of the central nervous system, as well as descending pathways that modulate that information (Bromm & Desmedth, 1995). The body's peripheral nociceptors (first-order neurons) project to second-order neurons in the spinal cord and medulla, which then carry the sensory information in the form of electrical impulse to the thalamus, where it synapses with third-order neurons that transmit the impulse to the cortex. Second-order neurons send their sensory inputs to the thalamus via two ascending pathways: the spinothalamic and spinoparabrachial pathways. The former transmits impulse involving position, sense, touch, and pressure. The latter pathway is involved in pain transmission (Karoly & Jensen 1987). Once the stimulus has been interpreted as painful, an endogenous analgesic descending inhibitory pain pathway is activated that allows the brain to modulate the severity of the pain sensation. Hence, the primarily role of the descending inhibitory pain pathway is to send chemical messages from the brain to close the gates in the spinal cord to ascending messages (Catalano, 1987, Wells & Nown, 1998). This transmission is thought to originate from neurons located in the amygdalar and hypothalamic brain regions and is relayed through the periaqueductal grey and rostral ventromedial medulla to the dorsal horn of the spinal cord, which exerts an inhibitory effect at the dorsal horn laminae resulting in the suppression of the ascending pain pathway.
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Fig. (1). The two main ascending spinal pathways are the spinothalamic and spinoparabrachial pathways.
PAG: periaqueductal grey; RVM: rostroventral medulla
1.2 Suppression of Nociceptive Behaviour by Stress or Fear
Neural systems exist within the mammalian central nervous system, that are capable of activating descending inhibitory pain pathway, which suppress the transmission of noxious information from the periphery to the CNS. Studies in rodents showed a reduction in nociceptive behaviours upon exposure to aversive stimuli (Akil et al., 1976a; Madden et al., 1977) including stimuli associated with learned fear (Calcagnetti et al., 1987; Chance et al.,1978; Finn et al., 2004, 2006; Harris and Westbrook, 1995; Helmstetter and Fanselow, 1987a, b; Roche et al., 2007). This antinociceptive response, termed stress-induced analgesia, may be activated by a wide range of environmental stressors including the presence of predators or aggressive conspecifics, (Fanselow et al., 1986, Miczek et al., 1985), as well as electric shock, (Maier et al., 1990, Terman et al., 1984, Watkins et al., 1986). Aversive behaviours (e.g. fear, anxiety, panic), or behaviours to avoid pain, may be viewed as part of an organism's defence system against stimuli that could cause pain (Bolles and Fanselow, 1980). Brain regions critically involved in the descending inhibitory pain pathway include the basolateral amygdaloid complex (BLA), central nucleus of the amygdala (CeA), periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM) and neuronal activity in these regions also subserves expression of fear. SIA is also expressed in humans (Flor et al., 2002; Flor and Grusser, 1999; Willer et al., 1981) and studies of the mechanisms involved in rodent models of SIA are likely to be relevant in humans. From an evolutionary perspective, SIA may be thought of as a component of the fight or flight response. Tending to a painful injury would not be conducive to the survival of an organism if further injury or death were threatened. Once the organism is no longer in danger, however, elevated nociception, expressed upon extinction of the aversive response, could be beneficial as normal behaviours may aggravate the injury. Predator-prey interactions most likely played a major role in the evolutionary development of SIA. Mice selectively bred for either a high sensitivity (HA) or a low sensitivity (LA) to SIA initiated by swim tests followed by the tail flick test of nociception have been the primary models utilised to find a genetic basis for this phenomenon (Panocka et al., 1986b). Two stimuli are needed to model SIA, a noxious and an aversive stimulus, and there are two main models of SIA:
In unconditioned SIA, an unconditioned stressful stimulus or environmental stressor is used to induce physiological changes to neural pathway that affect the subsequent noxious stimuli. Unconditioned aversive stimuli that have been used in these models include, foot shock, forced swim test, elevated plus maze, exposure to novel arena, social conflict or predators such as biting mice, biting flies, cats or snakes. Noxious stimuli used also vary depending on the model and species. These include injection of the chemical irritants such as formalin, carageenan, complete freunds adjuvant, radiant heat, insect bites, tail pinches, and intracutaneous electrical current. One study carried out by Helmstetter et al., 1993 demonstrated that microinjections of a benzodiazepine into the amygdala will attenuate hypoalgesic responses by antagonizing conditioned fear. They did this by shocking rats in a novel environment and measuring the subsequent time they spent freezing and responding to the pain produced by a subcutaneuous injection of formalin into the hind paw. Animals shocked and tested in a single session, under the influence of the benzodiazepine, spent more time responding to pain and less time freezing because the drug had prevented either learning about the context/shock association or the fear resulting from that learning (Harris et al., 1995; Fanselow & Helmstetter, 1988; Izquierdo, Cunha, & Medina, 1990; Westbrook et al., 1991). The SIA is an acute model as the pain test is carried almost immediately after the unconditioned aversive stimulus has been applied.
Conditioned SIA or Fear-Conditioned Analgesia
Fear-conditioned analgesia (FCA) is the phenomenon by which re-exposure to a neutral, non-aversive stimulus which has previously been paired with a noxious stimulus results in conditional analgesia, (Finn et al., 2006). Thus the memory of the noxious stimulus is enough to cause neurochemical changes in the animal, which result in the expression of fear behaviour. FCA is characterised by a robust decrease in nociception in rodent models that have been exposed to Pavlovian fear-conditioning, which at its peak can suppress behaviours associated with pain by over 90%, (Finn et al., 2004, Harris and Westbrook, 1995). The ability to induce FCA in rodents provides an excellent model with which to study the physiology of endogenous analgesic and aversive systems (Finn et al., 2006). Commonly used experimental models of FCA involve the exposure of rats to noxious thermal stimuli (Harris et al., 1993; Harris and Westbrook., 1994) or foot shock (Calcagnetti et al., 1987; Helmstetter and Fanselow, 1987; Fanselow and Helmstetter, 1988; Finn et al., 2004a, Finn et al., 2006), and the measurement of formalin-evoked nociceptive responses 24 h later in the same apparatus in the absence of further aversive noxious thermal stimuli or footshocks.
The Neural Circuitry of Fear-Conditioned Analgesia
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A series of experiments have determined that the amygdala is critical for the learning and expression of classical fear-conditioning (Blanchard and Blanchard, 1972; Kellicut and Schwartzbaum 1963; Spevack et al., 1975). The amygdala is a major component of the limbic system and as such plays an essential role in the processing and expression of emotional responses including, pain, fear, and fear-conditioned analgesia. Helmstetter et al., (1992) investigated amygdala-mediated hypoalgesia in rats as a Pavlovian conditioned response and found that electrolytic lesions of both the central nucleus and basolateral nucleus of the amygdala in separate rat groups resulted in attenuated freezing behaviour and also conditioned hypoalgesia compared to control groups. Similar effects have also been seen with chemical lesions produced by ibotenic acid Helmstetter et al., (1992), adding further weight to the theory that the amygdala is important in the expression of both fear and pain. The periaqueductal gray (PAG) also plays a role in the descending modulation of pain and in defensive behaviour. The ascending fibres of the spinothalamic tract also send information to the PAG via the spinomesencephalic tract. Stimulation of the dorsal and lateral aspects of the PAG (in the rat) can provoke defensive responses characterised by freezing immobility, running, jumping, tachycardia, and increases in blood pressure and muscle tonus. In contrast, stimulation of the caudal ventrolateral PAG can result in an immobile, relaxed posture known as quiescence, whereas its inhibition leads to increased locomotor activity. The stimulation at sites throughout the periaqueductal gray matter (PAG) can evoke behavioral analgesia in rats, cats, monkeys, and humans (Reynolds, 1969; Mayer et al., 1971; Liebeskind et al., 1973; Richardson and Akil, 1977; Fardin et al., 1984). The analgesia is mediated, at least in part, by descending pathways that inhibit the responses of dorsal horn neurons to noxious stimuli (Liebeskind et al., 1973; Willis, 1982). Thus the PAG is heavily involved in the fight or flight mechanism.
Figure 2: Proposed Neural Circuitry in the Amygdala
Paré et al., 2004, proposed that in the current experimental model of fear-conditioning, the convergence of neutral sensory inputs (conditioned stimulus, CS), and noxious unconditioned stimulus (US) inputs, increases the efficacy of synapses conveying information about the conditioned stimulus, to the lateral nucleus of the amygdala ( Le-Doux, 2000; Walker and Davis 2000). As a result, subsequent presentations of the conditioned stimulus alone evoke larger responses in the basolateral amygdala, (Collins and Paré, 2000; Quirk et al., 1995; Repa et al., 2001). The BLA, in turn, evokes conditioned fear responses via its projections to the central amygdaloid nucleus, which is the main source of amygdalar outputs to the PAG, brainstem and hypothalamic sites that produce fear responses. Thus, in the current model, the BLA is seen as the major site of plasticity, whereas the central nucleus of the amygdala CE is viewed as a passive relay to downstream structures, (Paré et al., 2004). At the core of this model are direct projections from the BLA to the CE to the brainstem. However in 1978 Krettek and Price published a study on the internuclear projections of the rat and cat amygdala that shows that the BLA has little if any projection to the medial sector of the central nucleus, (CEm), but rather projects to the lateral or amygdalo-striatal sectors of the CE, which was later replicated in the rat (Pitkanen et al. 1995), cat (Smith and Paré 1994), and monkey (Pitkanen and Amaral 1998). Thus, there is an apparent disconnect in the amygdala between the site of plasticity and site of expression. Paré et al. 2004, proposed a revised model whereby the BLA facilitates the activity of brain stem projecting CE neurons via intercalated cell masses (ITC). ITC cell masses are dense clusters of GABAergic neurons located between the basolateral amygdaloid complex and the CE (Mc-Donald and Augustine 1993; Nitecka and Ben Ari 1987; Paré and Smith 1993). ITC cells also receive glutamatergic inputs from the BLA and generate feed-forward inhibition in the CE, (Royer et al. 1999). Hence subsequent re-exposure to the context where the aversive events occurred results in more pronounced responses in neurons in the BLA (Rodriguez Manzanares et al., 2005), which then further relay the information through GABAergic intercalated cells (Royer and Pare, 2002, Pare et al., 2004), disinhibiting medially located CE neurons to facilitate CEm output (Paré et al. 2004). The medial sector of the CeA is thought to be the main source of amygdalar outputs to the PAG and hypothalamic sites responsible for fear behaviour (Bellgowan and Helmstetter, 1996, De Oca et al., 1998, Davis and Shi, 2000, LeDoux, 2007). Indeed, neuronal projections from the CeA to the PAG (Hopkins and Holstege, 1978, Mantyh, 1982, Li et al., 1990, Rizvi et al., 1991, Vianna and Brandao, 2003) are strongly involved in the endogenous aversive and analgesic systems. The manipulation of this pathway results in both the expression of fear-related behaviour (e.g. freezing and 22-kilohertz ultrasonic vocalisations) (Pitkanen et al., 1997, Davis et al., 2003) and robust analgesia - as a consequence of activating the descending inhibitory pain pathway (Helmstetter et al., 1998, Manning, 1998, Pavlovic and Bodnar, 1998, Oliveira and Prado, 2001, Millan, 2002). The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization (Oka et al., 2008). These researchers examined how the amygdaloid complex influences the PAG-NRA pathway by examining the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA using anterograde and retrograde tract-tracing techniques in the rat. Their results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the PAG-projecting GABAergic CeA neurons.
1.4 The Endocannabinoid Systems
The endocannabinoid system refers to a group of neuromodulatory lipids and their receptors that are involved in a variety of physiological processes including appetite, pain-sensation, mood and memory. It is comprised of the cannabinoid1 (CB1) receptor, cannabinoid2 (CB2) receptor, other putative receptors such as PPAR receptors, GPR55, TRPV1 receptors, endogenous cannabinoid ligands, their metabolizing enzymes and a putative anandamide uptake site, (Rea et al., 2007). Twenty-four years of pharmacological research separate the identification of (-)-âˆ†9-tetrahydrocannabinol (THC) (Gaoni and Mechoulam, 1964; Mechoulam 1970), from the characterization, (Devane et al., 1988, Herkenham et al, 1991) and molecular cloning (Matsuda et al., 1990) of its cellular target, the cannabinoid receptor (CB1). The discovery of the endocannabinoid receptor and the availability of highly selective and potent cannabimimetics lead to the rapid identification of a family of lipid transmitters that serve as natural ligands for the CB1 receptor. The most studied of these endocannabinoids are arachidonnylethanolamide (AEA) named anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (2-AG), (Mechoulam et al., 1995; Suguira et al., 1995). Endocannabinoids are derivatives of arachidonic acid conjugated with ethanolamine or glycerol, (F.R. De Fonseca et al., 2004), and are most likely synthesized on demand and function as retrograde messengers, (Kreitzer et al, 2004).
The CB1 and CB2 receptors are the most studied target receptors for the endocannabinoids. Both are members of the family of Gi/o-protein coupled receptors and are negatively coupled to adenylyl cyclase, (Finn and Chapman, 2004). CB1 receptors are encoded by the CNR1 gene and are believed to be the most widely expressed G-protein coupled receptors in the brain. They are expressed presynaptically on neurons in both the peripheral and central nervous system, as well as on a wide range of peripheral tissues. CB2 receptors are coded for by the CNR2 gene are expressed largely in non-neural tissues including immune cells, although there is a growing body of evidence that CB2 receptor protein and mRNA is also expressed in the brain, (Van Sickle et al., 2005), and spinal cord, (Beltramo et al.m 2006). Activation of the CB1 receptor leads to multiple intracellular signal transduction pathways being activated. Initially it was thought that cannabinoid receptors mainly activated the G protein Gi, which inhibits the enzyme adenyl cyclase, (Demuth et al., 2006). However, a much more complex picture has emerged in different cell types, implicating other potassium ion channels, calcium channels, protein kinase A and C, Raf-1, ERK, JNK, p38 c-fos, c-jun and many more, (Pagotto et al., 2006). It follows that by altering the conductance of these two ions into selected neurons, CB1 receptors may modulate the release of a variety of neurotransmitters in the body, including GABA and Glutamate.
Finn and Chapmann, 2004, provide evidence that suggests that endocannabinoids exert tonic control of nociception and the distribution of CB1 receptors in the brain suggests several anatomical regions where endocannabinoid action could modulate FCA. These include the dorsal root ganglia, spinal cord, thalamus, periaqueductal grey, rostroventromedial medulla and the amygdala. As we have already seen the amygdala is implicated in both fear conditioning and pain. Herkenham et al., 1991, found that CB1 immunoreactivity is dense in the BLA, but reportedly absent in the central nucleus of the amygdala, a result replicated by Katona et al., 2001. The anatomical localization of CB1 receptors in the BLA is consistent with electrophysiological data demonstrating that activation of these receptors presynaptically modulates GABAergic transmission and thus an endocannabinoid-mediated reduction of GABA release would disinhibit principal neurons innervating the central nucleus of the amygdala, to control information processing in the amygdala (Connel et al., 2006). Martin et al., 1999, found that unilateral microinjection of cannabinoid agonists into the amygdala suppressed nociceptive behavioural responses in the tail-flick test and unilateral or bilateral lesions of the CeA suppress the antinociception effects elicited by systemic cannabinoids, (Manning et al., 2003). Connel et al., 2006 found pro-nociceptive effects, following intra-BLA administration of the CB1 receptor antagonist SR141716A (rimonobant), in a stress-induced analgesic model. Mariscano et al., 2002, also found that endocannabinoid signalling in the BLA also mediates extinction of aversive memories. All of these studies supported a role for the endogenous cannabinergic system in the modulation of pain sensitivity in the BLA, most likely through the modulation of CB1 receptor containing GABAergic neurons in the BLA. In the FCA model, it was shown that the intraperitoneal administration of the SR141716A (1mg/kg) attenuated the expression of FCA in fear-conditioned formalin-treated rats, (Finn et al., 2004). In a follow up study it was found that the intraperitoneal administration of a fatty acid amide hydrolase inhibitor and endocannabinoid catabolism inhibitor (URB597), enhanced FCA expression in fear-conditioned formalin-treated rats. This effect was attenuated by intraperitoneal co-administration of URB597 with CB1 and CB2 receptor antagonists SR121716A and SR144528 respectively, (Butler et al., 2008). These studies implicate an important role for the CB1 receptor in FCA. Interestingly, Roche et al., 2007, found that the unilateral administration of SR141716A into the right BLA of fear-conditioned formalin-treated rats, failed to attenuate the expression of FCA. It was also found to cause a suppression of formalin-evoked nociceptive behaviour and attenuated the formalin-induced decrease in freezing and 22-kHz ultrasonic vocalization. However further work in this lab found that the bilateral administration of AM251, another CB1 receptor antagonist, into the BLA of fear-conditioned formalin treated rats resulted in the suppression of FCA, which conversely enhanced the nociceptive response when compared with controls. This adds further weight to the theory that CB1 receptors in the BLA mediate FCA, but as of yet the precise mechanisms that underlie the modulation of pain by cannabinoids are unclear. However, extensive experimental and clinical evidence suggests a presynaptic location of cannabinoid receptors on GABAergic and glutamatergic neurons in brain regions associated with pain modulation. Moreover, a large body of evidence implicates GABA and glutamate in the regulation of pain, and functional studies have demonstrated that the body's own endogenous cannabinoids control the release of these neurotransmitters.
1.5 The GABAergic system
Gamma-aminobutyric acid (GABA) is the chief inhibitory neurotransmitter in the mammalian central nervous system and it plays a key role in regulating neuronal excitability throughout the nervous system in humans. GABA is synthesized in vivo from glutamate using the enzyme L-glutamic acid decarboxylase and pyridoxal phosphate and exerts its effects on two principal classes of GABA receptors: GABAA and GABAB. It has long been recognised that the fast response of neurons to GABA is due to the direct opening of chloride ion channels, an event that is blocked by bicuculline and picrotoxin. Thus the GABAA receptors are ligand-gated ion channel receptors. GABAA receptors have a pentameric structure composed of seven different subtype families (Whiting et al 1999). Thus, there is a wide array of GABAA receptors with differing subunit compositions. GABAB receptors are G-protein coupled receptors that mediate slower pharmacological responses to GABA. Presynaptically located GABAB receptors are negatively coupled to adenylyl cyclase and decrease the conductance of calcium ion into the cell, which results in hyperpolarisation. Post-synaptic GABAB receptors regulate potassium channels and their activation results in slow cellular hyperpolarisation due to the opening of these channels, (Kerr and Ong 1995).
Previous exposure to both acute and chronic stressful events can positively affect classical conditioning tasks, including fear conditioning (Shors et al., 1992; Beylin and Shors, 1998; Shors, 2001; Cordero et al., 2003). Pharmacological studies support this view, because drugs acting on the GABAA receptor complex critically influence the behavioral responses to environmental challenges (Petersen et al., 1985; Biggio et al., 1990; Fanselow and Kim, 1992; Cancela et al., 1995; Cole et al., 1995; Jasnow and Huhman, 2001; Maren, 2001). Together, these studies suggest a functional association between changes in the activation of GABAA sites in selected corticolimbic areas and the occurrence of fear and anxiety. One candidate structure that could be a substrate for such function is the amygdala (Ledoux, 1993; Maren, 2001; McGaugh et al., 2002). A substantial number of studies have demonstrated that the basolateral complex of the amygdala (BLA) contains a powerful inhibitory circuit that uses GABA as a neurotransmitter (Takagi and Yamamato, 1981; Washburn and Moises, 1992a,b). Evidence suggests a critical role for GABA signalling in the BLA in the expression of pain, fear and fear-conditioned analgesia, as the basolateral amygdala (BLA) is a key component of descending modulatory pain pathways, and it has been shown that CB1 receptors are widely expressed on GABAergic neurons in the BLA, (Katona et al., 2001).
Early work implicating a role for the GABAA receptor in the mediation of fear behaviour and associated analgesia in rats, used the GABAA receptor agonistic drugs, benzodiazepines. (Fanselow and Helmstetter 1988) found that the intraperitoneal administration of benzodiazepines (Midazolam, Chloridiazepoxide and Diazepam) resulted in the significant attenuation of fear-conditioned analgesia. In a follow up study, (Helmstetter 1992) studied the effects of bilateral injections of diazepam directly into the basolateral amygdala and found that it resulted in an attenuation of both defensive freezing behaviour and hypoalgesia in fear-conditioned rats. They also found that the same dose of diazepam injected into the central amygdala attenuated freezing behaviour without the consequent suppression of hypoalgesia. These studies provide further evidence implicating a critical role for GABAergic transmission in the BLA in mediating FCA.
In experiments designed to investigate the relationship between stress and the acquisition of new fear memories, it was found that previous exposure to a restraint session increased fear conditioning in a contextual fear paradigm. The infusion of bicuculline, a competitive antagonist of GABAA receptors, into the basolateral amygdala complex (BLA), but not into the central amygdaloid nucleus, induced the same behavioral effect (Manzanares et al 2005). Pretreatment with midazolam (MDZ), prevented the facilitating influence on fear memory of stress (Manzanares et al 2005). This data suggest that facilitation of fear conditioning could be causally related to increased neuronal excitability attributable to depressed GABAergic inhibition in the BLA. All of these findings support the hypothesis that previous stress attenuates inhibitory GABAergic control in the BLA, leading to neuronal hyperexcitability and increased plasticity that facilitates fear learning.
A recent microdialysis study from our lab, reported a significant suppression of GABA levels in the BLA of fear-conditioned rats compared with non fear-conditioned controls, suggesting that reduced GABAergic signalling in the BLA may facilitate the expression of conditioned fear (Rea et al., 2009). In a follow up study, Rea et al., 2009, investigated the effects of intra-BLA administration of the GABAA receptor agonist, muscimol, on the expression of conditioned fear, formalin-evoked nociception and fear-conditioned analgesia in rats, and they found that intra-BLA microinjection of muscimol had no effect on formalin-evoked nociceptive behaviour but prevented the expression of fear-conditioned analgesia. Therefore it can be concluded that the GABAA receptor plays a role in FCA. The fact that muscimol, a GABAA agonist prevented the expression of FCA, and bicuculline, a GABAA antagonist appeared to have no effect, suggests that GABA levels are reduced in FCA.
1.6 Glutamatergic system
Glutamate is the most abundant excitatory neurotransmitter in the vertebrate nervous system. Because of its role in synaptic plasticity, glutamate is involved in cognitive functions like learning and memory in the brain, (McEntee and Crook 1993). The form of plasticity known as long-term potentiation takes place at glutamatergic synapses in the hippocampus, neocortex, and other parts of the brain, including the amygdala. Glutamate receptors are synaptic receptors located primarily on the membranes of neuronal cell and are involved in neural communication, memory formation, learning, and regulation. Glutamate receptors can be divided into two groups according to the mechanism by which their activation gives rise to a postsynaptic current. Ionotropic glutamate receptors (iGluRs) form the ion channel pore that activates when glutamate binds to the receptor. Metabotropic glutamate receptors (mGluRs) indirectly activate ion-channels on the plasma membrane through a signaling cascade that involves G proteins. Ionotropic receptors tend to be quicker in relaying information but metabotropic are associated with a more prolonged stimulus, G-protein coupled receptors often cause a signalling cascade. There are currently eight known mGluRs that can be further divided into three sub-groups, with varying reported effects on nociceptive behaviours (see review, Rea et al. 2009).
There is an accumulating body of neurochemical, pharmacological, electrophysiological and behavioural evidence implicating glutamate, and more specifically type 1 mGluR's in fear conditioning in rats, (for recent reviews see Bleakman et al., 2006; Enna and McCarson, 2006). Lu et al., 1997 and Xu et al., 2009, both showed that mGlu5R ''knockout mice'' demonstrate less contextual fear in a test carried out 24 hours after fear conditioning in rats, suggesting a role for mGlu5 receptors in the expression of conditioned fear. Rodrigues et al., 2002, showed using MPEP, that the activation of amygdalar mGluR5 is required for the acquisition of fear conditioning. Using immunocytochemical processes, Rodriguez confirmed the localization of mGlu5 receptor in the lateral nucleus of the amygdala, and showed that the intra-amygdaloid administration of MPEP, an mGlu5 receptor antagonist, dose-dependently blocked the acquisition of auditory and contextual fear conditioning in rats, but did not affect their expression or consolidation. These results were reinforced by the findings of Fendt et al, (2002), who found similar effects with a much lower dose of MPEP (5ÂµM/ 0.5ÂµL versus 40ÂµM/ 0.5ÂµL). Palazzo et al., 2001, found that MPEP, an mGlu5 receptor antagonist reduced the latency to respond to a noxious stimulus and reversed the analgesic effects observed following the microinjection of DHPG, a selective mGlu5 receptor agonist, into the PAG.
The most recent published study by Rudy et al., 2009, verifies the involvement of glutamatergic transmission in the acquisition of fear conditioning. They found that the intra-BLA microinjection of the group 1 mGlu receptor agonist DHPG, enhanced freezing behaviour during re-exposure to an environment previously paired with a noxious stimulus, (i.e. footshock). Furthermore, they discovered that the co-administration of DHPG with either a group 1 mGLU receptor antagonist AIDA, or with the selective mGlu5 receptor antagonist MPEP, suppressed any enhancement of stereotypical fear behaviour induced by DHPG administration. These findings further implicate a role for the mGlu5 receptor in the expression of conditioned fear. Initially it was thought that activated mGluR's, caused their effects solely through the activation of Phospholipase C (PLC) and adenylate cyclase. However recent data from studies such as Davies et al., (2002), have begun to uncover alternative signalling pathways on which many of the mGluR-mediated physiological functions depend and one such novel signalling mechanisms is the endocannabinoid system.
1.7 Rationale, Aims and Hypothesis
There is a large body of research implicating the cannabinergic, glutamatergic and GABAergic systems in FCA, yet very few studies have examined potential relationships that may exist between these endogenous systems in the mediation of this effect. In previous studies performed in this lab, intra-basolateral amygdala administration of the CB1 receptor antagonist AM251 suppressed fear and associated fear-conditioned analgesia in rats. Similarly, intra-basolateral amygdala administration of muscimol, resulted in the elimination of fear-conditioned analgesia in rats. This suggests that there is an overlap between the two systems in the processing of nociception in the BLA. Our hypothesis is that upon re-exposing a rat to an environment previously paired with a noxious stimulus (i.e. foot-shock), endocannabinoids are produced which utilise the GABAergic system to mediate FCA. To test this hypothesis we are using antagonists of the CB1 receptor and the GABAA receptor. We postulate, that the systemic administration of AM251, causing a CB1 receptor blockade, would lead to an enhancement of GABA release, and suppression of FCA. By co-administering bicuculline with AM251 we aim to cause a reversal of these effects by preventing the activation of the GABAA receptor located post-synaptically to the neuron and maintaining FCA expression in rats.
Figure 3:Possible mechanism for endocannabinoid-mediated control of nociception.
(A) Diagrammatical representation of some of the interactions between various brain regions of the descending pain pathway. The PAG receives critical input from various cortical areas as well as from the hypothalamus and amygdala. The net input of afferent neurons to the PAG determines the firing of the various PAG cell types. (B) (C) The circled section of (B), illustrates the possible mechanism behind cannabinoid-mediated antinociception. The activation of various receptor subtypes leads to an increase in intracellular calcium by various pathways. This increase in calcium concentration initiates endocannabinoid synthesis and release. The released endocannabinoids can then prevent the presynaptic release of neurotransmitters possibly by inhibiting calcium influx or vesicular release of neurotransmitters.
There is also evidence to suggest that the activation of amygdalar mGluR5 is required for the acquisition of fear conditioning and studies have shown that mGlu5 receptor antagonists, dose-dependently block the acquisition of auditory and contextual fear conditioning in rats, but do not affect their expression or consolidation. To this end, we aim to investigate the effect of systemic AM251 administration on FCA in the presence and absence of the mGlu5 receptor selective antagonist, MPEP.
2.0 Materials and Methods
Experiments were carried out on male Lister-hooded rats (225-250 g, Charles River, Margate Kent, UK), housed in plastic bottomed cages (45 x 25 x 20 cm), with wood shavings as bedding. Rats were housed in groups of three prior to surgery and singly housed post-surgery. The rats were maintained at a constant temperature (20 Â± 2 Â° C) under standard lighting conditions (12 : 12 h light : dark, lights on from 08.00 to 20.00 h). All of the experiments were carried out during the light phase between 09.00 h and 18.00 h. Food and water were ad-libitum and the bedding was changed twice weekly. The experimental procedure was carried out in accordance with the guidelines of the Animal Welfare Committee, national University of Ireland, Galway, under licence from the Irish Department of Health and Children, and in compliance with the European Communities Council directive 86/609.
The CB1 receptor antagonist AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) was supplied by Ascent scientific, Bristol, UK (Asc-088), and administered systemically through intraperitoneal injection at a dose of 3mg/kg. 50mg AM251 was first dissolved in 2.49999mL of pure ethanol, and then 330ïL aliquots of the solution were removed and stored in the freezer. The freezing process causes AM251 to come out of solution and so on test days it is gently heated and sonicated to dissolve it, before being further dissolved in a mixture of ethanol/AM251 mixture: cremaphore: saline solution of 1:1:18. 330ïL of ethanol/AM251 solution is mixed with 330ïL of cremaphore and 5.94mL of sterile saline. Due to the difficulty in dissolving this compound we had to make the drug up in a concentration of 1mg/mL, and so 3 times the body weight of the rat was administered to equate to 3mg/mL. e.g. A 350g rat received 1.05mL of a 1mg/mL AM251 solution.
The GABAA receptor antagonist Bicuculline Methbromide ([R-(R*,S*)]-5-(6,8-Dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-gisoquinolinium bromide), was supplied by Sigma-Aldrich, Airton Rd., Tallaght, Dublin (B7561-25MG). The solution was made up by dissolving 3.1461mg in 50mL of sterile saline to equate to a final volume of 25ng in each 0.5ïL aliquot. The solution was vortex, aliqoutted and then stored in the freezer and thawed out on the test days as required. The final dose of bicuculline being microinjected into the basolateral amygdala was 25ng/0.5ïL.
The mGlu5 receptor antagonist MPEP (2-Methyl-6-(phenylethynyl) pyridine), was supplied by Sigma-Aldrich, Airton Rd., Tallaght, Dublin (M5435-5MG). 20mM of stock solution was first made up by adding 1.088mL sterile saline to 5.0mg MPEP, which was vortexed and then further dissolved 1:100 again in sterile saline to give a final concentration of 200ïïï€®ï€ Stock solutions were stored in a freezer and thawed on the test day as required. 0.5ïL of a 200ïM solution of MPEP was microinjected into the basolateral amygdala on the test day.
50ïL of 37% formaldehyde solution, supplied by Sigma-Aldrich, Airton Rd., Tallaght, Dublin, was mixed with 690ïL of sterile saline, diluting the solution to 2.5%. On the test day, each animal received a 50ïL subcutaneous formalin injection into the right hind paw. All of the compounds were administered on the test day while the animals were under a brief (2-3min) isoflurane anaesthetic 30 minutes prior to re-exposure to the arena.
2.3 Cannulae Implantation
A stainless steel guide cannula (18mm) was stereotaxically implanted above both the right and left basolateral amygdala (AP -0.26 cm, ML -0.48 cm, DV -0.69 cm; Paxinos and Watson, 1986) under isoflurane anaesthetic. A stylet made from stainless steel tubing (18mm) was inserted into the guide cannula to prevent blockage by debris. A non-steroidal anti-inflammatory agent, carprofen (300 ïL, 0.5%, s.c., Rimadyl, Pfizer, Kent, UK), and 250ÂµL of the broad spectrum antibiotic, enrofloxacin (0.5% s.c.) (Baytril, Bayer Ltd., Dublin, Ireland) were administered duringing the surgery to manage postoperative analgesia. Following cannula implantation, the rats were placed in a cylindrical recovery chamber placed on a heating pad and monitored for an hour, before being returned to a home cage where they were singly housed. At least 6 days were allowed for post-surgery recovery. During this period the rats were handled, weighed, and their general health monitored on a daily basis. They also
2.4 Treatment Groups
The animals were randomly assigned to one of ten treatment groups, and the order of testing was similarly randomised. The treatment groups included:
No. Per group
2.5 Formalin Test
The formalin test is a procedure commonly used to characterize nociceptive processes and analgesic drug effects (Dubuisson and Dennis, 1977). This paradigm involves administration of dilute formalin into an animal's paw and subsequent observation of various pain responses. The formalin test has been used across a wide range of species, including rats and cats (Dubuisson and Dennis, 1977), mice (Hunskaar et al., 1985), rabbits (Carli et al., 1981), guinea-pigs (Takahashi et al., 1984), domestic fowl (Hughes and Sufka, 1990) and primates (Alreja et al., 1984). Formain solution consists of aqueous formaldehyde and is usually administered in 50ïL volumes, with concentrations varying from 1-5% depending on the study. In rats, the formalin reaction is biphasic and comprises an initial or acute phase of activity, lasting approximately 5-10 minutes, and an inflammatory phase which lasts for 60-90 minutes (Sawynok et al., 2003).
2.6 Fear-Conditioning and Testing
On each conditioning day, 6-7 days post-surgery, rats were placed in a square Perspex observation chamber with a metal grid base (30x30x30 cm). The rats in the fear conditioning groups received the first of 10 foot shocks (0.4mA, 1 second duration; LE85XCT Programmer and Scrambled Shock Generator, Linton Instrumentation, Diss, Norfolk UK), each spaced 60 seconds apart, 15 seconds after entering the chamber. A video camera placed beneath the chamber recorded animal behaviours. A bat detector located beside the chamber detected ultrasonic vocalisations at 22kHz emitted by rats expressing fear. One minute after the last foot shock, the rats were returned to their home cage. Control rats, not receiving foot-shock were placed in the arena for the same time period. The number of faecal pellets produced by each animal was recorded.
On each test day, the rats were placed under a brief isoflurane anaesthetic, during which time they received an intra-plantar injection of 50ïL formalin solution into the right hind-paw; an i.p. injection of either AM251 (3mg/kg) or vehicle; and an intra-BLA microinjection of bicuculline (25ng/0.5ïL), MPEP (200ïM) or vehicle (sterile saline) depending on their respective groups. Animals were returned to their home cage until 30 minutes post formalin injection to allow the effects of the anaesthetic to wear off. They were then re-exposed to the same Perspex arena as on the conditioning day, where they remained for 30 minutes. During this time their physical behaviour was recorded with the video recorded, their ultra sounding behaviour recorded with a bat detector, and the number of faecal pellets were recorded for each animal.
2.7 Behavioural Analysis
Behaviour was analysed using the Ethovision XT software package (Noldus, Wageningen, Netherlands), which allowed for continuous event recording over each 30-min trial. A trained scorer blind to the experimental conditioned assessed the behaviour. The event-recording technique was employed to assess the duration of freezing (defined as the cessation of all visible movement except that necessary for respiration), 22-kHz ultrasound emission, walking, rearing and grooming.
2.8 Composite Pain Score (CPS)
Formalin-evoked nociceptive behaviour was scored according to the weighted composite pain scoring technique (CPS-WST0,1,2), (Watson et al., 1997). Typically, a composite pain score (CPS) is derived by applying the amount of time the animal spends in a given behavioural category to a pre-assigned category, and summing the products. The scoring method of weight by time in a behavioural category is termed the weighted scores technique (WST). In this technique, elevation of the formalin-treated paw was regarded as pain 1 behaviour and licking/biting/shaking/flinching of the same paw were regarded as pain 2 behaviour.
CPS = (Duration of Pain 1 + 2*Duration of Pain 2) / Total Duration
2.9 Statistical Analysis
The statistical analysis was all done using the SPSS software package, with all data being expressed as means Â± SEM. P<0.05 was considered to be significant. Behavioural data from the test day were analysed using a three-way ANOVA, with FC, i.p. drug treatment, and intra-BLA drug administration as factors. Student-Newman Keuls and Fischers LSD tests were carried as Post-hoc analysis when appropriate.