Kieran Rea

Microdialysis of GABA and glutamate : Analysis, interpretation and comparison with microsensors

GABA and glutamate sampled from the brain by microdialysis do not always fulfill the classic criteria for exocytotic release. In this regard the origin (neuronal vs. astroglial, synaptic vs. extrasynaptic) of glutamate and GABA collected by microdialysis as well as in the ECF itself, is still a matter of debate. In this overview microdialysis of GABA and glutamate and the use of microsensors to detect extracellular glutamate are compared and discussed. During basal conditions glutamate in microdialysates is mainly derived from non-synaptic sources. Indeed recently several sources of astrocytic glutamate release have been described, including glutamate derived from gliotransmission. However during conditions of (chemical, electrical or behavioral) stimulation a significant part of glutamate might be derived from neurotransmission. Interestingly accumulating evidence suggests that glutamate determined by microsensors is more likely to reflect basal synaptic events. This would mean that microdialysis and microsensors are complementary methods to study extracellular glutamate. Regarding GABA we concluded that the chromatographic conditions for the separation of this transmitter from other amino acid-derivatives are extremely critical. Optimal conditions to detect GABA in microdialysis samples--at least in our laboratory--include a retention time of approximately 60 min and a careful control of the pH of the mobile phase. Under these conditions it appears that 50-70% of GABA in dialysates is derived from neurotransmission.

Augmentation of SSRI effects on serotonin by 5-HT2C antagonists: Mechanistic studies

The treatment of depression may be improved by using an augmentation approach involving selective serotonin reuptake inhibitors (SSRIs) in combination with compounds that focus on antagonism of inhibitory serotonin receptors. Using microdialysis coupled to HPLC, it has recently been shown that the systemic co-administration of 5-HT2C antagonists with SSRIs augmented the acute effect of SSRIs on extracellular 5-HT. In this paper, we have investigated the mechanism through which this augmentation occurs. The increase in extracellular 5-HT was not observed when both compounds were locally infused. However, varying the route of administration for both compounds differentially revealed that an augmentation took place when the 5-HT2C antagonist was locally infused into ventral hippocampus and the SSRI given systemically, but not when systemic 5-HT2C antagonist was co-administered with the local infusion of citalopram. This suggests that the release of extracellular serotonin in ventral hippocampus may be controlled by (an)other brain area(s). As 5-HT2C receptors are not considered to be autoreceptors, this would implicate that other neurotransmitter systems are involved in this process. To investigate which neurotransmitter systems were involved in the interaction, systemic citalopram was challenged with several glutamatergic, GABA-ergic, noradrenergic, and dopaminergic compounds to determine their effects on serotonin release in ventral hippocampus. It was determined that the involvement of glutamate, norepinephrine, and dopamine in the augmentation did not seem likely, whereas evidence implicated a role for the GABA-ergic system in the augmentation.

Supraspinal modulation of pain by cannabinoids: the role of GABA and glutamate

Recent physiological, pharmacological and anatomical studies provide evidence that one of the main roles of the endocannabinoid system in the brain is the regulation of γ‐aminobutyric acid (GABA) and glutamate release. This article aims to review this evidence in the context of its implications for pain. We first provide a brief overview of supraspinal regulation of nociception, followed by a review of the evidence that the brains endocannabinoid system modulates nociception. We look in detail at regulation of supraspinal GABAergic and glutamatergic neurons by the endocannabinoid system and by exogenously administered cannabinoids. Finally, we review the evidence that cannabinoid‐mediated modulation of pain involves modulation of GABAergic and glutamatergic neurotransmission in key brain regions.

Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype-dependent hyper-responsivity to noxious stimuli.

Summary Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins hyper‐responsivity to a noxious inflammatory stimulus in the Wistar–Kyoto rat, a genetic background prone to heightened stress/affect. ABSTRACT Pain is both a sensory and an emotional experience, and is subject to modulation by a number of factors including genetic background modulating stress/affect. The Wistar–Kyoto (WKY) rat exhibits a stress‐hyper‐responsive and depressive‐like phenotype and increased sensitivity to noxious stimuli, compared with other rat strains. Here, we show that this genotype‐dependent hyperalgesia is associated with impaired pain‐related mobilisation of endocannabinoids and transcription of their synthesising enzymes in the rostral ventromedial medulla (RVM). Pharmacological blockade of the Cannabinoid1 (CB1) receptor potentiates the hyperalgesia in WKY rats, whereas inhibition of the endocannabinoid catabolising enzyme, fatty acid amide hydrolase, attenuates the hyperalgesia. The latter effect is mediated by CB1 receptors in the RVM. Together, these behavioural, neurochemical, and molecular data indicate that impaired endocannabinoid signalling in the RVM underpins hyper‐responsivity to noxious stimuli in a genetic background prone to heightened stress/affect.

Endocannabinoid-mediated enhancement of fear-conditioned analgesia in rats: Opioid receptor dependency and molecular correlates

Abstract The opioid and endocannabinoid systems mediate analgesia expressed upon re‐exposure to a contextually aversive stimulus (fear‐conditioned analgesia; FCA), and modulate the mitogen‐activated protein kinase (MAPK) pathway. However, an interaction between the opioid and endocannabinoid systems during FCA has not been investigated at the behavioural or molecular level. FCA was modeled in male Lister‐hooded rats by assessing formalin‐evoked nociceptive behaviour in an arena previously paired with footshock. Administration of the fatty acid amide hydrolase and endocannabinoid catabolism inhibitor, URB597 (0.3 mg/kg, i.p.), enhanced expression of FCA. The opioid receptor antagonist, naloxone, attenuated FCA and attenuated the URB597‐induced enhancement of FCA. SR141716A (CB1 antagonist) and SR144528 (CB2 antagonist) also attenuated the URB597‐mediated enhancement of FCA. Expression of FCA was associated with increased relative phospho‐ERK2 expression in the amygdala, an effect blocked by naloxone, SR141716A, and SR144528. Furthermore, URB597‐mediated enhancement of FCA was associated with reduced phospho‐ERK1 and phospho‐ERK2 in the amygdala. Phospho‐ERK1/2 expression in the hippocampus, prefrontal cortex, and thalamus was unchanged following FCA and drug treatment. None of the drugs affected formalin‐evoked nociceptive behaviour or phospho‐ERK1/2 expression in non‐fear‐conditioned rats. These data suggest that endocannabinoid‐mediated enhancement of FCA is abolished by pharmacological blockade of opioid receptors as well as CB1 or CB2 receptors. Both pharmacological enhancement (with URB597) and attenuation (with naloxone) of this form of endogenous analgesia were associated with reduced expression of phospho‐ERK1/2 in the amygdaloid complex arguing against a causal role for ERK1/2 signaling in the amygdala during expression of FCA or its modulation by opioids or cannabinoids.

Evidence for a role of GABAergic and glutamatergic signalling in the basolateral amygdala in endocannabinoid-mediated fear-conditioned analgesia in rats

Summary CB1 receptors in the basolateral amygdala facilitate the expression of fear‐conditioned analgesia through a mechanism involving the modulation of GABAergic and glutamatergic signalling. Abstract The basolateral amygdala (BLA) is a key substrate facilitating the expression of fear‐conditioned analgesia (FCA). However, the neurochemical mechanisms in the BLA which mediate this potent suppression of pain responding during fear remain unknown. The present study investigated the role of cannabinoid1 (CB1) receptors and interactions with GABAergic (GABAA receptor) and glutamatergic (metabotropic glutamate receptor type 5; mGluR5) signalling in the BLA in formalin‐evoked nociceptive behaviour and FCA in rats. Reexposure to a context previously paired with foot shock significantly reduced formalin‐evoked nociceptive behaviour. Systemic or intra‐BLA microinjection of the CB1 receptor antagonist/inverse agonist AM251 prevented this expression of FCA, while injection of AM251 into the central nucleus of the amygdala did not. The suppression of FCA by systemic AM251 administration was partially attenuated by intra‐BLA administration of either the GABAA receptor antagonist bicuculline or the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl) pyridine, (MPEP). Bilateral microinjection of MPEP, but not bicuculline, alone into the BLA enhanced formalin‐evoked nociceptive behaviour. Postmortem analyses revealed that FCA was associated with a significant increase in tissue levels of anandamide in the BLA side contralateral to intraplantar formalin injection. In addition, fear‐conditioned rats exhibited a robust formalin‐induced increase in levels of 2‐arachidonyl glycerol and N‐palmitoylethanolamide in the ipsilateral and contralateral BLA, respectively. These data suggest that CB1 receptors in the BLA facilitate the expression of FCA, through a mechanism which is likely to involve the modulation of GABAergic and glutamatergic signalling.

Alterations in extracellular levels of gamma-aminobutyric acid in the rat basolateral amygdala and periaqueductal gray during conditioned fear, persistent pain and fear-conditioned analgesia.

UNLABELLED Evidence suggests an important role for supraspinal gamma-aminobutyric acid (GABA) in conditioned fear and pain. Using dual probe microdialysis coupled to HPLC, we investigated alterations in extracellular levels of GABA simultaneously in the rat basolateral amygdala and dorsal periaqueductal gray during expression of conditioned fear, formalin-evoked nociception, and fear-conditioned analgesia. Re-exposure to a context previously paired with footshock significantly increased the duration of freezing and 22-kilohertz ultrasonic vocalization, and reduced formalin-evoked nociceptive behavior. Upon re-exposure to the context, GABA levels in the basolateral amygdala were significantly lower in fear-conditioned animals compared with non-fear-conditioned controls, irrespective of intraplantar formalin/saline injection. GABA levels in the dorsal periaqueductal gray were lower in rats receiving intraplantar injection of formalin, compared with saline-treated controls. GABA levels sampled were sensitive to nipecotic acid and calcium infusion. No specific fear-conditioned analgesia-related alterations in GABA efflux were observed in these regions despite the ability of rats undergoing dual probe microdialysis to express this important survival response. In conclusion, expression of contextually induced fear- and pain-related behavior are accompanied by suppression of GABA release in the basolateral amygdala and dorsal periaqueductal gray, respectively, compared with non-fear, non-pain controls. PERSPECTIVE This study investigates alterations in levels of the neurotransmitter GABA simultaneously in the rat amygdala and periaqueductal grey during expression of pain- and fear-related behavior and fear-induced analgesia. The results enhance our understanding of the role of this neurotransmitter in pain, memory of pain and control of pain during fear.

Modulation of Conditioned Fear, Fear-Conditioned Analgesia, and Brain Regional C-Fos Expression Following Administration of Muscimol into the Rat Basolateral Amygdala

UNLABELLED Evidence suggests that gamma-aminobutyric acid (GABA) signalling in the basolateral amygdala (BLA) is involved in pain, fear, and fear-conditioned analgesia (FCA). In this study, we investigated the effects of intra-BLA administration of the GABA(A) receptor agonist muscimol on the expression of conditioned-fear, formalin-evoked nociception, and fear-conditioned analgesia in rats, and the associated alterations in brain regional expression of the immediate early gene product and marker of neuronal activity, c-Fos. Formalin-evoked nociceptive behavior, conditioned-fear and fear-conditioned analgesia were apparent in animals receiving intra-BLA saline. Intra-BLA muscimol suppressed fear behavior and prevented fear-conditioned analgesia, but had no significant effect on the expression of formalin-evoked nociception. The suppression of fear behavior by intra-BLA muscimol was associated with increased c-Fos expression in the central nucleus of the amygdala (CeA) and throughout the periaqueductal grey (PAG). These intra-BLA muscimol-induced increases in c-Fos expression were abolished in rats receiving intraplantar formalin injection. These data suggest that alterations in neuronal activity in the CeA and PAG as a result of altered GABAergic signalling in the BLA may be involved in the behavioral expression of fear and associated analgesia. Furthermore, these alterations in neuronal activity are susceptible to modulation by formalin-evoked nociceptive input in a state-dependent manner. PERSPECTIVE The expression of learned fear and associated analgesia are under the control of GABA(A) receptors in the basolateral amygdala, through a mechanism which may involve altered neuronal activity in key components of the descending inhibitory pain pathway. The results enhance our understanding of the neural mechanisms subserving fear-pain interactions.

A role for PPARα in the medial prefrontal cortex in formalin‐evoked nociceptive responding in rats

The nuclear hormone receptor, PPARα, and its endogenous ligands, are involved in pain modulation. PPARα is expressed in the medial prefrontal cortex (mPFC), a key brain region involved in both the cognitive‐affective component of pain and in descending modulation of pain. However, the role of PPARα in the mPFC in pain responding has not been investigated. Here, we investigated the effects of pharmacological modulation of PPARα in the rat mPFC on formalin‐evoked nociceptive behaviour and the impact of formalin‐induced nociception on components of PPARα signalling in the mPFC.

Microinjection of 2-arachidonoyl glycerol into the rat ventral hippocampus differentially modulates contextually induced fear, depending on a persistent pain state

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear‐related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin‐evoked nociceptive tone. Fear‐conditioned rats showed significantly increased freezing and a reduction in formalin‐evoked nociceptive behaviour upon re‐exposure to a context previously paired with footshock. Bilateral microinjection of 2‐AG into the vHip significantly reduced contextually induced freezing in non‐formalin‐treated rats, and reduced formalin‐evoked nociceptive behaviour in non‐fear‐conditioned rats. In contrast, 2‐AG microinjection had no effect on fear responding in formalin‐treated rats, and no effect on nociceptive behaviour in fear‐conditioned rats. The inhibitory effect of 2‐AG on fear‐related behaviour, but not pain‐related behaviour, was blocked by co‐administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N‐arachidonoylethanolamide (anandamide, AEA) and 2‐AG were similar in the vHip of fear‐conditioned rats receiving formalin injection and the vHip of fear‐conditioned rats receiving saline injection. However, the levels of AEA and 2‐AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin‐treated fear‐conditioned rats than in that of their saline‐treated counterparts. These data suggest that 2‐AG–CB1 receptor signalling in the vHip has an anti‐aversive effect, and that this effect is abolished in the presence of a persistent pain state.