Formalin-induced Conditioned Place Avoidance Research Articles

BDNF Activates mTOR to Upregulate NR2B Expression in the Rostral Anterior Cingulate Cortex Required for Inflammatory Pain-Related Aversion in Rats.

The mechanistic target of rapamycin (mTOR) has been demonstrated to mediate pain-related aversion induced by formalin in the rostral anterior cingulate cortex (rACC). However, it remains unclear the signaling pathways and regulatory proteins involved. In the rACC, brain-derived neurotrophic factor (BDNF), an activity-dependent neuromodulator, has been shown to play a role in the development and persistence of chronic pain. In this study, we used a rat formalin-induced inflammatory pain model to demonstrate BDNF up-regulation in the rACC. Stimulation with exogenous BDNF up-regulated mTOR, whilst cyclotraxin B (CTX-B), a tropomyosin receptor kinase B (TrkB) antagonist, down-regulated mTOR. Our results suggest BDNF could activate an mTOR signaling pathway. Subsequently, we used formalin-induced conditioned place avoidance (F-CPA) training in rat models to investigate if mTOR activation was required for pain-related aversion. We demonstrated that BDNF/mTOR signaling could activate the NMDA receptor subunit episilon-2 (NR2B), which is required for F-CPA. Our results reveal that BDNF activates mTOR to up-regulate NR2B expression, which is required for inflammatory pain-related aversion in the rACC of rats.

Increasing Pain Sensation Eliminates the Inhibitory Effect of Depression on Evoked Pain in Rats.

Although previous studies have suggested that depression may be associated with inhibition of evoked pain but facilitation of spontaneous pain, the mechanisms underlying these relationships are unclear. The present study investigated whether the difference between evoked and spontaneous pain on sensory (descending inhibition) and affective (avoidance motivation) components contributes to the divergent effects of depression on them. Depressive-like behavior was produced in male Wistar rats by unpredictable chronic mild stress (UCMS). Tone-laser conditioning and formalin-induced conditioned place avoidance (F-CPA) were used to explore avoidance motivation in evoked and spontaneous pain, respectively. Behavioral pharmacology experiments were conducted to examine descending inhibition of both evoked (thermal stimulation) and spontaneous pain behavior (formalin pain). The results revealed that the inhibitory effect of depression on evoked pain was eliminated following repeated thermal stimuli. Avoidance behavior in the tone-laser conditioning task was reduced in UCMS rats, relative to controls. However, avoidance motivation for formalin pain in the UCMS group was similar to controls. 5-HT1A receptor antagonism interfered with inhibition of pain responses over time. The present study demonstrated that the inhibitory effect of depression on evoked pain dissipates with increased nociception and that the sensory-discriminative and affective-motivational components of pain are jointly involved in the divergent effects of depression on pain.

Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats

Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion.

GABAergic neurons of the medial septum play a nodal role in facilitation of nociception-induced affect.

The present study explored the functional details of the influence of medial septal region (MSDB) on spectrum of nociceptive behaviours by manipulating intraseptal GABAergic mechanisms. Results showed that formalin-induced acute nociception was not affected by intraseptal microinjection of bicuculline, a GABAA receptor antagonist, or on selective lesion of septal GABAergic neurons. Indeed, the acute nociceptive responses were dissociated from the regulation of sensorimotor behaviour and generation of theta-rhythm by the GABAergic mechanisms in MSDB. The GABAergic lesion attenuated formalin-induced unconditioned cellular response in the anterior cingulate cortex (ACC) and blocked formalin-induced conditioned place avoidance (F-CPA), and as well as the contextual fear induced on conditioning with brief footshock. The effects of lesion on nociceptive-conditioned cellular responses were, however, variable. Interestingly, the lesion attenuated the conditioned representation of experimental context in dorsal hippocampus field CA1 in the F-CPA task. Collectively, the preceding suggests that the MSDB is a nodal centre wherein the GABAergic neurons mediate nociceptive affect-motivation by regulating cellular mechanisms in ACC that confer an aversive value to the noxious stimulus. Further, in conjunction with a modulatory influence on hippocampal contextual processing, MSDB may integrate affect with context as part of associative learning in the F-CPA task.

Effects of intra-periaqueductal gray injection of zeta pseudosubstrate inhibitory peptide on sensory and affective components of pain

To explore the effects of intra-PAG injection of ZIP on sensory and affective components of pain. For determining the role of ZIP on pain-induced aversion, the effects of intra-PAG injection of ZIP on formalin-induced conditioned place avoidance (F-CPA) was investigated. To determine the role of ZIP on pain perception, formalin-induced inflammatory pain model was established and the effects of intra-PAG injection of ZIP on formalin-induced nociceptive behaviors was investigated. In the NS-treated rats, the time in the pain-paired compartment during the test session was significantly shorter than that during the preconditioning session 2 and 24 hours after administration of the drug at both 1 and 7 day post-training (Group NS-1 d-2 h: (465.1 ± 40.6) vs (133.8 ± 29.4) s (P < 0.001); Group NS-7 d-2 h: (432.3 ± 43.7) vs (150.5 ± 26.6) s (P < 0.01); Group NS-1 d-24 h: (500.5 ± 20.6) vs (107.0 ± 15.7) s (P < 0.001); Group NS-7 d-24 h: (450.8 ± 27.4) vs (129.4 ± 21.1) s (P < 0.001)). On the contrary, in the ZIP-treated rats, no significant differences were observed in the time in the pain-paired compartment between the post-conditioning and pre-conditioning sessions at the same time-points. CPA scores also showed the attenuation of F-CPA by intra-PAG injection of ZIP in comparison to the saline-injected rats (P < 0.05). Compared with the intra-PAG saline-injected group, intra-PAG microinjection of ZIP did not affect the formalin-induced nociceptive behaviors (P > 0.05). The study suggests that PAG contributes to pain-related aversion in rats, and the mechanism of pain emotion encoding in PAG may attribute to the activation of targets of ZIP.

Intra-periaqueductal gray infusion of zeta inhibitory peptide attenuates pain-conditioned place avoidance in rats

Pain is a complex experience that made up of sensory, emotional and cognitive dimensions, and the emotional factors have an important influence on intensity of pain perception. The role of periaqueductal gray (PAG) in sensory component of pain has been extensively studied, while data about pain affect are quite limited. Using formalin-induced conditioned place avoidance (F-CPA) test and inflammatory pain model, present study investigated the effect of intra-PAG infusion of zeta inhibitory peptide (ZIP) on noxious stimulation induced aversion, and the sensory component of pain. Intra-PAG injection of ZIP is sufficient to disrupt pain-induced aversion, but the ZIP infusion did not change inflammation induced pain hypersensitivity in rats. These findings suggest that PAG contributes to pain-related aversion in rats, and the mechanism of pain emotion encoding in PAG may attribute to the activation of targets of ZIP.

Inactivation of the prelimbic rather than infralimbic cortex impairs acquisition and expression of formalin-induced conditioned place avoidance

Conditioned place avoidance (CPA) paradigm has been used to investigate the affective component of pain. Although the anterior cingulate cortex (ACC) has been demonstrated to play an important role in the affective aspect of pain, whether the other prefrontal subdivisions are involved in pain-related aversion is unknown. The present study investigated the role of the prelimbic cortex (PL) and infralimbic cortex (IL) in the acquisition and expression of formalin-induced CPA (F-CPA) in rats. GABAA receptor agonist muscimol was bilaterally microinjected into PL/IL before or after the formalin-paired training, to explore the effect of temporary inactivation of PL/IL on the acquisition and expression of F-CPA, respectively. The results showed that inactivation of PL rather than IL impaired the acquisition and expression of F-CPA. Moreover, the PL inactivation did not block the acquisition of LiCl-induced CPA, suggesting that PL may be specifically implicated in the pain-emotion related encoding. These results indicate that PL but not IL is involved in the aversive dimension of pain.

Ovariectomy Results in Variable Changes in Nociception, Mood and Depression in Adult Female Rats

Decline in the ovarian hormones with menopause may influence somatosensory, cognitive, and affective processing. The present study investigated whether hormonal depletion alters the nociceptive, depressive-like and learning behaviors in experimental rats after ovariectomy (OVX), a common method to deplete animals of their gonadal hormones. OVX rats developed thermal hyperalgesia in proximal and distal tail that was established 2 weeks after OVX and lasted the 7 weeks of the experiment. A robust mechanical allodynia was also occurred at 5 weeks after OVX. In the 5th week after OVX, dilute formalin (5%)-induced nociceptive responses (such as elevating and licking or biting) during the second phase were significantly increased as compared to intact and sham-OVX females. However, chronic constriction injury (CCI) of the sciatic nerve-induced mechanical allodynia did not differ as hormonal status (e.g. OVX and ovarian intact). Using formalin-induced conditioned place avoidance (F-CPA), which is believed to reflect the pain-related negative emotion, we further found that OVX significantly attenuated F-CPA scores but did not alter electric foot-shock-induced CPA (S-CPA). In the open field and forced swimming test, there was an increase in depressive-like behaviors in OVX rats. There was no detectable impairment of spatial performance by Morris water maze task in OVX rats up to 5 weeks after surgery. Estrogen replacement retrieved OVX-induced nociceptive hypersensitivity and depressive-like behaviors. This is the first study to investigate the impacts of ovarian removal on nociceptive perception, negative emotion, depressive-like behaviors and spatial learning in adult female rats in a uniform and standard way.

Pain-related aversion induces astrocytic reaction and proinflammatory cytokine expression in the anterior cingulate cortex in rats

Pain involves sensory and affective dimensions. It is well-known that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the pathogenesis of pain sensation. However, the role of glial cells and proinflammatory cytokines in pain affect is unclear. Several lines of evidence indicate that the anterior cingulate cortex (ACC) is a key structure for pain affect. Using the formalin-induced conditioned place avoidance (F-CPA) model, which reflects the pain-related negative affective state induced by nociceptive stimuli, we examined the mRNA and protein expression levels of astrocytic markers and proinflammatory cytokines in the ACC. F-CPA produced robust aversion-like behaviors in rats. In parallel, a significant increase of mRNA of astrocytic markers (GFAP and S100B), and proinflammatory cytokines (IL-1β and TNF-α) were observed in the ACC. The protein level of GFAP, IL-1β and TNF-α were also enhanced in the ACC. The results showed for the first time that astrocytes and proinflammatory cytokines are associated with the processing of pain-related aversion and may be crucial players in the affective dimension of pain in rats.

NMDA NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-related aversion in male rats

NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, Western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not of NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significantly greater in formalin-injected rats than in naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but it did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.

Is endogenous d‐serine in the rostral anterior cingulate cortex necessary for pain‐related negative affect?

Functional activation of NMDA receptors requires co-activation of glutamate- and glycine-binding sites. D-serine is considered to be an endogenous ligand for the glycine site of NMDA receptors. Using a combination of a rat formalin-induced conditioned place avoidance (F-CPA) behavioral model and whole-cell patch-clamp recording in rostral anterior cingulate cortex (rACC) slices, we examined the effects of d-amino acid oxidase (DAAO), an endogenous D-serine-degrading enzyme, and 7-chlorokynurenate (7Cl-KYNA), an antagonist of the glycine site of NMDA receptors, on pain-related aversion. Degradation of endogenous D-serine with DAAO, or selective blockade of the glycine site of NMDA receptors by 7Cl-KYNA, effectively inhibited NMDA-evoked currents in rACC slices. Intra-rACC injection of DAAO (0.1 U) and 7Cl-KYNA (2 and 0.2 mM, 0.6 microL per side) 20 min before F-CPA conditioning greatly attenuated F-CPA scores, but did not affect formalin-induced acute nociceptive behaviors and electric foot shock-induced conditioned place avoidance. This study reveals for the first time that endogenous D-serine plays a critical role in pain-related aversion by activating the glycine site of NMDA receptors in the rACC. Furthermore, these results extend our hypothesis that activation of NMDA receptors in the rACC is necessary for the acquisition of specific pain-related negative emotion. Thus a new and promising strategy for the prevention of chronic pain-induced emotional disturbance might be raised.

GABAergic disinhibition facilitates polysynaptic excitatory transmission in rat anterior cingulate cortex

Various studies implicate the anterior cingulate cortex (ACC) in processing pain. Combining whole-cell patch clamp recordings in rat ACC slices and a formalin-induced conditioned place avoidance (F-CPA) behavioral model, the present study was to address the effect of GABA A receptors on excitatory transmission to ACC layer V neurons and its possible functional significance related to pain. Removal of GABA A inhibition by bicuculline (10 μM) induced a novel long-lasting response in layer V neurons, which could be blocked by high divalent extracellular solution and was sensitive to relatively higher rate stimuli. Co-application of NMDA receptor antagonist APV (50 μM) and non-NMDA receptor antagonist DNQX (10 μM) completely blocked the responses. Enhancement of inhibition by intra-ACC microinjection of muscimol abolished the acquisition of F-CPA without affecting formalin-induced acute nociceptive responses. These results suggest that GABA A inhibition may be involved in pain-related aversion by modulating glutamate-mediated excitatory transmission in the ACC.

NMDA receptors in the anterior cingulate cortex mediate pain-related aversion

Using a rat formalin-induced conditioned place avoidance (F-CPA) model and Fos immunohistochemistry, the present study observed the effect of N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-isoxozole propionic acid/kainite (AMPA/KA) receptors on pain-related aversion. Adult Sprague–Dawley rats were implanted with cannula in the anterior cingulate cortex (ACC) or the lateral ventricle. Before (10 min) the injection of formalin into a hindpaw on days 2 and 4 of place-conditioning trials, vehicle (0.01 M PBS), the NMDA receptors antagonist, 2-amino-5-phosphonovalerate (AP5), or the AMPA/KA receptors antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), was injected through the cannula. F-CPA was effectively eliminated by both intracerebroventricular (icv) and intra-ACC microinjection of AP5. In contrast, the intra-ACC or icv injection of DNQX failed to alter the conditioning scores of F-CPA compared with vehicle control group ( P > 0.05). Intra-ACC or icv injection of AP5 or DNQX had no effect on formalin-induced acute nociceptive behaviors. Fos immunoreactivity in the ACC was activated by retrieval of pain-related aversion, and this activation was significantly suppressed by preadministration of AP5, but not DNQX ( P < 0.001). These results suggest that NMDA receptors in the ACC are preferentially involved in the processing of the affective dimension of pain.

Contributions of the anterior cingulate cortex and amygdala to pain- and fear-conditioned place avoidance in rats

The pain experience includes a sensory-discriminative and an affective-emotional component. The sensory component of pain has been extensively studied, while data about the negative affective component of pain are quite limited. The anterior cingulate cortex (ACC), and amygdala are thought to be key neural substrates underlying emotional responses. Using formalin-induced conditioned place avoidance (F-CPA) and electric foot-shock conditioned place avoidance (S-CPA) models, the present study observed the effects of bilateral excitotoxic (quinolinic acid 200 nmol/μl) lesions of the ACC and amygdala on pain and fear induced negative emotion, as well as on sensory component of pain. In the place-conditioning paradigm, both intraplantar (i.pl.) injection of formalin and electric foot-shock produced conditioned place avoidance. Excitotoxin-induced lesion of either the ACC or amygdala significantly reduced the magnitude of F-CPA. However, the decrease in the magnitude of S-CPA occurred only in the amygdala, but not ACC lesioned animals. Neither ACC nor amygdala lesion significantly changed formalin-induced acute nociceptive behaviors. These results suggest that the amygdala is involved in both pain- and fear-related negative emotion, and the ACC might play a critical role in the expression of pain-related negative emotion.

Pain-related aversion and Fos expression in the central nervous system in rats

Patients with chronic pain suffer from much more affective emotional disturbance than pain sensation. The present study examined Fos expression associated with pain-related aversion in rats, using formalin-induced conditioned place avoidance (F-CPA) test, which could distinguish pain emotion from pain sensation. When pain experience was retrieved, the rats with F-CPA produced rigorous emotion-like behaviors. As a result, more Fos-LI neurons were observed in anterior cingulate cortex, retrosplenial cortex, insular cortex, parietal cortex area 2, frontal cortex area 1-3, claustrum, lateral septal area, amygdala, dorsomedial hypothalamic nucleus, central medial nucleus, paraventricular nucleus, superior colliculus, inferior colliculus and periaqueductal gray. The results for the first time mapped the brain regions associated with processing of pain affect and emotion in rats.

The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex.

Numerous human and animal studies indirectly implicate neurons in the anterior cingulate cortex (ACC) in the encoding of the affective consequences of nociceptor stimulation. No causal evidence, however, has been put forth linking the ACC specifically to this function. Using a rodent pain assay that combines the hind-paw formalin model with the place-conditioning paradigm, we measured a learned behavior that directly reflects the affective component of pain in the rat (formalin-induced conditioned place avoidance) concomitantly with "acute" formalin-induced nociceptive behaviors (paw lifting, licking, and flinching) that reflect the intensity and localization of the nociceptive stimulus. Destruction of neurons originating from the rostral, but not caudal, ACC reduced formalin-induced conditioned place avoidance without reducing acute pain-related behaviors. These results provide evidence indicating that neurons in the ACC are necessary for the "aversiveness" of nociceptor stimulation.