- Open Access
The blockade of the transient receptor potential vanilloid type 1 and fatty acid amide hydrolase decreases symptoms and central sequelae in the medial prefrontal cortex of neuropathic rats
- Vito de Novellis†1,
- Daniela Vita†1,
- Luisa Gatta1,
- Livio Luongo1,
- Giulia Bellini1,
- Maria De Chiaro1,
- Ida Marabese1,
- Dario Siniscalco1,
- Serena Boccella1,
- Fabiana Piscitelli2,
- Vincenzo Di Marzo2,
- Enza Palazzo1,
- Francesco Rossi1 and
- Sabatino Maione1Email author
© de Novellis et al; licensee BioMed Central Ltd. 2011
Received: 3 August 2010
Accepted: 17 January 2011
Published: 17 January 2011
Neuropathic pain is a chronic disease resulting from dysfunction within the "pain matrix". The basolateral amygdala (BLA) can modulate cortical functions and interactions between this structure and the medial prefrontal cortex (mPFC) are important for integrating emotionally salient information. In this study, we have investigated the involvement of the transient receptor potential vanilloid type 1 (TRPV1) and the catabolic enzyme fatty acid amide hydrolase (FAAH) in the morphofunctional changes occurring in the pre-limbic/infra-limbic (PL/IL) cortex in neuropathic rats.
The effect of N-arachidonoyl-serotonin (AA-5-HT), a hybrid FAAH inhibitor and TPRV1 channel antagonist, was tested on nociceptive behaviour associated with neuropathic pain as well as on some phenotypic changes occurring on PL/IL cortex pyramidal neurons. Those neurons were identified as belonging to the BLA-mPFC pathway by electrical stimulation of the BLA followed by hind-paw pressoceptive stimulus application. Changes in their spontaneous and evoked activity were studied in sham or spared nerve injury (SNI) rats before or after repeated treatment with AA-5-HT. Consistently with the SNI-induced changes in PL/IL cortex neurons which underwent profound phenotypic reorganization, suggesting a profound imbalance between excitatory and inhibitory responses in the mPFC neurons, we found an increase in extracellular glutamate levels, as well as the up-regulation of FAAH and TRPV1 in the PL/IL cortex of SNI rats. Daily treatment with AA-5-HT restored cortical neuronal activity, normalizing the electrophysiological changes associated with the peripheral injury of the sciatic nerve. Finally, a single acute intra-PL/IL cortex microinjection of AA-5-HT transiently decreased allodynia more effectively than URB597 or I-RTX, a selective FAAH inhibitor or a TRPV1 blocker, respectively.
These data suggest a possible involvement of endovanilloids in the cortical plastic changes associated with peripheral nerve injury and indicate that therapies able to normalize endovanilloid transmission may prove useful in ameliorating the symptoms and central sequelae associated with neuropathic pain.
There is increasing evidence that the unpleasantness or affective component of pain, similarly to other high-order cognitive and emotional functions (i.e. decision making, goal-directed behavior, and working memory) [1, 2], are driven by specific forebrain areas, and, among these, the prefrontal cortex (PFC) plays a pivotal role. In particular, the medial prefrontal cortex (mPFC) participates in signalling the unpleasantness of pain in humans [3, 4], being the affective component of pain under the control of the anterior cingulate cortex [5, 6]. Supraspinal brain regions are profoundly affected by peripheral nerve injury or spinal nerve transection in rodents [7, 8]. Accordingly, patients with chronic back pain showed cortex morpho-functional frontal atrophy . Neural reorganization of the mPFC might occur and account for the impaired performance of emotional decision making tasks (i.e. the Iowa Gambling Task)  in patients suffering from complex region pain syndrome type I (CRPS I) or chronic back pain similarly to patients with frontal cortex lesions. The extent of activation of the mPFC during spontaneous pain and the extent of emotional and cognitive impairment correlates to the intensity and the duration of the pain condition in patients suffering from chronic back pain . Human brain imaging studies have thus revealed that chronic pain is associated with the activation of excitatory and inhibitory neurotransmission, neurotrophic factor transcription and synthesis of proteins involved in glutamate receptor expression, along with GABAergic neuron apoptosis and new cortical connection establishment . Enhanced pain perception [13–15] has been shown to be associated with over-expression of the NR2B subunit of the NMDA receptor and morphological reorganization in the anterior cingulate cortex . Larger NMDA-mediated currents were also observed in pyramidal cells of the infralimbic cortex in neuropathic rats, corresponding to the mPFC of primates . Moreover, in a more recent study, local application of D-cycloserine, an NMDA partial agonist, generated an anti-allodynic effect closely correlated with the infusion site in a way that the maximum effect was observed in the prelimbic (PL) cortex. Chronic pain can clearly interfere with the mPFC which plays a critical role in the neurophysiological processes such as a reorganization of synaptic and neural functioning [17, 18], which in turn, could be responsible for the impaired effectiveness of emotional decision making test.
The basolateral amygdala (BLA) can modulate cortical functions, and interactions between the BLA and mPFC are important for integrating emotionally salient information [19–24]; indeed the activation of BLA can modulate the activity of separate subpopolations of mPFC neurons [25–28]. Recent works have shown that pain-related plasticity in the central nucleus of the amigdala (CeA) contributes critically to the emotional affective component of pain [29–34]. Among the novel targets identified for chronic pain therapy, the transient receptor potential vanilloid subtype 1 (TRPV1) is attracting increasing interest, since it plays a central role in the transduction of pain and the initiation of the neurogenic inflammatory responses including cancer pain [35–38]. The expression and sensitivity of TRPV1 are enhanced during inflammation and neuropathic pain leading to a lowering of the pain threshold . Apart from peripheral sensory neurons , TRPV1 is also expressed in the brain [40–44], including those areas involved in pain processing, such as the periaqueductal grey (PAG) and cingulate cortex [45, 46]. TRPV1 has been shown to be physiologically active in some nuclei of the central nervous system [47, 48]. Based on recent evidence that N-arachidonoyl-serotonin (AA-5-HT, a unique compound with the "dual" ability to inhibit fatty acid amide hydrolase [FAAH], the catabolic enzyme of endocannabinoids/endovanilloids, and antagonize TRPV1), shows analgesic activity in acute or chronic pain models in rodents [49, 50], in this study we have investigated the effect of repeated systemic administration of AA-5-HT on: i) inhibitory and excitatory activity of the perilimbic/infra-limbic (PL/IL) cortex neurons, be it spontaneous, or evoked by electrical stimulation of the BLA, or by mechanical stimulation of the hind paw; ii) extracellular glutamate and GABA levels in PL/IL cortex in awake rats; and iii) phenotypic changes of inhibitory and excitatory PL/IL cortex neurons in SNI rats. Moreover, we assessed FAAH and TRPV1 expression and endovanilloid levels in the PL/IL cortex of sham and neuropathic rats, and the mechanical allodynia associated with neuropathic pain after a single intra mPFC administration of vehicle or AA-5-HT.
Materials and methods
Animals and surgery
Male Wistar rats (220-250 g) were housed 3 per cage under controlled illumination (12:12 h light:dark cycle; light on 06.00 h) and standard environmental conditions (ambient temperature 20-22°C, humidity 55-60%) for at least 1 week before the commencement of experiments. Rat chow and tap water were available ad libitum. The experimental procedures were approved by the Animal Ethics Commitee of the Second University of Naples. Animal care was in compliance with Italian (D.L. 116/92) and EEC (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals. All efforts were made to minimise animal suffering and to reduce the number of animals used.
Mononeuropathy was induced through spinal nerve ligation (SNI) according to the method of Decostered and Woolf . Rats were anaesthetized with sodium pentobarbital (50 mg/kg i.p.). The sciatic nerve was exposed at mid-thigh level distal to the trifurcation and freed of connective tissue; the three peripheral branches (sural, common peroneal, and tibial nerves) of the sciatic nerve were exposed without stretching nerve structures. Both tibial and common peroneal nerves were ligated and transected together. The sham procedure consisted of the same surgery without ligation and transection of the nerves.
For in vivo extracellular recording experiments groups (n = 10) of sham and SNI rats were treated for 7 days with vehicle (0.5% DMSO in ACSF) or AA-5-HT (5 mg/kg i.p.). Groups (n = 8-10) of sham and SNI rats were used for the assessment of mechanical allodynia 7 days after surgery before and after a single intra-cortex microinjections of vehicle (0.5% DMSO in ACSF), AA-5-HT (0.1-0.25-1 nmol), URB597 (1-2-4 nmol), I-RTX (0.25-0.5-1 nmol) or AM251 (0.25-0.5 nmol). Moreover, additional groups of sham and SNI rats treated with vehicle and following behavioural tests for ascertaining the occurrence of allodynia in SNI rats, were divided into three further groups (n = 3) for RT-PCR, western blot and immunohistochemistry. Finally, for the microdialysis experiments sham (n = 7) and SNI rats (n = 8) have been tested 7 days after surgery.
Nociceptive behaviour (allodynia)
Mechanical allodynia was measured by using Dynamic Plantar Aesthesiometer (Ugo Basile, Varese, Italy). Rats were allowed to move freely in one of the two compartments of the enclosure positioned on the metal mesh surface. Rats were adapted to the testing environment before any measurement was taken. The mechanical stimulus was then delivered to the plantar surface of the hind paw of the rat from below the floor of the test chamber by an automated testing device. A steel rod (2 mm) was pushed with ascending force (0-30 g in 10 sec). When the animal withdrew its hind paw, the mechanical stimulus was automatically withdrawn and the force recorded to the nearest 0.1 g. Nociceptive responses for mechanical sensitivity were expressed as mechanical withdrawal threshold (MWT) in grams.
Sham and SNI rats received a single administration of vehicle, AA-5-HT (0.1-0.25-1 nmol), URB597 (1-2-4 nmol), I-RTX (0.25-0.5-1 nmol) or AM251 (0.25-0.5 nmol) into the PL/IL cortex 7 day after the sciatic nerve insult. The AA-5-HT dose was chosen based on our previous study in which it proved to be effective in several pain models in rodents . Each rat served as its own control, the responses being measured both before and after vehicle or drug administration. MWT was quantified by an observer who was blind to the treatment.
In vivo single unit extracellular recording
This study only included neurons with a regular spiking pattern and a spontaneous firing rate between 0.4 and 1.5 Hz, which were classified as pyramidal neurons [53–55]. Once a neuron was encountered in the mPFC, the position of the microelectrode was adjusted to maximize the spike amplitude relative to background noise. We then delivered electrical stimuli into the BLA (700 μA) at 2 sec intervals. At least 50 single pulses were delivered to generate peristimulus time histograms (PSTHs). Once the cell was identified, mechanical stimuli were applied to the hind paw (contralateral to the mPFC) by using a home made spring-operated forceps that closed with a force (>500 and <2000 g/10 mm2) calibrated with a tension spring balance and delivered for 5 sec . By using electrical (BLA) or mechanical (hind paw) stimuli we were able to determine whether each individual neuron was inhibited, excited or showed no response to stimulation. We did not record data for neurons that displayed no change in firing as a result of stimulation and continued the cell-searching procedure.
Characterization of BLA-evoked responses and stimulation protocol
We observed that BLA stimulation could evoke two distinct types of firing changes in separate populations of mPFC responding neurons. The more commonly observed response was a robust inhibition of neural activity. We characterized these responses accordingly with previously established criteria used by Ishikawa and Nakamura . Specifically, a cell was considered to be inhibited by BLA stimulation if it displayed a complete cessation of spontaneous firing after BLA stimulation. Neurons displaying this type of response are referred to hereafter as BLA→mPFC(-) neurons . Only neurons that displayed a spontaneous firing rate of between 0.5 and 1.5 Hz were used for the data analysis.
Characterization of mechanical-evoked responses
In vivo microdialysis
Brain microdialysis experiments were performed in awake and freely moving rats. In brief, rats were anaesthetised with pentobarbital (50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the mPFC using coordinates: AP: +3.8-2.7 mm, L: 0.4-0.7 mm from bregma and V: 5.3 mm below the dura. Microdialysis concentric probes were constructed as described by Hutson et al.,  with 25 G (0.3 mm I.D., 0.5 mm O.D.) stainless steel tubing: inlet and outlet cannulae (0.04 mm I.D., 0.14 mm O.D.) consisted of fused silica tubing (Scientific Glass Engineering, Melbourne, Australia). The microdialysis probe had a tubular active membrane (Enka AG, Wuppertal, Germany) of 3 mm in length. Following a post-operative recovery period of approximately 24 hrs, probes were perfused with artificial cerebrospinal fluid (ACSF, composition in mM: NaCl, 125; KCl, 2.5; MgCl2, 1.18 and CaCl2, 1.26) at a rate of 0.8 μl/min using a Harvard Apparatus infusion pump (mod. 22). After an initial 60 min equilibration period, dialysate samples were collected every 30 min for and 2.5 hrs to establish baseline release of glutamate and GABA in sham and SNI rats. Groups of rats received tetrodotoxin (TTX, 1 μM), or calcium-free ACSF, by reverse microdialysis to assess the synaptic nature of glutamate and GABA in mPFC cortex dialysates. On completion of experiments, rats were anaesthetised with pentobarbital and their brains perfused-fixed via the left cardiac ventricle with heparinised paraformaldehyde saline (4%). Brains were dissected out and fixed in a 10% formaldehyde solution for 2 days. Each brain was cut in 40 micron thick slices and observed under a light microscope to identify the probe location. Dialysates were analysed for amino acid content using a high-performance liquid chromatography (HPLC) method. The system comprised a Varian ternary pump (mod. 9010), a C18 reverse-phase column, a refrigerated autoinjector (mod. 9100), a fluorimetric detector (mod. PS363). Dialysates were pre-column derivatised with o-pthaldialdehyde (10 microliter dialysate + 10 microliter o-pthaldialdehyde) and amino acid conjugates resolved using a gradient separation. The detection limit of GABA and glutamate in 10 microliter samples was approximately 0.5-1 and 2-3 pmol, respectively. The mobile phase consisted of two components: (A) 0.1 M sodium acetate buffer (pH 6.95), 25% tetrahydrofuran and 10% methanol and (B) 100% methanol; gradient composition was determined with a Dell PC installed with Varian Star gradient management software, and the mobile phase flow rate was maintained at 1.0 ml/min. Data were collected by a Dell Corporation PC system 310 interfaced with Varian Star 6.2 control data and acquisition software. The mean dialysate concentration of amino acids in the first five samples represents the basal release in the two different groups of rats.
RNA extraction and RT-PCR
Total RNA was extracted from homogenized mPFC using an RNA Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer's protocol. The extracted RNA was subjected to DNase I treatment at 37°C for 30 min. The total RNA concentration was determined by UV spectrophotometer. The mRNA levels of the genes under analysis were measured by RT-PCR amplification, as previously reported . RT minus controls were carried out in order to check potential genomic DNA contamination. These RT minus controls were performed without using the reverse transcriptase enzyme in the reaction mix. Sequences for the mouse mRNAs from GeneBank (DNASTAR INC., Madison, WI) were used to design primer pairs for RT-PCRs (OLIGO 4.05 software, National Biosciences Inc., Plymouth, MN). Each RT-PCR was repeated at least four times so as to achieve optimal reproducibility data. A semi-quantitative analysis of mRNA levels was carried out using the "Gel Doc 2000 UV System" (Bio-Rad, Hercules, CA). The measured mRNA levels were normalised with respect to β-actin chosen as housekeeping gene. The β-actin gene expression values were expressed as arbitrary units ± SE. Amplification of genes of interest and β-actin were performed simultaneously.
For the protein extraction, the mPFC was minced into small pieces with a blender, then was suspended in lysis buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% blue-bromophenol, Tris-HCl, pH 6.8, containing 6 M urea, 50 μM Na3VO4, 50 μM PMSF (Sigma Chemical Co., St. Louis, MO). The total protein concentration was determined using the method described by Bradford . Each sample was loaded, electrophoresed in a 12% polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Primary antibodies were used to detect TRPV1 and FAAH according to the manufacturer's instruction at 1:500 dilution (Santa Cruz; USA). Immunoreactive signals were detected with a horseradish peroxidase-conjugated secondary antibody and reacted with an ECL system (Amersham Pharmacia, Uppsala, Sweden). Protein levels were normalized with respect to the signal obtained with anti-beta-actin monoclonal antibodies (Sigma Chemical Co., St. Louis, MO, 1:1000 dilution).
Under pentobarbital anaesthesia, animals were perfused transcardially with saline solution (0.9% NaCl) and 4% paraformaldheyde fixative. The brain was taken out and kept in the fixative for 24 h at 4°C. The tissue was kept in 30% sucrose in PBS and frozen in cryostat embedding medium (Bio-Optica, Milano, Italy). Serial 15 μm sections of the brain were cut using a cryostat and thaw-mounted onto glass slides. After washing in PBS, non-specific antibody binding was inhibited by incubation for 30 min in blocking solution (1% BSA, 0.2% powdered skim milk, 0.3% Triton-X 100 in PBS). Primary antibodies were diluted in PBS blocking buffer and slides were incubated overnight at 4°C in primary antibodies to goat polyclonal TRPV1 (1:100, Santa Cruz; USA) or goat polyclonal FAAH (1:100, Santa Cruz; USA). Fluorescent-labelled secondary antibodies (1:500; Alexa Fluor 488, Molecular Probe, Invitrogen, Carlsbad, CA) specific to the IgG specie used as a primary antibody were used to locate the specific antigens in each section. Sections were counterstained with bisbenzimide (Hoechst 33258, Hoechst, Frankfurt, Germany) and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluorescently labelled sections were viewed with a fluorescence microscope (Leica, Wetzlar, Germany) to locate the cells and identify the area of the brain.
Analysis of endocannabinoid levels
In order to perform the endocannabinoid analysis, a different cohort of rats was used. Decapitation was performed and brains were rapidly removed and embedded in oxigenated ice-cold artificial cerebrospinal fluid. A PFC slice of 1.30-1.35 mm was cut throughout the PFC by using a vibrotome (Vibratome 1500, Warner Instruments, CT, USA) (interaural from +1.9 mm to +0.7 mm) . The obtained slice of tissue containing the mPFC was then further dissected under optical microscope for microsurgery to isolate the PL-IL cortex (M650, Wild Heerbrugg, Switzerland) to be homogenized accordingly to our protocol. In brief, tissues were homogenized in 5 volumes of chloroform/methanol/Tris HCl 50 mM (2:1:1) containing 20 pmol of d8-AEA and d5-2-AG. Deuterated standards were synthesized from commercially available deuterated arachidonic acid and ethanolamine or glycerol, as described, respectively, in Devane et al.  and Bisogno et al. . Homogenates were centrifuged at 13,000 × g for 16 min (4°C), the aqueous phase plus debris was collected and extracted again twice with 1 volume of chloroform. The organic phases from the 3 extractions were pooled and the organic solvents evaporated in a rotating evaporator. Lyophilized extracts were re-suspended in chloroform/methanol 99:1 by volumes. The solutions were then purified by open bed chromatography on silica as described in Bisogno et al. . Fractions eluted with chloroform/methanol 9:1 by volume (containing AEA and 2-AG) were collected, the excess solvent was evaporated with a rotating evaporator, and aliquots were analysed by isotope dilution-liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC-APCI-MS) carried out under conditions described previously  and allowing the separation of the four compounds. Mass spectrometric (MS) detection was carried out in the selected ion monitoring mode using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated AEA) and 384.35 and 379.35 (molecular ions +1 for deuterated and undeuterated 2-AG). The area ratios between the signals of the deuterated and undeuterated compounds varied linearly with varying amounts of undeuterated compounds (30 fmol-100 pmol). AEA and 2-AG levels in unknown samples were therefore calculated on the basis of their area ratios with the internal deuterated standard signal areas. For 2-AG, the areas of the peaks corresponding to 1(3)-and 2-isomers were added together. The amounts of endocannabinoids were expressed as pmol/g or nmol/g of wet tissue weight.
N-arachidonoyl-serotonin (AA-5-HT) was synthesized in V. Di Marzo's laboratory as previously described . 5'-Iodoresiniferatoxin (I-RTX), TTX and AM251 were purchased from Tocris Bioscience (Bristol, UK). 3'-carbamoylbiphenyl-3yl-cyclohexylcarbamate (URB597) was purchased from Cayman Chemical Co. (Germany). All drugs were dissolved in 0.5% DMSO in ACSF.
Microdialysis, behavioural and electrophysiology data are represented as means ± SE and statistical analysis of these data were performed by two way ANOVA for repeated measured followed by the Student-Newman-Keul for multiple comparisons to determine statistical significance between different treated groups of rats. For biomolecular analysis, protein quantification and immunohistochemistry the Student-Newman-Keuls and the Tukey tests have been used, respectively.
Characterization of electrical stimulation-evoked responses of mPFC neurons
Single-unit extracellular recording in anesthetized rats was made from individual neurons in the prelimbic or infralimbic part of the mPFC (Figure 1B). Action potential duration (540 ± 20 μsec peak-to-valley) and firing rate (1.1 ± 0.5 spikes/s) from recorded neurons were consistent with presumed pyramidal cells rather than fast-spiking interneurons, the latter having a higher baseline firing rate (>10 Hz) and narrower spike waveform (< 300 microsec) [66, 22, 34].
Electrophysiological properties of BLA-mPFC neurons in sham and SNI rats
We first investigated the proportion of mPFC neurons (n = 3-5 per rat) with ongoing activity that responded with inhibition [BLA→mPFC(-)], or excitation, [BLA→mPFC(+)]. In these studies, we first isolated mPFC neurons and thereafter stimulated the BLA at 0.5 Hz using an initial stimulation current of 700 μA. Whenever a neuron that was responsive to BLA stimulation was encountered, the BLA was stimulated with 100-200 pulses to determine whether the neuron responded with inhibition or excitation. The same procedure has been used for four groups of rats: 1) sham rats treated for 7 days with vehicle (sham/veh); 2) sham rats treated for 7 days with AA-5-HT (5 mg/kg, i.p.) (sham/AA-5-HT); 3) SNI rats treated for 7 days with vehicle (SNI/veh) and 4) SNI rats treated for 7 days with AA-5-HT (5 mg/kg, i.p.). In the control group (sham/veh), 80% of encountered neurons (n = 32) displayed an inhibition of spontaneous activity after BLA stimulation or mechanical stimulation, with the remaining proportion of cells showing an excitatory response (n = 8). In SNI/veh rats, 70% of neurons (n = 28) displayed excitation after BLA or mechanical stimulation, while 30% of cells (n = 12) showed an inhibitory response. The 7 day period of repeated treatment with AA-5-HT (5 mg/kg, i.p.) in SNI altered the proportion between BLA→mPFC(-) and BLA→mPFC(+) neurons. Indeed 67.8% of BLA→mPFC(-) (n = 27) and 32.2% of BLA→mPFC(+) neurons (n = 13) were encountered.
In BLA→mPFC(-) neurons the spontaneous firing rate was 1.1 ± 0.2 spikes/sec, the onset of BLA-evoked inhibition was 83.3 ± 7 ms and the duration of the inhibition was 463 ± 23 ms in sham/veh rats (2A, D and E). Treatment with AA-5-HT (5 mg/kg, i.p.) did not affect either the firing rate (1.12 ± 0.3 spikes/sec), the duration of the inhibition or the onset of inhibition of BLA→mPFC(-) neurons in the shams (not shown).
SNI/veh rats showed an increased firing rate (2.2 ± 0.5 spikes/sec) of BLA→mPFC(-) neurons. The onset of BLA-evoked inhibition was significantly (P < 0.05) reduced (32 ± 7.5 ms) in BLA→mPFC(-) neurons of this group of rats although no statistically significant changes were observed in the duration of the inhibition (455 ± 15 ms) (Figure 2B, D and 2E).
Treatment with AA-5-HT (5 mg/kg i.p.) for 7 days in SNI rats (SNI/AA-5-HT) decreased firing rate (1.2 ± 0.5 spikes/sec), caused a significant increase in the onset (175 ± 5 ms) of BLA-evoked inhibition and a significant (P < 0.05) decrease in the duration (250 ± 15 ms) (Figure 2C, D and 2E).
BLA→mPFC(+) neurons had a firing rate of 0.5 ± 0.2 spikes/sec in sham/veh group. The onset, the frequency and the duration of excitation were 47 ± 2.12 ms, 6.7 ± 0.8 spikes/sec and 380 ± 13.3 ms, respectively (3A, D, E and F). Sham rats treated for 7 days with AA-5-HT (5 mg/kg i.p.) did not show changes in the onset, the frequency and the duration of excitation (not shown) with respect to sham/veh. Rats which underwent SNI (SNI/veh,) showed a firing rate of 0.8 ± 0.2 spikes/sec. The onset of BLA-evoked excitation was significantly (P < 0.05) reduced (26.4 ± 3.4 ms) in this group of rats. The duration and the frequency of evoked excitation of BLA→mPFC(+) increased significantly (672.5 ± 13.43 ms and 10.3 ± 1.16 spikes/sec, respectively) in this group of rats (SNI/veh) (Figure 3B, D, E and 3F). Treatment with AA-5-HT (5 mg/kg i.p.) for 7 days in SNI rats caused a significant increase in the onset (46.8 ± 7.8 ms) and a significant reduction in the duration and the frequency of evoked excitation of BLA→mPFC(+) neurons (375 ± 16.37 ms and 7.7 ± 0.31 spikes/sec, respectively) (Figure 3C, D, E and 3F).
Mechanical stimulation-evoked responses of BLA-mPFC neurons in sham or SNI rats
This cell population, previously identified by BLA electrical stimulation as BLA→mPFC(-) neurons, responded accordingly to noxious mechanical stimuli with an inhibition. The onset and duration of the mechanical stimulation-evoked inhibition was 93.7 ± 4.7 and 480 ± 23 ms, respectively (Figure 4A, D, E) in sham/veh rats. Treatment with AA-5-HT (5 mg/kg) did not affect either the duration or the onset of mechanical stimulation-evoked inhibition in the shams (sham/AA-5-HT). In the SNI/veh group of rats the onset of mechanical stimulation-evoked inhibition and its duration (60 ± 10 and 385 ± 9.5 ms, respectively), were significantly reduced (Figure 4B, D and 4E). Treatment with AA-5-HT (5 mg/kg i.p.) for 7 days in SNI rats caused a significant increase in the onset (210 ± 5.7 ms) and in the duration (550 ± 10 ms) of the mechanical stimulation-evoked inhibition (Figure 4C, D and 4E).
Mechanical stimulation-evoked responses of mPFC BLA→mPFC(+)neurons
This cell population, preliminarily identified by BLA electrical stimulation as BLA→mPFC(+) neurons, responded accordingly to noxious mechanical stimuli with an excitatory response. The onset, the frequency and the duration of excitation of these mPFC neurons were 97 ± 8.5 ms, 400 ± 15 ms and 7.4 ± 1.2 spikes/sec, respectively in the sham/veh (Figure 5A, D, E and 5F) group of rats. Treatment with AA-5-HT (5 mg/kg, i.p.) did not affect either duration, onset and frequency of mechanical stimulation-evoked excitation in the shams (sham/AA-5-HT) (not shown).
SNI/veh rats showed a significantly reduced onset (65 ± 12.2 ms) and an increased duration and frequency of mechanical stimulation-evoked excitation (600 ± 10 ms and 12 ± 1.1 spikes/sec, respectively) (Figure.5B, D, E, F). Treatment for 7 days with AA-5-HT (5 mg/kg i.p.) caused a significant reduction in the duration (280 ± 12.2 ms) and frequency (6.2 ± 2.2 spikes/sec) of excitation in SNI/veh rats, whereas no change was observed in the onset (70 ± 2.8 ms) of mechanical stimulation-evoked excitation (Figure 5C, D, E and 5F).
TRPV1 and FAAH expression in sham and neuropathic rats
Intra-cortex microinjections of AA-5-HT, AM251, I-RTX or URB597 transiently inhibited allodynia in SNI rats
Endocannabinoid and endovanilloid levels are altered in the PL/IL cortex of SNI rats
SNI was accompanied by a slight but statistically significant decrease in anandamide levels (from 27.2 ± 0.9 to 23.4 ± 0.4 pmol/g wet tissue weight, P < 0.05) in the PL/IL cortex, whereas the levels of 2-AG were slightly increased (from 2.1 ± 0.3 to 2.5 ± 0.2 nmol/g wet tissue weight, P < 0.05) (means ± SEM, N = 5).
Most studies on pain-related synaptic plasticity have focused on long-term changes at the peripheral and spinal dorsal horn neurons [67–74]. Pain-related synaptic reorganization in cortical areas, including the mPFC, anterior cingulate cortex (ACC), insular cortex [17, 53, 75–78] and BLA , and its contribution to pain processing or to the emotional-affective aspects of pain , has been less investigated. More recently however, some contrasting data have emerged on the phenotypic changes of mPFC pyramidal neurons which may depend on the pain models used. For instance, a large increase in the NMDA/AMPA ratio of the synaptic currents in layers II-III of PL/IL neurons, together with specific dendritic spine proliferation has been found in the SNI of the sciatic nerve model , whereas a massive mPFC neural deactivation and depression were recently observed in the arthritic pain model .
In the present study, by using integrative methods, we demonstrate that BLA→mPFC(-) inhibitory and BLA→mPFC(+) excitatory neurons, which concurrently respond to the hind-paw pressoceptive stimuli, show phenotypic changes in SNI-induced mono-neuropathy in the rat, suggesting that the mPFC may undergo profound reorganization related to chronic pain. Consistent with data by Metz et al. , the current study shows that SNI can shift the balance of excitatory and inhibitory responses in the BLA→mPFC pathway, resulting in a net increase in the excitatory influence that the BLA exerts over the PL/IL neuron population of the mPFC [30, 79, 80]. Indeed, whilst in sham rats we found that the majority (about 80%) of the pyramidal neurons belongs to the inhibitory BLA→mPFC(-)subtype, with the remaining part being excitatory neurons of the BLA→mPFC(+) subtype, in SNI rats such a ratio was nearly the opposite. One of the main reasons for the strong presence of inhibitory cells in physiological conditions might be that GABAergic interneurons are mainly interfaced between BLA-driven excitatory input and the PL/IL pyramidal neurons of the mPFC [81, 82]. Moreover, cortex GABAergic interneurons show a very strong responsiveness to excitatory inputs because of the faster AMPA-mediated cationic gating in such interneurons than in excitatory pyramidal cells under basal conditions [83, 84]. Intriguingly, in this study a critical difference was detected between neuropathic and sham rats in the excitatory BLA→mPFC(+) neurons. A decrease in the onset, enhancement of frequency and a longer duration of evoked excitation following ipsilateral BLA electrical stimulation was observed in this study. However, our electrophysiological parameters, in particular as it regards the duration of evoked excitation are extremely longer than those found by Floresco and Tse  and Laviolette and Grace . Differences among the anaesthetics used during the electrophysiological procedures could be responsible of the discrepant results observed. In particular, Floresco and Tse and Laviolette and Grace [53, 22] have used a high dose of urethane (1.5 g/kg, i.p. instead of the more conventional 1.2 g/kg, i.p.) for maintaining anaesthesia. Urethane has a complex multi-target (still poorly known) mechanism of action such as a non-selective positive modulation of GABAA and GlyR receptors and a depression of the NMDA and AMPA glutamate receptors. Indeed, these anaesthetics very rarely allows a complete recovery from the anaesthesia. Collectively, the high dose and the multi-target mechanism of action of it may justify the decreased duration of the evoked excitation in the pyramidal neurons observed by Floresco and Tse and Laviolette and Grace [53, 22]. In BLA→mPFC(-) neurons, the onset of the inhibition decreased in SNI rats, suggesting that inhibitory neurotransmission might be down-regulated in this cortex area during a pathological painful condition. Indeed, in vivo microdialysis experiments performed here in awake rats showed that the extracellular levels of glutamate increased in the contralateral mPFC cortex of SNI rats, with no measurable change in GABA levels under the same experimental conditions. Overall, these data suggest an SNI-induced imbalance between the excitatory and inhibitory amino-acidergic neurotransmissions, resulting in the increased excitability of the layers II/III pyramidal cells of the mPFC cortex. Consistently, mechanical noxious stimulation applied to the contralateral paw evoked excitatory or inhibitory responses in the cell populations previously identified by BLA electrical stimulation. The application of noxious stimuli to the contralateral paw of SNI rats resulted in a decreased onset of burst or pause for the excitatory or inhibitory cells, respectively. As far as the other analyzed functional parameters are concerned, an increased frequency and duration of excitation were observed following paw mechanical stimulation. Collectively, these in vivo physiological data support recent ex vivo findings indicating that mPFC pyramidal neurons undergo profound morpho-functional reorganization associated with SNI-induced neuropathic pain, supporting the possibility of major involvement of the layers II/III of PL/IL cortex in the patho-physiological processes associated with the unpleasant or the affective component of pain . Although the details of the pain-related BLA-driven changes justifying the enhanced excitatory synaptic activity on PL/IL pyramidal cells are yet to be determined, the pharmacological, electrophysiological, biochemical and morphological data from the current and previous studies seem consistent with a polysynaptic pathway.
Indeed, even if the glutamatergic BLA projection to GABAergic mPFC inter
could explain the BLA-driven inhibitory responses in about 80% of the PL/IL pyramidal neurons in normal conditions [81, 82], it remains to be determined why the excitatory/inhibitory cell populations ratio shifted dramatically in favour of the excitatory cells in the SNI pain model. One possibility could be a strengthened direct connection between excitatory glutamatergic BLA impinging on pyramidal neurons of PL/IL cortex rather than on the inhibitory interneurons [85–87], caused by SNI-induced proliferation of mPFC pyramidal neuron dendrites . Alternatively, another possible explanation might be that the increased SNI-induced endovanilloid tone, i.e. the over-expression of the TRPV1 channel, may lead to the increased release of glutamate in the PL/IL cortex, since TRPV1 activation is well known to be coupled to enhanced glutamate release in the brain . Indirect evidence supporting this possibility comes from the present finding that SNI was also accompanied by increased FAAH expression and the subsequent decrease of the levels of the endocannabinoid/endovanilloid anandamide in the PL/IL cortex, as well as by an increase in the levels of the endocannabinoid 2-AG. These latter events might represent an adaptive mechanism aiming at providing a negative feed-back control on the putative TRPV1-mediated stimulation of glutamate release, since anandamide is an endogenous activator of TRPV1, and 2-AG, which is inactive at TRPV1, may instead inhibit glutamate release by acting as a retrograde signal at pre-synaptic CB1 receptors. Alternatively, the stimulation by 2-AG of presynaptic CB1 receptors on GABAergic fibers might contribute to reduce inhibitory signalling in the PL/IL cortex, even though we did not observe here any reduction in extracellular GABA levels in microdyalisates from this brain region of SNI rats. Mechanistic studies in mice with SNI are under way in order to investigate the role of the endocannabinoid and endovanilloid system in the enhanced excitatory vs. inhibitory signalling observed here in SNI rats.
It is worth noting that a relatively short temporal window (7 days after SNI) was sufficient to produce the observed morphological, neurochemical and functional changes. These data are consistent with previous evidence of increased NMDA receptor subunit NR2B in the cingulate cortex of mice with persistent pain , as well as with the reported synaptic proliferation on basal dendrites of pyramidal neurons in the mPFC cortex in SNI rats . Such a morpho-functional reorganization at the neuron basal dendrite level would indicate a specific long-lasting neuro-adaptive process aiming at straightening the intra-cortical circuits, more than the extra-cortical ones, in a way that such an increased local spine density would wrongly integrate inputs converging in this area.
A further consideration concerns the evidence that the current data from 7-day treated SNI rats do not seem to be consistent with data obtained in patients suffering from neuropathic pain, in whom atrophy of the limbic prefrontal cortex was reported . However, as previously suggested by Metz et al. , it is possible that this neuropathic pain model and the short period of observation (which in fact requires a longer interval of time in order to induce apoptosis or glutamate-mediated excitotoxicity) did not allow us to note any apparent neurodegeneration. Indeed there is no reason to exclude the possibility that following a longer period of over-excitation, increased spine number and NMDA receptor currents may lead to increased glutamatergic excitotoxicity and apoptosis.
Based on the over-expressed endocannabinoid/endovanilloid biomarkers and elevated glutamate levels, we decided to perform in SNI rats a repeated daily systemic treatment with AA-5-HT, which we had previously demonstrated to produce anti-allodynic and anti-hyperalgesic effects in the chronic constriction injury (CCI) neuropathic pain model via both indirect activation/desensitization of TRPV1 and activation of cannabinoid CB1 receptors, following elevation of AEA and 2-AG levels, and direct TRPV1 receptor antagonism . Indeed, AA-5-HT exerts its analgesic effect in three ways: i) antagonism of TRPV1 receptors involved in thermal hyperalgesia, ii) desensitization of TRPV1-expressing nociceptors involved in mechanical allodynia and iii) indirect agonism at cannabinoid CB1 receptors in the CCI model of neuropathic pain . Here we report that AA-5-HT treatment was able to prevent mechanical allodynia and modulate both inhibitory and excitatory transmission in the BLA-mPFC pathway. Intriguingly, the relative efficacy of the effects of intra-PL/IL cortex microinjections of selective vanilloid (I-RTX) or FAAH (URB597) inhibitors or of the "hybrid" compound, AA-5-HT, underline the importance of concentrating two activities in one single molecule to significantly reduce allodynia and further validate the critical role played by TRPV1 and FAAH in specific mPFC sub-regions involved in pain modulation. Several previous studies have reported that the PL/IL cortex, which corresponds to the dorsal-lateral prefrontal cortex in humans, plays a crucial role in pain processing [10, 89–93], and emerging imaging studies show that this brain region is involved in pain inhibition in humans 
Furthermore, since the treatment with AA-5-HT led to restore normal neuronal activity in the BLA-mPFC pathway, these data support our hypothesis that the over-expressed TRPV1 channel, which seems to be mainly present in glutamatergic neurons, is one of the mechanisms that in SNI rats activate pathways (likely calcium-dependent) associated with cell plasticity [94, 95]. In agreement with the TRPV1-induced neural plasticity described previously [42, 96–98], it is possible that functional re-organization mediated by glutamate/endovanilloid and GABA/endocannabinoid signalling can also occur following TRPV1 channel over-stimulation and the consequent increased release of glutamate in the PL-IL cortex of SNI rats in both identified populations of mPFC neurons.
In conclusion, this study shows that in pressoceptive responding populations of mPFC neurons, the previously described BLA→mPFC(-) and BLA→mPFC(+) neurons , may undergo profound reorganization related to neuropathic pain. The present findings indicate that a relatively short period of SNI-induced neuropathy (7 days) may be sufficient to up-regulate the endovanilloid/endocannabinoid machinery in the PL/IL cortex, implying that disruptions in the mPFC endovanilloid/endocannabinoid system might impair behaviours mediated by the BLA-mPFC circuits. Since similar alterations have been shown in corresponding neural circuitries in chronic pain subjects, it may be conceivable to speculate that the changes we have observed here could be a contributing factor to emotional and cognitive disturbances associated with chronic pain disorders. Local or systemic pharmacological manipulation of the TRPV1 channel and the enzyme FAAH with the hybrid drug AA-5-HT proves to inhibit allodynia/hyperalgesia and normalize the imbalance between excitatory and inhibitory responses in the mPFC neurons. As such, psychopharmacological therapies designed to normalize endovanilloid/endocannabinoid transmission in the mPFC glutamatergic terminals may prove useful in alleviating the symptoms and central sequelae of neuropathic pain syndromes.
- Gusnard DA, Akbudak E, Shulman GL, Raichle ME: Medial prefrontal cortex and self-referential mental activity: relation to a default mode of brain function. Proc Natl Acad Sci USA 2001, 98: 4259–4264. 10.1073/pnas.071043098PubMed CentralPubMedView ArticleGoogle Scholar
- Phelps EA, Delgado MR, Nearing KI, LeDoux JE: Extinction learning in humans: role of the amygdala and vmPFC. Neuron 2004, 43: 897–905. 10.1016/j.neuron.2004.08.042PubMedView ArticleGoogle Scholar
- Lorenz J, Cross DJ, Minoshima S, Morrow TJ, Paulson PE, Casey KL: A unique representation of heat allodynia in the human brain. Neuron 2002, 35: 383–393. 10.1016/S0896-6273(02)00767-5PubMedView ArticleGoogle Scholar
- Porro CA, Baraldi P, Pagnoni G, Serafini M, Facchin P, Maieron M, Nichelli P: Does anticipation of pain affect cortical nociceptive systems? J Neurosci 2002, 22: 3206–3214.PubMedGoogle Scholar
- Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC: Pain affect encoded in human anterior cingulate but not somatosensory cortex. Science 1997, 277: 968–971. 10.1126/science.277.5328.968PubMedView ArticleGoogle Scholar
- Wager TD, Rilling JK, Smith EE, Sokolik A, Casey KL, Davidson RJ, Kosslyn SM, Rose RM, Cohen JD: Placebo-induced changes in FMRI in the anticipation and experience of pain. Science 2004, 303: 1162–1167. 10.1126/science.1093065PubMedView ArticleGoogle Scholar
- Apkarian AV, Lavarello S, Randolf A, Berra HH, Chialvo DR, Besedovsky HO, del Rey A: Expression of IL-1beta in supraspinal brain regions in rats with neuropathic pain. Neurosci Lett 2006, 407: 176–81. 10.1016/j.neulet.2006.08.034PubMedView ArticleGoogle Scholar
- Xie W, Liu X, Xuan H, Luo S, Zhao X, Zhou Z, Xu J: Effect of betamethasone on neuropathic pain and cerebral expression of NF-kappaB and cytokines. Neurosci Lett 2006,30;393(2–3):255–9. 10.1016/j.neulet.2005.09.077View ArticleGoogle Scholar
- Apkarian AV, Sosa Y, Sonty S, Levy RM, Harden RN, Parrish TB, Gitelman DR: Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. J Neurosci 2004,24(46):10410–5. 10.1523/JNEUROSCI.2541-04.2004PubMedView ArticleGoogle Scholar
- Apkarian AV, Sosa Y, Krauss BR, Thomas PS, Fredrickson BE, Levy RE, Harden RN, Chialvo DR: Chronic pain patients are impaired on an emotional decision-making task. Pain 2004, 108: 129–136. 10.1016/j.pain.2003.12.015PubMedView ArticleGoogle Scholar
- Baliki MN, Chialvo DR, Geha PY, Levy RM, Harden RN, Parrish TB, Apkarian AV: Chronic pain and the emotional brain: Specific brain activity associated with spontaneous fluctuations of intensity of chronic back pain. J Neurosci 2006, 26: 12165–12173. 10.1523/JNEUROSCI.3576-06.2006PubMed CentralPubMedView ArticleGoogle Scholar
- Zhuo M: Cortical excitation and chronic pain. Trends Neurosci 2008, 31: 199–207. 10.1016/j.tins.2008.01.003PubMedView ArticleGoogle Scholar
- Calejesan AA, Kim SJ, Zhuo M: Descending facilitatory modulation of a behavioural nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain 2000, 4: 83–96. 10.1053/eujp.1999.0158PubMedView ArticleGoogle Scholar
- Hood WF, Compton RP, Monahan JB: D-cycloserine: A ligand for the N-methyl-D-aspartate coupled glycine receptor has partial agonist characteristics. Neurosci Lett 1989, 98: 91–95. 10.1016/0304-3940(89)90379-0PubMedView ArticleGoogle Scholar
- Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Tkatch T, Apkarian AV: D-cycloserine reduces neuropathic pain behavior through limbic NMDA-mediated circuitry. Pain 2007, 132: 108–123. 10.1016/j.pain.2007.03.003PubMed CentralPubMedView ArticleGoogle Scholar
- Metz AE, Yau HJ, Centeno MV, Apkarian AV, Martina M: Morphological and functional reorganization of rat medial prefrontal cortex in neuropathic pain. Proc Natl Acad Sci USA 2009, 106: 2423–8. 10.1073/pnas.0809897106PubMed CentralPubMedView ArticleGoogle Scholar
- Xu H, Wu LJ, Wang H, Zhang X, Vadakkan KI, Kim SS, Steenland HW, Zhuo M: Presynaptic and postsynaptic amplifications of neuropathic pain in the anterior cingulate cortex. J Neurosci 2008, 28: 7445–53. 10.1523/JNEUROSCI.1812-08.2008PubMed CentralPubMedView ArticleGoogle Scholar
- Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Tkatch T, Apkarian AV: D-cycloserine reduces neuropathic pain behavior through limbic NMDA-mediated circuitry. Pain 2007, 132: 108–23. 10.1016/j.pain.2007.03.003PubMed CentralPubMedView ArticleGoogle Scholar
- Garcia R, Vouimba RM, Baudry M, Thompson RF: The amygdala modulates prefrontal cortex activity relative to conditioned fear. Nature 1999, 402: 294–6. 10.1038/46286PubMedView ArticleGoogle Scholar
- Holland PC, Gallagher M: Amygdala-frontal interactions and reward expectancy. Curr Opin Neurobiol 2004, 14: 148–55. Review 10.1016/j.conb.2004.03.007PubMedView ArticleGoogle Scholar
- McGaugh JL: The amygdala modulates the consolidation of memories of emotionally arousing experiences. Annu Rev Neurosci 2004, 27: 1–28. 10.1146/annurev.neuro.27.070203.144157PubMedView ArticleGoogle Scholar
- Laviolette SR, Grace AA: Cannabinoids Potentiate Emotional Learning Plasticity in Neurons of the Medial Prefrontal Cortex through Basolateral Amygdala Inputs. J Neurosci 2006, 26: 6458–68. 10.1523/JNEUROSCI.0707-06.2006PubMedView ArticleGoogle Scholar
- Herry C, Ciocchi S, Senn V, Demmou L, Müller C, Lüthi A: Switching on and off fear by distinct neuronal circuits. Nature 2008, 454: 600–6. 10.1038/nature07166PubMedView ArticleGoogle Scholar
- Roozendaal B, McReynolds JR, Van der Zee EA, Lee S, McGaugh JL, McIntyre CK: Glucocorticoid effects on memory consolidation depend on functional interactions between the medial prefrontal cortex and basolateral amygdala. J Neurosci 2009, 29: 14299–308. 10.1523/JNEUROSCI.3626-09.2009PubMed CentralPubMedView ArticleGoogle Scholar
- Pérez-Jaranay JM, Vives F: Electrophysiological study of the response of medial prefrontal cortex neurons to stimulation of the basolateral nucleus of the amygdala in the rat. Brain Res 1991, 564: 97–101.PubMedView ArticleGoogle Scholar
- Ishikawa A, Nakamura S: Convergence and interaction of hippocampal and amygdalar projections within the prefrontal cortex in the rat. J Neurosci 2003, 23: 9987–95.PubMedGoogle Scholar
- Dilgen JE, O'Donnell P: In vivo intracellular recordings from the medial prefrontal cortex reveal an inhibitory response to basolateral amygdala stimulation. Soc Neurosci Abstr 2005, 31: 271.7.Google Scholar
- Dilgen JE, O'Donnell P: Basolateral amygdala projections to the medial prefrontal cortex: an inhibitory pathway? Soc Neurosci Abstr 2006, 32: 730.7.Google Scholar
- Neugebauer V, Li W, Bird GC, Han JS: The amygdala and persistent pain. Neuroscientist 2004, 10: 221–34. 10.1177/1073858403261077PubMedView ArticleGoogle Scholar
- Neugebauer V, Galhardo V, Maione S, Mackey SC: Forebrain pain mechanisms. Brain Res Rev 2009, 60: 226–42. 76 10.1016/j.brainresrev.2008.12.014PubMed CentralPubMedView ArticleGoogle Scholar
- Carrasquillo Y, Gereau RW: Hemispheric lateralization of a molecular signal for pain modulation in the amygdala. Mol Pain 2008, 23: 4:24.Google Scholar
- Ikeda R, Takahashi Y, Inoue K, Kato F: NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain. Pain 2007, 127: 161–72. 10.1016/j.pain.2006.09.003PubMedView ArticleGoogle Scholar
- Myers B, Greenwood-Van Meerveld B: Corticosteroid receptor-mediated mechanisms in the amygdala regulate anxiety and colonic sensitivity. Am J Physiol Gastrointest Liver Physiol 2007, 292: G1622–9. 10.1152/ajpgi.00080.2007PubMedView ArticleGoogle Scholar
- Ji G, Sun H, Fu Y, Li Z, Pais-Vieira M, Galhardo V, Neugebauer V: Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation. J Neurosci 2010, 30: 5451–64. 10.1523/JNEUROSCI.0225-10.2010PubMed CentralPubMedView ArticleGoogle Scholar
- Hautkappe M, Roizen MF, Toledano A, Roth S, Jeffries JA, Ostermeier AM: Review of the effectiveness of capsaicin for painful cutaneous disorders and neural dysfunction. Clin J Pain 1998, 14: 97–106. 10.1097/00002508-199806000-00003PubMedView ArticleGoogle Scholar
- Szallasi A, Szabó T, Bíró T, Modarres S, Blumberg PM, Krause JE, Cortright DN, Appendino G: Resiniferatoxin-type phorboid vanilloids display capsaicin-like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1. Br J Pharmacol 1999, 128: 428–34. 10.1038/sj.bjp.0702810PubMed CentralPubMedView ArticleGoogle Scholar
- Malmberg NJ, Falke JJ: Use of EPR power saturation to analyze the membrane-docking geometries of peripheral proteins: applications to C2 domains. Annu Rev Biophys Biomol Struct 2005, 34: 71–90. 10.1146/annurev.biophys.34.040204.144534PubMed CentralPubMedView ArticleGoogle Scholar
- Prevarskaya N, Zhang L, Barritt G: TRP channels in cancer. Biochim Biophys. Acta 2007, 1772: 937–46.Google Scholar
- Puntambekar P, Van Buren J, Raisinghani M, Premkumar LS, Ramkumar V: Direct interaction of adenosine with the TRPV1 channel protein. J Neurosci 2004, 24: 3663–71. 10.1523/JNEUROSCI.4773-03.2004PubMedView ArticleGoogle Scholar
- Mezey E, Tóth ZE, Cortright DN, Arzubi MK, Krause JE, Elde R, Guo A, Blumberg PM, Szallasi A: Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous system of the rat and human. Proc Natl Acad Sci USA 2000, 97: 3655–60. 10.1073/pnas.060496197PubMed CentralPubMedView ArticleGoogle Scholar
- Roberts JC, Davis JB, Benham CD: [3H] Resiniferatoxin in autoradiography in the CNS of wild type and TRPV1 null mice defines TRPV1 (VR-1) protein distribuition. Brain Res 2004, 995: 176–183. 10.1016/j.brainres.2003.10.001PubMedView ArticleGoogle Scholar
- Cristino L, de Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo V: Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience 2006, 139: 1405–15. 10.1016/j.neuroscience.2006.02.074PubMedView ArticleGoogle Scholar
- Palazzo E, Rossi F, Maione S: Role of TRPV1 receptors in descending modulation of pain. Mol Cell Endocrinol 2008, 286: S79–83. 10.1016/j.mce.2008.01.013PubMedView ArticleGoogle Scholar
- Maione S, Starowicz K, Cristino L, Guida F, Palazzo E, Luongo L, Rossi F, Marabese I, de Novellis V, Di Marzo V: Functional interaction between TRPV1 and micro-opioid receptors in the descending antinociceptive pathway activates glutamate transmission and induces analgesia. J Neurophysiol 2009, 101: 2411–22. 10.1152/jn.91225.2008PubMedView ArticleGoogle Scholar
- Maione S, Bisogno T, de Novellis V, Palazzo E, Cristino L, Valenti M, Petrosino S, Guglielmotti V, Rossi F, Di Marzo V: Elevation of endocannabinoid levels in the ventrolateral periaqueductal grey through inhibition of fatty acid amide hydrolase affects descending nociceptive pathways via both cannabinoid receptor type 1 and transient receptor potential vanilloid type-1 receptors. J Pharmacol Exp Ther 2006, 316: 969–82. 10.1124/jpet.105.093286PubMedView ArticleGoogle Scholar
- Steenland HW, Ko SW, Wu LJ, Zhuo M: Hot receptors in the brain. Mol Pain 2006, 8: 2–34.Google Scholar
- Marinelli S, Di Marzo V, Berretta N, Matias I, Maccarrone M, Bernardi G, Mercuri NB: Presynaptic facilitation of glutamatergic synapses to dopaminergic neurons of the rat substantia nigra by endogenous stimulation of vanilloid receptors. J Neurosci 2003, 23: 3136.PubMedGoogle Scholar
- Starowicz K, Maione S, Cristino L, Palazzo E, Marabese I, Rossi F, de Novellis V, Di Marzo V: Tonic endovanilloid facilitation of glutamate release in brainstem descending antinociceptive pathways. J Neurosci 2007, 27: 13739–49. 10.1523/JNEUROSCI.3258-07.2007PubMedView ArticleGoogle Scholar
- Maione S, De Petrocellis L, de Novellis V, Moriello AS, Petrosino S, Palazzo E, Rossi FS, Woodward DF, Di Marzo V: Analgesic actions of N-arachidonoyl-serotonin, a fatty acid amide hydrolase inhibitor with antagonistic activity at vanilloid TRPV1 receptors. Br J Pharmacol 2007, 150: 766–81. 10.1038/sj.bjp.0707145PubMed CentralPubMedView ArticleGoogle Scholar
- de Novellis V, Palazzo E, Rossi F, De Petrocellis L, Petrosino S, Guida F, Luongo L, Migliozzi A, Cristino L, Marabese I, Starowicz K, Di Marzo V, Maione S: Endocannabinoid Research Group. The analgesic effect of N-arachidonoyl-serotonin, a FAAH inhibitor and TRPV1 receptor antagonist, associated with changes in rostral ventromedial medulla and locus coeruleus cell activity in rats. Neuropharmacology 2008, 55: 1105–13. 10.1016/j.neuropharm.2008.06.023PubMedView ArticleGoogle Scholar
- Decosterd I, Woolf CJ: Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87: 149–58. 10.1016/S0304-3959(00)00276-1PubMedView ArticleGoogle Scholar
- Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates. Academic Press, London; 1986.Google Scholar
- Floresco SB, Tse MT: Dopaminergic regulation of inhibitory and excitatory transmission in the basolateral amygdala-prefrontal cortical pathway. J Neurosci 2007, 27: 2045–57. 10.1523/JNEUROSCI.5474-06.2007PubMedView ArticleGoogle Scholar
- Jung MW, Qin Y, McNaughton BL, Barnes CA: Firing characteristics of deep layer neurons in prefrontal cortex in rats performing spatial working memory tasks. Cereb Cortex 1998, 8: 437–450. 10.1093/cercor/8.5.437PubMedView ArticleGoogle Scholar
- Tierney PL, Dégenètais E, Thierry AM, Glowinski J, Gioanni Y: Influence of the hippocampus on interneurons of the rat prefrontal cortex. 1. Eur J Neurosci 2004,20(2):514–24. 10.1111/j.1460-9568.2004.03501.xPubMedView ArticleGoogle Scholar
- Hoheisel U, Unger T, Mense S: Sensitization of rat dorsal horn neurons by NGF-induced subthreshold potentials and low-frequency activation. A study employing intracellular recordings in vivo. Brain Res 2007, 1169: 34–43. 10.1016/j.brainres.2007.06.054PubMedView ArticleGoogle Scholar
- Laviolette SR, Lipski WJ, Grace A: A subpopulation of neurons in the medial prefrontal cortex encodes emotional learning with burst and frequency codes through a dopamine D4 receptor-dependent basolateral amygdala input. J Neurosci 2005, 25: 6066–75. 10.1523/JNEUROSCI.1168-05.2005PubMedView ArticleGoogle Scholar
- Hutson PH, Sarna GS, Kantamaneni BD, Curzon G: Monitoring the effect of a tryptophan load on brain indole metabolism in freely moving rats by simultaneous cerebrospinal fluid sampling and brain dialysis. J Neurochem 1985, 44: 1266–73. 10.1111/j.1471-4159.1985.tb08753.xPubMedView ArticleGoogle Scholar
- Galderisi U, Di Bernardo G, Cipollaro M, Peluso G, Cascino A, Cotrufo R, Melone MA: Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product. J Cell Biochem 1999, 73: 97–105. 10.1002/(SICI)1097-4644(19990401)73:1<97::AID-JCB11>3.0.CO;2-MPubMedView ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248–54. 10.1016/0003-2697(76)90527-3PubMedView ArticleGoogle Scholar
- Franklin KBJ, Paxinos G: The mouse brain in the stereotaxic coordinates. Academic Press, San Diego, USA; 1997.Google Scholar
- Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R: Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258: 1946–9. 10.1126/science.1470919PubMedView ArticleGoogle Scholar
- Bisogno T, Sepe N, Melck D, Maurelli S, De Petrocellis L, Di Marzo V: Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells. Biochem J 1997, 322: 671–677.PubMed CentralPubMedGoogle Scholar
- Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG, Hermann H, Tang J, Hofmann C, Zieglgänsberger W, Di Marzo V, Lutz B: The endogenous cannabinoid system controls extinction of aversive memories. Nature 2003, 418: 530–534. 10.1038/nature00839View ArticleGoogle Scholar
- Bisogno T, Melck D, De Petrocellis L, Bobrov MYu, Gretskaya NM, Bezuglov VV, Sitachitta N, Gerwick WH, Di Marzo V: Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase. Biochem Biophys Res Commun 1998, 248: 515–22. 10.1006/bbrc.1998.8874PubMedView ArticleGoogle Scholar
- Constantinidis C, Goldman-Rakic PS: Correlated discharges among putative pyramidal neurons and interneurons in the primate prefrontal cortex. J Neurophysiol 2002, 88: 3487–97. 10.1152/jn.00188.2002PubMedView ArticleGoogle Scholar
- Boucher TJ, McMahon SB: Neurotrophic factors and neuropathic pain. Curr Opin Pharmacol 2001, 1: 66–72. 10.1016/S1471-4892(01)00010-8PubMedView ArticleGoogle Scholar
- Coull JA, Boudreau D, Bachand K, Prescott SA, Nault F, Sík A, De Koninck P, De Koninck Y: Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 2003, 424: 938–42. 10.1038/nature01868PubMedView ArticleGoogle Scholar
- Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y: BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 2005, 438: 1017–21. 10.1038/nature04223PubMedView ArticleGoogle Scholar
- Ikeda H, Heinke B, Ruscheweyh R, Sandkühler J: Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 2003, 299: 1237–40. 10.1126/science.1080659PubMedView ArticleGoogle Scholar
- Kohno T, Moore KA, Baba H, Woolf CJ: Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn. J Physiol 2003, 548: 131–8. 10.1113/jphysiol.2002.036186PubMed CentralPubMedView ArticleGoogle Scholar
- Balasubramanyan S, Stemkowski PL, Stebbing MJ, Smith PA: Sciatic chronic constriction injury produces cell-type-specific changes in the electrophysiological properties of rat substantia gelatinosa neurons. J Neurophysiol 2006, 96: 579–90. 10.1152/jn.00087.2006PubMedView ArticleGoogle Scholar
- Ikeda H, Stark J, Fischer H, Wagner M, Drdla R, Jäger T, Sandkühler J: Synaptic amplifier of inflammatory pain in the spinal dorsal horn. Science 2006, 312: 1659–62. 10.1126/science.1127233PubMedView ArticleGoogle Scholar
- Nassar MA, Baker MD, Levato A, Ingram R, Mallucci G, McMahon SB, Wood JN: Nerve injury induces robust allodynia and ectopic discharges in Nav1.3 null mutant mice. Mol Pain 2006, 19: 2–33.Google Scholar
- Johansen JP, Fields HL, Manning BH: The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci USA 2001, 98: 8077–82. 10.1073/pnas.141218998PubMed CentralPubMedView ArticleGoogle Scholar
- Zhuo M: Molecular mechanisms of pain in the anterior cingulate cortex. J Neurosci Res 2006, 84: 927–33. 10.1002/jnr.21003PubMedView ArticleGoogle Scholar
- McGaraughty S, Heinricher MM: Microinjection of morphine into various amygdaloid nuclei differentially affects nociceptive responsiveness and RVM neuronal activity. Pain 2002, 96: 153–62. 10.1016/S0304-3959(01)00440-7PubMedView ArticleGoogle Scholar
- Jasmin L, Rabkin SD, Granato A, Boudah A, Ohara PT: Analgesia and hyperalgesia from GABA-mediated modulation of the cerebral cortex. Nature 2003, 424: 316–20. 10.1038/nature01808PubMedView ArticleGoogle Scholar
- Ji G, Neugebauer V: Differential effects of CRF1 and CRF2 receptor antagonists on pain-related sensitization of neurons in the central nucleus of the amygdala. J Neurophysiol 2007, 97: 3893–904. 10.1152/jn.00135.2007PubMedView ArticleGoogle Scholar
- Fu Y, Neugebauer V: Differential mechanisms of CRF1 and CRF2 receptor functions in the amygdala in pain-related synaptic facilitation and behavior. J Neurosci 2008, 28: 3861–76. 10.1523/JNEUROSCI.0227-08.2008PubMed CentralPubMedView ArticleGoogle Scholar
- Ferrante M, Migliore M, Ascoli GA: Feed-forward inhibition as a buffer of the neuronal input-output relation. Proc Natl Acad Sci USA 2009, 106: 18004–18009. 10.1073/pnas.0904784106PubMed CentralPubMedView ArticleGoogle Scholar
- Silberberg G, Markram H: Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells. Neuron 2007, 53: 735–746. 10.1016/j.neuron.2007.02.012PubMedView ArticleGoogle Scholar
- Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C: Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 2004, 5: 793–807. 10.1038/nrn1519PubMedView ArticleGoogle Scholar
- Cruikshank SJ, Lewis TJ, Connors BW: Synaptic basis for intense thalamocortical activation of feedforward inhibitory cells in neocortex. Nat Neurosci 2007, 10: 462–8.PubMedGoogle Scholar
- Kita H, Kitai ST: Amygdaloid projections to the frontal cortex and the striatum in the rat. J Comp Neurol 1990, 298: 40–9. 10.1002/cne.902980104PubMedView ArticleGoogle Scholar
- Bacon SJ, Headlam AJ, Gabbott PL, Smith AD: Amygdala input to medial prefrontal cortex (mPFC) in the rat: a light and electron microscope study. Brain Res 1996, 720: 211–9. 10.1016/0006-8993(96)00155-2PubMedView ArticleGoogle Scholar
- Gabbott PL, Warner TA, Busby SJ: Amygdala input monosynaptically innervates parvalbumin immunoreactive local circuit neurons in rat medial prefrontal cortex. Neuroscience 2006, 139: 1039–48. 10.1016/j.neuroscience.2006.01.026PubMedView ArticleGoogle Scholar
- Starowicz K, Cristino L, Di Marzo V: TRPV1 receptors in the central nervous system: potential for previously unforeseen therapeutic applications. Curr Pharm Des 2008,14(1):42–54. 10.2174/138161208783330790PubMedView ArticleGoogle Scholar
- Porro CA, Cettolo V, Francescato MP, Baraldi P: Temporal and intensity coding of pain in human cortex. J Neurophysiol 1998, 80: 3312–20.PubMedGoogle Scholar
- Derbyshire SW, Jones AK, Collins M, Feinmann C, Harris M: Cerebral responses to pain in patients suffering acute post-dental extraction pain measured by positron emission tomography (PET). Eur J Pain 1999, 3: 103–113. 10.1053/eujp.1998.0102PubMedView ArticleGoogle Scholar
- Gündel H, Valet M, Sorg C, Huber D, Zimmer C, Sprenger T, Tölle TR: Altered cerebral response to noxious heat stimulation in patients with somatoform pain disorder. Pain 2008, 137: 413–21.PubMedView ArticleGoogle Scholar
- Fuccio C, Luongo C, Capodanno P, Giordano C, Scafuro MA, Siniscalco D, Lettieri B, Rossi F, Maione S, Berrino L: A single subcutaneous injection of ozone prevents allodynia and ecreases the over-expression of pro-inflammatory caspases in the orbito-frontal cortex of neuropathic mice. Eur J Pharmacol 2009, 603: 42–9. 10.1016/j.ejphar.2008.11.060PubMedView ArticleGoogle Scholar
- Borckardt JJ, Smith AR, Reeves ST, Weinstein M, Kozel FA, Nahas Z, Shelley N, Branham RK, Thomas KJ, George MS: Fifteen minutes of left prefrontal repetitive transcranial magnetic stimulation acutely increases thermal pain thresholds in healthy adults. Pain Res Manag 2007, 12: 287–90.PubMed CentralPubMedGoogle Scholar
- Hong SH, Kim MJ, Ahn SC: Glutamatergic transmission is sustained at a later period of development of medial nucleus of the trapezoid body-lateral superior olive synapses in circling mice. J Neurosci 2008, 28: 13003–7. 10.1523/JNEUROSCI.3002-08.2008PubMedView ArticleGoogle Scholar
- Czaja K, Burns GA, Ritter RC: Capsaicin-induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats. Neuroscience 2008, 154: 621–30. 10.1016/j.neuroscience.2008.03.055PubMed CentralPubMedView ArticleGoogle Scholar
- Szallasi A: Vanilloid (capsaicin) receptors in health and disease. Am J Clin Pathol 2002, 118: 110–21. 10.1309/7AYY-VVH1-GQT5-J4R2PubMedView ArticleGoogle Scholar
- Gibson HE, Edwards JG, Page RS, Van Hook MJ, Kauer JA: TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 2008, 57: 746–59. 10.1016/j.neuron.2007.12.027PubMed CentralPubMedView ArticleGoogle Scholar
- Maione S, Cristino L, Migliozzi AL, Georgiou AL, Starowicz K, Salt TE, Di Marzo V: TRPV1 channels control synaptic plasticity in the developing superior colliculus. J Physiol 2009, 587: 2521–35. 10.1113/jphysiol.2009.171900PubMed CentralPubMedView ArticleGoogle Scholar
- Vertes RP: Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience 2006, 142: 1–20. 10.1016/j.neuroscience.2006.06.027PubMedView ArticleGoogle Scholar
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