Abstract

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1−/− mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1−/− mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB1 agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

Introduction

Neuropathic pain is caused by injury of the peripheral or central nervous system (Bonica 1970; Zimmermann 2001; Siniscalco et al. 2005). For its treatment, drugs targeting the transient receptor potential vanilloid type-1 (TRPV1) channel are attracting interest (Hautkappe et al. 1998; Szallasi and Blumberg 1999; Malmberg and Bley 2005; Prevarskaya et al. 2007). TRPV1 expression/activity in sensory fibers is enhanced in neuropathies leading to sustained hyperalgesia (Puntambekar et al. 2004; Rashid, Inoue, Bakoshi, et al. 2003; Rashid, Inoue, Kondo, et al. 2003). TRPV1 is also expressed in higher brain structures (Mezey et al. 2000; Roberts et al. 2004; Cristino et al. 2006), including those involved in pain processing, like the periaqueductal gray (PAG) and cortex (Maione et al. 2006; Steenland et al. 2006; Palazzo et al. 2008; de Novellis et al. 2011). The endocannabinoid anandamide (AEA), which is inactivated by the fatty acid amide hydrolase (FAAH) (Lichtman et al. 2004), stimulates this channel (Zygmunt et al. 1999) and shows analgesic activity (Bradshaw and Walker 2005) exerted via either cannabinoid CB1 and CB2 receptor activation or TRPV1 receptor activation/desensitization (Di Marzo et al. 2002). Central TRPV1 blockade participates in broad-spectrum analgesia (Cui et al. 2006), while producing hyperalgesic effects in the ventrolateral PAG (Starowicz et al. 2007), where capsaicin and AEA cause TRPV1-mediated analgesia (Palazzo et al. 2002; Maione et al. 2006). TRPV1 overactivation might also cause neurodegeneration via accumulation of intracellular Ca2+, caspase-3 release, and neuronal apoptosis (Shin et al. 2003; Kim et al. 2005).

Peripheral nociceptive inputs trigger events that propagate to the limbic and cortical areas of the brain (Millecamps et al. 2007; Nagai et al. 2007; Rea et al. 2007). Some prefrontal cortical areas participate in the affective–cognitive consequences of chronic pain (Apkarian et al. 2005; Ochsner et al. 2006; Kulkarni et al. 2007; Tracey and Mantyh 2007). Activation of the anterior cingulate and agranular insular cortex (Calejesan et al. 2000; Jasmin et al. 2003; Zhuo 2008; Cao et al. 2009; Alter et al. 2010) facilitates pain-related emotional and cognitive responses, in which the basolateral amygdala (BLA)-medial prefrontal cortex (mPFC) circuit is also involved (Floresco and Ghods-Sharifi 2007). Stimulation of the mPFC inhibits nocifensive responses (Ohara et al. 2005). Activation of cortical genes for neurotrophic factors and proteins involved in glutamate receptor formation/trafficking occurs during pain conditions (Cao et al. 2009; Alter et al. 2010). Patients with chronic back pain exhibit 5–10% less neocortical gray matter volume than control subjects, accompanied by neuronal or glial apoptosis (Apkarian et al. 2004; Xu et al. 2008). Activation of cortical caspases in neuropathic mice (Fuccio et al. 2009) may represent an index of degenerative processes leading to cognitive deficits during chronic pain (Thornberry and Lazebnik 1998; Metz et al. 2009; Neugebauer et al. 2009; Ji et al. 2010). Depending on the caspase and cell-type involved, this may lead to apoptosis, neuromodulation, or interleukin-1β (IL-1β)-mediated inflammation and excitotoxicity (for reviews, see D’Amelio et al. 2010; Dinarello 2011). Here, we investigated in the mouse limbic cortex: 1) whether neuropathic pain induced by injury of the sciatic nerve results in caspase release, possibly as a consequence of alterations the BLA-mPFC circuit and glutamate release and 2) the role of TRPV1 channels in these putative changes.

Materials and Methods

Drugs

N-arachidonoyl-serotonin (AA-5-HT) was synthesized in Dr Di Marzo’s laboratory as previously described (Bisogno et al. 1998; De Petrocellis et al. 2000). DL-2-amino-5-phosphonovalerate (APV) was purchased from Sigma-Aldrich (Milano, Italy). 2-Amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA), 2-methyl-6-(phenylethynyl)-pyridine (MPEP), 5′-iodoresiniferatoxin (I-RTX), N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), Capsaicin tetratoxin (TTX) and WIN55,212-2 were purchased from Tocris Bioscience (Bristol, UK). 3′-carbamoylbiphenyl-3yl-cyclohexylcarbamate (URB597) was purchased from Alexis Bio-chemicals. DEVD-CHO was purchased from Biosource International (California). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was purchased from Research Biochemical International (Natik, MA). All drugs were dissolved in 0.5% DMSO in ACSF for intra-PL-IL cortex microinjection and in saline for intraperitoneal (i.p.) administration.

Animals and Surgery

Male C57BL/6N (25-30 g) and Trpv1 −/− mice were housed 3 per cage under controlled illumination (12:12 h light:dark cycle; light on 06.00 h) and environmental conditions (room temperature 20–22° C, humidity 55–60%) for at least 1 week before the commencement of experiments. Mouse chow and tap water were available ad libitum. The experimental procedures were approved by the Animal Ethics Committee of the Second University of Naples. Animal care was in compliance with the IASP and European Community (E.C. L358/1 18/12/86) guidelines on the use and protection of animals in experimental research. All efforts were made to minimize animal suffering and the number of animals used.

Behavioral testing was performed before surgery to establish a baseline for comparison with postsurgical values. Spared nerve injury (SNI) and sham mice were tested for thermal and mechanical hyperalgesia and after that they were used for further studies. Mononeuropathy was induced according to the method of Bourquin et al. (2006). Mice were anaesthetized with sodium pentobarbital (50 mg/kg i.p.). The sciatic nerve was exposed at midthigh level distal to the trifurcation and freed of connective tissue; the 3 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 leaving the sural nerve intact. The sham procedure consisted of the same surgery without ligation and transection of the nerves. For repeated-treatment, SNI and sham mice were daily treated with vehicle (0.5% DMSO in saline, i.p.) (n = 9) or AA-5-HT (5 mg/kg i.p.) for 3, 7, and 14 days starting the day after surgery (day = 0). On days 3, 7, and 14 after surgery, vehicle and AA-5-HT-treated SNI or sham mice were divided into further groups at each time point for reverse transcriptase-polymerase chain reaction (RT-PCR) (n = 9), for western blot (n = 9), and histological examination (n = 9). For histological examination, naıve, sham, and SNI mice, treated with vehicle or AA-5-HT (5 mg/kg, i.p.) (n = 3) for 3 and 7 days after surgery, were used. Repeated-treatment experiments in SNI and sham mice were also carried out with daily vehicle (n = 5), I-RTX (0.2 mg/kg i.p.), or URB597 (3 mg/kg i.p.) for 7 and 14 days, starting the day after surgery (day = 0). For in vivo single-unit extracellular recording and nociceptive response combined experiments, 7-day vehicle or AA-5-HT-treated SNI and sham mice were used (n = 10). Only mice that showed mechanical allodynia (3.8 ± 0.3 vs. 9.5 ± 0.5 g/s) monitored by Dynamic Plantar Anesthesiometer test were used for electrophysiological experiments.

Sham and SNI mice (n = 6) were used for the assessment of mechanical allodynia 7 days after surgery also before and after a single intra-PL-IL cortex microinjection of 200 nL of vehicle (0.5% DMSO in artificial cerebrospinal fluid, ACSF, in mM: 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose, oxygenated and equilibrated to pH 7.4.ACSF), DEVD-CHO (1–5–10 nmol), AA-5-HT (0.25–1 nmol), URB597 (1–2–4 nmol), I-RTX (0.5–1 nmol), capsaicin (1.5–3–6 nmol), WIN55,212-2 (25–100 nmol), AM251 (0.25–0.5 nmol), and AA-5-HT (1 nmol) in combination with AM251 (0.25–0.5 nmol). CNQX (100–200 pmol), AMPA (100–200 pmol), MPEP (5–10–20 nmol), APV (2.5–5–10 nmol), MPEP (10–20 nmol) in combination with APV (10 nmol), capsaicin (1.5–6 nmol) in combination with I-RTX (0.5–1 nmol), I-RTX (0.5 nmol) in combination with capsaicin (–6 nmol). The drug doses were chosen based on our previous studies in which they were found to be effective in several pain models in rodents (Aimone and Gebhart 1986; Bruno et al. 2000; Maione et al. 2007; de Novellis et al. 2008; Merkina et al. 2009).

RNA Extraction and RT-PCR

Total RNA was extracted from homogenized PL-IL cortex using an RNA Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s protocol. Since pilot analyses showed no significant differences between ipsilateral and contralateral sections, these latter were pooled to carry out the bulk of the experiments. 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 (Galderisi et al. 1999). RT minus controls were carried out to check potential genomic DNA contamination. These RT minus controls were performed without using the RT 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 4 times to achieve best reproducibility data. A semiquantitative analysis of mRNA levels was carried out by the “Gel Doc 2000 UV System” (Bio-Rad, Hercules, CA). The measured mRNA levels were normalized with respect to hypoxanthine-guanine phosphoribosyltransferase (HPRT), chosen as housekeeping gene. The HPRT gene expression did not change in several experimental conditions (Siniscalco et al. 2007). To our knowledge, there is no molecular evidence for variation in HPRT mRNA-levels in SNI model of neuropathic pain. The gene expression values were expressed as arbitrary units ± standard error (SE). Amplification of genes of interest and HPRT were performed simultaneously.

Western Blotting

For the protein extraction, the PL-IL cortex was minced into small pieces with a blender, then 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]). Since pilot analyses showed no significant differences between ipsilateral and contralateral sections, these latter were pooled to carry out the bulk of the experiments. The total protein concentration was determined by the method described by Bradford (1976). Each sample was loaded and electrophoresed in a 8, 12, or 15% polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Primary antibodies to detect FAAH (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), caspase-1 (1:1000, Abcam, Cambridge, UK), IL-1beta (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), IL-1R (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and active caspase-3 (1:500, Chemicon/Millipore, Billerica, MA) were used according to the manufacturer’s instruction at 1:500 dilution (Chemicon/Millipore). 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. 1:1000 dilution).

Immunohistochemistry

Under pentobarbital anesthesia (50 mg/kg, i.p), animals were perfused transcardially with 50 mL of saline solution (0.9% NaCl) and 100 mL of 4% paraformaldehyde fixative. The brain of wild type and Trpv1−/− naïve (n = 3 per group), sham (n = 3 per group), and SNI mice sacrificed at 3 days (n = 3 wt only) and 7 days (n = 3 per group) after vehicle administration was taken out and kept in the fixative for 2 h at 4 °C. The tissue was kept in 30% sucrose in PBS and frozen in cryostat embedding medium (Bio-Optica, Milan, Italy). Serial 10-μm sections were cut using a cryostat and thaw mounted onto glass slides (Menzel, Germany) in several alternate series to be processed for single immunoperoxidase and single or multiple immunofluorescence techniques. For single immunoperoxidase staining, the sections were reacted for 10 min in 0.1% H2O2 to inactivate endogenous peroxidase activity and preincubated for 1 h at room temperature in 10% normal goat serum (NGS; Vector Laboratories, Burlingame, CA) in 0.1 M Tris-buffered saline, pH 7.6 (TBS), containing 0.3% Triton X-100 and 0.05% sodium azide (Sigma). The sections were then incubated for 2 days at 4 °C with NGS diluted (range 1:200–1:400) rabbit polyclonal anti-NAPE-PLD or anti-DAGL (either diluted 1:200 and kindly provided from Kenneth Mackie Laboratory, Indianapolis) or anti-FAAH or anti MAGL (Santa Cruz Biotechnology). After several rinses, the sections were incubated at room temperature for 2 h in biotinylated goat anti-rabbit IgGs (Vector Laboratories) followed 1 h by incubation in the avidin–biotin complex (ABC Kit; Vectastain, Vector) diluted in TBS according to the supplier indications and then in 0.05‰ 3-3′diaminobenzidine for 10 min (DAB Sigma Fast, Sigma-Aldrich). The same procedure was followed for TRPV1 immunostaining using goat polyclonal antibody that recognizes the N-terminus of TRPV1 receptor (Santa Cruz Biotechnology; diluted 1:200), normal rabbit serum (NRS; Dako, Glostrup, Denmark) and biotinylated rabbit anti-goat IgGs (Vector Laboratories). The specificity of the anti-TRPV1 had been previously validated in TRPV1 null mice (Cristino et al. 2006). For single anti-caspase immunoreactivity, the nonspecific 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). Thereafter, the slides were incubated overnight at 4 °C with anti-caspase3 antibody (Chemicon/Millipore, diluted 1:200 in PBS blocking buffer). The sections processed for double immunofluorescence were incubated with anti-caspase3 primary antibody together with GFAP (rabbit anti-GFAP, Cytomation, Denmark) or Iba-1 (rabbit anti-ionized calcium binding adapter molecule 1; 1:1000; Wako Chemicals, Germany).

Alexa Fluor 568 specific to the IgG species used as a primary antibody was 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). The sections processed for double immunofluorescence were preincubated for 1 h in 10% normal donkey serum (NDS Jackson Immunoresearch Laboratories, West Grove, PA) in phosphate buffer, pH 7.4 containing 0.3% Triton X-100 and 0.05% sodium azide (Sigma) for 2 days in a mixture of anti-TRPV1 receptor coupled to anti-VGluT1 and anti-CB1 reactivities (diluted 1:100 in NDS), at room temperature. After 3 washes in PB, double immunofluorescence was revealed by incubation for 4 h in a mixture of Alexa Fluor 488 anti donkey anti-rabbit and Alexa Fluor 546 anti donkey anti-guinea pig and Alexa Fluor 350 anti-goat diluted 1:200 in NDS. Thereafter, sections were washed with PB and coverslipped with Aquatex mounting medium (Merck, Darmstadt, Germany). Controls of single and double immunoreactivities included: 1) preabsorption of diluted antibodies with their respective immunizing peptides; 2) omission of either the primary antisiera or the secondary antibodies; and 3) for double immunofluorescence only, to make sure of no cross-reaction between the secondary and primary antibodies, incubation of the section with the first primary antibody and adding the second secondary antibody. In all controls, no immunostaining was detected. The sections processed for immunofluorescence were studied with an epifluorescence microscope equipped with the appropriate filters; all the other material was investigated at the microscope under bright-field illumination (Leica DM-IRB, Germany). Images were acquired by using the digital camera Leica DFC 320 connected to the microscope and the image analysis software Leica IM500 for Windows which allows both single and merged pictures. Digital images were processed in Adobe Photoshop, with contrast and brightness being the only adjustments made.

Controls included: 1) preabsorption of diluted antibodies with their respective immunizing peptides (the anti-DAGL-α and NAPE-PLD antibodies were kindly supplied from Prof. Kenneth Mackie) and 2) omission of either the primary antisera or the secondary antibodies. These control experiments did not show staining.

Analysis of Immunoreactivity

Analysis of immunoreactivity (ir) was performed in the laminae II/III and V/VI of PL-IL cortex on 15 sections per animal (wild-type sham and SNI mice at 3 days and 7 days after nerve injury and Trpv1−/− sham and SNI mice at 7 days after nerve injury) for each single immunoperoxidase (densitometric analysis of optical immunodensity) and multiple immunfluorescence (the mean percentage value of the number of neurons multiple labeled) signal.

Densitometric analysis of TRPV1, NAPE/PLD, DAGL-α, FAAH, and MAGL optical immunodensity was performed using a digital camera working on gray levels (JCV FC 340FX, Leica) and the software Image Pro Plus 6.0 MediaCybernetics for Windows, working on logarithmic values scale of absorbance for densitometric evaluation. All densitometric measures were performed on tissue or cells processed for immunoperoxidase reaction by an observer who did not know the experimental treatment of the sections being analyzed. A sample of 100 immunopositive cells with nuclei (unstained or lightly stained) in the focal plane were randomly identified per each animal. The images were acquired under constant light illumination and at the same magnification. In each section, the zero value of optical density was assigned to the background, that is, a portion of sample devoid of stained cell bodies.

Quantification of the mean percentage value of the number of GluR2-ir interneurons, GluR2-ir microglial cells, multiple-labeled neurons for TRPV1/vesicular glutamate transporter 1 (VGluT1), and CB1 and TRPV1/DAGL-α, in the laminae II/III of PL-IL cortex was performed on a mean of 200 neurons randomly selected with respect to each adjacent section labeled with cresyl violet and whose nuclei, unstained or lightly stained, were in the focal plane (by an observer blind to the experimental protocols). The level of the section evaluated for immunohistochemistry covered the entire extension of the PL-IL cortex. The histological identification of lamina II/III and V/VI for the analysis of immunoreactivity was grounded on cytoarchitectonic features of the limbic cortex according to criteria adopted by Van De Werd et al. (2010). Briefly, in the PL-IL cortex the cells of the layer V appear in disorderly arranged while in the layer VI are mainly arranged horizontal rows; layer II was a little broader with its cells more equally spread over the layer III. Moreover in the PL-IL cortex, we discerned a dorsal and a ventral part based on a different aspect of layer II, which was narrow and compact in the dorsal part of PL but broad and less compact in the ventral part. Layers III and V were more compact in the ventral part of the PL-IL cortex (Van De Werd et al. 2010).

In Vivo Single-Unit Extracellular Recording

Mice for electrophysiological recordings were anaesthetized with pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA). Body temperature was maintained at 37 °C with a temperature-controlled heating pad. In all surgical preparations, the scalp was incised and holes were drilled in the skull overlying the site of recording, mPFC (AP: +1–2.9, L: 0.2–0.3 from bregma and V: 1.2–3 mm below dura), and the site of stimulation, BLA (AP: −0.5–2.06, L: 2.8–3.0 from bregma and V: 4.2–5 below dura) according to the coordinates from the Atlas of Franklin and Paxinos (1997) and contralateral with respect to the nerve insult.

Anesthesia was maintained with a constant continuous infusion of propofol (5–10 mg/kg/h, i.v.) and a bipolar concentric electrode (NEX-100; Rhodes Medical Instruments Inc., Summerland, CA) connected to A320 stimulator (World Precision Instruments England) was lowered into the caudal region of the BLA. After lowering of the stimulating electrode into the BLA, a glass-insulated tungsten filament electrode (3–5 MΩ) (FHC Frederick Haer & Co., ME) was stereotaxically lowered into the mPFC. The recorded signals were amplified and displayed on a digital storage oscilloscope to ensure that the unit under study was unambiguously discriminated throughout the experiment. Signals were processed by an interface CED 1401 (Cambridge Electronic Design Ltd., UK) and analyzed through Spike2 software (CED, version 4) to create peristimulus rate histograms online and to store and analyze digital records of single-unit activity off-line. Configuration, shape, and height of the recorded action potentials were monitored and recorded continuously. This study only included neurons with a regular spiking pattern and a spontaneous firing rate between 0.4 and 1.5 Hz that were classified as pyramidal neurons in rodents (Jung et al. 1998; Tierney et al. 2004; Floresco and Tse 2007). Once a neuron was encountered in PL-IL cortex, the position of the microelectrode was adjusted to maximize the spike amplitude relative to background noise. After that we delivered electrical stimuli into the BLA (200 μA) at 2-s intervals. At least 50 single pulses were delivered to generate peristimulus time histograms (PSTHs). Mechanical stimuli were also applied to hind paw (contralateral to the mPFC) by von Frey filament with bending force of 97.8 mN (noxious stimulation) for 2 s with it slightly buckled (Simone et al. 2008). By using electrical (BLA) or mechanical (von Frey filament) stimuli, we could determine whether each individual neuron was inhibited, excited, or showed no response to stimulations. For neurons that displayed no change in firing in response to stimulation, we did not record data from that cell and continued the cell-searching procedure.

Characterization of BLA-Evoked Responses and Stimulation Protocol

We observed that BLA stimulation could evoke 2 distinct types of firing changes in separate populations of mPFC neurons. We characterized these responses accordingly with previously established criteria used by Ishikawa and Nakamura (2003). Whenever a neuron that was responsive to BLA stimulation was encountered, the BLA was stimulated with 100–200 pulses to determine whether the cell responded with inhibition or excitation. Specifically, a cell was considered to be inhibited by BLA stimulation if it displayed a complete cessation of spontaneous firing after BLA stimulation. Hereafter, neurons that displayed this type of response are referred to as “BLA→mPFC (−)” neurons (Floresco and Tse 2007). Only neurons that displayed a spontaneous firing rate between 0.5 and 1.5 Hz were used for the data analysis.

Once a neuron that was inhibited by BLA stimulation was isolated, single-pulse stimulation was delivered at 0.5 Hz. We typically used 100–250 sweeps, and PSTHs were generated on-line. We used 2 parameters derived from the PSTHs to asses differences between different groups of mice. Our primary measure was the “duration” of inhibition (in milliseconds) as defined by Ishikawa and Nakamura (2003). The duration was calculated from the longest period when spontaneous firing was completely suppressed after BLA stimulation. The second measure we have here considered was the “onset” of this period of inhibition (in milliseconds) after BLA stimulation (i.e., the time interval between the stimulus application and last spike before a complete cessation of neuronal activity). By using these parameters, we could have a reliable index of BLA-evoked inhibition and changes in the inhibitory influences that BLA inputs exert over the mPFC neuron firing (Ishikawa and Nakamura 2003; Laviolette and Grace 2006; Floresco and Tse 2007). Moreover, changes in timing of the onset and duration of inhibition to either BLA or mechanical stimuli inversely correlate with neuropathic pain symptoms.

A second group of neurons displaying a fast-onset increase of firing were classified as “BLA→ mPFC(+)” neurons (Floresco and Tse 2007). This group of neurons showed a cluster of spikes with an increased frequency showing typically a Gaussian pattern of distribution after BLA stimulation (200 μA). From the PSTHs, we measured the duration of excitation (in ms) as the period of the increased firing activity which exceeds the average baseline value +2 standard deviations (SDs). Moreover, we measured the frequency of evoked excitation and the onset of excitation which was considered as the time from the application of the stimulus (artifact) to the first-evoked spike which exceeds the average baseline value +2 SD. These criteria were used as an index of changes in the excitatory influence that BLA inputs exert over mPFC neuron firing. Moreover, changes in timing of the onset and duration of excitation to either BLA or mechanical stimuli inversely or directly correlate with neuropathic pain symptoms, respectively.

Characterization of Mechanically Evoked Responses

Mechanical stimuli were applied to the hind paw (contralateral to the mPFC) by von Frey filaments with bending force of 97.8 mN (noxious stimulation) for 2 s (Sergey et al. 2007; Simone et al. 2008) The mechanical stimulus evoked inhibitory or excitatory response in separate populations of mPFC neurons. The same parameters of the inhibitory and excitatory responses were measured and considered as an index of mPFC neuron firing response to mechanical noxious stimuli.

Nociceptive Behavior

Mechanical allodynia was measured by using Dynamic Plantar Aesthesiometer (Ugo Basile, Varese, Italy). Mice were allowed to move freely in 1 of the 2 compartments of the enclosure positioned on the metal mesh surface. Mice were adapted to the testing environment for 30 min before any measurement was taken. After that, the mechanical stimulus was delivered to the plantar surface of the hindpaw of the mouse from below the floor of the test chamber by an automated testing device. A steel rod (2 mm) was pushed with electronical ascending force (0–30 g in 10 s). When the animal withdrawn its hindpaw, 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 thresholds (PWT) in grams.

Each mouse served as its own control, the responses being measured both before and after surgical procedures. PWT were quantified by an observer blind to the treatment.

Motor Coordination Behavior

Possible motor coordination impairment was evaluated by Rotarod test (Ugo Basile). Mice were measured for the time (in s) of equilibrium before falling on a rotary cylinder by a magnet that, activated from the fall of the mouse on the plate, allows to record the time of permanence on the cylinder. After a period of adaptation of 30 s, the spin speed gradually increased from 5 to 40 rpm for a maximum time of 5 min. The animals were analyzed by 2 separate tests at 1-h interval in the same day. The experiment was performed for every group of animals: the day before the surgical procedure and the days before the behavioral tests in order to avoid useless stress. The time of permanence of the mouse on the cylinder was expressed as latency time (s).

In Vivo Microdialysis

Brain microdialysis experiments were performed in awake and freely moving mice. In brief, mice were anaesthetized with pentobarbital (50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the PL-IL cortex using coordinates: AP: 1.5–1.8 mm, L: 0.3–05 mm from bregma and V: 3.3 mm below the dura. Microdialysis concentric probes were constructed as described by Hutson et al. (1985) with 25G (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 dialysis membrane (Enka AG, Wuppertal, Germany) 1.3 mm in length. Following a postoperative recovery period of approximately 24 h, dialysis was commenced with ACSF (pH 7.2) perfused at a rate of 0.8 μL/min using a Harvard Apparatus infusion pump (mod. 22). Following an initial 60-min equilibration period, 12 consecutive 30-min dialysate samples were collected. Mice received drugs by reverse microdialysis (30-min perfusion). A group of mice received tetrodotoxin (TTX, 1 μM) to assess the synaptical nature of glutamate and γ-aminobutyric acid (GABA) released in PL-IL cortex dialysate. On completion of experiments, mice were anaesthetized 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-μm thick slices and observed under a light microscope to identify the probe locations.

Dialysates were analyzed for amino acid content using an high-performance liquid chromatography method. The system comprised a Varian ternary pump (mod. 9010), a C18 reverse-phase column, a Varian refrigerated autoinjector (mod. 9100), a Varian fluorimetric detector (mod. PS363). Dialysates were precolumn derivatized with O-pthaldialdehyde (10 μL dialysate + 10 μL o-pthaldialdehyde) and amino acid conjugates resolved using a gradient separation. The detection limit of GABA and glutamate in 10-μL samples was approximately 0.5–1 and 2–3 pmol, respectively. The mobile phase consisted of 2 components: 1) 0.1 M sodium acetate buffer (pH 6.95), 25% tetrahydrofuran, and 10% methanol and 2) 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 by Varian Star 6.2 control data and acquisition software. The mean dialysate concentration of amino acids in the first 5 samples before any drug treatment represented the basal release, and the results were expressed as percentage of this value.

N-Acylethanolamine and 2-Arachidonoylglycerol (2-AG) Measurement in the PL-IL Cortex of Sham or SNI Mice

In order to perform the endocannabinoid analysis, a different cohort of mice was used. Mouse decapitation was performed, and brains were rapidly removed and embedded in oxygenated 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) (interaural from +1.9 to +0.7 mm, Franklin and Paxinos 1997). 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. (1992) and Bisogno et al. (1997). 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 resuspended in chloroform/methanol 99:1 by volumes. The solutions were then purified by open bed chromatography on silica as described in Bisogno et al. (1997). Fractions eluted with chloroform/methanol 9:1 by volume (containing AEA, OEA, PEA, and 2-AG) were collected, the excess solvent was evaporated with a rotating evaporator, and aliquots were analyzed by isotope dilution-liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC-APCI-MS) carried out under conditions described previously (Marsicano et al. 2003) and allowing the separation of the 4 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), 304 and 300 (molecular ions +1 for deuterated and undeuterated PEA), 330 and 326 (molecular ions +1 for deuterated and undeuterated PEA), and 384.3 and 379.3 (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.

Statistics

Behavioral, electrophysiology, endocannabinoid level, and biomolecular data are represented as means ± SE, and statistical analysis of these data was performed by two way analysis of variance (ANOVA) for repeated measured followed by the Student Newman–Keuls for multiple comparisons to determine statistical significance between different treated groups of mouse. In addition, the differences in proportions were analyzed with the χ2 test. For analysis of immunoreactivity, data are represented as means ± SD and statistical analysis of these data were performed by one-way ANOVA followed by the Bonferroni’s test.

Results

Overexpression of Caspases-1 and 3, IL-1β and AMPA-Like (GluR2) Receptors in Glial Cells of the PL-IL Cortex of SNI Mice

For all genes and proteins examined, naive and sham mice treated with vehicle or AA-5-HT at 3, 7, and 14 days showed no differences in the expression levels (data not shown). To check activation of the apoptotic regulative caspases, expression of the caspase-8 and caspase-9 genes were analyzed. The semiquantitative analysis of mRNA levels measured by RT-PCR amplification, in the PL-IL cortex of mice, showed an increase in the mRNA levels of proapoptotic caspase-8 and caspase-9 in SNI/vehicle with respect to sham/vehicle mice 7 days after SNI (Table 1). To check for the presence of inflammation, changes in the expression of inflammation marker genes such as caspase-1 and caspase-12 were analyzed. The mRNA levels of the caspase-1 and caspase-12 genes were increased at day 7 and 14 in SNI/vehicle with respect to the sham/vehicle mice (Table 1).

Table 1

The mRNA levels (mean ± SE) of the genes under analysis measured by RT-PCR amplification are reported

Gene Sham/vehicle SNI + vehicle 3 days SNI + AA-5-HT 3 days SNI + vehicle 7 days SNI + AA-5-HT 7 days SNI + vehicle 14 days SNI + AA-5-HT 14 days 
Caspase-1/HPRT 0.99 ± 0.03 1.01 ± 0.03 0.75 ± 0.02* 1.83 ± 0.03° 0.80 ± 0.02* 1.37 ± 0.03° 0.82 ± 0.02* 
Caspase-3/HPRT 0.79 ± 0.02 0.87 ± 0.04 1.03 ± 0.06 0.93 ± 0.05 0.96 ± 0.02 0.69 ± 0.04 0.74 ± 0.01 
Caspase-8/HPRT 0.96 ± 0.01 1.06 ± 0.02 0.95 ± 0.03 1.48 ± 0.06° 0.85 ± 0.03* 1.00 ± 0.03 0.88 ± 0.03 
Caspase-9/HPRT 0.70 ± 0.03 0.63 ± 0.01 0.59 ± 0.01 0.89 ± 0.04° 0.72 ± 0.01* 0.67 ± 0.02 0.42 ± 0.02* 
Caspase-12/HPRT 0.52 ± 0.01 0.61 ± 0.03 0.28 ± 0.01* 0.85 ± 0.01° 0.23 ± 0.01* 0.91 ± 0.01° 0.26 ± 0.02* 
Gene Sham/vehicle SNI + vehicle 3 days SNI + AA-5-HT 3 days SNI + vehicle 7 days SNI + AA-5-HT 7 days SNI + vehicle 14 days SNI + AA-5-HT 14 days 
Caspase-1/HPRT 0.99 ± 0.03 1.01 ± 0.03 0.75 ± 0.02* 1.83 ± 0.03° 0.80 ± 0.02* 1.37 ± 0.03° 0.82 ± 0.02* 
Caspase-3/HPRT 0.79 ± 0.02 0.87 ± 0.04 1.03 ± 0.06 0.93 ± 0.05 0.96 ± 0.02 0.69 ± 0.04 0.74 ± 0.01 
Caspase-8/HPRT 0.96 ± 0.01 1.06 ± 0.02 0.95 ± 0.03 1.48 ± 0.06° 0.85 ± 0.03* 1.00 ± 0.03 0.88 ± 0.03 
Caspase-9/HPRT 0.70 ± 0.03 0.63 ± 0.01 0.59 ± 0.01 0.89 ± 0.04° 0.72 ± 0.01* 0.67 ± 0.02 0.42 ± 0.02* 
Caspase-12/HPRT 0.52 ± 0.01 0.61 ± 0.03 0.28 ± 0.01* 0.85 ± 0.01° 0.23 ± 0.01* 0.91 ± 0.01° 0.26 ± 0.02* 

Note: Each RT-PCR was repeated at least 4 times. The semiquantitative analysis of mRNA levels was carried out by the Gel Doc 2000 UV System (Bio-Rad). The measured mRNA levels were normalized with respect to HPRT (housekeeping gene), and gene expression values were expressed as arbitrary units ± SE. °P < 0.05 versus the corresponding naive, *P < 0.05 versus the SNI/vehicle mice, as analyzed by ANOVA, followed by Student–Neuman–Keuls test. Naive or sham mice treated with AA-5-HT were not reported in this table because they had same values of gene expression respect to sham/vehicle.

We investigated the protein levels of the active form of executioner caspase-3 in the PL-IL cortex following SNI. Western blotting, carried out using an antibody able to detect endogenous levels of the large fragments of activated caspase-3 without recognizing full length caspase-3 or other cleaved caspases, confirmed that caspase-3 was overexpressed in the PL-IL cortex (Fig. 1A); a strong protein reactivity for the active form of caspase-3 was observed 3 (194 ± 2% vs. sham/veh), 7 (164 ± 3% vs. sham/veh), and 14 (167 ± 2% vs. sham/veh) days after SNI. Immunofluorescence showed that caspase-3 was present in this brain area only in SNI mice and colocalized with a marker of microglia (Iba-1) but not of astrocytes (GFAP) (Fig. 1B). We also investigated the expression levels of caspase-1, which differs from capsase-3 because of its more direct role in inflammation via the interleukin-1β (IL-1β) signaling pathway. Western blotting carried out using an antibody able to detect endogenous levels of the large fragments of activated caspase-1 without recognizing full-length caspase-1 or other cleaved caspases, confirmed that caspase-1 was overexpressed in the PL-IL cortex (184 ± 5 vs. sham/veh) (Fig. 2A). We found that, unlike capsase-3, this protein was mostly elevated in astrocytes of the PL-IL cortex of SNI mice, as assessed by costaining with GFAP (Fig. 2B). Accordingly, also IL-1β was found to be expressed in reactive astrocytes of this brain area (Fig. 3A,B). Importantly, when SNI was induced in Trpv1−/− mice, no elevation of capsase-3 in microglia was detected, whereas caspase-1 and IL-1β were still overproduced in astrocytes of the PL-IL cortex (Figs 1C–3C).

Figure 1.

Caspase-3 activation in the PL-IL cortex of SNI mice. (A) Western blot analysis for activated caspase-3 in the PL-IL cortex of SNI mice 3, 7 and 14 days after sciatic nerve SNI. The histogram indicates percent variations in activated caspase-3 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-3 and ß-actin proteins are shown. (B) Caspase-3 immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of caspase-3 was found in Iba-1-ir microglia of the PL-IL cortex 7 days after SNI. (C) Caspase-3 protein levels did not change in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 1.

Caspase-3 activation in the PL-IL cortex of SNI mice. (A) Western blot analysis for activated caspase-3 in the PL-IL cortex of SNI mice 3, 7 and 14 days after sciatic nerve SNI. The histogram indicates percent variations in activated caspase-3 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-3 and ß-actin proteins are shown. (B) Caspase-3 immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of caspase-3 was found in Iba-1-ir microglia of the PL-IL cortex 7 days after SNI. (C) Caspase-3 protein levels did not change in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 2.

Caspase-1 activation in the PL-IL cortex of SNI mice. (A) Caspase-1 western blot analysis for activated caspase-1 in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in activated caspase-1 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-1 and ß-actin proteins are shown. (B) Caspase-1 immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of caspase-1 was found in the GFAP-ir profiles in PL-IL cortex 7 days after SNI. (C) Caspase-1 protein levels were increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 2.

Caspase-1 activation in the PL-IL cortex of SNI mice. (A) Caspase-1 western blot analysis for activated caspase-1 in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in activated caspase-1 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-1 and ß-actin proteins are shown. (B) Caspase-1 immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of caspase-1 was found in the GFAP-ir profiles in PL-IL cortex 7 days after SNI. (C) Caspase-1 protein levels were increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 3.

Interleukin 1-β in the PL-IL cortex of SNI mice. (A) Western blot analysis for activated interleukin 1-β in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in activated interleukin 1-beta protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated interleukin 1-β and ß-actin proteins are shown. (B) Interleukin 1-β immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of interleukin 1-β was found in the GFAP-ir profiles in PL-IL cortex 7 days after SNI. (C) Interleukin 1-β protein levels were increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 3.

Interleukin 1-β in the PL-IL cortex of SNI mice. (A) Western blot analysis for activated interleukin 1-β in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in activated interleukin 1-beta protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated interleukin 1-β and ß-actin proteins are shown. (B) Interleukin 1-β immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of interleukin 1-β was found in the GFAP-ir profiles in PL-IL cortex 7 days after SNI. (C) Interleukin 1-β protein levels were increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

A significant increase of AMPA-like receptor (GluR2)-ir was observed on the perisomatic compartment of GABAergic interneurons, mainly in the IL cortex (identified as GAD65-67 positive) of wild-type SNI mice and much less so in Trpv1−/− SNI mice (Fig. 4A–D) compared with the respective sham mice. In particular, more than a half of the interneuron population belonging to laminae II/III of the PL-IL cortex in wild-type SNI 7 day mice expressed GluR2-ir (28.5 ± 4.1%; 63.2 ± 9.8%; 37.5 ± 4.8%; 35.3± 3.3% mean of percent ± SD of the mean for wild-type sham, wild-type SNI 7d, Trpv1−/− sham and Trpv1−/− SNI 7d, respectively). A strong increase of GluR2-expressing microglial cells was also observed in the laminae II/III of wild-type brains (Fig. 4E) compared with wild-type sham-operated and Trpv1−/− mice, either in sham or after 7 days of SNI conditions (Fig. 4F). In particular, more than half of the microglial population belonging to laminae II/III of the PL-IL cortex in wild-type SNI 7 day mice expressed GluR2-ir (26.2 ± 4.5%; 59.7 ± 11.2%; 32.3 ± 5.5%; 30.2± 4.3% mean of percent ± SD of the mean for wild-type sham, wild-type SNI 7d, Trpv1−/− sham and Trpv1−/− SNI 7d, respectively). GluR2-ir was observed on the perisomatic compartment of microglial cells as identified by Iba1-ir (for high magnification, see Fig. 4E1).

Figure 4.

GluR2 immunoreactivity in the PL-IL cortex of SNI mice. (AD) GAD 65-67/GluR2 immunoreactive neuronal cells in the lamine II/III of the PL-IL cortex of wild-type sham mice (A), wild-type SNI mice at 7d (B), Trpv1−/− sham mice (C), and Trpv1−/− SNI mice at 7d (D). Note the increase of the GluR2 signal after nerve injury both in wild-type SNI mice at 7d and, to a much smaller extent, Trpv1−/− SNI mice at 7d. (E and F) Iba1/GluR2 immunoreactive microglial cells in the lamine II/III of the PL-IL cortex of wild-type SNI mice at 7d (EE1) and Trpv1−/− SNI mice at 7d (F). Note the typical perisomatic feature of the GluR2 signal (see in E1 the enlargements of the boxed areas in E). The histograms show the quantification of interneuronal cells in wild-type SNI mice at 7d compared with wild-type sham and Trpv1−/− sham mice at 7d (P < 0.05 wild-type SNI 7d vs. wild-type sham, Trpv1−/− mice sham and Trpv1−/− mice SNI 7d) and of microglial cells in wild-type SNI mice at 7d compared with wild-type sham and Trpv1−/− sham mice at 7d (P < 0.05 wild-type SNI 7d vs. wild-type sham, Trpv1−/− mice sham and Trpv1−/− mice SNI 7d). Scale bars: AD = 30 μm, E and F =10 μm, E1 =5 μm.

Figure 4.

GluR2 immunoreactivity in the PL-IL cortex of SNI mice. (AD) GAD 65-67/GluR2 immunoreactive neuronal cells in the lamine II/III of the PL-IL cortex of wild-type sham mice (A), wild-type SNI mice at 7d (B), Trpv1−/− sham mice (C), and Trpv1−/− SNI mice at 7d (D). Note the increase of the GluR2 signal after nerve injury both in wild-type SNI mice at 7d and, to a much smaller extent, Trpv1−/− SNI mice at 7d. (E and F) Iba1/GluR2 immunoreactive microglial cells in the lamine II/III of the PL-IL cortex of wild-type SNI mice at 7d (EE1) and Trpv1−/− SNI mice at 7d (F). Note the typical perisomatic feature of the GluR2 signal (see in E1 the enlargements of the boxed areas in E). The histograms show the quantification of interneuronal cells in wild-type SNI mice at 7d compared with wild-type sham and Trpv1−/− sham mice at 7d (P < 0.05 wild-type SNI 7d vs. wild-type sham, Trpv1−/− mice sham and Trpv1−/− mice SNI 7d) and of microglial cells in wild-type SNI mice at 7d compared with wild-type sham and Trpv1−/− sham mice at 7d (P < 0.05 wild-type SNI 7d vs. wild-type sham, Trpv1−/− mice sham and Trpv1−/− mice SNI 7d). Scale bars: AD = 30 μm, E and F =10 μm, E1 =5 μm.

Overexpression of TRPV1, Vesicular Glutamate Transporter-1 (VGluT1), and IL-1β Receptor-1 (IL-1R1) in Neurons of the PL-IL cortex

The values of TRPV1-ir optical density were expressed in the log scale; the data are presented as means ± SD of n = 3 mice for each group on a total of n = 100 cells per group. The identification of region of interest (laminae) was performed according to the cytoarchitectonic organization of prelimbic cortex as reported by Van De Werd et al. (2010). All the statistic densitometric analyses were referred to each immunological marker in the laminae II/III or in the laminae V/VI. No differences were observed in the immunoreactivity of the analyses proteins between the ipsilateral and contralateral sides of sham and SNI mice, either in wild-type or Trpv1−/− mice. Hereafter, therefore, only the data for the contralateral side are reported. We found TRPV1 immunoreactivity exclusively in the wild-type animals, wherein it is located in the somata of roughly half of the neuronal population belonging to laminae II/III of the PL-IL cortex (48.2 ± 4.2%; 57.3 ± 10.2%; 49.3 ± 8.7% mean of percent ± SD of the mean for sham, 3 and 7 days after SNI, respectively, Fig. 5A). Despite the fact that the number of TRPV1-expressing neurons did not change much following SNI, the density of the expression did. A significant increase of TRPV1 immunoreactivity was quantified in the laminae II/III after SNI (0.40 ± 0.03; 0.81 ± 0.07; 0.73 ± 0.05 mean optical density ± SD of the mean in sham, 3 and 7 days after SNI, respectively, Fig. 5B). Similar results were obtained in laminae V/VI (0.19 ± 0.02; 0.50 ± 0.06; 0.30 ± 0.03 mean optical density ± SD of the mean in sham, 3 and 7 days after SNI, respectively, Fig. 5B). Noteworthy, almost half of TRPV1-ir neurons in the laminae II/III of sham mice were glutamatergic since TRPV1/VGlut1 colocalization was shown in 51.6 ± 8.8% (mean of percentage ± SD of the mean) of somata of TRPV1-immunoreactive neurons and, to a lesser extent, in the prefrontal interlimbic cortical neuropil (Fig. 6A,B,D,E). A striking TRPV1-ir and VGluT1-ir rise was observed in the neuropils of laminae II/III already at 3 days after SNI compared with the sham. At the same time, TRPV1/VGlut1 colocalization increased in the neuropils and in 73.7 ± 10.2% of TRPV1 immunoreactive neurons, as compared with 64.5 ± 9.6% 7 days after SNI (Fig. 6A1,B1,D,E). These increases were lower in Trpv1−/− mice (Fig. 6A2,B2,F,G).

Figure 5.

TRPV1 immunoreactivity in the PL-IL cortex of SNI mice. (A) Immunohistochemical detection of TRPV1. Lamina II/III of PL-IL cortex in sham mice and in mice at 3 and 7 days after SNI. TRPV1-ir is localized in the soma of many cortical neurons with the highest expression near to the external border of Lamina II. Note the increase of the TRPV1 signal after nerve injury and the absence of signal in Trpv1−/− mouse brain. Scale bar = 100 μm. (B) Histogram of TRPV1-ir densitometric analysis. Note that optical density units are expressed in the log scale; data are means ± SD of n = 3 separate determinations on n = 100 cells. The identification of region of interest (laminae) was performed according to the cytoarchitectonic organization of prelimbic cortex as reported by Van De Werd et al. (2010). Statistical differences in densitometric analyses were performed for each immunological marker in the laminae II/III. TRPV1-ir significantly increases in wild types 3 days after SNI (P < 0.05, sham vs. SNI 3 days) and then slightly decreases 7 days after SNI (P < 0.01, sham vs. SNI 7 days and SNI 3 days vs. SNI 7 days).

Figure 5.

TRPV1 immunoreactivity in the PL-IL cortex of SNI mice. (A) Immunohistochemical detection of TRPV1. Lamina II/III of PL-IL cortex in sham mice and in mice at 3 and 7 days after SNI. TRPV1-ir is localized in the soma of many cortical neurons with the highest expression near to the external border of Lamina II. Note the increase of the TRPV1 signal after nerve injury and the absence of signal in Trpv1−/− mouse brain. Scale bar = 100 μm. (B) Histogram of TRPV1-ir densitometric analysis. Note that optical density units are expressed in the log scale; data are means ± SD of n = 3 separate determinations on n = 100 cells. The identification of region of interest (laminae) was performed according to the cytoarchitectonic organization of prelimbic cortex as reported by Van De Werd et al. (2010). Statistical differences in densitometric analyses were performed for each immunological marker in the laminae II/III. TRPV1-ir significantly increases in wild types 3 days after SNI (P < 0.05, sham vs. SNI 3 days) and then slightly decreases 7 days after SNI (P < 0.01, sham vs. SNI 7 days and SNI 3 days vs. SNI 7 days).

Figure 6.

Triple, TRPV1 (blue), VGluT1 (red) and CB1 (green) immunofluorescence in the Lamina II of wild-type sham-operated (AC and D), wild-type mice 7 days after SNI (A1C1 and E), sham Trpv1−/− mice (F) and Trpv1−/− mice 7d after SNI (A2C2 and G). VGluT1-ir is expressed in numerous TRPV1 neurons receiving CB1 afferences in wild-type sham mice (AC). Noteworthy, in wild types, a strong TRPV1-ir increase is found in the soma and neuropil and of VGlut1 in the neuropil, 7 days after nerve injury, which underlies the observed higher TRPV1/VGluT1/CB1 coexpression (A1C1 and E). In the SNI Trpv1−/− mice, a moderate VGluT1/CB1 colocalization is observed (A2C2 and F). AC: scale bar = 100 μm. Note in D and E the numerous white perisomatic puncta as merge of CB1-ir on many glutamatergic fibers surrounding TRPV1-ir cells in the whole laminae II/III of the wild-type mice, sham and SNI at 7d, respectively. A strong TRPV1-VGluT1 immunocolocalization is shown as pink–violet signal in the neuropil of laminae II/III in wild-type SNI mice at 7d (E), while a general reduction of VGluT1–ir occurs in F and G compared with D and E, respectively (in particular, see G as enlargement of the A2C2 boxed areas). DG: scale bar = 50 μm.

Figure 6.

Triple, TRPV1 (blue), VGluT1 (red) and CB1 (green) immunofluorescence in the Lamina II of wild-type sham-operated (AC and D), wild-type mice 7 days after SNI (A1C1 and E), sham Trpv1−/− mice (F) and Trpv1−/− mice 7d after SNI (A2C2 and G). VGluT1-ir is expressed in numerous TRPV1 neurons receiving CB1 afferences in wild-type sham mice (AC). Noteworthy, in wild types, a strong TRPV1-ir increase is found in the soma and neuropil and of VGlut1 in the neuropil, 7 days after nerve injury, which underlies the observed higher TRPV1/VGluT1/CB1 coexpression (A1C1 and E). In the SNI Trpv1−/− mice, a moderate VGluT1/CB1 colocalization is observed (A2C2 and F). AC: scale bar = 100 μm. Note in D and E the numerous white perisomatic puncta as merge of CB1-ir on many glutamatergic fibers surrounding TRPV1-ir cells in the whole laminae II/III of the wild-type mice, sham and SNI at 7d, respectively. A strong TRPV1-VGluT1 immunocolocalization is shown as pink–violet signal in the neuropil of laminae II/III in wild-type SNI mice at 7d (E), while a general reduction of VGluT1–ir occurs in F and G compared with D and E, respectively (in particular, see G as enlargement of the A2C2 boxed areas). DG: scale bar = 50 μm.

In the brain of sham mice, most of the TRPV1/VGluT1 expressing neuropil of laminae II/III of the PL-IL cortex which innervated TRPV1-positive neurons, coexpressed cannabinoid CB1 receptors-positive afferents. TRPV1, VGluT1, and CB1 colocalized also on the plasma membrane. Triple colocalization was found in 34.4 ± 4.6% of the neurons (mean of percentage ± SD of the mean, Fig. 6C–E). After spinal nerve injury, the TRPV1/VGluT1/CB1 colocalization persisted on the plasma membrane of many TRPV1-ir neurons (38.4 ± 8.1% and 39.5 ± 7.6% mean of percentage ± SD of the mean, respectively, at 3 and 7 days after SNI) (Fig. 6C–E and data not shown). In sham Trpv1−/− mice, some VGluT1 neurons of laminae II/III in the PL-IL cortex brain of still received unchanged CB1 afferents (32.8 ± 3.2% mean of percentage ± SD of the mean, Fig. 6F,G). After 7 days of SNI in these mice, VGluT1 expression and as a consequence, VGluT1/CB1 colocalization, significantly decreased (18.5 ± 4.6% mean of percentage ± SD of the mean, Fig. 6A2,B2,F,G).

Finally, immunoreactivity for the IL-1R1 was also found to be upregulated in the TRPV1-positive neurons of the PL/IL cortex of WT (228 ± 22% vs. sham/veh) (Fig. 7A,B) but not Trpv1−/− mice (93 ± 3% vs. sham/veh) (Fig. 7C).

Figure 7.

Interleukin 1−β receptor 1 (IL-1R) in the PL-IL cortex of SNI mice. (A) Western blot analysis for IL-1R in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in IL-1R protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: IL-1R and ß-actin proteins are shown. (B) IL-1R immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of interleukin 1-beta was found in the TRPV1-ir profiles in PL-IL cortex 7 days after SNI. (C) IL-1R protein levels were not increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Figure 7.

Interleukin 1−β receptor 1 (IL-1R) in the PL-IL cortex of SNI mice. (A) Western blot analysis for IL-1R in the PL-IL cortex of SNI mice 7 days after sciatic nerve SNI. The histogram indicates percent variations in IL-1R protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: IL-1R and ß-actin proteins are shown. (B) IL-1R immunostaining in PL-IL cortex of mice. Strong immunohistochemical reactivity for the active form of interleukin 1-beta was found in the TRPV1-ir profiles in PL-IL cortex 7 days after SNI. (C) IL-1R protein levels were not increased in SNI Trpv1−/− mice as compared with sham Trpv1−/− mice. Scale bar: 40 μm (B). Legends: sham mice treated with vehicle (sham/veh); SNI mice treated with vehicle (SNI/veh). Data in naive mice are not reported because they had very similar protein expression levels as compared with sham/veh. Numbers indicate the days of sciatic nerve injury and drug treatment. ° indicates significant difference versus sham/vehicle. P < 0.05 was considered as threshold of significance.

Effect of SNI on Glutamate and GABA Levels

The mean basal extracellular GABA and glutamate levels in the PL/IL portion of the mPFC (not corrected for probe recovery of 21 ± 4% and 26 ± 6% for GABA and glutamate, respectively) were 3.2 ± 0.5 and 7.8 ± 0.4 pmol/10 μL of dialysate (mean ± SE), respectively. These values are similar to those we found in our previous studies in the PAG of mouse (Oliva et al. 2006). Each animal was used only once, and the reported basal values of glutamate and GABA are the mean concentrations obtained from all experiments pooled as controls. IL-PL cortex perfusion with TTX (1 μM) for 30 min decreased the extracellular level of glutamate and GABA (48 ± 6% and 53 ± 7% of basal release), respectively. In the SNI mice, the extracellular level of glutamate resulted increased (85.9 ± 0.5% of basal release, F1–14 = 98,98; P < 0.01). The same surgical procedure did not affect dialysate GABA level (not shown).

Alterations of Endovanilloid and Endocannabinoid Metabolism in the PL-IL Cortex of Sham and SNI Mice

In the laminae II/III of wild-type mice, SNI induced a significant increase of immunoreactivity of the enzymes NAPE-PLD and FAAH, which respectively biosynthesize and degrade the N-acylethanolamines, proposed to act as TRPV1 ligands (Zygmunt et al. 1999; Movahed et al. 2005; Cristino et al. 2008) (0.19 ± 0.007; 0.25 ± 0.009; 0.39 ± 0.01 for NAPE-PLD and 0.17 ± 0.01; 0.63 ± 0.12; 0.55 ± 0.11 for FAAH as mean optical density ± SD of the mean in the sham, 3 and 7 days after SNI, respectively; Fig. 8A). These changes were observed also in the laminae V/VI (0.08 ± 0.002; 0.18 ± 0.04; 0.25 ± 0.06 for NAPE-PLD and 0.15 ± 0.02; 0.33 ± 0.04; 0.30 ± 0.03 for FAAH mean optical density ± SD of the mean in the sham, 3 and 7 days after SNI, respectively; Fig. 8B). On the contrary, in the Trpv1−/− laminae II/III and V/VI, NAPE-PLD-ir and FAAH-ir exist in slightly higher levels when compared with the corresponding laminae of wild-type animals, with no difference between sham and SNI 7d (0.29 ± 0.02 and 0.27 ± 0.03 for NAPE-PLD in laminae II/III, 0.18 ± 0.01 and 0.22 ± 0.03 for NAPE-PLD in laminae V/VI; 0.29 ± 0.03 and 0.33 ± 0.04 for FAAH in laminae II/III; 0.34 ± 0.04 and 0.35 ± 0.05 for FAAH in laminae V/VI; the values were expressed as mean optical density ± SD of the mean; Fig. 8A,B). The lack of FAAH upregulation was also confirmed by western blot (Fig. 8C).

Figure 8.

Immunohistochemical detection of endocannabinoid metabolic enzymes in the lamina II/III of PL-IL cortex in sham mice and in mice at 3 and 7 days after SNI. (A) NAPE-PLD signal is poorly expressed in the neurons of the whole area both of wild-type and Trpv1−/− sham and injured mice and slightly increases after SNI 7d, whereas FAAH-ir strongly increases after nerve 3 and 7 days from injury, in wild-type but not in Trpv1−/− mice. DAGLα-ir is faintly localized in neurons and puncta (somatodendritic compartment) of lamina II/III both in wild-type and Trpv1−/− mice and significantly increases after SNI 3d only in the former animals. Scale bar = 100 μm. (B) Histograms of immunoreactivity densitometric analysis. Note that optical density units are expressed in the log scale; data are means ± SD of n = 3 separate determinations on n = 100 cells. The identification of region of interest (laminae) was performed according to the cytoarchitectonic organization of prelimbic cortex as reported by Van De Werd et al. (2010). Statistical differences in densitometric analyses were performed for each immunological marker in the laminae II/III. NAPE-PLD-ir significantly increases only 7 days after SNI exclusively in wild types (P < 0.05, SNI 7 days vs. sham). FAAH-ir and DAGLα-ir both significantly increase at 3 days after SNI (P < 0.001, sham vs. SNI 3 days) and then decrease at 7 days (P < 0.05, SNI 3 days vs. SNI 7 days for FAAH and P < 0.001, SNI 3 days vs. SNI 7 days for DAGLα-ir). MAGL-ir slightly increases after 7 days of SNI (P < 0.01, vs. SNI 3 days, P < 0.05, vs. sham). FAAH-ir, DAGLα-ir, and MAGL-ir did not change in Trpv1−/− mice after 7d of SNI compared with sham Trpv1−/− mice. FAAH protein levels were also detected by western blot analysis, confirming no differences between SNI Trpv1−/− mice as compared with sham Trpv1/− mice (as indicated by histograms).

Figure 8.

Immunohistochemical detection of endocannabinoid metabolic enzymes in the lamina II/III of PL-IL cortex in sham mice and in mice at 3 and 7 days after SNI. (A) NAPE-PLD signal is poorly expressed in the neurons of the whole area both of wild-type and Trpv1−/− sham and injured mice and slightly increases after SNI 7d, whereas FAAH-ir strongly increases after nerve 3 and 7 days from injury, in wild-type but not in Trpv1−/− mice. DAGLα-ir is faintly localized in neurons and puncta (somatodendritic compartment) of lamina II/III both in wild-type and Trpv1−/− mice and significantly increases after SNI 3d only in the former animals. Scale bar = 100 μm. (B) Histograms of immunoreactivity densitometric analysis. Note that optical density units are expressed in the log scale; data are means ± SD of n = 3 separate determinations on n = 100 cells. The identification of region of interest (laminae) was performed according to the cytoarchitectonic organization of prelimbic cortex as reported by Van De Werd et al. (2010). Statistical differences in densitometric analyses were performed for each immunological marker in the laminae II/III. NAPE-PLD-ir significantly increases only 7 days after SNI exclusively in wild types (P < 0.05, SNI 7 days vs. sham). FAAH-ir and DAGLα-ir both significantly increase at 3 days after SNI (P < 0.001, sham vs. SNI 3 days) and then decrease at 7 days (P < 0.05, SNI 3 days vs. SNI 7 days for FAAH and P < 0.001, SNI 3 days vs. SNI 7 days for DAGLα-ir). MAGL-ir slightly increases after 7 days of SNI (P < 0.01, vs. SNI 3 days, P < 0.05, vs. sham). FAAH-ir, DAGLα-ir, and MAGL-ir did not change in Trpv1−/− mice after 7d of SNI compared with sham Trpv1−/− mice. FAAH protein levels were also detected by western blot analysis, confirming no differences between SNI Trpv1−/− mice as compared with sham Trpv1/− mice (as indicated by histograms).

Since one of the N-acylethanolamines acting as TRPV1, that is, N-arachidonoylethanolamine (anandamide) is also a ligand for cannabinoid CB1 receptors in the brain (Devane et al. 1992), and the other such ligand, 2-arachidonoylglycerol (2-AG) is known to be activated following enhanced glutamate release (Maejima et al. 2005), we also analyzed the expression levels of 2-AG metabolic enzymes. A statistically significant increase of immunoreactivity was found exclusively for DAGL-α in the wild-type mouse laminae II/III after SNI, whereas, the 2-AG hydrolytic enzyme, MAGL, did not change much both in wild-type and Trpv1−/− mice (in wild-type: 0.13 ± 0.01; 0.66 ± 0.12; 0.31 ± 0.17 for DAGL-alpha in laminae II/III and 0.09 ± 0.01; 0.26 ± 0.06; 0.35 ± 0.06 for DAGL-α in laminae V/VI; 0.16 ± 0.01; 0.13 ± 0.04; 0.21 ± 0.06 for MAGL in the laminae II/III and 0.10 ± 0.03; 0.05 ± 0.01; 0.11 ± 0.04 for MAGL in the laminae V/VI, the values being expressed as mean optical density ± SD of the mean in the sham, 3 and 7 days after SNI, respectively; in Trpv1−/− mice: 0.22 ± 0.01; 0.12 ± 0.001 for DAGL-α in laminae II/III and 0.3 ± 0.01; 0.3 ± 0.008 for DAGL-α in the laminae V/VI; 0.18 ± 0.01; 0.16 ± 0.009 for MAGL in the laminae II/III; and 0.15 ± 0.01; 0.1 ± 0.007 for MAGL in the laminae V/VI; the values being expressed as mean optical density ± SD of the mean; Fig. 8A,B).

Interestingly, DAGL-α was found to be coexpressed in the majority of TRPV1-ir neurons, prevalently in laminae II/III of the PL-IL cortex of wild-type SNI 7d mice compared with sham mice, that is, in 81.5 ± 11.1% and 30.2 ± 5.3% of the TRPV1-ir neurons, respectively (mean of percent ± SD of the mean, Supplementary Fig. 1). In agreement with the DAB-staining data mentioned above, DAGL-α increased in wild-type but not Trpv1−/− SNI 7d mice compared with sham mice (Supplementary Fig. 1C,D).

Since there was no difference in the effects of SNI on DAGLα, MAGL, NAPE-PLD, and FAAH expression between the ipsilateral and contralateral sides and in order to improve the sensitivity of the measurement, the 2 sides were pooled for endocannabinoid measurement. In agreement with the above finding of increased expression of a 2-AG biosynthesizing enzyme, we found a statistically significant increase of 2-AG levels, in the PL-IL cortex of SNI mice (Table 2). Interestingly, however, the levels of anandamide decreased, in agreement with the above finding of increased FAAH but not with the observation of increased NAPE-PLD expression. On the other hand, the levels of 2 other N-acylethanolamine with activity at TRPV1, OEA, and PEA, did not change, thus reflecting the increases of both NAPE-PLD and FAAH expression (Table 2).

Table 2

Tissue concentrations of anandamide (AEA), 2-arachidonoylglycerol (2-AG), N-oleoylethanolamide (OEA), and N-palmitoylethanolamide (PEA) in the PL-IL cortex (laminae II. III, IV, V, VI) of mice with sham or SNI operation, with or without 7 day treatment with arachidonoyl serotonin (AA-5-HT, 5 mg/kg, i.p.)

  AEA (pmol/g) 2-AG (nmol/g) OEA (pmol/g) PEA (pmol/g) 
Sham Vehicle 36.0 ± 2.5 3.3 ± 0.3 258.9 ± 25.6 95.6 ± 7.9 
AA-5-HT 32.4 ± 3.2 5.2 ± 0.4* 243.8 ± 21.8 107.6 ± 7.1 
SNI Vehicle 28.0 ± 0.5* 4.9 ± 0.2* 261.7 ± 32.5 100.3 ± 6.2 
AA-5-HT 35.7 ± 2.5 3.4 ± 0.5 269.7 ± 19.9 105.8 ± 5.9 
  AEA (pmol/g) 2-AG (nmol/g) OEA (pmol/g) PEA (pmol/g) 
Sham Vehicle 36.0 ± 2.5 3.3 ± 0.3 258.9 ± 25.6 95.6 ± 7.9 
AA-5-HT 32.4 ± 3.2 5.2 ± 0.4* 243.8 ± 21.8 107.6 ± 7.1 
SNI Vehicle 28.0 ± 0.5* 4.9 ± 0.2* 261.7 ± 32.5 100.3 ± 6.2 
AA-5-HT 35.7 ± 2.5 3.4 ± 0.5 269.7 ± 19.9 105.8 ± 5.9 

Note: Data means ± SE of n = 4 determinations and were compared by ANOVA followed by the Bonferroni’s test. * P < 0.05 versus sham/vehicle.

TRPV1-Dependent and -Independent Caspase Release in the PL-IL Cortex Contributes to Mechanical Allodynia in SNI Mice

All drugs tested for acute effect were administered into the PL-IL cortex 7 days after SNI. SNI induced a significant decrease of mechanical withdrawal threshold (3.51 ± 0.30 g). Intra-PL-IL cortex single administration of vehicle did not produce a significant change in mechanical withdrawal threshold of SNI mice 7 days after surgery (Fig. 9). A single microinjection of the pan-caspase inhibitor DEVD-CHO at the highest dose (10 nmol) significantly decreased mechanical allodynia from 10 to 85 min with maximum effect in SNI mice postdrug at 15 min (6.50 ± 0.46 g, F1–10 = 26.05; P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g). The intermediate dose (5 nmol) of DEVD-CHO significantly decreased mechanical allodynia in SNI mice only at 10 min postadministration (4.75 ± 0.35 g, F1–10 = 10.17; P < 0.01) with respect to SNI/vehicle mice (3.48 ± 0.19). At the lowest dose (1 nmol), DEVD-CHO did not change mechanical withdrawal threshold of SNI mice (Fig. 9A). Importantly, DEVD-CHO produced a smaller and shorter lasting, but still statistically significant, effect when injected into the PL-IL of SNI Trpv1−/− mice (6.65 ± 0.45 g, F1–10 = 37.44; P < 0.01) (Fig. 9B).

Figure 9.

Effects of a single microinjection on mechanical withdrawal threshold (g ± SE) of vehicle, DEVD-CHO (1–5–10 nmol) in WT SNI mice (A) and SNI Trpv1−/− mice (B), I-RTX (0.5–1 nmol) (C), capsaicin 6 nmol with or without I-RTX (0.5 nmol) (D), capsaicin (1.5–3–6 nmol) (E) into the contralateral PL-IL cortex of SNI mice 7 days after surgery (each group n = 6). *P < 0.05 versus SNI/vehicle and °P < 0.05 versus SNI/I-RTX. (F) Brain section showing the PL-IL divisions of the mPFC and a representative intra-PL/IL microinjection trace.

Figure 9.

Effects of a single microinjection on mechanical withdrawal threshold (g ± SE) of vehicle, DEVD-CHO (1–5–10 nmol) in WT SNI mice (A) and SNI Trpv1−/− mice (B), I-RTX (0.5–1 nmol) (C), capsaicin 6 nmol with or without I-RTX (0.5 nmol) (D), capsaicin (1.5–3–6 nmol) (E) into the contralateral PL-IL cortex of SNI mice 7 days after surgery (each group n = 6). *P < 0.05 versus SNI/vehicle and °P < 0.05 versus SNI/I-RTX. (F) Brain section showing the PL-IL divisions of the mPFC and a representative intra-PL/IL microinjection trace.

A single microinjection of the selective TRPV1 antagonist I-RTX at the highest dose (1 nmol) significantly decreased mechanical allodynia from 10 to 25 min with a maximum effect in SNI mice at 15 min postdrug (5.80 ± 0.56 g, F1–10 = 11.61; P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g). At the lowest dose, I-RTX (0.5 nmol) significantly decreased mechanical allodynia in SNI mice only at 25 min postdrug (6.00 ± 0.35 g, F1–10 = 28.24; P < 0.01) with respect to SNI/veh mice (3.48 ± 0.32 g) (Fig. 9C). The effect of I-RTX (0.5 nmol) was antagonized by microinjection of capsaicin (6 nmol) into the PL/IL cortex in SNI mice, which, however, was active per se (Fig. 9D). Indeed, a single microinjection of capsaicin significantly decreased mechanical allodynia at this dose (6 nmol) from 10 to 85 min postinjection, with a maximum effect at 15 min postdrug (7.05 ± 0.68 g, F1–10 = 20.94; P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g). The intermediate dose of capsaicin (3 nmol) significantly decreased mechanical allodynia in SNI mice from 10 to 40 min postdrug with a maximum effect at 15 min (5.70 ± 0.09 g, F1–10 = 35.39, P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g). At the lowest dose, capsaicin (1.5 nmol), did not show any change in mechanical allodynia as compared with SNI/vehicle mice (Fig. 9E). The effect of capsaicin (6 nmol) was antagonized by microinjection of I-RTX (0.5 nmol) into the PL/IL cortex in SNI mice (Fig. 9D).

Figure 12.

BLA stimulation evokes inhibitory responses in PL-IL cortex neurons indicated as BLA→PL-IL (-) neurons. The figure shows different parameters of BLA-evoked inhibition, including the onset and duration of the inhibition, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show representative ratemater records of a BLA → PL-IL (-) neurons of sham/veh, sham/AA-5-HT, SNI/veh, or SNI/AA-5-HT groups of mice, respectively. “E” and “F” show the mean ± standard error of the mean of the onset and the duration of the inhibition in the different groups of mice (n = 20, n = 18, n = 24, n = 21 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and 10 mice were used for each group. A schematic illustration of the PL-IL cortex recording sites is also shown, in which filled circles represent mPFC(+) neurons responding with excitation and open triangles those ones responding with inhibition mPFC(−) cell sites. Numbers indicate distance to bregma or dura.

Figure 12.

BLA stimulation evokes inhibitory responses in PL-IL cortex neurons indicated as BLA→PL-IL (-) neurons. The figure shows different parameters of BLA-evoked inhibition, including the onset and duration of the inhibition, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show representative ratemater records of a BLA → PL-IL (-) neurons of sham/veh, sham/AA-5-HT, SNI/veh, or SNI/AA-5-HT groups of mice, respectively. “E” and “F” show the mean ± standard error of the mean of the onset and the duration of the inhibition in the different groups of mice (n = 20, n = 18, n = 24, n = 21 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and 10 mice were used for each group. A schematic illustration of the PL-IL cortex recording sites is also shown, in which filled circles represent mPFC(+) neurons responding with excitation and open triangles those ones responding with inhibition mPFC(−) cell sites. Numbers indicate distance to bregma or dura.

AMPA-Like Receptor Activity in the PL-IL Cortex Underlies both Caspase-1 and -3 Release and Contributes to Mechanical Allodynia in SNI Mice

A single microinjection into the PL-IL cortex of AMPA at the highest dose (200 pmol) significantly increased mechanical allodynia from 10 to 25 min with maximum effect in SNI mice postdrug at 10 min (2.30 ± 0.34 g, F1–10 = 9.18; P < 0.05) with respect to SNI/vehicle mice (3.48 ± 0.19 g). At the lowest dose (100 pmol), AMPA did not change mechanical withdrawal threshold of SNI mice (Fig. 10A).

Figure 10.

Effects of a single microinjection on mechanical withdrawal threshold (g ± SE) of vehicle, AMPA (100–200 pmol) (A), CNQX (100-200 pmol) (B), APV (2.5–5–10 nmol) (D) MPEP (5–10–20 nmol) alone or in combination with APV (10 nmol) (E and F), WIN55,212-2 (25–100 nmol) (G), URB 597 (1–2–4 nmol) (H), AA-5-HT (0.25–1 nmol) (I) alone or in combination with AM251 (0.25–0.5 nmol) (J) into the contralateral PL-IL cortex of SNI mice 7 days after surgery (each group n = 6). *P < 0.05 versus SNI/vehicle and °P < 0.05 versus SNI/AA-5-HT. Effects of a single microinjection on protein levels of vehicle and CNQX (200 pmol) (C) into the PL-IL cortex of SNI mice 7 days after surgery (each group n = 3) of sham/vehicle, SNI/vehicle and SNI/CNQX mice, as analyzed by western blotting for the proteins: caspase-1, caspase-3, IL-1β and IL-1RI with respect to the housekeeping protein β-actin.

Figure 10.

Effects of a single microinjection on mechanical withdrawal threshold (g ± SE) of vehicle, AMPA (100–200 pmol) (A), CNQX (100-200 pmol) (B), APV (2.5–5–10 nmol) (D) MPEP (5–10–20 nmol) alone or in combination with APV (10 nmol) (E and F), WIN55,212-2 (25–100 nmol) (G), URB 597 (1–2–4 nmol) (H), AA-5-HT (0.25–1 nmol) (I) alone or in combination with AM251 (0.25–0.5 nmol) (J) into the contralateral PL-IL cortex of SNI mice 7 days after surgery (each group n = 6). *P < 0.05 versus SNI/vehicle and °P < 0.05 versus SNI/AA-5-HT. Effects of a single microinjection on protein levels of vehicle and CNQX (200 pmol) (C) into the PL-IL cortex of SNI mice 7 days after surgery (each group n = 3) of sham/vehicle, SNI/vehicle and SNI/CNQX mice, as analyzed by western blotting for the proteins: caspase-1, caspase-3, IL-1β and IL-1RI with respect to the housekeeping protein β-actin.

A single microinjection into the PL-IL cortex of the AMPA-like receptor antagonist, CNQX, significantly decreased mechanical allodynia at the highest dose (200 pmol) from 10 to 85 min with a maximum effect in SNI mice at 40 min postdrug (6.51 ± 0.35 g, F1–10 = 50.23; P < 0.01) with respect to SNI/vehicle mice (3.58 ± 0.22 g). The lower dose of CNQX (100 pmol) still significantly decreased mechanical allodynia in SNI mice postdrug from 10 to 55 min with a maximum effect at 25 min (6.60 ± 0.38 g, F1–10 = 39.44, P < 0.01) with respect to SNI/vehicle mice (3.48 ± 0.32 g) (Fig. 10B). Importantly, the anti-hyperalgesic effect of CNQX was accompanied by a strong reduction of caspase-1, caspase-3, IL-1β, and IL-1R in the PL-IL cortex of SNI mice (Fig. 10C).

NMDA, mGluR5, and CB1 Receptor Activity in the PL-IL Cortex Counteract Mechanical Allodynia in SNI Mice

A single microinjection into the PL-IL of APV, a selective NMDA antagonist, at the highest dose (10 nmol) significantly increased mechanical allodynia from 15 to 55 min with maximum effect in SNI mice postdrug at 25 min (1.26 ± 0.25 g, F1–10 = 29.89; P < 0.01) with respect to SNI/vehicle mice (3.48 ± 0.32 g). Intermediate dose (5 nmol) of APV still significantly increased mechanical allodynia in SNI mice from 25 to 40 min with maximum effect in SNI mice postdrug at 25 min (2.54 ± 0.24 g, F1–10 = 5.52; P < 0.05) with respect to SNI/vehicle mice (3.48 ± 0.32 g). At the lowest dose (2.5 nmol), APV did not change mechanical withdrawal threshold of SNI mice (Fig. 10D).

A single microinjection of MPEP, a selective mGluR5 antagonist, at the highest (20 nmol) or intermediate (10 nmol) doses into the PL/IL cortex significantly increased mechanical allodynia from 10 to 55 min with maximum effect in SNI mice postdrug at 15 min (1.97 ± 0.14 g, F1–10 = 17.57; P < 0.01 and 2.20 ± 0.11 g, F1–10 = 13.54; P < 0.01, respectively) with respect to SNI/vehicle mice (3.55 ± 0.35 g). At the lowest dose (5 nmol), MPEP did not change mechanical withdrawal threshold of SNI mice (Fig. 10E). Coadministration of APV (10 nmol) and MPEP (10–20 nmol) further increased the mechanical allodynia in SNI mice postdrug with respect to SNI/vehicle mice (Fig. 10F).

A single microinjection of the CB1 agonist, WIN55,212-2, significantly decreased mechanical allodynia at the highest dose (100 nmol) from 10 to 70 min with a maximum effect in SNI mice at 25 min postdrug (7.50 ± 0.79 g, F1–10 = 22.24; P < 0.01) with respect to SNI/vehicle mice (3.48 ± 0.32 g). At the lowest dose, WIN55,212-2 (25 nmol) significantly decreased mechanical allodynia in SNI mice from 10 to 70 min with a maximum effect at 15 min postdrug (6.45 ± 0.17 g, F1–10 = 55.55, P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g) (Fig. 10G).

A single microinjection of the selective FAAH inhibitor, URB597, significantly decreased mechanical allodynia at the highest dose (4 nmol) from 15 to 40 min with a maximum effect in SNI mice at 25 min postdrug (7.00 ± 0.50 g, F1–10 = 35.16; P < 0.01) with respect to SNI/vehicle mice (3.48 ± 0.32 g). The intermediate dose of URB597 (2 nmol) significantly decreased mechanical allodynia in SNI mice postdrug from 15 to 40 min with a maximum effect at 40 min (4.88 ± 0.24 g, F1–10 = 15.94, P < 0.01) with respect to SNI/vehicle mice (3.58 ± 0.22 g). At the lowest dose, URB597 (1 nmol) significantly decreased mechanical allodynia in SNI mice postdrug at 15 min (5.25 ± 0.14 g, F1–10 = 20.34; P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g) (Fig. 10H).

Dual Pharmacological Inhibition of TRPV1 and FAAH in the PL-IL Cortex Produces a Stronger Inhibition of Allodynia in SNI Mice

A single microinjection into the PL-IL of a dual TRPV1 and FAAH blocker, AA-5-HT, at the highest dose (1 nmol) into the PL-IL cortex significantly decreased mechanical allodynia from 10 to 85 min, with a maximum effect in SNI mice postdrug at 15 min (6.75 ± 0.48 g, F1–10 = 29.02; P < 0.01) with respect to SNI/vehicle mice (3.55 ± 0.35 g). At the lowest dose (0.25 nmol), AA-5-HT significantly decreased mechanical allodynia from 10 to 25 min with a maximum effect in SNI mice at 15 min postdrug (7.75 ± 0.75 g, F1–10 = 25.75; P < 0.05) compared with predrug SNI/vehicle mice (3.55 ± 0.35 g) (Fig. 10I). The effect of AA-5-HT (1 nmol) was antagonized by microinjection of AM251 (0.25–0.5 nmol) into the PL-IL cortex in SNI mice (Fig. 10J). AM251 was completely inactive per se at both doses (0.25 and 0.5 nmol) in SNI mice (data not shown).

Daily Systemic I-RTX or URB597 Only Transiently Prevent Mechanical Allodynia Induced by SNI

Nociceptive responses to mechanical stimuli was measured every 30 min for 3 h before surgery or days after SNI. The mean of nociceptive response was calculated from 6 consecutive trials (each performed every 30 min) and averaged for each group of mice. Sham mice treated with vehicle did not show any change in mechanical allodynia compared with the naïve mice (Fig. 11A). SNI mice treated with vehicle developed mechanical allodynia in the ipsilateral side at 7 and 14 days from surgery (3.40 ± 0.20 g and 3.46 ± 0.22 g, F1–8 = 230.42 and F1–8 = 288.50; P < 0.01, respectively), compared with sham mice treated with vehicle (9.00 ± 0.31 g and 8.99 ± 0.24 g, respectively) (Fig. 11A) but not in the contralateral side (data not shown). A daily systemic administration of I-RTX (0.2 mg/kg i.p.) or URB597 (3 mg/kg i.p.) in SNI mice significantly reduced mechanical allodynia in the ipsilateral side 7 days after SNI (5.00 ± 0.02 g and 5.89 ± 0.26 g, F1–8 = 32.00 and F1–8 = 57.62; P < 0.01, respectively) with respect to SNI/vehicle (3.40 ± 0.20 g), but not 14 days after peripheral injury (Fig. 11A), without any change in the contralateral side (data not shown), with respect to SNI mice treated with vehicle.

Figure 11.

(A) Effects of I-RTX (0.2 mg/kg i.p) or URB597 (3 mg/kg i.p) on mechanical withdrawal threshold (g, mean ± SE) in SNI mice. Mechanical allodynia was evaluated in ipsilateral sides 7 and 14 days post-SNI. I-RTX or URB 597 treatment reduced the mechanical allodynia 7 days after SNI (*P < 0.05 vs. SNI/vehicle mice) but not 14 days after peripheral injury (n = 5). (B) Effects of vehicle or AA-5-HT (5 mg/kg, i.p.) on mechanical withdrawal threshold (g, mean ± SE) in SNI mice. Mechanical allodynia was evaluated in ipsilateral hind paw 3, 7, and 14 days post-SNI. SNI mice showed a significant reduction in the threshold to mechanical stimulation in the ipsilateral paw (°P < 0.05 vs. sham/vehicle mice) after SNI surgery. AA-5-HT reduced mechanical allodynia 7 and 14 days after SNI (*P < 0.05 vs. SNI/vehicle mice). (C) Effects of vehicle or AA-5-HT (5 mg/kg, i.p.) on mechanical withdrawal threshold (g, mean ± SE) in SNI TRPV1 knockout mice. Mechanical allodynia was evaluated in ipsilateral hind paw 3 and 7 days post-SNI. SNI TRPV1 knockout mice showed a significant reduction in the threshold to mechanical stimulation in the ipsilateral paw (°P < 0.05 vs. sham-operated mice) after SNI surgery. AA-5-HT reduced mechanical allodynia 3 and 7 days after SNI (*P < 0.05 vs. SNI/vehicle mice). (D) Effects of vehicle, AA-5-HT (5 mg/kg i.p) on motor performance in the rotarod test. Systemic administration of AA-5-HT in SNI-mice had no effect on motor coordination compared with SNI/vehicle mice. Results are expressed as the mean ± SE of the latency (s) (n = 9 mice/group). °P < 0.05 versus sham/vehicle. (E) Caspase activation and western blot analysis for activated caspase-3 in the PL-IL cortex of SNI mice after vehicle or AA-5-HT treatment 3, 7, and 14 days after sciatic nerve SNI. (F) The histogram indicates percent variations in activated caspase-3 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-3 and ß-actin proteins are shown (°P < 0.05 vs. sham/vehicle mice; *P < 0.05 vs. SNI/vehicle mice).

Figure 11.

(A) Effects of I-RTX (0.2 mg/kg i.p) or URB597 (3 mg/kg i.p) on mechanical withdrawal threshold (g, mean ± SE) in SNI mice. Mechanical allodynia was evaluated in ipsilateral sides 7 and 14 days post-SNI. I-RTX or URB 597 treatment reduced the mechanical allodynia 7 days after SNI (*P < 0.05 vs. SNI/vehicle mice) but not 14 days after peripheral injury (n = 5). (B) Effects of vehicle or AA-5-HT (5 mg/kg, i.p.) on mechanical withdrawal threshold (g, mean ± SE) in SNI mice. Mechanical allodynia was evaluated in ipsilateral hind paw 3, 7, and 14 days post-SNI. SNI mice showed a significant reduction in the threshold to mechanical stimulation in the ipsilateral paw (°P < 0.05 vs. sham/vehicle mice) after SNI surgery. AA-5-HT reduced mechanical allodynia 7 and 14 days after SNI (*P < 0.05 vs. SNI/vehicle mice). (C) Effects of vehicle or AA-5-HT (5 mg/kg, i.p.) on mechanical withdrawal threshold (g, mean ± SE) in SNI TRPV1 knockout mice. Mechanical allodynia was evaluated in ipsilateral hind paw 3 and 7 days post-SNI. SNI TRPV1 knockout mice showed a significant reduction in the threshold to mechanical stimulation in the ipsilateral paw (°P < 0.05 vs. sham-operated mice) after SNI surgery. AA-5-HT reduced mechanical allodynia 3 and 7 days after SNI (*P < 0.05 vs. SNI/vehicle mice). (D) Effects of vehicle, AA-5-HT (5 mg/kg i.p) on motor performance in the rotarod test. Systemic administration of AA-5-HT in SNI-mice had no effect on motor coordination compared with SNI/vehicle mice. Results are expressed as the mean ± SE of the latency (s) (n = 9 mice/group). °P < 0.05 versus sham/vehicle. (E) Caspase activation and western blot analysis for activated caspase-3 in the PL-IL cortex of SNI mice after vehicle or AA-5-HT treatment 3, 7, and 14 days after sciatic nerve SNI. (F) The histogram indicates percent variations in activated caspase-3 protein levels normalized with respect to the signal obtained for ß-actin (housekeeping protein). Western blot lanes: activated caspase-3 and ß-actin proteins are shown (°P < 0.05 vs. sham/vehicle mice; *P < 0.05 vs. SNI/vehicle mice).

Figure 14.

Mechanical nociceptive stimulation evokes inhibitory responses in BLA→PL-IL (-) neurons. The figure shows different parameters of mechanical nociceptive stimulation-evoked inhibition, including the onset and duration of the inhibition, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT groups of mice, respectively. “E” and “F” show the onset and the duration (mean ± standard error of the mean) of the inhibition in the different groups of mice (n = 20, n = 19, n = 20, n = 22 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and n = 10 mice were used for each group.

Figure 14.

Mechanical nociceptive stimulation evokes inhibitory responses in BLA→PL-IL (-) neurons. The figure shows different parameters of mechanical nociceptive stimulation-evoked inhibition, including the onset and duration of the inhibition, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT groups of mice, respectively. “E” and “F” show the onset and the duration (mean ± standard error of the mean) of the inhibition in the different groups of mice (n = 20, n = 19, n = 20, n = 22 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and n = 10 mice were used for each group.

Daily Systemic AA-5-HT Treatment Long-Lastingly Reduce Mechanical Allodynia in SNI Mice

A daily systemic administration of AA-5-HT (5 mg/kg i.p.) in SNI mice significantly reduced mechanical allodynia in the ipsilateral side at 7 and 14 days after SNI (7.40 ± 0.19 g and 8.00 ± 0.22 g, F1–10 = 69.51 and F1–10 = 172.06; P < 0.01, respectively) with respect to SNI/vehicle (5.10 ± 0.20 g and 4.10 ± 0.20 g), without any change in the contralateral side (data not shown), with respect to SNI mice treated with vehicle (Fig. 11B).

Sham Trpv1/− mice treated with vehicle did not show any change in mechanical allodynia compared with the naïve mice (Fig. 11C). SNI Trpv1−/− mice treated with vehicle developed mechanical allodynia in the ipsilateral side at 3 and 7 days from surgery (5.10 ± 0.20 g and 4.10 ± 0.20 g, F1–10 = 105.54 and F1–10 = 200.69; P < 0.01, respectively), compared with sham mice treated with vehicle (8.89 ± 0.31 g and 8.86 ± 0.27 g, respectively) (Fig. 11C) but not in the contralateral side (data not shown).

Chronic daily treatment of these mice with AA-5-HT produced a stronger antiallodynic effect than in wild-type SNI mice (Fig. 11C).

The motor activity of SNI mice treated with vehicle showed a significant reduction at 2, 6, and 13 days after SNI (201.83 ± 4.11 s, F1–16 = 40.73; 204.33 ± 11.26 s, F1–16 = 12.75; 198.1 ± 8.4 s, F1–16 = 33.27, P < 0.01, respectively) compared with sham/vehicle (256.3 ± 6.2 s, 257.4 ± 9.7 s, 255.7 ± 5.4 s, respectively) (Fig. 11D). This fact was attributed to the permanent injury of the sciatic nerve that prevented the right posture on the rotarod. AA-5-HT treatment did not affect motor coordination in neuropathic mice (Fig. 11D).

Daily Systemic AA-5-HT Treatment Reequilibrates Caspase and Endovanilloid/Endocannabinoid Levels in the PL-IL Cortex of SNI Mice

Systemic daily treatment with AA-5-HT (5 mg/kg, i.p.) inhibited caspase-9 gene overexpression 7 and 14 days after SNI with respect to SNI/vehicle mice. AA-5-HT also inhibited caspase-8 gene overexpression 7 days after SNI with respect to SNI/vehicle mice (Table 1). In SNI mice, AA-5-HT also decreased caspase-1 expression after 3 days with respect to SNI/vehicle mice and abolished its overexpression after 7 and 14 days with respect to SNI/vehicle mice (Table 1). Furthermore, AA-5-HT dramatically decreased caspase-12 expression at day 3, 7, and 14 with respect to SNI/vehicle (Table 1). SNI did not affect caspase-3 gene expression at all the days examined (Table 1). Systemic daily treatment with AA-5-HT (5 mg/kg, i.p.) also reduced caspase-3 activation at 3 (77 ± 2% vs. SNI/vehicle), 7 (73 ± 2% vs. SNI/vehicle), and 14 (47 ± 3% vs. SNI/vehicle) days after SNI (Fig. 11E,F).

The effect of AA-5-HT (5 mg/kg i.p.) on endocannabinoid levels in the PL-IL (all laminae) is reported in Table 2. A 7 day daily treatment with AA-5-HT (5 mg/kg i.p.) significantly elevated 2-AG levels, with no effect on AEA levels, in sham-operated mice. AA-5-HT treatment, instead, normalized 2-AG and AEA levels in mice with SNI.

Electrophysiological Characterization of mPFC Neurons

The increased VGluT1 expression (and the increased extracellular glutamate levels) in the PL-IL cortex following SNI was suggestive of increased glutamatergic signaling in this area. Therefore, we performed single-unit extracellular recording in anaesthetized mouse from individual neurons in this part of the mPFC. Features such as long action potential duration (330 ± 25 μs peak-to-valley) and slow firing rate (0.5–1.2 ± 0.4 spikes/s) from recorded neurons were consistent with presumed pyramidal cells rather than fast-spiking interneurons, the latter exhibiting higher baseline firing rate (>10 Hz) and narrower spike waveform (<300 μs) in rats (Constantinidis and Goldman-Rakic 2002; Laviolette and Grace 2006; Ji et al. 2010).

Electrophysiological Properties of BLA–mPFC Neurons in Sham and SNI Mice

We first investigated the proportion of mPFC neurons with ongoing activity that responded with inhibition, denoted with BLA→mPFC(−) or excitation, denoted with BLA→mPFC(+). In these studies, we first isolated mPFC neurons and thereafter we stimulated the BLA at 0.5 Hz, using an initial stimulation current of 200 μA. This same procedure was used for 4 groups of mice: 1) sham mice treated for 7 days with vehicle (sham/veh); 2) sham mice treated for 7 days with AA-5-HT (5 mg/kg, i.p.) (sham/AA-5-HT); 3) SNI mice treated for 7 days with vehicle (SNI/veh); 4) SNI mice treated for 7 days with AA-5-HT (5 mg/kg, i.p.) (SNI/AA-5-HT). In the control group (sham/veh), the 60.6% of encountered neurons displayed excitation after BLA stimulation or hind paw mechanical stimulation, with the remaining proportion of cells showing inhibition (33.3%) (Koga et al. 2010). Moreover, a low percentage of encountered neurons (6.1%) did not display response after BLA electrical or hind paw mechanical stimulation. In SNI/veh mice, a significant change in neurons showing inhibition or excitation was found. Indeed, 84.5% neurons displayed an excitation after BLA or mechanical stimulation, while about 11.1% remaining cells showed inhibition of their spontaneous activity (P < 0.01, χ2 test vs. sham/veh). The percentage of nonresponding cell was in this case 4.4%. The 7 day repeated treatment with AA-5-HT (5 mg/kg, i.p.) in SNI changed the proportion of BLA→mPFC(−) and BLA→mPFC(+). Indeed, 62.8% of BLA→mPFC(+), 31.4% of BLA→mPFC(−) and 4.9% of nonresponding neurons were encountered (P < 0.01, χ2 test vs. SNI/veh). Sham mice treated with AA-5-HT (5 mg/kg, i.p.) did not show any change in proportion of BLA→mPFC(−) and BLA→mPFC(+) compared with the sham mice treated with vehicle.

BLA→mPFC(−) Neurons

In BLA→mPFC(−) neurons, the spontaneous firing rate was 0.6 ± 0.2 Hz, the onset of BLA-evoked inhibition was 32 ± 0.7 ms and the duration of the inhibition was 245 ± 8.6 ms in sham/veh mice (Fig. 12A,E,F) (n = 20 neurons recorded from 10 mice). The 7 day treatment with AA-5-HT (5 mg/kg, i.p.) did not affect either the firing rate (0.5 ± 0.2 Hz), the duration of the inhibition and the onset of inhibition in BLA→mPFC(−) neurons in the shams (n = 18 neurons recorded from 10 mice) (Fig. 12B,E,F). SNI/veh mice showed an increased firing rate (1.7 ± 0.4 Hz, F1–42 = 5.36; P < 0.05) of BLA→mPFC(−) neurons. The onset of BLA-evoked inhibition was significantly reduced (22.2 ± 1.03 ms, F1–42 = 57.01; P < 0.01) in BLA→mPFC(−) neurons of this group of mice while the duration of the inhibition was significantly increased (364 ± 22.7 ms, F1–42 = 20.76; P < 0.01) (n = 24 neurons recorded from 10 mice) (Fig. 12C,E,F). Treatment with AA-5-HT (5 mg/kg i.p.) for 7 days in SNI mice (SNI/AA-5-HT) decreased firing rate (0.5 ± 0.03 Hz, F1–43 = 7.82; P < 0.01), caused a significant increase of the onset (33.2 ± 1.9 ms, F1–43 = 27.73; P < 0.01), and a significant decrease in the duration (262.5 ± 16.5 ms, F1–43 = 12.44; P < 0.01) of BLA-evoked inhibition (Fig. 12D–F).

BLA→mPFC(+) neurons

In BLA→mPFC(+) neurons, the spontaneous firing rate was 0.4 ± 0.02 Hz in sham/veh group. The onset, the frequency, and the duration of excitation were 26.17 ± 2.89 ms, 4.5 ± 0.4 Hz, and 263.3 ± 24.2 ms, respectively (Fig. 13A,E–G) (n = 20 neurons recorded from 10 mice) in sham/veh group. Sham mice treated with AA-5-HT (5 mg/kg i.p.) for 7 days did not show changes in the onset, frequency, and duration of excitation (n = 22 neurons recorded from 10 mice, Fig. 13B,E–G) with respect to sham/veh. Mice which underwent SNI (SNI/veh) showed an increased firing rate of 1.4 ± 0.2 Hz (F1–43 = 19.78; P < 0.01). The onset of BLA-evoked excitation was significantly reduced (18.3 ± 1.85 ms, F1–43 = 5.66; P < 0.05) in this group of mice, whereas the duration and the frequency of excitation of BLA→mPFC(+) proved significantly increased (407 ± 16.67 ms, F1–43 = 25.34; P < 0.01 and 6.4 ± 0.2 Hz, F1–43 = 20.34; P < 0.01, respectively) (Fig. 13C,E–G) (n = 25 neurons recorded from 10 mice). Treatment with AA-5-HT (5 mg/kg i.p.) for 7 days in SNI mice, decreased firing rate (0.5 ± 0.07 Hz, F1–46 = 16.86; P < 0.01), caused a significant increase in the onset (27.2 ± 1.6 ms, F1–46 = 13.03; P < 0.01) and a decrease in duration and frequency of excitation in BLA→mPFC(+) neurons (270 ± 17.8 ms, F1–46 = 31.62; P < 0.01, 4.7 ± 0.2 Hz, F1–46 = 36; P < 0.01, respectively) (Fig. 13D,E–G) (n = 23 neurons recorded from 10 mice).

Figure 13.

BLA stimulation evokes excitatory responses in PL-IL cortex neurons indicated as BLA→PL-IL (+) neurons. The figure shows different parameters of BLA-evoked excitation, including the onset, the frequency, and the duration of the excitation, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT mice. “E,” “F,” and “G” show the onset, the frequency, and the duration (mean ± standard error of the mean) of the excitation in the different groups of mice (n = 20, n = 22, n =25, n = 23 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance and n = 10 mice were used for each group.

Figure 13.

BLA stimulation evokes excitatory responses in PL-IL cortex neurons indicated as BLA→PL-IL (+) neurons. The figure shows different parameters of BLA-evoked excitation, including the onset, the frequency, and the duration of the excitation, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT mice. “E,” “F,” and “G” show the onset, the frequency, and the duration (mean ± standard error of the mean) of the excitation in the different groups of mice (n = 20, n = 22, n =25, n = 23 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance and n = 10 mice were used for each group.

Mechanical Stimulation-Evoked Responses of BLA–mPFC Neurons in Sham or SNI Mice

This cell population preliminarily identified by BLA electrical stimulation as BLA→mPFC(−) neurons, responded to noxious mechanical stimuli with an inhibition, the onset and the duration of the mechanical stimulation-evoked inhibition were 32.25 ± 1.3 and 273.7 ± 13.3 ms, respectively (Fig. 14A,E,F) in sham/veh mice (n = 20 neurons recorded from 10 mice). Treatment with AA-5-HT (5 mg/kg i.p.) did not affect either the duration or the onset of mechanical stimulation-evoked inhibition in the shams (sham/AA-5-HT, n = 19 neurons recorded from 10 mice, Fig. 14B,E–G). In SNI/veh group of mice (n = 20 neurons recorded from 10 mice), the onset of mechanical stimulation-evoked inhibition was significantly reduced (11.7 ± 1.2 ms, F1–38 = 134.92; P < 0.01), whereas the duration was significantly increased (404.8 ± 8.1, F1–38 = 70.88; P < 0.01) (Fig. 14C,E,F). Treatment with AA-5-HT (5 mg/kg i.p.) (n = 22 neurons recorded from 10 mice) for 7 days in SNI mice caused a significant increase in the onset (31.5 ± 1.4 ms, F1–40 = 113.09; P < 0.01) and a significant reduction in the duration (272.8 ± 11.1 ms, F1–40 = 89.20; P < 0.01) of mechanical stimulation-evoked inhibition (Fig. 14D–F).

The cell population preliminary identified by BLA electrical stimulation as BLA→mPFC(+) neurons, responded to noxious mechanical stimulus with an excitation. The onset, duration, and frequency of mechanical stimulation-evoked excitation of these mPFC neurons were 40.17 ± 1.6 ms, 189 ± 13.7 ms, and 5.7 ± 0.2 Hz, respectively, in sham/veh mice (Fig. 15A,E–G) (n = 18 neurons recorded from 10 mice). 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 sham (sham/AA-5-HT, n = 17 neurons recorded from 10 mice; Fig. 15B,E–G). In SNI/veh mice (n = 22 neurons recorded from 10 mice), the onset was significantly reduced (24.7 ± 2.05 ms, F1–38 = 33.04; P < 0.01) while duration and frequency of mechanical stimulation-evoked excitation were significantly increased (490.6 ± 11.8 ms, F1–38 = 281.04; P < 0.01, 7.2 ± 0.4 Hz, F1–38 = 9.82; P < 0.01, respectively) (Fig. 15C,E–G). Treatment for 7 days with AA-5-HT (5 mg/kg i.p.) (n = 19 neurons recorded from 10 mice) caused a significant decrease in the duration (350.5 ± 12.2 ms, F1–39 = 67.73; P < 0.01) and the frequency (5.6 ± 0.6 Hz, F1–39 = 5.17; P < 0.05) in SNI mice, whereas no change was observed on the onset (30.3 ± 2.5 ms, F1–39 = 3.06; P = 0.088) of mechanical stimulation-evoked excitation (Fig. 15D–G).

Figure 15.

Mechanical stimulation evokes excitatory responses in PL-IL cortex neurons in BLA→PL-IL (+) neurons. The figure shows different parameters of mechanical stimulus-evoked excitation, including the onset, the frequency, and the duration of the excitation, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT groups of mice. “E”,“F,” and “G” show the onset, the frequency, and the duration (mean ± standard error of the mean) of the excitation in the different groups of mice (n = 18, n = 17, n = 22, n = 19 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and n=10 mice were used for each group.

Figure 15.

Mechanical stimulation evokes excitatory responses in PL-IL cortex neurons in BLA→PL-IL (+) neurons. The figure shows different parameters of mechanical stimulus-evoked excitation, including the onset, the frequency, and the duration of the excitation, in sham and SNI mice treated for 7 days with vehicle (veh) or AA-5-HT (5 mg/kg, i.p.). “A,” “B,” “C,” and “D” show a representative ratemater record of a single neuron of sham/veh, sham/AA-5-HT, SNI/veh, and SNI/AA-5-HT groups of mice. “E”,“F,” and “G” show the onset, the frequency, and the duration (mean ± standard error of the mean) of the excitation in the different groups of mice (n = 18, n = 17, n = 22, n = 19 cells, respectively). PSTH show responses of a single neuron to a pulse (200 μA intensity) delivered at 0.5-Hz stimulation (vertical black bar). * indicates statistically significant difference versus sham/veh and ° versus SNI/veh. P < 0.05 was considered as value of significance, and n=10 mice were used for each group.

Discussion

Stimulation of brain TRPV1 channels enhances glutamatergic signaling (Szallasi 2002; Starowicz et al. 2008), intracellular Ca2+ elevation (Sindreu et al. 2007; Cao et al. 2009), and caspase release (Czaja et al. 2008; Hong et al. 2008). We investigated here whether SNI-induced neuropathic pain induces a functional reorganization in the PL-IL cortex, mediated by glutamate and caspase signaling following TRPV1 overactivity. Our data indicate that, following SNI, this brain area shows increased TRPV1 expression in glutamatergic neurons, excitatory signaling of BLA→mPFC neurons, glutamate spillover, and caspase activation in both ipsi- and contralateral sides. These events appear to stimulate glutamate, cannabinoid CB1 and, possibly, IL-1R receptor-mediated cross-talk between astrocytes, microglia, and glutamatergic neurons, with the result of either reinforcing or counteracting pain (Fig. 16).

Figure 16.

Schematic representation of the SNI-induced alterations in the mouse limbic cortex and their contribution to mechanical allodynia perception. Through as yet unidentified mechanism, SNI is accompanied by enhanced excitatory signaling in the PL-IL cortex from the BLA as well as by upregulation of TRPV1 channels in glutamatergic terminals and neurons in this area. These 2 events concur at causing glutamate spillover, which might then stimulate both pro-nociceptive and anti-nociceptive mechanisms. The former mechanisms are mostly due to AMPA-like receptor stimulation and consist of caspase-1, and subsequent IL-1β, release in astrocytes, activation of IL-1R1 in TRPV1-expressing neurons, and caspase-3 release and possible apoptosis in microglia. Glutamate might also stimulate AMPA-like receptors in GABAergic interneurons and hence inhibit the descending anti-nociceptive pathways originating in the PL-IL cortex. Such pathways, however, might, instead, be activated by glutamate via NMDA receptors. Finally, again as part of the anti-nociceptive mechanisms triggered by glutamate, activation of mGluR5 receptors might trigger the release of 2-AG from TRPV1-expressing perykaria and retrogradely activate CB1 receptors to reduce spillover onto astrocytes and microglia. This latter mechanism would be facilitated by TRPV1-dependent overexpression of the major 2-AG biosynthesizing enzyme, DAGLα, which colocalizes in somas with TRPV1 Red and blue arrows and type denote mechanisms that are only pro- or anti-nociceptive, respectively, the targeting of which produces the strongest and more long-lasting effects on mechanical allodynia (see Figs 9–11). Pink arrows and type denote pathways that are both pro- and anti-nociceptive, the targeting of only one of which produce only weak or short-lasting effects on mechanical allodynia (see Figs 9–11). Broken arrows denote inhibition/degradation.

Figure 16.

Schematic representation of the SNI-induced alterations in the mouse limbic cortex and their contribution to mechanical allodynia perception. Through as yet unidentified mechanism, SNI is accompanied by enhanced excitatory signaling in the PL-IL cortex from the BLA as well as by upregulation of TRPV1 channels in glutamatergic terminals and neurons in this area. These 2 events concur at causing glutamate spillover, which might then stimulate both pro-nociceptive and anti-nociceptive mechanisms. The former mechanisms are mostly due to AMPA-like receptor stimulation and consist of caspase-1, and subsequent IL-1β, release in astrocytes, activation of IL-1R1 in TRPV1-expressing neurons, and caspase-3 release and possible apoptosis in microglia. Glutamate might also stimulate AMPA-like receptors in GABAergic interneurons and hence inhibit the descending anti-nociceptive pathways originating in the PL-IL cortex. Such pathways, however, might, instead, be activated by glutamate via NMDA receptors. Finally, again as part of the anti-nociceptive mechanisms triggered by glutamate, activation of mGluR5 receptors might trigger the release of 2-AG from TRPV1-expressing perykaria and retrogradely activate CB1 receptors to reduce spillover onto astrocytes and microglia. This latter mechanism would be facilitated by TRPV1-dependent overexpression of the major 2-AG biosynthesizing enzyme, DAGLα, which colocalizes in somas with TRPV1 Red and blue arrows and type denote mechanisms that are only pro- or anti-nociceptive, respectively, the targeting of which produces the strongest and more long-lasting effects on mechanical allodynia (see Figs 9–11). Pink arrows and type denote pathways that are both pro- and anti-nociceptive, the targeting of only one of which produce only weak or short-lasting effects on mechanical allodynia (see Figs 9–11). Broken arrows denote inhibition/degradation.

The PL-IL cortex corresponds to the dorsolateral prefrontal cortex in humans. It plays a crucial role in pain processing (Porro et al. 1998; Derbyshire et al. 1999; Apkarian et al. 2004; Borckardt et al. 2007; Gündel et al. 2008; Fuccio et al. 2009), and chronic pain alters its connectivity in humans (Apkarian et al. 2004; Baliki et al. 2008). NMDA-induced neural sensitization and increased dendritic plasticity, but not neural death, occur in the PL-IL cortex of SNI rats (Metz et al. 2009), whereas neural mPFC deactivation was described in arthritic rats (Ji et al. 2010). Both worsening (Neugebauer et al. 2003, 2004; Wu et al. 2005; Carrasquillo and Gereau 2007) and relief (Maione et al. 2000; Millecamps et al. 2007) of pain can be mediated by supraspinal glutamate. Following increased glutamate tone, caspase pathways can be activated and contribute to pain in models of nerve injury (Joseph and Levine 2004; Chee et al. 2005; Fuccio et al. 2009; Joseph and Levine 2009). By employing multidisciplinary approaches for the measurement of TRPV1, endovanilloid/endocannabinoid, glutamatergic, caspase and IL-1β signaling, pharmacological tools injected into the PL-IL cortex, and Trpv1−/− mice, we suggest that a likely sequence of SNI-induced events in the PL-IL cortex is: 1) TRPV1 overexpression, 2) enhanced glutamate spillover, 3) activation of AMPA-like receptors in astrocytes and overexpression and overactivation of such receptors in microglia, and 4) caspase-1 and 3 release in these 2 cell types. This latter event, as indicated by pharmacological experiments, then contributes to mechanical allodynia possibly also via IL-1β release and feed-forward actions on TRPV1-expressing neurons. Furthermore, we suggest that SNI-induced enhancement of TRPV1 activity causes also adaptive anti-hyperalgesic responses, including: 1) enhancement of the degradation of the endocannabinoid/endovanilloid AEA; 2) stimulation by glutamate of anti-nociceptive NMDA and mGluR5 receptors in glutamatergic neurons, and 3) elevation of the biosynthesis of the endocannabinoid 2-AG and activation of CB1 receptors. In favor of a pivotal role for both enhanced TRPV1 and excitatory signaling in these adaptive and maladaptive events, we observed that all of them, except for caspase-1 and IL-1β release in reactive astrocytes, were absent in Trpv1−/− mice. Furthermore, we observed that acute antagonism of AMPA-like receptors in the PL-IL cortex abolished caspase-1 and -3 and IL-1β release and reduced mechanical allodynia in SNI mice, that blockade of caspase action also reduced mechanical allodynia in these mice, and that this latter effect was significantly less marked and shorter lasting, but still present, in SNI Trpv1−/− mice. We hypothesize that SNI is accompanied by parallel 1) overall enhancement of BLA→mPFC neuron activity and 2) overexpression of TRPV1 in the PL-IL cortex, which then concur at causing glutamate spillover and triggering the above events (Fig. 16). That enhanced glutamatergic signaling in this brain area is not uniquely a consequence of TRPV1 overexpression and activation is suggested by the finding that AMPA-like receptor blockade by CNQX abolished also those events that were not reduced in SNI Trpv1−/− mice, that is, caspase-1 and IL-1β release, and by the reasoning that enhanced BLA→mPFC neuron activity may arise also from alterations in the BLA.

Although neuronal functions of caspases beyond apoptosis are known, we observed here that caspase-3 release in the PL-IL cortex of SNI mice occurs in microglia and, therefore, is more likely to be connected with apoptotic death. Indeed, apoptosis in the spinal cord, and caspase-3 activation in microglia of the orbitofrontal cortex, were observed in neuropathic pain (Maione et al. 2002; de Novellis et al. 2004; Scholz et al. 2005; Siniscalco et al. 2007, 2008). Importantly, in the absence of pathological insults, resting microglia are far from being dormant as they actively monitor the normal state of the synapses and, therefore, guarantee the well being of neurons and their plasticity (Wake et al. 2009). On the other hand, caspase-1 release in astrocytes is likely connected to the observed production of IL-1β and subsequent targeting of TRPV1-expressing PL-IL excitatory neurons, where we found IL-1R1 to be mostly located. This effect of IL-1β might, in turn, contribute to TRPV1 sensitization (Camprubí-Robles et al. 2009) and/or cause stronger release of glutamate, whereas TRPV1 can feedforward on IL-1β neuromodulatory actions such as inhibition of GABAergic signaling (Musumeci et al. 2011). Thus, caspase-1 might act as a link between TRPV1-independent/glutamate-dependent and TRPV1/glutamate/caspase-3–dependent pronociceptive mechanisms triggered in the PL-IL cortex by SNI and described above. That both caspase-1 and 3 as well as possibly the other caspases that we found upregulated following SNI, provide a fundamental contribution to nociception, is strongly suggested by the fact that a pan-caspase inhibitor, injected locally in the PL-IL cortex, was among the most efficacious pharmacological tools tested here at counteracting allodynia.

The present finding of a net increase in the excitatory influence (more rapid onset of excitation and inhibition of excitatory and inhibitory cells, respectively, with frequency and duration of excitation being both increased) that the BLA exerts over the PL-IL pyramidal neurons of the mPFC (Neugebauer et al. 2009), an effect that was strengthened by paw mechanical stimulation, represents strong electrophysiological evidence for the aforementioned SNI-induced enhanced glutamate release from these neurons. However, depending on the receptor and cellular target involved, enhanced glutamatergic signaling in the PL-IL cortex, apart from contributing to neuropathic pain via enhanced caspase activity, may also provide adaptive mechanisms counteracting hyperalgesia. Indeed, we showed here that glutamate acting at non-AMPA receptors in excitatory neurons represents an anti-hyperalgesic response, since local injection of NMDA or mGluR5 antagonists, alone or in combination, strongly exacerbated allodynia (Millecamps et al. 2007; Centeno et al. 2009). We also found that AMPA-like receptors are overexpressed in GABAergic interneurons of the infralimbic cortex, where their stimulation might enhance the activity of these neurons and hence counteract the descending anti-nociceptive pathway (see Gabbott et al. 2006). However, the lack of GABA release in microdialysis experiments seems to argue against this possibility. Indeed, as mentioned above, TRPV1-dependent enhancement of IL-1β was recently shown to curb GABAergic signaling in the brain (Musumeci et al. 2011).

One possible way for excess glutamate to produce analgesia via mGluR5 receptors is through stimulation of the biosynthesis of 2-AG, which occurs postsynaptically following strong metabotropic glutamate receptor stimulation and acts at presynaptic CB1 receptors to reduce both inhibitory and excitatory neurotransmitter release (Szabo et al. 2006). We hypothesize that the observed increased 2-AG levels in the PL-IL cortex are caused by enhanced glutamatergic signaling at mGluR5 as an adaptive response to minimize pyramidal cell overactivity, similar to what observed during glutamate excitotoxicity (Maejima et al. 2005; Marcaggi and Attwell 2005; Rancz and Häusser 2006). This mechanism is supported by our finding of the: 1) TRPV1-dependent upregulation of the major 2-AG biosynthesizing enzyme, DAGLα, and by the large somatic coexpression of this enzyme with TRPV1 in the PL-IL cortex and 2) coexpression of CB1 and VGluT1 in terminals afferents to TRPV1-expressing somas. However, this mechanism may be insufficient to tonically counteract pain, since intra-PL-IL cortex injection of a CB1 antagonist per se did not modify allodynia. This suggests that SNI-induced 2-AG biosynthesis and AEA level reduction in this brain area, with no increase of CB1 receptor expression, result in a net null effect on endocannabinoid tone. However, in support of its potential importance, the pharmacological enhancement of this mechanism by administration into the PL-IL cortex of a CB1 agonist, such as WIN55,212-2, or an indirect CB1 agonist (via FAAH inhibition), such as URB597, did counteract allodynia.

In fact, SNI was accompanied also by upregulation of FAAH and reduction of AEA levels, in the PL-IL cortex. This effect was again not observed in Trpv1/− mice, suggesting that it is due to TRPV1 activation. Given the dual nature of AEA as endocannabinoid/endovanilloid, the reduction of its levels might contribute to pain by impairing either TRPV1 desensitization (Maione et al. 2006, 2007) or CB1 activity. However, it may also represent an adaptive response aiming at counteracting the effects of enhanced TRPV1 signaling. SNI-induced impaired CB1 activity via this mechanism is an unlikely possibility due to the concomitant elevation of 2-AG levels, as discussed above. On the other hand, SNI-induced impaired TRPV1 activity is also unlikely since the levels of 2 other endovanilloids, PEA and OEA, did not change, possibly because these compounds are also substrates for hydrolytic enzymes other than FAAH (Ueda et al. 2010).

A role of SNI-induced TRPV1 upregulation in glutamate spillover in the PL-IL cortex is suggested not only by the present and previous observation that the channel is largely coexpressed in glutamatergic fibers and stimulates glutamate release (Starowicz et al. 2008; Musella et al. 2009; Peters et al. 2010), but, more importantly, by the fact SNI-induced upregulation of VGluT1 is absent in SNI Trpv1−/− mice. Accordingly, we observed that local TRPV1 blockade with I-RTX, like antagonism of AMPA-like receptors, produced anti-allodynic effects, in a manner attenuated by the prototypical TRPV1 agonist, capsaicin. High doses of capsaicin, however, were more efficacious than I-RTX at reducing allodynia. This finding can be explained in 2 ways. First, when TRPV1 is overexpressed, as in case of SNI mice, its blockade is effected with higher efficacy through desensitization with an agonist than by antagonism (Rashid et al. 2003). Alternatively, if capsaicin only activates, without desensitizing, TRPV1, it will also enhance the glutamate-mediated adaptive anti-hyperalgesic responses mentioned above. The fact that TRPV1 activation, and subsequent glutamate spillover, may both contribute to and counteract pain is also suggested by the present finding that, contrary to blockade of caspase activity, I-RTX, when administered either directly and acutely into the PL-IL cortex or systemically over several days, only caused transient and partial anti-allodynic effects. Furthermore, we found here that Trpv1−/− mice did not exhibit lower allodynia following SNI, in agreement with previous reports in other models of chronic pain (Bölcskei et al. 2005; Christoph et al. 2008). This latter observation, apart from confirming that peripheral TRPV1 channels are involved in the transmission of thermal more than mechanical pain, also suggests that, on the long run, the cortical alterations in VGluT1 and capsase-3 activity found here to be dependent on the presence of TRPV1, are counterbalanced by TRPV1-triggered adaptive mechanisms that counteract allodynia. It also suggests, on the other hand, that the lack of these alterations in Trpv1−/− mice is due to the absence of TRPV1 and not to decreased nociception. Unlike I-RTX, AA-5-HT, which not only antagonizes TRPV1, but also inhibits FAAH, thus indirectly potentiating CB1 receptors and compensating for the lack of TRPV1-triggered 2-AG elevation and anti-nociceptive glutamate receptor stimulation, was more efficacious than I-RTX (as well as of a FAAH inhibitor) at reducing allodynia, both when administered systemically and chronically, and when directly injected into the PL-IL. Furthermore, this compound, was more, and not less, efficacious in Trpv1−/− mice, suggesting that when the TRPV1-mediated component of its mechanism of action, along with the TRPV1-dependent pro- and anti-nociceptive events, are missing, its inhibitory effect at FAAH, and indirect activation of CB1 receptors (confirmed by the antagonism of its action by AM251), are still sufficient to fully inhibit pain. Indeed, it can be noticed from our data that those interventions that strongly shift the balance between TRPV1-dependent pro- and antinociceptive effects in the PL-IL cortex (Fig. 16), more strongly modify allodynia (Figs 9–11).

Daily systemic treatment of SNI rats with AA-5-HT normalized both inhibitory and excitatory transmission in the BLA-mPFC pathway, inhibited caspase activation, and counteracted allodynia while restoring the baseline levels in the PL-IL cortex not only of AEA but also of 2-AG. This latter finding supports our hypothesis that SNI-induced 2-AG level elevation in the PL-IL cortex is an adaptive consequence, rather than a cause, of the alterations observed in this brain area, since, if anything, FAAH inhibition by AA-5-HT should have enhanced further, rather than decreasing, the tissue concentrations of this endocannabinoid (which is also a FAAH substrate) (Maione et al. 2006). These effects of chronic AA-5-HT cannot be taken per se as evidence that TRPV1 overactivity also underlies enhanced BLA→mPFC neuronal activity, since they might also be due to the fact that the compound normalized the effects of SNI in the PL-IL cortex merely as a consequence of its reduction of pain. However, they do not exclude this possibility nor our above hypothesis that CB1 activation, due to either mGluR5 activation of 2-AG biosynthesis or reduced degradation of AEA, inhibits the activity of BLA→mPFC neurons.

In conclusion, we have shown that the PL-IL cortex undergoes several changes following SNI, including enhanced TRPV1 expression on glutamatergic fibers and excitatory signaling by BLA→mPFC neurons, with subsequently increased extracellular levels of glutamate. This contributes to nociception via AMPA-like receptor-mediated glial caspase activation but produces also adaptive anti-nociceptive effects via neuronal CB1 and non-AMPA-like glutamate receptors. These findings support the hypothesis that functional reorganization of PL-IL cortex plays a key role in neuropathic pain through several mechanisms.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/

Funding

National Institutes of Health grant (DA009789 to V.D.) and Ministero dell'Universitàe della Ricerca (Italy) Grant PRIN 2009 to S.M.

Conflict of Interest : None declared.

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Author notes

Giordano, Cristino, and Luongo have contributed equally to this work
Vincenzo Di Marzo and Sabatino Maione share senior authorship of this work