Endogenous N-acetylaspartylglutamate (NAAG) inhibits synaptic plasticity/transmission in the amygdala in a mouse inflammatory pain model
© Adedoyin et al; licensee BioMed Central Ltd. 2010
Received: 22 June 2010
Accepted: 22 September 2010
Published: 22 September 2010
The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is widely expressed throughout the vertebrate nervous system, including the pain processing neuraxis. Inhibitors of NAAG peptidases are analgesic in animal models of pain. However, the brain regions involved in NAAG's analgesic action have not been rigorously defined. Group II metabotropic glutamate receptors (mGluR2/3) play a role in pain processing in the laterocapsular part of the central nucleus of the amygdala (CeLC). Given the high concentration of NAAG in the amygdala and its activation of group II mGluRs (mGluR3 > mGluR2), this study was undertaken using the mouse formalin model of inflammatory pain to test the hypothesis that NAAG influences pain processing in the amygdala. Evoked excitatory postsynaptic currents (eEPSCs) were studied in neurons in the CeLC of mouse brain slices following stimulation of the spinoparabrachial amygdaloid afferents.
Application of a NAAG peptidase inhibitor, ZJ43, dose dependently inhibited the amplitude of the eEPSCs by up to 50% in control CeLC demonstrating the role of NAAG in regulation of excitatory transmission at this synapse. A group II mGluR agonist (SLx-3095-1) similarly inhibited eEPSC amplitude by about 30%. Both effects were blocked by the group II mGluR antagonist LY341495. ZJ43 was much less effective than SLx in reducing eEPSCs 24 hours post inflammation suggesting an inflammation induced reduction in NAAG release or an increase in the ratio of mGluR2 to mGluR3 expression. Systemic injection of ZJ43 proximal to the time of inflammation blocked peripheral inflammation-induced increases in synaptic transmission of this pathway 24 hrs later and blocked the induction of mechanical allodynia that developed by this time point.
The main finding of this study is that NAAG and NAAG peptidase inhibition reduce excitatory neurotransmission and inflammation-induced plasticity at the spinoparabrachial synapse within the pain processing pathway of the central amygdaloid nucleus.
The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) has a positive role in animal models of traumatic brain injury, stroke, schizophrenia, inflammatory pain and peripheral neuropathy (reviewed in [1, 2]). NAAG is widely distributed in the brain and spinal cord, including the ascending and descending pain pathways [3, 4]. NAAG activates group II metabotropic glutamate receptors (mGluR3 > mGluR2) [5–7]. Two enzymes, glutamate carboxypeptidase II and III (GCPII and GCPIII), that inactivate synaptically released NAAG have been cloned and characterized [8–10] and a series of NAAG peptidase inhibitors have been developed [2, 11]. These inhibitors have been used to define the effects of synaptically released NAAG in vivo.
Systemic, local and central applications of the NAAG peptidase inhibitors are analgesic in inflammatory and neuropathic pain models, an effect that is reversed by systemic administration of the group II mGluR antagonist, LY341495 [12–16]. It is hypothesized that NAAG exerts its analgesic effects by reducing glutamate release via the presynaptic group II mGluRs . NAAG peptidase inhibition reduced synaptic release of glutamate at an identified synapse in the hippocampus, consistent with a study of the actions of NAAG in cell culture [17, 18]. However, there have been no direct demonstrations of the actions of endogenous NAAG at other identified synapses, including those in the pain processing pathway.
The amygdala is involved in affective processing of sensory information including pain-related responses [19–22]. The central nucleus (CeA) is the main output of the multinucleated amygdaloid complex; its connections make it critical for expression of pain-related responses [19, 21, 23, 24]. A glutamatergic synaptic pathway in the laterocapsular part of the central nucleus amygdala (CeLC) is involved in inflammatory pain processing . Activation of the group II mGluRs significantly inhibited the evoked excitatory postsynaptic current (eEPSCs) in the CeLC in the rat arthritic model of inflammatory pain [26, 27]. Given the expression of NAAG and NAAG peptidase activity in the amygdala [28–30], we speculated that NAAG activation of presynaptic group II receptors in the CeLC plays a role in regulating transmitter release and that elevation of synaptic levels of NAAG influences processing of inflammatory pain signals .
The NAAG peptidase inhibitor, ZJ43, was used to define the peptide's role in the spinoparabrachial amygdaloid afferent synapses in the CeA in brain slices from mice prior to and at different intervals after induction of footpad inflammation.
Prolonged nociceptive behaviors in formalin mice model
Thermal hypersensitivity in formalin model
Mechanical hypersensitivity in formalin model
Mechanical withdrawal threshold (allodynia) was repeatedly assessed in each mouse using the electronic von Frey anesthesiometer (EVF) system prior to and at 1, 6 and 24 hours post injection into the mouse's footpad (saline and formalin groups). The formalin treated mice exhibited prolonged mechanical allodynia in the injected left hindpaw that was significant at 1, 6 and 24 hours compared to baseline, while the contralateral hindpaw gave a significantly lower threshold response at 24 hours post injection compared to baseline but not at 1 and 6 hours. In contrast, the saline treated mice did not show any significant reduction in the threshold response (Figure 1B). The mechanical thresholds of the formalin injected hindpaws were significantly lower than those observed in the saline-injected animals at all time points post injection (p ≤ 0.05).
ZJ43 treatment prevents the development of mechanical allodynia in the formalin mice model
After baseline trials, two doses of NAAG peptidase inhibitor ZJ43 (150 mg/kg) was administered intraperitoneally (I.P.) 30 minutes prior to and approximately 8 hours after formalin injection into the mouse's footpad. This treatment inhibited peptidase activity for no more than 16 hours but not at 24 hour testing time (see Methods). The mechanical withdrawal threshold was then assessed at 24 hours post formalin treatment. NAAG peptidase inhibition blocked the development of mechanical allodynia in the injected hindpaw while the effect of ZJ43 failed to reach significance (p = 0.13) in the contralateral hindpaws at the 24 hr time point (Figure 1B).
Peripheral inflammation results in synaptic changes in the CeLC region of the formalin mice model
Enhanced synaptic transmission in brain slices from formalin-treated mice
NMDA/AMPA ratio remain unchanged in brain slices from formalin-treated mice
CPP (10 μM), a NMDA receptor antagonist, was used to pharmacologically evaluate the contribution of postsynaptic AMPA and NMDA receptors to the enhanced current observed in the input-output experiment at 24 hours post inflammation. The evoked currents in the presence of CPP were digitally subtracted offline from baseline control current to obtain the isolated NMDA component (Figure 2E). The observed current ranged from 10 to 800 pA but are not reported because the current output depended on the stimulus intensity. As a result, analysis of the NMDA/AMPA ratio was used because it allows for comparison between cells. The NMDA/AMPA ratio was not significantly changed 24 hours post inflammation compared to that obtained in uninjected control mice (Figure 2F).
Increased excitability of neurons in brain slices from formalin-treated mice
The increased firing rate and the enhanced evoked current suggest that in addition to an increase in transmitter release in the CeLC as a consequence of peripheral inflammation, there also were changes in the excitability of postsynaptic neurons (Figure 2D & 3C).
NAAG peptidase inhibition, NAAG and group II mGluR agonist affect synaptic transmission in the CeLC of control mouse
ZJ43 inhibits synaptic transmission in the CeLC
To validate the effect of ZJ43 at this synapse, repurified NAAG (1 μM) and the group II mGluR agonist SLx-3095-1 (0.5 μM) were applied locally to the slice in nominally Mg2+ free and standard ACSF recording conditions. NAAG and Slx-3095-1 caused a significant reduction in the amplitude of the evoked current (Figure 5C, left traces; 5D). To determine whether the group II mGluRs are tonically activated in the CeLC region of the uninjected control mice brain slice, the group II mGluR antagonist LY341495 (1 μM) was applied locally to the slice preparation in both Mg2+ free and standard ACSF solutions. The antagonist had no significant effect on the amplitude of the evoked current in these control preparations in either recording solutions (Figure 5E and 5F; MgCl2, p = 0.76; Mg2+ Free conditions p = 0.74) suggesting minimal activation of the group II mGluRs in the CeLC region of the brain slice at low stimulation frequency (0.1 Hz) in control mice.
Endogenous NAAG effect in the CeLC following inflammation in the formalin mouse model
Endogenous NAAG inhibits synaptic transmission in brain slices from formalin-treated mice 6 and 24 hours after peripheral inflammation
IP injection of peptidase inhibitor, ZJ43, reduced inflammation-induced enhanced current output in the central nucleus of amygdala
To test the conclusion that these inflammation-induced changes in spinoparabrachial input to the central nucleus were specific to this pain pathway rather than generalized changes due to stress or other factors, recordings also were made following stimulation of the external capsule (EC) that carries cortical afferents input to the amygdala. No change in eEPSC amplitude was observed with increasing stimulus intensity in this circuit 24 hours after induction of inflammation (lower panel formalin 24 hr EC vs. middle panel formalin 24 hr PB, Figure 9A & 9B). Instead a reduction in the current output was observed when compared to that obtained in control brain slices.
Peptidases that inactivate NAAG have been cloned, characterized and deleted in null mutant mice [9, 10, 31, 32]. Inhibitors of these peptidases and agonists of NAAG's mGluRs have been studied as promising targets for drug development [1, 2, 11, 33, 34]. In contrast to other neurotransmitters, the actions of endogenously released NAAG have been difficult to rigorously characterize at identified synapses due to its conversion to glutamate by peptidases in the extrasynaptic space. Circumventing this, the application of NAAG peptidase inhibitors to slice preparations provides an opportunity to study the physiology of released peptide . Importantly in this and previous pain studies, the effects of ZJ43 are blocked by the group II mGluR antagonist, LY341495, supporting the conclusion that the peptide was acting via these receptors. The data reported here represent the first to couple the synaptic action of NAAG to a discrete central sensory processing pathway in a basal state and as it changes in response to sensory input.
These data support the hypothesis that ZJ43-mediated increases in NAAG decreased glutamate release in the amygdala, although this remains to be directly demonstrated in this brain region via microdialysis studies. It is well established, however, that group II mGluRs located at presynaptic glutamatergic synapses decreased evoked transmitter release [35–40]. Consistent with this, we previously used ZJ43 to elevate extracellular NAAG levels and decrease glutamate flux from hippocampal neurons in vivo . Important to interpreting studies using ZJ43, this peptidase inhibitor has no direct action on group II or other mGluRs [12, 42]. An alternative interpretation of these data would be that the ZJ43-induced decrease in EPSCs resulted from decreased release of glutamate directly from NAAG hydrolysis. Militating against this interpretation are microdialysis data demonstrating that extracellular NAAG levels are insufficient to significantly contribute to basal or stimulated glutamate levels [41, 43].
Presynaptic action of endogenous NAAG
NAAG peptidase inhibition significantly reduced the frequency of the miniature excitatory currents in this pain processing synapse in the amygdala (Figure 6E) supporting a presynaptic action of NAAG. The consistent degree of inhibition of eEPSC amplitude by ZJ43 in the presence and absence of Mg2+ in the bathing medium (Figure 5) provides further evidence for a presynaptic action inasmuch as a post-synaptic action would be unlikely to affect NMDA and AMPA currents equally. A structurally different NAAG peptidase inhibitor similarly reduced eEPSCs and increased the paired pulse ratio (PPR) at the mossy fiber-Ca3 synapse .
The efficacy of NAAG in reducing transmitter release supports the hypothesis that NAAG expands the dynamic range of synapses across the nervous system by dampening release of its different co-transmitters at high levels of excitation. This would be a particularly important concept under pathological states that are associated with excessive glutamate release [1, 40, 41, 44]. The absence of an effect of the group II mGluR antagonist alone under the basal activity of the control CeLC (Figure 5E, F) would be predicted by such a dynamic range model as would the absence of neurological deficits in GCPII knock-out mice under unstressed conditions .
Peripheral formalin-induced inflammation enhances current output in CeLC region
Elevated glutamatergic transmission in this spinoparabrachial amygdaloid pathway is involved in inflammatory pain processing . The parabrachial nucleus (origin of PB-CeLC afferents) relays converging specific nociceptive information from the spinal lamina I to the forebrain. The spinal nociceptive inputs reach, both directly and via the parabrachial nucleus, all subcortical components of the central autonomic network, including the amygdala [45–48]. The amygdaloid nuclear complex provides emotional significance to sensory stimuli, including pain [25, 49, 50]. Synaptic plasticity observed in the CeLC region following formalin-induced inflammation (Figures 2 and 3) is consistent with data from the rat mono-arthritic [51, 52] and spinal nerve ligation models . A similar current increase was found in the cingulate cortex after footpad inflammation [54–56]. In addition, molecular changes, such as enhanced pERK levels, were reported in the right CeLC amygdala region of mouse brain slices as early as 3 hours after formalin injection into the left or right footpad .
The change in efficacy of ZJ43 from control to 24 hr post inflammation (Figure 8), while the group II agonist remained effective under both conditions, is consistent with either a decrease in release of NAAG at the 24 hr point or a change in the ratio of the group II receptors in favor of mGluR2.
In contrast to the change in the PB pathway to the central nucleus 24 hrs after formalin injection, the EC evoked EPSCs is substantially reduced (Figure 9A). While this may have been due to plasticity induced by decreased cortical input the amygdala following peripheral inflammation, there are insufficient published data on cortical input to the amygdala under these conditions to permit such speculation.
Endogenous NAAG's role in the PB-CeA circuit
NAAG, NAAG peptidase activity and group II mGluRs are present in the pain processing pathway [28, 37–39]. While the efficacy of NAAG peptidase inhibitors in modulating perception of inflammatory pain has been studied extensively [14–16, 58], discrete sites in the pain pathway have not been explored systematically for the peptide's action. The observation that ZJ43-induced elevation of NAAG has a role at the PB-CeLC synapse in control mice supports the view that this is one site where systemic ZJ43 might act to reduce acute perception of inflammatory pain. In contrast, the substantially lower effect of ZJ43 24 hours after induction of inflammation argues that NAAG peptidase inhibition would have less influence on the PB-CeLC contributions to long term inflammatory pain perception. However, systemic injections of the peptidase inhibitor proximal to the time of in formalin treatment blocked the enhanced current output observed in the CeLC region associated with inflammatory input (Figure 9B) and mechanical allodynia in the injected hindpaw (Figure 1B) at the 24 hour time point. This result supports the view that early induction of analgesia by NAAG peptidase inhibition sufficiently suppresses activity in the pain pathways of the CeLC as to preclude the induction of this dramatic change in the circuit's excitatory transmission.
Adult male C57BL/6 mice (postnatal day 17-25; Taconic, Germantown, NY) were housed with food and water available ad libitum in a temperature-controlled environment with light/dark cycle of 12:12 hour. All procedures were conducted in compliance with Georgetown University's Animal Care and Use regulations. All experiments were performed with the approval of the Georgetown University Animal Care and Use Committee. Effort was made to minimize the number of animals used to avoid unnecessary suffering.
Mice were anaesthetized via inhalation of 3% isoflurane and maintained with 1.5% isoflurane while the left footpad was injected. Mice recover with spontaneous activity promptly after removal of the anesthetic. Inflammation was induced by injecting 20 μL of 5% formalin (37% formaldehyde solution diluted in saline solution, 30 gauge needle) into the intraplantar surface of the mouse's hindpaw [59, 60]. Control mice received 20 μL of saline solution (0.9%) into their hindpaw. Animals were monitored regularly after injections.
Nociceptive testing: Thermal hyperalgesia
Hargreaves plantar apparatus (Ugo Basile, Camerio VA, Italy) was used to assess the thermal withdrawal latency (TWL) in the injected hindpaw . Briefly, mice were allowed to acclimate (20 minutes) within a Plexiglas enclosure on a clear glass plate maintained at 30°C. When the mice were stationary, a mobile radiant heat source (i.e., high intensity projector lamp) located under the glass table was activated with a timer and focused onto the plantar surface of the left hindpaw. Thermal paw-withdrawal latency was determined by a motion detector that halted both lamp and timer when the paw was withdrawn. A maximal cut-off of 30 s was employed to prevent tissue damage. Paw withdrawal latencies were recorded automatically. Naïve (uninjected control), saline-treated, and formalin-treated animals were tested. Trials were repeated in the same animals prior to injection (baseline) and at 1, 3, 6 and 24 hours post-injection of either formalin or saline into the left footpad. Each trial was repeated twice at 3 minutes intervals, and the TWL is the average of the two responses. Behavioral response of flinching that may or may not be accompanied by licking of the heated paw was taken as a response. The heat stimulus gave a basal TWL of 7.1 ± 0.1 seconds.
Nociceptive testing: Mechanical allodynia and threshold
For the evaluation of mechanical allodynia, mice were placed individually in clear Plexiglas enclosures on elevated mesh stand (large base 36" × 16") (IITC Life Science, Woodland hills, CA) to allow access to the surface of the hindpaw. The withdrawal threshold (WT) in grams was obtained by application of the rigid tip attached to the anesthesiometer transducer probe (2390 series Electronic von Frey Anesthiometer (EVF), Stoelting Co, Wood Dale, IL). The EVF system allows measurement and displays test readings upon reaction in grams based upon the amount of pressure applied. The EVF is zero calibrated before each use. When the mice were stationary, the stimuli were delivered from below, to the plantar surface of the hindpaw. The animals were acclimatized for 1 hour before behavioral testing and the mechanical allodynia was evaluated at several time-points (1, 6, and 24 hours). In order to determine the basal mechanical thresholds, all the groups were evaluated before the test procedures. Each trial was repeated twice at 5 minutes interval, the withdrawal threshold is an average of the two responses. Saline treated, formalin treated, and ZJ43- Formalin treated animals were tested.
ZJ43 (150 mg/kg) was administered intraperitoneally (IP) 30 minutes prior to and approximately 8 hours after formalin injection into the mouse's left hind paw. Unpublished data from our lab indicate that a single i.p. injection of 150 mg/kg of ZJ43 inhibits NAAG peptidase inhibition for no more than 6 hours (Olszewski R, Ball S, Wegorzewska M, Lee M, Janczura K, Neale JH in preparation). As a result, in this dual injection paradigm there would be no significant peptidase inhibition ongoing at the time of behavioral testing or assessment of the brain slices at 24 hours post inflammation. The ZJ43 treated animals were divided into two groups: (1) used in electrophysiology experiments and (2) used in mechanical threshold (von Frey) experiments 24 hours post formalin injection.
Coronal brain slices (300 μM) (Bregma -1.06 to -1.58 mm) containing the right central nucleus of amygdala, laterocapsular part (CeLC) were prepared using techniques that emulate those used in rats [52, 62]. Briefly, mice (i.e. postnatal day 17-25, C57BL/6, Taconic Lab.) were decapitated, and the brain rapidly removed and placed in ice cold sucrose containing artificial cerebrospinal fluid (ACSF) buffer. No anesthesia was used prior to decapitation to avoid contamination of the brain tissue. Brain slices were then prepared using a micro slicer vibratome 3000 (Vibratome Inc, St Louis MO) and incubated at 32°C for 30 minutes in a sucrose based (ACSF) (mM): (NaCl 86, KCl 3, MgCl2 4, NaH2PO4 1, sucrose 75, glucose 25, CaCl2 1, NaHCO3 25). Slices were then maintained at 32°C for 30 minutes in standard ACSF of the following composition (mM): (NaCl 124, KCL 4.5, MgCl2 1, NaH2PO4 1, glucose 10, CaCl2 2, NaHCO3 26). The slice bathing medium was bubbled with O2/CO2 at a composition of 95% and 5%. After the incubation period, slices were transferred to the recording chamber and, unless stated otherwise, were perfused at 2 ml/min with a nominally Mg2+ free extracellular solution (Mg2+ was omitted from the standard ACSF). All experiments were performed at 30 ± 2°C in the presence of picrotoxin (30 μM, Sigma Aldrich St Louis, MO) and CGP52432 (10 μM, Tocris Bioscience Ellisville, MO) to block GABAergic currents. D-serine a co-agonist at glycine site on NMDA receptor (10 μM, Sigma) was included in nominal Mg2+ free extracellular solution. Coronal brain slices were obtained from untreated control mice, saline treated mice (24 hours post injection), ZJ43 treated formalin mice, and formalin-treated mice at 1, 6 and 24 hours post footpad injection. Using video as a visual guide the stimulating electrode was positioned on the fibers dorsomedial to the CeA and ventral to but outside the caudaputamen, (see fig 8A in ). The spinoparabrachial amygdaloid (PB-CeA) afferents reaching the central nucleus of the amygdala were stimulated using a tungsten bipolar stimulating electrode at a frequency of 0.1 Hz. Stimulation of the afferents in the external capsule (EC), which sends cortical inputs that originate outside of the pain pathway (PB-CeA) to the amygdala was used as a control for input specificity. Recordings were made from neurons in the anterior part of the CeLC region.
Input-output experiments [63, 64] were used to assess the synaptic strength response to incremental increases in stimulus intensity (0.08-5 mA) in the CeLC region and to compare changes in synaptic transmission between treatment groups. The same intensities were used for each animal group; the maximum stimulus intensity was that which failed to further increase the eEPSCs size. Current clamp experiments, with increasing depolarizing current injections of 20 pA steps were performed. Rheobase current was defined as the first current step, within a series of increasing 20 pA steps that elicited an action potential.
Electrodes were pulled in two stages on a vertical pipette puller from borosilicate glass capillaries (Wiretrol II, Drummond, Broomall, PA). The patch pipette solution composition (mM) was: (Kgluconate 145, EGTA 5, MgCl2 5, HEPES 10, ATP-Na 5, GTP-Na 0.2, and pH 7.2 with KOH). For the input-output experiments, the local anesthetic QX-314 (5 mM) was included in the pipette solution to block voltage gated Na+ conductance. Typical pipette resistance was 5-7 MΩ. Whole-cell recordings at Vhold = -60 mV were performed with a patch-clamp amplifier (Axopatch 200B, Axon Instrument, Foster City, CA) under video visual control with a Nikon Eclipse E600FN microscope (Nikon Japan).
Urea based glutamate carboxypeptidase (GCP) inhibitor N-[N-[(S)]-1, 3-dicarboxypropyl] carbamoyl]-L-leucine (ZJ43) was provided by Dr Alan Kozikowski at University of Illinois Chicago. At concentrations as high as 10 μM, ZJ43 fails to act as an agonist or antagonist at group I, II and III mGluRs . The group II metabotropic glutamate receptor (mGluR) antagonist (LY341495), the GABAB antagonist (CGP 52432), the AMPA receptor antagonist [sodium-2, 3-dihydro-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX)], NMDA receptor antagonist receptor antagonist [3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP)], and tetrodotoxin (TTX) were from Tocris Bioscience (Ellisville, MO). N-acetylaspartylglutamate (NAAG) from Tocris. We have long recognized that commercial NAAG is contaminated with 0.1-0.4% glutamate. As a result for more than a decade, we have routinely re-purified by ion exchange chromatography to remove traces of glutamate contamination (final glutamate levels less than 0.1%). The group II mGluR agonist SLx-3095-1 [see  was the generous gift of Alessandra Bartolozzi at Surface Logix, Inc. Boston, MA. SLx-3095-1 is the racemate (+/- isomer as HCl salt) of the highly selective group II mGluR agonist LY379268 (- isomer). Its synthesis is described as compound 9 in .
Stock solutions of TTX, NBQX, picrotoxin and CPP were dissolved in water. ZJ43, LY341495, and SLx-3095-1 were dissolved in saline. NAAG was dissolved in equimolar NaOH. CGP52432 was dissolved in dimethylsulfoxide (DMSO) at 0.01% final concentration.
The recording chamber was perfused continuously with nominal Mg2+ free or standard ACSF in order to minimize spread of the drug beyond the application site. Drugs were diluted in the recording ACSF to obtain optimal concentrations. All drugs solutions were locally applied adjacent to the recording site via a Y-tube  for 5 minutes and modified for optimal solution exchange in brain slices . AMPA receptor antagonist (NBQX, 5 μM) or NMDA receptor antagonist (CPP, 10 μM) were used at the end each experiment based on the type of current being measured to confirm the proper Y tube placement relative to the recording site.
Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) were identified with semi-automated template based analysis software in Clampfit 9.2 or using a semi-automated threshold based mini detection software (Mini Analysis, Synaptosoft Inc., http://www.synaptosoft.com, Fort Lee, NJ) and visually confirmed. Event detection threshold was set at 5 times the root mean square level of baseline noise. mEPSC and sEPSC averages were based on ≥30 events in each cell studied and the decay kinetics were determined using exponential curve fitting. Evoked excitatory postsynaptic currents (eEPSCs) inhibited or enhanced by various pharmacological agents are represented as absolute magnitude and were determined by comparison of mean baseline before and immediately after drug application. Failures were excluded from evoked current analysis. The change in the peak of the Gaussians was calculated to determine the drug effect. Cells with > 20% change in access resistance were discarded. Off-line evoked current data analysis, curve fitting, and figure preparation was performed using Clampfit 9.2 (Molecular Devices, Inc. Sunnyvale CA) and Microsoft excel.
TWL and mechanical WT values were obtained from the digital readout of the Hargreaves plantar and EVF apparatus respectively.
Statistical significance was determined using a two-tailed Student's t-test unpaired when comparing two populations of cells, and paired when comparing conditions in the same cell population or animal group. In ZJ43 response experiments one way repeated measure-analysis of variance (RM-ANOVA) was followed by pairwise Tukey t-test post hoc analysis (Sigmaplot 11 software, http://www.sigmaplot.com).
In the von Frey experiments, differences between the three groups (saline, formalin and naïve) and changes over time were analyzed for ipsilateral and contralateral hindpaw with a two way RM-ANOVA, followed by Tukey post-hoc comparisons between groups. Significant changes post-injection were established by comparison with baseline values (pre-injection) using Student's t-test for paired data. In each group of the von Frey experiments, differences between contralateral and ipsilateral sides to injection were tested over time with a two way RM-ANOVA. Two way RM-ANOVA analysis was used to analysis changes in TWL over time. The Tukey t-test post-hoc analysis was used for multiple comparisons between groups. All data are presented as mean ± SEM. The significance levels were set at *p ≤ 0.05. In all figures, *p ≤ 0.05 and **p ≤ 0.005 compared to control values, ◊ or #p ≤ 0.05 across groups. The term "control" when applied to brain slices or treatment groups refers to untreated mice.
We thank Dr. Alan Kozikowski at University of Illinois Chicago for providing ZJ43, and Alessandra Bartolozzi at Surface Logix, Inc. Boston, MA for the generous gift of SLx-3095-1. We also thank Dr. Volker Neugebauer at the University of Texas Medical Branch at Galveston (UTMB) for his suggestions on experimental design and review of this manuscript.
The study was supported by NIH grant R01 NS 38080 to JHN and a fellowship supplement to this grant for MOA. Additional fellowship support (MOA) was provided by NIH grant T32 NS041218.
- Neale JH, Olszewski RT, Gehl LM, Wroblewska B, Bzdega T: The neurotransmitter N-acetylaspartylglutamate in models of pain, ALS, diabetic neuropathy, CNS injury and schizophrenia. Trends Pharmacol Sci 2005, 26: 477–484.
- Tsukamoto T, Wozniak KM, Slusher BS: Progress in the discovery and development of glutamate carboxypeptidase II inhibitors. Drug Discovery Today 2007, 12: 767–776. 10.1016/j.drudis.2007.07.010View Article
- Coyle JT: The nagging question of the function of N-acetylaspartylglutamate. Neurobiol Dis 1997, 4: 231–238. 10.1006/nbdi.1997.0153View Article
- Neale JH, Bzdega T, Wroblewska B: N-Acetylaspartylglutamate: The Most Abundant Peptide Neurotransmitter in the Mammalian Central Nervous System. J Neurochem 2000, 75: 443–452. 10.1046/j.1471-4159.2000.0750443.xView Article
- Schweitzer C, Kratzeisen C, Adam G, Lundstrom K, Malherbe P, Ohresser S, Stadler H, Wichmann J, Woltering T, Mutel V: Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors. Neuropharm 2000, 39: 1700–1706. 10.1016/S0028-3908(99)00265-8View Article
- Wroblewska B, Santi MR, Neale JH: N-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytes. Glia 1998, 24: 172–179. 10.1002/(SICI)1098-1136(199810)24:2<172::AID-GLIA2>3.0.CO;2-6View Article
- Wroblewska B, Wroblewski JT, Pshenichkin S, Surin A, Sullivan SE, Neale JH: N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J Neurochem 1997, 69: 174–181. 10.1046/j.1471-4159.1997.69010174.xView Article
- Luthi-Carter R, Barczak AK, Speno H, Coyle JT: Molecular characterization of human brain N-acetylated alpha-linked acidic dipeptidase (NAALADase). J Pharmacol Exp Ther 1998, 286: 1020–1025.
- Bzdega T, Crowe SL, Ramadan ER, Sciarretta KH, Olszewski RT, Ojeifo OA, Rafalski VA, Wroblewska B, Neale JH: The cloning and characterization of a second brain enzyme with NAAG peptidase activity. J Neurochem 2004, 89: 627–635. 10.1111/j.1471-4159.2004.02361.xView Article
- Bzdega T, Turi T, Wroblewska B, She D, Chung HS, Kim H, Neale JH: Molecular cloning of a peptidase against N-acetylaspartylglutamate from a rat hippocampal cDNA library. J Neurochem 1997, 69: 2270–2277. 10.1046/j.1471-4159.1997.69062270.xView Article
- Zhou J, Neale JH, Pomper MG, Kozikowski AP: NAAG peptidase inhibitors and their potential for diagnosis and therapy. Nat Rev Drug Discov 2005, 4: 1015–1026. 10.1038/nrd1903View Article
- Kozikowski AP, Zhang J, Nan F, Petukhov PA, Grajkowska E, Wroblewski JT, Yamamoto T, Bzdega T, Wroblewska B, Neale JH: Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents. J Med Chem 2004, 47: 1729–1738. 10.1021/jm0306226View Article
- Yamamoto T, Hirasawa S, Wroblewska B, Grajkowska E, Zhou J, Kozikowski A, Wroblewski J, Neale JH: Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model. Eur J Neurosci 2004, 20: 483–494. 10.1111/j.1460-9568.2004.03504.xView Article
- Yamamoto T, Kozikowski A, Zhou J, Neale JH: Intracerebroventricular administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory pain. Mol Pain 2008, 4: 31. 10.1186/1744-8069-4-31PubMed CentralView Article
- Saito O, Aoe T, Kozikowski A, Sarva J, Neale JH, Yamamoto T: Ketamine and N-acetylaspartylglutamate peptidase inhibitor exert analgesia in bone cancer pain. Can J Anaesth 2006, 53: 891–898. 10.1007/BF03022832View Article
- Yamamoto T, Saito O, Aoe T, Bartolozzi A, Sarva J, Zhou J, Kozikowski A, Wroblewska B, Bzdega T, Neale JH: Local administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in rats. European Journal of Neuroscience 2007, 25: 147–158. 10.1111/j.1460-9568.2006.05272.xView Article
- Sanabria ER, Wozniak KM, Slusher BS, Keller A: GCP II (NAALADase) inhibition suppresses mossy fiber-CA3 synaptic neurotransmission by a presynaptic mechanism. J Neurophysiol 2004, 91: 182–193. 10.1152/jn.00465.2003View Article
- Zhao J, Ramadan E, Cappiello M, Wroblewska B, Bzdega T, Neale JH: NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance. Eur J Neurosci 2001, 13: 340–346.
- Bernard JF, Bester H, Besson JM: Involvement of the spino-parabrachio-amygdaloid and -hypothalamic pathways in the autonomic and affective emotional aspects of pain. Prog Brain Res 1996, 107: 243–255. full_textView Article
- Bester H, Chapman V, Besson JM, Bernard JF: Physiological properties of the lamina I spinoparabrachial neurons in the rat. J Neurophysiol 2000, 83: 2239–2259.
- Bester H, Matsumoto N, Besson JM, Bernard JF: Further evidence for the involvement of the spinoparabrachial pathway in nociceptive processes: a c-Fos study in the rat. J Comp Neurol 1997, 383: 439–458. 10.1002/(SICI)1096-9861(19970714)383:4<439::AID-CNE4>3.0.CO;2-3View Article
- Buritova J, Besson JM, Bernard JF: Involvement of the spinoparabrachial pathway in inflammatory nociceptive processes: a c-Fos protein study in the awake rat. J Comp Neurol 1998, 397: 10–28. 10.1002/(SICI)1096-9861(19980720)397:1<10::AID-CNE2>3.0.CO;2-LView Article
- Neugebauer V, Galhardo V, Maione S, Mackey SC: Forebrain pain mechanisms. Brain Res Rev 2009, 60: 226–242. 10.1016/j.brainresrev.2008.12.014PubMed CentralView Article
- Pitkanen A, Savander V, LeDoux JE: Organization of intra-amygdaloid circuitries in the rat: an emerging framework for understanding functions of the amygdala. Trends Neurosci 1997, 20: 517–523. 10.1016/S0166-2236(97)01125-9View Article
- Neugebauer V, Li W, Bird GC, Han JS: The amygdala and persistent pain. Neuroscientist 2004, 10: 221–234. 10.1177/1073858403261077View Article
- Han JS, Fu Y, Bird GC, Neugebauer V: Enhanced group II mGluR-mediated inhibition of pain-related synaptic plasticity in the amygdala. Mol Pain 2006, 2: 18. 10.1186/1744-8069-2-18PubMed CentralView Article
- Li W, Neugebauer V: Differential changes of group II and group III mGluR function in central amygdala neurons in a model of arthritic pain. J Neurophysiol 2006, 96: 1803–1815. 10.1152/jn.00495.2006View Article
- Fuhrman S, Palkovits M, Cassidy M, Neale JH: The regional distribution of N-acetylaspartylglutamate (NAAG) and peptidase activity against NAAG in the rat nervous system. J Neurochem 1994, 62: 275–281. 10.1046/j.1471-4159.1994.62010275.xView Article
- Moffett JR, Namboodiri MA, Neale JH: Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N- acetylaspartate immunoreactivities in rat brain. J Histochem Cytochem 1993, 41: 559–570.View Article
- Slusher BS, Tsai G, Yoo G, Coyle JT: Immunocytochemical localization of the N-acetyl-aspartyl-glutamate (NAAG) hydrolyzing enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). J Comp Neurol 1992, 315: 217–229. 10.1002/cne.903150208View Article
- Bacich DJ, Ramadan E, O'Keefe DS, Bukhari N, Wegorzewska I, Ojeifo O, Olszewski R, Wrenn CC, Bzdega T, Wroblewska B, et al.: Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate. J Neurochem 2002, 83: 20–29. 10.1046/j.1471-4159.2002.01117.xView Article
- Luthi-Carter R, Barczak AK, Speno H, Coyle JT: Hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) by cloned human glutamate carboxypeptidase II. Brain Res 1998, 795: 341–348. 10.1016/S0006-8993(98)00244-3View Article
- Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS, Bardenstein LM, Gurovich IY, Morozova MA, et al.: Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial. Nat Med 2007, 13: 1102–1107. 10.1038/nm1632View Article
- Rojas C, Thomas AG, Majer P, Tsukamoto T, Lu XM, Vornov JJ, Wozniak KM, Slusher BS: Glutamate carboxypeptidase II inhibition as a novel therapeutic target. Adv Exp Med Biol 2003, 524: 205–213. full_textView Article
- Dietrich D, Kral T, Clusmann H, Friedl M, Schramm J: Presynaptic group II metabotropic glutamate receptors reduce stimulated and spontaneous transmitter release in human dentate gyrus. Neuropharmacology 2002, 42: 297–305. 10.1016/S0028-3908(01)00193-9View Article
- Mateo Z, Porter JT: Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex. Neuroscience 2007, 146: 1062–1072. 10.1016/j.neuroscience.2007.02.053PubMed CentralView Article
- Ohishi H, Neki A, Mizuno N: Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody. Neurosci Res 1998, 30: 65–82. 10.1016/S0168-0102(97)00120-XView Article
- Ohishi H, Shigemoto R, Nakanishi S, Mizuno N: Distribution of the messenger RNA for a metabotropic glutamate receptor (mGluR3) in the rat brain - an insitu hybridization study. Journal of Comparative Neurology 1993, 335: 252–266. 10.1002/cne.903350209View Article
- Ohishi H, Shigemoto R, Nakanishi S, Mizuno N: Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study. J Comp Neurol 1993, 335: 252–266. 10.1002/cne.903350209View Article
- Schoepp DD, Conn PJ: Metabotropic glutamate receptors in brain function and pathology. Trends Pharmacol Sci 1993, 14: 13–20. 10.1016/0165-6147(93)90107-UView Article
- Zhong C, Zhao X, Van KC, Bzdega T, Smyth A, Zhou J, Kozikowski AP, Jiang J, O'connor WT, Berman RF, et al.: NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat. J Neurochem 2006, 97: 1015–1025. 10.1111/j.1471-4159.2006.03786.xView Article
- Olszewski RT, Bukhari N, Zhou J, Kozikowski AP, Wroblewski JT, Shamimi-Noori S, Wroblewska B, Bzdega T, Vicini S, Barton FB, Neale JH: NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR. J Neurochem 2004, 89: 876–885. 10.1111/j.1471-4159.2004.02358.xView Article
- Slusher BS, Vornov JJ, Thomas AG, Hurn PD, Harukuni I, Bhardwaj A, Traystman RJ, Robinson MB, Britton P, Lu XC, et al.: Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury. Nat Med 1999, 5: 1396–1402. 10.1038/70971View Article
- Bradley SR, Marino MJ, Wittmann M, Rouse ST, Awad H, Levey AI, Conn PJ: Activation of group II metabotropic glutamate receptors inhibits synaptic excitation of the substantia Nigra pars reticulata. J Neurosci 2000, 20: 3085–3094.
- Benarroch EE: Pain-autonomic interactions. Neurol Sci 2006,27(Suppl 2):S130–133. 10.1007/s10072-006-0587-xView Article
- Cortelli P, Pierangeli G: Chronic pain-autonomic interactions. Neurol Sci 2003,24(Suppl 2):S68–70.
- Kitamura T, Yamada J, Sato H, Yamashita K: Cells of origin of the spinoparabrachial fibers in the rat: a study with fast blue and WGA-HRP. J Comp Neurol 1993, 328: 449–461. 10.1002/cne.903280310View Article
- Sarhan M, Freund-Mercier MJ, Veinante P: Branching patterns of parabrachial neurons projecting to the central extended amgydala: single axonal reconstructions. J Comp Neurol 2005, 491: 418–442. 10.1002/cne.20697View Article
- Gao YJ, Ren WH, Zhang YQ, Zhao ZQ: Contributions of the anterior cingulate cortex and amygdala to pain- and fear-conditioned place avoidance in rats. Pain 2004, 110: 343–353. 10.1016/j.pain.2004.04.030View Article
- Wiech K, Tracey I: The influence of negative emotions on pain: behavioral effects and neural mechanisms. Neuroimage 2009, 47: 987–994. 10.1016/j.neuroimage.2009.05.059View Article
- Neugebauer V, Li W: Differential sensitization of amygdala neurons to afferent inputs in a model of arthritic pain. J Neurophysiol 2003, 89: 716–727. 10.1152/jn.00799.2002View Article
- Neugebauer V, Li W, Bird GC, Bhave G, Gereau RWt: Synaptic plasticity in the amygdala in a model of arthritic pain: differential roles of metabotropic glutamate receptors 1 and 5. J Neurosci 2003, 23: 52–63.
- Ikeda R, Takahashi Y, Inoue K, Kato F: NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain. Pain 2007, 127: 161–172. 10.1016/j.pain.2006.09.003View Article
- Toyoda H, Zhao MG, Zhuo M: Enhanced quantal release of excitatory transmitter in anterior cingulate cortex of adult mice with chronic pain. Mol Pain 2009, 5: 4. 10.1186/1744-8069-5-4PubMed CentralView Article
- Wu LJ, Steenland HW, Kim SS, Isiegas C, Abel T, Kaang BK, Zhuo M: Enhancement of presynaptic glutamate release and persistent inflammatory pain by increasing neuronal cAMP in the anterior cingulate cortex. Mol Pain 2008, 4: 40. 10.1186/1744-8069-4-40PubMed CentralView Article
- Zhao MG, Ko SW, Wu LJ, Toyoda H, Xu H, Quan J, Li J, Jia Y, Ren M, Xu ZC, Zhuo M: Enhanced presynaptic neurotransmitter release in the anterior cingulate cortex of mice with chronic pain. J Neurosci 2006, 26: 8923–8930. 10.1523/JNEUROSCI.2103-06.2006View Article
- Carrasquillo Y, Gereau RWt: Activation of the extracellular signal-regulated kinase in the amygdala modulates pain perception. J Neurosci 2007, 27: 1543–1551. 10.1523/JNEUROSCI.3536-06.2007View Article
- Yamamoto T, Hirasawa S, Wroblewska B, Grajkowska E, Zhou J, Kozikowski A, Wroblewski J, Neale JH: Antinociceptive effects of N -acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model. European Journal of Neuroscience 2004, 20: 483–494. 10.1111/j.1460-9568.2004.03504.xView Article
- Rosland JH, Tjolsen A, Maehle B, Hole K: The formalin test in mice: effect of formalin concentration. Pain 1990, 42: 235–242. 10.1016/0304-3959(90)91167-HView Article
- Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K: The formalin test: an evaluation of the method. Pain 1992, 51: 5–17. 10.1016/0304-3959(92)90003-TView Article
- Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988, 32: 77–88. 10.1016/0304-3959(88)90026-7View Article
- Travagli RA, Gillis RA, Rossiter CD, Vicini S: Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol 1991, 260: G531–536.
- Han JS, Li W, Neugebauer V: Critical role of calcitonin gene-related peptide 1 receptors in the amygdala in synaptic plasticity and pain behavior. J Neurosci 2005, 25: 10717–10728. 10.1523/JNEUROSCI.4112-05.2005View Article
- McKernan MG, Shinnick-Gallagher P: Fear conditioning induces a lasting potentiation of synaptic currents in vitro. Nature 1997, 390: 607–611. 10.1038/37605View Article
- Monn JA, Valli MJ, Massey SM, Hansen MM, Kress TJ, Wepsiec JP, Harkness AR, Grutsch JLJ, Wright RA, Johnson BG, et al.: Synthesis, pharmacological characterization, and molecular modeling of heterobicyclic amino acids related to (+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY354740): identification of two new potent, selective, and systemically active agonists for group II metabotropic glutamate receptors. J Med Chem 1999, 42: 1027–1040. 10.1021/jm980616nView Article
- Murase K, Ryu PD, Randic M: Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci Lett 1989, 103: 56–63. 10.1016/0304-3940(89)90485-0View Article
- Hevers W, Luddens H: Pharmacological heterogeneity of gamma-aminobutyric acid receptors during development suggests distinct classes of rat cerebellar granule cells in situ. Neuropharmacology 2002, 42: 34–47. 10.1016/S0028-3908(01)00158-7View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.