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Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston, Texas
Submitted 9 May 2006; accepted in final form 30 May 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Maren 2005; Phelps and Ledoux 2005; Rodrigues et al. 2004; Zald 2003). Pain not only carries a negative affective valence but is also intimately related to depression and anxiety disorders (Gallagher and Verma 2004; Rome Jr and Rome 2000). Accumulating evidence now suggests that the amygdala is an important neural substrate of the reciprocal relationship between pain and negative affect (Neugebauer et al. 2004). The present study continues and advances the analysis of pain mechanisms in the amygdala as an approach to the better understanding of the pain–affect interaction.
The amygdala constitutes several anatomically and functionally distinct nuclei. The central nucleus of the amygdala (CeA) provides the output for major amygdaloid functions through widespread connections with forebrain and brain stem areas that are involved in emotional behavior and regulate autonomic and somatomotor functions (Neugebauer et al. 2004; Par é et al. 2004; Price 2003; Rhudy and Meagher 2001). The CeA can also modulate pain behavior through multisynaptic pathways involving the basal forebrain, as well as through projections by the ventral amygdaloid pathway, to brain stem areas such as the parabrachial nuclei (PB) and periaqueductal gray (PAG) (Bourgeais et al. 2001; McGaraughty and Heinricher 2002; Neugebauer et al. 2004; Price 2003).
Within the CeA the laterocapsular part (CeLC) is now defined as the "nociceptive amygdala" because of its high content of neurons that process pain-related information (Gauriau and Bernard 2002; Neugebauer et al. 2004). The CeLC receives nociceptive inputs directly from the spino–parabrachio–amygdaloid pain pathway (Gauriau and Bernard 2002) and from spino–amygdaloid projections (Braz et al. 2005), whereas affect-related information comes from the lateral and basolateral amygdala (LA and BLA) (see Neugebauer et al. 2004). The LA represents the initial site of sensory convergence. Associative learning and plasticity in the LA–BLA circuitry is important for attaching affective content to sensory information and plays a key role in fear and anxiety (Davis 1998; Par é et al. 2004; Phelps and Ledoux 2005; Rodrigues et al. 2004; Walker and Davis 2004). Therefore the CeLC is well positioned to integrate nociceptive information with affective content and serve as the neuronal interface for the reciprocal relationship between pain and affective state (see Neugebauer et al. 2004).
Electrophysiological studies of single neurons in the CeLC at the systems level in vivo (Li and Neugebauer 2004a,b; Neugebauer and Li 2003) and in brain slices in vitro (Bird et al. 2005; Han et al. 2004; Neugebauer et al. 2003) showed that CeLC neurons develop sensitization and synaptic plasticity in persistent pain. The two principal CeLC neurons are nociceptive-specific (NS) neurons, which receive exclusively nociceptive input, and multireceptive (MR) neurons, which respond to innocuous and noxious stimuli and integrate nociceptive signals with affective information from the LA–BLA circuitry (Neugebauer et al. 2004). Importantly, it is the MR neurons, but not NS neurons, that become sensitized to afferent inputs in the arthritis pain model, which is consistent with the concept that the CeLC attaches affective valence to a noxious event. Therefore the present study focused on MR neurons.
The mechanisms of pain-related plasticity in the amygdala are not fully understood. Sensitization and synaptic plasticity in CeLC neurons depend on presynaptic group I metabotropic glutamate receptor (mGluR) upregulation (Li and Neugebauer 2004a; Neugebauer et al. 2003) and on postsynaptic N-methyl-D-aspartate (NMDA) receptor phosphorylation through protein kinase A (PKA) but not protein kinase C (PKC) (Bird et al. 2005; Li and Neugebauer 2004b). PKA activation is accomplished through postsynaptic calcitonin gene-related (CGRP1) receptors (Han et al. 2005b). G-protein–coupled mGluRs have been implicated in neuroplasticity associated with normal brain functions, but also in a variety of nervous system disorders (Bordi and Ugolini 1999; De Blasi et al. 2001; Gasparini et al. 2002; Kingston et al. 1999; Schoepp et al. 1999; Swanson et al. 2005). There is now strong evidence for an important role of mGluRs in nociception and pain (Fundytus 2001; Lesage 2004; Neugebauer 2001, 2002; Neugebauer and Carlton 2002; Varney and Gereau 2002). Eight mGluR subtypes have been cloned to date and are classified into groups I (mGluR1 and -5), II (mGluR2 and -3), and III (mGluR4, -6, -7, and -8). Group I mGluRs couple to the activation of phospholipase C, resulting in calcium release from intracellular stores and PKC activation. In contrast, mGluRs of groups II and III are negatively coupled to adenylyl cyclase, thereby inhibiting cyclic AMP (cAMP) formation and cAMP-dependent PKA activation.
The roles of mGluRs of groups II and III in nociception are less well understood than those of group I mGluRs. Activation of group II mGluRs in peripheral tissues had antinociceptive effects in models of inflammatory (Neugebauer and Carlton 2002; Yang and Gereau 2002, 2003) and neuropathic pain (Jang et al. 2004). The role of peripheral group III mGluRs remains to be determined. Activation of spinal group II and III mGluRs inhibited behavior (Dolan and Nolan 2002; Soliman et al. 2005) and central sensitization (Neugebauer et al. 2000a; Stanfa and Dickenson 1998) related to inflammatory pain. Intracisternal and systemic, but not intrathecal, administration of group II agonists inhibited formalin-induced pain behavior (Jones et al. 2005; Simmons et al. 2002). Behavioral data suggest that group II mGluRs in the periaqueductal gray (PAG) inhibit descending facilitation of pain behavior or positively modulate descending pain inhibition, whereas group III mGluRs inhibit this antinociceptive pathway (Maione et al. 2000). Activation of group II and group III mGluRs in the ventrobasal thalamus resulted in the disinhibition of nociceptive processing through the presynaptic reduction of GABAergic inhibition (Salt 2002).
The function of group II and group III mGluRs in higher brain areas in prolonged or chronic pain states remains to be determined. However, potential clinical indications particular for group II mGluR agonists include most notably anxiety disorders (Marek 2004; Swanson et al. 2005), which critically involve the amygdala. The present electrophysiological study is the first to analyze at the systems level the roles of group II and group III mGluRs in brief and prolonged nociceptive processing in the amygdala under normal conditions and in a model of persistent pain.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Animal preparation and anesthesia
On the day of the electrophysiological experiment, the animal was anesthetized with pentobarbital sodium (50 mg/kg, administered intraperitoneally). A cannula was inserted into the trachea for artificial respiration and to measure end-tidal CO2 levels. A catheter was placed in the jugular vein for continuous administration of anesthetic (see following text) and for fluid support [3–4 ml · kg–1 · h–1 lactated Ringer solution, administered intravenously (iv)]. The carotid artery was catheterized for blood pressure monitoring. Depth of anesthesia was assessed by regularly testing the corneal blink, hindpaw withdrawal, and tail-pinch reflexes; by continuously monitoring the end-tidal CO2 levels (kept at 4.0 ± 0.2%), arterial blood pressure (kept at 130 ± 5 mmHg), heart rate, and electrocardiogram (ECG) pattern; and by checking for abnormal breathing patterns. Core body temperature was maintained at 37°C by means of a homeothermic blanket system.
Animals were mounted in a stereotaxic frame, paralyzed with pancuronium (induction: 0.3–0.5 mg, iv; maintenance: 0.3 mg/h, iv) and artificially ventilated (3–3.5 ml; 55–65 strokes/min). Constant levels of anesthesia were maintained by continuous iv infusion of pentobarbital (15 mg · kg–1 · h–1). A unilateral craniotomy was performed at the sutura frontoparietalis level for recording of CeLC neurons and for administration of drugs into the CeLC contralateral to the knee joint in which the arthritis was induced. Our previous studies showed that multireceptive CeLC neurons with input from the contralateral knee become sensitized after induction of arthritis in that knee (Li and Neugebauer 2004a,b; Neugebauer and Li 2003). The dura mater was opened and reflected; the pia mater was removed over the recording and drug administration sites to allow smooth insertion of the recording electrode and microdialysis probe, respectively.
Electrophysiological recording and identification of amygdala neurons
As previously described (Li and Neugebauer 2004a,b; Neugebauer and Li 2002, 2003) long-term extracellular recordings were made from single neurons in the CeLC with glass-insulated carbon-filament electrodes (4–6 M
) using the following stereotaxic coordinates (Paxinos and Watson 1998): 2.1–2.8 mm caudal to bregma; 3.8–4.2 mm lateral to midline; depth of 7,000–9,000 µm. The recorded signals were amplified and displayed on analog and digital storage oscilloscopes. Signals were also fed into a window discriminator, whose output was processed by an interface (CED 1401) connected to a Pentium 4 PC. Spike2 software (CED, version 3) was used to create peristimulus rate histograms on-line and to store and analyze digital records of single-unit activity off-line. Spike size and configuration were continuously monitored on the storage oscilloscopes and with the use of Spike2 software (see individual examples in Figs. 1 and 4). Once an individual CeLC neuron was identified by its background activity and/or responses to mechanical stimuli (see following text), we optimized spike size, searched carefully for a receptive field in the knee joint(s), and determined both size and threshold of its total receptive field in the deep tissue and skin. In this study we included only multireceptive (MR) neurons (see CLASSIFICATION OF CELC NEURONS AND RESPONSE THRESHOLDS), because they consistently and reliably become sensitized in the arthritis pain model (Li and Neugebauer 2004a; Li and Neugebauer 2004b; Neugebauer and Li 2003).
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Experimental protocol
RECEPTIVE FIELDS. Neurons were selected that had a receptive field in the knee. Size and thresholds of the receptive fields in deep tissue and skin were mapped using graded mechanical stimuli of innocuous and noxious intensities (see following text). Mechanical stimuli were considered to activate deep tissue (joints, muscles) if the stimulation of overlying skin evoked no or a clearly distinct response. Cutaneous input was distinguished from deep tissue input by selective stimulation of skin folds gently raised from the underlying deep tissue. The focus of this study was on the processing of nociceptive information from the deep tissue.
RESPONSE THRESHOLDS. Mechanical stimuli of gradually increasing intensity (steps of 50 g/30 mm2) were applied to the deep tissue (knee joint, ankle joint, and muscles) by means of a forceps with a force transducer, whose calibrated output was amplified and displayed (in g) on an LCD screen. The output signal was also fed into the CED interface and recorded on the Pentium 4 PC for on- and off-line analysis. The hindlimbs, including knee and ankle, were readily accessible to compression of the medial and lateral aspects with the forceps. The mechanical threshold was defined as the minimum stimulus intensity that evoked an excitatory response (spike frequency >upper 95% confidence interval of background activity) or an inhibitory response (spike frequency <lower 95% confidence interval of background activity). Only responses that were distinctly evoked by stimulation of deep tissue, but not of the skin, were included in the analysis of information processing from the deep tissue.
CLASSIFICATION OF CELC NEURONS AND RESPONSE THRESHOLDS.
All neurons selected for this study were multireceptive (MR) neurons according to our classification of CeLC neurons with deep tissue input (Neugebauer and Li 2002, 2003). MR neurons have been shown to develop increased responsiveness ("sensitization") in the arthritis pain model (Li and Neugebauer 2004a,b; Neugebauer and Li 2003). Our classification is primarily based on the neurons' responses to mechanical stimulation of the knee joint and other deep tissue. MR neurons respond consistently to low-intensity stimuli (<500 g/30 mm2) but are more strongly activated by noxious stimuli (>1,500 g/30 mm2). Stimulus intensities of 100–500 g/30 mm2 applied to the knee and other deep tissue are considered innocuous because they do not evoke hindlimb withdrawal reflexes in awake rats and are not felt to be painful when tested on the experimenters. An intensity of 1,000 g/30 mm2 represents a firm but nonpainful stimulus that does not evoke a hindlimb withdrawal reflex. Pressure stimuli >1,500 g/30 mm2 are noxious because they evoke hindlimb withdrawal reflexes and vocalizations in awake rats and are distinctly painful when applied to the experimenters (Han et al. 2005a; Neugebauer and Li 2002, 2003).
EXPERIMENTAL PROTOCOL AND TEST STIMULI. Background activity and responses to brief (15-s) graded mechanical stimuli of increasing intensity were measured before and during the development of the arthritis and before and during drug application. Stimulus–response relationships were measured by applying graded mechanical test stimuli of 100 and 500–2,500 g/30 mm2 intensity in increments of 500 g/30 mm2 (15-s duration each; 15-s intervals). Mechanical stimuli were applied with the calibrated forceps (see above). The test stimuli were applied to knee joint and at least one other area of the receptive field (typically the ankle and/or contralateral knee) to compare processing of input from arthritic (knee) and noninjured (ankle) tissue. Number of stimulations was kept at a minimum to avoid any "sensitization" that might be produced by repeated stimulation. Sufficiently long control periods were included in each experiment (see Figs. 2 and 5) to establish the baseline responses before drug application and/or arthritis induction.
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,b; Neugebauer and Li 2002, 2003). Arthritis
In each experiment, one CeLC neuron was recorded before and for several hours after the induction of arthritis in one knee joint as in our previous studies using this pain model (Li and Neugebauer 2004a,b; Neugebauer and Li 2003). Background activity, evoked responses, and receptive field size had to be stable for 
2 h before the arthritis was induced. Throughout the experiment we carefully monitored several physiological parameters (body temperature, blood pressure, heart rate, ECG, end-tidal CO2 levels) to ensure a stable recording situation (see Animal preparation and anesthesia above).
Arthritis was induced as previously described in detail (Li and Neugebauer 2004a,b; Neugebauer and Li 2003). A kaolin suspension (4%, 100 µl) was slowly injected into the joint cavity through the patellar ligament with the use of a syringe and needle (1 ml, 25G, 5/8-in.). After repetitive flexions and extensions of the knee for 15 min, a carrageenan solution (2%, 100 µl) was injected into the knee joint cavity and the leg was flexed and extended for another 5 min. This treatment paradigm reliably leads to inflammation and swelling of the knee within 1–3 h and persists for weeks (Min et al. 2001; Neugebauer and Li 2003).
Drugs
The following selective receptor agonists and antagonists were used (Han et al. 2004; Neugebauer 2001; Schoepp et al. 1999): L-(+)-2-amino-4-phosphonobutyrate (LAP4; group III agonist); alpha-methyl-4-phosphonophenylglycine (UBP1112; group III antagonist); (S)-alpha-ethylglutamic acid (EGLU; group II antagonist), all purchased from Tocris Bioscience (Ellisville, MO). (+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740; group II agonist) was a generous gift from Eli Lilly. Drug applications were 15 min (usually 20 min) to establish equilibrium in the tissue.
Drug application
Known concentrations of group II and group III agonists and antagonists were administered into the CeLC by microdialysis before and/or 5–6 h postinduction of arthritis. Maximum changes of response behavior occur after 5–6 h postinduction of arthritis when the sensitization process reaches a plateau. Several hours before the start of the electrophysiological recordings a microdialysis probe (CMA11; CMA/Microdialysis; membrane diameter: 250 µm, membrane length: 1 mm) was lowered vertically into the CeA and positioned stereotaxically anterior and ipsilateral to the recording electrode, using the following coordinates: 1.6 mm caudal to bregma; 4.0 mm lateral to midline; depth of tip 9.0 mm (Li and Neugebauer 2004a,b). The distance between microdialysis probe and recording electrode was 0.5–1.0 mm. Using PE-50 tubing, the microdialysis probe was connected to an infusion pump (Harvard) and perfused with artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 125.0, KCl 2.6, NaH2PO4 2.5, CaCl2 1.3, MgCl2 0.9, NaHCO3 21.0, and glucose 3.5; oxygenated and equilibrated to pH = 7.4. Before each drug application, ACSF was pumped through the fiber for 1 h to establish equilibrium in the tissue (Li and Neugebauer 2004a,b).
Drugs were dissolved in ACSF on the day of the experiment at a concentration 100-fold that predicted to be needed based on data from in vitro studies (Han et al. 2004; Neugebauer et al. 2000b) as described before (Neugebauer et al. 2000a). The concentration gradient across the dialysis membrane is 10:1 for these molecules (Neugebauer et al. 2000a). The concentration is reduced by another order of magnitude as a result of diffusion in the tissue (Li and Neugebauer 2004a,b).
Drugs were administered into the CeLC at a rate of 5 µl/min for 15–30 min to establish equilibrium in the tissue. Different concentrations were administered in a cumulative fashion. When concentrations were tested individually in some experiments, no difference was found compared with the cumulative concentration–response data. Drug effects on background and evoked activity were measured every 5–10 min during drug application. The numbers given in this article refer to the drug concentrations in the microdialysis fiber. ACSF served as a vehicle control.
Histology
At the end of each experiment the recording site in the CeLC was marked by injecting DC (250 µA for 3 min) through the carbon-filament recording electrode. The brain was removed and submerged in 10% formalin and potassium ferrocyanide. Tissues were stored in 20% sucrose before they were frozen, sectioned at 50 µm. Sections were stained with Neutral Red, mounted on gel-coated slides, and coverslipped. The boundaries of the different amygdala nuclei were easily identified under the microscope. Lesion/recording sites were verified histologically and plotted on standard diagrams adapted from Paxinos and Watson (1998) (see Figs. 1, 2, 4, and 5).
Data analysis
Extracellularly recorded single-unit action potentials were analyzed off-line from peristimulus rate histograms using Spike2 software (CED, version 3). Responses to mechanical stimuli were measured and expressed as spikes per second (Hz). Background activity, if present, was subtracted from the total activity during the stimulus. Concentration–response relationships were measured for each neuron and then averaged across a sample of neurons. IC50 values and maximum effects were calculated from sigmoid curves fitted to the cumulative concentration–response data by nonlinear regression using the formula y = A + (B – A)/[1 + (10C/10X)D], where A = bottom plateau, B = top plateau, C = log (IC50), D = slope coefficient (Prism 3.0, GraphPad Software). Concentration–response functions under normal conditions and in arthritis were compared statistically using a two-way ANOVA followed by Bonferroni posttests (GraphPad Prism 3.0). Effects of antagonists alone and in combination with agonists were compared with predrug control values using the paired t-test. All averaged values are given as the means ± SE. Statistical significance was accepted at the level P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Extracellular single-unit recordings were made from 60 neurons in the laterocapsular division of the central nucleus of the amygdala (CeLC) in anesthetized rats (recording sites are shown in Fig. 9). All CeA neurons in this study were multireceptive (MR) neurons, which responded significantly to innocuous but more strongly to noxious stimuli (see METHODS, CLASSIFICATION OF CELC NEURONS AND RESPONSE THRESHOLDS). This study focused on MR neurons because they represent the class of CeLC neurons that consistently become sensitized to afferent inputs in the arthritis pain model (Neugebauer et al. 2004). In agreement with our previous studies (Li and Neugebauer 2004a,b; Neugebauer and Li 2003) all MR neurons developed enhanced background activity and responses to stimulation of the knee and ankle after induction of the localized monoarthritis in the knee (see examples in Figs. 1, 2, 4, and 5). Figures 1 and 4 also display extracellularly recorded action potentials of individual CeLC neurons in response to mechanical stimulation of the knee to illustrate that single-unit activity was recorded and that the recording conditions remained constant during these long-term experiments.
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A selective group III mGluR agonist (LAP4; Han et al. 2004; Neugebauer 2001; Schoepp et al. 1999; see METHODS) was tested under normal conditions (no arthritis) in 14 neurons and 5–6 h postinduction of arthritis in seven neurons. Maximum changes of responsiveness occur after 5–6 h postinduction of arthritis when the sensitization process reaches a plateau that lasts at least until 18 h postinduction (Li and Neugebauer 2004a,b; Neugebauer and Li 2003). In four of these neurons, drug effects were assessed before and after induction of arthritis in the same neuron; in three neurons drugs were tested only in the arthritic state as a control for any pretreatment effect. Because no difference was found these data were pooled.
Figure 1 shows an individual example. In the control period before arthritis induction (Fig. 1A), this multireceptive (MR) CeLC neuron responded with increasing magnitude to graded mechanical stimulation of the knee in the innocuous (100 and 500 g/30 mm2) and noxious range (1,500–2,500 g/30 mm2; see METHODS). Administration of LAP4 (100 µM, concentration in the microdialysis probe; 20 min) into the CeLC inhibited the responses to innocuous and noxious stimuli. The activity of the same neuron was continuously recorded during development of the knee joint arthritis (see METHODS). Size, shape, and configuration of action potentials ("spikes") were closely monitored to ensure that activity from only one and the same neuron was recorded (see individual spikes in Fig. 1, A and B). In the arthritis pain state (6 h postinduction; Fig. 1B) background activity and evoked responses of this neuron had increased. LAP4 (100 µM) diminished the increased activity. The histologically verified sites of the microdialysis probe and recording electrode in the CeLC are illustrated in Fig. 1, C and D (see METHODS).
The time course of arthritis- and drug-induced changes in one individual CeLC neuron is shown in Fig. 2A. LAP4 (100 µM) inhibited the responses of this neuron to innocuous (500 g/30 mm2) and noxious (2,000 g/30 mm2) stimulation of the knee in the control period before induction of the arthritis. Responses to stimulation of the arthritic knee increased after arthritis induction. In agreement with our previous studies (Li and Neugebauer 2004a,b; Neugebauer and Li 2003; Neugebauer et al. 2004) there was also an increased responsiveness to stimulation of noninjured parts of the body such as ankle (data not shown), indicating central sensitization. A lower concentration of LAP4 (10 µM) strongly reduced the enhanced responses. A higher concentration (100 µM) further reduced or reversed the increased responses to noxious and innocuous stimuli, respectively. Each symbol represents the response (number of action potentials/s) during stimulation. If present, background activity preceding the stimulus was subtracted from the total activity during stimulation. Positions of the microdialysis probe and recording electrode in the CeLC were verified histologically (Fig. 2, B and C; see METHODS).
Concentration–response data in Fig. 3 summarize the effects of LAP4. Under control conditions, LAP4 concentration-dependently inhibited the responses of CeLC neurons (n = 14) to innocuous (Fig. 3A, 500 g/30 mm2) and noxious (Fig. 3B, 2,000 g/30 mm2) stimulation of the knee and ankle and background activity (Fig. 3C). In the arthritis pain state (5–6 h postinduction; n = 7), LAP4 became more potent in inhibiting evoked activity (Fig. 3, A and B), but not background activity (Fig. 3C), as evidenced by the leftward shift of cumulative concentration–response curves. Statistical analysis (two-way ANOVA; see METHODS) revealed significant changes of LAP4 effects in arthritis compared with normal controls before arthritis [Fig. 3A, innocuous knee, P < 0.0001, F(1,62) = 21.22; innocuous ankle, P < 0.001, F(1,61) = 15.02; Fig. 3B, noxious knee, P < 0.001, F(1,62) = 15.93; noxious ankle, P < 0.01, F(1,62) = 8.96].
The potency of LAP4 increased six- to eightfold (ankle input) and 12- to 24-fold (knee input) in the arthritis pain state. This is reflected in the change of IC50 values for inhibiting responses to innocuous stimulation of the knee (2.8 µM, arthritis; 24.0 µM, normal) and ankle (5.0 µM, arthritis; 43.1 µM, normal) as well as noxious stimulation of the knee (2.5 µM, arthritis; 60.2 µM, normal) and ankle (8.4 µM, arthritis; 71.2 µM, normal). These values refer to drug concentrations in the microdialysis probes (see METHODS). IC50 values were calculated from sigmoid curves fitted to the cumulative concentration–response data by nonlinear regression using the formula y = A + (B – A)/[1 + (10C/10X)D], where A = bottom plateau, B = top plateau, C = log (IC50), D = slope coefficient (Prism 3.0, GraphPad Software; see METHODS).
These data suggest a significant increase in potency of a group III mGluR agonist in the CeLC in the arthritis pain model. Changes were more pronounced for the inhibition of responses to stimulation of the knee than to that of the ankle.
Effects of group II mGluR activation
A selective group II mGluR agonist (LY354740; Neugebauer 2001; Schoepp et al. 1999; see METHODS) was tested under normal conditions (no arthritis) in 12 neurons and 5–6 h postinduction of arthritis in 10 neurons. In four of these neurons, drug effects were assessed before and after induction of arthritis in the same neuron; in eight neurons drugs were tested only in the arthritic state as a control for any pretreatment effect. Because no difference was found these data were pooled.
Figure 4 shows an individual example. In the control period before arthritis induction (Fig. 4A), this multireceptive (MR) CeLC neuron responded with increasing magnitude to graded mechanical stimulation of the knee. Administration of LY354740 (100 µM, concentration in the microdialysis probe; 20 min) into the CeLC inhibited the evoked responses. Arthritis was induced in one knee and the activity of the same neuron was continuously recorded during development of the arthritis (see METHODS). Size, shape, and configuration of action potentials ("spikes") were closely monitored to ensure that activity from only one and the same neuron was recorded (see individual spikes in Fig. 4, A and B). In the arthritis pain state (6 h postinduction; Fig. 4B) LY354740 (100 µM) reversed the increased responses to the prearthritis control levels. The histologically verified sites of the microdialysis probe and recording electrode in the CeLC are illustrated in Fig. 4, C and D (see METHODS).
The time course of arthritis- and drug-induced changes in one individual CeLC neuron is shown in Fig. 5A. LY354740 (100 µM) inhibited the responses of this neuron to innocuous and stimulation of the knee in the control period before induction of the arthritis. In agreement with our previous studies (Li and Neugebauer 2004a,b; Neugebauer and Li 2003; Neugebauer et al. 2004) responses to stimulation of the arthritic knee and noninjured parts of the body such as ankle increased, indicating central sensitization. After arthritis induction, a lower concentration of LY354740 (10 µM) reduced the enhanced responses to noxious stimuli but had less effect during stimulation of the knee with normally innocuous intensity. A higher concentration of LY354740 (100 µM) reversed the arthritis-related increased responses to noxious and (normally) innocuous stimuli. Each symbol represents the response (number of action potentials/s) during stimulation. If present, background activity preceding the stimulus was subtracted from the total activity during stimulation. Positions of the microdialysis probe and recording electrode in the CeLC were verified histologically (Fig. 5, B and C; see METHODS).
Concentration–response data in Fig. 6 summarize the effects of LY354740. Under control conditions, LY354740 concentration-dependently inhibited the responses of CeLC neurons (n = 12) to innocuous (Fig. 6A) and noxious (Fig. 6B) stimulation of the knee and ankle and background activity (Fig. 6C). In the arthritis pain state (5–6 h postinduction; n = 10), LY354740 became more potent in inhibiting the responses to noxious stimulation of the arthritic knee (Fig. 6B). The leftward shift of cumulative concentration–response curve was statistically significant [P < 0.001, F(1,69) = 7.91; two-way ANOVA; see METHODS]. In contrast, the potency of LY354740 for inhibiting responses to innocuous stimulation of the knee and innocuous and noxious stimulation of the ankle and background activity did not change significantly in arthritis (P > 0.05; two-way ANOVA; see METHODS; Fig. 6, A–C).
The potency of LY354740 for inhibiting responses to noxious stimulation of the arthritic knee increased about 10-fold (IC50 = 0.88 µM, arthritis; 8.1 µM, normal). The apparent IC50 values did not change for inhibiting responses to innocuous stimulation of the knee (IC50 = 14.0 µM, arthritis; 8.0 µM, normal), innocuous stimulation of the ankle (8.9 µM, arthritis; 10.9 µM, normal), noxious stimulation of the ankle (14.3 µM, arthritis; 20.0 µM, normal), and background activity (10.5 µM, arthritis; 17.0 µM, normal). These values refer to drug concentrations in the microdialysis probes (see METHODS).
These data show differential changes in the potency of a group II mGluR agonist in the CeLC in the arthritis pain model. Potency increased significantly for noxious stimulation of the arthritic knee, which represents the strongest input to the CeLC in the arthritis pain model. The potency of LY354740 for inhibiting the responses to stimulation of the nonarthritic ankle and innocuous stimulation of the knee did not change significantly, suggesting input- and activity-dependent modulation of group II mGluR function.
Effects of group III mGluR inhibition
To determine any endogenous receptor activation, we tested the effects of a selective group III mGluR antagonist (UBP1112; Han et al. 2004; Neugebauer 2001; Schoepp et al. 1999; see METHODS) under normal conditions (no arthritis) and 5–6 h postinduction of arthritis. Figure 7, A and B shows that administration of UBP1112 (100 µM, concentration in the microdialysis probe; 15–20 min) into the CeLC significantly increased the responses to normally innocuous (500 g/30 mm2) and noxious (2,000 g/30 mm2) stimulation of the knee and ankle in arthritis (n = 5 neurons; P < 0.01–0.05, paired t-test) but had no effect under normal conditions (n = 8 neurons). UBP1112 (100 µM) also significantly reversed the effects of LAP4 when coadministered with the agonist (Fig. 8A) in normal animals (n = 4 neurons). These data suggest that group III mGluRs are endogenously activated in arthritis.
Effects of group II mGluR inhibition
A selective group II mGluR antagonist (EGLU; Neugebauer 2001; Schoepp et al. 1999; see METHODS) was tested under normal conditions (no arthritis) and 5–6 h postinduction of arthritis. Administration of EGLU (100 µM, concentration in the microdialysis probe; 15–20 min) into the CeLC significantly increased the responses to normally innocuous (500 g/30 mm2) and noxious (2,000 g/30 mm2) stimulation of the knee, but not ankle, in arthritis (n = 5 neurons; P < 0.01–0.05, paired t-test) but had no effect under normal conditions (n = 9 neurons; Fig. 7, C and D). EGLU (100 µM) also significantly reversed the effects of LY354740 (100 µM) when coadministered with the agonist (Fig. 8B) in normal animals (n = 4 neurons). These data suggest that group II mGluRs are endogenously activated during stimulation of the arthritic knee but not of noninjured and/or normal tissue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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,b; Neugebauer and Li 2003) all multireceptive neurons in the laterocapsular part of the central nucleus of the amygdala (CeLC) developed enhanced responses to nociceptive afferent input ("sensitization"). 2) Activation of group II and group III mGluRs with LY354740 and LAP4, respectively, had a general inhibitory effect on the responses to innocuous and noxious stimulation under normal conditions. 3) The potency of LAP4 increased for the inhibition of all evoked responses in the arthritis pain model. 4) The potency of LY354740 increased selectively for the inhibition of responses to noxious stimulation of the arthritic knee but did not change for stimulation of the uninjured ankle and innocuous stimulation of the knee. 5) A group III antagonist (UBP1112) blocked the effect of LAP4 and increased all evoked responses in the arthritis pain model but not under normal conditions. 6) A group II antagonist (EGLU) inhibited the effect of LY354740 and increased only the responses to noxious stimulation of the arthritic knee. These data suggest that the arthritis pain state induces an activity- and input-dependent increase of group II mGluR function and activation and an overall increase of group III mGluR-mediated effects. Low concentrations of group II agonists may thus be useful in selectively targeting pain-related activity changes in the amygdala.
The role of group II and group III mGluRs in nociceptive processing is less well understood than that of group I mGluRs (Fundytus 2001; Lesage 2004; Neugebauer 2001, 2002; Neugebauer and Carlton 2002; Varney and Gereau 2002). Activation of group II mGluRs in peripheral tissues by 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC) had antinociceptive effects in models of inflammatory (Neugebauer and Carlton 2002; Yang and Gereau 2002, 2003) and neuropathic pain (Jang et al. 2004). However, APDC did not alter mechanical and thermal sensitivity in normal animals (Yang and Gereau 2002, 2003). The role of peripheral group III mGluRs remains to be determined.
Activation of spinal group III mGluRs with LAP4 inhibited formalin- and capsaicin-induced pain behavior, whereas spinally administered group II agonists such as LY379268 were antinociceptive in the capsaicin model but not in the formalin test (Dolan and Nolan 2002; Jones et al. 2005; Neugebauer and Carlton 2002; Simmons et al. 2002; Soliman et al. 2005; Varney and Gereau 2002). In the absence of tissue injury, activation of spinal group II or group III mGluRs appears to have no antinociceptive behavioral effects (Neugebauer 2001, 2002; Soliman et al. 2005; Varney and Gereau 2002). In electrophysiological studies, spinal administration of group II mGluR agonists such as LY379268 inhibited carrageenan- and capsaicin-induced central sensitization of dorsal horn neurons including spinothalamic tract cells but had no or mixed effects under normal conditions (Neugebauer et al. 2000a; Stanfa and Dickenson 1998). In contrast, LAP4 inhibited the responses of spinothalamic tract cells to brief innocuous and noxious under normal conditions as well as in capsaicin-induced central sensitization (Neugebauer et al. 2000a). Activation of group III mGluRs with LAP4 and of group II mGluRs with a nonselective agonist [LCCG, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine] inhibited synaptic transmission in the dorsal horn in spinal cord slices from normal animals (Gerber et al. 2000). These rather heterogeneous findings suggest that the spinal cord may not be the optimum target for therapeutic agents that act through increasing the inhibitory functions of group II and group III mGluRs for pain relief.
At supraspinal sites, activation of group II and group III mGluRs in the periaqueductal gray (PAG) facilitates pain behavior under normal conditions, whereas in the formalin test group II mGluRs are antinociceptive and group III mGluRs increase pain behavior through the modulation of descending inhibition or facilitation (Berrino et al. 2001; Maione et al. 1998, 2000). Agonists of group II mGluRs (LY354740 and APDC) and group III mGluRs (LAP4) in the thalamus had presynaptic disinhibitory effects under normal conditions, decreasing the GABAergic inhibitory transmission from the thalamic reticular nucleus to the ventrobasal thalamus (Salt 2002). The function of group II and group III mGluRs in higher brain areas in prolonged or chronic pain states is largely unknown. Administration of a group II antagonist (EGLU) in the thalamic reticular nucleus produced antinociceptive effects in monoarthritic rats, presumably by blocking the activation of group II mGluRs and their disinhibitory effect on thalamic relay neurons (Neto and Castro-Lopes 2000).
The diverse effects of inhibitory group II and group III mGluRs in the spinal cord and the potentially opposite (pronociceptive) effects in the brain stem and thalamus formed part of the rationale of this study to determine the function of these receptors in the amygdala, a brain area that is emerging as a neuronal interface between pain and affective disorders such as anxiety (Neugebauer et al. 2004). The nociceptive amygdala (CeLC) integrates affect-related information from the lateral–basolateral amygdala (LA and BLA) with subcortical nociceptive inputs from the spino–parabrachio–amygdaloid pain pathway (Gauriau and Bernard 2002) and from spino–amygdaloid projections (Braz et al. 2005). Associative learning and plasticity in the LA–BLA circuitry plays a key role in affective states and disorders such as fear and anxiety (Davis 1998; Paré et al. 2004; Phelps and Ledoux 2005; Rodrigues et al. 2004; Walker and Davis 2004). Importantly, anxiety disorders represent one of the major clinical indications for group II mGluR agonists and potentiators (Marek 2004; Swanson et al. 2005). Preclinical and clinical studies suggest that these compounds, including LY354740, have beneficial effects in generalized anxiety disorder or panic disorder and that the amygdala may be an important site of drug action (Grillon et al. 2003; Walker et al. 2002).
This study analyzed the function of group II and group III mGluRs in the amygdala in normal nociceptive processing and in a model of arthritic pain. Our previous studies showed plastic changes in the CeLC in an arthritis pain model (Bird et al. 2005; Li and Neugebauer 2004a,b; Neugebauer and Li 2003; Neugebauer et al. 2003). Plasticity was measured as increased synaptic transmission in the nociceptive parabrachio–amygdaloid pathway, enhanced responsiveness to incoming signals (sensitization), and increased neuronal excitability of CeLC neurons. These plastic changes were predominantly observed in so-called multireceptive (MR) neurons, which are activated more strongly by noxious than innocuous stimuli so that they encode and distinguish nociceptive and nonnociceptive information. MR neurons are believed to be a site of convergence and integration of nociceptive and polymodal inputs in the CeA (Neugebauer et al. 2004). Mechanisms of pain-related synaptic plasticity include the enhanced presynaptic release of glutamate through group I mGluRs (particularly the mGluR1 subtype) (Li and Neugebauer 2004a; Neugebauer et al. 2003). Glutamate acts postsynaptically to activate N-methyl-D-aspartate (NMDA) receptors, which are "silent" under normal conditions but become functional through receptor phosphorylation by PKA but not PKC (Bird et al. 2005). Change in NMDA rather than 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function contributes to synaptic plasticity and processing of pain-related information (Bird et al. 2005; Li and Neugebauer 2004b). Endogenous CGRP released from the parabrachio–amygdaloid tract activates postsynaptic CGRP1 receptors coupled to PKA, but not PKC, to increase NMDA, but not AMPA, receptor function (Han et al. 2005b).
The present study shows that exogenous activation of inhibitory group II and group III mGluRs can inhibit the responses of sensitized MR neurons in a model of arthritic pain. Importantly, the potency of agonists for group II and group III mGluRs increased in arthritis. The increased sensitivity of group II mGluRs appeared to be input- and activity-dependent, whereas group III mGluRs showed a rather general change. Likewise, group II and group III mGluRs showed a different profile for endogenous activation measured with antagonists. Group II mGluRs were activated only by stimulation of the arthritic knee, likely providing the strongest input to the amygdala. Group III mGluRs, however, were activated during stimulation the knee and noninjured ankle.
These differences may be a result of the differential subcellular distribution of group II and group III receptors. Anatomical data suggest that group II mGluRs are localized peri- and extrasynaptically and thus may require high levels of glutamate for activation (Lujan et al. 1997; Shigemoto et al. 1997). On the other hand, group III mGluRs can be found in (mGluR7) or near (mGluR6 and -8) the active zone of the synapse, making them more readily accessible to glutamate (Cartmell and Schoepp 2000; Corti et al. 2002; Ferraguti et al. 2005; Kinoshita et al. 1998; Shigemoto et al. 1997; Swanson et al. 2005). LAP4 activates mGluR4, -6, and -8 with low micromolar potency, whereas mGluR7 requires two to three orders of magnitude higher concentrations (Anwyl 1999
