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Department of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
Submitted 20 May 2003; accepted in final form 16 September 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Cahill 1999; Cardinal et al. 2002; Davidson 2002; Davis 1998; Gallagher and Schoenbaum 1999; LeDoux 2000; Rasia-Filho et al. 2000; Rolls 2000; Zald 2003). The amygdala is composed of several anatomically and functionally distinct nuclei. Particularly the lateral and basolateral amygdaloid nuclei (LA and BLA) have been shown to exhibit a high degree of plasticity in various models of long-term synaptic and behavioral modification (Blair et al. 2001; Keele et al. 2000; LeDoux 2000; Lin et al. 2000; Neugebauer et al. 1997; Zinebi et al. 2001).
The central nucleus of the amygdala (CeA) provides the output pathway for major amygdala functions, acting as a "controller of the brain stem" to regulate emotional responses (Cardinal et al. 2002). Electrophysiological changes have been recorded in CeA neurons during fear conditioning (Pascoe and Kapp 1985), but it is not clear yet if the critical plasticity underlying fear conditioning resides in the CeA and/or the lateral and basolateral nuclei (Fanselow and LeDoux 1999; Goosens and Maren 2001). More recently, synaptic plasticity has been shown in the CeA in the kindling model of epilepsy (Neugebauer et al. 2000b) and the chronic cocaine model of drug addiction (Neugebauer et al. 2000b).
Accumulating evidence now also implicates the amygdala in pain processing and pain-related neuroplasticity. Pain has a strong emotional component, and arthritis pain in particular is significantly associated with negative affective states such as depression and anxiety (Huyser and Parker 1999). The relationship between pain and depression is reciprocal (Ohayon and Schatzberg 2003; Wilson et al. 2001), and although not fully understood, this interaction is likely to be mediated through circuits involving the amygdala (McGaraughty and Heinricher 2002; Meagher et al. 2001; Rhudy and Meagher 2000). In fact, the latero-capsular part of the CeA is now defined as the "nociceptive amygdala" because of its high content of nociceptive neurons (Bourgeais et al. 2001; Gauriau and Bernard 2002; Neugebauer and Li 2002, 2003). The laterocapsular CeA may regulate pain and related behavior through multisynaptic pathways involving the basal forebrain, most notably the substantia innominata dorsalis, rather than through direct brain stem projections, including the parabrachial nuclei and periaqueductal gray (Bourgeois et al. 2001). Our recent electrophysiological in vivo and in vitro studies further showed that two major subpopulations of CeA neurons, so-called multireceptive (MR) neurons and nonresponsive (noSOM) neurons, but not nociceptive-specific (NS) neurons, exhibit substantial nociceptive and synaptic plasticity in a model of persistent arthritic pain (Neugebauer and Li 2003; Neugebauer et al. 2003).
The mechanisms of neuroplasticity in the CeA are not yet fully understood. Our previous electrophysiological recordings of CeA neurons in brain slices in vitro suggest an important role for group I metabotropic glutamate receptors (mGluRs) in the arthritis pain model (Neugebauer et al. 2003). Eight mGluR subtypes have been cloned to date and are classified into groups I (mGluR1,5), II (mGluR2,3), and III (mGluR4,6,7,8). Group I mGluRs couple through Gq/11 proteins to the activation of phospholipase C, resulting in phosphoinositide hydrolysis, release of calcium from intracellular stores, and protein kinase C activation. Group I mGluR activation can also result in stimulation of cyclic AMP (cAMP) formation, increased cyclic GMP (cGMP) accumulation, activation of phospholipase A2, and tyrosine phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, also referred to as extracellular signal-regulated kinases (ERK1/2) (Anwyl 1999; Conn and Pin 1997; De Blasi et al. 2001; Neugebauer 2002; Schoepp et al. 1999). Extensive research has implicated mGluRs in the neuroplasticity associated with normal brain functions and also in various neurological and psychiatric disorders (Bordi and Ugolini 1999; Gasparini et al. 2002; Kingston et al. 1999; Nicoletti et al. 1996; Spooren et al. 2001). There is now strong evidence that suggests an important role of mGluRs in nociception and pain (Fundytus 2001; Neugebauer 2002; Neugebauer and Carlton 2002; Varney and Gereau 2002).
Although mGluR1, but not mGluR5, is a key player in synaptic plasticity in the CeA in vitro (Neugebauer et al. 2003), the role of these group I subtypes in the processing of sensory, and in particular, nociceptive information in the amygdala, is not yet known. The present electrophysiological study is the first to analyze at the system level the differential roles of mGluR1 and mGluR5 subtypes in individual amygdala neurons in the processing of innocuous and noxious sensory information under normal conditions and in pain-related sensitization.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Adult male Sprague-Dawley rats (200–350 g) were anesthetized with pentobarbital sodium (50 mg/kg, ip). 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 next paragraph) and for fluid support (3–4 ml/kg/h lactated ringer solution, 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 135 ± 5 mmHg), heart rate, and electrocardiogram (ECG) pattern; and by checking for abnormal breathing patterns. Core body temperature was measured with a rectal thermometer and 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 intravenous infusion of pentobarbital (15 mg/kg/h). A unilateral craniotomy was performed at the sutura fronto-parietalis level for the recording of CeA neurons in the amygdala contralateral to the knee joint in which the arthritis was induced. Nociceptive information reaches the CeA through the spino-parabrachio-amygdaloid pain pathway, which originates from lamina I neurons in the contralateral spinal dorsal horn (Gauriau and Bernard 2002). Accordingly, our previous study showed that multireceptive CeA neurons with input from the contralateral knee become sensitized after induction of arthritis in that knee (Neugebauer and Li 2003). The dura mater was opened and reflected; the pia mater was removed over the recording site to allow smooth insertion of the recording electrode.
Electrophysiological recording and identification of amygdala neurons
As described previously (Neugebauer and Li 2003) long-term extracellular recordings were made from single neurons in the CeA with glass insulated carbon filament electrodes (3–5 M
) using the following stereotaxic coordinates (Paxinos and Watson 1998): 2.1–3.3 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 III 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. Once an individual CeA neuron was identified by its background activity and/or responses to nonnoxious mechanical stimuli (see Experimental protocol), we optimized spike size, searched carefully for a receptive field in the knee joint(s), and determined size and threshold of its total receptive field in the deep tissue and skin. In this study we included only MR neurons, because they consistently and reliably become sensitized in the arthritis pain model (Neugebauer and Li 2003).
Configuration, shape, and height of the recorded action potentials were monitored and recorded continuously, using a window discriminator and Spike2 software for on-line and off-line analysis. Only those neurons were included in this study whose spike configuration remained constant and could be clearly discriminated from activity in the background throughout the experiment, indicating that the activity from one neuron only and from the same one neuron was measured (see examples in Figs. 2, 3, 5, 6, and 8).
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Background activity of the neuron was recorded for 
10 min to calculate means ± SE and 95% confidence intervals (CIs), using Prism 3.0 software (GraphPad Software, San Diego, CA). Size and thresholds of the receptive fields in deep tissue and skin were mapped. 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.
Response thresholds for mechanical stimulation of the knee joint and other deep tissue (e.g., ankle joint and muscles) were determined as follows: mechanical stimuli of gradually increasing intensity (steps of 50 g/30 mm2) were applied to the deep tissue (joints and muscles) by means of a forceps with a force transducer, whose calibrated output was amplified and displayed in g on a LCD screen. The output signal was also fed into the CED interface and recorded on the Pentium III PC for on- and off-line analysis. The mechanical threshold was defined as the minimum stimulus intensity that evoked an excitatory response (spike frequency higher than the upper 95% CI of background activity) or an inhibitory response (spike frequency less than the lower 95% CI of background activity). The threshold stimulus intensity was tested again three times to verify the presence of a response in 50% of trials. Only responses that were distinctly evoked by selective stimulation of deep tissue were included in the analysis of information processing from the deep tissue. Stimulus–response relationships were measured by applying graded mechanical test stimuli of 100 and 500–3,000 g/30 mm2 intensity in increments of 500 g/30 mm2 (15-s duration; 15-s intervals). Mechanical stimuli were applied with a forceps with a force transducer, whose calibrated output was amplified, displayed in grams on a LCD screen, and recorded and monitored on a PC for on- and off-line analysis. Cutaneous receptive fields were mapped using the following stimuli: BRUSH (brushing the skin with a soft-hair artist's brush in a stereotyped manner), PRESS (firm pressure using a large arterial clip to apply 1,005 g/8 mm2, which is marginally painful when applied to the skin in humans), and PINCH (using a small arterial clip to apply 2,660 g/4 mm2, which is clearly painful without causing overt damage to the skin). The threshold was defined as the minimum stimulus intensity that evoked a response in 50% of trials (tested 3 times). Cutaneous input was distinguished from deep tissue input by selective stimulation of skin folds gently raised from the underlying deep tissue.
Background activity and responses to graded mechanical stimuli were measured before and during the development of the arthritis and before and during drug application. The effects of drugs were tested only on a limited number of parameters, including background activity and responses evoked by stimulation of the deep tissue to avoid overly long drug applications (maximum 15–20 min) and to ensure drug effects were measured at comparable time points.
Classification of neurons and thresholds
According to our classification of CeA neurons with deep tissue input (Neugebauer and Li 2002, 2003), all CeA neurons selected for this study were MR neurons, which have been shown to become sensitized in the arthritis pain model (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 (deep tissue, <500 g/30 mm2; skin, BRUSH) but are more strongly activated by noxious stimuli (deep tissue, >1,500 g/30 mm2; skin, PRESS and PINCH). 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. Pressure stimuli >1,500 g/30 mm2 are noxious because they evoke hindlimb withdrawal reflexes in awake rats and are distinctly painful when applied to the experimenters (Neugebauer and Li 2002, 2003).
Arthritis
In each experiment, background activity of one CeA neuron and its responses to graded mechanical stimuli were recorded before and for several hours (6 h; maximum 12 h) after the induction of arthritis in one knee joint as described previously (Neugebauer and Li 2003). Background activity, evoked responses, and receptive field size had to be stable for several hours before the arthritis was induced. Throughout the experiment, we carefully monitored a variety of parameters (body temperature, blood pressure, heart rate, ECG, end-tidal CO2 levels) to ensure a stable recording situation.
Arthritis was induced as described in detail previously (Neugebauer and Li 2003; Neugebauer et al. 1993). A kaolin suspension (4%, 80 µl) was slowly injected into the joint cavity through the patellar ligament with the use of a syringe and needle (1 ml, 25G5/8''). After repetitive flexions and extensions of the knee for 15 min, a carrageenan solution (2%, 80 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 (Neugebauer and Li 2003; Neugebauer et al. 1993) and persists for weeks (Min et al. 2001).
Drug application
Known concentrations of mGluR1 and mGluR5 agonists and antagonists (see next paragraph) were administered into the CeA by microdialysis before and 6 h after induction of arthritis. Maximum changes of the neurons' response behavior occur after 5–6 h after induction of arthritis, when the sensitization process reaches a plateau that lasts at least until 18 h after induction (Neugebauer and Li 2003). 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: 1.8 mm caudal to bregma; 4.0 mm lateral to midline; depth of tip 9.0 mm (Paxinos and Watson 1998). 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) 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose and 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 (Neugebauer et al. 1999, 2000a).
Drugs were dissolved in ACSF on the day of the experiment at a concentration 100 times that predicted to be needed based on data from in vitro studies (Neugebauer et al. 1999). The concentration gradient across the dialysis membrane is 100:1 for these molecules. The concentration is reduced by another order of magnitude due to diffusion in the tissue (Neugebauer et al. 1999). Drugs were administered into the CeA at a rate of 5 µl/min for 10–20 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 3–5 min during drug application. Steady-state drug effects were determined from the time course of drug effects on the evoked responses. If no (further) change was measured between two consecutive responses, it was assumed that maximum drug effect was reached. The numbers given in this article refer to the drug concentrations in the microdialysis fiber. The following drugs were used: 2-chloro-5-hydroxyphenyl-glycine (CHPG; mGluR5 agonist); (S)-3,5-dihydroxyphenylglycine (DHPG; mGluR1 and 5 agonist); 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; mGluR1 antagonist); and 2-methyl-6-(phenylethynyl)pyridine (MPEP; mGluR5 antagonist); these were purchased from Tocris Cookson (Bristol, UK).
Histology
At the end of each experiment, the recording site in the CeA 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 cover-slipped. 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) (Fig. 1).
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Extracellularly recorded single-unit activity (action potentials) was analyzed off-line from peristimulus rate histograms using Spike2 software (CED, version 3). The neurons' responses to mechanical stimuli were measured and expressed as spikes/s (Hz). Unless stated otherwise, evoked responses were measured by subtracting background activity, if present, from the total activity during the stimulus. Ratios of evoked activity over background activity were also calculated to assess changes of drug effects in the arthritis pain model. The neurons' activity in the arthritic state was compared statistically to the same neurons' activity before arthritis using a paired t-test (Prism 3.0, GraphPad Software Inc.). Concentration–response relationships were measured for each neuron and then averaged across a sample of neurons. EC50s 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(EC50), and D = slope coefficient (GraphPad Prism 3.0). Unpaired t-tests were used for statistical comparison of EC50s and maximum effects under normal conditions and in arthritis. Concentration–response functions under normal conditions and in the arthritic pain state were compared statistically using a two-way ANOVA followed by Bonferroni posttests (GraphPad Prism 3.0). Linear regression analysis with runs test was used to analyze if the slope of the concentration–response curve was significantly different from zero (GraphPad Prism 3.0). All averaged values are given as the means ± SE. Statistical significance was accepted at the level P < 0.05.
The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and were consistent with ethical guidelines of NIH and ISAP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Extracellular single-unit recordings were made from 65 neurons in the CeA in anesthetized rats. Histological verification of the recording site was successful in 56 cases. All the recording sites were located in the latero-capsular subdivision of the CeA (Fig. 1), which is now defined as the "nociceptive" amygdala (Neugebauer and Li 2002, 2003). Data from experiments in which histological analysis revealed recording sites outside the CeA were not included in this study. All CeA neurons in this study were MR neurons, which responded significantly to innocuous but more strongly to noxious stimuli (see METHODS for classification). This study focuses on MR neurons because they represent the class of CeA neurons that consistently become sensitized to afferent inputs in the arthritis pain model (Neugebauer and Li 2003). In agreement with our previous studies (Neugebauer and Li 2002, 2003), all MR neurons had large symmetrical receptive fields in the deep tissue of both hind limbs including the knee joints (n = 65), ankles (n = 61), and paws (n = 54).
Drug effects were measured in a total of 65 CeA neurons: drugs were tested under normal conditions (no arthritis) in 56 neurons and 6 h after induction of the arthritis in 32 neurons. Maximum changes occur after 5–6 h after induction of arthritis, when the sensitization process reaches a plateau that lasts at least until 18 h after induction (Neugebauer and Li 2003). In 23 of these neurons, drug effects were assessed before and after induction of arthritis in the same neuron; in 9 neurons, drugs were only tested in the arthritic state as a control for any pretreatment effect. Since no difference was found, these data were pooled. Recordings were continued only if drug effects were reversible. These 32 neurons were recorded continuously before and during the development of the arthritis. Consistent with the results of our previous study (Neugebauer and Li 2003), this sample of neurons showed significantly increased background activity and responses to mechanical stimulation of the arthritic knee and of intact tissue such as the ankle (Fig. 2A). Figure 2, B and C, displays the extracellularly recorded action potentials of an individual CeA neuron in response to mechanical stimulation of the knee before and 6 h after induction of arthritis to illustrate that single-unit activity was recorded and that the recording conditions remained constant during these long-term experiments.
Effects of mGluR1 and mGluR5 agonists on the responsiveness of CeA neurons
A selective group I mGluR1 and 5 agonist (DHPG; 1 µM–10 mM, concentration in microdialysis probe corresponding to tissue concentrations of 1 nM–10 µM; see METHODS) (Neugebauer 2002; Schoepp et al. 1999) was administered into the CeA for 15 min under normal conditions (no arthritis; n = 10) and in the arthritis pain state (6 h after induction; n = 9). DHPG enhanced the evoked responses of all CeA neurons under normal conditions, and this effect increased in arthritis, suggesting a functional change of group I mGluRs.
Figure 3 shows an individual example. This MR CeA neuron responded with increasing magnitude to graded mechanical stimulation of the knee in the innocuous and noxious range (Fig. 3A). Administration of DHPG (100 µM, concentration in the microdialysis probe) into the CeA increased the responses to innocuous (100 and 500 g/30 mm2) and noxious stimuli (1,500–2,500 g/30 mm2). The activity of the same neuron was continuously recorded during development of the knee joint arthritis (see METHODS). Spike size, shape, and configuration were closely monitored to ensure that activity from only one and the same neuron was recorded (see spikes in Fig. 3C taken from the histograms in A and B, where indicated by a–d). In the arthritis pain state (6 h after induction; Fig. 3B), background activity and evoked responses of this neuron had increased. A 10-fold lower concentration of DHPG (10 µM; 10 min) now potentiated the neuron's increased responses with a magnitude comparable to that under normal conditions before arthritis, suggesting DHPG had become more potent in the arthritic state.
Figure 4A summarizes the data for the sample of neurons in which DHPG was tested. Under control conditions, DHPG concentration-dependently enhanced the responses of CeA neurons to innocuous and noxious innocuous (100 g/30 mm2) and noxious (2,500 g/30 mm2) stimulation of the knee with EC50 values of 19.4 ± 0.3 and 63.0 ± 2.2 µM, respectively (n = 10). In the arthritis pain state, DHPG showed a 5-fold and 12-fold increased potency in enhancing the responses to innocuous and noxious stimuli, respectively (n = 9), with EC50 values of 4.2 ± 0.13 (innocuous) and 5.4 ± 0.15 µM (noxious), which were significantly different from controls (P < 0.0001; unpaired t-test). In addition to the increase in potency, DHPG also became more efficacious in enhancing the evoked responses. Maximum potentiation by DHPG as measured from the concentration–responses curves (GraphPad Prism 3.0 software) increased significantly from 148 ± 1.1% to 170 ± 3.3% (innocuous; P < 0.001, unpaired t-test) and from 141 ± 2.6% to 152 ± 2.3% (noxious; P < 0.01, unpaired t-test). Similar changes in potency and maximum effect of DHPG were detected when analysis of the ratio of total activity during stimulus over background activity was used instead of subtracting background activity from total activity during stimulation.
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; Schoepp et al. 1999); selective mGluR1 agonists are not available as yet. Figure 5 illustrates the effects of CHPG in an individual CeA neuron under normal conditions and in arthritis. Like the mixed mGluR1 and mGluR5 agonist DHPG, CHPG (10 mM; concentration in microdialysis probe; administration into the CeA for 15 min) enhanced the responses of this neuron to innocuous and noxious mechanical stimuli (Fig. 5A). Unlike DHPG, however, the effects of CHPG did not change in the arthritis pain state (Fig. 5B), suggesting that it is not the mGluR5 subtype, but rather the mGluR1 subtype, that undergoes functional changes. Analysis of the concentration–response relationships of CHPG effects before and after induction of arthritis (Fig. 4B) showed that neither the potency nor the efficacy (maximum effect) of CHPG changed significantly. The EC50 values for CHPG enhancing the responses to innocuous (100 g/30 mm2) and noxious (2,500 g/30 mm2) stimuli, respectively, were 178 ± 9.3 and 193 ± 4.3 µM under normal conditions and 298 ± 14.8 and 297 ± 3.4 µM in the arthritis pain state (P > 0.05, unpaired t-test). Similarly, the maximum potentiation by CHPG calculated from the concentration–response curves (GraphPad Prism 3.0 software) was not significantly different in arthritis compared with control (P > 0.05, unpaired t-test). A similar lack of change in potency and maximum effect of CHPG in arthritis was also detected using the analysis of the ratio of total activity during stimulus over background activity instead of the difference between activity during stimulation and preceding background activity.
Effects of mGluR1 and mGluR5 antagonists on the responsiveness of CeA neurons
Given the enhanced mGluR1 receptor function in prolonged pain, we analyzed if there was also a change of intrinsic activation of group I mGluR subtypes. To test this hypothesis, we administered selective mGluR1 (CPCCOEt) and mGluR5 (MPEP) antagonists (Neugebauer 2002; Schoepp et al. 1999) into the CeA under normal conditions (no arthritis) and in the arthritis pain state (5.5–9 h after induction) to measure intrinsic activation of these receptors. The main result was that, under normal conditions, only MPEP had any effects, inhibiting the responses to brief noxious stimuli, whereas in arthritis CPCCOEt and MPEP inhibited the enhanced responses of sensitized neurons.
Figure 6 shows an individual example of CPCCOEt effects in a MR CeA neuron that responded with increasing magnitude to graded mechanical stimulation of the knee in the innocuous and noxious range (Fig. 6A). Administration of CPCCOEt into the CeA (1 mM, concentration in the microdialysis probe corresponding to 1 µM in the tissue; see METHODS; for 15 min) had no effect under normal conditions (Fig. 6A) but inhibited the enhanced responses of the sensitized neuron 6 h after induction of arthritis (Fig. 6B). CPCCOEt had no significant effect on background activity under normal conditions or in arthritis.
This change of endogenous activation of mGluR1 in the arthritis pain state is summarized in Fig. 7A for the sample of neurons in which the effects of the mGluR1 antagonist CPCCOEt administered into the CeA were measured (1 µM–10 mM, concentration in microdialysis probe corresponding to tissue concentrations of 1 nM–10 µM; see METHODS; for 15 min). Analysis of the concentration–response relationships showed that CPCCOEt inhibited the responses of CeA neurons to innocuous (500 g/30 mm2) and noxious (2,500 g/30 mm2) mechanical stimulation of the knee in the arthritis pain state (with EC50 values of 19.9 ± 0.50 and 29.4 ± 0.70 µM, respectively; n = 8), whereas CPCCOEt had no significant effect on the evoked responses under normal conditions, i.e., the slope of the concentration–response curve was not significantly different from zero [P > 0.05, F = 5.1431,3 (innocuous) and 8.1081,3 (noxious), n = 12; linear regression analysis, GraphPad Prism 3.0 software]. A similar change of CPCCOEt effects was measured when calculating ratios of evoked activity over background activity instead of subtracting background activity from evoked activity.
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Data suggesting the endogenous activation of mGluR5 during brief and prolonged nociceptive processing are summarized in Fig. 7B for the sample of neurons in which the effects of the mGluR5 antagonist MPEP administered into the CeA were measured (1 µM–10 mM, concentration in microdialysis probe corresponding to tissue concentrations of 1 nM–10 µM; see METHODS). Analysis of the concentration–response relationships showed that, under normal conditions, MPEP inhibited the responses of CeA neurons (n = 19) to brief noxious stimuli (2,500 g/30 mm2; EC50 = 93.3 ± 10.1 µM) but not to innocuous stimuli (500 g/30 mm2; P > 0.05, F = 8.7611,3, linear regression analysis, GraphPad Prism 3.0 software). In the arthritis pain state, MPEP inhibited the responses to innocuous and noxious stimuli with EC50 values of 57.6 ± 1.6 and 62.8 ± 5.7 µM, respectively (n = 8). There was no significant change of potency of MPEP in inhibiting brief noxious responses before and after induction of arthritis (P > 0.05, unpaired t-test, GraphPad Prism 3.0). Similar results were obtained when using the ratio of evoked activity over background activity instead of the difference between activity during stimuli and background activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), all multireceptive neurons in the CeA developed enhanced responses to nociceptive afferent input (sensitization). 2) Data obtained with group I agonists show that activation of mGluR5 under normal conditions can mimic the effects of arthritis; it is mGluR1, however, that undergoes functional changes in the arthritis pain state. 3) Antagonist data suggest that endogenous activation of mGluR1 occurs only in the state of pain-related sensitization, whereas mGluR5 is endogenously activated by brief nociceptive inputs under normal conditions and in prolonged pain states. Our data suggest important but differential roles of group I mGluR subtypes in brief and prolonged nociceptive processing in the amygdala.
Accumulating evidence points to the amygdala as a neural substrate of the reciprocal relationship between pain and emotion (McGaraughty and Heinricher 2002; Meagher et al. 2001). The amygdala plays a key role in the emotional-affective aspects of behavior, the emotional evaluation of sensory stimuli, emotional learning and memory, and related disorders (Aggleton 2000; Cahill 1999; Davidson et al. 1999; Davis 1998; LeDoux 2000; Rasia-Filho et al. 2000). It has become clear now that the amygdala is also part of the pain system, receiving nociceptive input from the spino-parabrachio-amygdaloid pathway, which connects the spinal cord and pontine parabrachial area with the CeA (Bernard et al. 1996; Gauriau and Bernard 2002; Jasmin et al. 1997). Polymodal sensory information reaches the amygdala from thalamic and cortical areas through connections with the lateral (LA) and basolateral amygdaloid nuclei (BLA), which then project to the CeA, the output nucleus for major amygdala functions (Doron and LeDoux 1999; LeDoux 2000; Linke et al. 1999; Pitkanen et al. 1997; Shi and Davis 1999; Smith et al. 2000). These highly integrated inputs from the LA and BLA to the CeA are part of the fear- and anxiety-related circuitry. The amygdala thus is well positioned to play a key role in the emotional-affective component of pain (Bellgowan and Helmstetter 1996; Bernard and Bandler 1998; Bernard et al. 1996; Fields 2000; Helmstetter 1992; Manning 1998; McGaraughty and Heinricher 2002; Meagher et al. 2001; Neugebauer and Li 2003; Neugebauer et al. 2003; Schneider et al. 2001).
Further evidence for an important role of the amygdala in pain comes from neuroimaging pain studies, such as PET and functional MRI (fMRI), which have repeatedly, although inconsistently, identified pain-related signal changes in the amygdala in animals and humans (Becerra et al. 1999; Bingel et al. 2002; Bonaz et al. 2002; Bornhovd et al. 2002; Derbyshire et al. 1997; Paulson et al. 2002; Schneider et al. 2001). Although these studies detected a correlation between amygdala responses and pain behavior in animals and pain experienced in humans, both activation and deactivation ("negative activation") were observed. The basis of these discrepancies is yet to be understood. The amygdala is a small structure with many diverse clusters of nuclei, subdivisions, and circuitry. Therefore our in vivo electrophysiological analysis of single neurons is well suited to study pain processing in the amygdala.
Our recent electrophysiological in vivo and in vitro studies showed that two major subpopulations of CeA neurons, so-called MR neurons and noSOM neurons, but not NS neurons, exhibit substantial nociceptive and synaptic plasticity in a model of persistent arthritic pain (Neugebauer and Li 2003; Neugebauer et al. 2003). This study focused on MR neurons, which are activated by noxious as well as innocuous stimuli in a graded fashion so that they process, but also distinguish between nociceptive and nonnociceptive information (see METHODS). MR neurons have been shown to become sensitized to afferent inputs evoked by peripheral mechanical, but not thermal, stimuli and to constant electrically evoked orthodromic activity in the arthritis model of persistent pain (Neugebauer and Li 2003). MR neurons are believed to be a site of convergence and integration of nociceptive and polymodal inputs in the CeA (Neugebauer and Li 2003). The pain-related sensitization of MR amygdala neurons would be consistent with the well-documented critical role of the amygdala in associative learning and memory to link sensory information and affective significance (Aggleton 2000; Blair et al. 2001; Davis 1998; Everitt et al. 1999; LeDoux 2000; Post et al. 1998; Rolls 2000).
The mechanisms of neuroplasticity in the CeA and of the sensitization of MR neurons are not yet fully understood. Our previous electrophysiological recordings of CeA neurons in brain slices in vitro suggest an important role for group I mGluRs in the arthritis pain model (Neugebauer et al. 2003). There we determined the enhanced receptor sensitivity and upregulation of presynaptic mGluR1 rather than mGluR5 in the CeA in the arthritis pain model (Neugebauer et al. 2003). This study takes the analysis to a new level by addressing the functional roles of the different group I mGluR subtypes in the processing of nonnociceptive and brief and prolonged nociceptive information in amygdala neurons. The novel finding is that, although activation of mGluR5 can mimic arthritis pain-related changes, it is the receptor sensitivity and intrinsic activation of mGluR1 that change in the arthritis pain state. Our data suggest distinct roles of mGluR1 and mGluR5 in the nociceptive amygdala. Nonnociceptive signaling, including highly integrated polymodal inputs from thalamic and cortical areas via the LA and BLA, does not involve group I mGluR activation in the CeA; mGluR5 plays a role in the processing of both brief and prolonged nociceptive information, whereas mGluR1 appears to be exclusively concerned with nociceptive plasticity, at least in the arthritis pain model.
It should be noted that the agonists and antagonists used in this study are selective for group I mGluR subtypes (CHPG, CPCCOEt, MPEP) or subgroup (DHPG) (Anwyl 1999; Cartmell and Schoepp 2000; De Blasi et al. 2001; Neugebauer 2002; Schoepp et al. 1999). Drugs were administered into the CeA by microdialysis, and concentrations were adjusted based on our previous analysis (Neugebauer et al. 1999). Importantly, the EC50 values of drugs in the microdialysis probe measured in the present in vivo study are 500–1,000 times higher (micromolar range) than the EC50 values for drugs directly superfused onto the amygdala slices in our previous in vitro study (nanomolar range) (Neugebauer et al. 2003). This is consistent with the previously established fact that the tissue concentrations of these agents are approximately three orders of magnitude lower than the concentrations within the microdialysis fiber (Neugebauer et al. 1999). It should be noted that due to this method of drug application and the experimental systems approach, drug effects measured in this study may involve other sites of action than the CeA, including the adjacent intercalated cell masses, which are known to inhibit CeA neurons (Royer et al. 1999).
Defining the differential roles of group I mGluR subtypes is currently a hot topic in neuroscience research because of new potential therapeutics that would selectively target certain disorders and syndromes (Karim et al. 2001; Neugebauer 2002; Neugebauer and Carlton 2002; Poisik et al. 2003; Spooren et al. 2001; Varney and Gereau 2002). Although there is evidence to suggest differential roles of group I mGluR subtypes in pain-related sensitization in the periphery (mGluR5 > mGluR1) and spinal cord (mGluR1 > mGluR5) (Neugebauer 2002; Neugebauer and Carlton 2002; Varney and Gereau 2002), this study is the first to provide evidence for a unique pattern of mGluR1 and mGluR5 involvement in nociceptive plasticity in the brain, in particular the amygdala.
The cellular and molecular mechanisms involved in the segregation of mGluR1 and mGluR5 function are not clear. Both receptors have similar affinities for L-glutamate and can couple to the same G-proteins and effector systems, which mainly include modulation of potassium currents, inositol trisphosphate receptors, protein kinases, and interaction with surface receptors (e.g., N-methyl-D-aspartate receptors) (De Blasi et al. 2001; Neugebauer 2002; Neugebauer and Carlton 2002; Schoepp et al. 1999; Varney and Gereau 2002). Functional differences of mGluR1 and mGluR5 could be due to differences in receptor protein expression levels, synaptic and perisynaptic distribution, affinities for the endogenous ligand(s), and effector mechanisms. Currently, the only explanation rooted in experimental evidence is that, under normal conditions, there is comparatively little mGluR1 protein expression in the CeA but significant amounts of mGluR5. In the arthritis pain state, mGluR1 and mGluR5 protein expression increase significantly (Neugebauer et al. 2003). Therefore group I mGluR effects are mediated through mGluR5 under normal conditions, whereas both mGluR1 and mGluR5 contribute to nociceptive plasticity. The enhanced function of group I mGluRs in the CeA in pain-related plasticity is paralled by enhanced synaptic transmission at the nociceptive PB-CeA synapse (Neugebauer et al. 2003) and increased background activity of CeA neurons (see Fig. 2), which may be the consequence of enhanced afferent input to the CeA. Thus enhanced transmission of incoming signals and subsequent transmitter release may result in the stronger inhibitory effects of group I mGluRs in arthritis pain measured in the present study. However, increased mGluR effects are probably not simply the reflection of increased activity at the presynaptic (input) site. If this were the case, increased effects of both mGluR1 and mGluR5 would be expected rather than the differential changes of mGluR1 versus mGluR5 actions that we observed in this study.
Importantly, group I mGluRs have been shown to be located at the margin of the synaptic specialization, away from the active zone, so that activation of these receptors would require conditions of high glutamate release (Cartmell and Schoepp 2000; Lujan et al. 1997; Ottersen and Landsend 1997). The extrasynaptic localization could explain why mGluR5, despite its abundant expression in the CeA, is activated during noxious but not innocuous stimuli. It will be interesting to analyze in future studies if coupling to different signal transduction pathways, specific G protein subunits and/or interaction with scaf-folding proteins such as Homer, Shank, and others might contribute to differences in mGluR1 and mGluR5 function as has been suggested for other brain areas including the hippocampus (Mannaioni et al. 2001) and globus pallidus (Poisik et al. 2003).
In conclusion, this study is the first to demonstrate pharmacological mechanisms underlying the neuroplastic changes of nociceptive transmission in amygdala neurons in a model of prolonged pain. As the output nucleus for major amygdala functions, the CeA modulates various effector systems involved in the expression of emotional responses through widespread connections with the forebrain and brain stem (Aggleton 2000; Bourgeais et al. 2001; Cassell et al. 1986; Gray 1993; Krettek and Price 1978; LeDoux 2000; LeDoux et al. 1988; Price and Amaral 1981). MR neurons in the nociceptive amygdala (latero-capsular CeA), which integrate nociceptive and polymodal inputs, may serve to evaluate sensory information in the context of prolonged pain. Thus the differential roles of mGluR1 and mGluR5 shown in this study could provide the basis for novel important drugs to target the emotional component of pain.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
This work was supported by John Sealy Memorial Endowment Fund for Biomedical Research Grants 2528-99 and 2521-04 and National Institute of Neurological Disorders and Stroke Grant NS-38261.
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FOOTNOTES |
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Address for reprint requests and other correspondence: V. Neugebauer, Dept. of Anatomy and Neurosciences and Marine Biomedical Institute, The Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail: voneugeb{at}utmb.edu).
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REFERENCES |
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