The role of neuropeptides in synaptic plasticity is less well understood than that of classical transmitters such as glutamate. Here we report the importance of the G-protein-coupled calcitonin gene-related peptide (CGRP1) receptor as a critical link between amygdala plasticity and pain behavior. A key player in emotionality and affective disorders, the amygdala has been implicated in the well documented, but mechanistically unexplained, relationship between pain and affect. Our electrophysiological and pharmacological in vitro (patch-clamp recordings) and in vivo (extracellular single-unit recordings) data show that selective CGRP1 receptor antagonists (CGRP 8 -37 and BIBN4096BS) in the amygdala reverse arthritis pain-related plasticity through a protein kinase A (PKA)-dependent postsynaptic mechanism that involves NMDA receptors. CGRP1 receptor antagonists inhibited synaptic plasticity in the laterocapsular division of the central nucleus of the amygdala (CeLC) in brain slices from arthritic rats compared with normal controls. The effects were accompanied by decreased neuronal excitability and reduced amplitude, but not frequency, of miniature EPSCs; paired-pulse facilitation was unaffected. The antagonist effects were occluded by a PKA inhibitor. CGRP1 receptor blockade also directly inhibited NMDA-evoked, but not AMPA-evoked, membrane currents. Together, these data suggest a postsynaptic site of action. At the systems level, the antagonists reversed the sensitization of nociceptive CeLC neurons in anesthetized rats in the arthritis pain model. Importantly, CGRP1 receptor blockade in the CeLC inhibited spinal (hindlimb withdrawal reflexes) and supraspinal pain behavior of awake arthritic rats, including affective responses such as ultrasonic vocalizations. This study provides direct evidence for the critical dependence of pain behavior on CGRP1-mediated amygdala plasticity.

The assessment of pain is of critical importance for mechanistic studies as well as for the validation of drug targets. This review will focus on knee joint pain associated with arthritis. Different animal models have been developed for the study of knee joint arthritis. Behavioral tests in animal models of knee joint arthritis typically measure knee joint pain rather indirectly. In recent years, however, progress has been made in the development of tests that actually evaluate the sensitivity of the knee joint in arthritis models. They include measurements of the knee extension angle struggle threshold, hind limb withdrawal reflex threshold of knee compression force, and vocalizations in response to stimulation of the knee. A discussion of pain assessment in humans with arthritis pain conditions concludes this review. ReviewArthritis represents one of the most prevalent chronic health problems and is a leading cause of disability. More than 40 million people in the United States have arthritis or chronic joint symptoms that are often accompanied by joint pain [1]. By the year 2020, this number is expected to reach 60 million. The most common form of arthritis is osteoarthritis affecting an estimated 21 million adults in the United States. Other common arthritic conditions include rheumatoid arthritis (about 2.1 million people in the United States) and gout [2]. The assessment of arthritic pain is of critical importance for the better understanding of underlying mechanisms and for the evaluation of therapeutic targets. Different animal models of arthritis are available for the assessment of joint pain and analgesic drug effects. This review will focus on arthritis models of knee joint pain and on behavioral tests used in these models. Information about the assessment of knee joint pain in humans with arthritis will also be provided. Arthritis pain modelsArthritis is the inflammation of a joint, which can include infiltration of inflammatory cells (monocytes), synovial hyperplasia, bone erosion and new bone formation, nar-

Mechanisms of pain-related plasticity in the amygdala, a key player in emotionality, were studied at the cellular and molecular levels in a model of arthritic pain. The influence of the arthritis pain state induced in vivo on synaptic transmission and N -methyl-D-aspartate (NMDA) receptor function was examined in vitro using whole-cell voltage-clamp recordings of neurones in the latero-capsular part of the central nucleus of the amygdala (CeA), which is now defined as the 'nociceptive amygdala'. Synaptic transmission was evoked by electrical stimulation of afferents from the pontine parabrachial area (part of the spino-parabrachio-amygdaloid pain pathway) in brain slices from control rats and from arthritic rats. This study shows that pain-related synaptic plasticity is accompanied by protein kinase A (PKA)-mediated enhanced NMDA-receptor function and increased phosphorylation of NMDA-receptor 1 (NR1) subunits. Synaptic plasticity in the arthritis pain model, but not normal synaptic transmission in control neurones, was inhibited by a selective NMDA receptor antagonist. Accordingly, an NMDA receptor-mediated synaptic component was recorded in neurones from arthritic animals, but not in control neurones, and was blocked by inhibition of PKA but not protein kinase C (PKC). Exogenous NMDA evoked a larger inward current in neurones from arthritic animals than in control neurones, indicating a postsynaptic effect. Paired-pulse facilitation, a measure of presynaptic mechanisms, was not affected by an NMDA-receptor antagonist. Increased levels of phosphorylated NR1 protein, but not of total NR1, were measured in the CeA of arthritic rats compared to controls. Our results suggest that pain-related synaptic plasticity in the amygdala involves a critical switch of postsynaptic NMDA receptor function through PKA-dependent NR1 phosphorylation.

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Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury.Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 μM) or a PKA inhibitor (KT5720, 1 μM), but not by a PKC inhibitor (GF109203X, 1 μM). Stereotaxic administration of CGRP (10 μM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 μM) but not by GF109203X (100 μM).The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.