Contribution of Corticotropin-Releasing Factor Receptor 1 (CRF1) to Serotonin Receptor 5-HT2CR Function in Amygdala Neurons in a Neuropathic Pain Model

The amygdala plays a key role in emotional-affective aspects of pain and in pain modulation. The central nucleus (CeA) serves major amygdala output functions related to emotional-affective behaviors and pain modulation. Our previous studies implicated the corticotropin-releasing factor (CRF) system in amygdala plasticity and pain behaviors in an arthritis model. We also showed that serotonin (5-HT) receptor subtype 5-HT2CR in the basolateral amygdala (BLA) contributes to increased CeA output and neuropathic pain-like behaviors. Here, we tested the novel hypothesis that 5-HT2CR in the BLA drives CRF1 receptor activation to increase CeA neuronal activity in neuropathic pain. Extracellular single-unit recordings of CeA neurons in anesthetized adult male rats detected increased activity in neuropathic rats (spinal nerve ligation model) compared to sham controls. Increased CeA activity was blocked by local knockdown or pharmacological blockade of 5-HT2CR in the BLA, using stereotaxic administration of 5-HT2CR short hairpin RNA (shRNA) viral vector or a 5-HT2CR antagonist (SB242084), respectively. Stereotaxic administration of a CRF1 receptor antagonist (NBI27914) into the BLA also decreased CeA activity in neuropathic rats and blocked the facilitatory effects of a 5-HT2CR agonist (WAY161503) administered stereotaxically into the BLA. Conversely, local (BLA) knockdown of 5-HT2CR eliminated the inhibitory effect of NBI27914 and the facilitatory effect of WAY161503 in neuropathic rats. The data suggest that 5-HT2CR activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.


Introduction
Serotonin (5-HT) plays an important role in pain modulation and can have facilitatory and inhibitory effects depending on the site of action in the nervous system, cell type, and receptor subtype and affinity [1][2][3][4]. The 14 5-HT receptor subtypes are classified into seven groups based on their structural and functional characteristics [5][6][7]. Selective serotonin reuptake inhibitors (SSRIs) are commonly used in the treatment of major depression and related disorders, and they can relieve neuropathic pain but have limited efficacy and adverse side effects [8][9][10][11][12].

Results
The experiments described here were designed to analyze the interaction between serotonin receptor 5-HT 2C R in the basolateral amygdala (BLA) and corticotropin-releasing factor receptor CRF1 signaling downstream of 5-HT 2C R and their contribution to the increased activity of the central amygdala (CeA) neurons in a neuropathic pain model. Single-unit recordings of CeA neurons in sham control rats and in neuropathic rats (spinal nerve ligation model, SNL) were used. Extracellular single-unit recordings were made from 44 CeA neurons (n = 15 neurons in 9 sham rats; n = 29 neurons in 11 SNL rats) in anesthetized (isoflurane) adult male rats ( Figure 1) as described in the materials and methods section. Neurons were selected that had a receptive field in the left hindpaw (side of sham or SNL surgery), and responded more strongly to brief noxious than innocuous test stimuli; these are so-called "multireceptive" (MR) neurons according to our classification of amygdala neurons [31,45,48,62]. The general experimental protocol was as follows: Induction of pain model (spinal nerve ligation) or sham surgery (Day 0), 5-HT 2C R short hairpin RNA (shRNA)-enhanced green fluorescence protein (eGFP) for knockdown) or shRNA-eGFP (control) adeno-associated viral vector (AAV) vectors injections (Day 14), and electrophysiological experiments with or without drug applications (Day 28). the materials and methods section. Neurons were selected that had a receptive field in the left hindpaw (side of sham or SNL surgery), and responded more strongly to brief noxious than innocuous test stimuli; these are so-called "multireceptive" (MR) neurons according to our classification of amygdala neurons [31,45,48,62]. The general experimental protocol was as follows: Induction of pain model (spinal nerve ligation) or sham surgery (Day 0), 5-HT2CR short hairpin RNA (shRNA)-enhanced green fluorescence protein (eGFP) for knockdown) or shRNA-eGFP (control) adeno-associated viral vector (AAV) vectors injections (Day 14), and electrophysiological experiments with or without drug applications (Day 28).

5-HT2CR Knockdown in the BLA Inhibits Activity of CeA Neurons in Neuropathic Rats
For local (BLA) knockdown of 5-HT2CR, recombinant AAV2 vectors expressing a short hairpin RNA (shRNA) directed at the 5-HT2CR or a control hairpin were used [45,63,64]. Either 5-HT2CR or a control shRNA-eGFP AAV2 vector was injected stereotaxically into the BLA two weeks after neuropathic or sham surgery, as described in the materials and methods section. Electrophysiology studies were done two weeks after viral vector injection. Compared to CeA neurons in sham controls treated with a control vector (n = 18 neurons), CeA neurons in SNL rats treated with control vector (n = 10 neurons) showed significantly (p < 0.05, ANOVA with Bonferroni post hoc tests) increased background activity and responses to innocuous and noxious stimuli (mechanical compression of the hindpaw with a calibrated forceps, see the materials and methods section; Figure 2G). Individual examples are shown in Figure 2A-C. There was also a significant increase in burst-like activity ( Figure 2H; p < 0.01) and irregular firing ( Figure 2I; p < 0.001, ANOVA with Bonferroni post hoc tests) of CeA neurons in SNL rats (n = 10 neurons) compared to sham controls

5-HT 2C R Knockdown in the BLA Inhibits Activity of CeA Neurons in Neuropathic Rats
For local (BLA) knockdown of 5-HT 2C R, recombinant AAV2 vectors expressing a short hairpin RNA (shRNA) directed at the 5-HT 2C R or a control hairpin were used [45,63,64]. Either 5-HT 2C R or a control shRNA-eGFP AAV2 vector was injected stereotaxically into the BLA two weeks after neuropathic or sham surgery, as described in the materials and methods section. Electrophysiology studies were done two weeks after viral vector injection. Compared to CeA neurons in sham controls treated with a control vector (n = 18 neurons), CeA neurons in SNL rats treated with control vector (n = 10 neurons) showed significantly (p < 0.05, ANOVA with Bonferroni post hoc tests) increased background activity and responses to innocuous and noxious stimuli (mechanical compression of the hindpaw with a calibrated forceps, see the materials and methods section; Figure 2G). Individual examples are shown in Figure 2A-C. There was also a significant increase in burst-like activity ( Figure 2H; p < 0.01) and irregular firing ( Figure 2I; p < 0.001, ANOVA with Bonferroni post hoc tests) of CeA neurons in SNL rats (n = 10 neurons) compared to sham controls (n = 14 neurons). Individual examples are shown in Figure 2D-F. Details of the analysis of neuronal activity are described in the materials and methods section. CeA neurons in SNL rats with local 5-HT 2C R knockdown in the BLA (n = 6 neurons) showed significantly lower background and evoked activity ( Figure 2G; p < 0.01), less burst-like activity ( Figure 2H; p < 0.01), and less irregular firing ( Figure 2I; p < 0.001, ANOVA with Bonferroni post hoc tests) compared to CeA neurons in control vector treated SNL rats. Our previous study showed that 5-HT 2C R knockdown had no effect in sham controls [45]. The results of the present study validate the neuropathic pain-related neuronal changes and the effectiveness of 5-HT 2C R knockdown observed in our previous study [45], allowing us to use the knockdown strategy to link 5-HT 2C R and CRF1 receptor function and to confirm the selectivity of pharmacological agents tested here.  Figure 2D-F. Details of the analysis of neuronal activity are described in the materials and methods section. CeA neurons in SNL rats with local 5-HT2CR knockdown in the BLA (n = 6 neurons) showed significantly lower background and evoked activity ( Figure 2G; p < 0.01), less burst-like activity ( Figure 2H; p < 0.01), and less irregular firing ( Figure 2I; p < 0.001, ANOVA with Bonferroni post hoc tests) compared to CeA neurons in control vector treated SNL rats. Our previous study showed that 5-HT2CR knockdown had no effect in sham controls [45]. The results of the present study validate the neuropathic pain-related neuronal changes and the effectiveness of 5-HT2CR knockdown observed in our previous study [45], allowing us to use the knockdown strategy to link 5-HT2CR and CRF1 receptor function and to confirm the selectivity of pharmacological agents tested here.

Inhibitory Effects of Antagonists for 5-HT 2C R and CRF1 Receptor on CeA Neurons in Neuropathic Rats
The effects of selective antagonists for 5-HT 2C R (SB242084) and the CRF1 receptor (NBI27914) were tested in neuropathic rats 2 weeks after control shRNA was injected into the BLA ( Figure 3). Control shRNA was used to allow the comparison with 5-HT 2C R knockdown effects shown in Figure 4. Background activity and evoked responses of CeA neurons to innocuous (100 g/6 mm 2 ) and noxious (500 g/6 mm 2 ) stimulation of the left hindpaw were recorded before (predrug control) and after drug administration. Administration of SB242084 (100 µM, concentration in microdialysis fiber; 15 min) into BLA in SNL rats decreased background activity and evoked responses of CeA neurons significantly (n = 8 neurons, p < 0.01-0.05, compared to predrug, paired t-test; Figure 3C). The activity of an individual CeA neuron was shown before (control, ACSF; Figure 3A) and during antagonist administration ( Figure 3B). Administration of NBI27914 (100 µM, concentration in microdialysis fiber; 15 min) into the BLA also decreased background activity and evoked responses significantly (n = 5 neurons, p < 0.05, compared to predrug, paired t-test; Figure 3F). An individual example is shown before ( Figure 3D) and during ( Figure 3E) antagonist administration.

5-HT 2C R Knockdown Eliminates the Inhibitory Effects of Antagonists for 5-HT 2C R and CRF1 Receptor on CeA Neurons in Neuropathic Rats
CeA neurons were recorded in neuropathic rats 2 weeks after 5-HT 2C R shRNA was injected into the BLA to knock down 5-HT 2C R, i.e., 4 weeks post SNL surgery. In contrast to SNL rats without 5-HT 2C R knockdown (see Figure 3), administration of SB242084 (100 µM, concentration in microdialysis fiber; 15 min) into the BLA of SNL rats with 5-HT 2C R knockdown had no significant effect on background activity and on evoked responses of CeA neurons (n = 6 neurons, p > 0.05, compared to predrug, paired t-test; Figure 4C). Activity of an individual CeA neuron before (control, ACSF) and during antagonist administration is shown in Figure 4A,B. Administration of a CRF1 receptor antagonist (NBI27914, 100 µM, concentration in microdialysis fiber; 15 min) into the BLA also had no effect on background and evoked activity of CeA neurons recorded in SNL rats with 5-HT 2C R knockdown (n = 6 neurons, Figure 4F). Figure 4D,E shows an individual example before (control, ACSF) and during antagonist administration. The data suggest that 5-HT 2C R knockdown in BLA eliminated the inhibitory effects of antagonists for 5-HT 2C and CRF1 receptors on CeA neurons in neuropathic pain.

5-HT2CR Knockdown Eliminates the Inhibitory Effects of Antagonists for 5-HT2CR and CRF1 Receptor on CeA Neurons in Neuropathic Rats
CeA neurons were recorded in neuropathic rats 2 weeks after 5-HT2CR shRNA was injected into the BLA to knock down 5-HT2CR, i.e., 4 weeks post SNL surgery. In contrast to SNL rats without 5-HT2CR knockdown (see Figure 3), administration of SB242084 (100 µM, concentration in microdialysis fiber; 15 min) into the BLA of SNL rats with 5-HT2CR knockdown had no significant effect on background activity and on evoked responses of CeA neurons (n = 6 neurons, p > 0.05, compared to predrug, paired t-test; Figure 4C). Activity of an individual CeA neuron before (control, ACSF) and during antagonist administration is shown in Figure 4A,B. Administration of a CRF1 receptor antagonist (NBI27914, 100 µM, concentration in microdialysis fiber; 15 min) into the

Excitatory Effects of a 5-HT 2C R Agonist (WAY161503) Are Blocked by a CRF1 Receptor Antagonist (NBI27914) in Neuropathic Rats
CeA neurons were recorded in neuropathic rats 4 weeks after SNL surgery. Administration of a 5-HT 2C R agonist (WAY161503; 100 µM, concentration in microdialysis fiber; 15 min) into the BLA increased responses of CeA neurons to innocuous (100 g/6 mm 2 ) and noxious (500 g/6 mm 2 ) stimulation of the left hindpaw ( Figure 5). A selective CRF1 receptor antagonist (NBI27914; 100 µM, concentration in microdialysis fiber) reversed the effect of WAY161503. An individual neuron is shown in Figure 5A-D. Data are summarized in Figure 5E. The facilitatory effects of the 5-HT 2C R agonist on the responses to innocuous and noxious stimuli, but not background activity, were significant, and the CRF1 receptor antagonist inhibited the facilitation significantly (n = 5 neurons; background activity, p > 0.05, F ( BLA also had no effect on background and evoked activity of CeA neurons recorded in SNL rats with 5-HT2CR knockdown (n = 6 neurons, Figure 4F). Figure 4D,E shows an individual example before (control, ACSF) and during antagonist administration. The data suggest that 5-HT2CR knockdown in BLA eliminated the inhibitory effects of antagonists for 5-HT2C and CRF1 receptors on CeA neurons in neuropathic pain.

Excitatory Effects of a 5-HT2CR Agonist (WAY161503) Are Prevented by a CRF1 Receptor Antagonist (NBI27914) in Neuropathic Rats
Next, we determined if pre-treatment with a CRF1 receptor antagonist would prevent the 5-HT2CR agonist effect. CeA neurons were recorded in SNL rats 4 weeks after SNL surgery. Administration of a selective CRF1 receptor antagonist (NBI27914; 100 µM, concentration in microdialysis fiber) into the BLA decreased background and evoked activity of CeA neurons ( Figure 6). In the presence of the CRF1 receptor antagonist, a 5-HT2CR agonist (WAY161503; 100 µM concentration in microdialysis fiber; 15 min) had no effect on background activity and evoked responses of CeA neurons. An individual neuron is shown in Figure 6A-D. Data are summarized in

Excitatory Effects of a 5-HT 2C R Agonist (WAY161503) Are Prevented by a CRF1 Receptor Antagonist (NBI27914) in Neuropathic Rats
Next, we determined if pre-treatment with a CRF1 receptor antagonist would prevent the 5-HT 2C R agonist effect. CeA neurons were recorded in SNL rats 4 weeks after SNL surgery. Administration of a selective CRF1 receptor antagonist (NBI27914; 100 µM, concentration in microdialysis fiber) into the BLA decreased background and evoked activity of CeA neurons ( Figure 6). In the presence of the CRF1 receptor antagonist, a 5-HT 2C R agonist (WAY161503; 100 µM concentration in microdialysis fiber; 15 min) had no effect on background activity and evoked responses of CeA neurons. An individual neuron is shown in Figure 6A-D. Data are summarized in Figure 6E. The inhibitory effects of NBI27914 on background activity and responses to innocuous stimuli were significant, and the inhibitory effects persisted during co-administration of WAY161503, suggesting that the CRF1 receptor antagonist prevented the effects of the 5-HT 2C R agonist (n = 6 neurons; background activity, p < 0.01, F (2,10) = 14.54; innocuous, p < 0.05, F (2,10) = 6.073; noxious, p < 0.01, F (2,10) = 10.31; ANOVA). Figure 6E. The inhibitory effects of NBI27914 on background activity and responses to innocuous stimuli were significant, and the inhibitory effects persisted during co-administration of WAY161503, suggesting that the CRF1 receptor antagonist prevented the effects of the 5-HT2CR agonist (n = 6 neurons; background activity, p < 0.01, F(2,10) = 14.54; innocuous, p < 0.05, F(2,10) = 6.073; noxious, p < 0.01, F(2,10) = 10.31; ANOVA).

Excitatory Effects of a 5-HT 2C R Agonist (WAY161503) Are Eliminated by 5-HT 2C R Knockdown in Neuropathic Rats
CeA neurons were recorded in SNL rats 2 weeks after 5-HT 2C R shRNA was injected into the BLA to knock down 5-HT 2C R, i.e., 4 weeks post SNL surgery. In contrast to neuropathic rats without knockdown ( Figures 5 and 6), WAY161503 (100 µM concentration in microdialysis fiber; 15 min) had no significant facilitatory effects (p > 0.05, paired t-test; n = 6 neurons) in SNL rats with 5-HT 2C R knockdown ( Figure 7C). Activity of an individual CeA neuron before (control, ACSF) and during agonist administration is shown in Figure 7A Figure 7C). Activity of an individual CeA neuron before (control, ACSF) and during agonist administration is shown in Figure 7A,B.

Discussion
Serotonin 5-HT2CR has emerged as an important target for the treatment of neurological and psychiatric disorders, including substance use disorders, schizophrenia, impulsive/compulsive disorders, anxiety, depression, and epilepsy. 5-HT2CR can modulate multiple neurotransmitter systems to modulate network functions and behaviors, and effects may depend on the site of action in the nervous system, differential coupling to intracellular signaling pathways, and condition/disease (see reference [65]).
While there is evidence for pro-nociceptive and anxiogenic effects of 5-HT2CR (see the introduction section and [66]), its role in pain modulatory centers in the brain is an understudied area of research. Evidence from our recent study suggests that 5-HT2CR in the basolateral amygdala (BLA) increases neuronal activity in the amygdala output region (central nucleus, CeA) to drive neuropathic pain-like behaviors [45]. Knockdown of 5-HT2CR in the BLA decreased the excitatory drive of CeA neurons more strongly than synaptic inhibition and attenuated nociceptive and averse

Discussion
Serotonin 5-HT 2C R has emerged as an important target for the treatment of neurological and psychiatric disorders, including substance use disorders, schizophrenia, impulsive/compulsive disorders, anxiety, depression, and epilepsy. 5-HT 2C R can modulate multiple neurotransmitter systems to modulate network functions and behaviors, and effects may depend on the site of action in the nervous system, differential coupling to intracellular signaling pathways, and condition/disease (see reference [65]).
While there is evidence for pro-nociceptive and anxiogenic effects of 5-HT 2C R (see the introduction section and [66]), its role in pain modulatory centers in the brain is an understudied area of research. Evidence from our recent study suggests that 5-HT 2C R in the basolateral amygdala (BLA) increases neuronal activity in the amygdala output region (central nucleus, CeA) to drive neuropathic pain-like behaviors [45]. Knockdown of 5-HT 2C R in the BLA decreased the excitatory drive of CeA neurons more strongly than synaptic inhibition and attenuated nociceptive and averse affective behaviors in a neuropathic pain model, whereas 5-HT 2C R knockdown in sham controls only decreased synaptic inhibition without behavioral effects [45]. Further supporting an important role of 5-HT 2C R in amygdala-dependent behaviors, a 5-HT2CR antagonist in the BLA, but not CeA, enabled a systemic SSRI to inhibit pain behaviors in an arthritis model [61].
The present study advanced this line of research by testing the novel hypothesis that the contribution of 5-HT 2C R in the BLA to neuropathic pain-related hyperactivity in the CeA involves activation of CRF1 receptors. Increased CeA activity was blocked by local knockdown of 5-HT 2C R in the BLA (Figure 2) or by a 5-HT 2C R antagonist (SB242084) administered into the BLA (Figure 3). Stereotaxic administration of a 5-HT 2C R agonist (WAY161503) had facilitatory effects in the neuropathic pain model ( Figure 5) but not in sham controls (see Section 2.4), which is consistent with the results of our previous brain slice physiology study showing a contribution of 5-HT 2C R in the BLA to excitatory transmission in the pain model but synaptic inhibition under control conditions [45].
There is evidence to suggest that in the amygdala CRF acts downstream of 5-HT 2C R [26]. Elimination of 5-HT 2C R in knockout mice blocked the neurochemical (FOS) activation of CRF CeA neurons in response to an anxiogenic stimulus (open field) [26]. The midbrain dorsal raphe nucleus is a major source of serotonergic input to the amygdala, and 5-HT axons innervate pyramidal cells and interneurons in the BLA, but not CeA [20,[78][79][80]). 5-HT 2C R is expressed postsynaptically to serotonergic axons [17,18]. Pharmacologic activation of 5-HT 2C R in the BLA, but not CeA, increased measures of anxiety-like behaviors in the elevated plus maze [81], elevated T-maze [15], light-dark transition test [15], and open-field test [82]. These lines of evidence point to the BLA as an important site of 5-HT 2C R action to modulate CeA function.
In the present study, we recorded from CeA neurons to determine their modulation by 5-HT 2C R through CRF1 receptors in the BLA. The BLA receives polymodal, including nociceptive, information and provides synaptic excitation as well as feedforward inhibition to CeA neurons [30][31][32]83]. While the CeA is generally considered the output region for major amygdala function [70], including emotional-affective aspects of pain and pain modulation [30][31][32]84], the BLA has also been implicated in top-down pain modulation [85,86], averse affective aspects of pain [83], and cognitive deficits associated with pain [56]. Our recent study showed a 5-HT 2C R-driven shift towards excitatory influences of the BLA on CeA activity and behaviors in a neuropathic pain model [45].
Here, we report that a CRF1 receptor antagonist administered into the BLA blocks or prevents ( Figures 5 and 6) the increase of CeA neuronal activity by a 5-HT 2C R agonist (WAY161503) administered into the BLA in the neuropathic pain model ( Figure 5), suggesting that 5-HT 2C R activation generates endogenous activation of CRF1 receptors, presumably through the activation of CRF neurons in the CeA [67][68][69][70][71] and release of CRF into the CeA and BLA. 5-HT 2C R knockdown in the BLA eliminated the agonist effects (Figure 7), confirming that they were mediated by 5-HT 2C R. 5-HT 2C R knockdown in the BLA of neuropathic rats also blocked the inhibitory effects of a CRF1 receptor antagonist (Figure 4), providing further support of our hypothesis that 5-HT 2C R activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.
As a note of caution, we did not confirm the effects of 5-HT 2C R knockdown in this study. The viral vector that we used has been validated thoroughly [63,64] and we showed previously that focal (BLA) injection of AAV-expressing 5-HT 2C R shRNA, but not control shRNA, decreased 5-HT 2C R mRNA and immunoreactivity [45]. Using the same tools, assays, and protocol, there was no reason to expect different results. Another consideration is drug application by microdialysis. This technique offers several advantages, such as avoidance of volume effect and steady state application, but the exact tissue concentration can only be estimated (see the materials and methods section). We selected concentrations based on our previous studies using antagonists for the CRF1 receptor (NBI27914) [36,56,87,88] and 5-HT 2C R (SB242084), which was supported by data in the literature (see the materials and methods section). Importantly, local (BLA) knockdown of 5-HT 2C R eliminated the inhibitory effect of SB242084 ( Figure 4) and the facilitatory effects of a 5-HT 2C R agonist (WAY161503) (Figure 7) in neuropathic rats, which strongly argues for specific actions on 5-HT 2C R. Finally, we did not perform off-site drug injections in this study. However, we showed previously that 5-HT 2C R knockdown in the BLA, but not the CeA, had inhibitory neuronal and behavioral effects [45]. Data in the literature also support an action of 5-HT 2C R in the BLA, but not the CeA (see the introduction and discussion sections). In this study, 5-HT 2C R knockdown in the BLA blocked the effects of pharmacological agents (Figures 4 and 7). While CRF1 receptors are expressed and functional both in BLA and CeA [36,56], offsite injections of CRF or CRF1 and CRF2 receptor antagonists into the neighboring striatum had no effect [36,87]. These findings suggest that 5-HT 2C R in the BLA and CRF1 in the amygdala contributed to the observed effects on the CeA neurons, but the site of action of CRF1 in the BLA versus CeA could not be distinguished here.
In summary, the new results of this study link CRF1 receptor activation to 5-HT 2C R in the BLA in a neuropathic pain model. The 5-HT 2C R and CRF1 receptor interaction results in increased activity of CeA neurons. Our previous work showed an important role of 5-HT 2C R in the BLA for CeA neuronal hyperactivity in neuropathic pain. The new data show that 5-HT 2C R knockdown eliminates the inhibitory effects of a CRF1 receptor antagonist and the facilitatory effects of a 5-HT 2C R agonist, and that the agonist effects are also blocked or prevented by a CRF1 receptor antagonist.

Animals
Male Sprague Dawley rats (250-350 g, Harlan Laboratories) were housed in a temperaturecontrolled room in the animal facility under a 12 h light/dark cycle with unrestricted access to water and food. On the day of the experiment, rats were transferred to the laboratory and allowed to acclimate for at least 1 h. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Texas Tech University Health Sciences Center, TX, USA (Protocol 14006, approved 23 June 2014) and conformed to the guidelines of the International Association for the Study of Pain and of the National Institutes of Health.

Neuropathic Pain Model
The spinal nerve ligation (SNL) model [89] was used as described in our previous study [45]. Rats were anesthetized with isoflurane (3-4% induction, 2% maintenance) while the L5 spinal nerve was tightly ligated using 6-0 silk, using sterile techniques. In the sham control group, the spinal nerve was exposed but not ligated. The SNL model generates stable neuropathic pain behaviors that last for at least 4-5 weeks [45]. While the experimenter was not blinded to the experimental condition for technical reasons, the analysis involved a blinded experimenter.

Viral Vector for 5-HT 2C R Knockdown
For local (basolateral amygdala, BLA) knockdown of 5-HT 2C R, recombinant AAV2 vectors expressing a short hairpin RNA (shRNA) directed at the 5-HT 2C R or a control hairpin were used [45,63,64]. Vectors were obtained from the Vector Core at the University of North Carolina at Chapel Hill (UNC Vector Core). 5-HT 2C R or control shRNA-eGFP AAV2 vector (1 µL) was injected stereotaxically into the BLA of rats anesthetized with isoflurane (3-4% induction, 2% maintenance; precision vaporizer, Harvard Apparatus), two weeks after neuropathic or sham surgery. The following coordinates were used: 2.5 mm caudal to bregma, 4.8 mm lateral, 8.0-8.5 mm depth. Two weeks after neuropathic surgery, control shRNA-eGFP AAV vector (1 µL) or 5-HT 2C R shRNA-eGFP AAV vector (for 5-HT 2C R knockdown; 1 µL) was injected stereotaxically into the BLA. Rats recovered for 2 weeks before the electrophysiological recordings to allow for stable transgene expression. 5-HT 2C R knockdown was validated with qPCR, western blot analysis and immunohistochemistry in our previous study [45].

Systems Electrophysiology
Extracellular single-unit recordings were made from neurons in the laterocapsular division of the CeA (CeLC) as described previously ( [90], for recent references see [45]). Rats were anesthetized with isoflurane (3-4% induction, 2% maintenance). Core body temperature was maintained at 37 • C with a homeothermic blanket system. The animal was mounted in a stereotaxic frame (Kopf Instruments) and a craniotomy was performed at the sutura frontoparietalis level to allow the insertion of the recording electrode and microdialysis probe for drug or vehicle administration.
Neurons were recorded with glass-insulated carbon filament electrodes (4-6 MΩ) using the following coordinates: 2.3-2.8 mm caudal to bregma, 3.8-4.2 mm lateral to midline, depth 7-8 mm. The recorded signals were amplified, band-pass filtered (300 Hz to 3 kHz), displayed on an analog oscilloscope, and processed by an interface (1401 Plus; CED). Spike2 software (version 4; CED) was used for spike sorting, data storage, and analysis of neuronal activity. Spike size and configuration were monitored continuously. For each neuron, a spike template was created during a 5 min baseline recording period. Only those neurons were included in the study that showed a spike configuration that matched the preset template and could be clearly discriminated from activity in the background throughout the experiment. Neurons were identified by monitoring background activity and responses to search stimuli, i.e., compression of the hindpaw at innocuous (100 g/6 mm 2 ) and noxious (500 g/6 mm 2 ) intensities with calibrated forceps. Noxious stimuli were used sparingly. Neurons included in this study had a receptive field in the hindpaw (L5 territory) and were activated more strongly by noxious than innocuous mechanical stimuli. One neuron was recorded per animal to allow for verification of the recording with electrolytic lesion at the end of the experiment (see "verification of recording site") and to avoid confounding effects of drug applications.

Neuronal Activity Analysis
Background activity (in the absence of intentional stimulation) was measured for 5 min and evoked activity was recorded during brief (15 s) innocuous and noxious test stimuli applied to the hindpaw. Net evoked activity was calculated by subtracting background activity (mean value of 1-3 min) preceding the stimulus from the total activity during stimulation. Neuronal activity was expressed as spikes per second. Spike2 software burst analysis script was used to analyze interspike interval (ISI) distribution and burst-like activity for each CeA neuron, as described previously [45]. Burst-like activity was defined as in our previous study [45]-a silent period of at least 100 ms preceded the first spike in a burst, which was followed by a second spike with an ISI of ≤10 ms. The coefficient of variation (CV2) was calculated to detect irregular firing based on the variation in the ISIs. CV2 was calculated as described previously [45] by assessing the SD and mean firing for two adjacent ISIs (CV2 = 2 (ISI2−ISI1)/(ISI2+ISI1)). The CV2 for each pair of adjacent ISIs was plotted against the mean of those two ISIs. Small CV2 values indicate regular firing, whereas large CV2 values (≥1) indicate irregular firing.

Drugs and Drug Application by Microdialysis
A 5-HT 2C receptor agonist (WAY161503), a 5-HT 2C receptor antagonist (SB242084), and a CRF1 receptor antagonist (NBI27914) were purchased from Tocris Bioscience. Several hours before the start of the electrophysiological recordings, a microdialysis probe (CMA11/Microdialysis; 250 µm membrane diameter; 1 mm membrane length) was lowered vertically into the BLA, ipsilateral to the recording electrode, using the following coordinates: 2.5 mm caudal to bregma, 4.8 mm lateral to midline, 9.0 mm depth. The distance between the microdialysis probe and recording electrode was~0.5 mm. Before each drug application, artificial cerebrospinal fluid (ACSF) was pumped through the microdialysis fiber for about 1 h at a rate of 5 µL/min to establish equilibrium in the tissue. The microdialysis probe was connected to an infusion pump (Harvard) using PE-50 tubing. The ACSF contained (in mM) 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose oxygenated and equilibrated to pH ± 7.4. Drugs were dissolved in ACSF on the day of the experiment at a concentration of 100 times that was predicted to be needed based on data from our previous studies [36,48,61,91,92] and data in the literature [93][94][95]. Drugs were administered into the BLA at a rate of 5 µL/min.

Verification of Recording Site
At the end of each experiment, the recording site in the CeA was marked by an electrolytic lesion with DC (250 mA for 3 min) injected through the recording electrode. The brain was removed and submerged in 10% formalin and potassium ferrocyanide. Tissues were stored in 30% sucrose before they were frozen sectioned at 50 µm and stained with hematoxylin and eosin. The boundaries of the different amygdala nuclei were easily identified under the microscope. Lesion/recording sites were verified histologically and plotted on standard diagrams.

Statistical Analysis
All averaged values are given as the mean ± SE. Statistical significance was accepted at the level p < 0.05. GraphPad Prism 5.0 software was used for all statistical analyses. Statistical analysis was performed on the raw data.
Author Contributions: G.J. carried out the experiments, analyzed the data, created figures, and provided a first draft of the manuscript. V.N. conceived the study, developed the experimental design, assisted with data analysis and presentation, and finalized the manuscript.