Regional Differences Within the Anterior Cingulate Cortex in the Generation Versus Suppression of Pain Affect in Rats

Accepted Manuscript

Regional differences within the Anterior Cingulate Cortex in the Generation versus Suppression of Pain Affect in Rats Casey A. Mussio , Steven E. Harte , George S. Borszcz PII: DOI: Reference:

S1526-5900(19)30742-4 https://doi.org/10.1016/j.jpain.2019.06.003 YJPAI 3758

To appear in:

Journal of Pain

Received date: Revised date: Accepted date:

5 April 2017 22 May 2019 2 June 2019

Please cite this article as: Casey A. Mussio , Steven E. Harte , George S. Borszcz , Regional differences within the Anterior Cingulate Cortex in the Generation versus Suppression of Pain Affect in Rats, Journal of Pain (2019), doi: https://doi.org/10.1016/j.jpain.2019.06.003

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Regional differences within the Anterior Cingulate Cortex in the Generation versus Suppression of Pain Affect in Rats Casey A. Mussio1, Steven E. Harte2, and George S. Borszcz1

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Behavioral and Cognitive Neuroscience Program, Department of Psychology, Wayne State

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University, Detroit, MI 48202

Chronic Pain and Fatigue Research Center, Department of Anesthesiology, University of

Disclosures: Authors report no conflicts of interest

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Michigan, Ann Arbor, MI 48109

This work was supported by the National Institutes of Health grant: R01 NS045720

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Running Title: NMDA in Anterior Cingulate Cortex and Pain Affect

Corresponding Author: George S. Borszcz

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Department of Psychology Wayne State University

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Detroit, MI 48202

Tel.: +1 313 577 2895

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fax: +1 313 577 7636

E-mail address: [email protected] URL: http://sun.science.wayne.edu/~gborszcz/

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Abbreviations: rACC (rostral anterior cingulate cortex) cACC (caudal anterior cingulate cortex) VAD (vocalization afterdischarge) VDS (vocalizations during shock)

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SMR (spinal motor reflex) NMDA (N-Methyl-D-Aspartate)

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AP-5 (D-2-amino-5-phosphonovalerate)

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Abstract The anterior cingulate cortex (ACC) modulates emotional responses to pain. Whereas, the caudal ACC (cACC) promotes expression of pain affect, the rostral ACC (rACC) contributes to its suppression. Both subdivisions receive glutamatergic innervation, and the present study evaluated the contribution of NMDA receptors within these subdivisions to rats’ expression of

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pain affect. Vocalizations that follow a brief noxious tail shock (vocalization afterdischarges, VAD) are a validated rodent model of pain affect. The threshold current for eliciting VAD was increased in a dose-dependent manner by injecting NMDA into the rACC, but performance (latency, amplitude and duration) at threshold was not altered. Alternately, the threshold current

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for eliciting VAD was not altered following injection of NMDA into the cACC, but its amplitude and duration at threshold were increased in a dose-dependent manner. These effects were limited to Cg1 of the rACC and cACC, and blocked by pre-treatment of the ACC with the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP-5). These findings demonstrate that NMDA receptor agonism within the cACC and rACC either increase or

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decrease emotional responses to noxious stimulation, respectively.

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Perspective: NMDA receptor activation of the rostral and caudal anterior cingulate cortex respectively inhibited or enhanced rats’ emotional response to pain. These findings mirror those

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obtained from human neuroimaging studies; thereby, supporting the use of this model system in

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evaluating the contribution of ACC to pain affect.

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Key Words: emotion; analgesia; hyperalgesia; glutamate; vocalization; ACC; NMDA

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Introduction The anterior cingulate cortex (ACC) regulates the unpleasantness that accompanies acute and chronic pain with different subdivisions implicated in its generation versus suppression, and glutamate signaling may govern both processes. Activation of the caudal ACC (cACC) contributes to pain affect in humans and rodents 2, 32, 71, 88, 99 via glutamate acting at NMDA and

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non-NMDA glutamate receptors 67, 115. Vocalizations of guinea pigs elicited by noxious

electrical stimulation are increased following microinjection of the NMDA receptor agonist homocysteic acid into cACC 120. In healthy human subjects, cACC glutamate levels are elevated in response to acute painful stimulation and positively correlated with baseline pain sensitivity 31, . Chronic pain patients also exhibit elevated glutamate levels in cACC that are positively

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correlated with pain severity 51 38. Alternately, reductions of glutamate levels in cACC correlated positively with relief of chronic pain, and healthy subjects administered sub-anesthetic doses of ketamine (noncompetitive NMDA antagonist) exhibited suppression of both pain affect and pain-induced activation of cACC to an acute noxious stimulus. 100, 103

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Conversely, the rostral ACC (rACC) contributes to suppression of pain affect. Human neuroimaging studies demonstrated activation of rACC, and associated subcortical

10, 70, 82, 83, 105

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antinociceptive circuitry, during a variety of interventions reported to reduce pain unpleasantness . Functional connectivity of rACC with subcortical antinociceptive circuits

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correlated positively with glutamate levels in rACC, and chronic pain in humans and rats corresponded with decreased levels of glutamate and glutamate signaling in rACC, reduced

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connectivity of rACC with subcortical antinociceptive circuits, and reduced rACC volume 43, 47, 54, 56, 57, 63, 90, 94, 96

. Pain relief in humans and rats is accompanied by recovery of rACC volume,

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and restoration of its’ functional connectivity with subcortical antinociceptive circuits 70, 78, 86, 90. In the rACC, downregulation of NMDA receptors is observed in a rat model of neuropathic pain with pain relief associated with normalization of NMDA receptor concentrations 75. We reported that microinjection of morphine into nucleus parafascicularis (PF = an intralaminar thalamic nucleus that provides glutamatergic afferents to ACC) suppressed vocalization afterdischarges (VAD) of rats elicited by noxious tail shock that was blocked by pretreatment of rACC with a

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NMDA receptor antagonist 49. VAD provides a direct measure of pain affect as its production is necessary for tail shock to support aversive conditioning12, 14, 20. In contrast, several studies reported that NMDA receptor activation of the rat rACC promotes pain affect as it is required for acquisition of conditioned place aversion supported by formalin injected into the paw (F-CPA) 24, 44, 60, 69. Yet, pain-induced aversive conditioning in rats is

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enhanced with downregulation of NMDA receptors in rACC and normalized with recovery of NMDA receptor concentrations72. If rats are to be used to elucidate the role of ACC in pain processing in humans then this discrepancy regarding the contribution of rACC NMDA receptors to suppression versus generation of pain affect needs to be resolved. Here we evaluated the

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effects of NMDA receptor agonism in rACC versus cACC on production versus suppression of VAD.

Experimental Procedures Animals

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Male Long-Evans rats (Charles River, Portage, MI) between 100 and 150 days old at the time of surgery were used. Rats were housed as pairs in polycarbonate cages in a climate-controlled

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vivarium (lights on 0700 h to 1900 h), and given ad libitum access to food and water. Testing occurred during the light portion of the cycle. Rats were handled daily for one week prior to

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surgery to minimize the effects of stress from human contact. All procedures were approved by the IACUC of Wayne State University and conform to the NIH guidelines (Guide for the Care

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and Use of Laboratory Animals, NIH Publication 86-23).

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Anatomical Definition of ACC subdivisions The rACC was identified as the area encompassing both the precallosal cingulate gyrus that

lies immediately anterior and ventral to the genu of the corpus callosum (approximately bregma 4.80 mm to 1.40 mm) and the supracollosal cingulate gyrus at the level of the genu (approximately bregma 1.40 mm to 0.00 mm). The cACC (or midcingulate cortex) was defined as the cingulate gyrus superior to the body of the corpus callosum (approximately bregma 0.00

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mm to -2.20 mm). This delineation is in general agreement with the regional segregation of the rodent ACC used elsewhere (see inset in Figure 5) 62, 108.

Stereotaxic Surgery and Histology Under aseptic conditions, rats were anesthetized with sodium pentobarbital (60 mg/kg, ip)

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following pretreatment with atropine sulfate (1 mg/kg, ip). Stereotaxic coordinates (Paxinos and Watson 81) were measured relative to the bregma suture and the top of the level skull. Stainless steel 26-gauge double-cannulae (Plastics One, Roanoke, VA) were implanted above either the rACC (AP: + 2.70 mm, L: + 0.6 mm, DV: -1.2 mm), or cACC (AP: -0.50 mm, L: + 0.4 mm, DV:

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-1.0 mm). Cannulae were affixed to the skull with bone screws and cranioplastic dental cement, and fitted with an obturator that extended the length of the cannulae to maintain patency. Rats were given 7-10 days to recover before testing. During recovery rats were handled each day to assess wound healing, general health, and recovery of body weight. At the end of the recovery period all rats had recovered their pre-surgical weight, their surgical wounds were healed, and

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they exhibited no sensitivity to palpation of the wound.

At the conclusion of testing, rats were sacrificed by carbon dioxide asphyxiation. Injection

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sites were marked by safranin-O dye (0.25 µl) and brains were extracted and placed in a 20% (w/v) sucrose formalin solution for 48-72 hours. Brains were sectioned at 40 µm on a freezing

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microtome, and injection sites were localized using the rat brain atlas of Paxinos and Watson 81.

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Histological analysis was done without knowledge of the behavioral results.

Assessment of Pain Affect

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Research in this laboratory validated vocalization afterdischarges (VAD) as a rodent model of pain affect. These vocalizations occur immediately following application of noxious tailshock, are organized within the forebrain, and have distinct spectrographic characteristics compared to vocalizations that occur during shock (VDS) 14, 16, 20, 25, 50. Systemically administered drug treatments that preferentially suppress the emotional response of humans to pain 27, 42, 84 also preferentially suppress production of VAD 17. Generation of VAD is suppressed by damage of or drug treatments into forebrain sites known to contribute to

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production of the affective response of humans to clinical and experimental pain 15, 20, 50, 74, 77, 104, 119

. Additionally, the capacity of noxious tail shock to support Pavlovian conditioned

vocalizations and avoidance conditioning is directly related to its production of VAD 12, 14, 16, 20. The effects of experimental treatments on current intensity that elicits VAD (VAD threshold)

thresholds and performance of VDS and SMR.

Testing Apparatus

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and VAD performance (amplitude, latency and duration) were compared with their effects on the

Testing was controlled by custom computer programs via a multifunction interface board

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(DT-2801, Data Translation, Marlboro, MA) installed in a PC. Rats were placed into custom made Velcro body suits and restrained on a Plexiglas pedestal using Velcro strapping that passes through loops located on the underside of the suits. This design maintains the rat in a crouching posture throughout testing, permits normal respiration and vocalizing, and allows unobstructed access to the head for intracerebral injections (see photograph in 14). Testing was conducted

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within a sound attenuating, lighted, and ventilated isolation chamber equipped with a small window that enabled visual monitoring of rats during testing.

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Tail shock (20 ms pulses at 25 Hz for 1,000 ms) was delivered by a computer controlled constant current shocker (STIMTEK, Arlington, MA) through electrodes (0-gauge stainless steel

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insect pins) placed intracutaneously on opposite sides of the tail, 7.0 cm (cathode) and 8.5 cm (anode) from the base. The intensity, duration, and timing of tail shocks were controlled by the

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computer. Current intensity was monitored by an A-to-D converter that digitized (500 Hz sampling rate) an output voltage of the shocker that was proportional to the current delivered. Spinal motor reflexes (SMRs) were measured with a semi-isotonic displacement transducer

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(Lafayette Instruments Model 76614, Lafayette, IN) attached to the rat’s tail with cotton thread. The arm of the transducer was positioned behind and perpendicular to the tail such that the thread extended in a straight line directly behind the rat. The output voltage of the transducer was amplified (x50) and then digitized (500 Hz sampling rate) by an analog-to-digital converter of the interface board. SMR was defined as movement of the transducer arm by at least 1.0 mm following shock onset. The computer recorded the latency from tail shock onset (ms), peak

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amplitude (mm), and magnitude (cm x ms) of tail movement on each trial. Displacements up to 100 mm can be detected, and latencies in 2 ms increments can be measured. Vocalizations were measured by a pressure-zone microphone (Realistic model 33-1090, Tandy, Ft. Worth, TX) located on the wall of the testing chamber 15 cm from the rat’s head. The microphone was connected to an audio amplifier (Technics model SA-160, Tandy, Ft. Worth,

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TX) and a 10-band frequency equalizer adjusted to selectively amplify frequencies above 1500 Hz. The filtering of low frequencies prevented extraneous noise (i.e., rats’ respiration and

movement artifacts) from contaminating vocalization records. The output of the amplifier was integrated by a Coulbourn Instruments (Allentown, PA) contour following integrator (2 ms time

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base) and digitized (500 Hz sampling rate) by a separate analog-to-digital converter of the

interface board. The peak intensity (in decibels: SPL, B scale), latency (ms), and duration (ms) of vocalizations during the shock epoch (VDS) and for the 2,000 ms interval following shock termination (VAD) were recorded by the computer. VDS and VAD latencies were measured from shock onset and offset, resepectively. The ambient background noise level in the isolation

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chamber was 54.0 dB, and sounds above 57.0 dB were considered vocalizations. See13, 14, 16 for

VDS.

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Behavioral Testing

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digitized oscilloscope tracings of VAD, VDS and SMR, and spectrographic records of VAD and

On 3 consecutive days prior to testing, rats were adapted to the testing apparatus for 20-25

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min/day to minimize the effects of restraint stress. Test sessions consisted of 20 randomly presented trials. On 16 trials different intensities of tail shocks between 0.02 mA and 2.50 mA

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were delivered once, and on 4 trials no tail shocks were delivered so as to assess false alarm rates. Trials were presented with a minimum inter-trial interval of 30 seconds, and each test session concluded within 20 minutes. Following each test session, the testing apparatus was cleaned with 5% ammonia hydroxide to eliminate stress odors 37. Rats do not exhibit signs that they are in pain or distress following testing, they are not agitated or aggressive. Monitoring of their behavior in the home cage following testing revealed no pain behaviors or enduring stress reactions such as autotomy of the tail or tenderness of the

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tail (i.e., vocalizing when the tail is palpated), or behavioral changes related to stress (i.e., weight loss, difficulty handling). These results are consistent with earlier histological studies that were made of rats’ tails following the testing procedure. Evaluation of rats’ tails by a veterinary pathologist (Dr. Jack Hoopes, DVM, Ph.D. of Dartmouth Medical School) confirmed that tissue damage is not produced during testing. Interstitial edema, hemorrhage of blood vessels or

observed either immediately after testing or 24 hr later.

Drug Injections

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capillaries, necrosis of muscle, and nerve damage (loss of myelin, atrophy of axons) are not

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Intracerebral injections were administered in a constant volume of 0.25 µl via 33-guage injectors that extended 1.2 mm beyond the cannula tip. Injections were made at a constant rate over 1 min via an infusion pump (Harvard Model PHD 2000), and injectors were left in place for 2 min after the completion of injections to aid the diffusion of drugs into the tissue. NMDA (Nmethyl-d-aspartic acid) and safranin-O were purchased from Sigma-Aldrich (St. Louis, MO) and

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AP-5 (2-amino-5-phosphonovaleric acid) was purchased from Tocris (Ellisville, MO). Drugs were dissolved in sterile isotonic saline. Pain testing began 5 – 7 minutes following completion

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of intracerebral injections. The injection volume precluded restriction of treatments to particular

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cortical layers so no attempt was made to relate drug effects to cortical layer.

Experimental Design

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Experiment 1: Dose-Response Analysis. NMDA treatment groups received bilateral microinjection of 1 dose of NMDA (.25, .5 or 1.0

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g/side) and saline prior to 2 separate test sessions into either the rACC (n = 8 –10/group = 27 total) or cACC (n = 6/group = 18 total). Doses of NMDA were based on our previous findings101, 102 and the results of preliminary studies. The order of NMDA and saline injections was counter-balanced for each group. Test sessions were separated by 5-7 days.

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Experiment 2: Pharmacology Specificity Pharmacological specificity of the effects of NMDA was assessed by administering the selective NMDA receptor antagonist AP-5 into the rACC (8 – 9/group = 26 total) or cACC (6/group = 18 total) immediately prior to injection into these sites of the dose of NMDA that produced the greatest effects in Experiment 1 (1.0 µg/side). Doses of AP-5 (rACC = 4 µg/side,

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cACC = 2 µg/side) were chosen based on the results of preliminary experiments, and reports that these doses were most effective in blocking the antinociceptive effects of NMDA49, 111. Separate groups were administered: AP-5 + NMDA, saline + NMDA or AP-5 + saline. All groups also received control injections of saline + saline. Control and drug treatments were given in a

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counter-balanced manner in two separate test sessions. Test sessions were separated by 5-7 days.

Experiment 3: Anatomical Specificity

Anatomical specificity was assessed by administering doses of NMDA (1.0 µg/side) that produced the greatest effects in Experiment 1 into sites surrounding the rACC (n = 8) and cACC

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(n = 9), ventral and lateral to the primary target regions of the ACC (Cg1). Test sessions were

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separated by 5-7 days.

Data Analysis

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Following each test session, data were reorganized in ascending order according to tail shock intensity. VAD, VDS, and SMR thresholds were calculated as the lesser current intensity from a

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string of at least two consecutive intensities that generated the response. Performance (latency, amplitude, magnitude, and duration) of each response at threshold was also analyzed.

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For dose-response analyses, threshold of each response and performance of each response at threshold were compared across doses of NMDA using one-way ANOVA with drug dose as a between subject variable. Significant ANOVA was followed by post-hoc Dunnett’s test that compared thresholds and performance at threshold following saline treatment and treatment with each dose of drug. Analysis of pharmacological specificity used two-factor (antagonist and agonist) ANOVA to assess the effects of pre-treatment of rACC or cACC with AP-5 on the effects exerted by NMDA

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injections at these sites. Significant two-factor ANOVA was followed by planned pair-wise comparisons using Student’s t-test for independent or paired groups. The alpha level was 0.05 for all analyses.

Results

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Response Profile

As demonstrated by Carroll and Lim 25, SMR, VDS, and VAD reflect nociceptive processing at progressively higher levels of the neuraxis. Their analysis of rats that received transections of the neuraxis revealed that SMR is organized at the spinal level, VDS within the medulla below

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the pontomedullary border, and VAD within the forebrain. Consistent with our previous reports, rostrally organized responses were rarely generated without those integrated more caudally within the CNS. Across all experiments, VAD generation without concomitant generation of VDS or SMR occurred on 0.44% of trials. Similarly, VDS was generated without SMR on 0.36% of trials in which only VDS and SMR were elicited. False alarm rates for each response

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were low (SMR = 2.01%, VDS = 1.81%, VAD = 0.92%) indicating that responses were not induced by drug administration, were not occurring spontaneously, and were not conditioned

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responses to the context, but instead were generated by tail shock.

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Experiment 1: Dose-Response Analysis.

Threshold currents were evenly distributed throughout the testing sequence indicating that

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drug effects were maintained throughout the testing session. Thresholds and performance variables following saline treatment did not differ across groups that also received different

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doses of NMDA (rACC: threshold and performance, Fs(2,26) < 1.29, ps > 0.29; cACC: threshold and performance, Fs(2,17) < 1.57, ps > 0.24); therefore, thresholds and performance following saline treatment were combined across groups for comparison with thresholds and performance following NMDA treatments. The dose-dependent effects of NMDA administered into the rACC on response thresholds and performance of responses at threshold are shown in Figure 1. Microinjection of NMDA into the rACC produced a dose-dependent increase in the intensity of tail shock needed to elicited

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VAD (F(3,53) = 35.47, p < .001) and VDS (F(3,53) = 32.31, p < .001), but did not affect threshold for eliciting SMR, F(3,53) < 1.0. Post-hoc pair-wise comparisons of VAD and VDS thresholds following saline treatment and treatment with each dose of NMDA revealed that VAD and VDS thresholds were initially elevated following administration of NMDA doses of 0.50 µg/side and 1.0 µg/side, respectively (Dunnett’s, ps < .05). No performance variable for any of

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the responses was affected by NMDA treatment of the rACC, all Fs(3,53) < 1.0. ----------------------------------------Insert Figure 1 about here

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On the other hand, NMDA injected into the cACC failed to alter the tail shock intensity needed to elicit any response (Figure 2, all Fs(3,35) < 1.0). However, NMDA administration produced dose-dependent increases at threshold of VAD amplitude (F(3,35) > 55.71, p < .001) and duration (F(3,35) > 3.32, p < .05), and VDS amplitude (F(3,35) > 13.50, p < .001) but did not alter latency of either response, F(3,35) < 1.0. Post-hoc pair-wise comparison of VAD and

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VDS performance following saline and NMDA treatments revealed that amplitude and duration of VAD were respectively enhanced following administration of NMDA in doses of 0.5 µg/side

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and 1.0 µg/side, and VDS amplitude following injection of 1.0 µg/side (Dunnett’s, ps < .05). Performance at threshold of SMR was not affected by NMDA treatments, all Fs(3,35) < 1.0.

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Experiment 2: Pharmacology Specificity Thresholds and performance variables following saline + saline treatment did not differ

across groups that also received different experimental treatments (rACC: threshold and performance, Fs(2,25) < 1.0, ps > 0.45; cACC: threshold and performance, Fs(2,17) < 1.0, ps > 0.40); therefore, thresholds and performance following saline + saline treatment were combined across groups for comparison with thresholds and performance following experimental treatments.

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The capacity to block the effects of NMDA administered rACC or cACC by pre-treating these sites with the NMDA receptor antagonist AP-5 is depicted in Figures 3 and 4, respectively. For the rACC, pretreatment with AP-5 (4 µg/side) blocked the increases in VAD and VDS thresholds that result from injection of NMDA (1.0 µg/side) into the rACC. Two-factor ANOVA across treatment groups revealed significant main effects of Agonist (Fs(1,52) > 27.76,

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ps < .001) and Antagonist (Fs(1,52) > 25.58, ps < .001), and a significant Agonist x Antagonist interaction (Fs(1,52) > 27.82, ps < .001) for both VAD and VDS. These findings demonstrate that, consistent with Experiment 1, the injection of NMDA into rACC significantly elevated VAD (t(8) = 6.18, p < .001) and VDS (t(8) = 5.03, p < .001) thresholds compared to following

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saline treatment. Pre-treatment with AP-5 blocked these increases in thresholds, as thresholds of VAD and VDS following AP-5 + NMDA treatment were significantly reduced compared to thresholds following saline + NMDA treatment (ts(15) > 3.92, ps < .001), and did not differ from thresholds observed following saline + saline treatment, (ts(7) < 1.0). Administration of AP-5 had no effect on baseline thresholds of VAD or VDS (saline + saline versus AP-5 + saline, ts(8)

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< 1.0). Also consistent with the findings of Experiment 1, no effects of NMDA treatment were observed on any performance variable for any of the responses (all Fs(1,52) < 1.0), or on the

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threshold of SMR, Fs(1,52) < 1.05, ps > .320

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Insert Figures 3 and 4 about here

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For the cACC, pre-treatment with a dose of AP-5 (2 µg/side) was effective in blocking increases in VAD amplitude and duration and VDS amplitude generated by injection of NMDA

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(1.0 µg/side). Two-factor ANOVA across treatment groups revealed significant main effects of Agonist (Fs(1,36) > 7.38, ps < .015) and Antagonist (Fs(1,36) > 6.63, ps < .015), and a significant Agonist x Antagonist interaction (Fs(1,36) > 6.79, ps < .015) for both amplitude and duration of VAD and amplitude of VDS. Consistent with the findings of Experiment 1, these results show that the injection of NMDA into cACC significantly increased VAD and VDS amplitude (t(5) = 7.06, p < .001) and VAD duration (t(5) = 3.38, p < .001) compared to following saline treatment. Pre-treatment with AP-5 blocked these increases in performance, as

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performance of VAD and VDS following AP-5 + NMDA treatment were significantly reduced compared to following saline + NMDA treatment (ts(10) > 3.84, ps < .001), and did not differ from that observed following saline + saline treatment, (ts(5) < 1.0). Administration of AP-5 had no effect on baseline amplitude or duration of either VAD or VDS (saline + saline versus AP-5 + saline, ts(5) < 1.0). Also consistent with the findings of Experiment 1, no effects of NMDA

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treatment were observed on thresholds of any of the responses (all Fs(1,36) < 1.0), or on performance of SMR, all Fs(1,36) < 1.0.

Experiment 3: Anatomical Specificity

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Schematics and representative photomicrographs depicting injection sites within the rACC and cACC that received the highest doses of NMDA (1.0 µg/side) in Experiment 1 and extraACC sites that also received this dose of NMDA are shown in Figure 5. Anatomical specificity of NMDA administered into the rACC and cACC was evaluated by examining effects following injections into sites lateral (secondary motor cortex = M2) and ventral (prelimbic cortex = Prl or

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Cg2) to the target region of Cg1.

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Insert Figure 5 about here

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Within rACC and cACC, thresholds and performance observed following injection of saline into sites surrounding Cg1 did not differ and these data were combined for comparison of the

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effects of NMDA. Increases in VAD and VDS thresholds observed following administration of NMDA into rACC was not observed with its injection into Prl or M2 (Figure 6). Comparison of

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thresholds following saline and NMDA injections into sites surrounding Cg1 of rACC revealed no effects of drug treatments (ts < 1.0), and these thresholds were significantly reduced compared to those observed following NMDA treatments into Cg1 of rACC (data from Experiment 1, Figure 1). One-way ANOVA of VAD and VDS thresholds following NMDA injections into Cg1, Prl and M2 revealed significant differences, Fs(2,16) > 10.6, ps < .002, with reduced thresholds following injections into Prl and M2 compared to Cg1 (Dunnett’s, ps < .05). Similarly, increases in the amplitude and duration of VAD and amplitude of VDS generated by

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injection of NMDA into Cg1 of cACC was not observed when it was administered into Cg2 or M2 (Figure 7). These performance variables did not differ following injection of saline and NMDA into these surrounding sites (ts < 1.0), and were significantly reduced compared to following administration of NMDA into Cg1 of cACC (data from Experiment 1, Figure 2). Oneway ANOVA of amplitude and duration of VAD and amplitude of VDS following NMDA

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injections into Cg1, Cg2 and M2 revealed significant differences, Fs(2,14) > 8.6, ps < .005, with reduced performance following injections into Cg2 and M2 compared to Cg1 (Dunnett’s, ps < .05).

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Insert Figures 6 & 7 about here

----------------------------------------Discussion

NMDA receptor activation within different subdivisions of the ACC differentially modulated rats’ affective response to a noxious stimulus. VAD is an established model of pain affect in rats

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and administration of NMDA into the rACC produced dose-dependent increases in VAD threshold. Conversely, injection of NMDA into the cACC failed to alter VAD threshold, but

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produced a dose-dependent increase in the amplitude and duration of VAD. These antinociceptive and pronociceptive actions were limited to Cg1, and blocked by local

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pretreatment with the NMDA receptor antagonist AP-5. Less robust effects of NMDA on threshold and performance of VDS were noted, and all treatments failed to alter SMR threshold

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or performance. These findings are consistent with those from human investigations showing the respective contributions of rACC and cACC to suppression and generation of pain affect, 82, 87, 93, 113

with the neurochemical, cytoarchitectural, hodological and functional homologies between

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these divisions of the cingulate cortex in human and rat,3, 5-8, 35, 40, 80, 92, 107, 108 and support the use of this model system in analyzing cortical contributions to emotional responses to pain. NMDA receptor activation in rACC also attenuates aversion associated with neuropathic

pain in rats. Long-term reduction in mechanical hypersensitivity in the spared nerve injury model (SNI) was observed with repeated oral administration of D-cycloserine, a partial NMDA agonist, which reversed the downregulated NR2B expression in rACC. Infusion of D-

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cycloserine or NMDA into rACC also suppressed mechanical hypersensitivity and prevented development of conditioned placed aversion induced by mechanical stimulation of the injured paw75. Also, in the SNI model, optogenetic activation of rACC projection neurons reduced paw withdrawal latencies to thermal and mechanical stimuli in injured and non-injured paws, and supported conditioned place preference indicating pain relief 68; whereas, optogenetic inhibition

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of rACC projections neurons exacerbated pain behaviors and conditioned place aversion118. Electrical or homocysteic acid stimulation of rACC also suppressed defense responses elicited by aversive physical stimulation or electrical stimulation of the ventromedial hypothalamus or

basolateral amygdala1, 33 - structures that contribute to generation of pain-induced vocalizations16, . Stimulation of rACC also suppressed bar pressing to avoid noxious footshock without

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59, 77

altering bar pressing for food reward1, 117. As noted earlier, chronic pain in humans and rats is accompanied by loss of rACC volume, decreased basal levels of glutamate and glutamate signaling in rACC, and reduced connectivity of rACC with subcortical antinociceptive circuits43, 54, 56, 57, 63, 90, 94, 96

that are reversed with pain relief 70, 78, 86, 90.

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Contrary to these findings are reports that NMDA receptor agonism within the rACC generates or facilitates pain affect. Johansen and colleagues reported that lesions of the rACC,

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but not cACC, prior to F-CPA training prevented aversive conditioning, and injection of homocysteic acid into rACC supported development of CPA in the absence of noxious

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peripheral stimulation 60, 61. Correspondingly, antagonism of NMDA receptors, NMDA receptor subunits (NR2A or NR2B), or the NMDA intracellular signaling pathway in rACC reportedly

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suppressed F-CPA 11, 24, 44, 60, 69, 89. Yet, lesions of the rACC or down regulation of NMDA receptors in rACC enhance aversive conditioning72, 76, 109; whereas, normalization of NMDA

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receptor concentrations in rACC normalizes aversive conditioning 72, 75. Additionally, neuroimaging studies of PTSD and human fear conditioning report rACC inhibition (with reduced levels of glutamate) coupled with cACC activation (with elevated levels of glutamate) 39, 97, 114, 116

. Alternately, safety signals to noxious stimuli reduce pain ratings, activate rACC and

increase its’ functional connectivity with ventrolateral periaqueductal gray (vPAG) 112- effects that are deficient in PTSD 53, 91. Although procedural and strain differences26 may account for these contradictory findings in rats no obvious reason is discernable.

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The ACC receives glutamatergic afferents from medial and intralaminar thalamic nuclei (MITN) that interact with NMDA and non-NMDA receptors67. We reported that morphine injected into PF increased VAD and VDS thresholds similarly to that observed in the present study, and pretreatment of rACC with AP-5 prevented these threshold increases49. Within the PF, morphine suppresses the response of nociceptive neurons to noxious stimulation and

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activates non-nociceptive neurons 34, 85. The latter set of PF neurons were hypothesized to be glutamatergic and project preferentially to the rACC thereby activating antinociceptive

projections from the rACC 49, 115. Increases in VAD and VDS thresholds observed in the present study following injection of NMDA into rACC presumably reflect the direct activation of these

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antinociceptive projections.

Projections of rACC stimulate the release of met-enkephalin in the vPAG 9, 46 – a nodal structure in the endogenous antinociceptive circuit 4, 15. We reported that increasing the level of -opioid receptor activation in vPAG, via microinjection of increasing doses of morphine, generates progressive increases in VAD, VDS, and SMR thresholds that are mediated by

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successive recruitment of antinociceptive projections to the limbic forebrain and thalamus, medulla, and spinal dorsal horn 15, 18, 19, 22. Administration of NMDA into rACC presumably

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activates antinociceptive projections of the vPAG that suppress nociceptive processing in limbic forebrain sites that contribute to the production of VAD, and medullary sites that contribute to

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generation of VDS 15, 16, 21, 48, 77, 101, 102. Medullary projections also suppress nociceptive throughput to forebrain sites responsible for producing VAD. The dual inhibition of nociceptive

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processing at medullary and forebrain levels could account for the greater effect of rACC administered NMDA on VAD versus VDS thresholds. Similarly, placebo analgesia in humans that increases the connection of rACC with vPAG and the release of endogenous opiates in

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vPAG, also enhances its connection (via vPAG) with the rostral ventromedial medulla 36, 65, 110. Noxious stimulation generates bursting activity in glutamatergic neurons in MITN that

project to the cACC engaging glutamate mediated intracingulate circuits and reciprocal connections with MITN 98. This reverberatory activity yields NMDA-dependent short-term plasticity as reflected in enhanced amplitude and duration of evoked activity within cACC to subsequent glutamatergic input from MITN 98. In the present study, administration of NMDA

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into cACC prior to noxious stimulation may potentiate the expression of short-term plasticity leading to the observed increases in the amplitude and duration of VAD (hyperalgesia). Similarly, hyperalgesia in humans is associated with increased activation of cACC 64. Maintenance of short-term plasticity by persistent noxious input to the MITN is posited to promote the transition of short-term plasticity to long-term potentiation of noxious-evoked

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activity in cACC that underlies development of chronic pain 11, 98.

NMDA injected into ACC failed to alter SMR threshold or performance suggesting that spinopetal inhibitory and facilitatory projections to the spinal dorsal horn were not activated. Similarly, electrical stimulation of the rACC did not alter paw withdrawal thresholds in normal

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or nerve-injured rats 66. Hardy 45, however, reported increases in tail flick latencies during rACC stimulation, and rACC stimulation inhibits noxious-evoked neural activity in the dorsal horn 73, 95

. Nociceptive reflexes are also inhibited in humans during transcranial motor cortex

stimulation that remits neuropathic pain, activates the rACC, and increases glutamate levels in rACC and its functional connectivity with the vPAG41. Additionally, pain relief generated by

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motor cortex stimulation is blocked by pretreatment with the NMDA receptor antagonist ketamine30. Conversely, electrical or optogenetic stimulation of cACC reduced tail flick

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latencies and paw withdrawal thresholds, and increased the frequency and amplitude of sEPSCs of dorsal horn neurons 23, 28. In the present study, SMR threshold or performance may have been

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altered if higher doses of NMDA were utilized; however, doses above 2 µg cause neuronal death in rodents which precluded evaluation of doses above those used 79. Importantly, spinal neurons

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involved in segmental reflex generation do not contribute to spinothalamic and spinomesencephalic pathways52, and spinopetal inhibitory projections to these separate pools of

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dorsal horn neurons are engaged by different doses of morphine injected into the vPAG 15, 58. Consequently, changes in spinal reflexes or noxious-evoked activity in unidentified dorsal horn neurons provide little insight into changes in nociceptive processing at supraspinal levels of the neuraxis.

The cACC and rACC may interact directly in modulating pain affect. The rACC and cACC are highly interconnected 106, and nociceptive input to the cACC from MITN is relayed to rACC. Cho et al. 29 reported that during painful thermal stimulation of humans the MITN was initially

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activated, followed shortly thereafter by the cACC, and eventually the rACC. Activation of the cACC  rACC projections only occurred if noxious input reached a certain threshold. rACC activation required higher temperatures compared to cACC activation, and only occurred when stimulation produced “stressful pain”. Activation of the rACC by the cACC may reflect engagement of endogenous homeostatic antinociceptive mechanisms that provide compensatory

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actions following the detection of a noxious stimulus that elicits stress, and dysregulation of this system may contribute to chronic pain. Fibromyalgia patients exhibit reduced activation of the rACC and its related antinociceptive subcortical circuits in response to noxious peripheral

stimulation that is correlated with increased pain perception 55. Conversely, re-engagement of

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the rACC may produce relief of chronic pain. For patients with fibromyalgia, transcranial

stimulation of the motor cortex, that activates and increases glutamate levels in rACC 41, reduced ongoing pain and negative affect, and reduced glutamate levels in cACC 38.

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Acknowledgements: The authors acknowledge the dedicated and excellent technical assistance

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of Amanda B. Flack.

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References 1.

al Maskati HA, Zbrozyna AW. Stimulation in prefrontal cortex area inhibits cardiovascular and motor components of the defence reaction in rats. J Auton Nerv Syst. 28:117-125, 1989

2.

Amirmohseni S, Segelcke D, Reichl S, Wachsmuth L, Gorlich D, Faber C, Pogatzki-

tools of MRI. Neuroimage. 127:110-122, 2016 3.

CR IP T

Zahn E. Characterization of incisional and inflammatory pain in rats using functional

Ash ES, Heal DJ, Clare Stanford S. Contrasting changes in extracellular dopamine and glutamate along the rostrocaudal axis of the anterior cingulate cortex of the rat following

4.

AN US

an acute d-amphetamine or dopamine challenge. Neuropharmacology. 87:180-187, 2014 Basbaum AI, Fields HL. Endogenous pain control systems: Brainstem spinal pathways and endorphin circuitry. Annual Reviews in Neuroscience. 7:309-338, 1984 5.

Becerra L, Navratilova E, Porreca F, Borsook D. Analogous responses in the nucleus accumbens and cingulate cortex to pain onset (aversion) and offset (relief) in rats and

6.

M

humans. J Neurophysiol. 110:1221-1226, 2013

Becerra L, Pendse G, Chang PC, Bishop J, Borsook D. Robust reproducible resting state

7.

ED

networks in the awake rodent brain. PLoS One. 6:e25701, 2011 Becerra L, Upadhyay J, Chang PC, Bishop J, Anderson J, Baumgartner R, Schwarz AJ,

PT

Coimbra A, Wallin D, Nutile L, George E, Maier G, Sunkaraneni S, Iyengar S, Evelhoch JL, Bleakman D, Hargreaves R, Borsook D. Parallel buprenorphine phMRI responses in

8.

CE

conscious rodents and healthy human subjects. J Pharmacol Exp Ther. 345:41-51, 2013 Beckmann M, Johansen-Berg H, Rushworth MF. Connectivity-based parcellation of

AC

human cingulate cortex and its relation to functional specialization. J Neurosci. 29:11751190, 2009

9.

Behbehani M. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobiol . 46:575-605, 1995

10.

Bingel U, Lorenz J, Schoell E, Weiller C, Buchel C. Mechanisms of placebo analgesia: rACC recruitment of a subcortical antinociceptive network. Pain. 120:8-15, 2006

ACCEPTED MANUSCRIPT

11.

Bliss TV, Collingridge GL, Kaang BK, Zhuo M. Synaptic plasticity in the anterior cingulate cortex in acute and chronic pain. Nat Rev Neurosci. 17:485-496, 2016

12.

Borszcz GS. The capacity of motor reflex and vocalization thresholds to support avoidance conditioning in the rat. Behav Neurosci. 107:678-693, 1993

13.

Borszcz GS. Increases in vocalization and motor reflex thresholds are influenced by the

CR IP T

site of morphine microinjection: Comparisons following administration into the

periaqueductal gray, ventral medulla, and spinal subarachnoid space. Behav Neurosci. 109:502-522, 1995 14.

Borszcz GS. Pavlovian conditional vocalizations of the rat: a model system for analyzing

15.

AN US

the fear of pain. Behav Neurosci.. 109:648-662, 1995

Borszcz GS. Differential contributions of medullary, thalamic, and amygdaloid serotonin to the antinociceptive action of morphine administered into the periaqueductal gray: A model of morphine analgesia. Behav Neurosci. 113:612- 631, 1999

16.

Borszcz GS. Contribution of the ventromedial hypothalamus to generation of the

17.

M

affective dimension of pain. Pain. 123:155-168, 2006

Borszcz GS, Johnson CP, Fahey KA. Comparison of motor reflex and vocalization

ED

thresholds following systemically administered morphine, fentanyl, and diazepam in the rat: assessment of sensory and performance variables. Pharmacol Biochem Behav.

18.

PT

49:827-834, 1994

Borszcz GS, Johnson CP, Thorp MV. The differential contribution of spinopetal

CE

projections to increases in vocalization and motor reflex thresholds generated by the microinjection of morphine into the periaqueductal gray. Behav Neurosci. 110:368-388,

AC

1996 19.

Borszcz GS, Johnson CP, Williams DH. Increases in vocalization and motor reflex thresholds generated by the intrathecal administration of serotonin or norepinephrine. Behav Neurosci.. 110:809 - 822, 1996

20.

Borszcz GS, Leaton RN. The effect of amygdala lesions on conditional and unconditional vocalizations in rats. Neurobiol Learn Mem. 79:212-225, 2003

ACCEPTED MANUSCRIPT

21.

Borszcz GS, Spuz CA. Hypothalamic control of pain vocalization and affective dimension of pain signaling. In: Handbook of Mammalian Vocalization.(Brudzynski, S.M., Ed.), Academic Press, Oxford, 2009, pp. 281 - 292.

22.

Borszcz GS, Streltsov NG. Amygdaloid-thalamic interactions mediate the antinociceptive action of morphine microinjected into the periaqueductal gray. Behav Neurosci. 114:574-

23.

CR IP T

584, 2000

Calejesan AA, Kim SJ, Zhuo M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain. 4:83-96, 2000

Cao H, Gao YJ, Ren WH, Li TT, Duan KZ, Cui YH, Cao XH, Zhao ZQ, Ji RR, Zhang

AN US

24.

YQ. Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction and expression of affective pain. J Neurosci. 29:3307-3321, 2009 25.

Carroll MN, Lim KS. Observations on the neuropharmacology of morphine and

26.

M

morphinelike analgesia. Arch Int Pharmacodyn Ther. 125:383-403, 1960 Chang CH, Maren S. Strain difference in the effect of infralimbic cortex lesions on fear

27.

ED

extinction in rats. Behav Neurosci. 124:391-397, 2010 Chapman CR, Feather BW. Effects of diazepam on human pain tolerance and pain

28.

PT

sensitivity. Psychosom Med. 35:330-340, 1973 Chen T, Taniguchi W, Chen QY, Tozaki-Saitoh H, Song Q, Liu RH, Koga K, Matsuda T,

CE

Kaito-Sugimura Y, Wang J, Li ZH, Lu YC, Inoue K, Tsuda M, Li YQ, Nakatsuka T, Zhuo M. Top-down descending facilitation of spinal sensory excitatory transmission

AC

from the anterior cingulate cortex. Nat Commun. 9:1886, 2018 29.

Cho ZH, Son YD, Kang CK, Han JY, Wong EK, Bai SJ. Pain dynamics observed by functional magnetic resonance imaging: differential regression analysis technique. J Magn Reson Imaging. 18:273-283, 2003

30.

Ciampi de Andrade D, Mhalla A, Adam F, Texeira MJ, Bouhassira D. Repetitive transcranial magnetic stimulation induced analgesia depends on N-methyl-D-aspartate glutamate receptors. Pain. 155:598-605, 2014

ACCEPTED MANUSCRIPT

31.

Cleve M, Gussew A, Reichenbach JR. In vivo detection of acute pain-induced changes of GABA+ and Glx in the human brain by using functional 1H MEGA-PRESS MR spectroscopy. Neuroimage. 105:67-75, 2015

32.

Cottam WJ, Condon L, Alshuft H, Reckziegel D, Auer DP. Associations of limbic-

trait anxiety. Neuroimage Clin. 12:269-276, 2016 33.

CR IP T

affective brain activity and severity of ongoing chronic arthritis pain are explained by

Coutinho MR, Menescal-de-Oliveira L. Role of homocysteic acid in the guinea pig (Cavia porcellus) anterior cingulate cortex in tonic immobility and the influence of NMDA receptors on the dorsal PAG. Behav Brain Res. 208:237-242, 2010

Dafny N, Gildenberg P. Morphine effects on spontaneous, nociceptive, antinociceptive

AN US

34.

and sensory evoked responses of parafasciculus thalami units in morphine naive and morphine dependent rats. Brain Res. 323:11-20, 1984 35.

Dou W, Palomero-Gallagher N, van Tol MJ, Kaufmann J, Zhong K, Bernstein HG, Heinze HJ, Speck O, Walter M. Systematic regional variations of GABA, glutamine, and

M

glutamate concentrations follow receptor fingerprints of human cingulate cortex. J Neurosci. 33:12698-12704, 2013

Eippert F, Bingel U, Schoell ED, Yacubian J, Klinger R, Lorenz J, Buchel C. Activation

ED

36.

of the opioidergic descending pain control system underlies placebo analgesia. Neuron.

37.

PT

63:533-543, 2009

Fanselow MS. Odors released by stressed rats produce opioid analgesia in unstressed rats.

38.

CE

Behav Neurosci. 99:589-592, 1985 Foerster BR, Nascimento TD, DeBoer M, Bender MA, Rice IC, Truong DQ, Bikson M,

AC

Clauw DJ, Zubieta JK, Harris RE, DaSilva AF. Excitatory and inhibitory brain metabolites as targets of motor cortex transcranial direct current stimulation therapy and predictors of its efficacy in fibromyalgia. Arthritis Rheumatol. 67:576-581, 2015

39.

Fullana MA, Harrison BJ, Soriano-Mas C, Vervliet B, Cardoner N, Avila-Parcet A, Radua J. Neural signatures of human fear conditioning: an updated and extended metaanalysis of fMRI studies. Mol Psychiatry. 21:500-508, 2016

ACCEPTED MANUSCRIPT

40.

Gabbott PL, Warner TA, Jays PR, Salway P, Busby SJ. Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol. 492:145-177, 2005

41.

Garcia-Larrea L, Peyron R, Mertens P, Gregoire MC, Lavenne F, Le Bars D, Convers P, Mauguiere F, Sindou M, Laurent B. Electrical stimulation of motor cortex for pain

42.

CR IP T

control: a combined PET-scan and electrophysiological study. Pain. 83:259-273, 1999 Gracely RH, McGrath P, Dubner R. Validity and sensitivity of ratio scales of sensory and affective verbal pain descriptors: manipulation of affect by diazepam. Pain. 5:19-29, 1978

Guida F, Luongo L, Marmo F, Romano R, Iannotta M, Napolitano F, Belardo C,

AN US

43.

Marabese I, D'Aniello A, De Gregorio D, Rossi F, Piscitelli F, Lattanzi R, de Bartolomeis A, Usiello A, Di Marzo V, de Novellis V, Maione S. Palmitoylethanolamide reduces pain-related behaviors and restores glutamatergic synapses homeostasis in the medial prefrontal cortex of neuropathic mice. Mol Brain. 8:47, 2015

Guo SG, Lv XH, Guan SH, Li HL, Qiao Y, Feng H, Cong L, Wang GM. Silencing the

M

44.

NR2B gene in rat ACC neurons by lentivirus-delivered shRNA alleviates pain-related

ED

aversion. Int J Clin Exp Med. 8:6725-6734, 2015 Hardy SG. Analgesia elicited by prefrontal stimulation. Brain Res. 339:281-284, 1985

46.

Hardy SG, Haigler HJ. Prefrontal influences upon the midbrain: a possible route for pain

PT

45.

modulation. Brain Res. 339:285-293, 1985 Harper DE, Ichesco E, Schrepf A, Hampson JP, Clauw DJ, Schmidt-Wilcke T, Harris

CE

47.

RE, Harte SE. Resting functional connectivity of the periaqueductal gray is associated

AC

with normal inhibition and pathological facilitation in conditioned pain modulation. J Pain. 19:635 e631-635 e615, 2018

48.

Harte SE, Kender RG, Borszcz GS. Activation of 5-HT1A and 5-HT7 receptors in the parafascicular nucleus suppresses the affective reaction of rats to noxious stimulation. Pain. 113:405-415, 2005

ACCEPTED MANUSCRIPT

49.

Harte SE, Spuz CA, Borszcz GS. Functional interaction between medial thalamus and rostral anterior cingulate cortex in the suppression of pain affect. Neuroscience. 172:460473, 2011

50.

Hoffmeister F: Effects of psychotropic drugs on pain. In: Pain.(Soulariarc, A., Cahn, J., Charpentier, J., Eds.), Academic Press, New York, 1968, pp. 309 - 319. Ito T, Tanaka-Mizuno S, Iwashita N, Tooyama I, Shiino A, Miura K, Fukui S. Proton

CR IP T

51.

magnetic resonance spectroscopy assessment of metabolite status of the anterior cingulate cortex in chronic pain patients and healthy controls. J Pain Res. 10:287-293, 2017 52.

Jasmin L, Carstens E, Basbaum AI. Interneurons presynaptic to rat tail-flick motoneurons

AN US

as mapped by transneuronal transport of pseudorabies virus: few have long ascending collaterals. Neuroscience. 76:859-876, 1997 53.

Jenewein J, Erni J, Moergeli H, Grillon C, Schumacher S, Mueller-Pfeiffer C, Hassanpour K, Seiler A, Wittmann L, Schnyder U, Hasler G. Altered pain perception and fear-learning deficits in subjects with posttraumatic stress disorder. J Pain. 17:1325-

54.

M

1333, 2016

Jensen KB, Kosek E, Petzke F, Carville S, Fransson P, Marcus H, Williams SC, Choy E,

ED

Giesecke T, Mainguy Y, Gracely R, Ingvar M. Evidence of dysfunctional pain inhibition in Fibromyalgia reflected in rACC during provoked pain. Pain. 144:95-100, 2009 Jensen KB, Kosek E, Petzke F, Carville S, Fransson P, Marcus H, Williams SC, Choy E,

PT

55.

Giesecke T, Mainguy Y, Gracely R, Ingvar M. Evidence of dysfunctional pain inhibition

56.

CE

in Fibromyalgia reflected in rACC during provoked pain. Pain. 144:95-100, 2009 Jensen KB, Loitoile R, Kosek E, Petzke F, Carville S, Fransson P, Marcus H, Williams

AC

SC, Choy E, Mainguy Y, Vitton O, Gracely RH, Gollub R, Ingvar M, Kong J. Patients with fibromyalgia display less functional connectivity in the brain's pain inhibitory network. Mol Pain. 8:32, 2012

57.

Jensen KB, Srinivasan P, Spaeth R, Tan Y, Kosek E, Petzke F, Carville S, Fransson P, Marcus H, Williams SC, Choy E, Vitton O, Gracely R, Ingvar M, Kong J. Overlapping structural and functional brain changes in patients with long-term exposure to fibromyalgia pain. Arthritis Rheum. 65:3293-3303, 2013

ACCEPTED MANUSCRIPT

58.

Jensen TS, Yaksh TL. Examination of spinal monoamine receptors through which brainstem opiate-sensitive systems act in the rat. Brain Res. 363:114-127, 1986

59.

Ji G, Sun H, Fu Y, Li Z, Pais-Vieira M, Galhardo V, Neugebauer V. Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation. J Neurosci. 30:5451-5464, 2010 Johansen JP, Fields HL. Glutamatergic activation of anterior cingulate cortex produces an aversive teaching signal. Nat Neurosci. 7:398-403, 2004

61.

CR IP T

60.

Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci. 98:8077-

62.

AN US

8082, 2001

Jones BF, Groenewegen HJ, Witter MP. Intrinsic connections of the cingulate cortex in the rat suggest the existence of multiple functionally segregated networks. Neuroscience. 133:193-207, 2005

63.

Kelly CJ, Huang M, Meltzer H, Martina M. Reduced glutamatergic currents and dendritic

M

branching of layer 5 pyramidal cells contribute to medial prefrontal cortex deactivation in a rat model of neuropathic pain. Front Cell Neurosci. 10:133, 2016 Kong J, Gollub RL, Polich G, Kirsch I, Laviolette P, Vangel M, Rosen B, Kaptchuk TJ.

ED

64.

A functional magnetic resonance imaging study on the neural mechanisms of

65.

PT

hyperalgesic nocebo effect. J Neurosci. 28:13354-13362, 2008 Kong J, Tu PC, Zyloney C, Su TP. Intrinsic functional connectivity of the periaqueductal

66.

CE

gray, a resting fMRI study. Behav Brain Res. 211:215-219, 2010 LaBuda CJ, Fuchs PN. Attenuation of negative pain affect produced by unilateral spinal

AC

nerve injury in the rat following anterior cingulate cortex activation. Neuroscience. 136:311-322, 2005

67.

Lee CM, Chang WC, Chang KB, Shyu BC. Synaptic organization and input-specific short-term plasticity in anterior cingulate cortical neurons with intact thalamic inputs. Eur J Neurosci. 25:2847-2861, 2007

ACCEPTED MANUSCRIPT

68.

Lee M, Manders TR, Eberle SE, Su C, D'Amour J, Yang R, Lin HY, Deisseroth K, Froemke RC, Wang J. Activation of corticostriatal circuitry relieves chronic neuropathic pain. J Neurosci. 35:5247-5259, 2015

69.

Li TT, Ren WH, Xiao X, Nan J, Cheng LZ, Zhang XH, Zhao ZQ, Zhang YQ. NMDA

related aversion in male rats. Pain. 146:183-193, 2009 70.

CR IP T

NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-

Li Z, Liu M, Lan L, Zeng F, Makris N, Liang Y, Guo T, Wu F, Gao Y, Dong M, Yang J, Li Y, Gong Q, Liang F, Kong J. Altered periaqueductal gray resting state functional connectivity in migraine and the modulation effect of treatment. Sci Rep. 6:20298, 2016 Lieberman MD, Eisenberger NI. The dorsal anterior cingulate cortex is selective for pain:

AN US

71.

Results from large-scale reverse inference. Proc Natl Acad Sci U S A. 112:15250-15255, 2015 72.

Liu G, Feng D, Wang J, Zhang H, Peng Z, Cai M, Yang J, Zhang R, Wang H, Wu S, Tan Q. rTMS ameliorates PTSD symptoms in rats by enhancing glutamate transmission and

55:3946-3958, 2018

Ma JH, Xiao TH, Chang CW, Gao L, Wang XL, Gao GD, Yu YQ. Activation of anterior

ED

73.

M

synaptic plasticity in the ACC via the PTEN/Akt signalling pathway. Mol Neurobiol.

cingulate cortex produces inhibitory effects on noxious mechanical and electrical stimuli-

74.

PT

evoked responses in rat spinal WDR neurons. Eur J Pain. 15:895-899, 2011 Mark VH, Ervin FR, Yakovlev PI. Correlation of pain relief, sensory loss, and anatomical

CE

lesion sites in pain patients treated with stereotactic thalamotomy. Trans Am Neurol Assoc. 86:86-90, 1961 Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Tkatch T, Apkarian

AC

75.

AV. D-cycloserine reduces neuropathic pain behavior through limbic NMDA-mediated circuitry. Pain. 132:108-123, 2007

76.

Morgan MA, LeDoux JE. Differential contribution of dorsal and ventral medial prefrontal cortex to the acquisition and extinction of conditioned fear in rats. Behav Neurosci. 109:681-688, 1995

ACCEPTED MANUSCRIPT

77.

Nandigama P, Borszcz GS. Affective analgesia following the administration of morphine into the amygdala of rats. Brain Res. 959:343-354, 2003

78.

Navratilova E, Atcherley CW, Porreca F. Brain Circuits Encoding Reward from Pain Relief. Trends Neurosci. 38:741-750, 2015

79.

Olney JW, Labruyere J, Wang G, Wozniak DF, Price MT, Sesma MA. NMDA antagonist

80.

CR IP T

neurotoxicity: mechanism and prevention. Science. 254:1515-1518, 1991

Palomero-Gallagher N, Vogt BA, Schleicher A, Mayberg HS, Zilles K. Receptor

architecture of human cingulate cortex: evaluation of the four-region neurobiological model. Hum Brain Mapp. 30:2336-2355, 2009

Paxinos G, Watson C: The rat brain in stereotaxic coordinates, Academic Press,

AN US

81.

Amsterdam, 2005. 82.

Petrovic P, Kalso E, Petersson KM, Ingvar M. Placebo and opioid analgesia-- imaging a shared neuronal network. Science. 295:1737-1740, 2002

83.

Peyron R, Faillenot I, Mertens P, Laurent B, Garcia-Larrea L. Motor cortex stimulation in

M

neuropathic pain. Correlations between analgesic effect and hemodynamic changes in the brain. A PET study. Neuroimage. 34:310-321, 2007 Price DD, Von der Gruen A, Miller J, Rafii A, Price C. A psychophysical analysis of

ED

84.

morphine analgesia. Pain. 22:261-269, 1985 Prieto-Gomez B, Dafny N, Reyes-Vazquez C. Dorsal raphe stimulation, 5-HT and

PT

85.

morphine microiontophoresis effects on noxious and nonnoxious identified neurons in the

86.

CE

medial thalamus of the rat. Brain Res Bull. 22:937-943, 1989 Qu C, King T, Okun A, Lai J, Fields HL, Porreca F. Lesion of the rostral anterior

AC

cingulate cortex eliminates the aversiveness of spontaneous neuropathic pain following partial or complete axotomy. Pain. 152:1641-1648, 2011

87.

Rainville P. Brain mechanisms of pain affect and pain modulation. Curr Opin Neurobiol. 12:195-204, 2002

88.

Rainville P, Carrier B, Hofbauer RK, Bushnell MC, Duncan GH. Dissociation of sensory and affective dimensions of pain using hypnotic modulation. Pain. 82:159-171., 1999

ACCEPTED MANUSCRIPT

89.

Ren WH, Guo JD, Cao H, Wang H, Wang PF, Sha H, Ji RR, Zhao ZQ, Zhang YQ. Is endogenous D-serine in the rostral anterior cingulate cortex necessary for pain-related negative affect? J Neurochem. 96:1636-1647, 2006

90.

Rodriguez-Raecke R, Niemeier A, Ihle K, Ruether W, May A. Brain gray matter decrease in chronic pain is the consequence and not the cause of pain. J Neurosci. 29:13746-

91.

CR IP T

13750, 2009

Rougemont-Bucking A, Linnman C, Zeffiro TA, Zeidan MA, Lebron-Milad K,

Rodriguez-Romaguera J, Rauch SL, Pitman RK, Milad MR. Altered processing of

contextual information during fear extinction in PTSD: an fMRI study. CNS Neurosci

92.

AN US

Ther. 17:227-236, 2011

Schafer SM, Geuter S, Wager TD. Mechanisms of placebo analgesia: A dual-process model informed by insights from cross-species comparisons. Prog Neurobiol. 160:101122, 2018

93.

Schrepf A, Harper DE, Harte SE, Wang H, Ichesco E, Hampson JP, Zubieta JK, Clauw

M

DJ, Harris RE. Endogenous opioidergic dysregulation of pain in fibromyalgia: a PET and fMRI study. Pain. 157:2217-2225, 2016

Seminowicz DA, Laferriere AL, Millecamps M, Yu JS, Coderre TJ, Bushnell MC. MRI

ED

94.

structural brain changes associated with sensory and emotional function in a rat model of

95.

PT

long-term neuropathic pain. Neuroimage. 47:1007-1014, 2009 Senapati AK, Lagraize SC, Huntington PJ, Wilson HD, Fuchs PN, Peng YB. Electrical

CE

stimulation of the anterior cingulate cortex reduces responses of rat dorsal horn neurons to mechanical stimuli. J Neurophysiol. 94:845-851, 2005 Shi H, Yuan C, Dai Z, Ma H, Sheng L. Gray matter abnormalities associated with

AC

96.

fibromyalgia: A meta-analysis of voxel-based morphometric studies. Semin Arthritis Rheum. 46:330-337, 2016

97.

Shin LM, Bush G, Milad MR, Lasko NB, Brohawn KH, Hughes KC, Macklin ML, Gold AL, Karpf RD, Orr SP, Rauch SL, Pitman RK. Exaggerated activation of dorsal anterior cingulate cortex during cognitive interference: a monozygotic twin study of posttraumatic stress disorder. Am J Psychiatry. 168:979-985, 2011

ACCEPTED MANUSCRIPT

98.

Shyu BC, Vogt BA. Short-term synaptic plasticity in the nociceptive thalamic-anterior cingulate pathway. Mol Pain. 5:51, 2009

99.

Spenger C, Josephson A, Klason T, Hoehn M, Schwindt W, Ingvar M, Olson L. Functional MRI at 4.7 tesla of the rat brain during electric stimulation of forepaw, hindpaw, or tail in single- and multislice experiments. Exp Neurol. 166:246-253, 2000 Sprenger T, Valet M, Woltmann R, Zimmer C, Freynhagen R, Kochs EF, Tolle TR,

CR IP T

100.

Wagner KJ. Imaging pain modulation by subanesthetic S-(+)-ketamine. Anesth Analg. 103:729-737, 2006 101.

Spuz CA, Borszcz GS. NMDA or non-NMDA receptor antagonism within the

AN US

amygdaloid central nucleus suppresses the affective dimension of pain in rats: evidence for hemispheric synergy. J Pain. 13:328-337, 2012 102.

Spuz CA, Tomaszycki ML, Borszcz GS. N-methyl-D-aspartate receptor agonism and antagonism within the amygdaloid central nucleus suppresses pain affect: differential contribution of the ventrolateral periaqueductal gray. J Pain. 15:1305-1318, 2014 Strigo IA, Duncan GH, Bushnell MC, Boivin M, Wainer I, Rodriguez Rosas ME, Persson

M

103.

J. The effects of racemic ketamine on painful stimulation of skin and viscera in human

104.

ED

subjects. Pain. 113:255-264, 2005

Sweet WH: Central mechanisms of chronic pain (neuralgias and certain other neurogenic

105.

PT

pain). In: Pain.(Bonica, J.J., Ed.), Raven Press, New York, 1980, pp. 287-303. Valet M, Sprenger T, Boecker H, Willoch F, Rummeny E, Conrad B, Erhard P, Tolle TR.

CE

Distraction modulates connectivity of the cingulo-frontal cortex and the midbrain during pain--an fMRI analysis. Pain. 109:399-408, 2004 Vogt BA. Pain and emotion interactions in subregions of the cingulate gyrus. Nat Rev

AC

106.

Neurosci. 6:533-544, 2005

107.

Vogt BA: Cingulate Cortex and Pain Architecture. In: The Rat Nervous System, Fourth Edition.(Paxinos, G., Ed.), Academic Press, New York, NY, 2015.

108.

Vogt BA, Paxinos G. Cytoarchitecture of mouse and rat cingulate cortex with human homologies. Brain Struct Funct. 219:185-192, 2014

ACCEPTED MANUSCRIPT

109.

Vouimba RM, Garcia R, Baudry M, Thompson RF. Potentiation of conditioned freezing following dorsomedial prefrontal cortex lesions does not interfere with fear reduction in mice. Behav Neurosci. 114:720-724, 2000

110.

Wager TD, Scott DJ, Zubieta JK. Placebo effects on human mu-opioid activity during pain. Proc Natl Acad Sci. 104:11056-11061, 2007 Wei HB, Jakeman LB, Hunter JC, Bonhaus DW. Pharmacological characterization of N-

CR IP T

111.

methyl-D-aspartate receptors in spinal cord of rats with a chronic peripheral mononeuropathy. Neuropharmacology. 36:1561-1569, 1997 112.

Wiech K, Edwards R, Moseley GL, Berna C, Ploner M, Tracey I. Dissociable neural

AN US

mechanisms underlying the modulation of pain and anxiety? An FMRI pilot study. PLoS One. 9:e110654, 2014 113.

Willoch F, Gamringer U, Medele R, Steude U, Tolle TR. Analgesia by electrostimulation of the trigeminal ganglion in patients with trigeminopathic pain: a PET activation study. Pain. 103:119-130, 2003

Wolf RC, Herringa RJ. Prefrontal-Amygdala dysregulation to threat in pediatric

M

114.

posttraumatic stress disorder. Neuropsychopharmacology. 41:822-831, 2016 Yang JW, Shih HC, Shyu BC. Intracortical circuits in rat anterior cingulate cortex are

ED

115.

activated by nociceptive inputs mediated by medial thalamus. J Neurophysiol. 96:3409-

116.

PT

3422, 2006

Yang ZY, Quan H, Peng ZL, Zhong Y, Tan ZJ, Gong QY. Proton magnetic resonance

CE

spectroscopy revealed differences in the glutamate + glutamine/creatine ratio of the anterior cingulate cortex between healthy and pediatric post-traumatic stress disorder

AC

patients diagnosed after 2008 Wenchuan earthquake. Psychiatry Clin Neurosci. 69:782790, 2015

117.

Zbrozyna AW, Westwood DM. Stimulation in prefrontal cortex inhibits conditioned increase in blood pressure and avoidance bar pressing in rats. Physiol Behav. 49:705-708, 1991

ACCEPTED MANUSCRIPT

118.

Zhang Z, Gadotti VM, Chen L, Souza IA, Stemkowski PL, Zamponi GW. Role of prelimbic GABAergic circuits in sensory and emotional aspects of neuropathic pain. Cell Rep. 12:752-759, 2015

119.

Zubieta JK, Smith YR, Bueller JA, Xu Y, Kilbourn MR, Jewett DM, Meyer CR, Koeppe

dimensions of pain. Science. 293:311-315., 2001 120.

CR IP T

RA, Stohler CS. Regional mu opioid receptor regulation of sensory and affective

Zugaib J, Coutinho MR, Ferreira MD, Menescal-de-Oliveira L. Glutamate/GABA

balance in ACC modulates the nociceptive responses of vocalization: an expression of affective-motivational component of pain in guinea pigs. Physiol Behav. 126:8-14, 2014 Zunhammer M, Schweizer LM, Witte V, Harris RE, Bingel U, Schmidt-Wilcke T.

AN US

121.

Combined glutamate and glutamine levels in pain-processing brain regions are associated

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PT

ED

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with individual pain sensitivity. Pain. 157:2248-2256, 2016

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Figure Captions

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Figure 1. Dose-dependent effects of bilateral administration of NMDA into the rostral anterior cingulate cortex (rACC) on response thresholds and performance of responses at threshold. Data are plotted as the mean (+/- S.E.M.) threshold and performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Thresholds following intra-rACC NMDA administration were compared to those attained following saline (sal)

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administered into rACC. Asterisk (*) indicates thresholds significantly elevated above rACC

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saline treatment (p < 0.05).

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Figure 2. Dose-dependent effects of bilateral administration of NMDA into the caudal anterior cingulate cortex (cACC) on response thresholds and performance of responses at threshold. Data are plotted as the mean (+/- S.E.M.) threshold and performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Thresholds following intra-cACC NMDA administration were compared to those attained following saline (sal)

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administered into rACC. Asterisk (*) indicates performance significantly elevated above cACC

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saline treatment (p < 0.05).

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Figure 3. The effects of the bilateral pretreatment of rACC with AP-5 (4 µg/side) on increases in vocalization thresholds produced by injection of NMDA (1.0 µg/side) into the rACC. Data are plotted as the mean ((+/- S.E.M.) threshold and performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Asterisk (*) indicates thresholds significantly elevated above saline + saline treatment (p < 0.05). Pound sign (#)

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indicates thresholds significantly reduced compared to saline + NMDA treatment (p < 0.05).

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Figure 4. The effects of the bilateral pretreatment of cACC with AP-5 (2 µg/side) on increases in vocalization performance produced by injection of NMDA (1.0 µg/side) into the cACC. Data are plotted as the mean (+/- S.E.M.) threshold and performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Asterisk (*) indicates performance significantly elevated above saline + saline treatment (p < 0.05). Pound sign (#)

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indicates performance significantly reduced compared to saline + NMDA treatment (p < 0.05).

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Figure 5. Representative distribution of injection sites in and around (A) rostral anterior cingulate cortex (rACC) and (B) caudal anterior cingulate cortex (cACC). Red circles = sites

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within rACC and cACC that received the highest dose of NMDA (1.0 µg/side) in Experiment 1. Black circles = NMDA (1.0 µg/side) injection sites outside rACC and cACC. Blue circles = injections sites that received AP-5 + NMDA in Experiment 2. Coordinates are in millimeters

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from bregma. Schematics were derived from the rat brain atlas of Paxinos and Watson81. Insets: Representative photomicrographs of injection sites in rACC and cACC. Flat map of rat cingulate cortex (modified from Vogt and Paxinos 108) depicting areas of injection into rACC (yellow: ACC) and cACC (green: MCC). RSC = retrosplenial cortex.

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Figure 6. Anatomical specificity of the effects of bilateral administration of NMDA (1.0 µg/side) into the rostral anterior cingulate cortex (rACC) on response thresholds and performance of responses at threshold. Injections of saline (sal) and NMDA were given prior to two separate test sessions into sites outside the rACC (M2 and Prl). Data from Cg1 are taken from Experiment 1, Figure 1. Data are plotted as the mean (+/- S.E.M.) threshold and

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performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Asterisk (*) indicates performance significantly elevated following NMDA

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treatment into Cg1 versus M2 or Cg2 (p < 0.05).

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Figure 7. Anatomical specificity of the effects of bilateral administration of NMDA (1.0 µg/side) into the caudal anterior cingulate cortex (cACC) on response thresholds and performance of responses at threshold. Injections of saline (sal) and NMDA were given prior to two separate test sessions into sites outside the cACC (M2 and Cg2). Data from Cg1 are taken from Experiment 1, Figure 2. Data are plotted as the mean (+/- S.E.M.) threshold and

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performance of vocalization afterdischarge (VAD), vocalization during shock (VDS), and spinal motor reflex (SMR). Asterisk (*) indicates performance significantly elevated following NMDA

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treatment into Cg1 versus M2 or Cg2 (p < 0.05).