Electroacupuncture attenuates visceral hyperalgesia and inhibits the enhanced excitability of colon specific sensory neurons in a rat model of irritable bowel syndrome

Abstract  The causes of irritable bowel syndrome remain elusive and there are few effective treatments for pain in this syndrome. Electroacupunture (EA) is used extensively for treatment of various painful conditions including chronic visceral hyperalgesia (CVH). However, mechanism of its analgesic effect remains unknown. This study was designed to investigate effect of EA on colon specific dorsal root ganglion (DRG) neurons in rats with CVH. CVH was induced by intracolonic injection of acetic acid (AA) in 10-day-old rats. Electromyography and patch clamp recordings were performed at age of 8–10 weeks. Colon DRG neurons were labelled by injection of DiI into the colon wall. EA was given at ST36 in both hindlimbs. As adults, neonatal AA-injected rats displayed an increased sensitivity to colorectal distension (CRD) and an enhanced excitability of colon DRG neurons. EA treatment for 40 min significantly attenuated the nociceptive responses to CRD in these rats; this attenuation was reversed by pretreatment with naloxone. EA treatment for 40 min per day for 5 days produced a prolonged analgesic effect and normalized the enhanced excitability of colon DRG neurons. Furthermore, in vitro application of [D-Ala², N -MePhe⁴, Gly⁵-Ol] enkephalin (DAMGO) suppressed the enhanced excitability of colon neurons from rats with CVH. These findings suggest that EA produced-visceral analgesia, which might be mediated in a large part by endogenous opioids pathways, is associated with reversal of the enhanced excitability of colon DRG neurons in rats with CVH.     

Multiple episodes of sodium depletion in the rat: a remodeling of the electrical properties of median preoptic nucleus neurons

In rat brain, the detection and integration of chemosensory and neural signals are achieved, inter alia, by the median preoptic nucleus (MnPO) during a disturbance of the hydromineral balance. This is allowed through the presence of the sodium (Na⁺) sensor neurons. Interestingly, enkephalins and mu‐opioid receptors (μ‐ORs) are known for their role in ingestive behaviors and have previously been shown to regulate the excitability of MnPO neurons following a single Na⁺ depletion. However, little is known about the role of these μ‐ORs in the response enhancement following repeated Na⁺ challenge. Therefore, we used whole‐cell recordings in acute brain slices to determine neuronal plasticity in the electrical properties of the MnPO Na⁺ sensor‐specific neuronal population following multiple Na⁺ depletions. Our results show that the population of Na⁺ sensor neurons was represented by 80% of MnPO neurons after a single Na⁺ depletion and was reduced after three Na⁺ depletions. Interestingly, the subpopulation of Na⁺ sensors responding to D‐Ala²,N‐MePhe⁴,Gly‐ol‐enkephalin (DAMGO), a specific μ‐OR agonist, represented 11% of MnPO neurons after a single Na⁺ depletion and the population doubled after three Na⁺ depletions. Moreover, Na⁺ sensor neurons displayed modifications in the discharge pattern distribution and shape of calcium action potentials after three Na⁺ depletions but these changes did not occur in Na⁺ sensors responding to DAMGO. Thus, the reinforced μ‐OR functionality in Na⁺ sensors might take place to control the neuronal hyperexcitability and this plasticity in opioid‐sensitive and Na⁺ detection MnPO networks might sustain the enhanced salt ingestion induced by repeated exposure to Na⁺ depletion.     

Glucagon‐like peptide‐1 inhibits voltage‐gated potassium currents in mouse nodose ganglion neurons

Background Glucagon‐like peptide‐1 (GLP‐1) is a major hormone known to regulate glucose homeostasis and gut function, and is an important satiety mediator. These actions are at least in part mediated via an action on vagal afferent neurons. However, the mechanism by which GLP‐1 activates vagal afferents remains unknown. We hypothesized that GLP‐1 acts on nodose ganglion neuron voltage‐gated potassium (KV) channels, increasing membrane excitability. Methods Employing perforated patch clamp recordings we examined the effects of GLP‐1 on membrane properties as well as voltage‐gated potassium currents. Extracellular recordings of jejunal afferents were performed to demonstrate the functional relevance of these effects at the nerve terminal. Key Results Glucagon‐like peptide‐1 depolarized a subpopulation of nodose neurons. This membrane depolarization was used to identify neurons containing functional GLP‐1 receptors. In these neurons, GLP‐1 decreased rheobase and broadened the action potential, and increased the number of action potentials elicited at twice rheobase. We identified a GLP‐1 sensitive current whose reversal potential shifted in a depolarizing direction when extracellular potassium was increased. We identified two macroscopic K currents, IA, an inactivating current and IK a sustained current. GLP‐1 caused inhibition of these currents, IK by 45%, P < 0.05 and IA currents by 52%P < 0.01, associated with a hyperpolarizing shift of steady‐state inactivation curves for both currents. In extracellular recordings of jejunal afferents, GLP‐1 increased firing rate, the effect blocked by the K⁺ channel antagonist 4‐AP. Conclusions & Inferences These experiments indicate that GLP‐1 receptor activation results in vagal afferent excitation, due at least in part to inhibition of sustained and inactivating potassium currents. This mechanism may be important in satiety and glucose homeostatic signals arising from the gastrointestinal tract.     

Inflammation without pain: Immune‐derived opioids hold the key

Visceral pain is commonly associated with acute or remitting inflammatory bowel disease (IBD). In marked contrast, chronic IBD is often painless, even in the presence of active inflammation. This suggests that inflammation in itself is insufficient to sustain altered nociceptive signaling and raises the possibility that there is an endogenous analgesic system in effect in chronic disease. A new study by Basso et al. published in this issue of Neurogastroenterology & Motility provides additional support for an immune‐mediated mechanism that suppresses visceral hypersensitivity. The authors examined visceral pain in the IL‐10‐piroxicam model of chronic colitis, which differs from other experimental IBD models in that it involves immune suppression. During active inflammation, responses by these mice to graded increases in colorectal distension were equivalent to healthy controls, consistent with normal afferent signaling. However, treatment with a peripherally restricted opioid receptor antagonist resulted in marked visceral hypersensitivity to the same stimuli. This effect was attributed to the production of endogenous opioids by colitogenic CD4⁺ T cells present in the mucosa. This mini‐review provides a brief overview of analgesia by immune‐derived opioids under inflammatory conditions and highlights how the work of Basso et al. contributes to this area of research. Potential pharmacological approaches to harness or mimic this system are provided. These strategies may prove to be an effective means through which targeted and sustained relief of IBD pain may be achieved.     

Neuronal Fc-gamma receptor I mediated excitatory effects of IgG immune complex on rat dorsal root ganglion neurons

Pain often accompanies antigen-specific immune-related disorders though little is known of the underlying neural mechanisms. A common feature among these disorders is the elevated level of antigen-specific immunoglobulin (Ig) G in the serum and the presence of IgG immune complex (IC) in the affected tissue. We hypothesize that IC may directly activate the Fc-gamma receptor type I (FcγRI) expressed in nociceptive dorsal root ganglion (DRG) neurons and increase neuronal excitability thus potentially contributing to pain. Immunofluorescent labeling indicated that FcγRI, but not FcγRIIB or FcγRIII, was expressed in a subpopulation of rat DRG neurons including those expressing nociceptive markers. Calcium imaging revealed that the IC, but neither of the antibody (IgG) or antigen alone, produced an increase in intracellular calcium. This effect was abolished by the removal of the IgG Fc portion in the IC or the application of an anti-FcγRI antibody, suggesting a key role of the FcγRI receptor. Removal of extracellular calcium or depletion of intracellular calcium stores prevented the IC-induced calcium response. In whole-cell current-clamp recordings, IC depolarized the resting membrane potential, decreased the rheobase, and increased the number of action potentials evoked by a depolarizing current at 2× rheobase. In about half of the responsive neurons, IC evoked action potential discharges. These results suggest that a subpopulation of nociceptive neurons expresses functional FcγRI and that the activation of this receptor by IC increases neuronal excitability.     

Prolonged hindlimb unloading leads to changes in electrophysiological properties of L5 dorsal root ganglion neurons in rats after 14 days

Introduction: The purpose of this study was to evaluate the electrophysiological changes observed in dorsal root ganglion (DRG) neurons in a simulated weightlessness rat model and to assess the mechanisms involved in these changes. Methods: The simulated weightlessness model was created by hindlimb unloading (HU). Whole‐cell patch‐clamp recordings, conduction velocity measurement, and ultrastructural observation were performed. Results: In the HU rats, the action potentials had a longer duration and slower falling rate, but there was no significant effect on amplitude or rate of rise. HU also induced lowering of rheobase and of the threshold potential, making the cells more excitable. The conduction velocities in the proximal branches of ganglion cells were also decreased, and some degenerative changes in the myelin sheath were noted. Conclusions: This study provides evidence of plasticity of DRG neurons induced by HU. The changes observed might contribute to impaired motor performance in rats submitted to HU. Muscle Nerve 45: 65–69, 2012     

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ‐opioid receptors and GABAB receptors,but not α2 adrenergic receptors

GABA_B, μ‐opioid and adrenergic α₂ receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ‐opioid and GABA_B receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage‐gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high‐frequency dorsal root stimulation was inhibited by the μ‐opioid agonist DAMGO and the GABA_B agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low‐frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α₂ adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low‐frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ‐opioid and GABA_B receptors, but not by α₂ receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels.     

Altered functional properties of satellite glial cells in compressed spinal ganglia

The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low‐back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole‐cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium‐sized neurons compared to non‐nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state. © 2009 Wiley‐Liss, Inc.     

Opioids excite rather than inhibit sensory neurons after chronic opioid exposure of spinal cord-ganglion cultures

Tests were carried out to determine if the tolerance that develops in dorsal-horn network responses of mouse dorsal root ganglion (DRG)-spinal cord explants after chronic exposure to opioids could be accounted for by alterations in the excitability and pharmacologic properties of the afferent DRG cells. Intracellular recordings were made from DRG neurons in organotypic DRG-cord explants after chronic treatment with 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 4 days in vitro. Acute application of 10 microM DADLE shortened the duration of the Ca2+ component of the somatic action potential (APD) in only 5% of the treated neurons (4 out of 79 cells), in contrast to about 50% of the cells in naive explants (36 out of 74). Thus many DRG neuron perikarya became tolerant to the APD-shortening effects of DADLE. Furthermore, 77% of the treated DRG cells (61 out of 79) showed prolongation of the APD in response to an acute increase in DADLE concentration vs 34% in naive explants (25 out of 74). However, when the DADLE responsivity tests were carried out in the presence of multiple K+ channel blockers, only 20% of the treated DRG neurons showed APD prolongation (3 out of 15 cells), whereas 73% showed APD-shortening responses (11 out of 15 cells). The results suggest that: (1) DADLE-induced APD prolongation of the treated DRG neurons is mediated by opioid receptor subtypes that decrease a voltage-sensitive K+ conductance; (2) the DADLE-induced APD-shortening effects which are unmasked during more complete K+ channel blockade are mediated by opioid-receptor subtypes in the same neuron that reduce a voltage-sensitive Ca2+ conductance (resembling kappa receptors). DRG neurons did not become tolerant to either of these two opioid effects after chronic exposure to DADLE. Opioid shortening of the APD of DRG neuron perikarya has been generally accepted to be a model of opioid inhibition of calcium influx and transmitter release at presynaptic DRG terminals6,52,53,65,75,76. It is postulated that the opioid-induced APD prolongation observed in the present study provides evidence that opioids can also evoke direct excitatory effects on neurons. The enhancement of DADLE-induced excitatory responses and attenuation of DADLE-induced inhibitory responses of DRG neurons after chronic exposure to this opioid show striking similarities to the effects of forskolin or pertussis toxin treatment. These in vitro studies may provide clues to compensatory mechanisms underlying physiologic expression of tolerance to opioid analgesic effects in primary afferent synaptic networks.     

Primary afferent neurons containing calcitonin gene‐related peptide but not substance P in forepaw skin,dorsal root ganglia,and spinal cord of mice

In mice dorsal root ganglia (DRG), some neurons express calcitonin gene–related peptide (CGRP) without substance P (SP; CGRP⁺SP^‐). The projections and functions of these neurons are unknown. Therefore, we combined in vitro axonal tracing with multiple‐labeling immunohistochemistry to neurochemically define these neurons and characterize their peripheral and central projections. Cervical spinal cord, DRG, and forepaw skin were removed from C57Bl/6 mice and multiple‐labeled for CGRP, SP, and either marker for the sensory neuron subpopulations transient receptor potential vanilloid type 1 (TRPV1), neurofilament 200 (NF200), or vesicular glutamate transporter 2 (VGluT1). To determine central projections of CGRP⁺SP^‐ neurons, Neurobiotin (NB) was applied to the C7 ventral ramus and visualized in DRG and spinal cord sections colabeled for CGRP and SP. Half (50%) of the CGRP‐immunoreactive DRG neurons lacked detectable SP and had a mean soma size of 473 ± 14 μm² (n = 5); 89% of the CGRP⁺SP^‐ neurons expressed NF200 (n = 5), but only 32% expressed TRPV1 (n = 5). Cutaneous CGRP⁺SP^‐ fibers were numerous within dermal papillae and around hair shafts (n = 4). CGRP⁺SP^‐ boutons were prevalent in lateral lamina I and in lamina IV/V of the dorsal horn (n = 5). NB predominantly labeled fibers penetrating lamina IV/V, 6 ± 3% contained CGRP (n = 5), and 21 ± 2% contained VGluT1 (n = 3). CGRP⁺SP^‐ afferent neurons are likely to be non‐nociceptive. Their soma size, neurochemical profile, and peripheral and central targets suggest that CGRP⁺SP^‐ neurons are polymodal mechanoceptors. J. Comp. Neurol. 523:2555–2569, 2015. © 2015 Wiley Periodicals, Inc.     

Interactions between the mu opioid agonist DAMGO and substance P in regulation of the ventral pallidum

Substance P (SP) increases, and the mu-specific opioid agonist DAMGO decreases neuronal firing within ventral pallidum (VP) of the basal forebrain. This study investigated a possibility that some VP neurons are oppositionally co-regulated by SP and DAMGO using microiontophoresis combined with the extracellular electrophysiological recordings from chloral hydrate-anesthetized rats. SP altered DAMGO's ejection current-response curve, decreasing E_max and slope, and increasing the E_cu50 (ejection current level at which 50% of the maximal response was obtained). The modulation was observed even at low ejection current levels that, when applied alone, were not sufficient to alter neuronal activity (i.e., subthreshold). Also, DAMGO altered the E_max and slope of SP's ejection current-response curve. DAMGO induced these effects even at subthreshold ejection current levels. The responses to each peptide were blocked by a receptor-specific antagonist. These findings demonstrate that SP and mu-activating opioids antagonize each other's effects on VP neuronal firing. Thus, they may interact as physiological antagonists in the regulation of VP-associated functions. © 1996 Wiley-Liss, Inc.     

Dopamine and opioids inhibit synaptic outputs of the main island of the intercalated neurons of the amygdala

Neural circuits in the amygdala are important for associating the positive experience of drug taking with the coincident environmental cues. During abstinence, cue re‐exposure activates the amygdala, increases dopamine release in the amygdala and stimulates relapse to drug use in an opioid dependent manner. Neural circuits in the amygdala and the learning that underlies these behaviours are inhibited by GABAergic synaptic inhibition. A specialised subtype of GABAergic neurons in the amygdala are the clusters of intercalated cells. We focussed on the main‐island of intercalated cells because these neurons, located ventromedial to the basolateral amygdala, express very high levels of dopamine D1‐receptor and μ‐opioid receptor, release enkephalin and are densely innervated by the ventral tegmental area. However, where these neurons project to was not fully described and their regulation by opioids and dopamine was incomplete. To address this issue we electrically stimulated in the main‐island of the intercalated cells in rat brain slices and made patch‐clamp recordings of GABAergic synaptics from amygdala neurons. We found that main‐island neurons had a strong GABAergic inhibitory output to pyramidal neurons of the basolateral nucleus and the medial central nucleus, the major output zones of the amygdala. Opioids inhibited both these synaptic outputs of the intercalated neurons and thus would disinhibit these target zones. Additionally, dopamine acting at D1‐receptors inhibited main‐island neuron synapses onto other main‐island neurons. This data indicates that the inhibitory projections from the main‐island neurons could influence multiple aspects of addiction and emotional processing in an opioid and dopamine dependent manner.     

Chronic opioid treatment of neuroblastoma X dorsal root ganglion neuron hybrid F11 cells results in elevated GM1 ganglioside and cyclic adenosine monophosphate levels and onset of naloxone-evoked decreases in membrane K+ currents

Prolongation of the action potential duration of dorsal root ganglion (DRG) neurons by low (nM) concentrations of opioids occurs through activation of excitatory opioid receptors that are positively coupled via G_s regulatory protein to adenylate cyclase. Previous results suggested GM1 ganglioside to have an essential role in regulating this excitatory response, but not the inhibitory (APD-shortening) response to higher (μM) opioid concentrations. Furthermore, it was proposed that synthesis of GM1 is upregulated by prolonged activation of excitatory opioid receptor functions. To explore this possibility we have utilized cultures of hybrid F11 cells to carry out closely correlated electrophysiological and biochemical analyses of the effects of chronic opioid treatment on a homogeneous population of clonal cells which express many functions characteristic of DRG neurons. We show that chronic opioid exposure of F11 cells does, in fact, result in elevated levels of GM1 as well as cyclic adenosine monophosphate (AMP), concomitant with the onset of opioid excitatory supersensitivity as manifested by naloxone-evoked decreases in voltage-dependent membrane K⁺ currents. Such elevation of GM1 would be expected to enhance the efficacy of excitatory opioid receptor activation of the G_s/adenylate cyclase/cyclic AMP system, thereby providing a positive feedback mechanism that may account for the remarkable supersensitivity of chronic opioid-treated neurons to the excitatory effects of opioid agonists as well as antagonists. These in vitro findings may provide novel insights into the mechanisms underlying naloxone-precipitated withdrawal syndromes and opioid-induced hyperalgesia after chronic opiatf addiction in vivo. © 1995 Wiley-Liss, Inc.     

Protein tyrosine phosphatase receptor type O regulates development and function of the sensory nervous system

The roles of protein tyrosine phosphatases (PTPs) in differentiation and axon targeting by dorsal root ganglion (DRG) neurons are essentially unknown. The type III transmembrane PTP, PTPRO, is expressed in DRG neurons, and is implicated in the guidance of motor and retinal axons. We examined the role of PTPRO in DRG development and function using PTPRO^−/− mice. The number of peptidergic nociceptive neurons in the DRG of PTPRO^−/− mice was significantly decreased, while the total number of sensory neurons appeared unchanged. In addition, spinal pathfinding by both peptidergic and proprioceptive neurons was abnormal in PTPRO^−/− mice. Lastly, PTPRO^−/− mice performed abnormally on tests of thermal pain and sensorimotor coordination, suggesting that both nociception and proprioception were perturbed. Our data indicate that PTPRO is required for peptidergic differentiation and process outgrowth of sensory neurons, as well as mature sensory function, and provide the first evidence that RPTPs regulate DRG development.     

Opioids at low concentration decrease openings of K+ channels in sensory ganglion neurons.

Previous studies showed that low concentrations of opioids prolong the calcium-dependent component of the action potential duration (APD) of dorsal root ganglion (DRG) neurons, whereas higher concentrations shorten the APD. In the present study whole-cell voltage-clamp, as well as cell-attached membrane-patch voltage-clamp, recordings demonstrate that application of picomolar to nanomolar concentrations of mu, delta or kappa opioid agonists (DAGO, DPDPE or dynorphin) to DRG neurons in dissociated cell cultures reversibly decreased the activities of voltage-sensitive K+ channels. Pretreatment of DRG neurons with the opioid receptor antagonists, naloxone (30 nM) or diprenorphine (1 nM) prevented mu/delta or kappa opioid-induced decreases in K+ channel activities, respectively. Since opioids added to the bath solution decreased the activities of K+ channels in the membrane patch sealed off by the pipette tip, our results provide strong evidence that some modes of excitatory modulation of the action potential of DRG neurons are mediated by diffusible second messengers. The data are consonant with our previous studies indicating that opioids can elicit excitatory effects on sensory neurons via cholera toxin-sensitive Gs-linked excitatory opioid receptors coupled to cyclic AMP-dependent ionic channels.     

Inhibition by morphine and its analogs of action potentials in adult rat dorsal root ganglion neurons

Although opioids inhibit action potential (AP) conduction in primary-afferent fibers, this has not yet been fully examined. We investigated by using the sharp glass microelectrode technique how opioids (morphine, codeine, and ethylmorphine) affect APs recorded from adult rat dorsal root ganglion (DRG) neurons in response to sciatic nerve stimulation. The DRG neurons were classified into three types, Aα/β, Aδ, and C, according to AP characteristics, including the fiber conduction velocity (CV) of the neuron. AP of the Aα/β neuron was reduced in peak amplitude by each of the opioids in a reversible and concentration-dependent manner. The potency sequence was ethylmorphine > codeine = morphine (IC(50) = 0.70, 2.5, and 2.9 mM, respectively), indicating that this AP inhibition is related to the chemical structure of the opioid. Each of the Aδ and C neuron APs was also inhibited by the opioids; ethylmorphine had a tendency to inhibit APs more effectively than codeine and morphine. This inhibition was variable in extent among neurons and was either comparable to or greater than that of the Aα/β neuron AP. The opioid-induced AP inhibitions were unaffected by nonspecific opioid-receptor antagonist naloxone; opioid-receptor agonists did not affect APs. In conclusion, the opioids inhibited APs in DRG neurons without opioid-receptor activation; this inhibition was different among neurons having different primary-afferent fiber CVs and also among the three kinds of opioid. The inhibition by opioid of primary-afferent fiber AP conduction is suggested to be distinct in extent among fibers conveying distinct types of nociceptive information.     

Localization of Mu and Delta Opioid Receptors to Anterior Cingulate Afferents and Projection Neurons and Input/Output Model of Mu Regulation

Anterior cingulate cortex (ACC) has one of the highest densities of opioid receptors in the CNS and it has been implicated in acute and chronic pain responses. Little is known, however, about which neurons express opioid receptors in their dendrites and axon terminals. The present studies employed experimental techniques to remove afferent axons or classes of projection neurons from ratACC area 24 followed by coverslip autoradiography to localize changes in binding of [³H]Tyr- -Ala-Gly-MePhe-Gly-ol (DAMGO) to mu receptors and 2-[³H] -penicillamine-5- -penicillamine-en-kephalin (DPDPE) to delta receptors. Removal of all afferents to area 24 with undercut lesions did not alter DPDPE binding, but significantly reduced binding of DAMGO in layers I, III, and V. In contrast, removal of all cortical neurons with the excitotoxin ibotenic acid almost abolished DPDPE binding in all layers. The same lesions reduced DAMGO binding in most layers; however, there was a postlesion bimodal distribution in binding with high levels of binding in layer I and moderate levels in layer VI. These data suggest that delta receptors are expressed by cortical neurons, while mu receptors are expressed by both cortical neurons and afferent axons. To explore the distribution of postsynaptic receptors, immunotoxin lesions were made in area 24 by injection of OX7-saporin into the caudate and/or thalamic nuclei. Almost complete removal of projection neurons to these targets in layers Vb and VIa did not alter DPDPE binding, while the lesions reduced DAMGO binding in all but layer II. Removal of layer Vb corticostriatal projection neurons with caudate OX7-saporin injections reduced binding only in this layer. It is proposed that opioidergic circuits in area 24 are organized according to an input/output model for mu opioid regulation. In this model mu receptors regulate axon terminal activity from the thalamus in layer Ia and the locus coeruleus in layers Ic and II, whereas cortical outputs to the thalamus are modulated via postsynaptic receptors expressed in all layers by thalamocortical projection neurons with somata in layer VI. These opioidergic circuits in ACC are of particular importance because they may regulate responses to chronic nociceptive activity and associated pain perceptions.