Ventral pallidal microinjections of receptor-selective opioid agonists produce differential effects on circling and locomotor activity in rats

Locomotor activity was investigated following microinjections of receptor-selective opioid agonists into the ventral pallidum (VP) of rats. In Expt. 1, male Long-Evans rats were treated with unilateral microinjections of the μ agonist [d-Ala²-MePhe⁴, Gly-ol⁵]-enkephalin (DAGO), the σ agonist [d-Pen²,d-Pen⁵]-enkephalin (DPDPE) or the κ agonist U50,488H, and the rate and duration of circling behavior were measured. DAGO (0.01, 0.1, 1.0 nmol) procedure a dose-dependent increse in contralateral circling; pretreatment with 1.0 mg/kg naltrexone blocked the circling induced by the highest dose. The behavioral effect was largest when injections were targeted at the VP rather than structures dorsal to the VP. In contrast to DAGO, intrapallidal DPDPE (0.01, 0.1, 1.0, 10.0 nmol) produced a slight increase in contralateral circling only at the highest dose and U50, 488H (0.01, 0.1, 1.0, 10.0 nmol) produced no effect. In Expt. 2, the effects of bilateral injections of DAGO, DPDPE and U50,488H were tested in photocell activity ☐es. DAGO produced a dose-dependent increase in locomotor activity and this increase was decreased by 1.0 mg/kg naltrexone. A slight increase in activity was observed with the highest dose of DPDPE, and a slight decrease was observed with the highest dose of U50,488H. These findings confirm that opiate actions in the VP contribute to opiate-induced locomotion and suggest that μ and to some extent σ receptors are involved in this behavior.     

Activation of transient receptor potential vanilloid 2‐expressing primary afferents stimulates synaptic transmission in the deep dorsal horn of the rat spinal cord and elicits mechanical hyperalgesia

Probenecid, an agonist of transient receptor vanilloid (TRPV) type 2, was used to evaluate the effects of TRPV2 activation on excitatory and inhibitory synaptic transmission in the dorsal horn (DH) of the rat spinal cord and on nociceptive reflexes induced by thermal heat and mechanical stimuli. The effects of probenecid were compared with those of capsaicin, a TRPV1 agonist. Calcium imaging experiments on rat dorsal root ganglion (DRG) and DH cultures indicated that functional TRPV2 and TRPV1 were expressed by essentially non‐overlapping subpopulations of DRG neurons, but were absent from DH neurons and DH and DRG glial cells. Pretreatment of DRG cultures with small interfering RNAs against TRPV2 suppressed the responses to probenecid. Patch‐clamp recordings from spinal cord slices showed that probenecid and capsaicin increased the frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents in a subset of laminae III–V neurons. In contrast to capsaicin, probenecid failed to stimulate synaptic transmission in lamina II. Intrathecal or intraplantar injections of probenecid induced mechanical hyperalgesia/allodynia without affecting nociceptive heat responses. Capsaicin induced both mechanical hyperalgesia/allodynia and heat hyperalgesia. Activation of TRPV1 or TRPV2 in distinct sets of primary afferents increased the sEPSC frequencies in a largely common population of DH neurons in laminae III–V, and might underlie the development of mechanical hypersensitivity following probenecid or capsaicin treatment. However, only TRPV1‐expressing afferents facilitated excitatory and/or inhibitory transmission in a subpopulation of lamina II neurons, and this phenomenon might be correlated with the induction of thermal heat hyperalgesia.     

Pre- and Post-synaptic Actions of 5-Hydroxytryptamine in the Rat Lumbar Dorsal Horn In Vitro: Implications for Somatosensory Transmission

Since the relative contribution of pre- versus post-synaptic actions of 5-hydroxytryptamine (5-HT) to modulation of somatosensory processing in the dorsal horn is not known, recordings fro m primary afferents and dorsal horn neurons from in vitro rat spinal cord were used to address this issue. 5-HT produced a depression of spontaneous dorsal root potentials and a slow primary afferent depolarization (PAD): the PAD versus 5-HT concentration-response curve was bell shaped (maximum at 5 μM; 250±C 41.5 μV). In 28/40 dorsal horn neurons, 5-HT elicited a slow depolarization not clearly associated with a specific input resistance change. Excitatory synaptic transmission from primary afferents to dorsal horn neurons was depressed by 5-HT in 40/45 neurons. 5-HT ≥ 5 μM significantly ( P ≤ 0.05) decreased the amplitude, shortened the total duration and half-decay time of the excitatory post-synaptic potential (EPSP). A dominant effect of 5-HT on longer latency EPSP components was evident. There was no direct relationship between the magnitude of PAD and the reduction of the EPSP by 5-HT. 5-Carboxamidotryptamine, an agonist for 5-HT¹ receptors, mimicked the depression of neurotransmission in the dorsal horn without producing PAD. A sample of dorsal horn neurons ( n = 8) was injected with biocytin and their morphology described. All had somata within laminae III-VI. In five of these neurons 5-HT depressed the EPSP but in one interneuron-like and one unclassed neuron the EPSP was potentiated. These data suggest that whilst depression of synaptic transmission is the predominant effect of 5-HT in the deep dorsal horn, this is not easily related to PAD or cellular actions of 5-HT on dorsal horn neurons.     

A two-compartment in vitro model for studies of modulation of nociceptive transmission

Here we present a two-compartment in vitro model in which embryonic rat dorsal root ganglia (DRG) neurons are cultured separately from their target dorsal horn neurons. Although separated, synaptic contact can be established between the peripheral and central neurons since the system allows the DRG axons to project into the other compartment, which contains a network of dorsal horn neurons. The efficacy of the model was evaluated by immunocytochemical, calcium imaging and electrophysiological experiments. The results showed that a subpopulation of the DRG neurons had nociceptor characteristics and that these made synaptic contact with the dorsal horn network. Application of current pulses, according to the stimulus paradigm used, evoked action potentials in DRG axons selectively. This in turn gave rise to increased postsynaptic activity in the network of dorsal horn neurons. The model offers a high degree of efficiency since large numbers of DRG axons can be stimulated simultaneously, thus permitting recording of strong output responses from the dorsal horn neurons. This in vitro model provides a means for studying the mechanisms by which modulatory factors, such as immunoregulatory molecules, applied at either the PNS or the CNS level, can affect synaptic activity and nociceptive transmission in single neurons or network of neurons in the dorsal horn.     

Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate

Intracellular recording techniques have been used to provide information on the identity of excitatory sensory transmitters released at synapses formed between dorsal root ganglion (DRG) and dorsal horn neurons maintained in cell culture. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurons and synaptic potentials were recorded intracellularly from dorsal horn neurons after DRG explant stimulation. More than 80% of dorsal horn neurons within 1 mm of DRG explants received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations, the acidic amino acid receptor agonists, L-glutamate, kainate, and quisqualate excited all dorsal horn neurons which received a monosynaptic DRG neuron input, whereas aspartate and N-methyl-D-aspartate (NMDA) had little or no action. Several compounds reported to antagonize the actions of acidic amino acids were tested for their ability to block DRG-evoked synaptic potentials and glutamate-evoked responses in dorsal horn neurons. 2-Amino-5-phosphonovalerate, a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurons. These findings indicate that glutamate, or a glutamate-like compound, but not aspartate, is the excitatory transmitter that mediates fast excitatory postsynaptic potentials at the DRG-dorsal horn synapses examined in this study.     

Actions of (-)-baclofen on rat dorsal horn neurons.

The actions of a gamma-aminobutyric acid B (GABAB) agonist, (-)-baclofen, on the electrophysiological properties of neurons and synaptic transmission in the spinal dorsal horn (laminae I-IV) were examined by using intracellular recordings in spinal cord slice from young rats. In addition, the effects of baclofen on the dorsal root stimulation-evoked outflow of glutamate and aspartate from the spinal dorsal horn were examined by using high performance liquid chromatography (HPLC) with flourimetric detection. Superfusion of baclofen (5 nM to 10 microM) hyperpolarized, in a stereoselective and bicuculline-insensitive manner, the majority (86%) of tested neurons. The hyperpolarization was associated with a decrease in membrane resistance and persisted in a nominally zero-Ca2+, 10 mM Mg(2+)- or a TTX-containing solution. Our findings indicate that the hyperpolarizing effect of baclofen is probably due to an increase in conductance to potassium ions. Baclofen decreased the direct excitability of dorsal horn neurons, enhanced accommodation of spike discharge, and reduced the duration of Ca(2+)-dependent action potentials. Baclofen depressed, or blocked, excitatory postsynaptic potentials evoked by electrical stimulation of the dorsal roots. Spontaneously occurring synaptic potentials were also reversibly depressed by baclofen. Whereas baclofen did not produce any consistent change in the rate of the basal outflow of glutamate and aspartate, the stimulation-evoked release of the amino acids was blocked. The present results suggest that baclofen, by activating GABAB receptors, may modulate spinal afferent processing in the superficial dorsal horn by at least two mechanisms: (1) baclofen depresses excitatory synaptic transmission primarily by a presynaptic mechanism involving a decrease in the release of excitatory amino acids, and (2) at higher concentrations, the hyperpolarization and increased membrane conductance may contribute to the depressant effect of baclofen on excitatory synaptic transmission in the rat spinal dorsal horn.     

Spinal dorsal horn neurons in elevated extracellular calcium: cell properties and spontaneous discharges

Recordings were made from neurons in the dorsal horn (DH), and from dorsal and ventral roots (DRs and VRs) of isolated spinal cords of infant mice. Raising calcium concentration ([Ca2+]) in the organ bath from 1.2 to 2.4 mmol/l resulted in a slight hyperpolarization, elevation of threshold current (rheobase), and augmentation of excitatory postsynaptic potentials (EPSPs). In many cells EPSPs acquired a much prolonged late phase. Orthodromic stimulation evoked in some DH neurons an action potential that had the same threshold as, and coincided in time with, the 'dorsal horn response' (DHR) recorded from DR. In spinal cords bathed in elevated [Ca2+], DR recordings showed irregularly recurring spontaneous waves, and DH neurons generated spontaneous EPSPs, often with spikes. Some neurons fired irregularly timed spontaneous action potentials that did not appear triggered by EPSPs. In less than 50% of the neurons the spontaneous EPSPs coincided in time with the spontaneous DR waves. The action potentials that appeared without EPSP were fired independently from DR activity. These observations confirm that elevation of interstitial free calcium concentration results in strong enhancement of excitatory transmission, especially of an EPSP of much extended duration. Virtually all neurons showed increased spontaneous activity in high [Ca2+], but only a minority appeared recruited into the synchronized discharges that are detectable as spontaneous waves in DR and VR recordings.     

Synaptic inputs to medullary respiratory neurons from superior laryngeal afferents in the cat.

Synaptic inputs from afferents in the superior laryngeal nerve (SLN) to medullary respiratory neurons (n = 154) in the dorsal respiratory group (DRG), ventral respiratory group (VRG) and the region of the B?tzinger complex (BOT) were studied in anesthetized cats. Single pulse stimulation of the SLN-evoked monosynaptic EPSPs in most inspiratory bulbospinal (I-BS) neurons in the DRG, and disynaptic or oligosynaptic chloride-dependent IPSPs in other I-BS neurons in the DRG and VRG. Stimulation of laryngeal afferents also inhibited oligosynaptically expiratory bulbospinal neurons in the VRG, and all types of respiratory neurons recorded in the BOT region. Oligosynaptic potentials (usually EPSPs) were recorded in inspiratory and expiratory laryngeal motoneurons. These results provide evidence of a processing of SLN-evoked synaptic responses by all tested groups of medullary respiratory neurons. The pathways mediating these synaptic responses are discussed.     

Modulation by muscarine and opioid receptors of acetylcholine release in slices from striato-striatal grafts in the rat

The accumulation of tritium during incubation with [³H]choline and the subsequent efflux of tritium were studied in striatal slices from non-operated rats, in striatal slices from animals which had received a contralateral striatal ibotenic acid lesion, and in slices from striato-striatal suspension grafts, 16–31 weeks after implantation into previously lesioned striata. In graft slices, the accumulation of tritium as well as the overflow of tritium evoked by electrical stimulation (360 pulses, 3 Hz) was much smaller than in slices from non-operated controls. The muscarine receptor agonist oxotremorine (0.1–1 μmol/l) inhibited the stimulation-evoked overflow, and this effect was blocked by the muscarine receptor antagonists atropine (0.1 μmol/l) and pirenzepine (1 μmol/l) in all experimental groups to the same extent. The δ-receptor selective opioid peptide [d-Pen², d-Pen⁵]enkephalin (0.3 μmol/l) inhibited [³H]acetylcholine release in all groups, although its effect was smaller in grafts than in normal tissue. The preferential μ-receptor agonist [d-Ala², N-methyl-Phe⁴,Gly-ol⁵]enkephalin also reduced [³H]acetylcholine release in all groups, but only at the high concentration of 10 μmol/l. The effect of both drugs was antagonized by naloxone (1 μmol/l). The preferential к-receptor agonist ethylketocyclazocine enhanced the stimulation-evoked overflow in non-operated animals, an effect abolished by naloxone and also by sulpiride. In grafts, ethylketocyclazocine caused no change. It is concluded that acetylcholine release in striato-striatal grafts can be modulated by muscarine autoreceptors and by opioid δ receptors. The enhancement by к-receptor activation of [³H]acetylcholine release in non-operated striata depends on a dopaminergic input to the cholinergic cells which does not exist in grafts.     

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.     

A stable prostacyclin analog enhances ectopic activity in rat sensory neurons following neuropathic injury

Prostanoids sensitize sensory afferents during inflammation. However, their role in neuropathic pain is still unclear. We analyzed the actions of prostanoids, non-selective (indomethacin) or selective (celecoxib and NS-398) cyclooxygenase-2 (COX or COX-2) inhibitors, on the ectopic activity of dorsal root ganglia (DRG) and dorsal horn (DH) neurons in a model of neuropathic injury. Extracellular recordings of DRG and DH neurons and cardiovascular measurements were performed on anesthetized, paralyzed and artificially ventilated adult male Sprague-Dawley rats whose sciatic nerve had been transected. PGD(2), PGE(2), PGF(2alpha), carbaprostacyclin (cPGI(2); a stable prostacyclin analog), and carbocyclic thromboxane (cTXA(2)) were administered at cumulative doses (0.0001-5 mg/kg, i.p.) at 5 or 10 min intervals. Only cPGI(2) significantly increased the DRG and DH activity in a dose-dependent manner, with ED(50) values of 0.05 (0.01-0.96) and 0.69 (0.11-1.04) mg/kg, respectively. The other prostanoids did not significantly increase activity, although they reduced heart rate for up to 5 min following administration. Time course experiments with single doses of cPGI(2) (1 mg/kg, i.v.) increased DH discharge rate 3-17 min after injection. Indomethacin (3 mg/kg, s.c.), but not celecoxib or NS-398 (both at 6 mg/kg, s.c.), reduced both DRG and DH activity. Our results indicate that cPGI(2) excites DRG and DH neurons of neuropathic rats, and may suggest a role for IP prostanoid receptors in pain episodes associated with nerve injury. The inhibitory effect of indomethacin, but not celecoxib or NS-398, on ectopic activity may suggest that a tonic generation of PGI(2) by COX-1 could contribute to neuropathic pain.     

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.     

Dynorphin reduces voltage-dependent calcium conductance of mouse dorsal root ganglion neurons

Dynorphin A (DYN) (1 microM) decreased somatic calcium-dependent action potential (CAP) duration of a portion of dorsal root ganglion (DRG) neurons in a naloxone reversible manner. Responses to DYN differed from responses to Leu-enkephalin in that only DYN decreases of somatic CAP duration were associated with decreased action potential after hyperpolarization and persisted after intracellular injection of the potassium channel blocker cesium. While Leu-enkephalin at 10 microM did not affect somatic CAP duration of DRG neurons impaled with cesium-filled micropipettes, dynorphin A (1-8), dynorphin B, and beta-neoendorphin were effective at 1 microM. During single electrode voltage clamp, DYN decreased inward current in a portion of DRG neurons under conditions that predominately isolated calcium current. Leak current was unaffected by dynorphin A. Therefore, we suggest that DYN decreases voltage-dependent calcium conductance. The action on calcium conductance appears specific for opioids with affinity for kappa-receptors.     

Reorganization of the primary afferent termination in the rat spinal dorsal horn during post-natal development

To study the reorganization of the primary afferent input in the spinal dorsal horn during post-natal development, synaptic responses evoked by large Abeta and fine Adelta afferents were recorded from substantia gelatinosa (SG) neurons in slices obtained from immature (post-natal days 21-23) and mature rats (post-natal days 56-60). Threshold stimulus intensities and conduction velocities (CVs) of Abeta and Adelta afferents were determined by intracellular recordings of the antidromic action potentials from dorsal root ganglion (DRG) neurons isolated from immature and mature rats. In immature rats, excitatory postsynaptic currents (EPSCs) were elicited by stimulation sufficient to activate Abeta afferents in the majority of SG neurons (64.9%, 24 of 37 neurons), while most EPSCs observed in mature rats were elicited by stimulation of Adelta afferents (62.5%, 25 of 40 neurons). These observations suggest that the primary afferents innervating SG neurons were reorganized following maturation; Abeta afferents were the predominant inputs to the SG neurons in the immature state, thereafter Adelta afferents were substituted for the Abeta afferents to convey sensory information to the SG neurons. This relatively slow reorganization of the sensory circuitry may correlate with slow maturation of the SG neurons and with a delay in the functional connections of C afferents to the SG neurons.     

Ultrastructural localization of δ-opioid receptor and Met-enkephalin immunoreactivity in rat insular cortex

The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the δ-opioid receptor (DOR) and the endogenous opioid ligand, Met⁵-enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex.     

F11 neuroblastoma × DRG neuron hybrid cells express inhibitory μ- and δ-opioid receptors which increase voltage-dependent K currents upon activation

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express μ- and δ-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of μ and δ, but not κ, receptor expression in F11 cells. Radioligand binding assaysshow that F11 cell membranes bind the μ- and δ-opioid receptor agonists, DAGO and DPDPE with K_d = 4.5 and 4.9 nM and B_max = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K⁺ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the κ-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, ß-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K⁺ current. The opioid-induced increase in voltage-dependent membrane K⁺ current in F11 cells resembles the inhibitory effect elicited by μ- and δ-opioid agonists in primary cultures of mouse DRG neurons.     

Opioids excite dopamine neurons by hyperpolarization of local interneurons.

Increased activity of dopamine-containing neurons in the ventral tegmental area is necessary for the reinforcing effects of opioids and other abused drugs. Intracellular recordings from these cells in slices of rat brain in vitro showed that opioids do not affect the principal (dopamine-containing) neurons but hyperpolarize secondary (GABA-containing) interneurons. Experiments with agonists and antagonists selective for opioid receptor subtypes indicated that the hyperpolarization of secondary cells involved the mu-receptor. Most principal cells showed spontaneous bicuculline-sensitive synaptic potentials when the extracellular potassium concentration was increased from 2.5 to 6.5 or 10.5 mM; these were prevented by TTX and assumed to result from action potentials arising in slightly depolarized local interneurons. The frequency of these synaptic potentials, but not their amplitudes, was reduced by opioids selective for mu-receptors. It is concluded that hyperpolarization of the interneurons by opioids reduces the spontaneous GABA-mediated synaptic input to the dopamine cells. In vivo, this would lead to excitation of the dopamine cells by disinhibition, which would be expected to contribute to the positive reinforcement seen with mu-receptor agonists such as morphine and heroin.     

Segmental disinhibition suppresses C‐fiber inputs to the rat superficial medullary dorsal horn via the activation of GABAB receptors

Specialized primary afferents, although they terminate in different laminae within the dorsal horn (DH), are known to interact through local circuit excitatory and inhibitory neurons. That a loss of segmental inhibition probably contributes to persistent pain hypersensitivity during chronic pain raises the question as to how disinhibition‐induced changes in cross‐modal interactions account for chronic pain symptoms. We sought to characterize how pharmacological blockade of glycine and gamma‐aminobutyric acid (GABA) receptors modifies synaptic transmission between primary afferent fibers and second‐order neurons by recording field potentials in the superficial medullary dorsal horn (MDH) of anesthetized rats. Transcutaneous electrical stimulation evokes three negative field potentials elicited by, from earliest to latest, Aβ‐, Aδ‐ and C‐fiber primary afferents. Blocking segmental glycine and/or GABA_A receptors, with strychnine and bicuculline, respectively, strongly facilitates Aβ‐ and Aδ‐fiber‐evoked polysynaptic field potentials but, conversely, inhibits, or even abolishes, the whole C‐fiber field potential. Blocking segmental GABA_B receptors, with phaclofen, reverses such suppression of C‐fiber field potentials. Interestingly, it also potentiates C‐fiber field potentials under control conditions. Finally, activation of segmental GABA_B receptors, with baclofen, preferentially inhibits C‐fiber field potentials. Our results suggest that activation of A‐fiber primary afferents inhibits C‐fiber inputs to the MDH by the way of polysynaptic excitatory pathways, last‐order GABAergic interneurons and presynaptic GABA_B receptors on C‐fiber primary afferents. Under physiological conditions, activation of such local DH circuits is closely controlled by segmental inhibition but it might contribute to paradoxically reduced pain hypersensitivity under pathological disinhibition.     

Electron microscopic localization of substance P and enkephalin in axon terminals related to dendrites of catecholaminergic neurons

Morphological and pharmacological data suggest that catecholaminergic neurons receive afferent axons positively labeled for the peptides, substance P and [Met⁵]enkephalin. In the present study, electron microscopic immunocytochemistry was used to determine whether a positive reaction for these peptides could be localized to axon terminals forming synapses with catecholaminergic neurons in the locus coeruleus and A2 regions of rat brain. Adjacent sections through these areas were incubated with antiserum to either substance P, [Met⁵]-enkephalin, or tyrosine hydroxylase, a specific marker for catecholaminergic neurons. The sections were subsequently processes by the peroxidase-antiperoxidase immunocytochemical technique. In both the locus coeruleus and A2 region, tyrosine hydroxylase was localized primarily to perikarya and dendrites of intrinsic neurons; whereas substance P and enkephalin-like immunoreactivity was localized to axons and axon terminals. The axon terminals showing positive reactions for substance P and [Met⁵]-enkephalin were morphologically similar to each other and to one type of axon terminal which formed synapses with dendrites labeled for tyrosine hydroxylase. This type of axon terminal always formed asymmetric synaptic junctions and contained 3–4 large (75–100 nm) dense vesicles (LDVs) and many small (40–60 nm) clear vesicles (SCVs). The reaction product for substance P and [Met⁵]-enkephalin was distributed throughout the lumen of the LDVs and formed a rim of labeling around the outer boundaries of the SCVs. These findings demonstrate that substance P and [Met⁵]-enkephalin-positive reactions are selectively localized to subcellular organelles in axon terminals in the locus coeruleus and A2 region of rat brain. They further suggest that the labeled axon terminals form synapses with dendrites of the catecholaminergic neurons.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.