After chronic opioid exposure sensory neurons become supersensitive to the excitatory effects of opioid agonists and antagonists as occurs after acute elevation of GM1 ganglioside

Mouse sensory dorsal-root ganglion (DRG) neurons chronically exposed to 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 1 week in culture become tolerant to opioid inhibitory effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD). Acute application of higher concentrations of DADLE (ca. 10 microM) to these treated neurons not only fails to shorten the APD but, instead, generally elicits excitatory effects, i.e. prolongation of the APD. The present study shows that chronic DADLE- or morphine-treated DRG neurons also become supersensitive to the excitatory effects of opioids. Whereas nM concentrations of dynorphin(1-13) are generally required to prolong the APD of naive DRG neurons, fM levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine do not alter the APD of naive DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the treated cells. Similar supersensitivity to the excitatory effects of opioid agonists and antagonists was previously observed after acute treatment of naive DRG neurons with GM1 ganglioside. Our results suggest that both chronic opioid and acute GM1 treatments of DRG neurons greatly enhance the efficacy of opioid excitatory receptor functions so that even the extremely weak agonist properties of naloxone and diprenorphine become effective in prolonging the APD of these treated cells when tested at low concentrations, whereas their antagonist properties at inhibitory opioid receptors do not appear to be altered. Furthermore, whereas cholera toxin-B subunit (CTX-B; 1-10 nM) blocks opioid-induced APD prolongation in naive DRG neurons (presumably by interfering with endogenous GM1 modulation of excitatory opioid receptors functions), even much higher concentrations of CTX-B were ineffective in chronic opioid-treated as well as acute GM1-elevated neurons. These and related data suggest that opioid excitatory supersensitivity in chronic opioid-treated DRG neurons may be due to a cyclic AMP-dependent increase in GM1 ganglioside levels. Our results may clarify mechanisms of opioid dependence and the paradoxical supersensitivity to naloxone which triggers withdrawal symptoms after opiate addiction.     

Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance.

We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.     

Cholera toxin-B subunit blocks excitatory opioid receptor-mediated hyperalgesic effects in mice, thereby unmasking potent opioid analgesia and attenuating opioid tolerance/dependence

In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine’s analgesic effects. This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions. By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng/kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia. Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors. Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE₁ receptor number (B_max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G_sα and S49 cyc⁻ reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE₁ receptors and G_s in situ by application of a C-terminal anti-G_sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE₁ receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G_sα within the PGE₁ receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE₁ receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Inhibitor of cyclic AMP-dependent protein kinase blocks opioid-induced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures

The duration of the calcium component of the action potential (APD) of dorsal root ganglion (DRG) neurons in mouse spinal cord-ganglion explants has been shown to be dually modulated via excitatory and inhibitory opioid receptors. In order to determine if opioid-induced APD prolongation is modulated by receptors that are positively coupled to the adenylate cyclase (AC)/cyclic AMP second messenger system, whole-cell recordings were made from mouse DRG neurons grown in dissociated cell cultures. Tests for opioid responsivity were carried out after intracellular dialysis of an inhibitor of cAMP-dependent protein kinase (PKI). In control recordings, both DADLE-induced APD prolongation as well as shortening were prevented by co-perfusion with the opioid antagonist, diprenorphine (10 nM). Intracellular dialysis of PKI in these neurons completely blocked opioid-induced APD prolongation but did not attenuate APD shortening generally elicited by higher opioid concentrations. Bath perfusion of 10 nM DADLE elicited APD prolongation in 59% of the DRG neurons (n = 34) tested with control solution in the recording pipette, whereas none showed APD prolongation when the pipette contained PKI (n = 18). In control tests with 1 microM DADLE, the APD was prolonged in 37% of the cells and shortened in 26% (n = 19); in contrast, a matched group of PKI-treated cells showed no APD prolongation, whereas 42% showed APD shortening (n = 26). The results support the hypothesis that opioid-induced APD prolongation in DRG neurons is mediated by opioid receptor subtypes that are positively coupled via Gs to AC/cAMP-dependent voltage-sensitive ionic conductances.     

Brief treatment of sensory ganglion neurons with GM1 ganglioside enhances the efficacy of opioid excitatory effects on the action potential

In previous studies, we showed that low (nM) concentrations of opioid prolong the action potential duration (APD) of many mouse dorsal root ganglion (DRG) neurons via Gs-linked excitatory opioid receptors, whereas micromolar opioid levels shorten the APD via Gi/Go-linked inhibitory receptors. In addition, cholera toxin-B subunit (CTX-B) selectively blocks opioid- but not forskolin-induced prolongation of the APD in DRG neurons. Since CTX-B binds with selective high affinity to GM1 ganglioside located on the cell surface, the results suggest that GM1 plays an essential role in regulating excitatory opioid receptor functions. This hypothesis was tested by treating DRG neurons in mouse DRG-cord explants with exogenous gangliosides and determining whether the efficacy of opioid agonists in prolonging the APD is enhanced. The threshold concentration of the opioids, dynorphin(1-13) and morphine required to prolong the APD in many DRG neurons was markedly decreased from nM to fM levels after bath exposure to 10 nM to 1 microM GM1 ganglioside for less than 5 min. In contrast, GM2 and GM3 gangliosides and asialo-GM1 ganglioside were ineffective, even when DRG neurons were exposed to high concentrations (1-10 microM) for periods greater than 1 h. Although GD1a, GD1b and GQ1b gangliosides appeared to be as effective as GM1 when tested at microM concentrations for 15 min, tests at lower concentrations, shorter periods, and/or at lower temperature (24 degrees vs 34 degrees C), showed that they were significantly less effective than GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera toxin-A subunit blocks opioid excitatory effects of sensory neuron action potentials indicating mediation by Gs-linked opioid receptors

Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin pretreatment desensitizes somatostatin receptors linked to adenylate cyclase and facilitates the stimulation of cyclic adenosine 3':5'-monophosphate accumulation in anterior pituitary tumor cells

Somatostatin-14 (SRIF) inhibits both hormone- and forskolin-stimulated cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation in tumor cells of the mouse anterior pituitary (AtT-20/D16-16). However, long-term pretreatment of cells with SRIF modifies the responsiveness of this system in two ways: The response of adenylate cyclase to stimulatory agents is enhanced, whereas the ability of SRIF to inhibit stimulated cyclic AMP formation is reduced. The supersensitive adenylate cyclase response and the SRIF desensitization were dependent on the concentration and duration of SRIF pretreatment. Enhancement of forskolin-stimulated cyclic AMP formation occurred within 4 hr, whereas that of corticotropin-releasing-factor-, (-)-isoproterenol-, and vasoactive intestinal peptide-induced cyclic AMP accumulation required 16 hr of pretreatment. The elevated responses to each of these stimulants were due to increases in their maximal ability to stimulate cyclic AMP formation. Cycloheximide treatment blocked the enhanced cyclic AMP response induced by SRIF pretreatment, suggesting a requirement for protein synthesis. In membrane preparations, SRIF pretreatment facilitated activation of adenylate cyclase by forskolin, sodium fluoride, and guanosine 5'-(beta,tau-imido)-triphosphate without affecting basal activity. These results suggest that desensitization of an inhibitory input to adenylate cyclase is accompanied by a supersensitivity of adenylate cyclase to stimulatory agents through a process requiring protein synthesis.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Etorphine elicits anomalous excitatory opioid effects on sensory neurons treated with GM1 ganglioside or pertussis toxin in contrast to its potent inhibitory effects on naive or chronic morphine-treated cells

The ultra-potent opioid analgesic, etorphine, elicits naloxone-reversible, dose-dependent inhibitory effects, i.e. shortening of the action potential duration (APD) of naive and chronic morphine-treated sensory dorsal root ganglion (DRG) neurons, even at low (pM-nM) concentrations. In contrast, morphine and most other opioid agonists elicit excitatory effects, i.e. APD prolongation, at these low opioid concentrations, require much higher (ca. 0.1–1 μM) concentrations to shorten the APD of naive neurons, and evoke only excitatory effects on chronic morphine-treated cells even at high > 1–10 wM concentrations. In addition to the potent agonist action of etorphine at μ-, δ- and κ-inhibitory opioid receptors in vivo and on DRG neurons in culture, this opioid has also been shown to be a potentantagonist of excitatory μ-, δ- and κ-receptor functions in naive and chronic morphine-treated DRG neurons. The present study demonstrates that the potent inhibitory APD-shortening effects of etorphine still occur in DRG neurons tested in the presence of a mixture of selective antagonists that blocks all μ-, δ- and κ-opioid receptor-mediated functions, whereas addition of the epsilon (ε)-opioid-receptor antagonist, β-endorphin(1–27) prevents these effects of etorphine. Furthermore, after markedly enhancing excitatory opioid receptor functions in DRG neurons by treatment with GM1 ganglioside or pertussis toxin, etorphine showsexcitatory agonist action onnon-μ-/δ-/κ-opioid receptor functions in these sensory neurons, in contrast to its usual potent antagonist action on μ-, δ- and κ-excitatory receptor functions in naive and even in chronic morphine-treated cells which become supersensitive to the excitatory effects of μ-, δ- and -opioid agonists. This weak excitatory agonist action of etorphine on non-μ-/δ-/κ-opioid receptor functions may account for the tolerance and dependence observed after chronic treatment with extremely high doses of etorphine in vivo.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Ontogeny of beta-adrenergic receptor-mediated cyclic AMP generating system in primary cultured neurons.

The developmental changes in the beta-adrenergic receptor/cyclic AMP generating system were examined using mouse cerebral cortical neurons in primary culture. During neuronal growth in vitro, the number of binding sites for [3H]dihydroalprenolol (DHA) showed a tendency to increase (Bmax), while the affinity (Kd) for [3H]DHA did not show any noticeable changes. Basal and isoproterenol-stimulated adenylate cyclase activities as well as the activation of adenylate cyclase by 5'-guanylylimidodiphosphate (GppNHp), NaF and forskolin showed progressive and parallel increases during neuronal growth on a polylysine-coated surface. The treatment of primary cultured neurons with islet-activating protein (IAP), one of the pertussis toxins, attenuated the inhibitory effect of carbachol, a muscarinic agonist, on isoproterenol-induced activation of adenylate cyclase activity. These results indicate that primary cultured neurons possess a cyclic AMP generating system coupled with beta-adrenergic and muscarinic receptors, which is regulated via stimulatory and inhibitory GTP-binding proteins, respectively. The results described above also suggest that the beta-adrenergic receptor, stimulatory and inhibitory types of GTP-binding proteins and adenylate cyclase may develop in a parallel fashion during neuronal growth on a polylysine-coated surface.     

Cyclic AMP-mediated potentiation of muscarinic hyperpolarization in neuroblastoma cells

Adenosine, 2-chloroadenosine and prostaglandin E₁ which are known to increase cyclic AMP in neuroblastoma cells potentiated the acetylcholine-induced muscarinic hyperpolarization of the cells without changing the resting membrane potential. The potentiation caused by 2-chloroadenosine was further augmented by Ro 20-1724, a phosphodiesterase inhibitor. A direct intracellular pressure application of cyclic AMP potentiated the muscarinic hyperpolarization without changing the resting membrane potential. Morphine which inhibits adenylate cyclase antagonized 2-chloroadenosine-induced potentiation of the muscarinic hyperpolarization. These results suggest that changes in cyclic AMP level modulate the muscarinic response of neuroblastoma cells.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release

Fractional [³H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC₅₀ ≥ 1 μM). Release was also stimulated by 3-isobutyl-l-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.     

Catecholamines and cyclic nucleotides in the modulation of superior cervical ganglia

Four hypotheses have been advanced to explain the relationships between the cholinergic presynaptic afferents, principal ganglionic neurons, and small, intensely fluorescent (SIF) cells in synaptic transmission in the mammalian superior cervical ganglion (SCG). The first hypothesis involves the role of the dopaminergic SIF cell and cyclic AMP in the modulation of ganglionic transmission through the generation of a slow inhibitory postsynaptic potential (s-IPSP). The second concerns the generation of a slow excitatory postsynaptic potential (s-EPSP), and the role of dopamine and cyclic AMP in potentiating it. The third postulates that a presynaptic α-adrenergic receptor is responsible for inhibiting f-EPSP generation. A fourth hypothesis concerns the localization of the β-adrenergic receptor—adenylate cyclase complex in SCG. This paper discusses the evidence for each hypothesis, with special emphasis on species variations in the modulation of ganglionic transmission by catecholamines, adrenergic receptors, and cyclic AMP.     

Pituitary adenylate cyclase-activating peptide (PACAP) induces differentiation in the neuronal F11 cell line through a PKA-dependent pathway

PACAP is a peptide with neuroprotective activity, which induces adenylate cyclase and protein kinase A (PKA) activity. PACAP has also been shown to induce neurite outgrowth in PC12 cells and dorsal root ganglion (DRG) neurons. Here, we report that exogenous PACAP38 promotes neurite outgrowth in the F11 neuroblastoma/dorsal DRG hybrid cell line. Using an automated microscopy system, we show that PACAP38 induces a 170-fold increase in neurite length, with an EC50 of 3.1 nM, compared to 3.7 microM for forskolin and 143.4 microM for dibutyril cyclic AMP (dbcAMP). PACAP38 induced a 4-fold increase in the level of phosphorylation of cAMP-responsive element binding protein (CREB) in F11 cells with an EC50 of 130 pM. In contrast a peptide related to PACAP, vasoactive intestinal peptide (VIP) failed to induce CREB phosphorylation or neurite outgrowth in F11 cells. Addition of the nonselective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX) increased the potency of PACAP at inducing neurite outgrowth by ten-fold. The PKA inhibitor, H89, was a potent inhibitor of PACAP38-induced neurite outgrowth. The delta-opioid receptor agonist, SNC 80, did not inhibit PACAP-induced neurogenesis even though it did reduce CREB phosphorylation. In contrast to previous studies in PC12 cells, PACAP38 failed to show MEK1 activation in F11 cells. PACAP is upregulated in DRG neurons as a result of injury, and F11 cells provide an easily accessible in vitro model for understanding mechanisms underlying PACAP differentiation and neurogenesis.     

Cholera toxin-induced Gsα down-regulation in neural tissue: studies on the pineal gland

Cholera toxin (CT) treatment (50 μg/ ml) was used to down regulate the α subunit of the stimulatory guanine nucleotide binding protein (Gsα) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces G_sα by ≈ 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of Gβ, G_iα or G_oα. This effect on G_sα was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of G_sα. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP (N⁶,2′-O-dibutyryl adenosine 3′,5′monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced α- or β-adrenergic stimulation. α₁-Adrenergic activation of the pineal gland elevates [Ca²⁺]_i, which potentiates effects of cyclic AMP; in these studies the response to α-adrenergic activation was markedly increased in 48-h CT-treated glands, reflecting Ca²⁺ potentiation of the effects of elevated levels of cyclic AMP. In contrast, the effects of the selective β-adrenergic agonist isoproterenol was reduced by ≈ 75%. These studies not only establish CT-induced G_sα down-regulation as a new tool for the study of adrenergic signal transduction in the pineal gland, but indicate that this paradigm is probably useful in all neural tissue.     

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.     

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.