Peregrine B. Osborne

Heteropolymeric potassium channels expressed in xenopus oocytes from cloned subunits

Voltage-dependent potassium currents were measured in Xenopus oocytes previously injected with RNAs generated in vitro from each of three cloned cDNAs (RBK1, RBK2, and RGK5). The currents differed in their sensitivities to blockade by tetraethylammonium (TEA; respective KDs 0.3, greater than 100, and 10 mM) and in their inactivation during a depolarizing pulse. Injections of RNA combinations (RBK1/RBK2 and RBK1/RGK5) caused currents that had TEA sensitivities different from those expected from the sum, in any proportion, of the two native channels. It is concluded that novel potassium channels are formed by the oocytes injected with two RNAs, presumably by heteropolymerization of subunits; such heteropolymerization would contribute functional diversity to voltage-dependent potassium channels in addition to that provided by a large gene family.

Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro.

1. Intracellular recordings were made from neurons of the nucleus raphe magnus (NRM) from rat (n = 128) and guinea‐pig (n = 115). Two types of cells were found in each, primary (103 in rat, 27 in guinea‐pig) and secondary cells (25 in rat, 88 in guinea‐pig). 2. Primary cells had input resistances of 186 +/‐ 9 M omega (n = 9) in rat and 255 +/‐ 50 M omega (n = 11) in guinea‐pig. The action potential in each was about 1.5 ms in duration. Synaptic potentials were evoked by focal electrical stimulation and consisted of both gamma‐aminobutyric acid (GABA) and excitatory amino acid components. 3. Morphine, [Met5]enkephalin (ME) and [D‐Ala2,N‐Me‐Phe4, Gly5‐ol]enkephalin (DAMGO) depressed the amplitude of the GABA‐mediated synaptic potential by a maximum of 50‐65% and had little effect on the excitatory amino acid‐mediated synaptic potential. There was no effect of these opioids on the resting membrane potential or input resistance of primary cells in rat or guinea‐pig. 4. Secondary cells had short duration action potentials (less than 1 ms) and an input resistance of 354 +/‐ 47 M omega in rat (n = 6) and 290 +/‐ 40 M omega in guinea‐pig (n = 15). The synaptic potential observed in the cells of this group was mediated by activation of only excitatory amino acid receptors. 5. ME hyperpolarized and/or abolished the spontaneous firing in sixteen out of twenty‐four neurons in the secondary group from rat and eight out of eighty‐four neurons from guinea‐pig. ME induced an outward current at ‐60 mV that reversed polarity at potentials more negative than ‐92 +/‐ 3 mV in rat (n = 6) and ‐98 +/‐ 2 mV in guinea‐pig (n = 18). The reversal potential of the opioid current was shifted to less negative potentials when the external potassium concentration was increased, as predicted by the Nernst equation. 6. The morphology of the two types of cells were distinguishable in that primary cells were oval (29 x 18 microns in rat; 36 x 19 microns in guinea‐pig) with two to four thick tapering dendrites that branched within 50 microns of the cell body. Secondary cells were generally round or oval (about 24 x 13 microns in rat; 27 x 17 microns in guinea‐pig) with two to five thin non‐tapering dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Where is the locus in opioid withdrawal

Identification of neuroadaptations in specific brain regions that generate withdrawal is crucial for understanding and perhaps treating opioid dependence. It has been widely proposed that the locus coeruleus (LC) is the nucleus that plays the primary causal role in the expression of the opioid withdrawal syndrome. MacDonald Christie, John Williams, Peregrine Osborne and Clare Bellchambers believe that this view and the interpretation of the literature on which it is based are at best controversial. Here, they suggest an alternative view in which regions close to the LC such as the periaqueductal grey, as well as other brain structures which are independent of the LC noradrenergic system, play a more important role in the expression of the opioid withdrawal syndrome.

Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro.

1. Rat caudal periaqueductal grey (PAG) output neurones containing rhodamine microspheres, retrogradely transported from an injection site in the rostral ventromedial medulla (RVM), were visualized in brain slices and recorded from using whole‐cell patch clamp techniques. 2. The specific GABAB receptor agonist baclofen (10 microM) produced an outward current or hyperpolarization in fifty out of fifty‐six caudal PAG output neurones. In 44% of these baclofen‐sensitive neurones, the opioid agonist methionine enkephalin (30 microM) also produced an outward current or hyperpolarization. The opioid current reversed polarity at ‐104 mV and could also be produced by DAMGO, an agonist selective for the mu‐subtype of opioid receptor. 3. Opioid‐responding output neurones were not distributed uniformly in the caudal PAG. In horizontal slices containing lateral PAG, 56% of output neurones were inhibited by opioids, as compared with only 14% of the output neurones in slices containing ventrolateral PAG. 4. These observations are consistent with opioid disinhibition of ventrolateral PAG neurones projecting to the RVM as the predominant mechanism underlying opioid‐induced analgesia in the PAG. The role of opioid receptors found on a major proportion of the output neurones in the lateral PAG remains to be established, but is assumed not be related to modulation of nociceptive function.

μ-Opioid receptor desensitization: Is morphine different?

Opioid tolerance and dependence are important phenomena. The contribution of acute μ‐opioid receptor regulatory mechanisms to the development of analgesic tolerance or physical dependence are unknown, and even the mechanisms underlying relatively rapid receptor desensitization in single cells are unresolved. To a large degree, the uncertainty surrounding the mechanisms and consequences of short‐term regulation of μ‐opioid receptors in single cells arises from the limitations in the experimental design in many of the studies that have investigated these events. Receptor overexpression and use of assays in which regulatory mechanisms are likely to blunt control determinations have led to measurements of opioid receptor activity that are likely to be insensitive to receptor uncoupling. Together with uncertainties concerning molecular details of μ‐opioid receptor interactions with potential regulatory molecules such as G protein‐coupled receptor kinases and arrestins, we are left with an incomplete picture crudely copied from the well‐worked‐out regulatory schema for β2‐adrenoceptors. As a consequence, suggestions that clinically relevant μ‐opioid receptor agonists may have different propensities to produce tolerance and dependence that arise from their differential recruitment of regulatory mechanisms are premature, and have not yet been appropriately assessed, nor explained in the context of a thoroughly established regulatory scheme. In this commentary, we outline the experimental limitations that have given rise to conflicting ideas about how μ‐opioid receptors are regulated, and identify the issues we feel still need to be addressed before we can understand why morphine promotes receptor trafficking differently to other opioids.

Coexpression of prodynorphin and corticotrophin-releasing hormone in the rat central amygdala: evidence of two distinct endogenous opioid systems in the lateral division.

The lateral subdivision of the central nucleus of the amygdala (CeA) comprises two groups of γ‐aminobutyric acid (GABA) neurons that express corticotrophin‐releasing hormone (CRH) and enkephalin. Regulation of the expression and release of these neuropeptides by glucocorticoids and other factors has been suggested to have a regulatory function on the diverse somatic, autonomic, and neuroendocrine responses that are coordinated by the CeA. Because another opioid peptide, dynorphin, has been reported to be also expressed by neurons in the lateral CeA, this study examined the neuronal expression of this kappa‐opioid (KOP) receptor‐preferring ligand by using immunohistochemistry for the precursor peptide prodynorphin. Prodynorphin neurons in the extended amygdala were observed mostly in the medial and central regions of the lateral CeA and the oval of the bed nucleus of the stria terminalis (BST). About one‐third of the prodynorphin neurons in the CeA coexpressed CRH, whereas no coexpression with CRH was detected in the BST. Prodynorphin was not expressed by calbindin neurons in the medial part of the lateral CeA, and indirect evidence suggested that it was not expressed by enkephalin neurons. Coexpression of prodynorphin in extrahypothalamic CRH neurons in the CeA could provide an anatomical basis for regulation of the stress responses and other CRH‐related functions by the brain dynorphin/KOP receptor system. J. Comp. Neurol. 504:702–715, 2007.

Characterization of neurons in the rat central nucleus of the amygdala: Cellular physiology, morphology, and opioid sensitivity

The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin‐filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin‐immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late‐firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low‐threshold bursting neurons. Mu‐opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR‐expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal‐like and pallidal‐like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid‐sensitive neurons. J. Comp. Neurol. 497:910–927, 2006.

17β-Estradiol Activates Estrogen Receptor β-Signalling and Inhibits Transient Receptor Potential Vanilloid Receptor 1 Activation by Capsaicin in Adult Rat Nociceptor Neurons

There is mounting evidence that estrogens act directly on the nervous system to affect the severity of pain. Estrogen receptors (ERs) are expressed by sensory neurons, and in trigeminal ganglia, 17beta-estradiol can indirectly enhance nociception by stimulating expression and release of prolactin, which increases phosphorylation of the nociceptor transducer transient receptor potential vanilloid receptor 1 (TRPV1). Here, we show that 17beta-estradiol acts directly on dorsal root ganglion (DRG) sensory neurons to reduce TRPV1 activation by capsaicin. Capsaicin-induced cobalt uptake and the maximum TRPV1 current induced by capsaicin were inhibited when isolated cultured DRGs neurons from adult female rats were exposed to 17beta-estradiol (10-100 nm) overnight. There was no effect of 17beta-estradiol on capsaicin potency, TRPV1 activation by protons (pH 6-4), and P2X currents induced by alpha,beta-methylene-ATP. Diarylpropionitrile (ERbeta agonist) also inhibited capsaicin-induced TRPV1 currents, whereas propylpyrazole triol (ERalpha agonist) and 17alpha-estradiol (inactive analog) were inactive, and 17beta-estradiol conjugated to BSA (membrane-impermeable agonist) caused a small increase. TRPV1 inhibition was antagonized by tamoxifen (1 microm), but ICI182870 (10 microm) was a potent agonist and mimicked 17beta-estradiol. We conclude that TRPV1 in DRG sensory neurons can be inhibited by a nonclassical estrogen-signalling pathway that is downstream of intracellular ERbeta. This affects the vanilloid binding site targeted by capsaicin but not the TRPV1 activation site targeted by protons. These actions could curtail the nociceptive transducer functions of TRPV1 and limit chemically induced nociceptor sensitization during inflammation. They are consistent with clinical reports that female pelvic pain can increase after reductions in circulating estrogens.

Localization of immunoreactivity for Deleted in Colorectal Cancer (DCC), the receptor for the guidance factor netrin-1, in ventral tier dopamine projection pathways in adult rodents

DCC (deleted in colorectal cancer)-the receptor of the netrin-1 neuronal guidance factor-is expressed and is active in the central nervous system (CNS) during development, but is down-regulated during maturation. The substantia nigra contains the highest level of netrin-1 mRNA in the adult rodent brain, and corresponding mRNA for DCC has also been detected in this region but has not been localized to any particular neuron type. In this study, an antibody raised against DCC was used to determine if the protein was expressed by adult dopamine neurons, and identify their distribution and projections. Significant DCC-immunoreactivity was detected in midbrain, where it was localized to ventrally displaced A9 dopamine neurons in the substantia nigra, and ventromedial A10 dopamine neurons predominantly situated in and around the interfascicular nucleus. Strong immunoreactivity was not detected in dopamine neurons found elsewhere, or in non-dopamine-containing neurons in the midbrain. Terminal fields selectively labeled with DCC antibody corresponded to known nigrostriatal projections to the dorsolateral striatal patches and dorsomedial shell of the accumbens, and were also detected in prefrontal cortex, septum, lateral habenular and ventral pallidum. The unique distribution of DCC-immunoreactivity in adult ventral midbrain dopamine neurons suggests that netrin-1/DCC signaling could function in plasticity and remodeling previously identified in dopamine projection pathways. In particular, a recent report that DCC is regulated through the ubiquitin-proteosome system via Siah/Sina proteins, is consistent with a potential involvement in genetic and sporadic forms of Parkinsons disease.

Characterization of acute homologous desensitization of μ-opioid receptor-induced currents in locus coeruleus neurones

1 Acute homologous desensitization of μ‐opioid receptor‐induced currents was pharmacologically characterized in locus coeruleus (LC) neurones by use of intracellular and whole cell recording in superfuised brain slices. 2 Following desensitization of opioid receptors by perfusion with a high concentration of [Met5] enkephalin (ME) for 5 min, there was a reduction in the maximum response and a rightward shift of the concentration ‐ response curves for ME, [D‐Ala2, N‐MePhe4, Gly ‐ ol] enkephalin (DAMGO) and nor‐morphine. 3 By simultaneously fitting the operational model to the paired pre‐ and post‐desensitization concentration‐response data for each agonist, estimates of the level of desensitization were obtained. The values obtained for the three agonists (between 88% and 96%) were similar and did not vary according to the efficacy of the agonist used. 4 Use of whole cell patch recording techniques caused a slow rundown in the amplitude of ME currents (approx. 40% reduction over 60 min) but did not greatly affect the expression of acute desensitization of opioid currents. 5 When included in the patch recording solution, the phosphatase inhibitors, microcystin (50 nM‐4 μm) and okadaic acid (1 μm) had no effect on the induction of desensitization or the normal ability of opioid or α2‐adrenoceptors to produce currents. Microcystin decreased the rate of recovery of the ME (300 nM) currents following desensitization; however, okadaic acid had little effect on the rate of recovery from desensitization. 6 Strong calcium buffering with BAPTA (10–20 mM) had no effect on desensitization or the recovery from desensitization. 7 These results suggest that acute homologous desensitization of μ‐opioid receptors in LC neurones entails a rapid loss of responsiveness that involves a majority of the receptor population. The mechanism by which desensitization is reversed may involve a non‐calcium‐dependent protein phosphatase but the processess that cause desensitization remain unclear.