Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Expression of a cloned rat brain potassium channel in Xenopus oocytes.

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.

Phosphorylation-deficient G-protein-biased µ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects.

Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the µ-opioid receptor and interaction with ß-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit ß-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that ß-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute µ-opioid receptor desensitization and long-term tolerance.

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.

Cellular neuroadaptations to chronic opioids: tolerance, withdrawal and addiction.

A large range of neuroadaptations develop in response to chronic opioid exposure and these are thought to be more or less critical for expression of the major features of opioid addiction: tolerance, withdrawal and processes that may contribute to compulsive use and relapse. This review considers these adaptations at different levels of organization in the nervous system including tolerance at the mu-opioid receptor itself, cellular tolerance and withdrawal in opioid-sensitive neurons, systems tolerance and withdrawal in opioid-sensitive nerve networks, as well as synaptic plasticity in opioid sensitive nerve networks. Receptor tolerance appears to involve enhancement of mechanisms of receptor regulation, including desensitization and internalization. Adaptations causing cellular tolerance are more complex but several important processes have been identified including upregulation of cAMP/PKA and cAMP response element-binding signalling and perhaps the mitogen activated PK cascades in opioid sensitive neurons that might not only influence tolerance and withdrawal but also synaptic plasticity during cycles of intoxication and withdrawal. The potential complexity of network, or systems adaptations that interact with opioid-sensitive neurons is great but some candidate neuropeptide systems that interact with mu-opioid sensitive neurons may play a role in tolerance and withdrawal, as might activation of glial signalling. Implication of synaptic forms of learning such as long term potentiation and long term depression in opioid addiction is still in its infancy but this ultimately has the potential to identify specific synapses that contribute to compulsive use and relapse.

Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins?

The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Enhanced Fos expression in glutamic acid decarboxylase immunoreactive neurons of the mouse periaqueductal grey during opioid withdrawal.

Previous studies using c-Fos immunohistochemistry suggest that a sub-population of neurons in the midbrain periaqueductal gray region is activated during opioid withdrawal. The neurochemical identity of these cells is unknown but cellular physiological studies have implicated GABAergic neurons. The present study investigated whether GABAergic neurons are activated in the mouse periaqueductal gray during opioid withdrawal using dual-antibody immunohistochemistry for Fos and glutamic acid decarboxylase. Both chronic opioid treatment and naloxone-precipitated opioid withdrawal increased Fos expression in the periaqueductal gray, with the greatest increase being four-fold in the caudal ventrolateral subdivision following withdrawal. Neurons stained for both Fos and glutamic acid decarboxylase were greatly enhanced in all subdivisions of the periaqueductal gray following withdrawal, particularly in the lateral and ventrolateral divisions where the increase was up to 70-fold. These results suggest that activation of a subpopulation of GABAergic interneurons in the periaqueductal gray plays a role in opioid withdrawal.

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.

Retrograde signalling by endocannabinoids.

The cannabinoid neurotransmitter system comprises cannabinoid G protein-coupled membrane receptors (CB1 and CB2), endogenous cannabinoids (endocannabinoids), as well as mechanisms for their synthesis, membrane transport and metabolism. Within the brain the marijuana constituent delta9-tetrahydrocannabinol (THC) produces its pharmacological actions by acting on cannabinoid CB1 receptors. THC modulates neuronal excitability by inhibiting synaptic transmission via presynaptic CB1-mediated mechanisms. More recently, it has been established that physiological stimulation of neurons can induce the synthesis of endocannabinoids, which also modulate synaptic transmission via cannabinoid CB1 and other receptor systems. These endogenously synthesised endocannabinoids appear to act as retrograde signalling agents, reducing synaptic inputs onto the stimulated neuron in a highly selective and restricted manner. In this review we describe the cellular mechanisms underlying retrograde endocannabinoid signalling.

ß-Arrestin-2 knockout prevents development of cellular µ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons.

BACKGROUND AND PURPOSE: Tolerance to the behavioural effects of morphine is blunted in ß-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from ß-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. EXPERIMENTAL APPROACH: µ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and ß-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. KEY RESULTS: MOPr inhibition of ICa in PAG neurons was unaffected by ß-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT ß-arrestin-2 knockouts. However, ß-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. CONCLUSIONS AND IMPLICATIONS: In ß-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by ß-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in ß-arrestin-2 knockouts. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Isolation and pharmacological characterisation of delta-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin.

delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.

Effects of sumatriptan on rat medullary dorsal horn neurons.

This study examined the cellular actions of the anti-migraine drug sumatriptan, on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis. Sumatriptan inhibited the miniature EPSC (mEPSC) rate in a dose dependent fashion, with an EC(50) of 250 nM. Sumatriptan (3 microM) inhibited the mEPSC rate by 36%, without altering the mEPSC amplitude. This effect was partially reversed by the 5HT(1D) specific antagonist BRL15572 (10 microM). In contrast, the 5HT(1B) agonist CP93129 (10 microm) did not alter the mEPSC rate. Furthermore, sumatriptan (3 microM) decreased the amplitude of electrically evoked EPSCs (eEPSC) by 40%. After incubating the slices in ketanserin (an antagonist which shows selectivity for 5HT(1D) over 5HT(1B) receptors) sumatriptan had little effect on eEPSC amplitude. In control conditions paired stimuli resulted in paired pulse depression (PPD; the ratio eEPSC(2)/eEPSC(1)=0.7+/-0.01), whilst in the presence of sumatriptan the PPD was blocked (ratio eEPSC(2)/eEPSC(1)=0.9+/-0.1). Sumatriptan produced no post-synaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (-60 to -130 mV). RT-PCR experiments revealed the presence of mRNA for both 5HT(1D) and 5HT(1B) receptor subtypes in the trigeminal ganglia and subnucleus caudalis. These data suggest that sumatriptan acts pre-synaptically on trigeminal primary afferent central terminals to reduce the probability of release of glutamate, and that this action is mediated through 5HT(1D) receptors.

Dye-coupling among neurons of the rat locus coeruleus during postnatal development.

Simultaneous recordings from pairs of locus coeruleus neurons in neonatal rat brain slices previously demonstrated synchronous, subthreshold oscillations of membrane potential (rats < 24 days old) and electronic-coupling between 40% of pairs of neurons from rats less than 10 days old. In the present study, slices from 1-21 day-old rats were stained with avidin-HRP-diaminobenzidine only if a single neuron per slice was impaled for longer than 10 min using an electrode containing biocytin. In slices from rats less than one week old, multiple stained neurons (3.8 +/- 0.6 neurons/slice) were observed in 10 of 11 slices. Apparent contacts between stained neurons were observed at varying distances along dendrites. In rats older than one week significantly fewer multiple stained neurons were observed (three of 20 slices). The proportion of neurons displaying spontaneous subthreshold oscillations of membrane potential decreased with age, and the frequencies of subthreshold oscillations of membrane potential and entrained action potentials increased with age. The presence of multiple stained neurons was not correlated with the occurrence of subthreshold oscillations, cell input resistance, or the number of coupled neurons predicted from the shape of electronic potentials. In recordings from neurons displaying subthreshold oscillations, input resistance was lower and the number of coupled neurons predicted from electrotonic potentials was greater than in those without oscillations. These results suggest that low resistance pathways are common between locus coeruleus neurons in brain slices from rats younger than about one week old, consistent with previous electrotonic-coupling studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Presynaptic delta opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat.

The specific role of the Delta opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure. Following injections of retrograde tracer into the spinal cord or intracellular labelling of respiratory neurons, in Sprague-Dawley rats, brainstem sections were processed for retrograde or intracellular labelling and DOR-ir. Bulbospinal neurons were apposed by DOR-ir boutons regardless of whether they projected to single (cervical or thoracic ventral horn) or multiple (cervical and thoracic ventral horn) targets in the spinal cord. In the VRG, a total of 24 +/- 5% (67 +/- 13/223 +/- 49) of neurons projecting to the cervical ventral horn, and 37 +/- 3% (96 +/- 22/255 +/- 37) of neurons projecting to the thoracic ventral horn, received close appositions from DOR-ir boutons. Furthermore, DOR-ir boutons closely apposed six of seven intracellularly labelled neurons, whilst the remaining neuron itself possessed boutons that were DOR-ir. DPDPE was microinjected (10 mM, 60 nl, unilateral) into regions of respiratory field activity in the VRG of anaesthetised, vagotomised rats, and the effects on phrenic nerve discharge and mean arterial pressure were recorded. DPDPE depressed phrenic nerve amplitude, with little effect on phrenic nerve frequency in the Bötzinger complex, pre-Bötzinger complex and rVRG, the greatest effects occurring in the Bötzinger complex. The results indicate that the DOR is located on afferent inputs to respiratory neurons in the VRG. Activation of the DOR in the VRG is likely to inhibit the release of neurotransmitters from afferent inputs that modulate the pattern of activity of VRG neurons.

Swim-stress but not opioid withdrawal increases expression of c-fos immunoreactivity in rat periaqueductal gray neurons which project to the rostral ventromedial medulla.

Expression of c-fos-like immunoreactivity has been used as a marker for neuronal activation and is elevated in the periaqueductal gray following stressful and noxious stimuli, and opioid withdrawal. The present study examined the staining of c-fos-like immunoreactivity following opiate withdrawal or swim-stress (2.5-3 min at 21 degrees C) in periaqueductal gray neurons of the rat which had projections to and through the rostral ventromedial medulla identified by microinjection of the retrograde tracer, Fast Blue, into the nucleus raphe magnus prior to development of morphine dependence. Both naloxone-precipitated withdrawal and swim-stress increased numbers of neurons expressing c-fos-like immunoreactivity in periaqueductal gray. Naloxone-precipitated withdrawal did not increase the number of double-labelled neurons in periaqueductal gray suggesting that neurons excited during opioid withdrawal do not project to the ventromedial medulla. In contrast, swim-stress produced increases in double-labelled neurons in periaqueductal gray suggesting that many periaqueductal gray neurons activated by swim-stress project to the ventromedial medulla. These findings suggest that naloxone-precipitated withdrawal does not activate ventrolateral periaqueductal gray neurons which are involved in descending inhibitory pathways, consistent with behavioural observations that naloxone-precipitated withdrawal is qualitatively opposite to electrical and chemical stimulation of the ventrolateral periaqueductal gray. The results are also consistent with a role of descending projections from periaqueductal gray in stress-induced antinociception.

Excitatory amino acid projections to the periaqueductal gray in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA.

The afferents to the periaqueductal gray utilizing excitatory amino acid transmitters have been described in rat brain by autoradiography following microinfusion and retrograde transport of D[3H]aspartate. Parallel experiments employing injections of [3H]GABA established that the retrograde labelling found with D[3H]aspartate was transmitter-selective. Following infusion of D[3H]aspartate, perikaryal labelling was found in nine subcortical areas, particularly infralimbic and cingulate cortices, with a predominance of ipsilateral labelled perikarya. Heaviest cortical labelling was localized in perirhinal cortex, in an extensive band of cells adjoining the rhinal sulcus. The hypothalamus contained the heaviest perikaryal labelling within brain: D[3H]aspartate labelled cells in 11 hypothalamic and mammillary nuclei. Intense bilateral labelling was obtained in ventromedial hypothalamus, although the number of perikarya was lower contralaterally. D[3H]Aspartate also produced heavy ipsilateral labelling of perikarya in posterior hypothalamus. Labelling patterns in cortex and hypothalamus were precise and topographic, and [3H]GABA never labelled cells in these regions. Other telencephalic and diencephalic areas containing prominent, retrogradely labelled cells were the lateral septum, amygdala, zona incerta and lateral habenula. The relative density of labelled cells in mesencephalic areas was much lower than that found in cortex and hypothalamus, although D[3H]aspartate labelled a moderate number of perikarya in the inferior colliculus and cuneiform nucleus. A smaller number of heavily labelled cells was found in the parabrachial nuclei, Kolliker-Fuse nucleus and laterodorsal tegmental nucleus. Only occasional labelled perikarya were observed in the myencephalon. Low densities of labelled cells were found after the injection of [3H]GABA into the periaqueductal gray, and the only regions in which a small number of perikarya were labelled by both [3H]GABA and D[3H]aspartate were the dorsal raphe and parabrachial nuclei. Overall, the retrograde transport of D[3H]aspartate revealed a complex topographic and convergent network of afferent pathways to the periaqueductal gray likely to utilize an excitatory amino acid transmitter. Our findings confirm the selectivity of this neurochemical mapping technique and provide evidence that hypothalamic, habenular, subthalamic and cuneiform afferents to the periaqueductal gray utilize an acidic amino acid as their transmitter. They also confirm that corticofugal afferents to periaqueductal gray utilize an excitatory amino acid.

Excitatory amino acid projections to the nucleus accumbens septi in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA.

Afferents to the nucleus accumbens septi utilizing glutamate or aspartate have been investigated in the rat by autoradiography following injection and retrograde transport of D[3H]aspartate. Parallel experiments with the intra-accumbal injection of [3H]GABA were employed to establish the transmitter-selective nature of the retrograde labelling found with D[3H]aspartate. The topography of cortical and thalamic perikarya labelled by D[3H]aspartate was extremely precise. D[3H]Aspartate labelled perikarya were found in layer V of agranular insular cortex; bilaterally within prelimbic and infralimbic subareas perikarya, but predominantly ipsilaterally. Ipsilateral labelling was observed in dorsal, ventral and posterior agranular insular cortices, and in perirhinal cortex. Injections into ventral accumbens labelled perikarya in ipsilateral entorhinal cortex, while infusion of D[3H]aspartate into anterior caudate-putamen resulted in labelling of perikarya in ipsilateral cingulate and lateral precentral cortices. Following infusion of D[3H]aspartate, ipsilateral midline thalamic nuclei contained the highest density of labelled perikarya; infusions centred on nucleus accumbens resulted in heavy retrograde labelling of the parataenial nucleus, but labelling was sparse from a lateral site and not observed after injection into anterior caudate-putamen. Less prominent labelling of perikarya was seen in other thalamic nuclei (mediodorsal, central medial, rhomboid, reuniens and centrolateral), mostly near the midline. Perikaryal labelling was also found in the ipsilateral amygdaloid complex, particularly in basolateral and lateral nuclei. Only weak labelling resulted in ventral subiculum. Numerous labelled cells were present bilaterally in anterior olfactory nucleus, although perikarya were more prominent ipsilaterally. Labelled perikarya were not consistently observed in other regions (ventral tegmental area, medial substantia nigra, raphe nuclei and locus coeruleus) known to innervate nucleus accumbens. Presumptive anterograde labelling was detected in ventral pallidum/substantia innominata, ventral tegmental area and medial substantia nigra. [3H]GABA was generally not retrogradely transported to the same regions labelled by D[3H]aspartate; an exception being the anterior olfactory nucleus, where large numbers of labelled perikarya were found. [3H]GABA failed to label perikarya in thalamus and amygdala, and a topographic distribution of label was absent in neocortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of occult infection following total joint arthroplasty using sequential technetium-99m HDP bone scintigraphy and indium-111 WBC imaging.

Preoperative exclusion or confirmation of periprosthetic infection is essential for correct surgical management of patients with suspected infected joint prostheses. The sensitivity and specificity of [111In]WBC imaging in the diagnosis of infected total joint prostheses was examined in 28 patients and compared with sequential [99mTc]HDP/[111In]WBC scintigraphy and aspiration arthrography. The sensitivity of preoperative aspiration cultures was 12%, with a specificity of 81% and an accuracy of 58%. The sensitivity of [111In]WBC imaging alone was 100%, with a specificity of 50% and an accuracy of 65%. When correlated with the bone scintigraphy and read as sequential [99mTc]HDP/[111In]WBC imaging, the sensitivity was 88%, specificity 95%, and accuracy 93%. This study demonstrates that [111In]WBC imaging is an extremely sensitive imaging modality for the detection of occult infection of joint prostheses. It also demonstrates the necessity of correlating [111In]WBC images with [99mTc]HDP skeletal scintigraphy in the detection of occult periprosthetic infection.

Hyperpolarization by opioids acting on mu-receptors of a sub-population of rat periaqueductal gray neurones in vitro.

1. The actions of opioids on membrane properties of rat periaqueductal gray neurones were investigated using intracellular recordings from single neurones in brain slices. Morphological properties and anatomical location of each impaled neurone were characterized by use of intracellular staining with biocytin. The present paper primarily considers neurones which were directly hyperpolarized by opioids. The accompanying paper considers inhibition of synaptic transmission by opioids. 2. Met-enkephalin (10-30 microM) hyperpolarized 29% (38/130) of neurones. The hyperpolarization was fully antagonised by naloxone (1 microM, n = 3). The response to Met-enkephalin was not affected by agents which block synaptic neurotransmission (1 microM tetrodotoxin, and 0.1 microM tetrodotoxin + 4 mM Co2+, n = 3). 3. The specific mu-receptor agonist, D-ala-met-enkephalin-glyol (3 microM, n = 17) produced hyperpolarizations of similar amplitude to those produced by Met-enkephalin (10-30 microM). The EC50 of D-ala-met-enkephalin-glyol was 80 nM and the maximum response was achieved at 1-3 microM. The delta-receptor (D-Pen-D-Pen-enkephalin, 3 microM, n = 7) and kappa-receptor (U50488H, 3 microM, n = 5) agonists had no effect on the membrane properties of these neurones. 4. The opioid-induced hyperpolarization was associated with an increased potassium conductance. Hyperpolarizations were accompanied by a significant decrease in membrane resistance between -70 and -80 mV, and a significantly greater decrease between -110 and -140 mV (n = 16). Hyperpolarizations reversed polarity at -111 +/- 3 mV (n = 16), close to the expected equilibrium potential for potassium ions. The reversal potential of outward currents increased by 24 mV when the extracellular potassium concentration was raised from 2.5 to 6.5 mM, which is close to the value predicted by the Nernst equation (25 mV) for a potassium conductance.5. Resting inward rectification (reduced input resistance at potentials more negative than - 100 mV in the absence of opioids) was significantly greater in neurones which were hyperpolarized by opioids than in those which were not hyperpolarized. The amplitude of action potential after hyperpolarizations was significantly smaller in neurones which were hyperpolarized by opioids. Other membrane properties did not differ significantly between opioid-sensitive and -insensitive neurones.6. Neurones hyperpolarized by opioids were multipolar (58%), triangular (21%) or fusiform (5%) in shape with a soma diameter of 22 +/- 1 microm (n = 19, longest axis). Dendritic spread was in a large radiating pattern, usually in all directions, with axons usually originating from primary dendrites. The axons were usually branched and projected in several directions. Morphological properties did not differ significantly between opioid-sensitive and -insensitive neurones.7. Neurones hyperpolarized by opioids were located predominantly in the lateral periaqueductal gray,as well as in the more dorsal areas of the ventrolateral periaqueductal gray, whereas neurones not hyperpolarized by opioids were located in the more ventral areas of the ventrolateral periaqueductal gray.8. These studies demonstrate that opioids acting on micro-receptors increase potassium conductance in a sub-population of large neurones located predominantly in the lateral column of the periaqueductal gray. The neurones hyperpolarized by opioids could be involved in the antinociceptive actions of opioids, but might also be involved in other functions because a large proportion lie outside of the main'antinociceptive zone' of the periaqueductal gray. It is also unlikely that these neurones are GABAergic,suggesting that they might not participate in the postulated antinociceptive action of opioids mediated via disinhibition of neurones which project to the ventral medulla.

Inhibition by opioids acting on mu-receptors of GABAergic and glutamatergic postsynaptic potentials in single rat periaqueductal gray neurones in vitro.

1. Membrane properties of rat periaqueductal gray neurones were investigated by use of intracellular recordings from single neurones in brain slices. Morphological properties and anatomical location of each impaled neurone were characterized by intracellular staining with biocytin. The present paper considers the properties of electrically-evoked and spontaneous postsynaptic potentials impinging on periaqueductal gray neurones, and the actions of opioids on postsynaptic potentials in neurones which were not directly hyperpolarized by opioids. The preceding paper considers neurones which were hyperpolarized by opioids. 2. Electrical stimulation in the vicinity of impaled neurones evoked postsynaptic potentials having fast (duration at half-maximal amplitude 37 +/- 2 ms, n = 65) and in some cases slow (duration at half-maximal amplitude 817 +/- 187 ms, n = 3) components. Amplitudes of evoked potentials were dependent on stimulus voltage, membrane potential, and were abolished during superfusion with solutions containing tetrodoxotoxin (100 nM to 1 microM, n = 5) or Co2+ (4 mM, n = 2). 3. Fast postsynaptic potentials were mediated predominantly by activation of glutamate and GABAA receptors. The GABAA-receptor antagonist, bicucuilline (30 microM), inhibited postsynaptic potentials by 44 +/- 8% (n = 14). The non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), inhibited postsynaptic potentials by 48 +/- 6% (n = 16). Combined superfusion of bicuculline (30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) inhibited postsynaptic potentials by 93 +/- 1% (n = 8). Additional superfusion of the NMDA-receptor antagonist, (+/-)-2-amino-5- phosphonovaleric acid (50 microM) inhibited synaptic potentials by 94 +/- 1% (n = 3). 5. Selective micro-receptor agonists inhibited fast postsynaptic potentials in all neurones tested which were not directly hyperpolarized by opioids. Met-enkephalin (30 micro M) and Tyr-D-Ala-Gly-MePhe-Glyol (3 microM)inhibited postsynaptic potentials by 53 +/- 3% and 49 +/- 3%, respectively. This effect was completely antagonised by naloxone (1 micro M, n = 3). A small inhibition produced by the selective delta-receptor agonist,Tyr-D-Pen-Gly-Phe-D-Pen-enkephalin (3 micro M, 26 +/- 4%, n = 14), was antagonized by naloxone (1 micro M), but not by the selective delta-receptor antagonist, naltrindole (10 nM), suggesting non-specific micro-receptor activation by this agonist. The selective K-receptor agonist, U50488H (3 micro M), also consistently inhibited postsynaptic potentials by 45 +/- 15% (n = 4). However, this effect was not fully reversed by naloxone(1 micro M) suggesting a non-specific action.6. Both glutamatergic and GABAergic components of fast postsynaptic potentials were inhibited by Met-enkephalin (10 or 30 micro M). Met-enkephalin inhibited postsynaptic potentials by 55 +/- 5% (n = 12) in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM, predominantly GABAergic component).Met-enkephalin did not affect the response to GABA applied directly by pressure ejection, indicating that opioids exclusively inhibited presynaptic release of GABA. Met-enkephalin (10-30 micro M) inhibited postsynaptic potentials by 48 +/- 6% (n = 11) in the presence of bicuculline (30 micro M, predominantly glutamatergic component). In the presence of both bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione,Met-enkephalin inhibited the small residual component of the synaptic potential by 42 +/- 15% (n = 2).7. Frequent spontaneous synaptic potentials were also observed in 11% (10/94) of the neurones which were not directly hyperpolarized by opioids. These were reversibly abolished by bicuculline (30 micro M,n = 5) and substantially inhibited by Met-enkephalin (30 micro M, n = 6), but were unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM, n = 2).8. In conclusion, fast glutamatergic and GABAergic synaptic potentials were evoked by electrical stimulation throughout the lateral and ventrolateral periaqueductal gray. Slow inhibitory synaptic potentials were also evoked in some neurones. Opioids acting on micro-receptors inhibited both GABAergic and glutamatergic components of synaptic potentials throughout this brain region.