R. Alan North

Neurobiology of opiate abuse

Opiates interact with cell surface receptors on neurons involved in the transmission of information along neural pathways that are related to behaviours essential for the life of the self and of the species. Opiates are provided with powerful and multifaceted rewarding properties that are fundamental for the acquisition, maintenance and relapse of opiate addiction. Gaetano Di Chiara and Alan North argue that both dopaminergic and non-dopaminergic systems are involved in opiate reward, and that opiate addiction results from adaptive and learning processes involving both positive reinforcing mechanisms related to the rewarding properties of opiates and negative reinforcing mechanisms related to the aversive properties of withdrawal in dependent subjects.

Calcium-activated potassium channels expressed from cloned complementary DNAs

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

Drug receptors and the inhibition of nerve cells

It would be trite indeed to say much to this audience of the contributions to pharmacology made by Sir John Gaddum. His quantitative approaches to the study of drug antagonism have found their way into most textbooks of pharmacology. I dare say that, if asked to define that aspect of the subject which is uniquely pharmacology-a task of increasing difficulty in these interdisciplinary days-many Society members would think first of those quantitative methods for studying drug-receptor interactions spawned by A.J. Clark, tested with antagonists by John Gaddum, and much popularized by Heinz Schild (Clark, 1933; Gaddum, 1937; 1957; Schild, 1949). I thank the Trustees for providing me with the opportunity to add my own small tribute to the memory of John Gaddums work; this is particularly so because-as you will see-my own research has been much influenced by his contributions. John Gaddum worked mostly with peripheral tissues. This was convenient because the tissues were readily accessible, easy to maintain in vitro, and in those days devites could be made to measure the appropriate response, such as contraction, relaxation or secretion; furthermore, it was generally not important to distinguish the drug effects on the individual cells within the syncytium. The first efforts to classify receptors on nerves were also made at their terminations in the periphery, following on from the well-known observations of Lindor Brown and John Gillespie (1957). That field, the study of autonomic presynaptic receptors, has matured and has led to novel therapies. But in the ganglia of the autonomic nervous system and in the central nervous system, the individual nerve cell is the functional unit, and information about drug receptors on nerve cells can best be obtained by recording from single cells. Conversely, the demonstration and characterization of drug receptors on nerve cells can itself be used as a way of classifying the cells, particularly when taken in concert with other information regarding the ion channels expressed, the transmitters synthesized and the targets to which the cells project.

Synaptic inputs to GABAA and GABAB receptors originate from discrete afferent neurons

gamma-Aminobutyric acid (GABA) inhibits neurons by acting at GABAA and GABAB receptors but it is not known whether the two receptors are associated with discretely separate afferent inputs or whether GABA released from a single presynaptic neuron activates both receptors. Intracellular recordings were used to show that, in the lateral amygdala and ventral tegmental area of the rat, distinct sets of GABA-containing neurons provide the synaptic input to GABAA and GABAB receptors. Synaptic potentials resulting from GABAA receptor activation (blocked by bicuculline) and from GABAB receptor activation (blocked by 2-hydroxysaclofen) occurred spontaneously but as unrelated events. Furthermore, the two components of evoked synaptic potentials were differentially inhibited by agonists acting presynaptically (muscarine and 5-hydroxytryptamine). The finding that GABA acting at GABAA and GABAB receptors originates from distinct sets of presynaptic fibers suggests that two groups of GABA-containing neurons might be generally distinguishable in the mammalian nervous system.

Actions of 5-hydroxytryptamine on ventral tegmental area neurons of the rat in vitro

Intracellular recordings were made with conventional microelectrodes and with whole-cell patch-clamp electrodes from neurons of the rat ventral tegmental area and substantia nigra zona compacta in vitro. Neurons were distinguished as principal cells and secondary cells; it is known from previous work that most principal cells contain dopamine whereas secondary cells do not. 5-Hydroxytryptamine (5-HT; 3-100 microM) depolarized (or evoked an inward current at -60 mV) 46% of 153 principal cells; a small proportion (11%) of cells were hyperpolarized (or showed outward current at -60 mV). Secondary cells were equally likely to be depolarized (or inward current at -60 mV, 30% of 80 cells) or hyperpolarized (or outward current at -60 mV, 28%). approximately 40% of each type of cell were unaffected by 5-HT. Depolarizing responses of 5-HT were mimicked by (+/-)-1-(2,5-dimethoxy-4-iodophenyl)- 2-aminopropane (DOI) and blocked by ketanserin. Hyperpolarizing responses were mimicked by dipropyl-5-carboxamidotryptamine and reversed polarity at the K+ equilibrium potential. Inhibitory postsynaptic potentials (or currents) mediated at GABAA receptors occurred spontaneously in some principal cells; they were reversibly blocked by tetrodotoxin and bicuculline. 5-HT either increased or decreased the frequency of these synaptic potentials but did not change their mean amplitude or decay time.(ABSTRACT TRUNCATED AT 250 WORDS)

Apamin increases NMDA-induced burst-firing of rat mesencephalic dopamine neurons.

Intracellular recordings made in vitro from rat midbrain dopamine neurons showed that apamin (100 nM) did not alter the regular spontaneous firing of the neurons, but it increased the occurrence of bursts of action potentials in N-methyl-D-aspartate. Apamin appeared to facilitate burst-firing induced by NMDA because, by blocking an outward calcium-activated potassium current, it increased the depolarizing action of NMDA.

Opioid, 5-HT1A and α2 receptors localized to subsets of guinea-pig myenteric neurons

The expression of α opioid, α2 and 5-hydroxytryptamine1A (5-HT1A) receptors on guinea-pig myenteric neurons was determined using receptor selective agonists during intracellular recordings in vitro. Agonists known to hyperpolarize myenteric neurons by increasing potassium conductance were tested: noradrenaline and UK 14304 (α2 agonists); 5-HT, 8-hydroxydipropylaminotctralin, 5-carboxamidotryptamine (5-HT1A agonists); normorphine, [Met5]-enkephalin and d-Ala2-Phe4, Gly-ol5 enkephalin (α agonists). The α2 agonists hyperpolarized 4667 AH cells; μ agonists hyperpolarized 1166 AH cells and 5-HT1A agonists inhibited 2857 AH cells. Hyperpolarizations to both α2 and μ agonists were observed in 1159 AH cells: hyperpolarizations to both α2 and 5-HT1A agonists were observed in 2349 AH cells. Hyperpolarizations mediated at α2 receptors were observed in 1154 S neurons and μ agonists hyperpolarized 1745 S cells. α2 and μ receptors were localized together on 1043 S cells tested with receptor selective agonists. 5-HT1A -mediated hyperpolarizations were not observed in 36 S cells. Presynaptic inhibition of fast excitatory post-synaptic potentials (fast e.p.s.p.s, S neurons) was observed in all cells tested with α2 agonists (n = 32); in 1423 cells tested with 5-HT1A agonists and in 822 cells tested with μ agonists. Both α2 and 5-HT1A agonists inhibited fast e.p.s.p.s in 1523 cells, while α2 and μ agonists both inhibited the fast e.p.s.p. in 821 cells. Inhibition of fast e.p.s.p.s by α and 5-HT1A agonists occurred together in 219 cells. Slow non-cholinergic e.p.s.p.s were inhibited by α2 agonists in 1919 cells and by 5-HT1A agonists in 1921 cells. α2- and 5-HT1A -mediated inhibition of slow e.p.s.p.s occurred together in 1214 cells. These data allow AH neurons to be divided into two groups: those expressing α2 and 5-HT1A receptors and those expressing α2 and μ receptors. α2 and μ receptors coexist on S neurons which do not express 5-HT1A receptors. Terminals that release acetylcholine express either α2 and μ or α2 and 5-HT1A receptors, consistent with the idea that they are provided by AH cells. Terminals that release mediators of the slow e.p.s.p. express primarily α2 and 5-HT1A receptors.

Hypotonic solution increases the slowly activating potassium current IsK expressed in Xenopus oocytes

A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network.

Cellular Actions of Opiates and Cocaine

The mesolimbic dopamine system has been increasingly implicated in the reinfbrcing actions of opiates and cocaine, implying that neurons within this system should be directly affected by these drugs.1-5 Such direct actions have been studied by removing tissue slices containing parts of the ventral tegmental area and nudeus accumbens, and measuring the actions of the drugs by electrophysiological methods. The neurons from which the recording are made can be identified, either histologically or by their unique membrane electrical properties. Two kinds of information have been sought. First, intracellular recording of membrane potential or current allows any direct actions of the drugs on the ion channels of the neuron to be assessed. Second, intracellular recording of synaptic potentials gives a sensitive measure of drug action on the neurons that provide synaptic inputs to the region.

Aromatic residues affecting permeation and gating in dSlo BK channels

Abstractu2002Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site.