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.