The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Ionic conductances underlying excitability in tonically firing neurons (TFNs) from substantia gelatinosa (SG) were studied by the patch-clamp method in rat spinal cord slices. Ca(2+)-dependent K(+) (K(CA)) conductance sensitive to apamin was found to prolong the interspike intervals and stabilize firing evoked by a sustained membrane depolarization. Suppression of Ca(2+) and K(CA) currents, however, did not abolish the basic pattern of tonic firing, indicating that it was generated by voltage-gated Na(+) and K(+) currents. Na(+) and K(+) channels were further analyzed in somatic nucleated patches. Na(+) channels exhibited fast activation and inactivation kinetics and followed two-exponential time course of recovery from inactivation. The major K(+) current was carried through tetraethylammonium (TEA)-sensitive rapidly activating delayed-rectifier (K(DR)) channels with a slow inactivation. The TEA-insensitive transient A-type K(+) (K(A)) current was very small in patches and was strongly inactivated at resting potential. Block of K(DR) rather than K(A) conductance by 1 mM TEA lowered the frequency and stability of firing. Intracellular staining with biocytin revealed at least three morphological groups of TFNs. Finally, on the basis of present data, we created a model of TFN and showed that Na(+) and K(DR) currents are sufficient to generate a basic pattern of tonic firing. It is concluded that the balanced contribution of all ionic conductances described here is important for generation and modulation of tonic firing in SG neurons.

Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.

Using tight-seal recordings from rat spinal cord slices, intracellular labelling and computer simulation, we analysed the mechanisms of spike frequency adaptation in substantia gelatinosa (SG) neurones. Adapting-firing neurones (AFNs) generated short bursts of spikes during sustained depolarization and were mostly found in lateral SG. The firing pattern and the shape of single spikes did not change after substitution of Ca 2+ with Co 2+ , Mg 2+ or Cd 2+ indicating that Ca 2+ -dependent conductances do not contribute to adapting firing. Transient K A current was small and completely inactivated at resting potential suggesting that adapting firing was mainly generated by voltage-gated Na + and delayed-rectifier K + (K DR ) currents. Although these currents were similar to those previously described in tonic-firing neurones (TFNs), we found that Na + and K DR currents were smaller in AFNs. Discharge pattern in TFNs could be reversibly converted into that typical of AFNs in the presence of tetrodotoxin but not tetraethylammonium, suggesting that lower Na + conductance is more critical for the appearance of firing adaptation. Intracellularly labelled AFNs showed specific morphological features and preserved long extensively branching axons, indicating that smaller Na + conductance could not result from the axon cut. Computer simulation has further revealed that down-regulation of Na + conductance represents an effective mechanism for the induction of firing adaptation. It is suggested that the cell-specific regulation of Na + channel expression can be an important factor underlying the diversity of firing patterns in SG neurones.

No abstract

It is suggested that tonic-firing neurons, presumably functioning as excitatory interneurons, are primary postsynaptic targets for administered and endogenous opioid agonists in spinal SG. Functional transition of cells in this group from tonic to adapting firing mode may represent an important mechanism facilitating opioidergic analgesia.

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 μM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (∼77%) but not DFNs (∼8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ∼5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.