Substantia gelatinosa (SG, lamina II) is a spinal cord region where most unmyelinated primary afferents terminate and the central nociceptive processing begins. It is formed by several distinct groups of interneurons whose functional properties and synaptic connections are poorly understood, in part, because recordings from synaptically coupled pairs of SG neurons are quite challenging due to a very low probability of finding connected cells. Here, we describe an efficient method for identifying synaptically coupled interneurons in rat spinal cord slices and characterizing their excitatory or inhibitory function. Using tight-seal whole-cell recordings and a cell-attached stimulation technique, we routinely tested about 1500 SG interneurons, classifying 102 of them as monosynaptically connected to neurons in lamina I-III. Surprisingly, the vast majority of SG interneurons (n = 87) were excitatory and glutamatergic, while only 15 neurons were inhibitory. According to their intrinsic firing properties, these 102 SG neurons were also classified as tonic (n = 49), adapting (n = 17) or delayed-firing neurons (n = 36). All but two tonic neurons and all adapting neurons were excitatory interneurons. Of 36 delayed-firing neurons, 23 were excitatory and 13 were inhibitory. We conclude that sensory integration in the intrinsic SG neuronal network is dominated by excitatory interneurons. Such organization of neuronal circuitries in the spinal SG can be important for nociceptive encoding.

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.

Serotonergic mechanisms play a central role in migraine pathology. However, the region-specific effects of serotonin (5-HT) mediated via multiple types of receptors in the nociceptive system are poorly understood. Using extracellular and patch-clamp recordings, we studied the action of 5-HT on the excitability of peripheral and central terminals of trigeminal afferents. 5-HT evoked long-lasting TTX-sensitive firing in the peripheral terminals of meningeal afferents, the origin site of migraine pain. Cluster analysis revealed that in majority of nociceptive fibers 5-HT induced either transient or persistent spiking activity with prevailing delta and theta rhythms. The 5-HT3-receptor antagonist MDL-72222 or 5-HT1B/D-receptor antagonist GR127935 largely reduced, but their combination completely prevented the excitatory pro-nociceptive action of 5-HT. The 5-HT3 agonist mCPBG activated spikes in MDL-72222-dependent manner but the 5HT-1 receptor agonist sumatriptan did not affect the nociceptive firing. 5-HT also triggered peripheral CGRP release in meninges, which was blocked by MDL-72222.5-HT evoked fast membrane currents and Ca transients in a fraction of trigeminal neurons. Immunohistochemistry showed expression of 5-HT3A receptors in fibers innervating meninges. Endogenous release of 5-HT from degranulated mast cells increased nociceptive firing. Low pH but not histamine strongly activated firing. 5-HT reduced monosynaptic inputs from trigeminal Aδ- and C-afferents to the upper cervical lamina I neurons and this effect was blocked by MDL-72222. Consistent with central inhibitory effect, 5-HT reduced CGRP release in the brainstem slices. In conclusion, 5-HT evokes powerful pro-nociceptive peripheral and anti-nociceptive central effects in trigeminal system transmitting migraine pain.

Voltage‐gated Na+ channels and their distribution were studied by the patch‐clamp technique in intact dorsal horn neurones in slices of newborn rat spinal cord and in neurones isolated from the slice by slow withdrawal of the recording pipette. This new method of neurone isolation was further used to study the roles of soma and axon in generation of action potentials. Tetrodotoxin (TTX)‐sensitive Na+ currents in intact neurones consisted of three components. A fast component with an inactivation time constant (τf) of 0.6–2.0 ms formed the major part (80–90%) of the total Na+ current. The remaining parts consisted of a slowly inactivating component (τs of 5–20 ms) and a steady‐state component. Single fast and slow inactivating Na+ channels with conductances of 11.6 and 15.5 pS, respectively, were identified in the soma of intact neurones in the slice. Steady‐state Na+ channels were not found in the soma, suggesting an axonal or dendritic localization of these channels. In the whole‐cell recording mode, the entire soma of a dorsal horn neurone could be isolated from the slice by slow withdrawal of the recording pipette, leaving all or nearly all of its processes in the slice. The isolated structure was classified as: (1) ‘soma’ if it lost all of its processes, (2) ‘soma+axon’ complex if it preserved one process and at least 85% of its original peak Na+ current or (3) ‘soma+dendrite’ complex if it preserved one process but the remaining Na+ current did not exceed those observed in the isolated ‘somata’. The spatial distribution of Na+ channels in the neurone was studied by comparing Na+ currents recorded before and after isolation. The isolated ‘soma’ contained 13.8 ± 1.3% of inactivating Na+ current but no steady‐state Na+ current. ‘Soma+axon’ complexes contained 93.6 ± 1.4% of inactivating and 46% of steady‐state Na+ current. In current‐clamp experiments, the intact neurones and isolated ‘soma+axon’ complexes responded with ‘all‐or‐nothing’ action potentials to current injections. In contrast, isolated ‘somata’ showed only passive or local responses and were unable to generate action potentials. It is concluded that dorsal horn neurones of the spinal cord possess three types of TTX‐sensitive voltage‐gated Na+ channels. The method of entire soma isolation described here shows that the majority of inactivating Na+ channels are localized in the axon hillock and only a small proportion (ca 1/7) are distributed in the soma. Steady‐state Na+ channels are most probably expressed in the axonal and dendritic membranes. The soma itself is not able to generate action potentials. The axon or its initial segment plays a crucial role in the generation of action potentials.

Pinto V, Derkach VA, Safronov BV. Role of TTX-sensitive and TTX-resistant sodium channels in A␦-and C-fiber conduction and synaptic transmission. J Neurophysiol 99: 617-628, 2008. First published December 5, 2007 doi:10.1152/jn.00944.2007. Thin afferent axons conduct nociceptive signals from the periphery to the spinal cord. Their somata express two classes of Na ϩ channels, TTXsensitive (TTX-S) and TTX-resistant (TTX-R), but their relative contribution to axonal conduction and synaptic transmission is not well understood. We studied this contribution by comparing effects of nanomolar TTX concentrations on currents associated with compound action potentials in the peripheral and central branches of A␦-and C-fiber axons as well as on the A␦-and C-fiber-mediated excitatory postsynaptic currents (EPSCs) in spinal dorsal horn neurons of rat. At room temperature, TTX completely blocked A␦-fibers (IC 50 , 5-7 nM) in dorsal roots (central branch) and spinal, sciatic, and sural nerves (peripheral branch). The C-fiber responses were blocked by 85-89% in the peripheral branch and by 65-66% in dorsal roots (IC 50 , 14 -33 nM) with simultaneous threefold reduction in their conduction velocity. At physiological temperature, the degree of TTX block in dorsal roots increased to 93%. The A␦-and C-fiber-mediated EPSCs in dorsal horn neurons were also sensitive to TTX. At room temperature, 30 nM blocked completely A␦-input and 84% of the C-fiber input, which was completely suppressed at 300 nM TTX. We conclude that in mammals, the TTX-S Na ϩ channels dominate conduction in all thin primary afferents. It is the only type of functional Na ϩ channel in A␦-fibers. In C-fibers, the TTX-S Na ϩ channels determine the physiological conduction velocity and control synaptic transmission. TTX-R Na ϩ channels could not provide propagation of full-amplitude spikes able to trigger synaptic release in the spinal cord.

SUMMARY1. Voltage-dependent potassium channels were investigated in rat axonal membrane by means of the patch-clamp recording technique. Three different types of channels (F, I and S) have been characterized on the basis of their single-channel conductance, activation, deactivation and inactivation properties.2. The fast (F) channels were activated smoothly at potentials (E) between -50 and 50 mV (E50 = 4-6 mV). They had a conductance of 55 pS for inward current and 30 pS for outward current in solutions containing 155 mm K+ (high K+) on both sides of the membrane at 21-23 'C. The F-channels demonstrated the fastest deactivation, within 1-2 ms, and inactivated in a few hundreds of milliseconds. The time constant of inactivation was 143 ms at E = +40 mV.3. The intermediate (I) channels activated steeply between E =-70 and -50 mV (E50 = -64-2 mv) and had a single-channel conductance of 33 pS for inward and 18 ps for outward currents. The I-channels deactivated with intermediate kinetics with the time constants of 20-4 ms and 10-1 ms at E = -80 mV and E = -100 mV, respectively. Complete inactivation of the channels developed over tens of seconds. The time constant of inactivation was 7-4 s at E = + 40 mV.4. The slow (S) channels were active at potentials positive to -90 mV. Their conductance was 10 pS for inward currents. The time constant of activation of the S-channels was strongly potential dependent. At a holding potential of -100 mV the channels deactivated during a long time interval between 30 ms and 1 s, producing long-lasting tail currents. The mean time constant of deactivation for S-channels was 129 ms.5. The conductances of F-and I-channels measured under normal physiological conditions (Ringer solution in bath) were 17 and 10 pS, respectively. 6. Tetraethylammonium (TEA), the classic blocker of potassium channels, suppressed F-, I-and

DR -channel currents were reduced to 50% in the presence of 1 mm TEA, DR2-channel currents were reduced to about 50 % by 5 mm TEA, whereas the amplitudes of currents through DR3-channels were almost unaffected by 5 mm TEA.5. Addition of external 1 and 5 mm TEA to whole cells under current-clamp condition depolarized the cell membrane, lowered the threshold for action potential firing, prolonged action potential duration and reduced the amplitude of after-hyperpolarization.6. It is concluded that potassium A-, DRF-DR1-, DR2-and DR3-channels play multiple roles in the excitability of DRG neurones.

1. Voltage-activated Na+ and K+ channels were investigated in the soma membrane of motoneurones using the patch-clamp technique applied to thin slices of neonatal rat spinal cord.2. One type of TTX-sensitive Nae channel, with a conductance of 14-0 pS, was found to underlie the macroscopic Na+ conductance in the somata of motoneurones. These channels activated within a potential range between -60 and -20 mV with a potential of halfmaximal activation (E50) of -38-9 mV and steepness factor (k) of 6-1 mV.3. Kinetics of Nae channel inactivation could be fitted with a single exponential function at all potentials investigated. The curve of the steady-state inactivation had the following parameters: a half-maximal potential (E,50) of -81'6 mV and k of -10-2 mV.4. Kinetics of recovery of Na+ channels from inactivation at a potential of -80 mV were double exponential with fast and slow components of 16-2 (76%) and 153-7 ms (24%), respectively. It is suggested that the recovery of Na+ channels from inactivation plays a major role in defining the limiting firing frequency of action potentials in motoneurones. 5. Whole-cell K+ currents consisted of transient (A)-and delayed-rectifier (DR)-components.The A-component activated between -60 and +20 mV with an E50 of -33-3 mV and k of 15'7 mV. The curve of steady-state inactivation was best fitted with an E 50 of -82-5 mV and k of -10-2 mV. The DR-component of K+ current activated smoothly at more positive potentials. E50 and k for DR-currents were +P14 and 16 9 mV, respectively. 6. The most frequent single K+ channel found in the somata of motoneurones was the fast inactivating A-channel with a conductance of 19-2 pS in external Ringer solution. In symmetrical high-K+ solutions the conductance was 50'9 and 39'6 pS for inward and outward currents, respectively. The channel activation took place between -60 and +20 mV. The curve of steady-state inactivation of single A-channels had an Eho50 of -87-1 mV and k of -12-8 mV. In high-Ko solution A-channels demonstrated a rapid deactivation at potentials between -110 and -60 mV. The time constant of the channel deactivation depended on the membrane potential and changed from 1 5 ms at -110 mV to 6-3 ms at -60 mV.7. Delayed-rectifier K+ channels were found in the soma membrane at a moderate density. The channel conductance in Ringer solution was 10-2 pS and in symmetrical high-K+ solutions was 31P1 and 22-5 pS for inward and outward currents, respectively. The activation of the channels took place at -60 to 0 mV with an E50 of -43-8 mV and k of 8-5 mV. In external high-K+ solution DR-channels showed a slow deactivation with a time constant of 5-9 ms at -110 mV and 60-0 ms at -60 mV. 8. Tetraethylammonium suppressed both A-and DR-components of whole-cell K+ conductance and reduced the amplitudes of the single-channel currents. The concentration giving 50% inhibition (IC50) values was 14 8 and 0 8 mm, respectively.9. ITIX-sensitive Nae channels, together with A-and DR-types of K+ channels, form the basis of voltage-activated conductance i...