In the spinal dorsal horn (DH), transmission and modulation of peripheral nociceptive (pain-inducing) messages involve classical neurotransmitters and neuropeptides. We show that approximately half of DH neurons use ATP as a fast excitatory neurotransmitter acting at ionotropic P2X postsynaptic receptors. ATP was not codetected with glutamate but was coreleased with the inhibitory neurotransmitter GABA. Moreover, adenosine, probably generated by extracellular metabolism of ATP, finely tuned GABAergic inhibitory postsynaptic currents. Differential modulation of excitatory versus inhibitory components of this mixed cotransmission may help to explain changes in sensory message processing in the DH during mechanical hyperalgesia and neuropathic pain.

Cerebrospinal fluid-contacting neurons (CSFcNs) occur in various brain regions of lower vertebrates. In mammals, they are restricted to medullospinal areas, and little is known about their projection sites. In the present work, we investigated some morphofunctional characteristics of such neurons in the rat spinal cord by light and electron microscopic immunocytochemistry. CSFcNs expressing the P2X(2) subunit of purinergic receptors were present throughout the spinal cord, though more numerous at lower thoracolumbar and sacral levels. These neurons coexpressed GAD and the polysialylated neural cell adhesion molecule (PSA-NCAM), a marker of cellular plasticity. From low thoracic levels downward, tiny amyelinic axons (less than 200 nm in diameter) were tightly packed in bundles, which ran along the ependyma and extended ventrally, eventually concentrating against the walls of the ventral median fissure. In addition to P2X(2), GAD, gamma-aminobutyric acid (GABA), and PSA, these axons expressed GAP-43 immunoreactivity. Moreover, they were labelled along their entire lengths with antibodies against synaptotagmin and synaptophysin, but these failed to reveal intraspinal terminal fields. Taken together, our observations indicate the presence in the rat spinal cord of a highly plastic system of GABAergic CSFcNs that express the P2X(2) subunit of purinergic receptors. The function of this original system remains open to question. In these neurons, the P2X(2) receptors may confer a sensitivity to ATP either present in the CSF or released by nearby neurons of the central autonomic area.

BackgroundRecent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception.ResultsWe show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages.ConclusionOur results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.

The superficial layers of the spinal cord dorsal horn (DH) express P2X2, P2X4, and P2X6 subunits entering into the formation of ionotropic (P2X) receptors for ATP. Using a culture system of laminae I-III from neonatal rat DH, we show that ATP induced a fast nonselective cation current in 38% of the neurons (postsynaptic effect). ATP also increased the frequency of miniature IPSCs (mIPSCs) mediated by GABA(A) receptors or by glycine receptors in 22 and 9%, respectively, of the neurons tested (presynaptic effect) but had no effect on glutamatergic transmission. The presynaptic effect of ATP on GABAergic transmission was not significantly affected by thapsigargin (1 microM) but was completely dependent on Ca(2+) influx. Presynaptic and postsynaptic effects were inhibited by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and reactive blue and were not reproduced by uridine 5'-triphosphate (UTP) or adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S), suggesting the implication of ionotropic P2X rather than of metabotropic P2Y receptors. alphabeta-methylene-ATP (100 microM) did not reproduce the effects of ATP. ATP reversibly increased the amplitude of electrically evoked GABAergic IPSCs and reduced paired-pulse inhibition or facilitation without affecting IPSC kinetics. This effect was preferentially, but not exclusively, observed in neurons coreleasing ATP and GABA. We conclude that in cultured DH neurons, the effects of ATP are mediated by P2X receptors having a pharmacological profile dominated by the P2X2 subunit. The presynaptic receptors might underlie a modulatory action of ATP on a subset of GABAergic interneurons involved in the spinal processing of nociceptive information.