Research reportExpression of P2X7 receptor immunoreactivity in distinct subsets of synaptic terminals in the ventral horn of rat lumbar spinal cord
Introduction
Extracellular adenosine 5′-triphosphate (ATP), released by a variety of central and peripheral neurons and glia [16], [21], [28], [50], elicits fast neurotransmitter-like responses by activating members of the P2X receptor family. Seven (P2X1–P2X7) cDNAs have been identified and characterized in this family of genes. Homo- and/or heteromultimeric expression of the corresponding proteins forms functional receptors that operate as ligand-gated ion channels with a variety of kinetic, pharmacologic and cationic selectivity (including calcium permeability) attributes [11], [39], [40]. Some purinergic ionotropic receptors, such as P2X7, have a broader spectrum of effects, including the induction of lytic pore formation and cell death in various cell types of the immune system [12], [39], [48] and signaling in glia [15], [38].
Several lines of evidence indicate that the activation of P2X receptors in the CNS may mediate both postsynaptic currents and presynaptic modulation of transmitter release [5], [7], [16], [17]. The presynaptic modulatory effects of P2X receptor activation are varied and dependent on the specific nature of the affected synapses and receptor subtype. For example, P2X7 receptor activation in the CNS induces effects ranging from the depression of synaptic transmission at mossy fiber-CA3 synapses in the hippocampus to the facilitation of glutamate release in the spinal cord; P2X7 receptor activation also promotes vesicular release at the neuromuscular junction (NMJ) [5], [14], [47]. In the context of presynaptic activation of P2X receptors in the spinal cord, there is strong evidence for P2X-mediated facilitation of transmitter release from both excitatory and inhibitory terminals in the dorsal horn [23], [36], [37], [43]. The recent demonstration that immunoreactivity (IR) against P2X7 receptors is localized in several types of presynaptic terminals in the spinal cord, including subsets of glutamatergic terminals (identified by immunoreactivity against the vesicular glutamate transporters VGLUT1 and VGLUT2), strongly supports the notion that the P2X7 receptor subtype may play a fundamental role in the regulation of synaptic transmission in spinal sensorimotor systems [6], [14].
Here, we systematically explore the distribution of P2X7 receptors in spinal synapses of diverse phenotype to determine quantitatively to what extent the receptor is present in ventral horn terminals that are likely involved in motor control circuits. Our quantitative dual-labeling analyses confirm the differential colocalization of P2X7 in VGLUT2-containing terminals [6] and extend these observations to demonstrate a differential colocalization, in particular, classes of cholinergic terminals apposed to spinal motoneurons. Furthermore, P2X7 receptors are expressed in only a minority of monoaminergic or inhibitory terminals.
Section snippets
Materials and methods
Adult Sprague–Dawley rats were deeply anesthetized with pentobarbital sodium (100–150 mg/kg) and then transcardially perfused with a vascular rinse (0.01 M phosphate buffer with 0.8% NaCl, 0.025% KCl and 0.05% NaHCO3, pH 7.4), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal cords and gastrocnemius/soleus muscles were quickly removed and postfixed in the same fixative for 2–4 h at room temperature. Tissue was stored in 15% sucrose at 4 °C until sectioning on a
Results
P2X7-IR was broadly distributed, in punctate fashion, throughout the neuropil of the ventral horn. In the lamina IX, P2X7-IR punctate was observed around large cell bodies (presumed motoneurons), as well as being more diffusely distributed in their vicinity (Fig. 1A). In ventral lamina VII, where Renshaw cells are located (e.g., [18]), the relationship of the punctate to cell bodies was harder to discern (but see Fig. 3D–F), although the overall density and labeling intensity of punctate
Discussion
Extracellular ATP can modulate presynaptic transmitter release via P2X receptor activation, as has been demonstrated at the neuromuscular junction and at a variety of central synapses in the brain and spinal cord [5], [14], [23], [27], [30], [35], [37], [43], [44], [46], [47]. Functional P2X7 receptors are implicated in some of these modulatory actions [33], and the involvement of these receptors is strongly supported by the targeting of P2X7 receptors to presynaptic terminals in the CNS and at
Acknowledgements
This work was supported by NIH grants NS25547 and NS40850 to REWF.
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