P2X receptors and their expression on primary afferent neurons

P2X receptors are cation channels on the plasma membranes that open in response to the binding of extracellular ATP. Seven P2X subunits have been identified and cloned [1, 2]. These subunits can form a number of functional subtypes of recombinant P2X receptors in a heterologous expression system [1, 2]. Biophysical and pharmacological characterization of these P2X receptor subtypes have been extensively reviewed [1, 2]. It should be pointed out that the two terms, P2X subunit and P2X subtype, have distinct meanings. The former only refers to a component of a functional P2X receptor; the later is used for a functional P2X receptor. A functional P2X receptor can be formed from same P2X subunits (i.e., homomeric P2X receptors) or different subunits (i.e., heteromeric P2X receptors).

All of the seven P2X receptor subunits appear to be expressed on primary afferent neurons in the spinal dorsal root ganglia (DRG) and trigeminal ganglia [3, 4]. ATP and other P2X receptor agonists can evoke membrane currents in many primary afferent neurons. The evoked currents show three distinct phenotypes: Fast current, slow current, and mixed current with both fast and slow components [5–10]. The fast current is manifested by rapid desensitization in the range of milliseconds in the presence of agonists. In contrast, the slow current displays weak or little desensitization in the range of seconds in the presence of agonists. By comparing the findings from primary afferent neurons with those recombinant P2X receptors, it has been suggested that homomeric P2X3 receptors (P2X3) account for fast currents, heteromeric P2X receptors composed of P2X2 and P2X3 subunits (P2X2 + 3 receptors) account for slow currents, and the co-expression of P2X3 and P2X2 + 3 receptors account for the mixed currents [11–16]. However, for many DRG neurons slow currents appear to also be mediated by P2X receptor subtypes other than P2X2 + 3 receptors [10].

Studies have shown that P2X receptors can be involved in both peripheral and central sensory signaling and processing [11, 12, 17–24]. In the periphery, ATP may be released as a result of tissue stretch, injury and inflammation, visceral distension, or sympathetic activation [25]. ATP release can excite afferent fibers by the activation of P2X receptors [25–27]. Behavioral studies indicate that ATP and P2X receptors are involved in both nociceptive and innocuous functions [28–32]. P2X receptors are suggested to play roles in nociception under conditions of acute tissue injury and inflammation. Furthermore, P2X receptors have been implicated in neuropathic pain conditions [19, 33–35]. At central sites in the spinal cord dorsal horn, sensory impulses can release ATP [23], which may arise from afferent central terminals, second-order neurons, or astrocytes [36]. ATP can also be released centrally during spinal cord tissue damage and inflammation as a result of numerous disorders in the spinal cord. Centrally released ATP [37] may act on the central terminals of afferent fibers, which may then modulate or directly evoke the release of neurotransmitters from afferent central terminals [23, 24, 38]. This action may represent a major function of ATP and P2X receptors at the central sites in sensory pathways. P2X receptors on afferent central terminals have novel and important implications in the centrally initiated sensory signals including neuropathic pain associated with disorders in the spinal cord. Furthermore, P2X receptor-mediated modulation of transmitter release at afferent central terminals can also be a novel mechanism for the sensitization of sensory inputs from the periphery [39].

P2X receptor-mediated sensory pathways to different regions of the spinal cord dorsal horn

Studies on the nociceptive functions of P2X receptors are still at an early stage. To understand the nociceptive functions of P2X receptors under both physiological and pathological conditions, it is essential to identify P2X-mediated nociceptive pathways and to know where and how P2X-mediated nociceptive inputs are transmitted and processed in the spinal cord dorsal horn (DH). The dorsal horn, comprising laminae I and II (superficial laminae), III and IV (intermediate part), and V and VI (deep laminae), is the primary central site for processing somatic sensory inputs [40, 41]. Both the superficial and the deep laminae of the DH are responsible in the reception, processing and transmission of nociceptive information [41–46]. In contrast, the intermediate part of the dorsal horn is mainly involved in processing non-nociceptive information [41].

P2X sensory pathways to the dorsal horn can be studied on spinal cord sections by immunochemistry with P2X antibodies and by synaptic physiology using the patch-clamp technique. These approaches are used to determine whether the central terminals of primary afferent fibers express P2X receptors and, if so, what types of P2X receptors are expressed and where these P2X-expressing terminals are located in the dorsal horn. These approaches for the study of P2X purinergic pathways are based on the assumption that if a type of P2X receptors is expressed at the central terminal of a primary afferent fiber, the peripheral site of the afferent fiber also expresses the same type of P2X receptors (Figure 1A). Thus, by studying P2X receptors at the central terminals of primary afferent fibers, one can map the sensory pathways of P2X purinergic inputs into the spinal cord dorsal horn. These approaches can help us understand the potential functions of different P2X subtypes in sensory transmission. However, although this assumption is likely to be true for most receptors, it should be noted that membrane receptors may not always be delivered to both ends of a primary afferent fiber.

Figure 1
figure 1

An assumption of P2X purinergic pathway to the spinal cord dorsal horn and the synaptic physiological approach for the study of P2X purinergic pathway. A) The diagram illustrates a dorsal root ganglion with both peripheral nerve endings and central terminals. The central terminals are within the spinal cord dorsal horn. It is proposed that the expression of a P2X receptor at the central terminal also predicts its presence in other part of the primary afferent fibers including peripheral nerve endings. Thus, P2X purinergic pathway to the dorsal horn can be mapped by studying P2X receptors at the central terminals through synaptic physiology or immunochemistry. B) A schematic diagram illustrates the use of synaptic physiology to study P2X purinergic pathway to the dorsal horn using spinal cord slice preparations. Synaptic transmission between afferent central terminals and dorsal horn (DH) neurons is recorded using patch-clamp technique. Activation of P2X receptors on the central terminals of primary afferent fibers results in the release of glutamate, which in turn activate glutamate receptors (GluR) on dorsal horn neurons and generate excitatory postsynaptic currents (EPSCs).

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In immunochemistry studies with P2X3 antibodies, P2X3-expressing afferent terminals [47–49] appear to be restricted to the inner part of lamina II (lamina IIi). Lamina distribution of afferent fibers that express other P2X subunits remains unclear, although there was a report that showed immunoreactivity of P2X1 and P2X2 subunits in superfacial lamina as well [50]. Except for P2X3 subunit antibodies, it appears that antibodies for other P2X receptor subunits have limited usefulness in the spinal cord sections. In addition, a further limitation of immunochemistry is that it only reveals P2X subunits rather than functional P2X receptors [36].

The use of synaptic physiology with patch-clamp recording technique for the study of P2X purinergic sensory pathways (Figure 1B) provides the opportunity to assess both function and structure in the system in several ways. First, this approach allows one to assess functional P2X receptors that are expressed on primary afferent central terminals. Second, it reveals the effects of P2X purinergic sensory inputs on dorsal horn neurons (i.e., the secondary-order sensory neurons within the dorsal horn). Third, it allows one to characterize neuronal circuits that are involved in processing P2X purinergic sensory inputs within the dorsal horn. To this end, we have applied patch-clamp recording technique to the spinal cord slice preparations to assess the effects of P2X receptor activation on monosynaptic and polysynaptic transmission from primary afferent fibers to dorsal horn neurons located in a number of lamina regions. We have found that P2X agonists increased monosynaptic transmission from afferent central terminals to the dorsal horn neurons located in lamina V (Figure 2) and lamina II [23, 24, 38]. The original aim of these studies was to explore the role of presynaptic P2X receptors, i.e., P2X receptors at the central terminal of primary afferent fibers in modulating glutamate release from P2X-expressing afferent central terminals. However, based on our assumption as illustrated in Figure 1, presynaptic P2X receptor-mediated increases of monosynaptic transmission have also revealed sensory pathways from some P2X-expressing afferent fibers to dorsal horn neurons in these regions (also see [36]). Recordings from lamina V neurons demonstrated that P2X receptor agonists produced a prolonged increase of monosynaptic transmission to the majority of lamina V neurons (Figure 2; [23, 24]). This finding suggests the wide expression of P2X receptors at the central terminals of these afferent fibers (Figure 3). These afferent fibers have been found to be Aδ afferent fibers and insensitive to capsaicin (Figures 2 and 3), a noxious stimulant that has been commonly used to identify nociceptive afferent fibers. Pharmacological studies have suggested that the P2X receptors expressed on these capsaicin-insensitive Ad afferent terminals were not P2X3 or P2X2 + 3 subtypes but were more likely to be P2X1 + 5 or P2X4 + 6 subtypes [10, 38]. One potential function of this P2X purinergic sensory pathway may be to transmit sensory information of mechanical stimuli, as based on sensory physiology of Aδ-afferent fibers that innervates lamina V of the spinal cord dorsal horn. Currently, it is unclear whether this P2X purinergic sensory pathway is directly involved in nociceptive transmission due to its lack of capsaicin sensitivity. However, nociceptive afferent fibers can also be capsaicin-insensitive. Interestingly, we have found that this ATP-sensitive/capsaicin-insensitive P2X purinergic pathway has convergence and temporal summation with a capsaicin-sensitive input that is polysynaptically transmitted to lamina V neurons (Figure 3; [24]). This convergence may suggest that this ATP-sensitive/capsaicin-insensitive P2X purinergic pathway has interaction with nociceptive input.

Figure 2
figure 2

Effects of αβmeATP on the frequency of miniature excitatory postsynaptic currents recorded from lamina V dorsal horn neurons. A–B) Spinal cord slice preparation viewed under IR-DIC microscope. Lamina regions were identified under 10× objective (A). A part of a patch electrode is also seen in panel A. The electrode tip is inside tissue about 70 µM from the surface, and its lamina location is indicated by a box. A neuron in the box region can be seen under 40× objective (B, center of the field). The patch electrode is to the left side. C) A trace (top) shows mEPSCs recorded from a lamina V neuron before and following application of 100 µM αβmeATP. Bottom traces show, at an expanded time scale, the mEPSCs before (left three traces) and following (right three traces) αβmeATP application. D) Histogram shows the time course and degree of the increases in mEPSC frequency after 100 µM αβmeATP. It also shows, in the same recording, 2 µM capsaicin did not have effect on mEPSCs. Modified and reprinted (with permission) from Nakatsuka et al. [38]. Copyright by the Society for Neuroscience.

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Figure 3
figure 3

P2X-purinergic sensory pathways to the dorsal horn of the spinal cord. The schematic diagram illustrates three P2X-purinergic sensory pathways to laminas I, II, and V of the dorsal horn. The pathway to lamina II is the afferent fibers expressing both P2X3 and VR1 receptors. The P2X receptor subtypes of two other pathways had pharmacological properties distinct from P2X3 containing receptors.

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In contrast to lamina V, many lamina II neurons are monosynaptically contacted by P2X3-expressing afferent fibers ([38], Figure 3). This result is consistent with immunochemistry of P2X3 subunit distribution in lamina II. These P2X3-expressing afferent fibers also express VR1 receptors [47, 51] and are sensitive to capsaicin. Thus, this P2X3 sensory pathway may be directly involved in transmitting noxious signals. Consistent with this idea, previous studies have shown the nociceptive function of P2X3 [35, 52–54]. The ATP-sensitive/capsaicin-sensitive pathway to lamina II was shown to converge to lamina V neurons through polysynaptic transmission ([24], Figure 3). This may mediate the spatial and temporal sensory summation and subsequent hyperactivity in deep laminae, a potential mechanism of hyperalgesia.

Another important region for pain transmission, processing, and the development of pathological pain is lamina I of the dorsal horn. Due to technical difficulties in performing electrophysiological recordings in lamina I region, there is no report on whether P2X-mediated sensory signals are transmitted to and processed in this important nociceptive region. Our recent experiments performed in lamina I show that some afferent fibers innervating lamina I neurons also express P2X receptors at their central sites (Figure 3, unpublished result, presented at the Purines 2004 meeting). These afferent fibers are capsaicin-sensitive fibers, suggesting that this is a nociceptive P2X purinergic pathway to the spinal cord dorsal horn. These P2X receptors are less likely to be P2X3 containing subtypes based on the restricted lamina IIi distribution of P2X3-expression afferent central terminals [47, 51]. It would be interesting to identify the P2X subtype or subtypes and to see whether it can be a selective target for the control of pain conditions.

Concluding remarks

The map of P2X purinergic sensory input to different laminas of the spinal cord dorsal horn helps in understanding the sensory functions of P2X receptors. Under pathological conditions, the destination of P2X purinergic sensory input in different laminas may be altered due to an aberrant expression of P2X receptors on primary afferent fibers [34, 55]. This can be a potential mechanism by which P2X receptors are involved in abnormal sensations such as mechanical allodynia.