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In intact sensory ganglia, the primary sensory neurones are ensheathed by satellite glial cells (SGCs) (Hanani, 2005). Dorsal root ganglia (DRG) cell cultures prepared from adult rats typically contain significantly more glial cells (SGCs, Schwann cells and some fibroblasts) than neurones and it is difficult to generate neurone-containing cultures of high purity (Ahmed et al., 2006; Ng et al., 2011). However, as SGCs may remain associated with neurones in dissociated cell cultures (Ceruti et al., 2011), this a useful model system to examine neurone–glial communication underlying responses to nerve injury (for reviews, see McMahon et al., 2005; Scholz and Woolf, 2007; Austin and Moalem-Taylor, 2010; Ren and Dubner, 2010). For example, in dissociated trigeminal ganglion cell cultures, bradykinin stimulates the release of calcitonin gene-related peptide (CGRP) from sensory neurones which activates CGRP receptors on SGCs, ultimately increasing the expression of purine P2Y receptors and affecting cytokine production (Ceruti et al., 2011); cytokines such as TNF-α then increase neuronal excitability (Zhang et al., 2007a).
Isolated cell cultures of DRG and trigeminal ganglia are frequently used as model systems to study neuronal responses relating to pain and migraine respectively (LaMotte, 2007; Ceruti et al., 2008). It has generally been assumed that if a GPCR has been identified on primary sensory neurones in intact DRG, then these receptors will similarly be expressed only on neurones in isolated cell cultures. However, although the PGE2 receptor (EP4) subtype and the prostacyclin (IP) receptor are localized to neurones in intact DRG (Oida et al., 1995), we have shown that the non-neuronal cell population in mixed DRG cell cultures also express these receptors (Ng et al., 2011). Because the majority of non-neuronal cells in our DRG cell cultures express glial fibrillary-acidic protein (GFAP) (Ng et al., 2010), we refer to these cells as DRG glial cells. What causes the up-regulation of EP4 and IP receptors in isolated DRG glial cells is presently unknown, but up-regulation of EP2 and EP4 receptors in spinal cord microglial cells in vivo occurs in response to chronic, but not acute, inflammatory conditions to provide a negative feedback system regulating neuroinflammation (Noda et al., 2007; Brenneis et al., 2011).
During our work to identify EP4 and IP receptors on glial cells of adult rat DRG cells in vitro (Ng et al., 2011), we noted that AC responses in pure glial cell preparations were significantly greater than in mixed DRG neurone–glial cell preparations, despite similar numbers of glial cells. These observations suggested that DRG neurones were inhibiting agonist-stimulated AC responses by DRG glial cells, so we have examined this interaction in more detail, focusing on EP4 receptors and IP receptors to determine if cell–cell contact is essential for neuronal inhibition of Gs-coupled GPCR responses in DRG glial cells. Activation of Gs-coupled prostanoid receptors is associated with hyper-nociceptive responses (Sachs et al., 2009), and we have previously shown that AC activity in mixed DRG cell cultures is also stimulated by CGRP, isoprenaline and salbutamol (Rowlands et al., 2001). Therefore, our goal here was also to pharmacologically characterize CGRP and β-adrenoceptor responses in mixed DRG cell cultures and to determine if these receptors were also present in pure glial cell cultures. We demonstrate here that in addition to Gs-coupled EP4 and IP receptors, CGRP receptors and β2-adrenoceptors are also functionally expressed in DRG glial cells. Furthermore, the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, appears to be subject to inhibition by DRG neurones.
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Damage to the peripheral nervous system often results in hyperexcitability of DRG neurones, and the enhanced sensitivity of these primary sensory neurones to cAMP is maintained in vitro (Zheng et al., 2007). Nevertheless, much remains to be understood about the role of cAMP in pain-related states (Inceoglu et al., 2011), and a more complete understanding of the factors regulating cAMP production in dorsal root ganglia is essential. Our discovery of EP4 and IP receptor expression in adult rat DRG glial cells in vitro in typical DRG cell preparations was unexpected (Ng et al., 2011). In the current study, we further demonstrated that EP4 and IP receptor-dependent AC responses in DRG neurone-enriched cell cultures were much weaker than those in typical mixed DRG cell cultures containing similar numbers of neurones but many more glial cells. In adult mouse DRG cell preparations, the expression of other pain-related signalling components falls in the first 2 days in culture and remains low unless NGF is added (Franklin et al., 2009). Both PGE2 and prostacyclin mediate their hyper-nociceptive responses in part by stimulating receptors on peptidergic neurones and potentiating neuropeptide release (Hingtgen et al., 1995). Even though the majority of peptidergic DRG neurones are responsive to NGF (Averill et al., 1995), we found that the addition of NGF did not enhance EP4 and IP receptor agonist-stimulated responses in neurone-enriched DRG cell cultures. The neurone-enriched DRG cell preparation was similarly least responsive to the other Gs-coupled GPCR agonists studied herein, i.e. CGRP, formoterol and isoprenaline. When IP receptor-stimulated responses in mixed DRG cells were previously measured over a 5 day period, the weak response after 16 h in culture was assumed to result from a lack of time for neurones to adopt the typical phenotype of primary sensory neurones (Wise, 2006). The results from the current study challenge this assumption and suggest that the bulk of cAMP generated in typical DRG cell cultures derives from glial cells rather than neurones, and time is needed for these glial cells to express EP4 and IP receptors.
CGRP responses are expected to be present in DRG glial cells, because CGRP receptor components have been identified in rat trigeminal ganglia in both neurones and SGCs in vivo and in vitro (Zhang et al., 2007b; Li et al., 2008). We found that the responses to CGRP in neurone-enriched cultures were relatively weak, but readily detectable in glial cell-containing DRG cell cultures, where they were significantly inhibited by CGRP8–37, suggesting the presence of classical CGRP1 receptors. The lack of complete inhibition of 1 μmol·L−1 CGRP-stimulated [3H]-cAMP production by CGRP8–37 (10 μmol·L−1) does not necessarily implicate a novel CGRP receptor in DRG cells, but results from using a near maximally effective concentration of CGRP. Nevertheless, the insurmountable antagonism displayed by CGRP8–37 against a complete log concentration–response curve for CGRP was unexpected and might be due to a slow dissociating property of the antagonist (Lew et al., 2000). The predominant second messenger system for CGRP receptors in neurones is reported to be cAMP (Ceruti et al., 2011), and our data suggest this might also be the case for glial cells.
Adrenaline produces β2-adrenoceptor-mediated mechanical hyperalgesia and sensitization of rat nociceptors in vitro (Khasar et al., 1999; Aley et al., 2001). Although we detected β2-adrenoceptors in our neurone-enriched cell cultures and pure glial cell cultures, evidence for the in vivo expression of β2-adrenoceptors in DRG is lacking (Nicholson et al., 2005; Leon et al., 2008). β-adrenoceptor-mediated responses in isolated rat DRG cells may be dependent on time in culture, with hyper-nociceptive β2-adrenoceptor responses dominating in short-term cultures (Pluteanu et al., 2002). The hypothesis that isolated DRG cells represent cells responding to axotomy is widely supported (LaMotte, 2007; Zheng et al., 2007), so the up-regulation (Ceruti et al., 2011; Ng et al., 2011) or down-regulation of GPCRs (Franklin et al., 2009) relative to expression in whole DRG may reflect responses to nerve injury. Therefore, both time-dependent and cell type-dependent receptor expression needs to be clarified in mixed DRG cell cultures to allow for accurate interpretation of experimental paradigms.
Not only were AC responses in neurone-enriched cell preparations lower than anticipated, the relative size of the EP4 and IP receptor-dependent responses in glial cells was also higher than responses of mixed DRG cells containing comparable numbers of glial cells. Furthermore, even though the IB4-positive and IB4-negative cell fractions contained similar numbers of neurones, the AC responses in the IB4-positive cell cultures were the smallest (Figure 1D), and this fraction contained the lowest proportion of glial cells (23% in IB4-positive cells vs. 37% in IB4-negative cells). The proportion of glial cells in a population of mixed DRG cells on the day of assay was >80% (Figure 2A). We therefore propose that most of the [3H]-cAMP response determined in mixed DRG cell cultures derived not from neurones but from glial cells. Furthermore, because [3H]-cAMP production decreased in neurone-containing cultures, we hypothesize that this glial response is inhibited by the presence of DRG neurones.
By selectively radiolabelling either neurones or glial cells with [3H]-adenine, we were able to confirm that EP4 and IP receptor-stimulated [3H]-cAMP production by DRG glial cells was indeed inhibited by the presence of DRG neurones, but responses to forskolin were unaffected. We hypothesized that if AC responses to any Gs-coupled GPCR were higher in glial cells compared with mixed DRG cells, then neurones were inhibiting glial cell responses. Thus, CGRP receptor responses in glial cells were inhibited by DRG neurones but β2-adrenoceptor-mediated responses were not. This pattern of responses with CGRP, EP4 and IP receptors having a different profile from those of β2-adrenoceptors and forskolin was reminiscent of our earlier study, where we had mistakenly presumed that these Gs-GPCRs were expressed only on DRG neurones (Rowlands et al., 2001).
The lack of effect of neurone-conditioned medium on glial cell responses to EP4 and IP receptor agonists suggested that cell–cell contact was required to observe this inhibitory phenomenon, and/or a highly unstable soluble factor was involved. But when glial cells were co-cultured with neurone-enriched cells, but separated in a transwell culture system, IP receptor responses in glial cells were still inhibited, but to a lesser extent than when the two cell groups were in direct contact. Thus, the inhibitory effect of DRG neurones on DRG glial cell responses to IP receptor agonists is most likely mediated through a combination of cell–cell interactions and the release of unstable soluble factors. In contrast, the forskolin-stimulated response of glial cells in the transwell system was not significantly inhibited by neurone-dependent factors; therefore, it is unlikely that this inhibitory effect on IP receptor signalling is mediated via activation of Gi/o-protein coupled receptors. By comparing the profile of DRG cell responses to agonist-dependent and agonist-independent activation of AC, we see that the ligands fall into two distinct groups (Figure 10). We propose that the AC stimulating properties of CGRP, EP4 and IP receptors in DRG glial cells are inhibited by the presence of DRG neurones, but we obtained no direct evidence for a similar inhibition of β2-adrenoceptors and forskolin-stimulated responses.
Potential candidates for the inhibitory factor derived from neurones are ATP and glutamate (Zhang et al., 2007a; Gu et al., 2010), but inhibition of purine receptors or glutamine receptors did not attenuate the inhibitory effect of neurones on cicaprost responses in glial cells, nor did we observe any effect of these antagonists on forskolin-stimulated responses. Rat SGCs express P2Y2 and P2Y4 receptors (Villa et al., 2010), and when a higher concentration of suramin was used to more effectively block P2Y4 receptors (Wildman et al., 2003) direct inhibition of cicaprost and forskolin-stimulated [3H]-cAMP production by glial cells was observed (data not shown). Therefore, at the present time, we cannot completely exclude a role for P2Y4 receptors because the classical antagonist Reactive Blue-2 (Wildman et al., 2003) is no longer commercially available.
The precise identity of our glial cells targeted by the inhibitory action of neurones currently remains uncertain. Glutamine synthetase is the classical marker of SGCs in DRG sections, but since both SGCs and Schwann cells express glutamine synthetase (Procacci et al., 2008), we cannot use this marker to distinguish SGCs from Schwann cells in the glial cell cultures. The SGCs in our glial cell preparation have properties similar to astrocytes and microglial cells (Hanani, 2005; van Velzen et al., 2009), and neurone–microglial cell inhibitory signalling is well established (for references, see Ransohoff and Cardona, 2010). Therefore, SGCs may similarly be restrained by inhibitory influences generated by DRG neurones. For the Schwann cell component of our glial cell preparation, axonal contact would be expected to increase cAMP production and drive Schwann cell differentiation into a myelinating phenotype (Morgan et al., 1991; Yoon et al., 2008). Our hypothesis that neurones might be inhibiting Schwann cell cAMP production is apparently contradictory to expectations. According to Yoon et al. (2008), the effect of cAMP on Schwann cell differentiation is very much concentration-dependent and is heavily influenced by the presence of growth factors. Our hypothesis is that two days in culture might be too early to observe axonal contact-induced increases in cAMP in Schwann cells in the conditions used for our mixed DRG cell cultures. Therefore, any neuronal inhibition of Schwann cell cAMP production in these short-term cultures could lead to inhibition of Schwann cell differentiation and contribute to the high proportion of GFAP-positive cells observed in our mixed DRG cell cultures.
In conclusion, the presence of CGRP receptors and the up-regulation of β2-adrenoceptors, EP4 receptors and IP receptors on DRG glial cells in vitro significantly contribute to the overall assessment of AC activity in mixed DRG cell cultures. In future, the role of GPCR-stimulated cAMP-dependent hyper-nociceptive responses in mixed DRG cell cultures can no longer be assumed to result solely from a direct effect of agonists on DRG neurones. Furthermore, receptor-specific inhibition of glial cell AC activity by DRG neurones adds a further complexity to the analysis of DRG cell responses. Because DRG neurones and SGCs form unique functional complexes in vivo, the physical relationship between these cell types in culture should be carefully controlled in studies of neurone–glia communication. The inhibitory action of neurones described herein depends on both neurone–glial cell contact and neurone-derived soluble factors, and is unlikely to involve activation of either purine or glutamine receptors. This inhibitory activity of DRG neurones on DRG glial cells may be an important issue to consider for studies of neurone–glial cell communication within intact dorsal root ganglia.