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    • br Expression of GlyT in the spinal cord

    2021-09-23


    Expression of GlyT in the spinal cord in response to neuropathic pain Twelve days after sham injury or CCI, GlyT1 and GlyT2 mRNA expression in rat spinal cord was analyzed using qPCR. Here, no significant changes in expression of the transporters in our CCI model of neuropathic pain compared with sham-operated controls were observed (Fig. 4A and B). Similarly, protein expression, as determined densitometric analysis of Western blots for GlyT1 (representative blot in Fig. 4C) and GlyT2 (representative blot in Fig. 4D), did not reveal any significant difference in the spinal cord of CCI-treated animals compared to sham controls (two membranes with n=3 per group, Fig. 4E and F).
    Expression of GlyT1 and NR1 in DRG tissue in response to neuropathic pain In DRG, no changes in mRNA expression for GlyT1 were observed in response to CCI 12 days after surgery when compared to sham controls (Fig. 5A). Similarly, GlyT1 protein expression was not differentially altered 12 days after CCI compared to sham operation (Fig. 5B and C). Additionally, no significant expression changes of NR1 were detected in the ipsilateral DRG (Fig. 6).
    Discussion In the present study, the expression of GlyTs and the glycine binding subunit NR1 of the NMDA receptor was investigated in the nervous system in the context of neuropathic pain. We confirmed expression of GlyT1 and GlyT2 in rat spinal cord as described previously [2]. Furthermore, we provide evidence that GlyT1, but not GlyT2, is also expressed in the PNS. GlyT1, but not GlyT2, was identified in DRG. Here, we show that GlyT1 localizes in the vicinity of peripheral NMDA receptors, as shown by co-localization with the NR1 subunit. A further result of our investigation is that GlyTs are not differentially regulated after induction of neuropathic pain, neither in the spinal cord, nor in the DRG. In Fosmidomycin sodium salt to previous findings in the spinal cord [7], no changes in NR1 expression were observed in DRG, indicating that the regulation of NR1 expression differs between spinal cord and DRG. At present, two glycine transporters have been cloned: GlyT1 and GlyT2, which both exist in multiple isoforms. GlyT1 is expressed in many parts of the CNS, predominantly by astrocytes. Additional expression was shown in a subset of glutamatergic neurons [14]. GlyT2 is exclusively expressed by glycinergic neurons of the CNS [15]. GlyT1 is found in areas without strychnine-sensitive glycine receptors as well [2], [3]. Consistently, GlyT1 has been shown to also affect NMDA receptor mediated glutamatergic neurotransmission in addition to glycine dependent inhibitory neurotransmission [15]. Consistent with the fact that the majority of DRG cells are glutamatergic, and glycinergic cells are absent [16], GlyT2 was not detected, whereas GlyT1 was identified in the vicinity of NR1-positive neurons of DRG. To our knowledge, up to now there are no studies reporting GlyT expression in the PNS. Our results from immunofluorescence microscopy demonstrate a neuronal localization of GlyT1 protein in DRG. However, the functional role of GlyT1 in the PNS remains unclear. Since GlyT1 seems to be co-localized with NR1, i.e., the subunit binding glycine of the NMDA receptor, it is conceivable that, similar to its function in the CNS, GlyT1 modulates glutamatergic transmission by regulating the glycine concentration at NMDA receptors [14]. Whether NMDA receptors expressed at DRG neuron somata receive synaptic input or whether they participate in chemical neurotransmission between DRG neurons is unclear. Furthermore, GlyT1 and NMDA receptors might also co-express at the spinal terminals of DRG neurons. These questions require further future research. Numerous studies have reported changes in expression of neurotransmitter transporters in pathological pain. For GABA transporters, however, the results appear inconsistent. Here both an up-regulation [17] as well as a significant down-regulation [18], [19] of the GABA transporter GAT-1 has been described in the context of neuropathic pain. Also the spinal expression of the glutamate/aspartate transporter (GLAST) and glutamate transporter GLT-1 was differentially regulated in animal models of neuropathic pain [20], [21].