2.1.4. pH Sensing by TRPA1

The relationship between oxygen and pH is physiologically important because hypoxic conditions caused by ischemia induce intracellular acidification. Neuronal death may occur upon low levels of oxygen and glucose, as well as excessive release of glutamic acid from astrocytes, which induces a fatal calcium influx in neurons. Intracellular pH of astrocytes is drastically reduced by lactic acid production due to anaerobic respiration in response to hypoxia. Subsequent acidification induces glutamic acid exocytosis led to brain damage [57]. Importantly, TRPA1 expressed in astrocytes may be activated by acidification [58–60]. In addition, activation of TRPA1 expressed in oligodendrocytes can damage myelin [61]. In contrast, intracellular alkalization also affects TRPA1 activity [26]. TRPA1 is activated at approximately pH 8.0, and the alkalization-induced pain-related behavior is significantly reduced in TRPA1 deficient mice. Consequently, the pH dependency of TRPA1 may be beneficial target for the treatment of central nervous system diseases, not only pain.

#### 2.1.5. Neural Networks Involving TRPA1-Mediated Pain Sensation

A-fiber and C-fiber primary sensory nerves govern fast and slow responses to pain, respectively. Aδ-fibers (mid-sized DRG neurons) innervate lamina I and V, whereas C-fibers (small-sized DRG neurons) innervate lamina I and II of the dorsal horn of the spinal cord [1]. C-fibers also contain peptidergic and nonpeptidergic neurons. Peptidergic neurons contain substance P and CGRP, with both peptides released upon neural excitation. TRPA1-positive neurons are immunoreactive for CGRP in DRG neurons [15]. In healthy mice, these CGRP-positive neurons enhance heat sensation and suppress cold sensation [62]. These findings suggest that TRPA1 in CGRP-positive DRG neurons contributes less significantly to noxious cold sensation.

Neural transmission in the spinal cord can modify pain perception. Substantia gelatinosa (SG) neurons in lamina II are important targets for investigation of how pain sensations are transmitted from the periphery to the central nervous system. Initial understanding on in vivo SG neuronal responses to peripheral stimulation is that SG neuronal activity mediated through non-*N*-methyl-*D*-aspartate (non-NMDA) receptors is enhanced by mechanical stimuli, such as pinch and air flow, but not thermal changes [63]. However, excitatory postsynaptic currents enhanced by capsaicin treatment are detected in approximately 80% of SG neurons in slice patch-clamp recordings [64]. Interestingly, there is no neuronal response to AITC alone (Figure 1).

Since AITC responses depend on both NMDA and non-NMDA receptors [65], TRPA1-mediated pain signals are likely integrated with TRPV1-mediated pain signals in lamina II of the spinal cord. Importantly, there are three types of DRG neurons: those that express both TRPA1 and TRPV1, TRPA1 alone, or TRPV1 alone. Meanwhile, one study demonstrated that spinal TRPA1 activation by intrathecal administration of the acetaminophen metabolite, N-acetyl-p-benzoquinone imine, enhanced anti-nociception in the spinal cord of mice [66]. Therefore, components of TRPA1-mediated neural systems may participate in pain reduction, while nociception by TRPA1 activation can function in central termini of DRG neurons. For instance, TRPA1 activation by hepoxilin causes mechanical allodynia in rats, whereas pinch-evoked SG neuronal excitation is reduced by increases in inhibitory postsynaptic currents mediated by TRPA1 activation in vivo [67,68]. Thus, consideration of TRPA1 activation in the central nervous system may also be important for investigating pain mechanisms.

**Figure 1.** Nociceptor populations in substantia gelatinosa (SG) neurons of lamina II, which receive nociceptive inputs from dorsal root ganglion (DRG) neurons, no neurons respond only to TRPA1-associated stimuli. Approximately 30% of SG neurons are double-positive to capsaicin (CAP) and cinnamaldehyde (CA), 45% of SG neurons response to only CAP, and 25% of neurons show no effect to either CAP or CA. There are calcitonin gene-related peptide (CGRP)-positive and -negative neurons in peripheral sensory nerves. Most TRPV1–TMEM100–TRPA1 complexes and TRPV1–TRPA1–TRPM3 trios are expressed in CGRP-positive neurons. Anoctamin 1 (ANO1) is also expressed in CGRP-positive neurons, however approximately 70% of ANO1-expressing neurons are CGRP-negative.
