CCI of sciatic nerve increased responses to glutamate and AMPA in small diameter neurons.

<p>A1. Example of glutamate-induced (1 mM, 200 ms) increases in inward currents in small diameter neurons after CCI compared to naïve. Bar indicates the duration of drug application. A2. Statistical analysis showed that glutamate induced significantly larger inward currents in the CCI group compared with naïve. A3. Population analysis showed that CCI increased the proportion of the neurons responsive to glutamate. B1. Example of AMPA-induced (100 µM, 200 ms) increases in inward current in small diameter neurons after CCI compared to naïve. Bar indicates the duration of drug application. B2. Statistical analysis showed that AMPA induced significantly larger inward currents in the CCI group compared with naïve. B3. Population analysis showed that CCI increased the proportion AMPA-responsive neurons. C. Western blot analysis showed increased GluA1 expression (normalized membrane/normalized total protein) in naïve and CCI DRGs. D. Western blot analysis showing decreased GluA2 expression in naïve and CCI DRGs. Numbers in each column represents recorded neurons. Mean ± SEM, * p<0.05, ** p<0.01.</p

Increased Response to Glutamate in Small Diameter Dorsal Root Ganglion Neurons after Sciatic Nerve Injury

<div><p>Glutamate in the peripheral nervous system is involved in neuropathic pain, yet we know little how nerve injury alters responses to this neurotransmitter in primary sensory neurons. We recorded neuronal responses from the <i>ex-vivo</i> preparations of the dorsal root ganglia (DRG) one week following a chronic constriction injury (CCI) of the sciatic nerve in adult rats. We found that small diameter DRG neurons (<30 µm) exhibited increased excitability that was associated with decreased membrane threshold and rheobase, whereas responses in large diameter neurons (>30 µm) were unaffected. Puff application of either glutamate, or the selective ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA), or the group I metabotropic receptor (mGluR) agonist (<i>S</i>)-3,5-dihydroxyphenylglycine (DHPG), induced larger inward currents in CCI DRGs compared to those from uninjured rats. N-methyl-D-aspartate (NMDA)-induced currents were unchanged. In addition to larger inward currents following CCI, a greater number of neurons responded to glutamate, AMPA, NMDA, and DHPG, <i>but not</i> to KA. Western blot analysis of the DRGs revealed that CCI resulted in a 35% increase in GluA1 and a 60% decrease in GluA2, the AMPA receptor subunits, compared to uninjured controls. mGluR1 receptor expression increased by 60% in the membrane fraction, whereas mGluR5 receptor subunit expression remained unchanged after CCI. These results show that following nerve injury, small diameter DRG neurons, many of which are nociceptive, have increased excitability and an increased response to glutamate that is associated with changes in receptor expression at the neuronal membrane. Our findings provide further evidence that glutamatergic transmission in the periphery plays a role in nociception.</p></div

CCI of sciatic nerve increased responses to NMDA and KA in small diameter neurons.

<p>A. Upper panel: Puff application of NMDA (100 µM, 200 ms) had no effect on inward currents in small DRG neurons from naïve or CCI rats. Bar indicates the duration of drug application. Lower panel: Statistical analysis showed no differences in inward currents induced by NMDA between CCI and naïve rats. B. Population analysis showed that the percentage of the NMDA-responsive neurons increased after CCI. C. Upper panel: Example of KA-induced (100 µM, 200 ms) increases in inward currents in CCI DRGs. Bar indicates the duration of drug application. Lower panel: Statistical analysis showed that KA induced a significantly larger response in CCI group compared with naïve. D. Population analysis showed that the percentage of the KA-responsive neurons did not change significantly after CCI. Numbers in each column represents recorded neurons. Mean ± SEM, * p<0.05.</p

CCI of sciatic nerve increased responses to DHPG in small diameter neurons.

<p>A. Upper panel: Example of DHPG-induced (100 µM, 200 ms) increases in currents in CCI vs. naïve DRGs. Bar indicates the duration of drug application. Lower panel: Statistical analysis showed that DHPG induced a much bigger response in CCI group compared with naïve. B. Population analysis showed that the percentage of DHPG-responsive neurons increased after CCI. C. Western blot analysis showed increased mGluR1 expression (normalized membrane/normalized total protein) in naïve and CCI DRGs. D. Western blot analysis showing that the expression of mGluR5 subunit in the membrane did not change significantly after CCI, compared to the naïve group. Numbers in each column represents recorded neurons. Mean ± SEM, * p<0.05, **p<0.01.</p

The effect of DHPG incubation on responses to AMPA, NMDA and KA in small diameter neurons.

<p>A. Incubation with 50 µM DHPG for 2 hours reduced AMPA-induced (100 µM) inward currents in small diameter neurons in both naïve and CCI groups. B. Incubation with 50 µM DHPG for 2 hours reduced NMDA-induced (100 µM) inward currents in small diameter neurons in both naïve and CCI groups. C. Incubation with 50 µM DHPG for 2 hours increased KA-induced (100 µM) inward currents in small diameter neurons in the naïve group, but had no effect on KA-induced currents in the CCI group. Numbers in each column represents recorded neurons. Mean ± SEM, * p<0.05, **p<0.01.</p

Lipoic-Based TRPA1/TRPV1 Antagonist to Treat Orofacial Pain

Inflammation of the trigeminal nerve is considered one of the most painful conditions known to humankind. The diagnosis is often difficult; moreover, safe and effective pharmacological treatments are lacking. A new molecule, ADM_12, formed by a lipoic and omotaurine residues covalently linked, is here reported. In vitro and in vivo tests showed that ADM_12 is a very attractive original compound presenting (i) a remarkable safety profile; (ii) a high binding constant versus TRPA1; (iii) an intriguing behavior versus TRPV1; and (iv) the ability to significantly and persistently reduce mechanical facial allodynia in rats. Noteworthy, by testing ADM_12, we shed light on the unprecedented involvement of TRPA1 and TRPV1 channels in orofacial pain