The role of spinal cord extrasynaptic α5 GABAA receptors in chronic pain.

Chronic pain is an incapacitating condition that affects a large population worldwide. Until now, there is no drug treatment to relieve it. The impairment of GABAergic inhibition mediated by GABAA receptors (GABAA R) is considered a relevant factor in mediating chronic pain. Even though both synaptic and extrasynaptic GABAA inhibition are present in neurons that process nociceptive information, the latter is not considered relevant as a target for the development of pain treatments. In particular, the extrasynaptic α5 GABAA Rs are expressed in laminae I-II of the spinal cord neurons, sensory neurons, and motoneurons. In this review, we discuss evidence showing that blockade of the extrasynaptic α5 GABAA Rs reduces mechanical allodynia in various models of chronic pain and restores the associated loss of rate-dependent depression of the Hoffmann reflex. Furthermore, in healthy animals, extrasynaptic α5 GABAA R blockade induces both allodynia and hyperalgesia. These results indicate that this receptor may have an antinociceptive and pronociceptive role in healthy and chronic pain-affected animals, respectively. We propose a hypothesis to explain the relevant role of the extrasynaptic α5 GABAA Rs in the processing of nociceptive information. The data discussed here strongly suggest that this receptor could be a valid pharmacological target to treat chronic pain states.

L5-6 Spinal Nerve Ligation-induced Neuropathy Changes the Location and Function of Ca2+ Channels and Cdk5 and Affects the Compound Action Potential in Adjacent Intact L4 Afferent Fibers.

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

γ-Aminobutyric acid (GABA) from satellite glial cells tonically depresses the excitability of primary afferent fibers.

Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.