Exome Sequencing Implicates Impaired GABA Signaling and Neuronal Ion Transport in Trigeminal Neuralgia.

Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated de novo mutation (p.Cys188Trp) in the GABAA receptor Cl- channel γ-1 subunit (GABRG1) exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na+ and Ca+ channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca2+ channel Cav3.2 (CACNA1H). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis.

Enhancing neuronal chloride extrusion rescues α2/α3 GABAA-mediated analgesia in neuropathic pain.

Spinal disinhibition has been hypothesized to underlie pain hypersensitivity in neuropathic pain. Apparently contradictory mechanisms have been reported, raising questions on the best target to produce analgesia. Here, we show that nerve injury is associated with a reduction in the number of inhibitory synapses in the spinal dorsal horn. Paradoxically, this is accompanied by a BDNF-TrkB-mediated upregulation of synaptic GABAARs and by an α1-to-α2GABAAR subunit switch, providing a mechanistic rationale for the analgesic action of the α2,3GABAAR benzodiazepine-site ligand L838,417 after nerve injury. Yet, we demonstrate that impaired Cl- extrusion underlies the failure of L838,417 to induce analgesia at high doses due to a resulting collapse in Cl- gradient, dramatically limiting the benzodiazepine therapeutic window. In turn, enhancing KCC2 activity not only potentiated L838,417-induced analgesia, it rescued its analgesic potential at high doses, revealing a novel strategy for analgesia in pathological pain, by combined targeting of the appropriate GABAAR-subtypes and restoring Cl- homeostasis.

Amiloride modulation of carbon dioxide hypersensitivity and thermal nociceptive hypersensitivity induced by interference with early maternal environment.

BACKGROUND: Early life adversities are risk factors for anxiety disorders and for pain syndromes, which are, in turn, highly comorbid with anxiety disorders. Repeated cross-fostering mouse pups to adoptive lactating females induces epigenetic modification and heightened mRNA-expression of the acid-sensing-ion-channel-1 gene, altered nociception, and hypersensitivity to 6% carbon dioxide air mixtures, a trait marker of specific human anxiety disorders such as, most clearly and prominently, panic disorder. AIMS: We hypothesized that the acid-sensing ion channel inhibitor amiloride can modulate repeated cross-fostering animals' exaggerated responses to carbon dioxide and nociceptive thermal stimulation. METHODS: Respiratory carbon dioxide sensitivity was assessed by plethysmography during 6% carbon dioxide air mixture challenges, and nociception was assessed by latency of paw withdrawal to thermal stimulation, in repeated cross-fostering and control animals. To circumvent the blood-brain barrier, prior to testing, amiloride was nebulized in a plethysmograph. Data were analyzed by general linear models. RESULTS: Analyses of tidal volume responses to 6% carbon dioxide of animals pre-treated with nebulized amiloride/saline in a randomized crossover design showed significant modulatory effect of amiloride, and amiloride×repeated cross-fostering interaction. In contrast, repeated cross-fostering animals' responses to 6% carbon dioxide after intraperitoneal amiloride, saline, or no treatment, were no different. Analyses of responses to thermal stimuli showed a significant modulatory effect of nebulized amiloride, and repeated cross-fostering×amiloride interaction. CONCLUSIONS: Single-dose nebulized amiloride decreased repeated cross-fostering animals' carbon dioxide sensitivity and nociception indices to levels that were no different from those of control animals. Inasmuch as these results pertain to human anxiety and/or pain hypersensitivity, our findings provide a rationale for studying inhaled amiloride in some anxiety disorders and/or pain syndromes.

Maintaining polarization in polarimetric multiphoton microscopy.

Polarimetric measurements in multiphoton microscopy can reveal information about the local molecular order of a sample. However, the presence of a dichroic through which the excitation beam propagates will generally scramble its polarization. We propose a simple scheme whereby a second properly-oriented compensation dichroic is used to negate any alteration regardless of the wavelength and the initial polarization. We demonstrate how this robust and rapid approach simplifies polarimetric measurements in second-harmonic generation, two-photon excited fluorescence and coherent anti-Stokes Raman scattering. Illustration of the polarization maintaining strategy with the compensating dichroic oriented such that its s- and p-axes are interchanged with these of the primary dichroic.

Chloride extrusion enhancers as novel therapeutics for neurological diseases.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Dorsal horn neurons presynaptic to lamina I spinoparabrachial neurons revealed by transynaptic labeling.

Sensory input to supraspinally projecting lamina I (LI) neurons arises both directly from primary afferents and via neurons intrinsic to the spinal dorsal horn. The types of neurons presynaptic to those projection neurons remain poorly known. To address this question we used retrogradely transported adenoviral vectors encoding green fluorescent protein (GFP) and a GFP-TTC (fragment C of the tetanus toxin) fusion protein, labeling respectively spinoparabrachial projection neurons and neurons presynaptic to them. The expression of GFP by infected neurons labeled the entire dendritic tree, enabling a more complete and quantitative morphological description of spinoparabrachial neurons than previous methods. These neurons were located in spinal LI, with dendritic arbors oriented extensively in the rostrocaudal axis (1,089.8 +/- 91.5 microm) and displaying low spine density. In contrast, their dendrites did not extend significantly ventrally (29.2 +/- 3.5 microm). The use of transynaptic tracer GFP-TTC revealed a population of local circuit LI neurons presynaptic to LI projection neurons. These local circuit LI neurons had distinct morphological properties, in particular significantly longer ventrally oriented dendrites (80.1 +/- 10.1 microm). The transynaptic tracer also revealed a population of stalked cells, some being highly spiny, directly in contact with spinal projection neurons. However, stalked cells were not the only lamina II cells in direct contact with projection neurons. Intracellular injections with Lucifer yellow in parasagittal slices of fixed tissue confirmed the above observations. Overall, these experiments demonstrated that neurons projecting to the parabrachial nucleus had their dendritic branching almost exclusively in LI and had sparse dendritic spines, in contrast with local circuit neurons that often extended ventrally and could be very spiny.

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain.

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.