Neuropathic pain changes the output of rat lamina I spino-parabrachial neurons.

• Spared nerve injury (SNI) altered the action potential (AP) output of lamina I spino-parabrachial neurons (SPNs) without affecting their resting potential or membrane resistance. • In one-third of SPNs, high-threshold dorsal root stimulation elicited persistent AP firing which was never observed in cells from naïve animals. • 38% of SPNs from SNI rats showed spontaneous persistent AP firing. • After SNI low- and high-output SPNs were no longer nociceptive-specific as part of them responded with APs to low-threshold stimulation. • These SNI-induced changes of SPN output might represent cellular mechanisms for neuropathy-associated allodynia, hyperalgesia, and spontaneous pain.

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Segmental and descending control of primary afferent input to the spinal lamina X.

ABSTRACT: Despite being involved in a number of functions, such as nociception and locomotion, spinal lamina X remains one of the least studied central nervous system regions. Here, we show that Aδ- and C-afferent inputs to lamina X neurons are presynaptically inhibited by homo- and heterosegmental afferents as well as by descending fibers from the corticospinal tract, dorsolateral funiculus, and anterior funiculus. Activation of descending tracts suppresses primary afferent-evoked action potentials and also elicits excitatory (mono- and polysynaptic) and inhibitory postsynaptic responses in lamina X neurons. Thus, primary afferent input to lamina X is subject to both spinal and supraspinal control being regulated by at least 5 distinct pathways.

Elucidating afferent-driven presynaptic inhibition of primary afferent input to spinal laminae I and X.

Although spinal processing of sensory information greatly relies on afferent-driven (AD) presynaptic inhibition (PI), our knowledge about how it shapes peripheral input to different types of nociceptive neurons remains insufficient. Here we examined the AD-PI of primary afferent input to spinal neurons in the marginal layer, lamina I, and the layer surrounding the central canal, lamina X; two nociceptive-processing regions with similar patterns of direct supply by Aδ- and C-afferents. Unmyelinated C-fibers were selectively activated by electrical stimuli of negative polarity that induced an anodal block of myelinated Aß/δ-fibers. Combining this approach with the patch-clamp recording in an ex vivo spinal cord preparation, we found that attenuation of the AD-PI by the anodal block of Aß/δ-fibers resulted in the appearance of new mono- and polysynaptic C-fiber-mediated excitatory postsynaptic current (EPSC) components. Such homosegmental Aß/δ-AD-PI affected neurons in the segment of the dorsal root entrance as well as in the adjacent rostral segment. In their turn, C-fibers from the L5 dorsal root induced heterosegmental AD-PI of the inputs from the L4 Aδ- and C-afferents to the neurons in the L4 segment. The heterosegmental C-AD-PI was reciprocal since the L4 C-afferents inhibited the L5 Aδ- and C-fiber inputs, as well as some direct L5 Aß-fiber inputs. Moreover, the C-AD-PI was found to control the spike discharge in spinal neurons. Given that the homosegmental Aß/δ-AD-PI and heterosegmental C-AD-PI affected a substantial percentage of lamina I and X neurons, we suggest that these basic mechanisms are important for shaping primary afferent input to the neurons in the spinal nociceptive-processing network.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.