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

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

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

Estimation of approach accuracy using a tandem of Cerulean and Venus.

<p>(A) Images of representative PC12 cell expressing the tandem: Cerulean fluorescence (a), Venus fluorescence (b), and a ratio image of Venus to Cerulean fluorescence (c). A color and intensity of each pixel in (c) represent the ratio of Venus/Cerulean fluorescence and averaged intensity of respective pixels in the images (a) and (b) <i>(F</i>_<i>avr</i> <i>= (F</i>_<i>C</i> <i>+ F</i>_<i>V</i><i>)/2)</i>, respectively. A scale bar in (a) is 5μm. (B) A linear regression of correlation plot between Cerulean and Venus fluorescence intensities for each pixel within the PC12 cell image shown in (A). A strong linear correlation between the intensities (slope = 3.922±0.004, intercept = 11.1±0.01, R² = 0.99; the slope is significantly different from zero at the 0.05 level) demonstrates co-localization of fluorescent protein labels. (<b>C</b>) Linear regressions of correlation plots similar to one shown in (B) for five PC12 cells. Cells having different levels of tandem expression were chosen for this plot in order to demonstrate that the ratio of fluorescence intensities remains unchanged in the wide range of tandem expression levels. (D) Expected (Expec.) and apparent (Appar.) ratios of Venus to Cerulean concentrations in the tandem ([<i>L</i>_<i>V</i>]/[<i>L</i>_<i>C</i>]). The histogram demonstrates that the apparent ratio of Venus to Cerulean concentrations estimated based on the <i>ratio factor</i> (1.33±0.06, mean±S.E.M., n = 5) is close to the expected ratio, which is equal to 1. It indicates that an error associated with inaccurate determination of spectral properties of labels and equipment is about 30%.</p

Parameters necessary for calculation of <i>ratio factor A</i> for Cerulean and Venus fluorescent proteins.

<p>(A) Spectra of the optical components in the excitation path of the imaging system. <i>S</i>_<i>src</i>(<i>λ</i>), <i>S</i>_<i>ex</i>(<i>λ</i>), <i>S</i>_<i>dichr</i>(<i>λ</i>), <i>S</i>_<i>obj</i>(<i>λ</i>) are spectra for the light source (Polychrome V monochromator, TILL Photonics, yellow), excitation filter (Chroma 69008x, blue), dichroic mirror (Chroma 69008bs, green) and objective (Olympus UAPO 40XOI3/340, black), respectively, mounted on Olympus IX71 microscope. Point-by-point multiplication of spectra for each optical element yields an optical function of microscope excitation light path shown by a violet trace. This excitation optical function of the particular imaging system can be used for calculation of a <i>ratio factor A</i> for a wide range of different fluorescent labels. (B) Further point-by-point multiplication of the optical function (violet), normalized Cerulean absorption spectrum (blue), and a spectrum of monochromator slit chosen for a given experiment (orange) gives a function of excitation path for Cerulean (black). Integration of this function and multiplication by the extinction coefficient for Cerulean absorbance results in , necessary for estimation of the <i>ratio factor A</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194031#pone.0194031.e010" target="_blank">Eq 1</a>). (C) Spectra of the optical components in the emission path of the imaging system. <i>S</i>_<i>obj</i>(<i>λ</i>),<i>S</i>_<i>dichr</i>(<i>λ</i>),<i>S</i>_<i>em</i>(<i>λ</i>),<i>S</i>_<i>det</i>(<i>λ</i>) are objective (Olympus UAPO 40XOI3/340, black), dichroic mirror (Chroma 69008bs, green), and emission filter (Chroma 69008m, dotted red) transmittance and detector (QImaging ExiBlue, brown) sensitivity, respectively. Point-by-point multiplication of spectra for each optical element gives an optical function of microscope emission path shown by a red bold trace. This emission optical function of the particular imaging system can be used for calculation of a <i>ratio factor A</i> for a wide range of different fluorescent labels. (D) Further point-by-point multiplication of the optical function (red dashed trace; left Y axes) and Cerulean emission spectrum, integral of which is normalized to 1, (blue dashed trace; right Y axes) gives the function of emission path for Cerulean (black trace, right Y axes). Integration of this function and multiplication by Cerulean quantum yield results in .</p

Reference data on optical elements of the imaging system.

<p>Reference data on optical elements of the imaging system.</p

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&nbsp;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&nbsp;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