Li WC et al. (2017), Mechanosensory Stimulation Evokes Acute Concuss...

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eNeuro 2017 Jan 01;42:. doi: 10.1523/ENEURO.0073-17.2017.

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Mechanosensory Stimulation Evokes Acute Concussion-Like Behavior by Activating GIRKs Coupled to Muscarinic Receptors in a Simple Vertebrate.

Li WC , Zhu XY , Ritson E .

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Most vertebrates show concussion responses when their heads are hit suddenly by heavy objects. Previous studies have focused on the direct physical injuries to the neural tissue caused by the concussive blow. We study a similar behavior in a simple vertebrate, the Xenopus laevis tadpole. We find that concussion-like behavior can be reliably induced by the mechanosensory stimulation of the head skin without direct physical impacts on the brain. Head skin stimulation activates a cholinergic pathway which then opens G protein-coupled inward-rectifying potassium channels (GIRKs) via postsynaptic M2 muscarinic receptors to inhibit brainstem neurons critical for the initiation and maintenance of swimming for up to minutes and can explain many features commonly observed immediately after concussion. We propose that some acute symptoms of concussion in vertebrates can be explained by the opening of GIRKs following mechanosensory stimulation to the head.

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Species referenced: Xenopus laevis
Genes referenced: asprv1 dtl

???displayArticle.disOnts??? spinocerebellar ataxia type 1

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	Figure 1. KO responses in free-swimming and restrained tadpoles. A, KO responses videoed in a dish (left) with swimming speeds plotted against post-clash swimming lengths (right, 18 responses in six tadpoles, p < 0.01, two-tailed Pearson correlation). Post-clash swimming lasts for 0.33 Â± 0.02 s (0.21â€“0.63 s). Tracing shows the tadpole swimming track. B, Simulating head-on clashes in a physically restrained tadpole (left) and the distribution of post-tap swimming lengths. Gray represents single taps (half-cycle sinewave at 20 Hz) and black represents multiple taps (five sinewave cycles at 20 Hz).
	Figure 2. Responses of dINs to head taps. A, A slow tap at 0.125 Hz ends swimming (motor nerve, m.n.) and evokes a mixture of EPSPs and IPSPs (arrow heads in inset) in a dIN on the right side of the hindbrain (r.dIN). B, A fast tap at 2.5 Hz briefly excites another dIN in the right side hindbrain () then stops swimming with delayed, prolonged inhibition in the RMP and decreased Rinp (test steps: â100 pA). C, D, RMPs and Rinp in control (unfilled columns) and at the trough period of inhibition after taps. While slow taps at 0.125â0.25 Hz decrease RMPs in 8 dINs (from â52.2 Â± 1.9 to â54.4 Â± 1.9 mV, gray) without changing the Rinp, fast taps at 2.5â10 Hz in 6 dINs decrease RMPs from â52.6 Â± 0.7 to â59.1 Â± 2 mV with a reduction in the Rinp by 29.6 Â± 8.5% (black, p < 0.05, paired t test or related sample Wilcoxon signed rank test). Dashed lines indicate RMP in A, B. Inset on the top shows experimental setup.
	Figure 3. KO responses evoked by electrical skin stimulation. A, Increasing skin stimulation (arrow, five pulses at 30 Hz) intensity shortens swimming in one tadpole. B, Summary of poststimulation swimming lengths at different stimulation current intensities, capped below 320 ÂµA, as shown in A. Swimming thresholds at rest (Thr.) are 2â€“14 ÂµA. C, Three stimuli at 40 Hz (arrow, 55 ÂµA) evokes a KO response in one tadpole without evoking any skin impulse, while a single stimulus (80 ÂµA) evokes a skin impulse (arrowhead) without shortening swimming. Insets are stretched from time around stimulation. D, Paired comparison of poststimulation swimming lengths as in C showing their dependence on the number of stimuli (n = 29 pairs from four tadpoles, p < 0.001, related samples Wilcoxon signed rank test). A single stimulus always evokes a skin impulse. E, Experimental setup for the direct stimulation of the trigeminal ganglion (tg). A dorsal view of the CNS and some swimming myotomes are shown (fb, forebrain; mb, midbrain; hb, hindbrain; nll, lateral line nerve; stim., stimulation electrode; oc, otic capsule; sc, spinal cord; m, myotome). Dashed lines indicated severed nerves and oc. F, KO responses evoked by stimulating the tg directly after removing nlls (p < 0.01 in each of five tadpoles, two-tailed independent sample t test). G, Bath application of SR95531, a GABAAR antagonist, does not affect the KO response in a tadpole. KO stimuli (arrow) consist of five pulses at 30 Hz. H, Summary of SR95531 experiments in six tadpoles (p > 0.05, related samples Friedmanâ€™s two-way ANOVA by ranks). I, KO sites mapped with electrical skin stimuli (10 at 30 Hz). Each site has been applied KO stimuli for more than five times intercalated with trials without KO stimuli. Filled circles indicate KO stimuli shortened swimming (p < 0.05, paired t test or related samples Wilcoxon signed rank test) and empty circles indicate KO stimuli have no effect.
	Figure 4. Properties of KO inhibition and their relation with the longitudinal location of neurons. A, KO inhibition in a dIN 468 Âµm from mid/hindbrain border following electrical skin stimulation (five pulses at 30 Hz, arrow). B, KO inhibition after 10 electrical skin stimuli (25 Hz, arrow) in a dIN 670 Âµm from mid/hindbrain border. Dashed lines indicate RMP in A, B. C, A dIN filled with neurobiotin in the hindbrain (hb, lateral view). Fb, forebrain; mb, midbrain; p, pineal eye. Black dashed lines outline the central nervous system. White dashed line marks the border between mb and hb (0 in E). D, Photo of the cell in C (white rectangle). Arrow head marks a short ascending axon. E, The distribution of somata (circles) and axons (lines) of 13 neurons with >100% peak conductance increases during KO. Dashed vertical line marks the position of obex. F, Longitudinal location of neurons plotted against their peak conductance increases during KO (48 dINs, 25 non-dINs, p < 0.001, two-tailed Spearman rank correlation coefficient 0.58). G, KO inhibition amplitudes plotted against conductance increases (p < 0.0001, correlation coefficient 0.834). In EâG, red symbols represent dINs; black symbols represent non-dINs. Hollow symbols are from tap experiments and solid ones from electrical skin stimulation. H, Increasing the number of electrical skin stimuli (numerals above bars) while maintaining the stimulation strengths and frequencies induces larger rises in the membrane conductance in 10 dINs. The recordings from one dIN are shown in insets.
	Figure 5. KO inhibition induced by electrical head skin stimulation (arrows) is mediated by GIRKs. A, A voltage-clamp recording shows the slow outward KO current and its onset (inset, ) after KO stimulation (five pulses at 30 Hz). A voltage ramp rising from â100 to 0 mV is applied at the peak of the current. Membrane potential is held at â60 mV at rest. B, I-V data points averaged from five recordings during the voltage ramp in A for estimating the reversal of the currents and their rectification. C, Effects of microperfusing 50 ÂµM Ba2+ on KO inhibition in a dIN. D, Summary of blockade of peak conductance rises by 50â100 ÂµM Ba2+ during KO in two dINs and six non-dINs (paired t tests). E, Microperfusing 3 ÂµM Tertiapin-Q (T-Q) weakened the peak conductance increase during KO in one dIN and five non-dINs (paired t tests). F, One dIN recording with a pipette solution containing 10 ÂµM GRK2i at the beginning (control) and after 28 min of equilibration. G, GRK2i weakened the peak conductance increase during KO inhibition (n = 8, paired t test). H, Summary of peak membrane conductance increase during KO in control and after BAPTA equilibration (n = 8, related samples Wilcoxon signed rank test, p = 0.33). , significance at p < 0.05 and ** at p < 0.01 in D, E, G. Dashed lines indicate RMP or clamping current at â60 mV in A, C, F.
	Figure 6. The pharmacological blockade of KO responses and KO inhibition. A, KO responses induced by KO stimulation (arrow, five pulses at 30 Hz) applied every 100 s. Swimming (m.n.) is evoked by dimming an LED. Gray bar indicates time of the application of 10 ÂµM methoctramine (methoc., 100 s). B, Example KO responses before (1), shortly after methoc. application (2), and recovery (3). C, KO responses are blocked 200 s after methoc. application (p < 0.01, n = 9 tadpoles, related samples Wilcoxon signed rank test) but without recovery 25 min after wash. D, KO inhibition following skin stimulation (arrows, five at 30Hz) in a dIN is reduced by the microperfused 10 ÂµM methoc. Swimming is initiated by dimming an LED light. E, Conductance increases during KO inhibition are weakened by methoc. (n = 6, p < 0.05, related samples Wilcoxon signed rank test). F, Summary of post-KO swimming lengths measured at 0, 200, and 2500 s from the application of different antagonists (numerals in brackets indicate number of tadpoles tested, related samples Wilcoxon signed rank tests). Atropine (10â50 ÂµM) is a nonselective muscarinic receptor blocker. PD102807 (0.5 ÂµM) is a selective M4 muscarinic receptor blocker. CGP54626 (10 ÂµM) is a GABAB receptor blocker and LY341495 (10 nM) blocks mGluR II and III. p < 0.05 and *p < 0.01 in C, E, F.
	Figure 7. Swimming activity after KO stimulation. A, Distribution of swimming lengths after KO stimulation (range: 0â3.81 s, n = 197 trials). Trials are chosen from 20 tadpoles where membrane conductance during KO has increased by >100% (50/197 without swimming preceding KO). B, Normalized swimming frequency before and after KO stimuli. Gray traces are six trials with frequency increases (p < 0.05, t test, 10 post-KO swimming cycles compared with control). C, Motor responses following KO stimulation (arrow). Four single stimuli (arrow heads, 220 ÂµA) are applied to evoke swimming during KO inhibition. Note failures in dIN spiking. D, Swimming thresholds (normalized to control, dashed line) tested with single skin stimulation after KO. E, Swimming thresholds are higher after KO stimulation and their recovery >5 min later (n = 10 sites in six tadpoles, p < 0.001, related samples Friedmanâs two-way ANOVA by ranks). F, Lengths of swimming evoked by single suprathreshold skin stimulation after KO stimulation (filled circles). Empty circles show the same normalized data (secondary axis).
	Figure 8. Locating neurons responsible for KO responses. A, A dIN recording after KO stimulation (arrow, 10 pulses at 30 Hz). Gray bar marks the Rinp-testing period. B, dIN firing reliability decreases after KO stimulation (**p < 0.001, n = 19 trials from 10 tadpoles, related sample Wilcoxon signed rank test). C, KO responses persist with lesions at forebrain and midbrain border (fb/mb). D, KO responses persist in three out of six tadpoles with lesions at the midbrain and hindbrain border (mb/hb). E, KO responses persist in three out of six tadpoles with lesions at the rostral edge of trigeminal nerve entry to hindbrain. Diagrams below bar charts in Câ€“E show lesion locations (dashed lines outline removed brain). Each line connects averaged swimming lengths from more than five trials in control and with KO stimuli (5 at 30 Hz and 320 ÂµA). Empty symbols show tadpoles where KO persists after lesions and filled symbols represent tadpoles with KO responses abolished by the lesion (independent samples t test or median test, p < 0.05 in each tadpole). F, Swimming of a tadpole with KO responses abolished by the lesion at the rostral edge of the trigeminal nerve entry to the hindbrain. Arrow indicates time of electrical skin stimuli. Swimming is initiated by a single electrical stimulus to the head skin. G, Diagrammatic summary of the KO pathway (dorsal view) mediating the loss of motor responses. Stimulating the head skin excites the peripherals of rapid-transient detectors (red) located in the trigeminal ganglion, which in turn activate the unidentified cholinergic interneurons (green with dashed processes) in the brainstem. The cholinergic cells inhibit the rostral dINs (blue) by opening the GIRKs coupled to M2 muscarinic cholinergic receptors. The non-dINs (black) then lose the excitatory inputs from dINs and tadpole motor responses are suppressed. For abbreviations, see Figure 3E.

References [+] :

Adams, Diffuse brain damage of immediate impact type. Its relationship to 'primary brain-stem damage' in head injury. 1977, Pubmed