Touch-evoked Responses Research Articles

Adverse effects of bifenthrin exposure on neurobehavior and neurodevelopment in a zebrafish embryo/larvae model

Bifenthrin, a third-generation synthetic pyrethroid, is widely used as an agricultural insecticide. However, it can flow into surface and groundwater, leading to adverse consequences such as immunotoxicity, hepatotoxicity, hormone dysregulation, or neurotoxicity. Nevertheless, the entire range of its neurotoxic consequences, particularly in aquatic organisms, remains unclear. In this study, we conducted an extensive examination of how exposure to bifenthrin affects the behavior and nervous system function of aquatic vertebrates, using a zebrafish model and multiple-layered assays. We exposed wild-type and transgenic lines [tg(elavl3:eGFP) and tg(mbp:mGFP)] to bifenthrin from <3 h post-fertilization (hpf) to 120 hpf. Our findings indicate that bifenthrin exposure concentrations of 103.9 and 362.1 μg/L significantly affects the tail-coiling response at 24 hpf and the touch-evoked responses at 72 hpf. Moreover, it has a significant effect on various aspects of behavior such as body contact, distance between subjects, distance moved, and turn angle. We attribute these effects to changes in acetylcholinesterase and dopamine levels, which decrease in a concentration-dependent manner. Furthermore, neuroimaging revealed neurogenesis defects, e.g., shortened brain and axon widths, and demyelination of oligodendrocytes and Schwann cells. Additionally, the transcription of genes related to neurodevelopment (e.g., gap43, manf, gfap, nestin, sox2) were significantly upregulated and neurotransmitters (e.g., nlgn1, drd1, slc6a4a, ache) was significantly downregulated. In summary, our data shows that bifenthrin exposure has detrimental effects on neurodevelopmental and neurotransmission systems in the zebrafish embryo/larvae model.

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish.

Although the increased risk of developing sporadic Alzheimer's disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C>R) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor.

L-bupivacaine Inhibition of Nociceptive Transmission in Rat Peripheral and Dorsal Horn Neurons.

Although the widely used single L-enantiomers of local anesthetics have less toxic effects on the cardiovascular and central nervous systems, the mechanisms mediating their antinociceptive actions are not well understood. The authors hypothesized that significant differences in the ion channel blocking abilities of the enantiomers of bupivacaine would be identified. The authors performed electrophysiologic analysis on rat dorsal root ganglion neurons in vitro and on spinal transmissions in vivo. In the dorsal root ganglion, these anesthetics decreased the amplitudes of action potentials. The half-maximum inhibitory concentrations of D-enantiomer D-bupivacaine were almost equal for Aβ (29.5 μM), Aδ (29.7μM), and C (29.8 μM) neurons. However, the half-maximum inhibitory concentrations of L-bupivacaine was lower for Aδ (19.35 μM) and C (19.5 μM) neurons than for A β (79.4 μM) neurons. Moreover, D-bupivacaine almost equally inhibited tetrodotoxin-resistant (mean ± SD: 15.8 ± 10.9% of the control, n = 14, P < 0.001) and tetrodotoxin-sensitive (15.4 ± 15.6% of the control, n = 11, P = 0.004) sodium currents. In contrast, L-bupivacaine suppressed tetrodotoxin-resistant sodium currents (26.1 ± 19.5% of the control, n = 18, P < 0.001) but not tetrodotoxin-sensitive sodium currents (74.5 ± 18.2% of the control, n = 11, P = 0.477). In the spinal dorsal horn, L-bupivacaine decreased the area of pinch-evoked excitatory postsynaptic currents (39.4 ± 11.3% of the control, n = 7, P < 0.001) but not touch-evoked responses (84.2 ± 14.5% of the control, n = 6, P = 0.826). In contrast, D-bupivacaine equally decreased pinch- and touch-evoked responses (38.8 ± 9.5% of the control, n = 6, P = 0.001, 42.9 ± 11.8% of the control, n = 6, P = 0.013, respectively). These results suggest that the L-enantiomer of bupivacaine (L-bupivacaine) effectively inhibits noxious transmission to the spinal dorsal horn by blocking action potential conduction through C and Aδ afferent fibers.

Dynamic causal modeling of touch-evoked potentials in the rubber hand illusion

The neural substrate of bodily ownership can be disclosed by the rubber hand illusion (RHI); namely, the illusory self-attribution of an artificial hand that is induced by synchronous tactile stimulation of the subject's hand that is hidden from view. Previous studies have pointed to the premotor cortex (PMC) as a pivotal area in such illusions. To investigate the effective connectivity between – and within – sensory and premotor areas involved in bodily perceptions, we used dynamic causal modeling of touch-evoked responses in 13 healthy subjects. Each subject's right hand was stroked while viewing their own hand (“REAL”), or an artificial hand presented in an anatomically plausible (“CONGRUENT”) or implausible (“INCONGRUENT”) position. Bayesian model comparison revealed strong evidence for a differential involvement of the PMC in the generation of touch-evoked responses under the three conditions, confirming a crucial role of PMC in bodily self-attribution. In brief, the extrinsic (forward) connection from left occipital cortex to left PMC was stronger for CONGRUENT and INCONGRUENT as compared to REAL, reflecting the augmentation of bottom-up visual input when multisensory integration is challenged. Crucially, intrinsic connectivity in the primary somatosensory cortex (S1) was attenuated in the CONGRUENT condition, during the illusory percept. These findings support predictive coding models of the functional architecture of multisensory integration (and attenuation) in bodily perceptual experience.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Touch Activates Mechanosensitive Ion Channels in Merkel Cells In Vitro

Merkel cell-neurite complexes are gentle touch receptors that mediate slowly adapting type I (SAI) responses. Since their description in 1875, Merkel cells have been proposed to be mechanosensory cells that transduce mechanical stimuli into electrical signals that activate somatosensory neurons. Consistent with this model, conditional knockout mice that lack Merkel cells show the loss of touch-evoked SAI responses. Moreover, in vitro studies on cultured Merkel cells report calcium elevation in Merkel cells in response to swelling or membrane stretch. Previous studies support the contribution of Merkel cells to touch sensation, however, the central question of whether Merkel cells are intrinsically touch sensitive is unanswered. To tackle this problem, we performed live-cell imaging and electrophysiological recordings from mouse Merkel cells. Merkel cells were dissociated from the epidermal skin of transgenic Atoh1/nGFP mice, whose Merkel cells selectively express green fluorescent protein. GFP-positive cells were purified using fluorescence-activated cell sorting and cultured for 1-5 days. Individual Merkel cells were stimulated by families of displacements (≦0.3-μm steps) with a glass probe driven by a piezoelectric actuator. Touch-evoked responses were monitored with either ratiometric calcium imaging or tight-seal, whole-cell recordings. Merkel cells displayed reversible calcium responses to focal displacements applied to somata. Moreover, electrophysiological recordings demonstrated mechanically activated inward currents at a negative holding potential. Peak inward currents ranged from 107.3-431.3 pA. The 10-90% operating range of these mechanically activated currents was 1.7±0.1 μm (N=10 cells, mean±SE). Like mechanosensitive currents in hair cells and somatosensory neurons, Merkel-cell currents adapted exponentially to sustained stimuli. Quantitative PCR indicated that Merkel cells expressed both Piezo1 and Piezo2 genes. Together, these data demonstrate that Merkel cells are intrinsically mechanosensitive in the absence of other skin cells or somatosensory neurons.

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch-evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA-type glutamate receptor activation. Transection experiments suggest that the circuits required for touch-evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA-type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation.

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca(2+) signaling and neurotransmission modulate touch-evoked responses in Merkel cell-neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca(2+) signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca(2+) channels (Ca(V)2.1) and voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels. Voltage-clamp recordings revealed small Ca(2+) currents, which produced Ca(2+) transients that were amplified sevenfold by Ca(2+)-induced Ca(2+) release. Merkel cells' voltage-activated K(+) currents were carried predominantly by BK(Ca) channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK(Ca) channels. Finally, blocking K(+) channels increased response magnitude and dramatically shortened Ca(2+) transients evoked by mechanical stimulation. Together, these results demonstrate that Ca(2+) signaling in Merkel cells is governed by the interplay of plasma membrane Ca(2+) channels, store release and K(+) channels, and they identify specific signaling mechanisms that may control touch sensitivity.

Does intravenous administration of GABA A receptor antagonists induce both descending antinociception and touch-evoked allodynia?

Effects of intravenous administration of picrotoxin (PTX), a GABA A receptor antagonist, upon activities of wide dynamic range (WDR) neurons in the lumbar spinal cord were studied in urethane–chloralose anesthetized cats. Intravenous PTX augmented tactile evoked responses of WDR neurons, but reduced nociceptive responses dose-dependently. Spinal transection reversed the suppression of nociceptive responses. In the spinal cat, intravenous PTX enhanced the tactile evoked response. Intravenous PTX enhanced the spontaneous firing of nucleus raphe dorsalis (NRD) and/or ventral periaqueductal gray (PAG) neurons projecting to nucleus raphe magnus. Lidocaine injected into NRD/PAG reversed the antinociceptive action of intravenous PTX. PTX injected into NRD/PAG reduced heat-evoked responses of WDR units. These data suggest that antinociceptive effects of intravenous PTX is primarily due to disinhibitory activation of the descending antinociceptive system originating from NRD and PAG, and that PTX reinforces touch-evoked responses in the spinal cord.

Noxious stimuli induce an N-methyl- d-aspartate receptor-dependent hypersensitivity of the flexion withdrawal reflex to touch: implications for the treatment of mechanical allodynia

Primary C-fiber afferents can induce a state of increased excitability in spinal cord neurons amplifying their responsiveness to noxious and innocuous stimuli — the phenomenon of central sensitization. We have examined whether the hypersensitivity to low-intensity stimuli (mechanical allodynia) evoked by C-afferent conditioning inputs is NMDA receptor dependent. The enhancement by C-afferent conditioning stimuli of the normally low or absent cutaneous touch-evoked responses of posterior biceps femoris/ semitendinosus flexor motoneurons in the decerebrate-spinal rat has been used as a model of touch-evoked allodynia. Three different noxious conditioning stimuli (electrical stimulation of the sural nerve at C-fiber strength, cutaneous application of mustard oil and the intramuscular (i.m.) injection of bradykinin) enhanced touch-evoked responses and decreased the threshold of the flexor reflex to mechanical stimulation. Intrathecal (i.t.) injections of NMDA (20 pmol) produced a similar effect. A non-competitive NMDA antagonist MK 801, administered prior to the conditioning input at doses that did not modify the baseline reflex, significantly attenuated both the enhancement of touch-evoked responses and the threshold decrease following the noxious conditioning stimuli. Treatment with MK 801 some time after the conditioning input when the mechanical hypersensitivity is fully established, also reduced the increased touch-evoked responses. The reduction in threshold and the increase in touch responsiveness induced by cutaneous and muscle noxious C-fiber conditioning stimuli in the rat spinal cord are, therefore, both prevented and reversed by NMDA receptor antagonism. NMDA antagonists may be potentially useful, therefore, in treating postinjury pain hypersensitivity.