Remyelination in humans due to a retinoid-X receptor agonist is age-dependent.

Remyelination efficiency declines with advancing age in animal models, but this has been harder to demonstrate in people with multiple sclerosis. We show that bexarotene, a putatively remyelinating retinoid-X receptor agonist, shortened the visual evoked potential latency in patients with chronic optic neuropathy aged under 42 years only (with the effect diminishing by 0.45 ms per year of age); and increased the magnetization transfer ratio of deep gray matter lesions in those under 43 years only. Addressing this age-related decline in human remyelination capacity will be an important step in the development of remyelinating therapies that work across the lifespan.

The role of nonmuscle myosin II in polydrug memories and memory reconsolidation.

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Influence of Cholecystokinin-8 on Compound Nerve Action Potentials from Ventral Gastric Vagus in Rats.

OBJECTIVE: Vagus Nerve Stimulation (VNS) has shown great promise as a potential therapy for a number of conditions, such as epilepsy, depression and for Neurometabolic Therapies, especially for treating obesity. The objective of this study was to characterize the left ventral subdiaphragmatic gastric trunk of vagus nerve (SubDiaGVN) and to analyze the influence of intravenous injection of gut hormone cholecystokinin octapeptide (CCK-8) on compound nerve action potential (CNAP) observed on the same branch, with the aim of understanding the impact of hormones on VNS and incorporating the methods and results into closed loop implant design. METHODS: The cervical region of the left vagus nerve (CerVN) of male Wistar rats was stimulated with electric current and the elicited CNAPs were recorded on the SubDiaGVN under four different conditions: Control (no injection), Saline, CCK1 (100[Formula: see text]pmol/kg) and CCK2 (1000[Formula: see text]pmol/kg) injections. RESULTS: We identified the presence of A[Formula: see text], B, C1, C2, C3 and C4 fibers with their respective velocity ranges. Intravenous administration of CCK in vivo results in selective, statistically significant reduction of CNAP components originating from A and B fibers, but with no discernible effect on the C fibers in [Formula: see text] animals. The affected CNAP components exhibit statistically significant ([Formula: see text] and [Formula: see text]) higher normalized stimulation thresholds. CONCLUSION: This approach of characterizing the vagus nerve can be used in closed loop systems to determine when to initiate VNS and also to tune the stimulation dose, which is patient-specific and changes over time.

Uridine-5'-Triphosphate Partially Blocks Differentiation Signals and Favors a more Repair State in Cultured rat Schwann Cells.

Schwann cells (SCs) play a key role in peripheral nerve regeneration. After damage, they respond acquiring a repair phenotype that allows them to proliferate, migrate and redirect axonal growth. Previous studies have shown that Uridine-5'-Triphosphate (UTP) and its purinergic receptors participate in several pathophysiological responses in the nervous system. Our group has previously described how UTP induces the migration of a Schwannoma cell line and promotes wound healing. These data suggest that UTP participates in the signaling involved in the regeneration process. In the present study we evaluated UTP effects in isolated rat SCs and cocultures of SCs and dorsal root ganglia neurons. UTP reduced cAMP-dependent Krox-20 induction in SCs. UTP also reduced the N-cadherin re-expression that occurs when SCs and axons make contact. In myelinating cocultures, a non-significant tendency to a lower expression of P0 and MAG proteins in presence of UTP was observed. We also demonstrated that UTP induced SC migration without affecting cell proliferation. Interestingly, UTP was found to block neuregulin-induced phosphorylation of the ErbB3 receptor, a pathway involved in the regeneration process. These results indicate that UTP could acts as a brake to the differentiation signals, promoting a more migratory state in the repair-SCs.

Urothelial bladder afferent neurons in the rat are anatomically and neurochemically distinct from non-urothelial afferents.

There is mounting evidence underscoring a role for the urothelium in urinary bladder sensation. Previous functional studies have identified bladder primary afferents with mechanosensitive properties suggesting urothelial innervation and/or communication. The current study identifies a group of urothelium-innervating afferent neurons in rat, and characterizes and compares the properties of these and non-urothelial afferent neuron populations. Lumbosacral (LS) primary afferent neurons were retrogradely labeled using intraparenchymal (IPar) microinjection or intravesical (IVes) infusion of tracer into the bladder. Using these techniques, separate populations of neurons were differentiated by dorsal root ganglion (DRG) somata labeling and dye distribution within the bladder. IPar- and IVes-labeled neurons accounted for 85.0% and 14.4% of labeled L6-S1 neurons (P < .001), respectively, with only 0.6% of neurons labeled by both techniques. Following IVes labeling, dye was contained only within the periurothelial bladder region in contrast to non-urothelial distribution of dye after IPar labeling. Electrophysiological characterization by in situ patch-clamp recordings from whole-mount DRG preparations indicated no significant difference in passive or active membrane properties of IPar and IVes DRG neurons. However, calcium imaging of isolated neurons indicates that a greater proportion of IPar- than IVes-labeled neurons express functional TRPA1 (45.7% versus 25.6%, respectively; P < .05). This study demonstrates that two anatomically distinct groups of LS bladder afferents can be identified in rat. Further studies of urothelial afferents and the phenotypic differences between non-/urothelial afferents may have important implications for normal and pathophysiological bladder sensory processing.

Membrane Mechanics of Primary Afferent Neurons in the Dorsal Root Ganglia of Rats.

Membrane mechanics is an important biological factor regulating many cellular functions including cell motility, intercellular and intracellular signaling, gene expression, and membrane ion channel activity. Primary afferent neurons transduce sensory information about temperature, touch, and pain. These sensory functions may be profoundly affected by the states of primary afferent neuron mechanics. However, membrane mechanics of primary afferent neurons is largely unknown. In this study, we established the optical trapping technique for determining membrane mechanics of cultured primary afferent neurons of the dorsal root ganglia (DRG). We further determined the roles of cytoskeleton and membrane lipids in DRG neuron mechanics. We found that DRG neurons had a plasma membrane tension of ∼54 pN/µm, and the tension was significantly decreased to ∼29 pN/µm by cytochalasin D treatment to disrupt actin cytoskeleton and increased to ∼79 pN/µm by methyl-ß-cyclodextrin treatment to sequester membrane cholesterol. DRG neuron membrane stiffness was not significantly affected by the cytoskeleton disruption but was significantly increased after cholesterol sequestration. Our findings elucidate membrane mechanical properties of primary afferent neurons, which provide, to our knowledge, a new perspective on their sensory functions.

Retinal gap junctions are involved in rhythmogenesis of neuronal activity at remote locations - Study on infra-slow oscillations in the rat olivary pretectal nucleus.

A subpopulation of olivary pretectal nucleus (OPN) neurons fire action potentials in a rhythmic manner with an eruption of activity occurring approximately every two minutes. These infra-slow oscillations depend critically on functional retinal input and are subject to modulation by light. Interestingly, the activity of photoreceptors is necessary for the emergence of the rhythm and while classic photoreceptors (rods and cones) are necessary in darkness and dim light, melanopsin photoreceptors are indispensable in bright light. Using pharmacological and electrophysiological approaches in vivo, we show that also blocking retinal gap junctions (GJs), which are expressed by multitude of retinal cells, leads to the disruption of oscillatory activity in the rat OPN. Intravitreal injection of carbenoxolone (CBX) quenched oscillations in a concentration-dependent manner with 1mM being ineffective, 5mM showing partial and 20mM showing complete effectiveness in disrupting oscillations. Moreover, the most effective CBX concentration depressed cone-mediated light-induced responses of oscillatory neurons suggesting that CBX is also acting on targets other than GJs. In contrast, intravitreal injection of meclofenamic acid (MFA, 20mM) led to disruption of the rhythm but did not interfere with cone-mediated light-induced responses of oscillatory neurons, implying that MFA is more specific toward GJs than CBX, as suggested before. We conclude that electrical coupling between various types of retinal cells and resultant synchronous firing of retinal ganglion cells is necessary for the generation of infra-slow oscillations in the rat OPN.

Ampakine CX717 potentiates intermittent hypoxia-induced hypoglossal long-term facilitation.

Glutamatergic currents play a fundamental role in regulating respiratory motor output and are partially mediated by α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors throughout the premotor and motor respiratory circuitry. Ampakines are pharmacological compounds that enhance glutamatergic transmission by altering AMPA receptor channel kinetics. Here, we examined if ampakines alter the expression of respiratory long-term facilitation (LTF), a form of neuroplasticity manifested as a persistent increase in inspiratory activity following brief periods of reduced O2 [intermittent hypoxia (IH)]. Current synaptic models indicate enhanced effectiveness of glutamatergic synapses after IH, and we hypothesized that ampakine pretreatment would potentiate IH-induced LTF of respiratory activity. Inspiratory bursting was recorded from the hypoglossal nerve of anesthetized and mechanically ventilated mice. During baseline (BL) recording conditions, burst amplitude was stable for at least 90 min (98 ± 5% BL). Exposure to IH (3 × 1 min, 15% O2) resulted in a sustained increase in burst amplitude (218 ± 44% BL at 90 min following final bout of hypoxia). Mice given an intraperitoneal injection of ampakine CX717 (15 mg/kg) 10 min before IH showed enhanced LTF (500 ± 110% BL at 90 min). Post hoc analyses indicated that CX717 potentiated LTF only when initial baseline burst amplitude was low. We conclude that under appropriate conditions ampakine pretreatment can potentiate IH-induced respiratory LTF. These data suggest that ampakines may have therapeutic value in the context of hypoxia-based neurorehabilitation strategies, particularly in disorders with blunted respiratory motor output such as spinal cord injury.

Stimulating the proliferation, migration and lamellipodia of Schwann cells using low-dose curcumin.

Transplantation of peripheral glia is being trialled for neural repair therapies, and identification of compounds that enhance the activity of glia is therefore of therapeutic interest. We have previously shown that curcumin potently stimulates the activity of olfactory glia. We have now examined the effect of curcumin on Schwann cell (SC) activities including proliferation, migration and the expression of protein markers. SCs were treated with control media and with different concentrations of curcumin (0.02-20 µM). Cell proliferation was determined by MTS assay and migration changes were determined by single live cell migration tracking. We found that small doses of curcumin (40 nM) dramatically increased the proliferation and migration in SCs within just one day. When compared with olfactory glia, curcumin stimulated SC proliferation more rapidly and at lower concentrations. Curcumin significantly increased the migration of SCs, and also increased the dynamic activity of lamellipodial waves which are essential for SC migration. Expression of the activated form of the MAP kinase p38 (p-p38) was significantly decreased in curcumin-treated SCs. These results show that curcumin's effects on SCs differ remarkably to its effects on olfactory glia, suggesting that subtypes of closely related glia can be differentially stimulated by curcumin. Overall these results demonstrate that the therapeutically beneficial activities of glia can be differentially enhanced by curcumin which could be used to improve outcomes of neural repair therapies.

Blockade of dorsolateral pontine 5HT1A receptors destabilizes the respiratory rhythm in C57BL6/J wild-type mice.

The neurotransmitter serotonin (5HT) acting via 5HT1a receptors (5HT1aR) is a potent determinant of respiratory rhythm variability. Here, we address the 5HT1aR-dependent control of respiratory rhythm variability in C57BL6/J mice. Using the in situ perfused preparation, we compared the effects of systemic versus focal blockade of 5HT1aRs. Blocking 5HT1aRs in the Kölliker-Fuse nucleus (KFn) increased the occurrence of spontaneous apneas and accounted for the systemic effects of 5HT1aR antagonists. Further, 5HT1aRs of the KFn stabilized the respiratory rhythm's response to arterial chemoreflex perturbations; reducing the recovering time, e.g., the latency to return to the baseline pattern. Together, these results suggest that the KFn regulates both intrinsic and sensory determinants of respiratory rhythm variability.

Rapamycin-Resistant mTOR Activity Is Required for Sensory Axon Regeneration Induced by a Conditioning Lesion.

Neuronal mammalian target of rapamycin (mTOR) activity is a critical determinant of the intrinsic regenerative ability of mature neurons in the adult central nervous system (CNS). However, whether its action also applies to peripheral nervous system (PNS) neurons after injury remains elusive. To address this issue unambiguously, we used genetic approaches to determine the role of mTOR signaling in sensory axon regeneration in mice. We showed that deleting mTOR in dorsal root ganglion (DRG) neurons suppressed the axon regeneration induced by conditioning lesions. To establish whether the impact of mTOR on axon regeneration results from functions of mTOR complex 1 (mTORC1) or 2 (mTORC2), two distinct kinase complexes, we ablated either Raptor or Rictor in DRG neurons. We found that suppressing mTORC1 signaling dramatically decreased the conditioning lesion effect. In addition, an injury to the peripheral branch boosts mTOR activity in DRG neurons that cannot be completely inhibited by rapamycin, a widely used mTOR-specific inhibitor. Unexpectedly, examining several conditioning lesion-induced pro-regenerative pathways revealed that Raptor deletion but not rapamycin suppressed Stat3 activity in neurons. Therefore, our results demonstrate that crosstalk between mTOR and Stat3 signaling mediates the conditioning lesion effect and provide genetic evidence that rapamycin-resistant mTOR activity contributes to the intrinsic axon growth capacity in adult sensory neurons after injury.

The role of the Kölliker-Fuse nuclei in the determination of abdominal motor output in a perfused brainstem preparation of juvenile rat.

The abdominal muscles are largely quiescent during normal breathing but may exhibit tonic activity or subtle respiratory modulation. The origin of baseline abdominal motor nerve activity (AbNA) if present remains uncharacterised. The contribution of the Kölliker-Fuse nucleus (KF) in the dorsolateral pons in the patterning and amplitude of AbNA was investigated using in situ perfused brainstem preparations of juvenile rats (n=12). Two types of AbNA were observed: Type I - expiratory-modulated (n=7), and Type II - weakly inspiratory/post-inspiratory-modulated (n=5). Despite this, all preparations exhibited the same bi-phasic late expiratory/postinspiratory bursts upon elicitation of the peripheral chemoreflex. Interestingly, the type of AbNA exhibited correlated with postinspiratory duration. Targeted microinjections of GABA-A receptor agonist isoguvacine (10mM; 70nl) into KF however did not significantly modify pattern or amplitude of baseline AbNA in either Type besides the selective abolition of the postinspiratory phase and, consequently, postinspiratory modulation in AbNAwhen present. In sum, the KF is not a major contributorin setting baseline abdominal motor output.

Effects of proinflammatory cytokines on axonal outgrowth from adult rat lumbar dorsal root ganglia using a novel three-dimensional culture system.

BACKGROUND CONTEXT: Degeneration of the intervertebral disc is often associated with low back pain and increased infiltration of nerve fibers originating from dorsal root ganglia (DRG). The degenerated disc is also characterized by the presence of proinflammatory cytokines, which may influence axonal outgrowth. Toward an improved understanding of the growth of DRG neurons into compliant extracellular matrices, we developed a novel experimental system to measure axonal outgrowth of adult rat lumbar DRG neurons within three-dimensional (3D) collagen hydrogels and used this system to examine the effects of interleukin 1ß (IL-1ß) and tumor necrosis factor (TNF)-α treatment. PURPOSE: The aim was to investigate the effects of proinflammatory cytokines on 3D neuronal growth into collagen matrices. STUDY DESIGN: This was an in vitro study of neurite outgrowth from adult rat lumbar DRG into collagen gels in response to IL-1ß and TNF-α. METHODS: Lumbar DRG were obtained from adult Sprague Dawley rats, bisected to expose cell bodies and placed onto collagen gel constructs prepared in 24-well Transwell inserts. Dorsal root ganglia were then treated with nerve growth factor (NGF)-free Neurobasal media (negative control) or NGF-supplemented media containing 0, 1, and 10 ng/mL of IL-1ß and TNF-α. After 7 days, collagen gel-DRG constructs were immunostained for phosphorylated neurofilament, an axonal marker. Simple Neurite Tracer (Fiji/ImageJ) was used to quantify 3D axonal outgrowth from confocal image stacks. Data were analyzed using one-way analysis of variance, with Tukey HSD post hoc correction at a level of p<.05. RESULTS: Immunostaining showed robust axonal outgrowth into collagen gels from all NGF-treated DRG. The negative control demonstrated very few and short neurites. Tumor necrosis factor-α (1 and 10 ng/mL) significantly inhibited axonal outgrowth compared with NGF-only media (p<.026 and p<.02, respectively). After IL-1ß treatment, average axon length was 10% lower at 1 ng/mL and 7.5% higher at 10 ng/mL, but these differences were not statistically significant. Among cytokine treatments, however, average axon length in the IL-1ß (10 ng/mL) group was significantly higher than that in the other groups (p<.05). CONCLUSIONS: A novel 3D collagen gel culture system was used to investigate factors modulating neuronal ingrowth. Our results showed that NGF was necessary to promote neurite growth into collagen gels. In the presence of proinflammatory cytokines, high concentrations of IL-1ß induced significantly higher axonal outgrowth than TNF-α and low levels of IL-1ß.

Trigeminal pathways for hypertonic saline- and light-evoked corneal reflexes.

Cornea-evoked eyeblinks maintain tear film integrity on the ocular surface in response to dryness and protect the eye from real or potential damage. Eyelid movement following electrical stimulation has been well studied in humans and animals; however, the central neural pathways that mediate protective eyeblinks following natural nociceptive signals are less certain. The aim of this study was to assess the role of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical cord (Vc/C1) junction regions on orbicularis oculi electromyographic (OOemg) activity evoked by ocular surface application of hypertonic saline or exposure to bright light in urethane anesthetized male rats. The Vi/Vc and Vc/C1 regions are the main sites of termination for trigeminal afferent nerves that supply the ocular surface, while hypertonic saline (saline=0.15-5M) and bright light (light=5k-20klux) selectively activate ocular surface and intraocular trigeminal nerves, respectively, and excite second-order neurons at the Vi/Vc and Vc/C1 regions. Integrated OOemg activity, ipsilateral to the applied stimulus, increased with greater stimulus intensities for both modalities. Lidocaine applied to the ocular surface inhibited OOemg responses to hypertonic saline, but did not alter the response to light. Lidocaine injected into the trigeminal ganglion blocked completely the OOemg responses to hypertonic saline and light indicating a trigeminal afferent origin. Synaptic blockade by cobalt chloride of the Vi/Vc or Vc/C1 region greatly reduced OOemg responses to hypertonic saline and bright light. These data indicate that OOemg activity evoked by natural stimuli known to cause irritation or discomfort in humans depends on a relay in both the Vi/Vc transition and Vc/C1 junction regions.

Responses of intact and injured sural nerve fibers to cooling and menthol.

Intact and injured cutaneous C-fibers in the rat sural nerve are cold sensitive, heat sensitive, and/or mechanosensitive. Cold-sensitive fibers are either low-threshold type 1 cold sensitive or high-threshold type 2 cold sensitive. The hypothesis was tested, in intact and injured afferent nerve fibers, that low-threshold cold-sensitive afferent nerve fibers are activated by the transient receptor potential melastatin 8 (TRPM8) agonist menthol, whereas high-threshold cold-sensitive C-fibers and cold-insensitive afferent nerve fibers are menthol insensitive. In anesthetized rats, activity was recorded from afferent nerve fibers in strands isolated from the sural nerve, which was either intact or crushed 6-12 days before the experiment distal to the recording site. In all, 77 functionally identified afferent C-fibers (30 intact fibers, 47 injured fibers) and 34 functionally characterized A-fibers (11 intact fibers, 23 injured fibers) were tested for their responses to menthol applied to their receptive fields either in the skin (10 or 20%) or in the nerve (4 or 8 mM). Menthol activated all intact (n = 12) and 90% of injured (n = 20/22) type 1 cold-sensitive C-fibers; it activated no intact type 2 cold-sensitive C-fibers (n = 7) and 1/11 injured type 2 cold-sensitive C-fibers. Neither intact nor injured heat- and/or mechanosensitive cold-insensitive C-fibers (n = 25) and almost no A-fibers (n = 2/34) were activated by menthol. These results strongly argue that cutaneous type 1 cold-sensitive afferent fibers are nonnociceptive cold fibers that use the TRPM8 transduction channel.

M2 muscarinic receptor activation regulates Schwann cell differentiation and myelin organization.

Glial cells express acetylcholine receptors. In particular, rat Schwann cells express different muscarinic receptor subtypes, the most abundant of which is the M2 subtype. M2 receptor activation causes a reversible arrest of the cell cycle. This negative effect on Schwann cell proliferation suggests that these cells may possibly progress into a differentiating program. In this study we analyzed the in vitro modulation, by the M2 agonist arecaidine, of transcription factors and specific signaling pathways involved in Schwann cell differentiation. The arecaidine-induced M2 receptor activation significantly upregulates transcription factors involved in the promyelinating phase (e.g., Sox10 and Krox20) and downregulates proteins involved in the maintenance of the undifferentiated state (e.g., c-jun, Notch-1, and Jagged-1). Furthermore, arecaidine stimulation significantly increases the expression of myelin proteins, which is accompanied by evident changes in cell morphology, as indicated by electron microscopy analysis, and by substantial cellular re-distribution of actin and cell adhesion molecules. Moreover, ultrastructural and morphometric analyses on sciatic nerves of M2/M4 knockout mice show numerous degenerating axons and clear alterations in myelin organization compared with wild-type mice. Therefore, our data demonstrate that acetylcholine mediates axon-glia cross talk, favoring Schwann cell progression into a differentiated myelinating phenotype and contributing to compact myelin organization.