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We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: To simulate 24-2 visual field (VF) using optical coherence tomographic angiography (OCTA) for glaucoma evaluation., Design: Cross-sectional study., Methods: One eye each of 39 glaucoma and 31 age-matched normal participants was scanned using 4.5-mm OCTA scans centered on the disc. The peripapillary retinal nerve fiber layer plexus capillary density (NFLP-CD, %area) was measured. The NFLP-CD and 24-2 VF maps were divided into 8 corresponding sectors using an extension of Garway-Heath scheme., Results: Sector NFLP-CD was transformed to a logarithmic dB scale and converted to sector simulated VF deviation maps. Comparing simulated and actual 24-2 VF maps, the worst sector was in the same or adjacent location in the same hemisphere 97% of the time. VF mean deviation (VF-MD) was simulated by NFLP mean deviation (NFLP-MD). The differences between NFLP-MD and VF-MD in early, moderate, and severe glaucoma stages were -0.9 ± 2.0, 0.9 ± 2.9, and 5.8 ± 3.2 dB. NFLP-MD had better (P = .015) between-visit reproducibility (0.63 dB pooled standard deviation) than VF-MD (1.03 dB). NFLP-MD had a significantly higher sensitivity than VF-MD (P < .001) and overall NFL thickness (P = .031)., Conclusions: OCTA-based simulated VF agreed well with actual 24-2 VF in terms of both the location and severity of glaucoma damage, with the exception of severe glaucoma in which the simulation tended to underestimate severity. The NFLP-MD had better reproducibility than actual VF-MD and holds promise for improving glaucoma monitoring. The NFLP-MD had better diagnostic accuracy than both VF-MD and overall NFL thickness and may be useful for early glaucoma diagnosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The perception of fine textures relies on highly precise and repeatable spiking patterns evoked in tactile afferents. These patterns have been shown to depend not only on the surface microstructure and material but also on the speed at which it moves across the skin. Interestingly, the perception of texture is independent of scanning speed, implying the existence of downstream neural mechanisms that correct for scanning speed in interpreting texture signals from the periphery. What force is applied during texture exploration also has negligible effects on how the surface is perceived, but the consequences of changes in contact force on the neural responses to texture have not been described. In the present study, we measure the signals evoked in tactile afferents of macaques to a diverse set of textures scanned across the skin at two different contact forces and find that responses are largely independent of contact force over the range tested. We conclude that the force invariance of texture perception reflects the force independence of texture representations in the nerve., (Copyright © 2017. Published by Elsevier Ltd.)

Objective: To determine the pathophysiological progresses following bilateral cavernous nerve crushing injury, as an index for a treatment point and establishment of adequate treatment strategies for neurogenic erectile dysfunction., Materials and Methods: Thirty-six rats were assigned to 1 of 6 groups, and bilateral cavernous nerve crushing or sham surgery was performed. Functional testing and ultrastructural analyses were performed immediately and at 7, 14, 21, and 28 days after the cavernous nerve (CN) injury (n = 6)., Results: Intracavernos pressure lowered progressively from 7 days to 14 days post-injury, and histologic staining revealed that the number of neuronal nitric oxide synthase-positive nerve fibers on the dorsal penile nerve decreased significantly and progressively from 7 days until 21 days post-injury. Furthermore, ultrastructural analyses revealed axon loss and demyelination of the CN at 7 and 14 days post-injury. However, it is followed by partial spontaneous recovery of erectile function and regeneration of the CN at 28 days post-injury, suggesting that these time points may be useful for evaluating the effects of drug treatments. Furthermore, we found that CN injury-induced damage to corporal smooth muscle cells was irreversible; therefore, focusing on protecting the corpus cavernosum from apoptosis may be more important than nerve protection when assessing treatment mechanisms in the CN injury model., Conclusion: Our study makes a significant contribution to the human diagnostic pathology literature because it describes characteristics of relevant tissue in the rat and provides information regarding time points that may be useful for future studies of pathologic mechanisms or treatment evaluations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 μg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Retinal nerve fiber layer defect (RNFLD) provides an early objective evidence of structural changes in glaucoma. RNFLD detection is currently carried out using imaging modalities like OCT and GDx which are expensive for routine practice. In this regard, we propose a novel automatic method for RNFLD detection and angular width quantification using cost effective redfree fundus images to be practically useful for computer-assisted glaucoma risk assessment. After blood vessel inpainting and CLAHE based contrast enhancement, the initial boundary pixels are identified by local minima analysis of the 1-D intensity profiles on concentric circles. The true boundary pixels are classified using random forest trained by newly proposed cumulative zero count local binary pattern (CZC-LBP) and directional differential energy (DDE) along with Shannon, Tsallis entropy and intensity features. Finally, the RNFLD angular width is obtained by random sample consensus (RANSAC) line fitting on the detected set of boundary pixels. The proposed method is found to achieve high RNFLD detection performance on a newly created dataset with sensitivity (SN) of 0.7821 at 0.2727 false positives per image (FPI) and the area under curve (AUC) value is obtained as 0.8733., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Afferent chorda tympani (CT) fibers innervating anterior tongue fungiform papillae have neuron cell bodies in the geniculate ganglion (GG). To characterize electrophysiological and receptive field properties, we recorded extracellular responses from single GG neurons to lingual application with chemical, thermal and mechanical stimuli. Receptive field size was mapped by electrical stimulation of individual fungiform papillae. Responses of GG neurons to room temperature chemical stimuli representing five taste qualities, and distilled water at 4 °C and mechanical stimulation were used. Based on responses to these stimuli, GG neurons were divided into CHEMICAL, CHEMICAL/THERMAL, THERMAL and TACTILE groups. Neurons in the CHEMICAL group responded to taste stimuli but not to either cold water or stroking stimuli. CHEMICAL/THERMAL neurons responded to both taste stimuli and cold water. THERMAL neurons responded only to cold water and TACTILE neurons responded only to light stroking stimuli. The receptive field sizes for CHEMICAL, and CHEMICAL/THERMAL neurons averaged five papillae exceeding the field size of THERMAL and TACTILE neurons which averaged about two papillae. Detailed analysis of the receptive field of CHEMICAL/THERMAL neurons revealed that within one field only a subset of the fungiform papillae making up the receptive field responded to the cold stimuli, whereas the other papillae responded only to chemical stimuli. These finding demonstrate that fungiform papilla are complex sensory organs with a multisensory function suggesting a unique role in detecting and sampling food components prior to ingestion., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Striatal-enriched phosphatase 61 (STEP61) is a member of intracellular protein tyrosine phosphatases, which is involved in the regulation of synaptic plasticity and a line of neuropsychiatric disorders. This protein tyrosine phosphatase is also abundant in pain-related spinal cord dorsal horn neurons. However, whether and how this tyrosine phosphatase modulates the nociceptive plasticity and behavioral hypersensitivity remain largely unknown. The present study recorded the long-term potentiation (LTP) of primary afferent C fiber-evoked field potentials in vivo in superficial dorsal horn of rats, and tested the possible role of STEP61 in spinal LTP. We found that LTP induction significantly increased STEP61 phosphorylation at Ser221 residue, a key molecular event that has been shown to impair the phosphatase activity. The STEP61 hypoactivity allowed for the activation of three substrates, GluN2B subunit-containing N-methyl-d-aspartate-subtype glutamate receptors, Src-family protein tyrosine kinase member Fyn and extracellular signal-regulated kinase 1/2, through which the thresholds for LTP induction were noticeably decreased. To reinstate STEP61 activity, we overexpressed wild-type STEP61 [STEP61(WT)] in spinal dorsal horn, finding that STEP61(WT) completely blunted LTP induction. Behavioral tests showed that LTP blockade by STEP61(WT) correlated with a long-lasting alleviation of thermal hypersensitivity and mechanical allodynia induced by chronic constriction injury of sciatic nerves. These data implicated that STEP61 exerted a negative control over spinal nociceptive plasticity, which might have therapeutic benefit in pathological pain., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Idiopathic rapid eye movement (REM) sleep behavior disorder (iRBD) has been increasingly acknowledged to be an initial specific manifestation of alpha-synucleinopathies such as Parkinson's disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Recent findings suggest that cutaneous abnormalities like small fiber neuropathy and alpha-synuclein deposition might reflect brain pathology and might function as early biomarkers in PD. This is the first study to elucidate whether iRBD patients already suffer from distinctive cutaneous features., Methods: We examined skin punch biopsies from the distal leg of 18 iRBD patients and 22 age- and sex-matched controls using immunohistochemistry and microscopy. Further clinical evaluation included structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. Unified Parkinson's disease rating scale [UPDRS], non-motor symptoms questionnaire [NMS-Quest] and Beck Depression Inventory, Epworth Sleepiness Scale, evaluation of cognitive and olfactory functioning as well as blood samples., Results: Intraepidermal nerve fiber density (IEFND) was reduced in iRBD patients compared to controls (p = 0.037), whereas the axon swelling ratio did not differ between groups. Patients with iRBD reported non-motor symptoms more frequently than controls (UPDRS I, NMS-Quest). Olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition., Conclusions: These in vivo findings demonstrate small fiber neuropathy in iRBD patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in iRBD. However, the prognostic value has to be further investigated in longitudinal studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Our recent studies have shown that the difference in basal pain sensitivity to mechanical and thermal stimulation between Dark-Agouti (DA) rats and a novel congenic DA.1U rats is major histocompatibility complex (MHC) genes dependent. In the present study, we further used DA and DA.1U rats to investigate the role of MHC genes in formalin-induced pain model by behavioral, electrophysiological and immunohistochemical methods. Behavioral results showed biphasic nociceptive behaviors increased significantly following the intraplantar injection of formalin in the hindpaw of DA and DA.1U rats. The main nociceptive behaviors were lifting and licking, especially in DA rats (P<0.001 and P<0.01). The composite pain scores (CPS) in DA rats were significantly higher than those in DA.1U rats in both phases of the formalin test (P<0.01). Electrophysiological results also showed the biphasic increase in discharge rates of C and Aδ fibers of L5 dorsal root in the two strains, and the net change of the discharge rate of DA rats was significantly higher than that of DA.1U rats (P<0.05). The mechanical thresholds decreased after formalin injection in both strains (P<0.01), and the net change in the mechanical threshold in DA was greater than that in DA.1U rats (P<0.05). The expression of RT1-B, representation of MHC class II molecule, in laminae I-II of L4/5 spinal cord in DA rats was significantly higher than that in DA.1U rats in the respective experimental group (P<0.05). These results suggested that both DA and DA.1U rats exhibited nociceptive responses in formalin-induced pain model and DA rats were more sensitive to noxious chemical stimulus than DA.1U rats, indicating that MHC genes might contribute to the difference in pain sensitivity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study investigated the vestibulospinal system which originates from the spinal vestibular nucleus (SpVe) with both retrograde and anterograde tracer injections. We found that fluoro-gold (FG) labeled neurons were found bilaterally with a contralateral predominance after FG injections into the upper lumbar cord. Anterogradely labeled fibers from the rostral SpVe traveled in the medial part of the ventral funiculus ipsilaterally and the dorsolateral funiculus bilaterally in the cervical cord. They mainly terminated in laminae 5-8, and 10 of the ipsilateral spinal cord. The contralateral side had fewer fibers and they were found in laminae 6-8, and 10. In the thoracic cord, fibers were also found to terminate in bilateral intermediolateral columns. In the lumbar and lower cord, fibers were mainly found in the dorsolateral funiculus bilaterally and they terminated predominantly in laminae 3-7 contralaterally. Anterogradely labeled fibers from the caudal SpVe did not travel in the medial part of the ventral funiculus but in the dorsolateral funiculus bilaterally. They mainly terminated in laminae 3-8 and 10 contralaterally. The present study is the first to describe the termination of vestibulospinal fibers arising from the SpVe in the spinal cord. It will lay the anatomical foundation for those who investigate the physiological role of vestibulospinal fibers and potentially target these fibers during rehabilitation after stroke, spinal cord injury, or vestibular organ injury., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Nicotine has been shown to have neuroprotective and neurotrophic actions in the central nervous system. To elucidate the peripheral neurotrophic effects of nicotine, we determined whether nicotine affected the reinnervation of mesenteric perivascular nerves following a topical phenol treatment. A topical phenol treatment was applied to the superior mesenteric artery proximal to the abdominal aorta in Wistar rats. We examined the immunohistochemistry of the distal small arteries 7 days after the treatment. The topical phenol treatment markedly reduced the density of tyrosine hydroxylase (TH)-LI and calcitonin gene-related peptide (CGRP)-LI fibers in these arteries. The administration of nicotine at a dose of 3 mg/kg/day (1.5 mg/kg/injection, twice a day), but not once a day or its continuous infusion using a mini-pump significantly increased the density of TH-LI nerves without affecting CGRP-LI nerves. A pretreatment with nicotinic acetylcholine receptor antagonists hexamethonium, mecamylamine, and methyllycaconitine, but not dextrometorphan, canceled the TH-LI nerve reinnervation induced by nicotine. Nicotine significantly increased NGF levels in the superior cervical ganglia (SCG) and mesenteric arteries, but not in the dorsal root ganglia, and also up-regulated the expression of NGF receptors (TrkA) in the SCG, which were canceled by hexamethonium. These results suggested that nicotine exhibited neurotrophic effects that facilitated the reinnervation of adrenergic TH-LI nerves by activating α7 nicotinic acetylcholine receptor and NGF in the SCG., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The spatial pattern of synapse activation may impact on synaptic plasticity. This applies to the synaptically-evoked endocannabinoid-mediated short-term depression at the parallel fiber (PF) to Purkinje cell synapse, the occurrence of which requires close proximity between the activated synapses. Here, we determine quantitatively this required proximity, helped by the geometrical organization of the cerebellar molecular layer. Transgenic mice expressing a calcium indicator selectively in granule cells enabled the imaging of action potential-evoked presynaptic calcium rise in isolated, single PFs. This measurement was used to derive the number of PFs activated within a beam of PFs stimulated in the molecular layer, from which the density of activated PFs (input density) was calculated. This density was on average 2.8 μm(-2) in sagittal slices and twice more in transverse slices. The synaptically-evoked endocannabinoid-mediated suppression of excitation (SSE) evoked by ten stimuli at 200 Hz was determined from the monitoring of either postsynaptic responses or presynaptic calcium rise. The SSE was significantly larger when recorded in transverse slices, where the input density is larger. The exponential description of the SSE plotted as a function of the input density suggests that the SSE is half reduced when the input density decreases from 6 to 2 μm(-2). We conclude that, although all PFs are truncated in an acute sagittal slice, half of them remain respondent to stimulation, and activated synapses need to be closer than 1.5 μm to synergize in endocannabinoid signaling., (Copyright © 2013 The authors. Published by Elsevier Ltd.. All rights reserved.)

Previous behavioral studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy (EM). Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed neurofilament 200 (NF200), a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF200. Further, EM revealed immuno-gold labeling of CaV3.2 preferentially in association with unmyelinated sensory fibers from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localization with Mrgpd-GFP-positive fibers. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recently it has been suggested that the neurohormone prolactin (PRL) could act on the afferent nociceptive neurons. Indeed, PRL sensitizes transient receptor potential vanilloid 1 (TRPV1) channels present in nociceptive C-fibers and consequently reduces the pain threshold in a model of inflammatory pain. Accordingly, high plasma PRL levels in non-lactating females have been associated with several painful conditions (e.g. migraine). Paradoxically, an increase of PRL secretion during lactation induced a reduction in pain sensitivity. This difference could be attributed to the fact that PRL secreted from the adenopituitary (AP) is transformed into several molecular variants by the suckling stimulation. In order to test this hypothesis, the present study set out to investigate whether PRL from AP of suckled (S) or non-suckled (NS) lactating rats affects the activity of the male Wistar wide dynamic range (WDR) neurons. The WDR neurons are located in the dorsal horn of the spinal cord and receive input from the first-order neurons (Ab-, Ad- and C-fibers). Spinal administration of prolactin variant from NS rats (NS-PRL) or prolactin variant from S rats (S-PRL) had no effect on the neuronal activity of non-nociceptive Ab-fibers. However, the activities of nociceptive Ad-fibers and C-fibers were: (i) increased by NS-PRL and (ii) diminished by S-PRL. Either NS-PRL or S-PRL enhanced the post-discharge activity. Taken together, these results suggest that PRL from S or NS lactating rats could either facilitate or depress the nociceptive responses of spinal dorsal horn cells, depending on the physiological state of the rats., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We demonstrated previously that the lysophosphatidic acid-1 (LPA1) receptor plays a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. The present study revealed that mild cerebral ischemia by left transient middle cerebral artery occlusion (tMCAO) for 15min causes the hypersensitive responses (paw withdrawal) to the nociception by electrical stimuli to the paw by the use of Neurometer Current Perception Threshold/C (CPT/C). The hypersensitivity or neuropathic pain was only observed by the stimulation with 250 and 2000, but not 5Hz, which are the characterized sine-wave frequencies of Aδ-, Aβ- or C-fibers, respectively. The significant neuropathic pain was observed from day 2 through week 2 on the right paw after tMCAO, while there was slight but significant pain sensitivity on the left paw at day 7. The neuropathic pain on the contralateral side at week 2 after tMCAO was completely abolished in LPA1(-/-) mice. These results suggest that LPA1 receptor signaling plays key roles in the development of central neuropathic pain following cerebral ischemia as well as the peripheral neuropathic pain following partial sciatic nerve injury., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

In adult macaque monkeys subjected to an incomplete spinal cord injury (SCI), corticospinal (CS) fibers are rarely observed to grow in the lesion territory. This situation is little affected by the application of an anti-Nogo-A antibody which otherwise fosters the growth of CS fibers rostrally and caudally to the lesion. However, when using the Sternberger monoclonal-incorporated antibody 32 (SMI-32), a marker detecting a non-phosphorylated neurofilament epitope, numerous SMI-32-positive (+) fibers were observed in the spinal lesion territory of 18 adult macaque monkeys; eight of these animals had received a control antibody infusion intrathecally for 1 month after the injury, five animals an anti-Nogo-A antibody, and five animals received an anti-Nogo-A antibody together with brain-derived neurotrophic factor (BDNF). These fibers occupied the whole dorso-ventral axis of the lesion site with a tendency to accumulate on the ventral side, and their trajectories were erratic. Most of these fibers (about 87%) were larger than 1.3 μm and densely SMI-32 (+) stained. In the undamaged spinal tissue, motoneurons form the only large population of SMI-32 (+) neurons which are densely stained and have large diameter axons. These data therefore suggest that a sizeable proportion of the fibers seen in the lesion territory originate from motoneurons, although fibers of other origins could also contribute. Neither the presence of the antibody neutralizing Nogo-A alone, nor the presence of the antibody neutralizing Nogo-A combined with BDNF influenced the number or the length of the SMI-32 (+) fibers in the spinal lesion area. In summary, our data show that after a spinal cord lesion in adult monkeys, the lesion site is colonized by fibers, a large portion of which presumably originate from motoneurons., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

While chronic pain is a main symptom in endometriosis, the underlying mechanisms and effective therapy remain elusive. We developed an animal model enabling the exploration of ectopic endometrium as a source of endometriosis pain. Rats were surgically implanted with autologous uterus in the gastrocnemius muscle. Within two weeks, visual inspection revealed the presence of a reddish-brown fluid-filled cystic structure at the implant site. Histology demonstrated cystic glandular structures with stromal invasion of the muscle. Immunohistochemical studies of these lesions revealed the presence of markers for nociceptor nerve fibers and neuronal sprouting. Fourteen days after surgery rats exhibited persistent mechanical hyperalgesia at the site of the ectopic endometrial lesion. Intralesional, but not contralateral, injection of progesterone was dose-dependently antihyperalgesic. Systemic administration of leuprolide also produced antihyperalgesia. In vivo electrophysiological recordings from sensory neurons innervating the lesion revealed a significant increase in their response to sustained mechanical stimulation. These results are consistent with clinical and pathological findings observed in patients with endometriosis, compatible with the ectopic endometrium as a source of pain. This model of endometriosis allows mechanistic exploration at the lesion site facilitating our understanding of endometriosis pain., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Spreading depolarization (SD) is a slowly propagating, coordinated depolarization of brain tissue, which is followed by a transient (5-10min) depression of synaptic activity. The mechanisms for synaptic depression after SD are incompletely understood. We examined the relative contributions of action potential failure and adenosine receptor activation to the suppression of evoked synaptic activity in murine brain slices. Focal micro-injection of potassium chloride (KCl) was used to induce SD and synaptic potentials were evoked by electrical stimulation of Schaffer collateral inputs to hippocampal area Cornu Ammonis area 1 (CA1). SD was accompanied by loss of both presynaptic action potentials (as assessed from fiber volleys) and field excitatory postsynaptic potentials (fEPSPs). Fiber volleys recovered rapidly upon neutralization of the extracellular direct current (DC) potential, whereas fEPSPs underwent a secondary suppression phase lasting several minutes. Paired-pulse ratio was elevated during the secondary suppression period, consistent with a presynaptic mechanism of synaptic depression. A transient increase in extracellular adenosine concentration was detected during the period of secondary suppression. Antagonists of adenosine A1 receptors (8-cyclopentyl-1,3-dipropylxanthine [DPCPX] or 8-cyclopentyl-1,3-dimethylxanthine [8-CPT]) greatly accelerated fEPSP recovery and abolished increases in paired-pulse ratio normally observed after SD. The duration of fEPSP suppression was correlated with both the duration of the DC shift and the area of tissue depolarized, consistent with the model that adenosine accumulates in proportion to the metabolic burden of SD. These results suggest that in brain slices, the duration of the DC shift approximately defined the period of action potential failure, but the secondary depression of evoked responses was in large part due to endogenous adenosine accumulation after SD., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The role of endothelial and neural factors as modulators of neurogenic- and noradrenaline-induced vasoconstriction was examined in healthy pig internal mammary artery (IMA). Tetrodotoxin-, guanethidine-sensitive electrical field stimulation (EFS)-, and noradrenaline-elicited contractions were significantly diminished by prazosin (n=8, P<0.001) and less so by rauwolscine, indicating functional α₁- and α₂-adrenoceptor-mediated noradrenergic innervation of the IMA. Endothelium removal reduced neurogenic (n=8, P<0.01) but augmented noradrenaline responses (n=8, P<0.01), suggesting the release of two endothelium-dependent factors with opposite effects. In the presence of endothelium, neurogenic and exogenous noradrenaline vasoconstrictions were enhanced by L-NOArg (n=7, P<0.05 and P<0.01 respectively) and ODQ (n=7, both P<0.05); in denuded arteries, nNOS inhibition with N(ω)-propyl-L-arginine increased neurogenic contraction (n=7, P<0.05). Western blotting indicated the presence of neural and endothelial origin NO (n=6, P<0.001). Tetraethylammonium (n=9, P<0.001), iberiotoxin (n=7, P<0.001) and 4-aminopyridine (n=8, P<0.01) enhanced vasoconstrictions revealing a modulatory role of big conductance Ca²⁺-activated K⁺ (BK(Ca)) and voltage-dependent K⁺ (K(v)) channels in noradrenergic responses. Bosentan pretreatment (n=8, P<0.05) suggested endothelin-1 as the inferred contractile neurogenic endothelial-dependent factor. Indomethacin-induced inhibition involved a muscular prostanoid (n=9, P<0.05), functionally and immunologically localized, and derived from cyclooxygenase (COX)-1 and COX-2, as revealed by Western blots (n=5, P=0.1267). Thus, noradrenergic IMA contractions are controlled by contractile prostanoid activation and endothelin-1 release, and offset by BK(Ca) and K(v) channels and neural and endothelial NO. These results help clarify the mechanisms of vasospasm in IMA, as the preferred vessel for coronary bypass., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fatty acid amide hydrolase (FAAH) is an enzyme that metabolizes endocannabinoids and fatty acid amides possibly linked to activation of the opioid system. To examine how this enzyme affects spinal signalling, electrophysiological recordings in the dorsal horn and qPCR on dorsal horn tissue following systemic administration of the FAAH inhibitor URB597 (0.3 and 1.0mg/kg i.v.) and spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.), were performed. The present data showed that the suppressive effect of the FAAH inhibitor URB597 (1.0mg/kg i.v.) on the spinal nociceptive responses was prevented by spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.). Moreover, the present findings demonstrated that the FAAH inhibitor URB597 (1.0mg/kg i.v.) partly reversed expression of spinal long-term potentiation (LTP) and also attenuated the LTP-associated increased Zif expression. We conclude that pharmacological inactivation of FAAH may be a promising strategy to inhibit the development of central hyperalgesia; thereby reinforcing the role of FAAH as a potential therapeutic target., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Doublecortin (DCX) is a microtubule-associated protein that is critical for neuronal migration and the development of the cerebral cortex. In the adult, it is expressed in newborn neurons in the subventricular and subgranular zones, but not in the mature neurons of the cerebral cortex. By contrast, neurogenesis and neuronal migration of cells in the cerebellum continue into early postnatal life; migration of one class of cerebellar interneuron, unipolar brush cells (UBCs), may continue into adulthood. To explore the possibility of continued neuronal migration in the adult cerebellum, closely spaced sections through the brainstem and cerebellum of adult (3-16 months old) Sprague-Dawley rats were immunolabeled for DCX. Neurons immunoreactive (ir) to DCX were present in the granular cell layer of the vestibulocerebellum, most densely in the transition zone (tz), the region between the flocculus (FL) and ventral paraflocculus (PFL), as well as in the dorsal cochlear nucleus (DCN). These DCX-ir cells had the morphological appearance of UBCs with oval somata and a single dendrite ending in a brush. There were many examples of colocalization of DCX with Eps8 or calretinin, UBC markers. We also identified DCX-ir elements along the fourth ventricle and its lateral recess that had labeled somata but lacked the dendritic structure characteristic of UBCs. Labeled UBCs were seen in nearby white matter. These results suggest that there may be continued neurogenesis and/or migration of UBCs in the adult. Another possibility is that UBCs maintain DCX expression even after migration and maturation, reflecting a role of DCX in adult neuronal plasticity in addition to a developmental role in migration., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Effectively bridging the lesion gap is still an unmet demand for spinal cord repair. In the present study, we tested our hypothesis if cograft of Schwann cells (SCs) and neural stem cells (NSCs) with genetically enhanced expression of neurotrophin-3 (NT-3) and its high affinity receptor TrkC, respectively, could strengthen neural repair through increased NSC survival and neuronal differentiation at the epicenter after complete T10 spinal cord transection in adult rats. Transplantation of NT-3-SCs + TrkC-NSCs in Gelfoam (1 × 10(6)/implant/rat; n = 10) into the lesion gap immediately following injury results in significantly improved relay of the cortical motor evoked potential (CMEP) and cortical somatosensory evoked potential (CSEP) as well as ameliorated hindlimb deficits, relative to controls (treated with LacZ-SCs + LacZ-NSCs, NT-3-SCs + NSCs, NSCs alone, or lesion only; n = 10/group). Further analyses demonstrate that NT-3-SCs + TrkC-NSCs cografting augments levels of neuronal differentiation of NSCs, synaptogenesis (including inhibitory/type II-like synapses) and myelin formation of SCs, in addition to neuroprotection and outgrowth of serotonergic fibers in the lesioned spinal cord. Compared with controls, the treated spinal cords also show elevated expression of laminin, a pro-neurogenic factor, and decreased presence of chondroitin sulfate proteoglycans, major inhibitors of axonal growth and neuroplasticity. Together, our data suggests that coimplantation of neurologically compatible cells with compensatorily overexpressed therapeutic genes may constitute a valuable approach to study, and/or develop therapies for spinal cord injury (SCI)., (Published by Elsevier Ltd.)

In the gut, transient receptor potential vanilloid (TRPV) 1 activation leads to release of neurotransmitters such as neuropeptides and nitric oxide. However, the distribution of TRPV1 nerve fibers and neurotransmitters released form sensory nerve endings in the enteric nervous system are currently not well understood. The present study investigated the immunohistochemical distribution of TRPV1 channels, sensory neuropeptides, and nitric oxide and their co-localization in mouse large intestine. Numerous TRPV1 and calcitonin gene-related peptide (CGRP) immunoreactivities were detected, mainly in the mucosa, submucosal layer, and myenteric plexus. Abundant substance P (SP), neurokinin A (NKA), and neuronal nitric oxide synthase (nNOS)-immunoreactivity were revealed in muscle layers. Motor function studies of circular and longitudinal muscles found that contractile responses to capsaicin in the rectum were most sensitive among the rectum, and distal, transverse, and proximal colon. Double labeling studies were carried out in horizontal sections of mouse rectum. TRPV1/protein gene product (PGP)9.5 double labeled axons were observed, but PGP9.5 and neuronal nuclear protein immunopositive cell bodies did not express TRPV1 immunoreactivity in the myenteric plexus. In the mucosa, submucosal layer, deep muscular plexus, circular muscle, myenteric plexus and longitudinal muscle layer, TRPV1 nerve fibers were found to contain CGRP, SP and nNOS. SP and NKA were almost entirely colocalized at the axons and cell bodies in all layers. Double labeling with c-Kit revealed that TRPV1 nerve fibers localized adjacent to the interstitial cells of Cajal (ICC). These results suggest that the TRPV1-expressing nerve and its neurotransmitters regulate various functions of the large intestine., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The deep cerebellar nuclei (DCN) are the terminal components of the cerebellar circuitry and constitute its primary output structure. Their activity is important for certain forms of motor learning as well as generation and control of movement. DCN neurons receive glutamatergic excitatory inputs from the pontine nuclei via mossy fibres (MFs) and concomitantly receive inputs from 5-HT-containing neurons of the raphe nuclei. We aimed to explore the roles of 5-HT at MF-DCN synapses by using cerebellar slices from 11 to 15-day-old rats. Bath application of 5-HT reversibly decreased the amplitude of stimulation-evoked excitatory postsynaptic currents (eEPSCs) via the activation of 5-HT1B receptors at the presynaptic terminals of the MFs. Burst stimulation of the MFs elicited long-term depression (LTD) at the MF-DCN synapses that require activation of the group I metabotropic glutamate receptor (mGluR). In the presence of 5-HT, the extent of burst-induced LTD of MF EPSCs was significantly reduced. Application of 5-HT also decreased the amplitude of mGluR-dependent slow EPSCs evoked by similar burst stimulation. Furthermore, (S)-3,5-dihydroxyphenylglycine (DHPG), a group I mGluR agonist, induced chemical LTD of MF EPSCs, and 5-HT had no significant effect on this LTD. Taken together, the results suggest that 5-HT not only has transitory inhibitory effects on MF EPSCs but also plays a role in regulating the long-term synaptic efficacy., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Alpha(1)-adrenoceptors (α(1)-ARs) are involved in neuro-thymic and thymic intercellular communications, and consequently modulation of T-cell development. Ageing is associated with a number of changes in noradrenergic neuro-effector transmission, and possibly intercellular noradrenaline (NA)-mediated communication resulting in altered responses of target cells to NA. Thus, in old animals an altered NA modulation of thymopoiesis via α(1)-ARs may be expected. To test this hypothesis, in old and young adult Wistar rats we examined: 1) thymic NA levels, density of noradrenergic innervation and NA synthesizing cells, as well as α(1)-AR expression, and 2) then the effects of 14-day-long treatment with the α(1)-AR blocker, urapidil, on thymocyte development. Overall, the first part of study suggested augmented NA signalling to thymic cells via α(1)-ARs due to increased NA availability and α(1)-AR thymocyte surface density in old rats. The second part of study supported this assumption. Namely, although in rats of both ages urapidil affected the same thymocyte developmental steps ultimately leading to changes in the relative number of the most mature single positive TCRαβ(high) thymocytes, its effects were generally more prominent in old animals. Following urapidil treatment, the percentages of CD4+CD8- cells, including those showing a regulatory CD4+CD25+RT6.1- phenotype, were increased, while CD4-CD8+ cells decreased. In old rats, an augmented thymic escape of immature CD4+CD8+ cells was also registered. In rats of both ages the thymic changes were accompanied by alterations in the proportions of major cell populations in the T-lymphocyte compartment of both peripheral blood and spleen, leading to an increase in the CD4+/CD8+ T-cell ratio. These alterations were also more pronounced in old rats. Moreover, in old rats following urapidil treatment the proportion of TCRαβ+cells in the periphery was slightly greater reflecting, most likely, partly enhanced thymic production of regulatory CD161+TCRαβ+cells. Thus, the study indirectly suggests an age-associated increase in the basal α(1)-AR-mediated inhibitory influence of NA on thymopoiesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We have examined the basic electrophysiology and pharmacology of cells in the anterior caudal lobe (CLa) of the mormyrid cerebellum. Intracellular recordings were performed in an in vitro slice preparation using the whole-cell patch recording method. The responses of cells to parallel fiber (PF) and climbing fiber (CF) stimulation and to somatic current injection were recorded, and then characterized by bath application of receptor and ion channel blockers. Using biocytin or neurobiotin, these cells were also morphologically identified after recording to ensure their classification. Efferent cells and two subtypes of Purkinje cells were identified on the basis of their physiology and morphology. While the majority of Purkinje cells fire a single type of spike that is mediated by Na(+), some fire a large broad spike mediated by Ca(2+) and a narrow spike mediated by Na(+) at resting potential levels. By patching one recording electrode to the soma and another to one of the proximal dendrites of the same cell simultaneously, it was found that the Na(+) spike has an axonal origin and the Ca(2+) spike is generated in the soma-dendritic region of Purkinje cells. Efferent cells fire a single type of Na(+) spike only. Despite variations in their physiology and morphology, all cell types responded to PF stimulation with graded excitatory postsynaptic potentials (EPSPs) mediated by AMPA receptors. However, none of the efferent cells and only some of the Purkinje cells responded to CF activation with a large, AMPA receptor-mediated all-or-none EPSPs. We conclude that the functional circuitry of the CLa resembles that of other regions of the mormyrid cerebellum and is largely similar to that of the mammalian cerebellum., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: We examined the emerging vertical component after cross-axis vestibulo-ocular reflex (VOR) adaptation., Methods: For the adaptive conditioning, the whole body of a subject was rotated horizontally while a visual pattern was rotated vertically for 1h., Results: During the conditioning period, the vertical component in response to the vertical visual stimulation was an up-down asymmetric, and its amplitude gradually increased as the stimulus progressed. After conditioning, the direction of VOR was changed via an emerging vertical component during horizontal head rotation in total darkness. Even though the retinal slip-velocity was greater for downward than for upward during the conditioning period, the emerging upward component was significantly larger than the emerging downward component., Conclusion: These results may be helpful for developing an optimal vestibular-compensation system by visual-vestibular mismatch/optokinetic training., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

A new fuzzy algorithm for assessing white matter connectivity in the brain using diffusion-weighted magnetic resonance images is presented. The proposed method considers anatomical paths as chains of linked neighbouring voxels. Links between neighbours are assigned weights using the respective fibre orientation estimates. By checking all possible paths between any two voxels, a connectedness value is assigned, representative of the weakest link of the strongest path connecting the voxel pair. Multiple orientations within a voxel can be incorporated, thus allowing the utilization of fibre crossing information, while fibre branching is inherently considered. Under the assumption that paths connected strongly to a seed will exhibit adequate orientational coherence, fuzzy connectedness values offer a relative measure of path feasibility. The algorithm is validated using simulations and results are shown on diffusion tensor and Q-ball images.

This study was focused on determining the possible role of corticotropin-releasing hormone (CRH) on play fighting in juvenile golden hamsters. As no specific neural sites have been proposed, we looked for changes in CRH innervations at the peak of play-fighting activity on postnatal day 35 (P-35) from a week before on P-28. We noted that the increase in play-fighting activity between these two dates was associated with a 100% increase of the density of CRH fibers within the lateral septum. We, then, tested the possible role of CRH receptors on play fighting within the lateral septum through microinjections of alpha-helical CRH, a CRH receptor antagonist (either 0, 30, or 300 ng), directly into the area. The treatments inhibited play-fighting attacks and pins as well as reduced the duration of time that the resident hamsters spent in contact with the intruders, though locomotor activity remained unaffected. The possible source of CRH release in the lateral septum was addressed by quantification of CRH neurons also labeled with a marker of cellular activity, c-Fos, after consummation of play fighting. CRH neurons in the horizontal part of the diagonal band, an area reciprocally connected with the lateral septum, showed a 75% increase in double labeling with c-Fos as compared to controls. Together, these data show that CRH receptors in the lateral septum have a general role on play fighting, not just facilitating its consummation, but also likely enhancing appetitive aspects as well. In addition, this effect is associated with enhanced CRH availability in the area and enhanced neuronal activity within interconnected areas., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Previous reports show that vagal afferent innervation of the stomach eventually regenerates from surviving nodose ganglion (NG) neurons after subdiaphragmatic vagotomy. Systemic capsaicin treatment destroys gastric vagal afferent neurons expressing vanilloid receptor 1 (VR1). However, it is not known whether gastric innervation lost after neuronal destruction can be restored. Here, we report that capsaicin-induced damage of NG neurons innervating the stomach in adult rats is followed by restoration of vagal afferent projections. Specifically, we compared measures of neuronal plasticity in NG and vagi after subdiaphragmatic vagotomy or capsaicin treatment. The numbers of VR1-immunoreactive neurons projecting to the stomach were significantly reduced 10 days after either capsaicin treatment or vagotomy. However, the VR1-immunoreactive afferent innervation of the stomach was restored to levels exceeding those of vagotomized rats by 37 days after capsaicin, whereas neither total afferent innervation nor VR1-immunoreactive innervation reached control levels, even by 67 days after vagotomy. Capsaicin treatment significantly increased NG neuronal nitric oxide synthase (nNOS) immunoreactivity at 10 days after capsaicin, and this increase was sustained for the duration of the study, indicating higher nNOS demand in restoration of vagal projections. Vagotomy was associated with a much smaller increase in the number of nNOS-immunoreactive NG neurons, detectable only at 10 days after surgery. The number of nNOS-immunopositive gastric-projecting neurons was dramatically reduced 10 days after either capsaicin treatment or vagotomy but returned to the control level in both groups at 67 days. We found a significantly higher number of growth cones in capsaicin-treated animals compared with controls. Capsaicin significantly increased the number of nNOS-immunopositive and nNOS-immunonegative growth cones in NG at all time points. Vagotomy did not increase the number of nNOS(-) growth cones in NG. We conclude that capsaicin treatment may result in more significant restorative capacities than vagotomy, mainly because of sprouting of capsaicin-insensitive nerve fibers., (Published by Elsevier Ltd.)

The development of neuromuscular junctions exhibits profound remodeling that brings from an immature state characterized by multiple motoneuronal inputs per muscle fiber, to a mature mononeuronal innervation. This striking elimination process occurs both perinatally and during adult reinnervation, and is also widely present in the developing CNS. The accelerating influence of the amount of impulse activity on this process, has been shown by various studies, but a more subtle role of the time correlation of action potential firing in the competing inputs, has also been suggested. Here we explore the latter influence using a rat adult model of neuromuscular junction formation, that is reinnervation following a motor nerve crush. This shares all important features with perinatal development, especially the strict juxtaposition of the competing inputs. In fact the regenerating axons converge on a single cluster of postsynaptic receptors, that is the original endplate of each muscle fiber. This focus on the spatial aspect of competition between nerve endings was missing in our previous experiments employing a similar paradigm. We impose a chronic synchronous firing to the competing terminals, by in vivo electrical stimulation of their axons distal to a sciatic nerve conduction block. Control preparations, with similar post-crush reinnervation, are left with their natural impulse activity unperturbed. We find that the experimental muscles display a prolonged duration of polyneuronal innervation with respect to controls, indicating that hebbian mechanisms participate in the synapse elimination process. Another aspect dealt with in our study is the genuine nature of the polyneuronal innervation occurring during adult muscle reinnervation, because it is supported by both confocal microscopy and by appropriate electrophysiological tests that exclude electrical coupling of myofibers by gap junctions., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Aims: Several types of gastric surgeries have been associated with early satiety, dyspepsia and food intolerances. We aimed to examine alterations in gastric vagal afferents following gastric surgery-fundus ligation., Methods: Six week old, male Sprague-Dawley rats underwent chronic ligation (CL) of the fundus. Sham rats underwent abdominal surgery, but without ligation. Another group of rats underwent acute ligation (AL) of the fundus immediately prior to experiments. CL and sham rats were allowed to grow to age 3-4 months. Food intake and weights were recorded post-operatively. Gastric compliance and gastric wall thickness was measured at baseline and during gastric distension (GD). Extracellular recordings were made to examine response characteristics of vagal afferent fibers to GD and to map the stomach receptive field (RF). The morphological structures of afferent terminals in the stomach were examined with retrograde tracings from the nodose ganglion., Results: The CL group consumed significantly less food and weighed less than sham control. The mean compliance of the CL group was significantly less than control, but higher than the AL group. The spontaneous firing and responses to GD of afferent fibers from the CL rats were significantly higher than AL rats. There was a marked expansion of the gastric RF in the CL rats with significant reorganization and regeneration of intramuscular array (IMA) terminals. There was no difference in total wall or muscle thickness among the groups., Conclusion: CL results in aberrant remodeling of IMAs with expansion of the gastric RF and alters the mechanotransduction properties of vagal afferent fibers. These changes could contribute to altered sensitivity following gastric surgery.

Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague-Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15-16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP(+)) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200(+)) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain-related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP(+) C-fibers) and capsaicin-insensitive (primarily NF200(+) A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.

The cerebellum can be viewed as supporting two distinct aspects of motor execution related to a) motor coordination and the sequence that imparts such movement temporal coherence and b) the reorganization of ongoing movement when a motor execution error occurs. The former has been referred to as "motor time binding" as it requires that the large numbers of motoneurons involved be precisely activated from a temporal perspective. By contrast, motor error correction requires the abrupt reorganization of ongoing motor sequences, on occasion sufficiently important to rescue the animal or person from potentially lethal situations. The olivo-cerebellar system plays an important role in both categories of motor control. In particular, the morphology and electrophysiology of inferior olivary neurons have been selected by evolution to execute a rather unique oscillatory pace-making function, one required for temporal sequencing and a unique oscillatory phase resetting dynamic for error correction. Thus, inferior olivary (IO) neurons are electrically coupled through gap junctions, generating synchronous subthreshold oscillations of their membrane potential at a frequency of 1-10 Hz and are capable of fast and reliable phase resetting. Here I propose to address the role of the olivocerebellar system in the context of motor timing and reset.

Elimination of redundant synapses and strengthening of the surviving ones are crucial steps in the development of the nervous system. Both processes can be readily followed at the climbing fiber to Purkinje cell synapse in the cerebellum. Shortly after birth, around five equally strong climbing fiber synapses are established. Subsequently, one of these five synaptic connections starts to grow in size and synaptic strength, while the others degenerate and eventually disappear. Both the elimination of the redundant climbing fiber synapses and the strengthening of the surviving one depend on a combination of a genetically coded blueprint and synaptic activity. Recently, it has been shown that synaptic activity affects the synaptic strength of developing climbing fibers. Remarkably, the same pattern of paired activity of the presynaptic climbing fiber and the postsynaptic Purkinje cell resulted in strengthening of already "large" climbing fibers and weakening of already "weak" climbing fibers. In this review, we will integrate the current knowledge of synaptic plasticity of climbing fibers with that of other processes affecting climbing fiber development.

The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with "hypogranular" cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF-PC synapses. Our understanding of how PF-PC synapse formation affects development of CF-PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor delta2 subunit (GluRdelta2), an orphan receptor expressed selectively in PCs. Deletion of GluRdelta2 results in impairment of PF-PC synapse formation, which leads to defects in development of CF-PC synapses. In this article, we review how impaired PF-PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluRdelta2 knockout mice. We propose a new scheme that CF-PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, "early phase" of CF synapse elimination from P7 to around P11, and "late phase" of CF synapse elimination from around P12. Normal PF-PC synapse formation is required for the "late phase" of CF synapse elimination.

The recently described bi-stability of Purkinje cells and the state-dependence of the complex spike waveform suggest that calcium currents may play a pivotal role in both the complex spike waveform and the state of the membrane voltage. Here we used Ca2+ imaging to record the changes in intracellular [Ca2+] that are elicited by either spontaneous or climbing fiber-evoked activity in rat Purkinje cells. We show that a continuous somatic Ca2+ influx occurs during an "UP" state. Furthermore Ca2+ transients that are evoked by climbing fiber stimulation are state-dependent. Somatic transients are smaller following an "UP" state, while dendritic transients are smaller following a "DOWN" state. The state-dependence of these signals should affect the intrinsic firing of Purkinje cells as well as plastic processes that modulate synaptic strength.

Long-term potentiation (LTP) of parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum has been suggested to underlie aspects of motor learning. Previous in vitro studies have primarily used low frequency PF stimulation conditioning paradigms to generate either presynaptic PF-PC LTP (4-8 Hz) or postsynaptic PF-PC LTP (1 Hz). Little is known about the conditions that evoke PF-PC LTP in vivo. High frequency stimulation in vivo increases PC responsiveness to peripheral stimuli; however, neither the site of action nor the signaling pathways involved have been examined. Using flavoprotein autofluorescence optical imaging in the FVB mouse in vivo, this report describes that a conditioning stimulation consisting of a high frequency burst of PF stimulation (100 Hz, 15 pulse trains every 3 s for 5 min) evokes a long-term increase in the response to PF stimulation. Following the conditioning stimulation, the response to PF stimulation increases over 20 min to approximately 130% above baseline and this potentiation persists for at least 2 h. Field potential recordings of the responses to PF stimulation show that the postsynaptic component is potentiated but the presynaptic, parallel fiber volley is not. Paired-pulse facilitation does not change after the conditioning stimulation, suggesting the potentiation occurs postsynaptically. Blocking non-NMDA (N-methyl-d-aspartic acid) ionotropic glutamate receptors with DNQX (6,7-dinitroquinoxaline-2,3-dione disodium salt, 50 muM, bath application) during the conditioning stimulation has no effect on the long-term increase in fluorescence. However, blocking subtype I metabotropic glutamate receptors (mGLuR(1)) with LY367385 (200 muM) during the conditioning stimulation abolishes the long-term increase in fluorescence. Blocking GABAergic neurotransmission is not required to evoke this long-term potentiation. Blocking GABA(A) receptors reduces but does not eliminate the long-term potentiation. Therefore, this study demonstrates that high frequency PF stimulation generates long-term potentiation of PF-PC synapses in vivo. This novel form of LTP is generated primarily postsynaptically and is mediated by mGluR(1) receptors.

The peculiar shape and disposition of Purkinje cell (PC) dendrites, planar and highly branched, offers an optimal model to analyze cellular and molecular regulators for the acquisition of neuronal dendritic trees. During the first 2 weeks after the end of the proliferation period, PCs undergo a 2-phase remodeling process of their dendrites. The first phase consists in the complete retraction of the primitive but extensive dendritic tree, together with the formation of multiple filopodia-like processes arising from the cell body. In the second phase, there is a progressive disappearance of the somatic processes along with rapid growth and branching of the mature dendrite. Mature Purkinje cell dendrites bear two types of spiny protrusions, named spine and thorn. The spines are numerous, elongated, located at the distal dendritic compartment and form synapses with parallel fibers, whereas the thorns are shorter, rounded, emerge from the proximal compartment and synapse with climbing fibers. Different culture models and mutant mice analyses suggest the identification of intrinsic versus extrinsic determinants of the Purkinje cell dendritic development. The early phase of dendritic remodeling might be cell autonomous and regulated by specific transcription factors such as retinoid-related orphan receptor alpha (RORalpha). Afferent fibers, trophic factors and hormones regulate the orientation and growth of the mature dendritic tree contributing, with still unknown intrinsic factors, to sculpt its general architecture. The formation of spines appears as an intrinsic phenomenon independent of their presynaptic partner, the parallel fibers, and confined to the distal compartment by inhibitory influences of the climbing fibers along the proximal compartment.

The cerebellar circuitry consists of two main excitatory glutamatergic pathways. The inputs of mossy fibers and climbing fibers converge on Purkinje cells and deep cerebellar nuclei. In this circuitry, Golgi interneurons suppress granule cell excitability via the inhibitory GABA transmitter. A novel technique termed reversible neurotransmission blocking (RNB) was genetically established, in which granule cell transmission to Purkinje cells was selectively and reversibly blocked in the mouse cerebellar circuitry. This study revealed that Purkinje cells are essential for expression of conditioned eye-blink motor learning but that this memory is acquired and stored in deep cerebellar nuclei. A different technique termed immunotoxin-mediated cell targeting (IMCT) was developed to selectively ablate Golgi cells from the mouse cerebellar network. The study disclosed that excitatory glutamate receptors and inhibitory GABA receptors cooperatively act at Golgi cell-mossy fiber-granule cell synapses and are indispensable for motor coordination and adaptation. Finally, gene targeting of mGluR2 displayed that the metabotropic glutamate receptor acts collaboratively with the ionotropic AMPA receptors at granule cell-Golgi cell synapses and is crucial for the spatiotemporal regulation in the mouse cerebellar circuitry. The neural information is thus hierarchically regulated and integrated at different levels of the cerebellar network.

The brain generates many rhythmic activities, and the olivo-cerebellar system is not an exception. In recent years, the cerebellum has revealed activities ranging from low frequency to very high-frequency oscillations. These rhythms depend on the brain functional state and are typical of certain circuit sections or specific neurons. Interestingly, the granular layer, which gates sensorimotor and cognitive signals to the cerebellar cortex, can also sustain low frequency (7-25 Hz) and perhaps higher-frequency oscillations. In this review we have considered (i) how these oscillations are generated in the granular layer network depending on intrinsic electroresponsiveness and circuit connections, (ii) how these oscillations are correlated with those in other cerebellar circuit sections, and (iii) how the oscillating cerebellum communicates with extracerebellar structures. It is suggested that the granular layer can generate oscillations that integrate well with those generated in the inferior olive, in deep-cerebellar nuclei and in Purkinje cells. These rhythms, in turn, might play a role in cognition and memory consolidation by interacting with the mechanisms of long-term synaptic plasticity.

Gastro-esophageal acid reflux can stimulate esophageal vagal sensory afferents by activating proton-sensitive ion channel transient receptor vanilloid one (TRPV1). The objective of this study was to investigate the response characteristics of vagal afferent fibers of rats to acid (0.1 N HCl) and capsaicin (CAP) following esophagitis and differential effects of two classes of TRPV1 antagonists on responses of vagal afferent fibers. The chronic reflux was induced by ligating the fundus of the stomach and partial constriction of pylorus. Extracellular single fiber recordings were made from the cervical vagal afferent fibers from naive control and fundus-ligated (FL) esophagitis rats. Innervations of fibers were identified to esophageal distension (ED) and subsequently tested to CAP and acid before and after injection of TRPV1 antagonist JYL1421 or AMG9810 (10 micromol/kg i.v.). Seventy-five vagal afferent fibers from 70 rats were identified to ED. Intra-esophageal CAP (0.1 ml of 1 mg/ml) excited 39.5% (17/43, 5/22 from naive and 12/21 from FL rats) fibers. In contrast, i.v. injection of CAP (0.03-0.3 micromol/kg) dose-dependently excited 72% (42/58) fibers. Responses to CAP were significantly greater for fibers from FL rats (n=32) than naive rats (n=25). TRPV1 antagonists JYL1421 and AMG9810 (10 micromol/kg) significantly blocked response to CAP. Intra-esophageal acid infusion stimulated 5/17 (29.4%) fibers from naive rats and 12/28 (42%) from FL rats. Effect of acid was significantly blocked by AMG9810, but not by JYL1421. Results indicate that following esophagitis the number of fibers responsive to CAP and acid is greater than noninflamed esophagus, which may contribute to esophageal hypersensitivity. Acid-induced excitation of vagal sensory afferents can be differentially attenuated by different classes of TRPV1 antagonists. Therefore, TRPV1 antagonists play a key role in attenuation of hypersensitivity following reflux-induced esophagitis. The use of TRPV1 antagonists could be an alternative to the traditional symptoms-based treatment of chronic acid reflux and esophageal hypersensitivity.

Skeletal muscle regenerative potential is reduced with aging. We hypothesized that in vitro activation of muscle satellite cells would be compromised, and that nitric oxide (NO) supplementation would improve satellite cell activity in old muscle. Single intact myofibers were isolated from the gastrocnemius muscles of young (2 mo), adult (10 mo), and aged (22 mo) mice. Fibers were centrifuged to stimulate satellite cells and incubated with L-arginine (2mM), the NO donor, diethylenetriamine NONOate (DETA-NO; 10 microM), or control media for 48 h. The number of activated satellite cells after centrifugation was reduced in aged fibers compared to young and adult. L-arginine or DETA-NO treatment increased satellite cell activation in all age groups. However, an age-dependent deficit in satellite cell activity persisted within treatment groups. In separate fibers, exogenous HGF was equally effective in activating satellite cells across age groups, indicating that events downstream of HGF release are intact in aged muscle. These data suggest that l-arginine bioavailability and NO production limit muscle satellite cell activity in response to a submaximal mechanical stimulus, regardless of age. Further, the decline in satellite cell activity in early senescence can be partially abrogated by exogenous L-arginine or an NO donor.

Recent studies suggest that tumor necrosis factor-alpha (TNF) sensitizes primary afferent neurons, and thus facilitates neuropathic pain. Here, we separately examined the roles of tumor necrosis factor receptor (TNFR) 1 and 2 by parallel in vivo and in vitro paradigms using proteins that selectively activate TNFR1 or TNFR2 (R1 and R2). In vivo, intrathecally injected R1, but not R2 slightly reduced mechanical and thermal withdrawal thresholds in rats, whereas co-injection resulted in robust, at least additive pain-associated behavior. In vitro, the electrophysiological responses of dorsal root ganglia (DRG) from rats with spinal nerve ligation were measured utilizing single-fiber recordings of teased dorsal root filaments. In naïve DRG, only R1 (10-1000 pg/ml) induced firing in Ass- and Adelta-fibers, whereas R2 had no effect. In injured DRG, both R1 and R2 at significantly lower concentrations (1 pg/ml) increased discharge rates of Adelta-fibers. Most interesting, in adjacent uninjured DRG, R2 and not R1, increased ectopic activity in both Ass- and Adelta-fibers. We conclude that TNFR1 may be predominantly involved in the excitation of sensory neurons and induction of pain behavior in the absence of nerve injury, TNFR2 may contribute in the presence of TNFR1 activation. Importantly, the effects of individually applied R1 and R2 on injured and adjacent uninjured fibers imply that the role of TNFR2 in the excitation of sensory neurons increases after injury.

The cerebellum is organized into parasagittal zones with respect to the topography of climbing fiber (CF) afferents and the expression of molecular markers such as zebrin II. Zebrin is expressed by a subset of Purkinje cells that are distributed as a parasagittal array of immunopositive and immunonegative stripes. Several studies in rodents suggest that, in general, CFs to the zebrin negative stripes convey somatosensory information, whereas CFs to the zebrin positive stripes convey information from visual and other sensory systems. The pigeon flocculus consists of four pairs of zebrin+/- stripes (P4 +/- through P7 +/-), however the CF input consists entirely of visual inputs. Thus, because the correspondence of zebrin expression and CF information must be different from that proposed for rodents, we investigated this relationship in the pigeon flocculus. Floccular Purkinje cells respond to patterns of optic flow resulting from self-rotation about one of two axes: either the vertical axis (zones 0 and 2), or a horizontal axis (zones 1 and 3). Visual CF afferents projecting to the flocculus arise from the medial column of the inferior olive (mcIO). Zones 0 and 2 receive input from the caudal mcIO, whereas zones 1 and 3 receive input from the rostral mcIO. We injected a fluorescent anterograde tracer into the rostral and/or caudal mcIO and visualized zebrin expression. There was a strict concordance between CF organization and zebrin labeling: caudal mcIO injections resulted in CFs in zebrin bands P4 +/- and P6 +/-, whereas rostral mcIO injections resulted in CFs in zebrin bands P5 +/- and P7 +/-. Thus, zebrin stripes P4 +/- and P6 +/- correspond to the vertical axis zones 0 and 2, whereas P5 +/- and P7 +/- correspond to the horizontal axis zones 1 and 3. This is the first explicit demonstration that a series of zebrin stripes corresponds with functional zones in the cerebellum.

A topic of high current interest and controversy is the basis of the homeostatic sleep response, the increase in non-rapid-eye-movement (NREM) sleep and NREM-delta activity following sleep deprivation (SD). Adenosine, which accumulates in the cholinergic basal forebrain (BF) during SD, has been proposed as one of the important homeostatic sleep factors. It is suggested that sleep-inducing effects of adenosine are mediated by inhibiting the wake-active neurons of the BF, including cholinergic neurons. Here we examined the association between SD-induced adenosine release, the homeostatic sleep response and the survival of cholinergic neurons in the BF after injections of the immunotoxin 192 immunoglobulin G (IgG)-saporin (saporin) in rats. We correlated SD-induced adenosine level in the BF and the homeostatic sleep response with the cholinergic cell loss 2 weeks after local saporin injections into the BF, as well as 2 and 3 weeks after i.c.v. saporin injections. Two weeks after local saporin injection there was an 88% cholinergic cell loss, coupled with nearly complete abolition of the SD-induced adenosine increase in the BF, the homeostatic sleep response, and the sleep-inducing effects of BF adenosine infusion. Two weeks after i.c.v. saporin injection there was a 59% cholinergic cell loss, correlated with significant increase in SD-induced adenosine level in the BF and an intact sleep response. Three weeks after i.c.v. saporin injection there was an 87% cholinergic cell loss, nearly complete abolition of the SD-induced adenosine increase in the BF and the homeostatic response, implying that the time course of i.c.v. saporin lesions is a key variable in interpreting experimental results. Taken together, these results strongly suggest that cholinergic neurons in the BF are important for the SD-induced increase in adenosine as well as for its sleep-inducing effects and play a major, although not exclusive, role in sleep homeostasis.