Normal Innervation Research Articles

The EGFR Is Required for Proper Innervation to the Skin

EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner.

Structural and functional cardiac cholinergic deficits in adult neurturin knockout mice

Previous work provided indirect evidence that the neurotrophic factor neurturin (NRTN) is required for normal cholinergic innervation of the heart. This study used nrtn knockout (KO) and wild-type (WT) mice to determine the effect of nrtn deletion on cardiac cholinergic innervation and function in the adult heart. Immunohistochemistry, confocal microscopy, and quantitative image analysis were used to directly evaluate intrinsic cardiac neuronal development. Atrial acetylcholine (ACh) levels were determined as an indirect index of cholinergic innervation. Cholinergic function was evaluated by measuring negative chronotropic responses to right vagal nerve stimulation in anaesthetized mice and responses of isolated atria to muscarinic agonists. KO hearts contained only 35% the normal number of cholinergic neurons, and the residual cholinergic neurons were 15% smaller than in WT. Cholinergic nerve density at the sinoatrial node was reduced by 87% in KOs, but noradrenergic nerve density was unaffected. Atrial ACh levels were substantially lower in KO mice (0.013 +/- 0.004 vs. 0.050 +/- 0.011 pmol/microg protein; P < 0.02) as expected from cholinergic neuron and nerve fibre deficits. Maximum bradycardia evoked by vagal stimulation was reduced in KO mice (38 +/- 6% vs. 69 +/- 3% decrease at 20 Hz; P < 0.001), and chronotropic responses took longer to develop and fade. In contrast to these deficits, isolated atria from KO mice had normal post-junctional sensitivity to carbachol and bethanechol. These findings demonstrate that NRTN is essential for normal cardiac cholinergic innervation and cholinergic control of heart rate. The presence of residual cardiac cholinergic neurons and vagal bradycardia in KO mice suggests that additional neurotrophic factors may influence this system.

Abstract 6128: Cardiac Sympathetic Denervation in Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia and Risk of Recurrent Ventricular Tachyarrhythmias

Background: Patients (pts) with arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) typically present with ventricular tachyarrhythmias often provokable by stress or catecholamines thereby indicating an involvement of the cardiac adrenergic system in the arrhythmogenesis. Previous studies demonstrated abnormalities of the presynaptic uptake-1 assessed by 123I-MIBG scintigraphy (MIBG-SPECT). In this study, we investigated the potential role of adrenergic dysfunction on the arrhythmia profile in pts with ARVC/D. Methods: In 42 pts with ARVC/D (mean age 43±14 years), MIBG-SPECT was performed. Results were compared to those obtained from 10 control subjects (n=10; mean age 43±12 years) without identifiable structural heart disease. MIBG images were acquired four hours post injection and analysed for regional 123I-MIBG uptake in a standardized 33-segment bull’s eye scheme. Results: Overall, an abnormal tracer uptake was detected in 25 pts with ARVC/D (59%). The extent of right ventricular dilatation and regional wall motion abnormalities as well as electrocardiographic markers of re-/depolarisation (e.g. T-wave inversion, epsilon-potential, maximal QRS-duration in right precordial leads) were not significantly different between pts with a normal and an abnormal MIBG-SPECT. However, during long-term follow-up of 9.2±3.4 years, pts with abnormal MIBG-SPECT experienced life-threatening ventricular tachyarrhythmias more often than those with a normal sympathetic innervation (21 of 25 pts (84%) vs. 7 of 17 pts (41%); P &lt;0.01). Conclusions: An impairment of adrenergic innervation appears to account for a higher risk for VT recurrences in pts with ARVC/D. These results suggest a future role of MIBG-SPECT for individualized risk stratification in pts with ARVC/D.

Deficient α-smooth muscle actin as a cause of functional intestinal obstruction in childhood

Most functional intestinal obstruction in childhood is due to defects in the enteric innervation. Functional intestinal obstruction due to smooth muscle disorders is rare. The aim of this study was to describe four patients with functional intestinal obstruction where the only histopathological abnormality was deficient, alpha-smooth muscle actin (alpha-SMA) in the circular muscle of their small bowel. Resected intestinal segments from four children with functional intestinal obstruction were either snap frozen or embedded in paraffin wax, and transversely sectioned. Sections were examined by routine H&E staining, AChE and NADPH-diaphorase histochemistry, and immunohistochemistry using PGP9.5 and alpha-SMA antibodies. Normal age-matched tissue was used as controls. All four patients demonstrated normal innervation in the small and large bowel. We observed striking differences in the expression of alpha-SMA within the intestinal muscle layers in all four patients. There was a markedly decreased level of alpha-SMA in the smooth muscle compared to the longitudinal muscle in the small bowel in each patient. All other regions of the intestine examined showed normal levels of alpha-SMA, with similar levels expressed in both the circular and longitudinal muscles. The decreased level of alpha-SMA in the smooth muscle of these patients causes a serious mechanical defect, thus interfering with normal intestine motility. These findings suggest that patients with functional intestinal obstruction should have a comprehensive investigation of the intestinal smooth muscle in addition to the assessment of the enteric nervous system.

Colonic dysmotility in postsurgical patients with Hirschsprung's disease. Potential significance of abnormalities in the interstitial cells of Cajal and the enteric nervous system

Purpose Normal gut muscular function depends on the coordinated activity of both the enteric nervous system (ENS) and the interstitial cells of Cajal (ICC). Hirschsprung's disease (HD) has long been considered a purely neuronal deficit but recent data point to abnormalities in ICC in the proximal ganglionated HD colon. We examined the labeling of ICC and neuronal cells in the proximal ganglionated colon in patients with HD to determine whether abnormalities of ICC and ENS might be associated with a poor clinical outcome. Methods Tissue from 11 patients with HD was studied using immunohistochemistry for ICC and neuronal identification in comparison to control tissue from patients without HD. Image data were evaluated quantitatively and interpreted relative to clinical outcome. Results Interstitial cells of Cajal in the ganglionated colon of the HD group did not differ from the control group, but nerve cells/fibers were decreased 40%. Paired decreases in both nerve fibers and ICC in individual patients were associated with normal bowel function. Poor postoperative outcome was observed in a patient with normal innervation but with a profound decrease in ICC in the ganglionated colon. Conclusions Nerve fibers are decreased in the proximal ganglionated colon in patients with HD without associated gut dysmotility. Poor clinical outcome was noted only in a patient with normal innervation and markedly decreased ICC. Collection of data from a much larger number of patients with poor clinical outcome will be necessary to determine the significance of this imbalance of ICC and innervation.

Abnormal sympathetic nervous system development and physiological dysautonomia in Egr3-deficient mice

Sympathetic nervous system development depends upon many factors that mediate neuron migration, differentiation and survival. Target tissue-derived nerve growth factor (NGF) signaling-induced gene expression is required for survival, differentiation and target tissue innervation of post-migratory sympathetic neurons. However, the transcriptional regulatory mechanisms mediated by NGF signaling are very poorly defined. Here, we identify Egr3, a member of the early growth response (Egr) family of transcriptional regulators, as having an important role in sympathetic nervous system development. Egr3 is regulated by NGF signaling and it is expressed in sympathetic neurons during development when they depend upon NGF for survival and target tissue innervation. Egr3-deficient mice have severe sympathetic target tissue innervation abnormalities and profound physiological dysautonomia. Unlike NGF, which is essential for sympathetic neuron survival and for axon branching within target tissues, Egr3 is required for normal terminal axon extension and branching, but not for neuron survival. The results indicate that Egr3 is a novel NGF signaling effector that regulates sympathetic neuron gene expression required for normal target tissue innervation and function. Egr3-deficient mice have a phenotype that is remarkably similar to humans with sympathetic nervous system disease, raising the possibility that it may have a role in some forms of human dysautonomia, most of which have no known cause.

Functional effects of cardiac sympathetic denervation in neurogenic orthostatic hypotension

Background Diseases characterized by neurogenic orthostatic hypotension (NOH), such as Parkinson disease (PD) and pure autonomic failure (PAF), are associated with cardiac sympathetic denervation, as reflected by low myocardial concentrations of 6-[ 18F]fluorodopamine-derived radioactivity. We studied the impact of such denervation on cardiac chronotropic and inotropic function. Methods Cardiac inotropic function was assessed by the pre-ejection period index and the systolic time ratio index in response to the directly acting beta-adrenoceptor agonist, isoproterenol, and to the indirectly acting sympathomimetic amine, tyramine, in patients with PD + NOH or PAF (PD + NOH/PAF group, N = 13). We compared the results to those in patients with multiple system atrophy, which usually entails NOH with normal cardiac sympathetic innervation (MSA, N = 15), and in normal control subjects ( N = 5). Results The innervated and denervated groups did not differ in baseline mean pre-ejection period index or systolic time ratio index. Tyramine increased cardiac contractility in the MSA patients and controls but not in the PD + NOH/PAF group. For similar heart rate responses, the PD + NOH/PAF group required less isoproterenol ( p < 0.01) and had lower plasma isoproterenol levels ( p < 0.01) than did the MSA group. Conclusions Among patients with NOH those with cardiac sympathetic denervation have an impaired inotropic response to tyramine and exaggerated responses to isoproterenol. This pattern suggests that cardiac denervation is associated with decreased ability to release endogenous norepinephrine from sympathetic nerves and with supersensitivity of cardiac beta-adrenoreceptors.

Neonatal 6-hydroxydopamine lesions of the frontal cortex in rats: Persisting effects on locomotor activity, learning and nicotine self-administration

Dopaminergic innervation of the frontal cortex in adults is important for a variety of cognitive functions and behavioral control. However, the role of frontal cortical dopaminergic innervation for neurobehavioral development has received little attention. In the current study, rats were given dopaminergic lesions in the frontal cortex with local micro-infusions of 6-hydroxydopamine (6-OHDA) at 1 week of age. The long-term behavioral effects of neonatal frontal cortical 6-OHDA lesions were assessed in a series of tests of locomotor activity, spatial learning and memory, and i.v. nicotine self-administration. In addition, neurochemical indices were assessed with tissue homogenization and HPLC in the frontal cortex, striatum, and nucleus accumbens of neonatal and adult rats after neonatal 6-OHDA lesions. In neonatal rats, frontal 6-OHDA lesions as intended caused a significant reduction in frontal cortical dopamine without effects on frontal cortical 5-HT and norepinephrine. The frontal cortical dopamine depletion increased 5-HT and norepinephrine levels in the nucleus accumbens. Locomotor activity assessment during adulthood in the figure-8 maze showed that lesioned male rats were hyperactive relative to sham-lesioned males. Locomotor activity of female rats was not significantly affected by the neonatal frontal 6-OHDA lesion. Learning and memory in the radial-arm maze was also affected by neonatal frontal 6-OHDA lesions. There was a general trend toward impaired performance in early maze acquisition and a paradoxical improvement at the end of cognitive testing. Nicotine self-administration showed significant lesion×sex interactions. The sex difference in nicotine self-administration with females self-administering significantly more nicotine than males was reversed by neonatal 6-OHDA frontal cortical lesions. Neurochemical studies in adult rats showed that frontal cortical dopamine and DOPAC levels significantly correlated with nicotine self-administration in the 6-OHDA-lesioned animals but not in the controls. Frontal cortical 5-HT and 5HIAA showed inverse correlations with nicotine self-administration in the 6-OHDA-lesioned animals but not in the controls. These results show that interfering with normal dopamine innervation of the frontal cortex during early postnatal development has persisting behavioral effects, which are sex-specific.

Confocal microscopy: when is it helpful to diagnose corneal and conjunctival disease?

In vivo confocal microscopy (IVCM) is able to demonstrate the characteristic corneal and conjunctival anatomy in vivo at the cellular level. Normal corneal innervation and cell distribution, as well as changes associated with age, contact lens wear and systemic disease such as diabetes, can be documented in vivo with this technique. IVCM is helpful to differentiate corneal dystrophies and inclusion keratopathies. Infectious agents, such as fungi and Acanthameba, may be visualized directly in the cornea in keratitis. Postsurgical applications include its use following refractive surgery, UV-crosslinking, keratoplasty and amniotic membrane transplantation to evaluate corneal wound healing. Conjunctivalization of the cornea in presumed limbal insufficiency can also be shown by IVCM. Laser scanning IVCM also allows the examination of conjunctival structures, as well as the lid margin. Thus, IVCM may help to establish a final diagnosis in atypical conjunctival inflammation and uncharacteristic conjunctival tumors. Furthermore, IVCM has proven useful in the follow-up of filtering blebs after trabeculectomy.

Structural and functional cardiac cholinergic deficits in adult neurturin knockout mice

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the mouse heart. This study used NRTN knockout (KO) and wild type (WT) mice to determine the impact of NRTN deletion on adult intracardiac cholinergic neurons and nerve fibers and on cardiac cholinergic function. Immunohistochemical analysis of atrial tissue stained for vesicular ACh transporter revealed that sinoatrial node cholinergic nerve density was reduced by more than 40% in 8 week KO hearts. Additionally, the number of cholinergic neurons in KO hearts was 80% less than in WT, and KO neurons were 22% smaller in size. Atrial ACh levels were significantly lower in 16 week KO mice compared to WT (1.8±0.3 vs 3.7±0.8 pmol/mg wt; P&lt;0.02), as expected from cholinergic neuron and nerve fiber deficits. Maximum heart rate (HR) responses to right vagal nerve stimulation (VNS) were decreased in 16 week KO (38±6 vs 81±3% decrease at 20Hz; P&lt;0.0001), with no difference in baseline HR. KO animals took longer to reach maximum bradycardia during VNS and longer to recover to baseline HR upon stimulus termination. Data from isolated atrial preparations showed no change in postjunctional cholinergic sensitivity. These results demonstrate that NRTN KO leads to structural and functional cholinergic deficits in the heart but residual cholinergic nerve fibers retain ability to modify cardiac function. Supported by the AHA SE Affiliate.

The Neurotrophic Effects of Glial Cell Line-Derived Neurotrophic Factor on Spinal Motoneurons Are Restricted to Fusimotor Subtypes

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

The α10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system

Although homomeric channels assembled from the alpha9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both alpha9 and alpha10 subunits. To gain insight into alpha10 subunit function in vivo, we examined olivo cochlear innervation and function in alpha10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in alpha10(+/+) and alpha10(+/-) mice, with no detectable responses to ACh in alpha10(-/-) mice. In contrast, a proportion of alpha10(-/-) outer hair cells showed small ACh-evoked currents. In alpha10(-/-) mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual alpha9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, alpha10(-/-) mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that alpha9(-/-) and alpha10(-/-) mice have overlapping but nonidentical phenotypes. Moreover, alpha10 nAChR subunits are required for normal olivocochlear activity because alpha9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in alpha10(-/-) mutant mice.

Morphologic analysis of the neuromuscular development of the anorectal unit in fetal rats with retinoic acid induced myelomeningocele

To investigate whether myelomeningocele (MMC) is associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution and staining intensity of non-neuronal (alpha-smooth-muscle-actin), neural crest cell (NCC, [Hoxb5]), and neuronal markers (PGP-9.5, synaptophysin, neurotubulin-beta-III) within the distal colon, rectum, and anal sphincters were analyzed by immunohistochemistry and Western blot in rat fetuses with retinoic acid (RA) induced MMC. At term (E22), no gross-morphological differences of the anorectal unit of OIL (n=21) MMC (n=31), and RA-exposed-non MMC (RA, n=19) fetuses were found. Smooth muscle cells were evenly distributed within the muscle layers of the rectum and the internal anal sphincter in OIL, MMC, and RA fetuses. Density and staining intensity of NCC and mature enteric neurons within the myenteric plexus of the distal colon and rectum and innervation pattern within anal sphincters in MMC fetuses were analogous to RA and OIL controls. Normal smooth muscle and myenteric plexus development of the rectum and normal innervation of the anal sphincters and pelvic floor suggests that MMC is not associated with a global neuromuscular maldevelopment of lower GI structures in this short-gestational model.

A simple slice culture system for the imaging of nerve development in embryonic mouse

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest.

EMLA and Water Immersion Cause Similar Vasodilatation in Replanted Fingers

Skin wrinkling on water immersion is a reliable and simple test of sympathetic innervation. The eutectic mixture of local anesthetic (EMLA) cream has been shown to induce near identical clinical wrinkling scores and reduction in digit blood flow as that following water immersion in people with normal sympathetic innervation. This study was designed to investigate the vasomotor response to EMLA in replanted fingers that had poor sympathetic innervation. Laser Doppler imaging (PeriScan PIM II; Perimed AB, Stockholm, Sweden) was used to detect perfusion changes in the pulps of fourteen replanted fingers before and after 0.5 g of 5% EMLA cream application and water immersion in a 40 degrees C normal saline for 30 min, respectively. Comparisons were made with the contralateral corresponding normal fingers. After water immersion and EMLA application, all of the normal fingers showed a considerable and similar decrease in blood perfusion that demonstrated in the absolute perfusion units (pU) (baseline: 1.57 +/- 0.33 pU, after water-immersion, 1.19 +/- 0.22 pU, P < 0.001; decrease: 23.6 +/- 7.7%, after EMLA application: 1.20 +/- 0.18 pU, P < 0.001; decrease: 22.4 +/- 8.9%). In contrast, all of the replanted fingers showed a statistically significant vasodilatatory response (baseline: 1.20 +/- 0.29 pU, after water-immersion: 1.36 +/- 0.28 pU, P < 0.001; increase: 15.2 +/- 9.1%, after EMLA application: 1.38 +/- 0.27 pU, P < 0.001; increase: 16.8 +/- 9.1%). EMLA and water immersion both cause vasodilatation and no skin wrinkling in replanted fingers. These results imply that intact sympathetic nerve function is required to induce the vasoconstrictive effect of EMLA.

Uterine innervation in fibroids: A qualitative study

SummaryThis study describes the innervation of uterine fibroids in a retrospective survey of archived material. A total of 24 uteri containing fibroids were identified; four nulliparous uteri with single large fundal fibroids (200 – 320 g) and 20 multiparous uteri with one, or more, fibroids (80 – 230 g). Tissue blocks from the uterine isthmus and the fibroids, were identified, sectioned and stained for nerves with anti-S100 using a standard immunohistochemical regimen. Normal uterine innervation includes concentrations of nerves in the subserosal layer and at the endometrial-myometrial interface with sparse neurovascular bundles distributed throughout the myometrial stroma. Nulliparous uteri with single large fibroids demonstrated relatively normal patterns of innervation with nerves distributed throughout the stroma of the fibroid. In multiparous uteri with fibroids, there were no nerves detectable in the substance of the fibroids. Increased numbers of nerve fibres were observed in the pseudo-capsule of some fibroids and may reflect compression of normal myometrial tissue and their contained nerves, or, a minor degree of nerve fibre proliferation. This study demonstrates that fibroids in multiparous uteri do not contain nerves. Single, large nulliparous fibroids situated at the uterine fundus appear to contain relatively normal patterns of innervation.

Uterine innervation in adenomyosis

SummaryThis study describes the innervation of the uterus with a histopathological diagnosis of adenomyosis in a retrospective survey of two groups of uteri. Group 1 consisted of 17 histologically-normal, parous uteri and eight nulliparous uteri. Group 2 consisted of 23 parous uteri with the histopathological diagnosis of adenomyosis. Tissue sections from the uterine isthmus were stained for nerves with PGP 9.5 using a standard immunohistochemical regimen. In group 1 (n = 25, normal histological report), normal innervation of the uterine isthmus included concentrations of nerves in the subserosal layers and at the endometrial-myometrial interface with sparse, neurovascular bundles distributed throughout the myometrial stroma. In group 2, (n = 23 with adenomyosis), there were no nerves in areas of adenomyosis and absence of nerves at the endometrial-myometrial nerve plexus. Focal proliferation of small-diameter nerve fibres was observed at the margins of adenomyosis in some uteri. Subserosal nerve fibres were still present in those sections that extended to include this region. Adenomyosis is associated with loss of nerve fibres at the endometrial-myometrial interface and absence of nerve fibres in the adenomyosis.

Progressive Deafness and Altered Cochlear Innervation in Knock-Out Mice Lacking Prosaposin

After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.

Is spinal cord isolation a good model of muscle disuse?

The patterns of normal daily activity that are required to maintain normal skeletal muscle properties remain unknown. The present study was designed to determine whether spinal cord isolation can be used as a reliable experimental model of neuromuscular inactivity, that is, as a baseline for the absence of activity. Electromyograms (EMGs) were recorded from selected hindlimb muscles of unanesthetized rats over 24-hour periods before and 7, 30, 60, and 90 days after surgical isolation of the lumbar spinal cord. Our data indicate that some rat slow muscle fibers pre-surgery were activated for less than 3 hours per day. Spinal cord isolation (SI) reduced the mean daily integrated EMG (IEMG) and daily EMG duration in the primary slow extensor muscle (soleus) to <1% of control, and in the primary fast extensor muscles [medial gastrocnemius (MG) and vastus lateralis (VL)] to <2% of control. These parameters were decreased to <8% and 3% of control, respectively, in a primary fast flexor muscle, the tibialis anterior (TA). From 30 to 90 days post-SI, the mean amplitudes of the spontaneous EMG bursts were relatively normal in the soleus, increased approximately 2-fold in the MG and VL, and increased approximately 4-fold in the TA. Some evidence of the normal antagonistic flexor-extensor relationship was apparent in the brief periods of recorded activity post-SI. These results indicate that SI eliminates nearly all of the normal EMG activity in the hindlimb muscles in the presence of relatively normal muscle innervation and functional intraspinal neural circuitry.

Comparison of Orbital Magnetic Resonance Imaging in Duane Syndrome and Abducens Palsy

To help resolve the clinical ambiguity between Duane syndrome with severe abduction deficit and abducens palsy, we performed orbital magnetic resonance imaging (MRI) to qualify abnormalities of the lateral rectus (LR) muscle in these entities. Prospective observational case series. Orbital MRI was performed in 13 subjects with Duane syndrome (19 eyes), 10 subjects with chronic abducens palsy (10 eyes), and 10 orthotropic control subjects (18 eyes). High-resolution, surface coil, T(1)-weighted MRI was used to obtain contiguous, 2-mm thick quasi-coronal images of the orbits in central gaze. Digital image analysis was used to quantify cross-sectional area of the ipsilesional and contralesional LR to provide comparison with control measurements. Mean maximum LR cross-sectional area in Duane syndrome was statistically similar to control (P = .454) and contralesional LR cross-sectional area (P = .227). However, in chronic abducens palsy, mean maximum ipsilesional LR cross-sectional area was markedly smaller than contralesional (P = .003) and control cross-sectional areas (P < .0001), as well as smaller than the LR in Duane syndrome (P= .0017). The LR muscle in abducens palsy exhibits profound atrophy. The sparing of the LR in Duane syndrome from denervation atrophy despite absence of normal abducens innervation suggests existence of alternative LR innervation. High-resolution MRI can noninvasively demonstrate LR muscle size and distinguish Duane syndrome from chronic abducens palsy in uncertain cases.