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Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2021. Published by Elsevier Inc.)

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes compromised function of motor neurons and neuronal death. However, oculomotor neurons are largely spared from disease symptoms. The underlying causes for sporadic ALS as well as for the resistance of oculomotor neurons to disease symptoms remain poorly understood. In this bioinformatic-analysis, we compared the gene expression profiles of spinal and oculomotor tissue samples from control individuals and sporadic ALS patients. We show that the genes GAD2 and GABRE (involved in GABA signaling), and CALB1 (involved in intracellular Ca 2+ ion buffering) are downregulated in the spinal tissues of ALS patients, but their endogenous levels are higher in oculomotor tissues relative to the spinal tissues. Our results suggest that the downregulation of these genes and processes in spinal tissues are related to sporadic ALS disease progression and their upregulation in oculomotor neurons confer upon them resistance to ALS symptoms. These results build upon prevailing models of excitotoxicity that are relevant to sporadic ALS disease progression and point out unique opportunities for better understanding the progression of neurodegenerative properties associated with sporadic ALS.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) Isl MN :GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Purpose: To further chemically characterize palisade endings in extraocular muscles in rhesus monkeys., Methods: Extraocular muscles of three rhesus monkeys were studied for expression of the calcium-binding protein calretinin (CR) in palisade endings and multiple endings. The complete innervation was visualized with antibodies against the synaptosomal-associated protein of 25 kDa and combined with immunofluorescence for CR. Six rhesus monkeys received tracer injections of choleratoxin subunit B or wheat germ agglutinin into either the belly or distal myotendinous junction of the medial or inferior rectus muscle to allow retrograde tracing in the C-group of the oculomotor nucleus. Double-immunofluorescence methods were used to study the CR content in retrogradely labeled neurons in the C-group., Results: A subgroup of palisade and multiple endings was found to express CR, only in the medial and inferior rectus muscle. In contrast, the en plaque endings lacked CR. Accordingly, within the tracer-labeled neurons of the C-group, a subgroup expressed CR., Conclusions: The study indicates that two different neuron populations targeting nontwitch muscle fibers are present within the C-group for inferior rectus and medial rectus, respectively, one expressing CR, one lacking CR. It is possible that the CR-negative neurons represent the basic population for all extraocular muscles, whereas the CR-positive neurons giving rise to CR-positive palisade endings represent a specialized, perhaps more excitable type of nerve ending in the medial and inferior rectus muscles, being more active in vergence. The malfunction of this CR-positive population of neurons that target nontwitch muscle fibers could play a significant role in strabismus.

Background: The cytoarchitecturally defined Edinger-Westphal nucleus (EW) is now referred to by many investigators as the centrally-projecting EW (EWcp) in humans. Although the mature structure is well-characterized, there have been few reports describing the precise morphology of this nucleus during the second half of gestation., Subjects/design: Eleven brains were examined from preterm infants, aged 20-39 postmenstrual weeks, who died of various causes. After fixation, the brains were embedded in celloidin and serial sections of 30-μm thickness were cut in the horizontal plane. Sections were stained using the Klüver-Barrera method. In addition to microscopic observations, computerized 3D reconstruction and morphometry were performed., Results: From 21 weeks, the EWcp had a distinctive, complex 3D structure comprising two or three parts. The dorsal part was arcuate, half encircling the oculomotor somatic nuclei (OSN). The rostral part was the most voluminous, ventral to the rostral OSN, extending anteriorly. The caudal part was the smallest, and was composed of several neuronal groups near the ventral tip of the OSN. In three cases, the caudal part was absent. It could also be joined to the rostral part, forming a ventral part. The total volume of the EWcp increased exponentially with age, and the ventral part grew more rapidly than the dorsal part. The mean neuronal profile area increased linearly with age, and the rate of increase was almost equal between the dorsal and ventral parts., Conclusions: This study suggests that a distinctive, complex, two- or three-part 3D structure of the EWcp is preserved after mid-gestation, and that the ventral part of the EWcp may expand in volume more rapidly than the dorsal part., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We investigated the microscopic structure of transverse sections of the oculomotor, trochlear and abducens nerves of Arabian foals using stereological methods. Bilateral nerve pairs from 2-month-old female Arabian foals were analyzed. The tissues were embedded in plastic blocks, then 1 µm thick sections were cut and stained with osmium tetroxide and methylene blue-azure II. Stereology was performed using light microscopy. Morphometry showed that the right and left pairs of nerves were similar. The transverse sectional areas of the oculomotor, trochlear and abducens nerves were 1.93 ± 0.19 mm 2 , 0.32 ± 0.06 mm 2 and 0.70 ± 0.08 mm 2 , respectively. The oculomotor nerve exhibited a significantly greater number of myelinated axons (16755 ± 1279) and trochlear (2656 ± 494) and the abducens nerves (4468 ± 447). The ratio of the axon diameter to myelinated nerve fiber diameter was 0.58, 0.55 and 0.55 for the oculomotor, trochlear and abducens nerves, respectively. Of the three nerves studied, the abducens nerve exhibited the greatest nerve fiber area, myelin area, nerve and axon diameters, and myelin thickness. The ratio of small myelinated nerve fibers was greatest in the oculomotor nerve.

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3β phosphorylation and β-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123-4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4-6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 μm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Technical developments have extensively promoted experimental and clinical studies on cranial nerve regeneration, but intracranial nerve recovery is still an unexplored research area compared to peripheral nerve repair. In this study, we researched whether neurogenesis occurs in adult oculomotor (OMN) and Edinger-Westphal nucleus (EWN) or not after oculomotor nerve injury. To assess cell proliferation in response to unilateral oculomotor nerve crush (ONC) in adult beagle dog, repetitive 5-bromo-2'-deoxyuridine (BrdU) intravenous injections were performed during 3 or 7 days before the dogs were euthanized 2 h after the last injection on days 3, 7, 14, and 28 post-ONC. The proliferating cell types were investigated with three cell phenotypic markers and confocal microscopy on serial sections throughout the whole extent of OMN and EWN. BrdU-positive nuclei were detected in bilateral OMNs and EWNs from 3 to 28 days after ONC with the peak value at 3 days. Confocal analysis revealed that partial BrdU-positive cells colocalized with nestin or βIII-tubulin or GFAP, and the number of every kind of double-labeled cell maintained an increased tendency from 3 to 28 days post-ONC. Neither single-labeled BrdU-positive nuclei nor double-labeled cells were detected in the subependymal layer of cerebral aqueduct (SELCA) of all unilateral ONC dogs; also, they were not observed in the OMNs, EWNs, and SELCA of intact and sham-operated dog. These findings demonstrate that ONC can trigger continual mitotic activity, proliferation of NSCs, neurogenesis, and astrogliogenesis in the OMN and EWN of adult dogs.

Perinatal hypoxia could cause long-term disturbances of the dopaminergic (DA) systems, leading to behavioral and/or neurological deficits later in life. Increased expression of tyrosine hydroxylase (TH) was shown in the substantia nigra (SN) and ventral tegmental area (VTA) of human neonates that suffered severe/acute perinatal hypoxic insults, but also in all neurons of the Edinger-Westphal nucleus (EW). Since EW, in humans, contains urocortin 1 (UCN1)/centrally projecting neurons (EWcp), we investigated: (a) the development of UCN1-positive neurons and the possible effect of neonatal hypoxic/ischemic encephalopathy on UCN1 expression and (b) the possible colocalization of UCN1 with TH in neonates with histological signs of acute hypoxic injury. Our results showed that in EWcp of the human neonate, UCN1-immunoreactivity was already evident from 34 weeks of gestation onwards at very low levels. No UCN1-immunoreactivity was found in neurons of SN or VTA. In EWcp, a positive correlation was found between UCN1 expression and the age of the neonates, but not with hypoxia neuropathological grade. UCN1 was colocalized with TH in most EWcp neurons. Since UCN1 in EWcp may play a significant role in stress adaptation and consequently in stress-related disorders, the role of catecholamine synthesis in this nucleus under acute hypoxic conditions must be further investigated., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

The ocular motor system consists of three nerves which innervate six muscles to control eye movements. In humans, defective development of this system leads to eye movement disorders, such as Duane Retraction Syndrome, which can result from mutations in the α2-chimaerin signaling molecule. We have used the zebrafish to model the role of α2-chimaerin during development of the ocular motor system. We first mapped ocular motor spatiotemporal development, which occurs between 24 and 72 h postfertilization (hpf), with the oculomotor nerve following an invariant sequence of growth and branching to its muscle targets. We identified 52 hpf as a key axon guidance "transition," when oculomotor axons reach the orbit and select their muscle targets. Live imaging and quantitation showed that, at 52 hpf, axons undergo a switch in behavior, with striking changes in the dynamics of filopodia. We tested the role of α2-chimaerin in this guidance process and found that axons expressing gain-of-function α2-chimaerin isoforms failed to undergo the 52 hpf transition in filopodial dynamics, leading to axon stalling. α2-chimaerin loss of function led to ecotopic and misguided branching and hypoplasia of oculomotor axons; embryos had defective eye movements as measured by the optokinetic reflex. Manipulation of chimaerin signaling in oculomotor neurons in vitro led to changes in microtubule stability. These findings demonstrate that a correct level of α2-chimaerin signaling is required for key oculomotor axon guidance decisions, and provide a zebrafish model for Duane Retraction Syndrome.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops after exposure to a life-threatening traumatic experience. Mental disorder appears after the traumatic stress incident and affects the movement of the eye muscle dominated by the oculomotor nucleus, an important nuclear group of the brainstem. It has been reported that dysfunction of the neurotransmitter 5-hydroxytryptamine (5-HT) can lead to the instability of the internal environment in response to stress and plays an important role in the pathology of PTSD and that the 5-HT1A receptor (5-HT1AR) is critically involved in regulating mood and anxiety levels. In this study, the 5-HT1AR expression in the oculomotor nucleus was examined in rats with single-prolonged stress (SPS), a well established post-traumatic stress disorder animal model. Our results show that the expression of 5-HT1AR in the oculomotor nucleus neurons gradually increased 1, 4, and 7 days after exposure to SPS in comparison to the normal control group, measured by immunohistochemistry, western blotting, and reverse transcription polymerase chain reaction (RT-PCR). The expression of 5-HT1AR reached its peak 7 days after the SPS exposure and then decreased 14 days after. There is also a change in the ultrastructure in the oculomotor nucleus neuron upon SPS treatment which was observed by transmission electron microscopy. These results suggest that SPS can induce a change of the 5-HT1AR expression in the oculomotor nucleus, which may be one of the molecular mechanisms that lead to PTSD.

Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2(max) duration of 2-12 s, velocity of 25-50 μm/s) and two fast "flash-like" types (Type 1, 30-90 ms; Type 2, 90-150 ms 1/2(max) duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

TDP-43 has been identified as a major component of the pathological inclusions in most forms of frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). In the present study, paraffin sections of the midbrain in 112 patients with various non-ALS disorders and 27 patients with sporadic ALS were immunostained with antibody against phosphorylated TDP-43 (pTDP-43). pTDP-43-positive inclusions in oculomotor neurons were detected in 18 of 112 patients with non-ALS disorders (16.1%). The appearance of the inclusions showed fine filamentous structures rather than the skein-like inclusions seen in the anterior horn cells of ALS spinal cords. The incidence was increased in the age range of 80-89 years old (10/37 cases; 27.0%), in which 6 of 10 cases demonstrated AD pathology in the temporal lobes. Twenty-seven ALS patients were examined and the findings were compared with those of non-ALS patients. There were 13 cases demonstrating pTDP-43-positive inclusions (48.1%) which showed stronger immunoreactivities in ALS cases. This is the first report demonstrating fine filamentous pTDP-43-positive inclusions in oculomotor neurons in non-ALS disorders. Although the mechanisms underlying pTDP-43 in oculomotor neurons are currently unknown, its detection is of interest, and the expression may occur not only in ALS but also during the aging process., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pituitary adenylate-cyclase activating polypeptide (PACAP) has been implicated in the (patho)physiology of stress-adaptation. PACAP deficient (PACAP(-/-)) mice show altered anxiety levels and depression-like behavior, but little is known about the underlying mechanisms in stress-related brain areas. Therefore, we aimed at investigating PACAP(-/-) mice in light-dark box, marble burying, open field, and forced swim paradigms. We also analyzed whether the forced swim test-induced c-Fos expression would be affected by PACAP deficiency in the following stress-related brain areas: magno- and parvocellular paraventricular nucleus of the hypothalamus (PVN); basolateral (BLA), medial (MeA), and central (CeA) amygdaloid nuclei; ventral (BSTv), dorsolateral (BSTdl), dorsomedial (BSTdm), and oval (BSTov) nuclei of the bed nucleus of stria terminalis; dorsal (dLS) and ventral parts (vLS) of lateral septal nucleus, central projecting Edinger-Westphal nucleus (EWcp), dorsal (dPAG) and lateral (lPAG) periaqueductal gray matter, dorsal raphe nucleus (DR). Our results revealed that PACAP(-/-) mice showed greatly reduced anxiety and increased locomotor activity compared with wildtypes. In forced swim test PACAP(-/-) mice showed increased depression-like behavior. Forced swim exposure increased c-Fos expression in all examined brain areas in wildtypes, whereas this was markedly blunted in the DR, EWcp, BSTov, BSTdl, BSTv, PVN, vLS, dPAG, and in the lPAG of PACAP(-/-) mice vs. wildtypes, strongly suggesting their involvement in the behavioral phenotype of PACAP(-/-) mice. PACAP deficiency did not influence the c-Fos response in the CeA, MeA, BSTdm, and dLS. Therefore, we propose that PACAP exerts a brain area-specific effect on stress-induced neuronal activation and it might contribute to stress-related mood disorders., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recent monkey studies showed that motoneurons of the oculomotor nucleus involved in upward eye movements receive a selective input from afferents containing calretinin (CR). Here, we investigated the sources of these CR-positive afferents. After injections of tract-tracers into the oculomotor nucleus (nIII) of two monkeys, the retrograde labeling was combined with CR-immunofluorescence in frozen brainstem sections. Three sources of CR inputs to nIII were found: the rostral interstitial nucleus of the medial longitudinal fascicle (RIMLF), the interstitial nucleus of Cajal, and the y-group. CR is not present in all premotor upward-moving pathways. The excitatory secondary vestibulo-ocular neurons in the magnocellular part of the medial vestibular nuclei contained nonphosphorylated neurofilaments, but no CR, and they received a strong supply of large CR-positive boutons. In conclusion, the present study presents evidence that only specific premotor pathways for upward eye movements--excitatory upgaze pathways--contain CR, but not the up vestibulo-ocular reflex pathways. This property may help to differentiate between premotor up- and downgaze pathways in correlative clinico-anatomical studies in humans., (© 2011 New York Academy of Sciences.)

Ataxia with oculomotor apraxia type1 (AOA1, MIM 208920) is a rare autosomal recessive disease caused by mutations in the APTX gene. We screened a cohort of 204 patients with cerebellar ataxia and 52 patients with early-onset isolated chorea. APTX gene mutations were found in 13 ataxic patients (6%). Eleven patients were homozygous for the known p.W279X, p.W279R, and p.P206L mutations. Three novel APTX mutations were identified: c.477delC (p.I159fsX171), c.C541T (p.Q181X), and c.C916T (p.R306X). Expression of mutated proteins in lymphocytes from these patients was greatly decreased. No mutations were identified in subjects with isolated chorea. Two heterozygous APTX sequence variants (p.L248M and p.D185E) were found in six families with ataxic phenotype. Analyses of coenzyme Q10 in muscle, fibroblasts, and plasma demonstrated normal levels of coenzyme in five of six mutated subjects. The clinical phenotype was homogeneous, irrespectively of the type and location of the APTX mutation, and it was mainly characterized by early-onset cerebellar signs, sensory neuropathy, cognitive decline, and oculomotor deficits. Three cases had slightly raised alpha-fetoprotein. Our survey describes one of the largest series of AOA1 patients and contributes in defining clinical, molecular, and biochemical characteristics of this rare hereditary neurological condition.

Unlabelled: The doublecortin-like kinase (DCLK) gene is crucially involved in neuronal plasticity and microtubule-guided retrograde transport of signaling molecules. We have explored the possibility that DCLK is involved in pain-induced signaling events in adult male Wistar rats. Our results show that both DCLK-short and DCLK-long splice variants are present in the cell body and proximal dendrites of neurons in stress-related nuclei, ie, the paraventricular nucleus of the hypothalamus (PVN) and the non-preganglionic Edinger-Westphal nucleus (npEW) in the rostroventral periaqueductal grey. We found that DCLK-long but not DCLK-short is phosphorylated in its serine/proline-rich domain. Furthermore, we demonstrate that phosphorylation of DCLK-long in the npEW is increased by acute pain, whereas DCLK-long phosphorylation in the PVN remains unaffected. This is the first report revealing that DCLK isoforms in the PVN and npEW occur in the adult mammalian brain and that pain differentially affects DCLK-long-mediated neuronal plasticity in these 2 stress-sensitive brain centers., Perspective: Pain is a burden for society and the individual, and although the mechanisms underlying pain are relatively well known, its treatment remains difficult and incomplete. Pain stress can lead to diseases like chronic pain and depression. The differential DCLK-phosphorylation in stress-sensitive brain areas is a potential novel therapeutic target in pain research., (Copyright © 2010 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Purpose: To search for proprioceptive nerve terminals in human ciliary muscle., Methods: In 48 human donor eyes, histologic and ultrathin sections cut in different planes and wholemounts of the ciliary muscle were studied. Immunohistochemical staining with antibodies against pan-neuronal antigens and antigens reported as markers for sensory terminals in other organs was performed., Results: Among the markers for proprioceptive terminals, only calretinin was present in the ciliary body. Calretinin-immunoreactive (IR) nerve terminals surrounded the posterior and reticular ciliary muscle tips and their elastic tendons. Terminals in that region contained mitochondria and neurofilaments. At the anterior tips larger terminals with numerous membrane-filled vesicles were located between the muscle fibers. The most elaborate network of calretinin-IR nerve fibers was present in the ground plate covering the circular muscle portion. Here calretinin-IR neurons with morphologic features of mechanoreception were present. Within the circular muscle portion numerous calretinin-IR ganglion cells were found. Their processes were connected to the calretinin-IR network but also surrounded ciliary muscle cells and NADPH-diaphorase-positive ganglion cells., Conclusions: These morphologic findings indicate that there are proprioreceptors in the ciliary muscle that morphologically and presumably functionally differ at different locations. At the posterior muscle tips, the receptors could measure stretch of the tendons, whereas the large receptor organs located at the anterior muscle tips morphologically resemble mechanoreceptors measuring shear stress. The presence of the numerous intrinsic nerve cells indicates that contraction of the circular muscle portion can be modulated locally via a self-contained reflex arc.

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed-end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti-Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.

Little is known about the cues controlling the generation of motoneuron populations in the mammalian ventral midbrain. We show that Otx2 provides the crucial anterior-posterior positional information for the generation of red nucleus neurons in the murine midbrain. Moreover, the homeodomain transcription factor Nkx6-1 controls the proper development of the red nucleus and of the oculomotor and trochlear nucleus neurons. Nkx6-1 is expressed in ventral midbrain progenitors and acts as a fate determinant of the Brn3a(+) (also known as Pou4f1) red nucleus neurons. These progenitors are partially dorsalized in the absence of Nkx6-1, and a fraction of their postmitotic offspring adopts an alternative cell fate, as revealed by the activation of Dbx1 and Otx2 in these cells. Nkx6-1 is also expressed in postmitotic Isl1(+) oculomotor and trochlear neurons. Similar to hindbrain visceral (branchio-) motoneurons, Nkx6-1 controls the proper migration and axon outgrowth of these neurons by regulating the expression of at least three axon guidance/neuronal migration molecules. Based on these findings, we provide additional evidence that the developmental mechanism of the oculomotor and trochlear neurons exhibits more similarity with that of special visceral motoneurons than with that controlling the generation of somatic motoneurons located in the murine caudal hindbrain and spinal cord.

Sonic hedgehog (Shh) is well known as the molecule responsible for the induction and maintenance of ventral neural tube structures. Recent data have shown that ventral neuronal populations react differentially to the amount of this morphogen not only in the spinal cord, but also in more rostral parts of the brain, like the midbrain. A dorsal expansion in the Shh expression domain modifies the differentiation program in this territory. The lack of Shh produces alterations in the development of this area as well. Here, for the first time, we analyze in detail the development of the different mesencephalic basal nuclei in the absence of Shh. We report that the oculomotor complex is lost, the dopaminergic populations are strongly affected but the red nucleus is maintained. These results point out that not all the midbrain neuronal populations are dependent on Shh for their maintenance, as previously thought. Based on our results and recently published data, we suggest the existence of a specific genetic pathway for the specification of the mesencephalic red nucleus. Foxa2 could be the candidate gene that might control this genetic pathway.

The prepositus hypoglossi (PH) nucleus has been proposed as a pivotal structure for horizontal eye-position generation in the oculomotor system. Recent studies have revealed that acetylcholine (ACh) in the PH nucleus could mediate the persistent activity necessary for this process, although the origin of this ACh remains unknown. It is also known that nitric oxide (NO) in the PH nucleus plays an important role in the control of velocity balance, being involved in a negative feedback control of tonic signals arriving at the PH nucleus. As it could be expected that neurons taking part in eye-position generation must control their tonic background inputs, the existence of a relationship between nitrergic and cholinergic neurons is hypothesized. In the present study we analyzed the distribution, size, and morphology of choline acetyltransferase-positive neurons, and their relationship with neuronal nitric oxide synthase in the PH nucleus of the cat. As presumed, some 96% of cholinergic neurons were also nitrergic in the PH nucleus, suggesting that NO is regulating the level of ACh released by cholinergic PH neurons. Furthermore, we studied the alterations induced by muscarinic-receptor agonists and antagonists on spontaneous and vestibularly induced eye movements in the alert cat and compared them with those induced in previous studies by modification of NO levels in the same animal preparation. The results suggest that ACh is necessary for the generation of saccadic and vestibular eye-position signals, whereas the NO is stabilizing the eye-position generator by controlling background activity reaching cholinergic neurons in the PH nucleus.

We have compared the expression pattern of NMDA receptor subunits (NR1 and NR2A-D) and NR1 splice variants (NR1-1a/1b,-2a/2b,-3a/3b,-4a/4b) in motor neuron populations from adult Wistar rats that are vulnerable (hypoglossal, XII) or resistant (oculomotor, III) to death in amyotrophic lateral sclerosis (ALS). The major finding was higher levels of expression of the NR2B subunit in the hypoglossal nucleus. Quantitative real-time PCR showed that NR1 was expressed at a greater level than any of the NR2 subunits (>15 fold greater, P

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.

The paralogous paired-like homeobox genes Phox2a and Phox2b are involved in the development of specific neural subtypes in the central and peripheral nervous systems. The different phenotypes of Phox2 knockout mutants, together with their asynchronous onset of expression, prompted us to generate two knock-in mutant mice, in which Phox2a is replaced by the Phox2b coding sequence, and vice versa. Our results indicate that Phox2a and Phox2b are not functionally equivalent, as only Phox2b can fulfill the role of Phox2a in the structures that depend on both genes. Furthermore, we demonstrate unique roles of Phox2 genes in the differentiation of specific motor neurons. Whereas the oculomotor and the trochlear neurons require Phox2a for their proper development, the migration of the facial branchiomotor neurons depends on Phox2b. Therefore, our analysis strongly indicates that biochemical differences between the proteins rather than temporal regulation of their expression account for the specific function of each paralogue.

The question whether general tetanus arises from the independent sum of multiple local tetani or results from the actions of the transynaptic tetanus neurotoxin (TeNT) in higher brain centres remains unresolved. Despite the blood-borne dissemination of TeNT from an infected wound, the access to the central nervous system is probably prevented by the blood-brain barrier. However, several long-term sequelae (e.g. autonomic dysfunction, seizures, myoclonus, and sleep disturbances) present after the subsidence of muscle spasms might be indicative of central actions that occur farther away from lower motoneurons. Subsequently, the obvious entry route is the peripheral neurons followed by the transynaptic passage to the brain. We aimed at describing the pathophysiological correlates of TeNT translocation using the oculomotor system as a comprehensive model of cell connectivity and neuronal firing properties. In this study, we report that injection of TeNT into the medial rectus muscle of one eye resulted in bilateral gaze palsy attributed to firing alterations found in the contralaterally projecting abducens internuclear neurons. Functional alterations in the abducens-to-oculomotor internuclear pathway resembled in part the classically described TeNT disinhibition. We confirmed the transynaptic targeted action of TeNT by analysing vesicle-associated membrane protein2 (VAMP2) immunoreactivity (the SNARE protein cleaved by TeNT). VAMP2 immunoreactivity decreased by 94.4% in the oculomotor nucleus (the first synaptic relay) and by 62.1% presynaptic to abducens neurons (the second synaptic relay). These results are the first demonstration of physiological changes in chains of connected neurons that are best explained by the transynaptic action of TeNT on premotor neurons as shown with VAMP2 immunoreactivity which serves as an indicator of TeNT activity.

Nerve transection induces complex changes in gene regulation and expression that can have profound phenotypic effects on the fate of axotomized neurons. The transcription factors c-Jun and ATF-2 (activating transcription factor-2) are components of a regulatory network that mediates survival, regeneration, and apoptosis following axotomy in rodents. The activation and function of c-Jun and ATF-2 after nerve injury have not been examined in primates. Using a novel model of cranial nerve injury in baboons, we have examined the temporality of c-Jun activation (phosphorylation) in cranial nerve (CN) III and CN VI neurons and ATF-2 activation in CN VI neurons at 2, 4, and 9 days post-injury by immunohistochemistry. Furthermore, we have addressed whether the activation of these factors is associated with apoptosis by the TUNEL assay. We report that activated c-Jun is present in CN III and CN VI neurons ipsilateral to axotomy at 2, 4, and 9 days post-injury, but not in neurons contralateral to injury. Additionally, CN VI neurons ipsilateral to injury at 4 and 9 days contain activated ATF-2. Furthermore, no evidence of TUNEL reactivity was observed in either nucleus, regardless of laterality, at any of the examined time points. These findings suggest that activation of both c-Jun and ATF-2 does not mediate apoptosis in axotomized primate CN III and CN VI neurons at time points up to 9 days. This report serves as a basic inquiry into the neuronal response to cranial nerve injury in primates.

Different lines of studies suggest that both the corticotropin-releasing hormone-related peptide Urocortin I (Ucn) and the neuromodulator nitric oxide (NO) are involved in the regulation of the complex mechanisms controlling feeding and anxiety-related behaviors. The aim of the present study was to investigate the possible interaction between Ucn and NO in the hypothalamic paraventricular nucleus (PVN), an area known to be involved in the modulation of these particular behaviors. Therefore, we mapped local mRNA and peptide/protein presence of both Ucn and the NO producing neuronal NO synthase (nNOS). This investigation was extended to include the hypothalamic supraoptic nucleus (SON) and the Edinger-Westphal nucleus area (EW), the latter being one of the major cellular Ucn-expressing sites. Furthermore, we compared the two predominantly used laboratory rat strains, Wistar and Sprague-Dawley. Ucn mRNA and immunoreactivity were detected in the SON and in the EW. A significant difference between Wistar and Sprague-Dawley rats was found in mRNA levels in the EW. nNOS was detected in all brain areas analyzed, showing a significantly lower immunoreactivity in the PVN and EW of Sprague-Dawley versus Wistar rats. Contrary to some previous reports, no Ucn mRNA and only a very low immunoreactivity were detectable in the PVN of either rat strain. Interestingly, double-labeling immunofluorescence revealed that in the SON approximately 75% of all cells immunoreactive for Ucn were colocalized with nNOS, whereas in the EW only approximately 2% of the Ucn neurons were found to contain nNOS. These findings suggest an interaction between Ucn and NO signaling within the SON, rather than the PVN, that may modulate the regulation of feeding, reproduction, and anxiety-related behaviors., (Copyright 2004 Wiley-Liss, Inc.)

To further our understanding of the role that voltage-activated Ca2+ channels play in the development, physiology and pathophysiology of motoneurones (MNs), we used whole-cell patch-clamp recording to compare voltage-activated Ca2+ currents in oculomotor (III) and hypoglossal (XII) MNs of neonatal [postnatal day (P)1-5] and juvenile (P14-19) rats. In contrast to III MNs that innervate extraocular muscles, XII MNs that innervate tongue muscles mature more rapidly, fire bursts of low frequency action potentials and are vulnerable to degeneration in amyotrophic lateral sclerosis. In neonates, low voltage-activated (LVA) Ca2+ current densities are similar in XII and III MNs but high voltage-activated (HVA) Ca2+ current densities are twofold higher in XII MNs. The HVA Ca2+ channel antagonists (nimodipine and nifedipine for L-type, omega-agatoxin-TK for P/Q-type and omega-conotoxin-GVIA for N-type) revealed that, while N- and P/Q-type HVA Ca2+ channels are present in both MN pools, a 3.5-fold greater P/Q-type Ca2+ current in XII MNs accounts for their greater HVA Ca2+ currents. Developmentally, LVA and HVA Ca2+ current densities decrease in III MNs but remain unchanged in XII MNs. Thus, the differences between these MN pools increase developmentally so that, in juveniles, the LVA Ca2+ current density is twofold greater and the HVA Ca2+ current density is threefold greater in XII compared with III MNs. We propose that this differential expression of LVA and HVA Ca2+ channels in XII and III MNs during development contributes to their distinct physiology and may also be a factor contributing to the greater susceptibility of XII MNs to degeneration as seen in amyotrophic lateral sclerosis.

We examined the expression of the three Trk receptors for neurotrophins (TrkA, TrkB, and TrkC) in the extraocular motor nuclei of the adult cat by using antibodies directed against the full-Trk proteins in combination with horseradish peroxidase retrograde tracing. The three receptors were present in all neuronal populations investigated, including abducens motoneurons and internuclear neurons, medial rectus motoneurons of the oculomotor nucleus, and trochlear motoneurons. They were also present in the vestibular and prepositus hypoglossi nuclei. TrkA, TrkB, and TrkC immunopositive cells were found in similar percentages in the oculomotor and in the trochlear nuclei. In the abducens nucleus, however, a significantly higher percentage of cells expressed TrkB than the other two receptors, among both motoneurons (81.8%) and internuclear neurons (88.4%). The percentages obtained for the three Trk receptors in identified neuronal populations pointed to the colocalization of two or three receptors in a large number of cells. We used confocal microscopy to elucidate the subcellular location of Trk receptors. In this case, abducens motoneurons and internuclear neurons were identified with antibodies against choline acetyltransferase and calretinin, respectively. We found a different pattern of staining for each neurotrophin receptor, suggesting the possibility that each receptor and its cognate ligand may use a different route for cellular signaling. Therefore, the expression of Trk receptors in oculomotor, trochlear, and abducens motoneurons, as well as abducens internuclear neurons, suggests that their associated neurotrophins may exert an influence on the normal operation of the oculomotor circuitry. The presence of multiple Trk receptors on individual cells indicates that they likely act in concert with each other to regulate distinct functions.

Central stress regulatory pathways utilize various neurotransmitters/neuropeptides, such as urocortin (Ucn) and catecholamines. Ucn is most abundantly expressed in the Edinger-Westphal nucleus (E-WN), co-distributed with catecholaminergic terminals. Acute stress recruits E-WN neurons, and ascending catecholaminergic pathways also contribute to the activation of various brain areas in response to stress. We hypothesized that catecholamine and Ucn interactions in the E-WN mediated the recruitment of these neurons in response to stress. Using double-labeling immunohistochemistry, we found close appositions between urocortin-immunoreactive nervous structures and dopaminergic terminals, however, depletion of them had no effect on the activation pattern of E-WN neurons upon acute immune challenge. From these results we conclude that dopaminergic terminals innervating E-WN Ucn neurons do not play a major role in mediating the responses of E-WN neurons upon acute immune challenge.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron survival factor. We show here that in the chick oculomotor system, endogenous GDNF is derived largely from extraocular muscle but less from glial cells and not from muscle spindles. Increased levels of GDNF exclusively in the target rescued 30% of oculomotor neurons that would normally die during developmental cell death, a rate of rescue similar to that with systemic GDNF application. Thus, GDNF supports motoneuron survival in a retrograde, target-derived fashion, as opposed to a local paracrine route or an indirect route via sensory afferents. Persephin, another member of the GDNF family, did not increase survival with target delivery, despite its retrograde transport from the target. Unlike GDNF, however, persephin increased neurite outgrowth from oculomotor nuclei in vitro. Thus, one GDNF family member acts as a muscle-derived retrograde survival factor, whereas another one has distinct functions on neurite outgrowth.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis (ALS), yet it remains unclear why some groups of motoneurons (MNs) are more vulnerable to degeneration than others. Our aim was to compare, in normal adult rats, the expression of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) in MNs normally affected in ALS (XII and spinal MNs) with those which are spared (III and IV MNs). RT-PCR analysis of tissue punches taken from III and XII motor nuclei revealed mRNA for both 'a' and 'b' splice variants of the mGluR1 and mGluR5 receptor subtypes, with expression of the 'a' variant dominant for both receptor subtypes in III and XII nuclei. Immunolabeling for mGluR1a protein was strong in vulnerable (XII and spinal) but negligible in the resistant (III and IV) MNs. Immunoreactivity for mGluR5 was not detected in the cell bodies or proximal dendrites of any MN pool examined. Greater expression of mGluR1a receptor protein within vulnerable MN pools may predispose these neurons to neurodegeneration as seen in ALS.

Nitric oxide (NO) production by specific neurons in the prepositus hypoglossi (PH) nucleus is necessary for the correct performance of eye movements in alert cats. In an attempt to characterize the morphological substrate of this NO function, the distribution of nitrergic neurons and NO-responding neurons has been investigated in different brainstem structures related to eye movements. Nitrergic neurons were stained by either immunohistochemistry for NO synthase I or histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The NO targets were identified by cyclic guanosine monophosphate (cGMP) immunohistochemistry in animals treated with a NO donor immediately before fixation of the brain. Connectivity between cells of the NO-cGMP pathway was analyzed by injections of the retrograde tracers horseradish peroxidase or fast blue in different structures. The motor nuclei commanding extraocular muscles did not contain elements of the NO-cGMP pathway, except for some scattered nitrergic neurons in the most caudal part of the abducens nucleus. The PH nucleus contained the largest number of nitrergic cell bodies and a rich neuropil, distributed in two groups in medial and lateral positions in the caudal part, and one central group in the rostral part of the nucleus. An abundant cGMP positive neuropil was the only NO-sensitive element in the PH nucleus, where no cGMP-producing neuronal cell bodies were observed. The opposite disposition was found in the marginal zone between the PH and the medial vestibular nuclei, with a large number of NO-sensitive cGMP-producing neurons and almost no nitrergic cells. Both nitrergic and NO-sensitive cell bodies were found in the medial and inferior vestibular nuclei and in the superior colliculus, whereas the lateral geniculate nucleus contained nitrergic neuropil and a large number of NO-sensitive cell bodies. Some of the cGMP-positive neurons in the marginal zone and medial vestibular nucleus projected to the PH nucleus, predominantly to the ipsilateral side. These morphological findings may help to explain the mechanism of action of NO in the oculomotor system., (Copyright 2001 Wiley-Liss, Inc.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration., (Copyright 2001 Academic Press.)

Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohistochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliable marker for resistance to degeneration in ALS.

Intravenous picrotoxin inhibits spinal nociceptive neurons through disinhibitory activation of neurons in the periaqueductal gray (PAG) and nucleus raphe dorsalis (NRD), where the descending antinociceptive system arises. We found Fos-like immunoreactivity in PAG/NRD neurons after intravenous injection of picrotoxin. This distribution of c-Fos expression is consistent with a role of PAG/NRD for antinociception; neurons with intense Fos-like immunoreactivity was also clustered in the Edinger-Westphal nucleus (EW). Double fluorescence immunohistochemistry for c-Fos and serotonin revealed that PAG/NRD/EW neurons expressing c-Fos were non-serotonergic. These data suggest that non-serotonergic PAG/NRD/EW neurons are involved in the picrotoxin-induced analgesia.

Motoneurones are particularly vulnerable both in human forms of amyotrophic lateral sclerosis (ALS) and corresponding animal models of the disease. While most motoneurone populations are selectively impaired, oculomotor neurones are essentially resistant to ALS-related damage. Motoneurone vulnerability has been closely linked to disruptions of calcium signalling. To investigate underlying events, we performed a quantitative analysis of calcium homeostasis in oculomotor neurones from mice by simultaneous patch-clamp recordings in sliced tissue and microfluorometric-calcium measurements. Somatic calcium dynamics were investigated by using a computer-controlled microfluorometric system. In oculomotor neurones, basal calcium concentrations were around 80 nM and depolarisation-induced calcium responses were observed for membrane voltages positive to -40 u1u1u approximately mV1 approximately . Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. The recovery phase of depolarisation-induced calcium transients was well approximated by a mono-exponential function with a decay time constant that showed a linear dependence on dye concentration. The extrapolated time constant in the absence of indicator dye was 1.7 +/- 0.2 s (n = 11 cells, 21C). Endogenous calcium binding ratios (kappa(s)) were found to be 264 +/- 25 (n = 11 cells), indicating that 99.6 % of cytosolic calcium ions were taken up by endogenous buffers. Recovery of calcium transients was characterised by an 'effective' extrusion rate gamma = 156 +/- 20 s-1 (n = 11 cells, 21 C). Endogenous calcium binding ratios in oculomotor neurones were 5- to 6-fold larger compared with those of more vulnerable motoneurones in the nucleus hypoglossus and spinal cord. In a first order approximation, they reduced the volume of local calcium elevations around open calcium channels, lowered peak amplitudes of global calcium transients for a given influx and prolonged calcium recovery times for a given set of uptake and extrusion mechanisms. With respect to motoneurone degeneration, our measurements suggest that the exceptional stability of oculomotor neurones partially results from a specialised calcium homeostasis based on high buffering capacities. Furthermore, they indicate that cellular adaptations that account for rapid calcium signalling in hypoglossal and spinal motoneurones enhance their vulnerability during ALS-related motoneurone disease.

SOD-1-deficient mice demonstrate no loss of motoneurons but are still vulnerable to axotomy and ischemic insults. To investigate possible reasons for vulnerability of motoneuron populations, we studied changes in ultrastructural calcium distribution during maturation in spinal- and oculomotor neurons in SOD-1(-/-) mice. Between 3 and 11 months the cytoplasmic component of the intracellular calcium changed at a lower rate in spinal motoneurons and motor axon terminals in the interosseus muscle of SOD-1(-/-) animals compared to wild-type controls. No such dissimilarities were noted in the oculomotor system, or in mitochondrial calcium contents of either cell type. These data suggest that the lack of SOD-1 may be associated with vulnerability to insult by depletion of non-mitochondrial calcium stores selectively in motoneurons lacking parvalbumin and/or calbindin D28K.

Except during blinks, movements of the upper eyelid are tightly coupled to vertical eye movements. The premotor source for the coordination of lid and eye movements is unknown. The present paper provides the anatomical identification of a new premotor cell group in the rostral mesencephalon of the monkey and human, which lies in close proximity to the premotor center for vertical saccades and is thought to participate in lid-eye coordination. After injections of a retrograde transsynaptic tracer (tetanus toxin fragment C or BII(b)) into the levator palpebrae (LP), the superior rectus (SR), or the inferior oblique (IO) muscle of macaque monkeys, a small circumscribed group of premotor neurons was labeled in the central gray of the rostral mesencephalon, but not after superior oblique or inferior rectus muscle injections. This group lies immediately rostral to the interstitial nucleus of Cajal and medial to the rostral interstitial nucleus of the medial longitudinal fasciculus, each of which contain premotor neurons for vertical saccades, and was termed the M-group. Injections of tritiated leucine into the M-group led to afferent labeling primarily over LP motoneurons. In addition, label was present over the SR- and IO-motoneuron subgroups in the oculomotor nucleus and frontalis muscle motoneurons in the facial nucleus. This projection pattern of the M-group suggests a role in the coordination of the upper eyelid and eyes during upgaze. Double-labeling experiments in macaque monkeys revealed that the M-group is strongly parvalbumin immunoreactive and contains high levels of cytochrome oxidase activity. With these two histochemical markers, the homologue of the M-group was identified in the human brain as well., (Copyright 2000 Wiley-Liss, Inc.)

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, SNAP-25a and SNAP-25b, which arise from alternate splicing and which are differentially expressed throughout the nervous system. In situ hybridization was used to examine the presence and subcellular localization of SNAP-25a and SNAP-25b RNA transcript expression in motor and parasympathetic nuclei associated with cranial nerves. SNAP-25a RNA transcripts were strongly expressed in the parasympathetic Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but weakly expressed in motor nuclei such as the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal and accessory nuclei and in motoneurons of mouse lumbar spinal cord. In contrast, SNAP-25b RNA transcripts were not detectable in the Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but were strongly expressed in the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal, and accessory nuclei and in the motoneurons of mouse lumbar spinal cord. In the parasympathetic cranial nerve nuclei, displaying high levels of SNAP-25a RNA transcripts, the labeling was cytoplasmic, whereas the labeling was nuclear in the cranial nerve motor nuclei, displaying lower levels of transcripts. In contrast, labeling of SNAP-25b RNA transcripts was cytoplasmic in cranial nerve motor nuclei and not detectable in parasympathetic cranial nerve nuclei. Possible explanations for the region-specific and differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts are discussed., (Copyright 1999 Wiley-Liss, Inc.)

Background: A Canadian family with oculoleptomeningeal amyloidosis with both central and peripheral nervous system disorders was described in 1988. Death of affected family members resulted from recurrent cerebral hemorrhage., Objective: To determine if oculoleptomeningeal amyloidosis is caused by a mutation in transthyretin (prealbumin)., Methods: DNA isolated from peripheral blood and archival tissues of affected members of the kindred was studied by direct DNA sequencing and restriction fragment length polymorphism analysis., Results: Direct DNA sequencing identified a thymine-to-cytosine transition at the second base of codon 64, which resulted in a replacement of serine for phenylalanine. This mutation, which creates an additional HinfI site was detected by restriction fragment length polymorphism analysis in each affected individual., Conclusion: In this kindred, oculoleptomeningeal amyloidosis is related to a mutation in transthyretin (Phe64Ser).

Certain species of anti-ganglioside antibodies are associated with specific clinical features in various neurologic diseases. Serum autoantibodies to these minor gangliosides were investigated in a number of neurological diseases in order to examine the biological functions of GD1alpha and GQ1beta. Eleven patients with Guillain-Barré syndrome had remarkably high IgG anti-GD1alpha antibody titers, but no GD1alpha was detected in human peripheral nerve. An absorption study showed that IgG anti-GD1alpha antibodies from eight of the 11 patients were significantly absorbed by GD1a and GM1b, indicative that the IgG anti-GD1alpha antibodies cross-react with GD1a and GM1b. Both GD1a and GM1b have been reported to be target molecules for serum antibodies in certain patients with Guillain-Barré syndrome. GD1alpha may induce the production of IgG anti-GD1alpha antibody which cross-reacts with GD1a or GM1b, and subsequently functions in the development of Guillain-Barré syndrome. The IgGs from six patients with Fisher's syndrome who had the anti-GQ1beta antibody had anti-GQ1b activity as well. All the patients had external ophthalmoplegia, but no GQ1beta was detected in the human oculomotor nerve, further evidence that GQ1b, not GQ1beta, is the molecule targeted by the autoantibody in Fisher's syndrome.

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons., (Copyright 1999 Elsevier Science B. V.)

The rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) contains excitatory and inhibitory burst neurons that are related to the control of vertical and torsional eye movements. In the present study, light microscopic examination of the immunohistochemical localization of amino acid neurotransmitters demonstrated that the riMLF in the cat contains overlapping populations of neurons that are immunoreactive to the putative inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the excitatory neurotransmitters glutamate and aspartate. By using a double-labelling paradigm, GABA-, glutamate-, and aspartate-immunoreactive neurons in the riMLF were retrogradely labelled by transport of horseradish peroxidase (HRP) from the oculomotor and trochlear nuclei. Electron microscopy showed that the oculomotor and trochlear nuclei contain synaptic endings that are immunoreactive to GABA, glutamate, or aspartate. Each neurotransmitter-specific population of synaptic endings has distinctive ultrastructural and synaptic features. Synaptic endings in the oculomotor and trochlear nuclei that are anterogradely labelled by transport of biocytin from the riMLF are immunoreactive to GABA, glutamate, or aspartate. Taken together, the findings from these complimentary retrograde and anterograde double-labelling studies provide rather conclusive evidence that GABA is the inhibitory neurotransmitter, and glutamate and aspartate are the excitatory neurotransmitters, utilized by premotor neurons in the riMLF that are related to the control of vertical saccadic eye movements.

To determine when the calcium-binding protein parvalbumin appears during development, neurons in the chick Edinger Westphal nucleus were examined for parvalbumin immunoreactivity at a variety of embryonic stages. Parvalbumin immunoreactivity appeared on embryonic day 14 (E14, Hamburger and Hamilton stage 40) in predominantly lateral Edinger Westphal neurons. Cytochrome oxidase activity within the nucleus was examined throughout development, as an indicator of physiological activity, and expression of cytochrome oxidase was compared with that of parvalbumin. Cytochrome oxidase activity was found to be uniformly high in all parts of the Edinger Westphal nucleus throughout development. Either the Edinger Westphal nucleus in physiologically active quite early in its development or other energy demands mask the correlation of cytochrome oxidase with electrical activity. Cytochrome oxidase was expressed well before parvalbumin immunoreactivity appeared. Voltage-activated calcium currents were characterized in E12 Edinger Westphal neurons. In both amplitude and composition, E12 calcium currents resemble those of E16 neurons, excluding the possibility that calcium currents appear de novo during or just prior to the appearance of parvalbumin. Both cytochrome oxidase activity and calcium currents are observed in Edinger Westphal neurons well before the appearance of parvalbumin during development. These findings do not exclude the possibility that physiological activity affects the expression of parvalbumin since other factors such as changing patterns of synaptic activity or the appearance of calcium conducting NMDA receptors have yet to be examined. However, they raise the possibility that additional factors such as an intrinsic developmental program or a change in the neuron's basal intracellular calcium requirements may also be involved.

In the present study we examined the possible colocalization of the inhibitory neurotransmitters glycine and GABA in the oculomotor nucleus of the rabbit. Serial sections were processed alternately for glycine and GABA postembedding immuno-cytochemistry. Ultrastructural analysis revealed that all terminals that showed glycine-positive immunoreactivity were also GABA positive; up to 5% of the GABA-positive terminals were also glycine positive.