Ion channel profiles of extraocular motoneurons and internuclear neurons in human abducens and trochlear nuclei.

Introduction: Extraocular muscles are innervated by two anatomically and histochemically distinct motoneuron populations: motoneurons of multiply-innervated fibers (MIF), and of singly-innervated fibers (SIF). Recently, it has been established by our research group that these motoneuron types of monkey abducens and trochlear nuclei express distinct ion channel profiles: SIF motoneurons, as well as abducens internuclear neurons (INT), express strong Kv1.1 and Kv3.1b immunoreactivity, indicating their fast-firing capacity, whereas MIF motoneurons do not. Moreover, low voltage activated cation channels, such as Cav3.1 and HCN1 showed differences between MIF and SIF motoneurons, indicating distinct post-inhibitory rebound characteristics. However, the ion channel profiles of MIF and SIF motoneurons have not been established in human brainstem tissue. Methods: Therefore, we used immunohistochemical methods with antibodies against Kv, Cav3 and HCN channels to (1) examine the human trochlear nucleus in terms of anatomical organization of MIF and SIF motoneurons, (2) examine immunolabeling patterns of ion channel proteins in the distinct motoneurons populations in the trochlear and abducens nuclei. Results: In the examination of the trochlear nucleus, a third motoneuron subgroup was consistently encountered with weak perineuronal nets (PN). The neurons of this subgroup had -on average- larger diameters than MIF motoneurons, and smaller diameters than SIF motoneurons, and PN expression strength correlated with neuronal size. Immunolabeling of various ion channels revealed that, in general, human MIF and SIF motoneurons did not differ consistently, as opposed to the findings in monkey trochlear and abducens nuclei. Kv1.1, Kv3.1b and HCN channels were found on both MIF and SIF motoneurons and the immunolabeling density varied for multiple ion channels. On the other hand, significant differences between SIF motoneurons and INTs were found in terms of HCN1 immunoreactivity. Discussion: These results indicated that motoneurons may be more variable in human in terms of histochemical and biophysiological characteristics, than previously thought. This study therefore establishes grounds for any histochemical examination of motor nuclei controlling extraocular muscles in eye movement related pathologies in the human brainstem.

Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin. METHODS: Human choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein-gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used. RESULTS: In choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity. CONCLUSION: UCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Consensus Paper: Ataxic Gait.

The aim of this consensus paper is to discuss the roles of the cerebellum in human gait, as well as its assessment and therapy. Cerebellar vermis is critical for postural control. The cerebellum ensures the mapping of sensory information into temporally relevant motor commands. Mental imagery of gait involves intrinsically connected fronto-parietal networks comprising the cerebellum. Muscular activities in cerebellar patients show impaired timing of discharges, affecting the patterning of the synergies subserving locomotion. Ataxia of stance/gait is amongst the first cerebellar deficits in cerebellar disorders such as degenerative ataxias and is a disabling symptom with a high risk of falls. Prolonged discharges and increased muscle coactivation may be related to compensatory mechanisms and enhanced body sway, respectively. Essential tremor is frequently associated with mild gait ataxia. There is growing evidence for an important role of the cerebellar cortex in the pathogenesis of essential tremor. In multiple sclerosis, balance and gait are affected due to cerebellar and spinal cord involvement, as a result of disseminated demyelination and neurodegeneration impairing proprioception. In orthostatic tremor, patients often show mild-to-moderate limb and gait ataxia. The tremor generator is likely located in the posterior fossa. Tandem gait is impaired in the early stages of cerebellar disorders and may be particularly useful in the evaluation of pre-ataxic stages of progressive ataxias. Impaired inter-joint coordination and enhanced variability of gait temporal and kinetic parameters can be grasped by wearable devices such as accelerometers. Kinect is a promising low cost technology to obtain reliable measurements and remote assessments of gait. Deep learning methods are being developed in order to help clinicians in the diagnosis and decision-making process. Locomotor adaptation is impaired in cerebellar patients. Coordinative training aims to improve the coordinative strategy and foot placements across strides, cerebellar patients benefiting from intense rehabilitation therapies. Robotic training is a promising approach to complement conventional rehabilitation and neuromodulation of the cerebellum. Wearable dynamic orthoses represent a potential aid to assist gait. The panel of experts agree that the understanding of the cerebellar contribution to gait control will lead to a better management of cerebellar ataxias in general and will likely contribute to use gait parameters as robust biomarkers of future clinical trials.

Delineation of biomarkers and molecular pathways of residual effects of fluoxetine treatment in juvenile rhesus monkeys by proteomic profiling.

Fluoxetine (Prozac™) is the only antidepressant approved by the US Food and Drug Administration (FDA) for the treatment of major depressive disorder (MDD) in children. Despite its considerable efficacy as a selective serotonin reuptake inhibitor, the possible long-term effects of fluoxetine on brain development in children are poorly understood. In the current study, we aimed to delineate molecular mechanisms and protein biomarkers in the brains of juvenile rhesus macaques (Macaca mulatta) one year after the discontinuation of fluoxetine treatment using proteomic and phosphoproteomic profiling. We identified several differences in protein expression and phosphorylation in the dorsolateral prefrontal cortex (DLPFC) and cingulate cortex (CC) that correlated with impulsivity in animals, suggesting that the GABAergic synapse pathway may be affected by fluoxetine treatment. Biomarkers in combination with the identified pathways contribute to a better understanding of the mechanisms underlying the chronic effects of fluoxetine after discontinuation in children.

Outcome Quality After Colorectal Cancer Resection in Certified Colorectal Cancer Centers­Patient-Reported and Short-Term Clinical Outcomes.

BACKGROUND: In this observational study, patient-reported outcomes and short-term clinical outcome parameters in patients with colorectal cancer were studied 12 months after the start of treatment. Outcomes were also compared across German Certified Colorectal Cancer Centres. METHODS: Data were collected from 4239 patients with colorectal cancer who had undergone elective tumor resection in one of 102 colorectal cancer centers and had responded to a quality-of-life questionnaire before treatment (EORTC QLQ-C30 and -CR29). 3142 (74.1%) of these patients completed a post-treatment questionnaire 12 months later. Correlation analyses were calculated and case-mix adjusted comparisons across centers were made for selected patient-reported outcomes, anastomotic insufficiency, and 30-day-mortality. RESULTS: At 12 months, mild improvements were seen in mean quality-of-life scores (66 vs. 62 points), constipation (16 vs. 19), and abdominal pain (15 vs. 17). Worsening was seen in physical function (75 vs. 82) and pain (22 vs. 19). Better patient-reported outcomes at 12 months were associated with better scores before treatment. Better results in at least three of the five scores were associated with male sex, higher educational level, higher age, and private health insurance. Major worsening of fecal incontinence was seen among patients with rectal cancer without a stoma. The largest differences across centers were found with respect to physical function. Anastomotic insufficiency was found in 4.3% of colon cancer patients and 8.2% of rectal cancer patients. 1.9% of patients died within 30 days after their resection. CONCLUSION: Clinicians can use these findings to identify patients at higher risk for poorer patient-reported outcomes. The differences among cancer centers that were found imply that measures for quality improvement would be desirable.

Saccadic premotor burst neurons and histochemical correlates of their firing patterns in rhesus monkey.

Bursting behavior of brainstem premotor burst neurons (BNs) is essential for initiation of saccades and calibrating their metrics. Several ion channel families such as voltage-gated potassium (Kv) channels, low-voltage-activated calcium (Cav3) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of the bursting in neurons. Therefore, it was speculated that ion channels with rapid kinematics are essential for characteristic firing patterns of the BNs and rapid saccade velocities. However, the expression patterns of ion channels are yet to be confirmed. Confirmation would not only support the neuromimetic model predictions for saccade generation in brainstem, but also contemporary views that channelopathies can cause saccade disorders in humans. As proof of concept, we examined excitatory BNs in the rostral interstitial nucleus of medial longitudinal fasciculus (RIMLF, vertical saccades) and inhibitory BNs in nucleus paragigantocellularis dorsalis (PGD, horizontal saccades) histochemically in macaque monkeys. We found strong expression of Kv channels, which enable rapid-firing, as well as HCN1&2 and Cav3.2&3.3, which enable post-inhibitory rebound bursting, in both BN populations. Moreover, PGD was found to host multiple neuron groups in terms of calretinin immunoreactivity. Our results provide histochemical evidence that supports models proposing post-inhibitory rebound facilitates bursting in BNs. Furthermore, our findings support the notion that deductions can be made about electrophysiological firing properties by histochemical examination of functional groups within the brainstem saccadic circuitry. This development is an important building block supporting the concept of channelopathies in saccadic disorders. Future histological studies in humans will confirm this approach for saccadic disorders.

The brainstem connectome database.

Connectivity data of the nervous system and subdivisions, such as the brainstem, cerebral cortex and subcortical nuclei, are necessary to understand connectional structures, predict effects of connectional disorders and simulate network dynamics. For that purpose, a database was built and analyzed which comprises all known directed and weighted connections within the rat brainstem. A longterm metastudy of original research publications describing tract tracing results form the foundation of the brainstem connectome (BC) database which can be analyzed directly in the framework neuroVIISAS. The BC database can be accessed directly by connectivity tables, a web-based tool and the framework. Analysis of global and local network properties, a motif analysis, and a community analysis of the brainstem connectome provides insight into its network organization. For example, we found that BC is a scale-free network with a small-world connectivity. The Louvain modularity and weighted stochastic block matching resulted in partially matching of functions and connectivity. BC modeling was performed to demonstrate signal propagation through the somatosensory pathway which is affected in Multiple sclerosis.

Functional Organization of Extraocular Motoneurons and Eye Muscles.

Eye movements are indispensable for visual image stabilization during self-generated and passive head and body motion and for visual orientation. Eye muscles and neuronal control elements are evolutionarily conserved, with novel behavioral repertoires emerging during the evolution of frontal eyes and foveae. The precise execution of eye movements with different dynamics is ensured by morphologically diverse yet complementary sets of extraocular muscle fibers and associated motoneurons. Singly and multiply innervated muscle fibers are controlled by motoneuronal subpopulations with largely selective premotor inputs from task-specific ocular motor control centers. The morphological duality of the neuromuscular interface is matched by complementary biochemical and molecular features that collectively assign different physiological properties to the motor entities. In contrast, the functionality represents a continuum where most motor elements contribute to any type of eye movement, although within preferential dynamic ranges, suggesting that signal transmission and muscle contractions occur within bands of frequency-selective pathways.

Long-Term Fluoxetine Administration Causes Substantial Lipidome Alteration of the Juvenile Macaque Brain.

Fluoxetine is an antidepressant commonly prescribed not only to adults but also to children for the treatment of depression, obsessive-compulsive disorder, and neurodevelopmental disorders. The adverse effects of the long-term treatment reported in some patients, especially in younger individuals, call for a detailed investigation of molecular alterations induced by fluoxetine treatment. Two-year fluoxetine administration to juvenile macaques revealed effects on impulsivity, sleep, social interaction, and peripheral metabolites. Here, we built upon this work by assessing residual effects of fluoxetine administration on the expression of genes and abundance of lipids and polar metabolites in the prelimbic cortex of 10 treated and 11 control macaques representing two monoamine oxidase A (MAOA) genotypes. Analysis of 8871 mRNA transcripts, 3608 lipids, and 1829 polar metabolites revealed substantial alterations of the brain lipid content, including significant abundance changes of 106 lipid features, accompanied by subtle changes in gene expression. Lipid alterations in the drug-treated animals were most evident for polyunsaturated fatty acids (PUFAs). A decrease in PUFAs levels was observed in all quantified lipid classes excluding sphingolipids, which do not usually contain PUFAs, suggesting systemic changes in fatty acid metabolism. Furthermore, the residual effect of the drug on lipid abundances was more pronounced in macaques carrying the MAOA-L genotype, mirroring reported behavioral effects of the treatment. We speculate that a decrease in PUFAs may be associated with adverse effects in depressive patients and could potentially account for the variation in individual response to fluoxetine in young people.

Superiority of Formalin-Fixed Paraffin-Embedded Brain Tissue for in vitro Assessment of Progressive Supranuclear Palsy Tau Pathology With [ 18 F]PI-2620.

Objectives: Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [18F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP). Methods: We applied [18F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities. Results: In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMRfrozen vs. CWMRFFPE: 2.5-fold, p < 0.001), whereas the opposite was observed in PSP tissue (CWMRfrozen vs. CWMRFFPE: 0.8-fold, p = 0.004). In FFPE samples, [18F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP (R = 0.641, p < 0.001) and AD tissue (R = 0.435, p = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD (R = 0.417, p = 0.014) but not in PSP tissue (R = -0.115, p = n.s.). Conclusion: Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [18F]PI-2620. The [18F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.

Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei.

Extraocular motoneurons initiate dynamically different eye movements, including saccades, smooth pursuit and vestibulo-ocular reflexes. These motoneurons subdivide into two main types based on the structure of the neuro-muscular interface: motoneurons of singly-innervated (SIF), and motoneurons of multiply-innervated muscle fibers (MIF). SIF motoneurons are thought to provoke strong and brief/fast muscle contractions, whereas MIF motoneurons initiate prolonged, slow contractions. While relevant for adequate functionality, transmitter and ion channel profiles associated with the morpho-physiological differences between these motoneuron types, have not been elucidated so far. This prompted us to investigate the expression of voltage-gated potassium, sodium and calcium ion channels (Kv1.1, Kv3.1b, Nav1.6, Cav3.1-3.3, KCC2), the transmitter profiles of their presynaptic terminals (vGlut1 and 2, GlyT2 and GAD) and transmitter receptors (GluR2/3, NMDAR1, GlyR1α) using immunohistochemical analyses of abducens and trochlear motoneurons and of abducens internuclear neurons (INTs) in macaque monkeys. The main findings were: (1) MIF and SIF motoneurons express unique voltage-gated ion channel profiles, respectively, likely accounting for differences in intrinsic membrane properties. (2) Presynaptic glutamatergic synapses utilize vGlut2, but not vGlut1. (3) Trochlear motoneurons receive GABAergic inputs, abducens neurons receive both GABAergic and glycinergic inputs. (4) Synaptic densities differ between MIF and SIF motoneurons, with MIF motoneurons receiving fewer terminals. (5) Glutamatergic receptor subtypes differ between MIF and SIF motoneurons. While NMDAR1 is intensely expressed in INTs, MIF motoneurons lack this receptor subtype entirely. The obtained cell-type-specific transmitter and conductance profiles illuminate the structural substrates responsible for differential contributions of neurons in the abducens and trochlear nuclei to eye movements.

Dysfunctional mode switching between fixation and saccades: collaborative insights into two unusual clinical disorders.

Voluntary rapid eye movements (saccades) redirect the fovea toward objects of visual interest. The saccadic system can be considered as a dual-mode system: in one mode the eye is fixating, in the other it is making a saccade. In this review, we consider two examples of dysfunctional saccades, interrupted saccades in late-onset Tay-Sachs disease and gaze-position dependent opsoclonus after concussion, which fail to properly shift between fixation and saccade modes. Insights and benefits gained from bi-directional collaborative exchange between clinical and basic scientists are emphasized. In the case of interrupted saccades, existing mathematical models were sufficiently detailed to provide support for the cause of interrupted saccades. In the case of gaze-position dependent opsoclonus, existing models could not explain the behavior, but further development provided a reasonable hypothesis for the mechanism underlying the behavior. Collaboration between clinical and basic science is a rich source of progress for developing biologically plausible models and understanding neurological disease. Approaching a clinical problem with a specific hypothesis (model) in mind often prompts new experimental tests and provides insights into basic mechanisms.

Histochemical Characterization of the Vestibular Y-Group in Monkey.

The Y-group plays an important role in the generation of upward smooth pursuit eye movements and contributes to the adaptive properties of the vertical vestibulo-ocular reflex. Malfunction of this circuitry may cause eye movement disorders, such as downbeat nystagmus. To characterize the neuron populations in the Y-group, we performed immunostainings for cellular proteins related to firing characteristics and transmitters (calretinin, GABA-related proteins and ion channels) in brainstem sections of macaque monkeys that had received tracer injections into the oculomotor nucleus. Two histochemically different populations of premotor neurons were identified: The calretinin-positive population represents the excitatory projection to contralateral upgaze motoneurons, whereas the GABAergic population represents the inhibitory projection to ipsilateral downgaze motoneurons. Both populations receive a strong supply by GABAergic nerve endings most likely originating from floccular Purkinje cells. All premotor neurons express nonphosphorylated neurofilaments and are ensheathed by strong perineuronal nets. In addition, they contain the voltage-gated potassium channels Kv1.1 and Kv3.1b which suggests biophysical similarities to high-activity premotor neurons of vestibular and oculomotor systems. The premotor neurons of Y-group form a homogenous population with histochemical characteristics compatible with fast-firing projection neurons that can also undergo plasticity and contribute to motor learning as found for the adaptation of the vestibulo-ocular reflex in response to visual-vestibular mismatch stimulation. The histochemical characterization of premotor neurons in the Y-group allows the identification of the homologue cell groups in human, including their transmitter inputs and will serve as basis for correlated anatomical-neuropathological studies of clinical cases with downbeat nystagmus.

Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue.

Ischemic stroke causes cellular alterations in the "neurovascular unit" (NVU) comprising neurons, glia, and the vasculature, and affects the blood-brain barrier (BBB) with adjacent extracellular matrix (ECM). Limited data are available for the zone between the NVU and ECM that has not yet considered for neuroprotective approaches. This study describes ischemia-induced alterations for two main components of the neurovascular matrix adhesion zone (NMZ), i.e., collagen IV as basement membrane constituent and fibronectin as crucial part of the ECM, in conjunction with traditional NVU elements. For spatio-temporal characterization of these structures, multiple immunofluorescence labeling was applied to tissues affected by focal cerebral ischemia using a filament-based model in mice (4, 24, and 72 h of ischemia), a thromboembolic model in rats (24 h of ischemia), a coagulation-based model in sheep (2 weeks of ischemia), and human autoptic stroke tissue (3 weeks of ischemia). An increased fibronectin immunofluorescence signal demarcated ischemia-affected areas in mice, along with an increased collagen IV signal and BBB impairment indicated by serum albumin extravasation. Quantifications revealed a region-specific pattern with highest collagen IV and fibronectin intensities in most severely affected neocortical areas, followed by a gradual decline toward the border zone and non-affected regions. Comparing 4 and 24 h of ischemia, the subcortical fibronectin signal increased significantly over time, whereas neocortical areas displayed only a gradual increase. Qualitative analyses confirmed increased fibronectin and collagen IV signals in ischemic areas from all tissues and time points investigated. While the increased collagen IV signal was restricted to vessels, fibronectin appeared diffusely arranged in the parenchyma with focal accumulations associated to the vasculature. Integrin α5 appeared enriched in the vicinity of fibronectin and vascular elements, while most of the non-vascular NVU elements showed complementary staining patterns referring to fibronectin. This spatio-temporal characterization of ischemia-related alterations of collagen IV and fibronectin in various stroke models and human autoptic tissue shows that ischemic consequences are not limited to traditional NVU components and the ECM, but also involve the NMZ. Future research should explore more components and the pathophysiological properties of the NMZ as a possible target for novel neuroprotective approaches.

Potassium channels in omnipause neurons.

Potassium (K+) channels are major contributors to fast and precise action potential generation. The aim of this study was to establish the immunoreactivity profile of several potassium channels in omnipause neurons (OPNs), which play a central role in premotor saccadic circuitry. To accomplish this, we histochemically examined monkey and human brainstem sections using antibodies against the voltage gated K+-channels KV1.1, KV3.1b and K+-Cl- cotransporter (KCC2). We found that OPNs of both species were positive for all three K+-antibodies and that the staining patterns were similar for both species. In individual OPNs, KV3.1b was detected on the somatic membrane and proximal dendrites, while KV1.1 was mainly confined to soma. Further, KCC2 immunoreactivity was strong in distal dendrites, but was weak in the somatic membrane. Our findings allow the speculation that the alterations in K+-channel expression in OPNs could be the underlying mechanism for several saccadic disorders through neuronal and circuit-level malfunction.

Extraocular muscles involved in convergence are innervated by an additional set of palisade endings that may differ in their excitability: A human study.

Palisade endings are located at the myotendinous junction of extraocular muscles in most mammals. Irrespective of their unclarified function as motor or sensory nerve endings, a specialized role in convergence is proposed, based on their high number in the medial rectus muscle (MR). Further support comes from a study in monkey demonstrating that only the MR and inferior rectus muscle (IR) contain an additional population of palisade endings that express the calcium-binding protein calretinin (CR) in addition to choline acetyltransferase (ChAT). Here we studied, whether CR-positive palisade endings are present in human as well and confined to extraocular muscles most active during convergence. The systematic analysis of all eye muscles of 17 human specimen revealed that only the MR and IR contain an additional population of CR-positive palisade endings and multiple en-grappe endings, which target non-twitch muscle fibers along their whole length. Approximately 80% of all palisade endings in the MR expressed CR. Furthermore, the intrafusal muscle fibers of some muscle spindles in the MR were innervated by CR-positive annulospiral nerve endings that transmit the signals of muscle length changes to the brain. All extraocular muscles contained few thin CR-positive, but ChAT-negative nerve fibers, possibly representing free sensory or autonomic endings arising from the trigeminal ganglion. As in monkey, in the medial periphery of the human oculomotor nucleus ChAT-positive neurons were found to co-express CR. Therefore these neurons most likely represent the cell bodies of CR-positive palisade endings in the MR. Unlike in monkey, these neurons do not lie within a compact cell group, but are more scattered. In conclusion, the MR and IR in human contain two histochemically different populations of palisade and multiple endings that may contribute to ocular alignment and convergence in a different way.

Continuous cuprizone intoxication allows active experimental autoimmune encephalomyelitis induction in C57BL/6 mice.

Oligodendrocyte degeneration is a hallmark of multiple sclerosis pathology, and protecting oligodendrocytes and myelin is likely to be of clinical relevance. Traditionally, oligodendrocyte and myelin degeneration are viewed as a direct consequence of an inflammatory attack, but metabolic defects might be equally important. Appropriate animal models to study the interplay of inflammation and metabolic injury are, therefore, needed. Here, we describe that in spite of its immunosuppressive effects, a continuous intoxication with cuprizone allows the induction of active experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG35-55) immunization. Although the clinical severity of EAE is ameliorated in cuprizone-intoxicated mice, the recruitment of granulocytes, and especially, CD3+ lymphocytes into the forebrain is triggered by the cuprizone insult. Such combined lesions are further characterized by oligodendrocyte apoptosis and microglia activation, closely mimicking type III multiple sclerosis lesions. In summary, we provide a protocol that allows to study the direct interplay of immune-mediated and metabolic oligodendrocyte injury and its consequences for the cerebral white and grey matters.

Adaptation of spatio-temporal convergent properties in central vestibular neurons in monkeys.

The spatio-temporal convergent (STC) response occurs in central vestibular cells when dynamic and static inputs are activated. The functional significance of STC behavior is not fully understood. Whether STC is a property of some specific central vestibular neurons, or whether it is a response that can be induced in any neuron at some frequencies is unknown. It is also unknown how the change in orientation of otolith polarization vector (orientation adaptation) affects STC behavior. A new complex model, that includes inputs with regular and irregular discharges from both canal and otolith afferents, was applied to experimental data to determine how many convergent inputs are sufficient to explain the STC behavior as a function of frequency and orientation adaptation. The canal-otolith and otolith-only neurons were recorded in the vestibular nuclei of three monkeys. About 42% (11/26 canal-otolith and 3/7 otolith-only) neurons showed typical STC responses at least at one frequency before orientation adaptation. After orientation adaptation in side-down head position for 2 h, some canal-otolith and otolith-only neurons altered their STC responses. Thus, STC is a property of weights of the regular and irregular vestibular afferent inputs to central vestibular neurons which appear and/or disappear based on stimulus frequency and orientation adaptation. This indicates that STC properties are more common for central vestibular neurons than previously assumed. While gravity-dependent adaptation is also critically dependent on stimulus frequency and orientation adaptation, we propose that STC behavior is also linked to the neural network responsible for localized contextual learning during gravity-dependent adaptation.

A Subset of Palisade Endings Only in the Medial and Inferior Rectus Muscle in Monkey Contain Calretinin.

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.