IGF-I specifically enhances axon outgrowth of corticospinal motor neurons

Corticospinal motor neurons (CSMN) are among the most complex CNS neurons; they control voluntary motor function and are prototypical projection neurons. In amyotrophic lateral sclerosis (ALS), both spinal motor neurons and CSMN degenerate; their damage contributes centrally to the loss of motor function in spinal cord injury. Direct investigation of CSMN is severely limited by inaccessibility in the heterogeneous cortex. Here, using new CSMN purification and culture approaches, and in vivo analyses, we report that insulin-like growth factor-1 (IGF-I) specifically enhances the extent and rate of murine CSMN axon outgrowth, mediated via the IGF-I receptor and downstream signaling pathways; this is distinct from IGF-I support of neuronal survival. In contrast, brain-derived neurotrophic factor (BDNF) enhances branching and arborization, but not axon outgrowth. These experiments define specific controls over directed differentiation of CSMN, indicate a distinct role of IGF-I in CSMN axon outgrowth during development, and might enable control over CSMN derived from neural precursors.     

Neurite outgrowth from mammalian CNS neurons on astrocytes in vitro may not be mediated primarily by laminin

Summary A number ofin vitro studies suggest that certain components of the extracellular matrix (ECM), such as the glycoprotein laminin, can promote neurite outgrowth. In the present study the presence of laminin and heparan sulphate proteoglycan, another ECM molecule, on CNS glial (astrocytes) and non-glial (leptomeningeal) cellsin vitro was examined. In addition, the ability of these cells and laminin-coated tissue culture substrates to promote neurite outgrowth from developing mammalian cerebellar cortical neurons was also assayed. Leptomeningeal cells were found to be labelled much more intensely with antibodies against laminin and heparin sulphate proteoglycan than were astrocytes. However, the proportion of neurons extending neurites was fourfold greater on astrocyte monolayers than on leptomeninges, and twofold greater than that on laminin. In addition, the length of the neurites growing on astrocyte monolayers was three- to fourfold greater than that on laminin or leptomeninges. Neurite outgrowth on leptomeninges and laminin could not be enhanced by culturing in astrocyte conditioned medium. The ability of various antibodies to block neurite outgrowth on these monolayers was also studied. These results suggest that the robust neurite outgrowth from mammalian CNS neurons plated on astrocytes may be mediated via an astrocyte surface molecule distinct from laminin.     

Changes in the ventral dendrite of Mauthner neurons in goldfish after optokinetic stimulation

Recent studies have established that following an optomotor drum rotating in the direction contralateral to that spontaneously preferred by goldfish fry inverts their motor asymmetry. Studies of the structure of Mauthner neurons (MN) using the histological technique of three-dimensional reconstruction along with measurement of the volumes of the right and left MN in these fish showed that the ipsilateral MN underwent a three-fold reduction in the volume of the ventral dendrite (VD), which was in inverse proportion to the functional activity of the MN. The total volume of the contralateral MN was 25% larger than the volume of the ipsilateral MN, the situation typical of intact fish. It is suggested that the decreases in the size of the VD receiving afferentation from the contralateral eye, which follows the moving band of the optomotor drum, results from the specific contralateral visual stimulation, and is the first evidence of the possible natural stimulation of the MN via the VD. __________ Translated from Morfologiya, Vol. 132, No. 6, pp. 29–34, November–December, 2007.     

Mouse semaphorin H inhibits neurite outgrowth from sensory neurons.

Mouse semaphorin H (M-semaH) was structurally similar to semaphorin III/D, a mammalian homologue of collapsin 1 which was identified as a collapsing factor for sensory nerves. In this study we investigated the expression patterns of M-semaH mRNA and the protein binding sites in the trunk of mouse embryos. M-semaH mRNA was expressed in the mesenchymal tissues surrounding each dorsal root ganglia. These tissues include the caudal sclerotome and perinotochordal mesenchyme, which were thought to express factors repulsive to axons. M-semaH binding was detected on the spinal nerves. We further investigated, using in vitro co-culture assay, whether M-semaH acted as a chemorepulsive molecule on sensory axons. The results suggested that M-semaH was a candidate for a chemorepellent expressed in the mesenchyme surrounding the sensory ganglia, which is involved in the axonal guidance mechanism of sensory nerves in the trunk.     

Absence of callosal collaterals derived from rat corticospinal neurons

Summary In rat the presence of axon collaterals from corticospinal neurons to the contralateral hemisphere has been investigated by means of anatomical and electrophysiological techniques. Anatomical Experiments. Several combinations of fluorescent retrograde tracers were used. In eight rats injections of Evans Blue, True Blue, Fast Blue or DAPI-Primuline were made in areas 10, 6, and 4 and in the most medial part of the S1 granular cortex of one hemisphere, 1.5 mm below cortical surface. These injections were combined with injections of Fast Blue, DAPI-Primuline, Granular Blue, Nuclear Yellow, or Bisbenzimide in the ipsilateral corticospinal tract in the C2 segment.Survival times of the animals varied according to the tracers used. In the non-injected hemisphere the retrogradely labeled corticospinal neurons were present in layer V of especially areas 10, 6, 4 and the medial portion of the S1 granular cortex. However, the retrogradely labeled callosal neurons in these areas were present in all layers except layer I. The labeled callosal and corticospinal neurons in layer V were intermingled and frequently situated very close to one another. However, with none of the tracer combinations were double labeled neurons observed. Electrophysiological Experiments. In six rats, layer V neurons of hindlimb-sensorimotor cortex were tested for antidromic responses to stimulation of contralateral corticospinal tract (CST) and corpus callosum (CC). Eighty-five CST neurons were identified, none of which responded antidromically to CC shocks. Eighty-two layer V neurons were identified which responded antidromically to CC shocks, but none of them responded antidromically to CST shocks. CC shocks elicited strong synaptic responses in CST neurons and vice versa. Depth measures indicated extensive intermingling of CST and CC neurons.From both sets of findings it was concluded that, in rat, CST neurons do not give rise to callosal collaterals.This study was in part supported by Grant 13-46-15 of the Fungo/ ZWO (Dutch Organization for Fundamental Research in Medicine)     

Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

We explored the contribution of inhibitory peanut-binding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules.     

Origins of postsynaptic potentials evoked in identified rat neostriatal neurons by stimulation in substantia nigra

Summary Responses of striatal neurons to stimulation in substantia nigra were recorded intracellularly in intact rats and after acute or chronic unilateral lesions of cerebral cortex or after combined cortical lesions and unilateral thalamic transections. Spiny striatal efferent neurons were identified by intracellular injection of horseradish peroxidase. In intact animals substantia nigra stimulation evoked a complex response with both excitatory and inhibitory phases. Acute unilateral decortication abolished the inhibitory phase of the response and reduced the amplitude of the initial EPSP. Thus, part of the excitatory phase and most or all of the inhibitory phase of the response result from polysynaptic routes to striatum involving cerebral cortex. The remaining EPSP observed in acute decorticate animals exhibited two components distinguished on the basis of their time courses. The latter of these was abolished by thalamic transections. The earlier component was shown to be a monosynaptic EPSP evoked by axon collaterals of cortical efferent neurons projecting to brainstem and was not observed in animals subjected to chronic decortication. After removal of all of these non-nigral response components a small long latency EPSP could be evoked by nigral stimulation. This EPSP is probably due to activation of dopaminergic nigro-striatal axons.A preliminary report of this work was presented at the 10th Annual Meeting of the Society for Neuroscience, November, 1980Supported by USPHS grant NS17294 (to C.J. Wilson), USPHS grant NS 14866 (to S.T. Kitai), and NIH BRSG RR 0572 to the College of Osteopathic Medicine, Michigan State University     

Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly

Dendrite branching has an important role in normal brain function. Here we report that overexpression of cypin, a protein that has guanine deaminase activity and is expressed in developing processes in rat hippocampal neurons, results in increased dendrite branching in primary culture. Mutant cypin proteins that lack guanine deaminase activity act in a dominant-negative manner when expressed in primary neurons. Furthermore, we knocked down cypin protein levels using a new strategy: expressing a 5' end-mutated U1 small nuclear RNA (snRNA) to inhibit maturation of cypin mRNA. Neurons that express this mutant snRNA show little or no detectable cypin protein and fewer dendrites than normal. In addition, we found that cypin binds directly to tubulin heterodimers and promotes microtubule polymerization. Thus, our results demonstrate a new pathway by which dendrite patterning is regulated, and we also introduce a new method for decreasing endogenous protein expression in neurons.     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells in vitro

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.     

Effects of serotonin on neurite outgrowth from thalamic neurons in vitro

Altering levels of serotonin in the primary somatosensory cortex during early postnatal life influences thalamocortical development. Recent in vivo experiments suggest that serotonin may have direct effects on the growth of thalamocortical axons, and the present study was undertaken to determine whether this amine influences process outgrowth from thalamic cells maintained in culture. Ventrobasal thalamic neurons were harvested from newborn rats and maintained in culture for eight days. At the end of this period, 0, 10, 25, 50 or 100 microM serotonin was added to the culture medium. After an additional six days, cultures were fixed and stained with neuron-specific enolase. Quantitative analysis of >500 cells from each condition indicated that 25 microM serotonin, but not the other concentrations of this amine, significantly increased the length of the primary (longest) process growing out from the cell body (P < 0.001), the total (summed) length of all processes (P < 0.0001), total neurites per cell (P < 0.05), number of branch points per cell (P < 0.01) and branch points on the primary neurite (P < 0.0005). These results demonstrate that exposing thalamic cells to serotonin increases process outgrowth from them in the absence of their cortical targets.     

Direct innervation of identified tectothalamic neurons in the inferior colliculus by axons from the cochlear nucleus.

The present study sought to identify tectothalamic neurons in the rat inferior colliculus that receive their innervation directly from the cochlear nuclei and to identify the axons that provide the innervation. A direct projection would bypass the binaural interactions of the superior olivary complex and provide the quickest route to the neocortex. Axons, primarily from the dorsal cochlear nucleus, were labeled with anterograde transport of dextran and terminated in the central nucleus of the inferior colliculus in a laminar pattern. Most labeled axons were thin and simply branched. Other axons were thicker, gnarly, less frequently observed and probably originated from the ventral cochlear nucleus. None had concentrated endbulbs or a nest of endings. Both types of axons terminated primarily in the central nucleus and layer 3 of the external cortex. This pattern suggests that the combination of these subdivisions in the rat are equivalent to the central nucleus as defined in other species. Tectothalamic neurons in the inferior colliculus in the same animals were identified by retrograde transport from the medial geniculate body and intracellular injection of Lucifer Yellow. A number of different cell types act as tectothalamic neurons and receive contacts from cochlear nucleus axons. These include flat cells (disc-shaped), less-flat cells and stellate cells. Two innervation patterns were seen: a combination of axosomatic and axodendritic contacts, and predominantly axodendritic contacts. Both patterns were seen in the central nucleus, but axosomatic contacts were seen less often in the other subdivisions. This is the first study to show direct connections between cochlear nuclear axons and identified tectothalamic neurons. The layers of axons from cochlear nuclei may provide convergent inputs to neurons in the inferior colliculus rather than the heavy inputs from single axons typical of lower auditory nuclei. Excitatory synapses made by axons from the cochlear nuclei on tectothalamic neurons may provide a substrate for rapid transmission of monaural information to the medial geniculate body.     

The origin of corticospinal projection neurons in rat

Summary The distribution of corticospinal projection neurons in adult rats was determined using a retrograde tracing technique. Horseradish peroxidase (HRP) and an emulsifier (Nonidet) were injected into the 5th and 6th segments of the cervical spinal cord. The greatest concentrations of HRP-positive neurons were distributed in area 4 and rostral area 6/8 (motor cortices) and medial area 3 and caudal area 2 (somatosensory cortices). The largest labeled neurons were in areas 4 and 3. HRP-positive neurons were absent or few in regions of motor and somatosensory fields which contained the face representation. Less dense concentrations of retrogradely labeled neurons were also in posterior parietal and association areas 14, 39 and 40, rostral occipital visual areas 18a and 18b, and anterior cingulate and prefrontal areas 24a, 24b, and 32. The topography of the corticospinal pathway was determined by injecting HRP without Nonidet into the cervical, upper thoracic, lower thoracic, or lumbar spinal cord. Although the distribution of labeled neurons decreased with distance down the spinal cord, the size of the corticospinal neurons in each cytoarchitectonic area was not significantly different regardless of where the injection was placed. For example, upper thoracic cord injections retrogradely labeled neurons in each of the regions containing neurons filled by cervical cord injections, however, lumbar injections retrogradely labeled neurons only in caudal areas 4 and 3 and in area 18b. The distribution of corticospinal neurons in rats is similar to the organization of the corticospinal system in higher animals. The origin of corticospinal neurons in occipital and cingulate cortices may be related to visuomotor and visceromotor control.     

Influences of sensory input from the limbs on feline corticospinal neurons during postural responses

The dorsal-side-up body posture of standing quadrupeds is maintained by coordinated activity of all limbs. Somatosensory input from the limbs evokes postural responses when the supporting surface is perturbed. The aim of this study was to reveal the contribution of sensory inputs from individual limbs to the posture-related modulation of pyramidal tract neurons (PTNs) arising in the primary motor cortex. We recorded the activity of PTNs from the limb representation of motor cortex in the cat maintaining balance on a platform periodically tilted in the frontal plane. Each PTN was recorded during standing on four limbs, and when two or three limbs were lifted from the platform and thus did not signal its displacement to motor cortex. By comparing PTN responses to tilts in different tests we found that the amplitude and the phase of the response in the majority of them were determined primarily by the sensory input from the corresponding contralateral limb. In a portion of PTNs, this input originated from afferents of the peripheral receptive field. Sensory input from the ipsilateral limb, as well as input from limbs of the other girdle made a much smaller contribution to the PTN modulation. These results show that, during postural activity, a key role of PTNs is the feedback control of the corresponding contralateral limb and, to a lesser extent, the coordination of posture within a girdle and between the two girdles.     

Noninvasive stimulation of human corticospinal axons innervating leg muscles

These studies investigated whether a single electrical stimulus over the thoracic spine activates corticospinal axons projecting to human leg muscles. Transcranial magnetic stimulation of the motor cortex and electrical stimulation over the thoracic spine were paired at seven interstimulus intervals, and surface electromyographic responses were recorded from rectus femoris, tibialis anterior, and soleus. The interstimulus intervals (ISIs) were set so that the first descending volley evoked by cortical stimulation had not arrived at (positive ISIs), was at the same level as (0 ISI) or had passed (negative ISIs) the site of activation of descending axons by the thoracic stimulation at the moment of its delivery. Compared with the responses to motor cortical stimulation alone, responses to paired stimuli were larger at negative ISIs but reduced at positive ISIs in all three leg muscles. This depression of responses at positive ISIs is consistent with an occlusive interaction in which an antidromic volley evoked by the thoracic stimulation collides with descending volleys evoked by cortical stimulation. The cortical and spinal stimuli activate some of the same corticospinal axons. Thus it is possible to examine the excitability of lower limb motoneuron pools to corticospinal inputs without the confounding effects of changes occurring within the motor cortex.     

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.     

Co-culture with astrocytes or microglia protects metabolically impaired neurons.

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism that leads to neurodegeneration. TD induces oxidative stress and death in neurons, but does not kill astrocytes, microglia or brain endothelial cells. TD primary hippocampal neurons were either cultured alone, or co-cultured with primary astrocytes or microglia. After 7 days of TD, 50% of the neurons died, and the processes of many of the surviving neurons were severely truncated. When TD neurons were co-cultured with astrocytes or microglia, neurons did not die nor show decreased neurite outgrowth. Thus, neuronal-glial interactions are critical for maintaining neuronal homeostasis during chronic metabolic impairment.