Further study of soma,dendrite, and axon excitation in single neurons

The present investigation continues a previous study in which the soma-dendrite system of sensory neurons was excited by stretch deformation of the peripheral dendrite portions. Recording was done with intracellular leads which were inserted into the cell soma while the neuron was activated orthodromically or antidromically. The analysis was also extended to axon conduction. Crayfish, Procambarus alleni (Faxon) and Orconectes virilis (Hagen), were used. 1. The size and time course of action potentials recorded from the soma-dendrite complex vary greatly with the level of the cell's membrane potential. The latter can be changed over a wide range by stretch deformation which sets up a "generator potential" in the distal portions of the dendrites. If a cell is at its resting unstretched equilibrium potential, antidromic stimulation through the axon causes an impulse which normally overshoots the resting potential and decays into an afternegativity of 15 to 20 msec. duration. The postspike negativity is not followed by an appreciable hyperpolarization (positive) phase. If the membrane potential is reduced to a new steady level a postspike positivity appears and increases linearly over a depolarization range of 12 to 20 mv. in various cells. At those levels the firing threshold of the cell for orthodromic discharges is generally reached. 2. The safety factor for conduction between axon and cell soma is reduced under three unrelated conditions, (a) During the recovery period (2 to 3 msec.) immediately following an impulse which has conducted fully over the cell soma, a second impulse may be delayed, may invade the soma partially, or may be blocked completely. (b) If progressive depolarization is produced by stretch, it leads to a reduction of impulse height and eventually to complete block of antidromic soma invasion, resembling cathodal block, (c) In some cells, when the normal membrane potential is within several millivolts of the relaxed resting state, an antidromic impulse may be blocked and may set up within the soma a local potential only. The local potential can sum with a second one or it may sum with potential changes set up in the dendrites, leading to complete invasion of the soma. Such antidromic invasion block can always be relieved by appropriate stretch which shifts the membrane potential out of the "blocking range" nearer to the soma firing level. During the afterpositivity of an impulse in a stretched cell the membrane potential may fall below or near the blocking range. During that period another impulse may be delayed or blocked. 3. Information regarding activity and conduction in dendrites has been obtained indirectly, mainly by analyzing the generator action under various conditions of stretch. The following conclusions have been reached: The large dendrite branches have similar properties to the cell body from which they arise and carry the same kind of impulses. In the finer distal filaments of even lightly depolarized dendrites, however, no axon type all-or-none conduction occurs since the generator potential persists to a varying degree during antidromic invasion of the cell. With the membrane potential at its resting level the dendrite terminals contribute to the prolonged impulse afternegativity of the soma. 4. Action potentials in impaled axons and in cell bodies have been compared. It is thought that normally the over-all duration of axon impulses is shorter. Local activity during reduction of the safety margin for conduction was studied. 5. An analysis was made of high frequency grouped discharges which occasionally arise in cells. They differ in many essential aspects from the regular discharges set up by the generator action. It is proposed that grouped discharges occur only when invasion of dendrites is not synchronous, due to a delay in excitation spread between soma and dendrites. Each impulse in a group is assumed to be caused by an impulse in at least one of the large dendrite branches. Depolarization of dendrites abolishes the grouped activity by facilitating invasion of the large dendrite branches.     

After-currents,after-potentials,excitability, and ventral root electrotonus in spinal motoneurons

The spinal cord constitutes a volume conductor. Potential changes are recorded therefrom only as current flows. During the period of the after-potentials current flows in significant density only if the after-polarization differs at different points of the active neurons. Thus one does not record after-potentials in volume; one may record after-currents which are defined as the resultants of differences in after-potentials. Measurable excitability change during the period of the after-potentials, in the event no current flows, might be regarded as approximating the change of intrinsic polarization status at the region tested. In the presence of after-current flow excitability change would approximate the sum of intrinsic change and extrinsic change due to current flow. In giving rise by differences to current flow after-potentials come to act as agents, and events in one part of a neuron help to determine excitability in other parts. Since the intramedullary after-current flow is not the after-potential of the soma, it follows that ventral root electrotonus which results from axonal after-current flow cannot be considered the counterpart of somatic after-potential. Following conduction of an antidromic volley after-current flows between somata and axons. According to the signs of the recorded potential changes, after-current flow initially, and for approximately 45 msec., is in the direction from somata to axons. Thereafter, and for approximately another 75 msec., the direction of flow is reversed. During the period of after-current flow following antidromic conduction the excitability of neighboring motoneurons is altered in a manner that reproduces the phases of after-current flow. The initial phase, depression, was first described by Renshaw. The after-potentials of ventral root fibers have been studied. In a single action and in usual form, they consist of a negative after-potential of considerable magnitude and of some 35 msec. duration, and a positive after-potential detectable for approximately 120 msec. Variants and the influence of temperature change are described. The recovery cycle of ventral root axons in general compares with the after-potential cycle. Recovery of intramedullary motor axons differs from that of their extramedullary projections as ventral root fibers in a manner that is accountable to intramedullary flow of after-current. Since the intrinsic recovery process of the motoneuron somata cannot be measured in the presence of current flow it must be estimated by correcting the observed recovery for the influence of known current flows. When this is done the resultant in simplest form provides for intrinsic somatic recovery from refractoriness through a single phase of subnormality lasting some 60 msec. Conditions for the relatively undistorted recording of antidromic ventral root electrotonus are described. They include provisions that the proximal ventral root electrode must be within 12 mm. of the root-cord junction and that the distal electrode must be located in excess of 30 mm. from the distal severed end of the ventral root. Antidromic ventral root electrotonus is a counterpart of the current flows in the intramedullary stretch of the axons. Initially, during the phase of metadromal postivity of the intramedullary axons, electrotonus is negative. During the period of deflections Sp-An, that signify after-current flow into the axons, electrotonus is positive. Finally during the period of deflections Sn-Ap, that signify after-current flow outwards through the intramedullary axon membranes, electrotonus is negative. Electrotonic showing is not of sufficient magnitude to make the time course of ventral root electrotonus palpably different from that of the generating intramedullary currents.     

Responses of motoneurons undergoing chromatolysis

The delayed and asynchronous firing of chromatolytic motoneurons in response to group I afferent volleys is shown to be evoked monosynaptically, there being an abnormally long and variable delay between onset of monosynaptic action and generation of impulse discharge. Intensity of monosynaptic excitatory action is reduced, and considerable variability in the form of successively evoked postsynaptic potentials is often observed. No evidence has been found for the development of excitatory group I polysynaptic pathways. Reduction in responsiveness of finer dendrites is indicated by the feeble "d" response evoked by an antidromic volley in a chromatolytic motor nucleus. Antidromic impulses appear to invade the cell bodies and coarse dendrites, but die out at points short of the normal extent of dendritic invasion. Vigorous firing of Renshaw cells can be elicited by antidromic volleys. Chromatolytic motoneurons appear to maintain reasonably normal resting membrane potentials, but are more susceptible to damage than are normal cells. Action potentials are large and usually overshoot the resting potential level. Post spike potentials are similar to those of normal cells except for a less prominent, or absent, early phase of depolarisation. In contrast with the reduced responsiveness of peripheral dendrites, there is a lowered threshold for antidromic and segmental reflex synaptic activation of the more central regions, probably the cell bodies and nearby coarse dendrites, of motoneurons undergoing chromatolysis.     

Synaptic inhibition in an isolated nerve cell

Following the preceding studies on the mechanisms of excitation in stretch receptor cells of crayfish, this investigation analyzes inhibitory activity in the synapses formed by two neurons. The cell body of the receptor neuron is located in the periphery and sends dendrites into a fine muscle strand. The dendrites receive innervation through an accessory nerve fiber which has now been established to be inhibitory. There exists a direct peripheral inhibitory control mechanism which can modulate the activity of the stretch receptor. The receptor cell which can be studied in isolation was stimulated by stretch deformation of its dendrites or by antidromic excitation and the effect of inhibitory impulses on its activity was analyzed. Recording was done mainly with intracellular leads inserted into the cell body. 1. Stimulation of the relatively slowly conducting inhibitory nerve fiber either decreases the afferent discharge rate or stops impulses altogether in stretched receptor cells. The inhibitory action is confined to the dendrites and acts on the generator mechanism which is set up by stretch deformation. By restricting depolarization of the dendrites above a certain level, inhibition prevents the generator potential from attaining the "firing level" of the cell. 2. The same inhibitory impulse may set up a postsynaptic polarization or a depolarization, depending on the resting potential level of the cell. The membrane potential at which the inhibitory synaptic potential reverses its polarity, the equilibrium level, may vary in different preparations. The inhibitory potentials increase as the resting potential is displaced in any direction from the inhibitory equilibrium. 3. The inhibitory potentials usually rise to a peak in about 2 msec. and decay in about 30 msec. After repetitive inhibitory stimulation a delayed secondary polarization phase has frequently been seen, prolonging the inhibitory action. Repetitive inhibitory excitation may also be followed by a period of facilitation. Some examples of "direct" excitation by the depolarizing action of inhibitory impulses are described. 4. The interaction between antidromic and inhibitory impulses was studied. The results support previous conclusions (a) that during stretch the dendrites provide a persisting "drive" for the more central portions of the receptor cell, and (b) that antidromic all-or-none impulses do not penetrate into the distal portions of stretch-depolarized dendrites. The "after-potentials" of antidromic impulses are modified by inhibition. 5. Evidence is presented that inhibitory synaptic activity increases the conductance of the dendrites. This effect may occur in the absence of inhibitory potential changes.     

Regeneration from crayfish phasic and tonic motor axons in vitro

An explant culture system is described that allows examination of axonal growth from the tonically and phasically active motoneurons of the abdominal nerve cord of the crayfish. In this preparation, growth occurs from the cut end of the axon while the remainder of the motoneuron is undisturbed. In vitro growth from the branches of the third roots, which contain the axons from the tonic and phasic motoneurons of abdominal ganglia one through four, was verified as axonal by retrograde labeling of axons and neuronal somata within the nerve cord. Growth from the axons of phasic and tonic cells was observed as early as 24 h after plating and continued for an additional 7–10 days. The morphology and growth rates of the motor terminals differed between the tonic and phasic axons. The phasic axons grew significantly faster and branched more often than did the tonic motor axons. These differences in growth may be related to differences in motoneuron size or, may result from differences in electrical activity. Tonic motoneurons show spontaneous impulse activity for up to 6 days in culture, whereas phasic motoneurons show no spontaneous impulse activity. In addition, the differences in growth may be related to the morphological differences in tonic and phasic motor terminals observed in situ. © 1993 John Wiley & Sons, Inc.     

Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.     

Continuous conduction of impulses in peripheral myelinated nerve fibers

1. Conduction of impulses in peripheral myelinated fibers of a nerve trunk is a continuous process, since with uninjured nerve fibers: (a) within each internodal segment the conduction time increases continuously and linearly with increasing conduction distance; (b) the presence of nodes of Ranvier does not result in any detectable discontinuity in the conduction of the impulse; (c) the ascending phase of the spike always has an S shape and never presents signs of fractionation; (d) the shape and magnitude of the spike are constant at all points of each internodal segment. 2. Records have been presented of the external logitudinal current that flows during propagation of an impulse in undissected single nerve fiber (Fig. 6). 3. Propagation of impulses across a conduction block occurs with a readily demonstrable discontinuity.     

Postsynaptic potentials of neck muscle motoneurons evoked by horizontal semicircular canal stimulation in cats

Postsynaptic potentials evoked by stimulation of ipsilateral and contralateral horizontal semicircular canals in motoneurons of muscles tilting and turning the head were investigated in acute experiments on cats anesthetized with chloralose and pentobarbital. Stimulation of the ipsilateral canal evoked EPSPs with latent periods varying from 1.8 to 10.0 msec in 25 of these motoneurons and IPSPs with latent periods varying from 1.9 to 3.9 msec in 10 of them. Calculation of the impulse conduction time from the ipsilateral semicircular canal through Deiters' nucleus to the cervical motoneurons indicates that EPSPs with latent periods of under 3.8 msec may be regarded as disynaptic, and those with latent periods of over 3.8 msec as polysynaptic. Stimulation of the contralateral canal evoked EPSPs with latent periods varying from 1.8 to 6.0 msec in 19 motoneurons and IPSPs with latent periods varying from 3.2 to 3.9 msec in two cells. The possible pathways of transmission of these influences and their functional role are discussed.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

Influence of asphyxia upon the responses of spinal motoneurons

Observations have been made upon asphyxial and postasphyxial changes in the electrical responses of motoneurons to antidromic stimulation. Analysis has been aided by the use of a simple method for locating conduction blocks in the circumstances of volume conduction. Asphyxiation has been produced by suspending artificial ventilation. Regular practice has been to restore ventilation immediately after complete conduction block is established. This has permitted study of the postasphyxial state, but not of the effects of prolonged asphyxiation with the latter of which this paper is not concerned. With asphyxiation produced in the manner outlined a latent period of approximately 1 minute precedes the onset of asphyxial change. The initial change, to judge by the work of others (6, 7), is beginning central depolarization. At the same time there is a severe loss of somatic after-potential (Fig. 1). Through this loss the dendrites acquire the ability to carry two volleys in rapid succession (Fig. 13). These changes appear to reach completion within approximately 30 seconds. There follows a period of convulsive activity during which reciprocal amplitude changes in the response of axons and dendrites prove that a fluctuation in somatic responsivity is taking place (Fig. 11). Intermittent impulse discharge in ventral roots is seen (Fig. 1). Conduction block may be developing slowly throughout the period of convulsive activity (Fig. 11). Frequently there is a rather definite instant at which convulsive activity ceases and a rapid development of block begins. Usually the recorded amplitude of the dendritic response then increases to a peak (the preterminal increment) before final disappearance (Figs. 9 to 11, 13 to 15). A variety of reasons has been advanced to show that this preterminal increment represents not increased response, but rather a developing block (Figs. 11 to 13). When fully established, asphyxial block is located at the junction of the initial and myelinated axon segments (Figs. 5 to 7). It is a depolarization or cathodal block. On restoring ventilation a latency of less than 20 seconds antecedes the onset of postasphyxial change. Within the span of a few seconds membrane potential recovers and overshoots the normal level. At a critical stage of repolarization motoneurons are capable of conducting impulses, but again lapse into block (Figs. 9, 10, 14, and 15). The newly established block is due to hyperpolarization and is anodal in type. It is a somatic rather than an axonal block. Final recovery from the postasphyxial block requires some 20 minutes. As soon as motoneurons perform the rapid transition from asphyxial block through normal to postasphyxial block they will, upon reasphyxiation, pass through a new and complete asphyxial cycle with the one difference that a marked phase of incrementing response is experienced due to asphyxial mitigation of the postasphyxial block (Fig. 14). Barbiturate narcosis depresses the response of dendrites in a manner that resembles anodal depression for it is relieved rather than reinforced by asphyxial depolarization (Fig. 15). Asphyxial augmentation of response may acquire spectacular dimensions when written upon a state of barbiturate depression. Blocking time of the spinal motoneurons is on the average about 3.5 minutes. It may be shortened by prior asphyxiation (Fig. 14) and is lengthened by cooling of the preparation. Narcotization has not been observed to alter survival time significantly (Fig. 15).     

Monosynaptic inhibitory postsynaptic potentials of cortical neurons

Monopolar intracortical stimulation of the auditory cortex was carried out in cats immobilized with D-tubocurarine. A macroelectrode (tip diameter 100 µ) or a microelectrode (tip diameter 10–15 µ) was used for stimulation. In both cases, besides excitatory responses, primary IPSPs with latent periods of 0.4–1.2 and 1.4–6.0 msec were recorded in cortical neurons close to the point of stimulation. The first group of IPSPs are considered to be generated in response to direct stimulation of bodies or axons of inhibitory cortical neurons, i.e., monosynaptically. The amplitude of these IPSPs varied in different neurons from 3 to 15 mV, and their duration from 4 to 150 msec. Additional later inhibitory responses were superposed on many of them. Of the IPSPs generated in auditory cortical neurons in response to stimulation of geniculocortical fibers 1.5% had a latency of 0.8–1.3 msec. They also are assumed to be monosynaptic. It is concluded that the duration of synaptic delay of IPSPs in cortical neurons and spinal motoneurons is the same, namely 0.3–0.4 msec. Axons of auditory cortical inhibitory neurons may be 1.5 mm long. The velocity of impulse conduction along these axons is 1.6–2.8 m/sec. The genesis of some special features of IPSPs of cortical neurons is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 7, No. 5, pp. 458–467, September–October, 1975.     

Diapause Formation and Downregulation of Insulin-Like Signaling via DAF-16/FOXO Delays Axonal Degeneration and Neuronal Loss

Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO–dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment.     

Neural Organization of the Median Ocellus of the Dragonfly : I. Intracellular electrical activity

Intracellular responses from receptors and postsynaptic units have been recorded in the median ocellus of the dragonfly. The receptors respond to light with a graded, depolarizing potential and a single, tetrodotoxin-sensitive impulse at "on." The postsynaptic units (ocellar nerve dendrites) hyperpolarize during illumination and show a transient, depolarizing response at "off." The light-evoked slow potential responses of the postsynaptic units are not altered by the application of tetrodotoxin to the ocellus. It appears, therefore, that the graded receptor potential, which survives the application of tetrodotoxin, is responsible for mediating synaptic transmission in the ocellus. Comparison of pre- and postsynaptic slow potential activity shows (a) longer latencies in postsynaptic units by 5–20 msec, (b) enhanced photosensitivity in postsynaptic units by 1–2 log units, and (c) more transient responses in postsynaptic units. It is suggested that enhanced photosensitivity of postsynaptic activity is a result of summation of many receptors onto the postsynaptic elements, and that transients in the postsynaptic responses are related to the complex synaptic arrangements in the ocellar plexus to be described in the following paper.     

Collaterals and Bifurcations of Axons of Spinal Cord Motoneurons of the Lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Structure of central projections of the motoneuron axons of the spinal cord of the lamprey Lampetra fluviatilis was studied using labeling with horseradish peroxidase in vitro. Axons of the lamprey spinal cord motoneurons were found to have collaterals terminating in ventral columns of the white matter, in which they establish contacts with dendrites of adjacent motoneurons, which can be considered as a substrate of the intermotoneuron interaction. Some axons of motoneurons give bifurcations to two equal branches connected with two neighboring ventral roots, which seems to facilitate propagation of rhythmic activity of locomotor generator in the rostro caudal direction for providing continuous wave of contraction of myotome muscles in the course of undulating movement.     

Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord

Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.     

Morphology and Intrinsic Excitability of Regenerating Sensory and Motor Neurons Grown on a Line Micropattern

Axonal regeneration is one of the greatest challenges in severe injuries of peripheral nerve. To provide the bridge needed for regeneration, biological or synthetic tubular nerve constructs with aligned architecture have been developed. A key point for improving axonal regeneration is assessing the effects of substrate geometry on neuronal behavior. In the present study, we used an extracellular matrix-micropatterned substrate comprising 3 µm wide lines aimed to physically mimic the in vivo longitudinal axonal growth of mice peripheral sensory and motor neurons. Adult sensory neurons or embryonic motoneurons were seeded and processed for morphological and electrical activity analyses after two days in vitro. We show that micropattern-guided sensory neurons grow one or two axons without secondary branching. Motoneurons polarity was kept on micropattern with a long axon and small dendrites. The micro-patterned substrate maintains the growth promoting effects of conditioning injury and demonstrates, for the first time, that neurite initiation and extension could be differentially regulated by conditioning injury among DRG sensory neuron subpopulations. The micro-patterned substrate impacts the excitability of sensory neurons and promotes the apparition of firing action potentials characteristic for a subclass of mechanosensitive neurons. The line pattern is quite relevant for assessing the regenerative and developmental growth of sensory and motoneurons and offers a unique model for the analysis of the impact of geometry on the expression and the activity of mechanosensitive channels in DRG sensory neurons.     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

Electrical characteristics of neurons in the cat motor cortex

Electrical characteristics of motor cortical neurons were studied in acute experiments on immobilized cats. Values of the input resistances varied from units to tens of megohms (mean 11.11±3.93 MΩ). The threshold current is a hyperbolic function of input resistance of the corresponding neurons and negative correlation was found between the axonal conduction velocity and input resistance. The time constant (τ₀) of the membrane was 7.1±3.46 msec. A time constant τ₁, of 1.65±0.36 msec, could also be distinguished in some neurons. Electrotonic lengths of dendrites of the cortical neurons were calculated by the use of Rall's model: mean 3.66±0.94 (in units of length constant).     

Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves In Vivo

Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction in vivo. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons in vivo, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.     

N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons

N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of ‘DYNLRB1 (74–96)’, a small peptide derived from DYNLRB1’s C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.