Comparative study of spinal motoneuron axon collaterals

Numerous spinal motoneurons in mammals possess recurrent axon collaterals included in a feedback loop for controlling motoneuron activity. For nonmammalian vertebrates, the data concerning the existence of collaterals and their intraspinal branching are fragmentary and contradictory. We focused on axonal branching of motoneurons in lampreys, frogs, turtles and young rats, using light microscopic analysis of HRP- or neurobiotin-labeled motoneurons. In lampreys, only a restricted portion of spinal motoneurons, related to the dorsal fins, showed recurrent collaterals. In frogs, a great complexity and high total length of collateral branches as well as a great number of axon swellings were found. In turtles, axon collateralization of spinal motoneurons was much more restricted, and present in particular in lumbar motoneurons innervating proximal hindlimb muscles. Young rat spinal motoneurons have rather abundant recurrent axon collaterals. It is likely that the presence of axon collaterals from spinal motoneurons is related to the level of complexity of locomotion.     

Mouse subthalamic nucleus neurons with local axon collaterals

The neuronal population of the subthalamic nucleus (STN) has the ability to prolong incoming cortical excitation. This could result from intra‐STN feedback excitation. The combination of inducible genetic fate mapping techniques with in vitro targeted patch‐clamp recordings, allowed identifying a new type of STN neurons that possess a highly collateralized intrinsic axon. The time window of birth dates was found to be narrow (E10.5–E14.5) with very few STN neurons born at E10.5 or E14.5. The fate mapped E11.5–12.5 STN neuronal population included 20% of neurons with profuse axonal branching inside the nucleus and a dendritic arbor that differed from that of STN neurons without local axon collaterals. They had intrinsic electrophysiological properties and in particular, the ability to generate plateau potentials, similar to that of STN neurons without local axon collaterals and more generally to that of classically described STN neurons. This suggests that a subpopulation of STN neurons forms a local glutamatergic network, which together with plateau potentials, allow amplification of hyperdirect cortical inputs and synchronization of the STN neuronal population.     

Frog sympathetic ganglion cells have local axon collaterals

Amphibian autonomic ganglia have been used as simple models for studies involving the physiology of synaptic transmission. These models assume an anatomical simplicity where the ganglion is a simple relay for central nervous system output to peripheral autonomic targets. Cholinergic preganglionic fibers innervate the soma and proximal axon of the unipolar ganglion cells, which were thought to relay the information to the periphery with little ganglionic processing. However, several different types of synaptic potentials occur in response to preganglionic stimulation. Also, a variety of neuropeptides are found in both preganglionic fibers and ganglion cells; at least one of the peptides found in preganglionic fibers is known to act as a neurotransmitter in the ganglion. Finally, there may be communication between ganglion cells. In the present study, we have explored the morphology of lumbar sympathetic chain ganglion cells by intracellular injection with horseradish peroxidase to determine whether an anatomical substrate exists for processing information within these ganglia. We have shown that 39% of these cells have axons that branch within the ganglion. While both major classes of ganglion cells (B cells and C cells) had intraganglionic axon collaterals, there was a marked difference in the frequency: 65% of the C cell axons had collaterals while only 19% of the B cell axons collateralized within the ganglion. Ultrastructural examination of labeled axon collaterals indicated that these collaterals receive synaptic input; whether the collaterals also make synapses has not been definitively established.     

Cellular and molecular features of axon collaterals and dendrites

Neural geometry is the major factor that determines connectivity and, possibly, functional output from a nervous system. Recently some of the proteins and pathways involved in specific modes of branch formation or maintenance, or both, have been described. To a variable extent, dendrites and axon collaterals can be viewed as dynamic structures subject to fine modulation that can result either in further growth or retraction. Each form of branching results from specific molecular mechanisms. Cell-internal, substrate-derived factors and functional activity, however, can often differ in their effect according to cell type and physiological context at the site of branch formation. Neural branching is not a linear process but an integrative one that takes place in a microenvironment where we have only a limited experimental access. To attain a coherent mechanism for this phenomenon, quantitative in situ data on the proteins involved and their interactions will be required.     

Spatial distribution of axon collaterals of single inferior olive neurons

The aim of this study was to define the overall distribution pattern of the axon collaterals of single inferior olive (IO) neurons in relation to the multiple somatotopic maps defined by the climbing fiber (CF) input through the cerebellar cortex. In a previous study (Rosina and Provini: Brain Res. 289:45-63, '83), it was shown that the IO neurons supply interlobar collaterals to pairs of somatotopically related areas in the intermediate part of the anterior lobe (PIAL), in the paramedian lobule (PML), in crus II, and in the simple lobule, within strips C1 to D2. The residual branches then could either distribute within single folia or to adjacent folia within each somatotopically defined cerebellar area or both. We studied whether or not the IO axons branch over neighboring folia of the face-forelimb (FL) areas of PIAL and PML and how this interfolial branching relates to the interlobar collateralization by using the multiple fluorescent retrograde tracing technique. The main results of the study were as follows: the axons from neurons in IO subdivisions that are related to strips C1-C3 give off two interfolial branches in the FL area of PIAL and practically no interfolial collaterals are given in the FL area of PML; and the neurons that give off interfolial collaterals also give interlobar branches. From these data we have inferred the general branching pattern of the IO neurons that convey FL information to PIAL and PML. Each neuron gives off two interlobar collaterals: the branch directed to PIAL splits again into two interfolial collaterals, while each of these three collaterals should give off about three branches within each target folium to account for the ten collaterals estimated to be present in the cat. The distribution pattern of IO axon collaterals proposed here suggests that the same CF-relayed information may interact, at the Purkinje cell level, with different sets of mossy fiber inputs. The effect of this interaction would be to modulate the motor commands forwarded to specific muscle groups in relation to the different conditions under which a given movement is executed.     

Excitation of perigeniculate neurones via axon collaterals of principal cells

Intra- and extracellular recordings were used to study the synaptic excitation of perigeniculate neurones in the cat. The cells were activated with a monosynaptic latency after stimulation of the primary visual cortex and with a disynaptic latency after optic tract stimulation. Collision tests revealed that this excitation was due to a common mechanism, namely an excitatory connexion via axon collaterals of principal cells in the lateral geniculate nucleus.     

Nerve growth factor-induced sprouting of mature, uninjured sympathetic axons.

The infusion of nerve growth factor (NGF) into the lateral ventricle of the mature rat brain elicits a sprouting response from axons associated with the intradural segment of the internal carotid artery. Using electron microscopic techniques, we observed a three-fold increase in the total number of perivascular axons. This NGF-elicited response is characterized by a dramatic reduction in glial cell ensheathment similar to that observed during development and by the presence of profiles devoid of organelles that may represent newly formed sprouts. In spite of the increase in axon number, no significant changes in the percentage of small, medium, or large axons were observed. The three-fold increase in the total number of axons was accompanied by an increase in the number of axons/fascicle but no change in the number of fascicles. This, along with the observation that a majority of sprouted axons were associated with other axons, supports the idea that the sprouted axons tend to associate preferentially with other axons. Bilateral superior cervical ganglionectomies following cytochrome C infusion indicate that approximately 60% of the axons associated with the internal carotid artery arise from the superior cervical ganglion and that the majority of axons contacting the smooth muscle layer arise from this ganglion. Sympathectomy following NGF infusion resulted in a 79% reduction in the total number of perivascular axons, demonstrating overwhelmingly that the majority of sprouted axons are sympathetic fibers. These results demonstrate that infusion of NGF into the mature rat brain results in the preferential sprouting of sympathetic axons associated with the internal carotid artery. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic cerebrovascular innervation in the adult animal and that mature, uninjured sympathetic neurons remain responsive to NGF.     

Systematic and differential myelination of axon collaterals in the mammalian auditory brainstem

A brainstem circuit for encoding the spatial location of sounds involves neurons in the cochlear nucleus that project to medial superior olivary (MSO) neurons on both sides of the brain via a single bifurcating axon. Neurons in MSO act as coincidence detectors, responding optimally when signals from the two ears arrive within a few microseconds. To achieve this, transmission of signals along the contralateral collateral must be faster than transmission of the same signals along the ipsilateral collateral. We demonstrate that this is achieved by differential regulation of myelination and axon caliber along the ipsilateral and contralateral branches of single axons; ipsilateral axon branches have shorter internode lengths and smaller caliber than contralateral branches. The myelination difference is established prior to the onset of hearing. We conclude that this differential myelination and axon caliber requires local interactions between axon collaterals and surrounding oligodendrocytes on the two sides of the brainstem. GLIA 2016;64:487–494     

Neurofilament spacing,phosphorylation, and axon diameter in regenerating and uninjured lamprey axons

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 μm. Antibodies specific for highly phosphorylated NFs labeled only large axons (>10 μm), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5–10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment. © 1996 Wiley-Liss, Inc.     

Spinal cord layer I neurons with axon collaterals that generate local arbors

The morphology and location of physiologically characterized neurons in layer I of the spinal cord dorsal horn were revealed by the intracellular deposition of horseradish peroxidase (HRP). This material showed that the axons of some layer I neurons issue varicosity-bearing collaterals that generate arbors that overlap the neurons' dendritic territories in layer I.     

Light and electron microscopic analyses of intraspinal axon collaterals of sympathetic preganglionic neurons

Experiments were performed in pigeons (Columba livia). Sympathetic preganglionic neurons (SPNs) in the first thoracic spinal cord segment (T1) were identified electrophysiologically using antidromic activation and collision techniques and then intracellularly labeled with horseradish peroxidase (HRP). In 6 of 10 HRP-labeled SPNs, the site of axon origin and intraspinal axonal trajectory could be specified. In 2 of the 6 HRP-labeled axons, the peripherally projecting process branched intraspinally. The presence or absence of SPN intraspinal axonal collateralization did not correlate with parent perikaryal subnuclear location or dendritic alignment. None of the collaterals were recurrent onto the SPN of origin. Light microscopically, the collateral branches appeared to end with punctate, bulbous swellings. The spinal regions of the terminal end swellings for the two axons did not overlap one another. In one instance the entire terminal field was confined within the principal preganglionic cell column (column of Terni). The other axon had collateral branches which terminated in the lateral white matter and in a ventrolateral region of lamina VII. A serial section, electron microscopic reconstructive analysis of the entire intraspinal collateral terminal field within the column of Terni revealed that: (a) the primary collateral process was unmyelinated and arose at a node of Ranvier; (b) after issuance of the collateral branch, the myelinated parent axon continued to increase its myelin wrapping throughout the spinal gray; (c) the bulbous swellings observed light microscopically corresponded to axon terminal boutons and regions of synaptic contact; (d) the axon collateral terminals were exclusively presynaptic to small caliber dendrites and formed only asymmetric specializations; and (e) the collateral terminals contained numerous mitochondria, and densely packed, electron-lucent, spherical vesicles.     

Transient retinal axon collaterals to visual and somatosensory thalamus in neonatal hamsters

We have studied the postnatal development of individual axons in the optic tract and thalamus of the Syrian hamster, concentrating attention on retinal ganglion cell axons that make a transient projection to the main somatosensory nucleus, the ventrobasal complex. We bulk-filled axons with horseradish peroxidase in hemithalami maintained en bloc, in vitro. After processing and reaction with diaminobenzidine, we reconstructed individual axons from serial sections. In hamsters and other rodents, the optic tract is composed of superficial and internal components, either or both being possible sources of the retino-ventrobasal projection. Both project to the midbrain, but in normal adults only the superficial optic tract maintains collaterals in the thalamus. We found that the axons of the internal component bear numerous transient thalamic collaterals on postnatal days 0, 1, and 2, and some of these extend into the ventrobasal complex. Axons in the superficial optic tract also bear collaterals on days 0 to 2, but these are confined to the superficial half of the dorsal lateral geniculate nucleus. Thus the transient retino-ventrobasal projection comprises solely transient collaterals originating from axon trunks in the internal optic tract. On days 1 and 2, some collaterals from the superficial optic tract appear to have begun to arborize in the lateral geniculate nucleus. In contrast, collaterals from internal optic tract axons to the ventrobasal complex branch little if at all as they traverse the lateral geniculate nucleus, and at no time prior to their elimination do they develop an appreciable terminal arbor. These long collaterals often terminate in growth cones that include lamellopodia. Our HRP-impregnation method also revealed some transient non-retinofugal axons that pass medially from the ventral lateral geniculate nucleus to the ventrobasal complex but then return without terminating or branching. By day 4, they are absent, as are collaterals from the internal optic tract to the ventrobasal complex.     

Organization of local axon collaterals of efferent projection neurons in rat visual cortex

We have studied the laminar origins of local long-range connections within rat primary visual cortex (area 17), by using retrograde tracing of nerve cell bodies with fluorescent markers. Injections throughout the thickness of cortex produce distinct laminar labeling patterns which indicate that a substantial number of cells in layers 2/3, 5, and 6 have wide local axon collateral arbors, while the local arbors of layer 4 cells are much narrower. Double labeling experiments which combined area 17 injections with injections into different projection targets of area 17 (opposite area 17, area 18a, and area 18b) show that many cortico-cortically projecting cells make widespread projections within area 17. In contrast, the overwhelming majority of subcortically projecting cells have narrow collateral arbors within area 17. Anterograde tracing of local projections within areas 17 with the lectin Phaseolus vulgaris leucoagglutinin shows an extensive system of horizontally running fibers which terminate in distinct 0.15-0.25 mm wide clusters up to 1.8 mm from the injection site. On horizontal sections the termination pattern resembles a closely spaced lattice. The results indicate that cortico-cortically projecting cells provide for long-range interactions between distant points of the visuotopic map, while subcortically projecting cells mediate information within a cortical column. Interestingly, subcortically projecting cells differ functionally from cortico-cortically projecting cells in that they are not orientation selective (Klein et al., Neurosci. 17:57-78, '86; Mangini and Pearlman, J. Comp. Neurol. 193:203-222, '80; Simmons and Pearlman, J. Neurophysiol. 50:838-848, '83). We therefore suggest that cortico-cortically projecting cells with wide collateral arbors are orientation selective and that clustered long-range projections within area 17 connect columns with similar functional specificity.     

Olivocochlear neurons sending axon collaterals into the ventral cochlear nucleus of the rat

The olivocochlear projection constitutes the last stage of the descending auditory system in the mammalian brain. Its neurons reside in the superior olivary complex (SOC) and project to the inner and outer hair cell receptors in the cochlea. Olivocochlear neurons were also reported to send axon collaterals into the cochlear nucleus, but controversies about their number and about species differences persist. By injecting the fluorescent retrograde axonal tracers diamidino yellow and fast blue into the cochlea and the ventral cochlear nucleus (VCN), we studied the distribution and number of olivocochlear neurons with and without axon collaterals into the VCN of the rat. We found that olivocochlear neurons residing in the lateral superior olive (LSO), the intrinsic lateral olivocochlear cells (intrinsic LOCs), do not send axon collaterals into the VCN. By contrast, a majority, and possibly all, olivocochlear neurons residing in the ventral nucleus of the trapezoid body (VNTB), the medial olivocochlear cells (MOCs), do have such axon collaterals. These cells may thus affect processing in the ascending auditory pathway at the level of the receptors and concurrently at the level of the secondary sensory neurons in the cochlear nucleus. Belonging to the lateral olivocochlear system, shell neurons reside around the LSO and form a third group of olivocochlear cells (shell LOCs). Like intrinsic LOCs, they innervate the inner hair cells, but like MOCs they do, by means of axon collaterals, project into the VCN. These findings have implications for understanding both auditory signal processing and the plasticity responses that occur following loss of cochlear function.     

Divergent axon collaterals of rat locu Demonstration by a fluorescent double labeling technique

The distribution of the noradrenaline-containing neurons of the rat locus coeruleus has been investigated with retrograde labeling techniques using two different fluorescent tracers. Injections were placed in the prefrontal cortex, the striatum, the thalamus, the hippocampus, the cerebellar cortex and the lumbar spinal cord.No evidence for locus coeruleus projections to the striatum was found. Injections in the cortex, thalamus and hippocampus revealed not only ipsilateral but also contralateral labeling of cells in the locus coeruleus. Following unilateral or bilateral homo- or heterotopic injections of the two tracers several cells of the locus coeruleus were double labeled. Combined injections of the two fluorophores in any of these forebrain areas and in the spinal cord also produced double labeled cells. The majority of double labeled cells was located in an area between the ventral and the dorsal parts of the locus coeruleus.These results indicate that individual neurons of the locus coeruleus have the possibility to influence adrenergic receptors at remote areas in the central nervous system.     

Raphespinal and reticulospinal axon collaterals to the hypoglossal nucleus in the rat.

Neurons in the medial tegmental field project directly to spinal somatic motoneurons and to cranial motoneuron pools such as the hypoglossal nucleus. The axons of these neurons may be highly collateralized, projecting to multiple levels of the spinal cord and to many diverse regions at different levels of the neuraxis. We employed a double fluorescent retrograde tracer technique to examine whether medial tegmental neurons that project to the spinal cord also project to the hypoglossal nucleus. Injections of Diamidino Yellow into the hypoglossal nucleus and Fast Blue into the spinal cord produced large numbers of double labeled neurons in the medial tegmental field, particularly in the caudal raphe nuclei and adjacent ventromedial reticular formation. In these structures the number of neurons projecting to both the hypoglossal nucleus and the spinal cord was equivalent to the number of neurons projecting to multiple levels of the spinal cord observed in control animals. Fewer neurons projecting to both the hypoglossal nucleus and the spinal cord were observed in several other nuclei and subregions of the medial tegmental field, while almost no such neurons were observed in the lateral tegmental field or other pontomedullary structures. These results demonstrate that neurons of the caudal raphe nuclei and adjacent ventromedial reticular formation project to both the spinal cord and the hypoglossal nucleus, and support the concept that the diffuse projections to motoneuron pools from the medial tegmental field globally modulate both spinal and cranial somatic motoneuron excitability.