Experience with the use of horseradish peroxidase for light and electron microscope studies of interneuronal connections]

By the method based on a retrograde axonal transport of exogenous horseradish peroxidase (HRP), the origins of afferentation of the motor cortex of adult cats, kittens and albino rats were studied. HRP-positive neurons were found by light and electron microscopy in the somatosensory cortex (C1) of the ipsilateral hemisphere and in the portions of the cortex of the contralateral hemisphere which were symmetrical to the site of injection of HRP. The disposition of neurons, marked by HRP, in the Vth layer of the motor cortex suggest that these neurons may send their axons into the bundles of comissural fibres going to the motor cortex of the opposite hemisphere. This method considerably expands possibilities of revealing the origins of afferentation of the investigated portion of the nervous system and allows more complete and reliable investigation of interneuronal connections.     

Selective binding, uptake, and retrograde transport of tetanus toxin by nerve terminals in the rat iris. An electron microscope study using colloidal gold as a tracer

A series of specific macromolecules (tetanus toxin, cholera toxin, nerve growth factor [NGF], and several lectins) have been shown to be transported retrogradely with high selectivity from terminals to cell bodies in various types of neurons. Under identical experimental conditions (low protein concentrations injected), most other macromolecules, e.g. horseradish peroxidase (HRP), albumin, ferritin, are not transported in detectable amounts. In the present EM study, we demonstrate selective binding of tetanus toxin to the surface membrane of nerve terminals, followed by uptake and subsequent retorgrade axonal transport. Tetanus toxin or albumin was adsorbed to colloidal gold particles (diam 200 A). The complex was shown to be stable and well suited as an EM tracer. 1-4 h after injection into the anterior eye chamber of adult rats, tetanus toxin-gold particles were found to be selectively associated with membranes of nerve terminals and preterminal axons. Inside terminals and axons, the tracer was localized mainly in smooth endoplasmic reticulum (SER)-like membrane compartments. In contrast, association of albumin-gold complexes with nervous structures was never observed, in spite of extensive uptake into fibroblasts. Electron microscope and biochemical experiments showed selective retrograde transport of tetanus toxin-gold complexes to the superior cervical ganglion. Specific binding to membrane components at nerve terminals and subsequent internalization and retrograde transport may represent an important pathway for macromolecules carrying information from target organs to the perikarya of their innervating neurons.     

Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles

The uptake and retrograde transport of noradrenaline (NA) within the axons of sympathetic neurons was investigated in an in vitro system. Dissociated neurons from the sympathetic ganglia of newborn rats were cultured for 3-6 wk in the absence of non-neuronal cells in a culture dish divided into three chambers. These allowed separate access to the axonal networks and to their cell bodies of origin. [3H]NA (0.5 X 10(-6) M), added to the axon chambers, was taken up by the desmethylimipramine- and cocaine-sensitive neuronal amine uptake mechanisms, and a substantial part was rapidly transported retrogradely along the axons to the nerve cell bodies. This transport was blocked by vinblastine or colchicine. In contrast with the storage of [3H]NA in the axonal varicosities, which was totally prevented by reserpine (a drug that selectively inactivates the uptake of NA into adrenergic storage vesicles), the retrograde transport of [3H]NA was only slightly diminished by reserpine pretreatment. Electron microscopic localization of the NA analogue 5-hydroxydopamine (5-OHDA) indicated that mainly large dense-core vesicles (700-1,200-A diam) are the transport compartment involved. Whereas the majority of small and large vesicles lost their amine dense-core and were resistant to this drug. It, therefore, seems that these vesicles maintained the amine uptake and storage mechanisms characteristic for adrenergic vesicles, but have lost the sensitivity of their amine carrier for reserpine. The retrograde transport of NA and 5-OHDA probably reflects the return of used synaptic vesicle membrane to the cell body in a form that is distinct from the membranous cisternae and prelysosomal structures involved in the retrograde axonal transport of extracellular tracers.     

Afferent connections in the ventromedial hypothalamus of rats demonstrated by retrograde transport of horseradish peroxidase

Projections into rat ventromedial hypothalamus were studied with retrograde transport of horseradish peroxidase (HRP). Following injection of HRP into ventromedial hypothalamus, labeled neurons were found in cortical and medial amygdaloid nuclei, ipsilateral mediodorsalis thalamus (MD), dorsal raphe nucleus, and contralateral sensorimotor cortex. Futhermore, labeled axons that connect directly amygdala with hypothalamus (DAH) also were found.     

Colloquium 10: 3

Previous work has shown that neurotrophins bind to and activate Trk receptors on distal axons, and that neurotrophin‐Trk complexes are internalized and retrogradely transported to cell bodies. Whether retrograde transport of neurotrophins and retrograde neurotrophin‐Trk signalling are necessary for survival remains unclear, and recently published findings are controversial. We are using compartmentalized cultures of sympathetic neurons to address the mechanism of retrograde NGF signalling and survival. We performed survival experiments using either the Trk kinase inhibitor K252a to inhibit TrkA activity in different cellular compartments, or a dominant‐negative form of dynamin, K44A dynamin, to block internalization of NGF‐TrkA complexes. We found that sympathetic neurons supported by NGF acting on distal axons undergo apoptosis when TrkA activity in either cell bodies or distal axons is inhibited by K252a, or when internalization is blocked by K44A dynamin. Results of experiments employing three‐compartment chambers indicate that TrkA signalling is required within cell bodies and distal axons, but not in proximal axons, for retrograde support of survival. Likewise, TrkA activity within distal axons, but not in proximal axons, is required for retrograde transport of [125I] NGF. Finally, peptide‐mediated delivery of affinity‐purified anti‐NGF into cell bodies results in apoptosis of neurons. Taken together, our results support a model in which NGF internalization and retrograde transport and retrograde TrkA signalling are necessary for survival of sympathetic neurons. This work is supported by the NIH and HHMI.     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Reversal of axonal transport at a nerve crush.

Abstract— —We have compared retrograde axonal transport of ³H-labeled protein in normal rat motor and sensory axons, and axons which were injured by a distal ligation of the sciatic nerve. After injection of L-[³H]leucine into the vicinity of the neuron cell bodies, labeled protein was transported into the axons. A premature return of protein towards the cell bodies occurred in the injured axons, which we interpret as a reversal of axonal transport occurring at the site of injury. We estimate that reversal of transport occurred within 1.9–2.4 h of the arrival of labeled protein at the injury, and that the minimum velocity of the subsequent retrograde transport was 112–133 mm day^?1. The ability of the injured axons to reverse transport developed about 0.8 h after making the injury. A large fraction of the orthograde transported protein was returned towards the cell body: it is estimated that by 28 h after labeled protein in sensory axons reached the injury, 46% of the³H-labeled protein originally transported to the injury site had been returned. In intact sensory nerves at this time only 15% of the transported protein had returned. It is suggested that axonal injury produces a sudden increase in the return of newly synthesized protein to the cell body, and that this might serve as a signal for chromatolysis.     

Different Patterns of Neuronal Infection after Intracerebral Injection of Two Strains of Pseudorabies Virus

Pseudorabies virus (PRV), a swine neurotropic alphaherpesvirus, is known to invade the central nervous system (CNS) of a variety of animal species through peripherally projecting axons, replicate in the parent neurons, and then pass transsynaptically to infect other neurons of a circuit. Studies of the human pathogen herpes simplex virus type 1 have reported differences in the direction of transport of two strains of this virus after direct injection into the primate motor cortex. In the present study we examined the direction of transport of virulent and attenuated strains of PRV, utilizing injections into the rat prefrontal cortex to evaluate specific movement of virus through CNS circuitry. The data demonstrate strain-dependent patterns of infection consistent with bidirectional (anterograde and retrograde) transport of virulent virus and unidirectional (retrograde) transport of attenuated PRV from the site of injection. The distribution of infected neurons and the extent of transsynaptic passage also suggest that a release defect in the attenuated strain reduces the apparent rate of viral transport through neuronal circuitry. Finally, injection of different concentrations of virus influenced the onset of replication within a neural circuit. Taken together, these data suggest that viral envelope glycoproteins and virus concentration at the site of injection are important determinants of the rate and direction of viral transport through a multisynaptic circuit in the CNS.     

Single Doses of Acrylamide Reduce Retrograde Transport Velocity

Abstract: Single doses of acrylamide (0–1.3 mmol/kg) produced a dose-dependent decrease in the transport of ¹²⁵I-tetanus toxin to the perikarya of sensory neurons in dorsal root ganglia and motor neurons in ventral spinal cord. Acrylamide was a more potent inhibitor of retrograde transport in sensory axons than in motor axons. Substantially greater doses of N,N '-methylene-bis-acryl-amide, a reportedly non-neurotoxic analog of acrylamide, were required to alter the axonal transport of ¹²⁵I-tetanus toxin. Velocity of retrograde transport was assessed by determining the position of the leading edge of transported¹²⁵I-tetanus toxin at times following single doses of acrylamide. Acrylamide reduced the velocity of ¹²⁵I-tetanus toxin transport in a dose-dependent manner by up to 75%. No change in neuronal uptake of ¹²⁵I-tet-anus toxin was detected. It is concluded that single doses of acrylamide produce profound alterations in retrograde transport which precede the appearance of structural changes in affected nerve fibers.     

Specific guidance of motor axons to duplicated muscles in the developing amniote limb

The effect of alteration of limb pattern upon motor axon guidance has been investigated in chick embryos. Following grafting of the zone of polarizing activity (ZPA) into the anterior margin of the early limb bud, limbs develop with forearms duplicated about the anteroposterior axis. The position of motoneurones innervating the duplicated posterior forearm extensor EMU was mapped by retrograde transport of horse radish peroxidase (HRP). The motor pool labelled from injection into the anteriorly duplicated EMU muscle is consistently similar to that supplying the posterior EMU muscle on the unoperated side of the embryo. In those cases where the axons are well filled, their trajectories from the injection site are observed to change position within the radial nerve to specifically innervate the duplicated muscle. The axons modify their trajectories proximal to the level of limb duplication in a region where there is no change in the pattern of overt differentiation of the limb cells. This suggests that axons may use a cell's positional value to navigate and provides significant support for the theory of positional information.     

Topical organization of the neuronal projections of the substantia nigra and ventral field of the tectum mesencephali onto the head of the caudate nucleus in the cat brain

The investigation performed on cats by means of retrograde axonal transport of horseradish peroxidase and luminophores has presented the data demonstrating spatial organization of separate part projections of the nigral complex and the tegmental ventral field to various segments of the caudate nucleus head. Terminal fields from neurons of various parts of the substantia nigra and the tegmental ventral field are demonstrated to overlap in segments of the caudate nucleus. Experiments with double fluorescent labelling demonstrate divergence of axons of the nigral neurons.     

NGF and the local control of nerve terminal growth

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response. 1994 John Wiley & Sons, Inc.     

Neurons forming striatonigral connections in the rat brain

Using the retrograde axonic transport of horseradish peroxidase method the striatal neurons projections to substance nigra have been studied in rats. After peroxidase injection into substance nigra a considerable number of small and medium sized neurons (10-20 mkm) become labelled in the ipsilateral striatum. Large labelled striatal cells (20-25 mkm) have been found. Among labelled striatal neurons multipolar cells with triangular and oval body prevailed. The number of cells with elongated multipolar or spindle-shaped body was less. The data obtained disprove the conception that only large ("giant") neurons form the efferent striatal pathways to substance nigra.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Retrograde axonal transport in lateral motor neurons of the chick embryo prior to limb bud innervation

Horseradish peroxidase was injected into the distal limb buds of 3-, 3.5-, and 4-day chick embryos and was allowed to diffuse into the path of outgrowing axons. In the majority of the embryos injected, reaction product was found in cells of the ventral spinal cord ipsilateral to the injection site. The reaction product in the most clearly stained cells appeared smooth and diffuse rather than the typical granular appearance found in later embryos and adults. A granular background was seen in 4-day embryos, however, although distinct granular cell outlines were infrequent. This study indicates that retrograde axonal transport is a very early cellular feature during neurogenesis, demonstrable almost from the inception of neurite outgrowth. It is speculated that this transport capacity might function in relaying positional information from growing fiber tips back to cell bodies to aid in the formation of specific synaptic connections or in guiding directed axonal growth, or it may provide a means for trophic interactions vital to the survival of the young neuroblast.     

Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons

NGF is a target-derived growth factor for developing sympathetic neurons. Here, we show that application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement for PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

Dopamine and noradrenaline neurons projecting to the septal area in the rat

Summary The organisation of the catecholamine innervation of the rat septal area was investigated by means of the glyoxylic acid fluorescence method in combination with dopamine uptake studies, lesions and retrograde tracing of horseradish peroxidase. The following catecholamine systems to the septum could be established:The Locus Coeruleus Noradrenergic System These axons are widespread in the septum forming a moderately dense innervation in the anterior hippocampus, the medial septal nucleus, the nucleus of the diagonal band, and the interstitial nucleus of the stria terminalis, and a sparse innervation in the lateral septal nucleus and the septofimbrial nucleus.The Medulla Oblongata Noradrenergic System This system originates in the Al, A2 or A3 cell groups, the axons forming a very dense innervation in the ventral part of the interstitial nucleus of the stria terminalis, a moderately dense innervation in the nucleus of the diagonal band and lateral septal nucleus, and a sparse innervation in the medial septal nucleus, the septofimbrial nucleus and the dorsal part of the interstitial nucleus of the stria terminalis.The Mesencephalic Dopaminergic System This system originates in the medial part of the A10 cell group, the axons forming two distinct terminal patterns. In the first type, smooth axons form pericellular arrangements around non-fluorescent neurons in the lateral septal nucleus. The second type is formed by fine-varicose axons which form a dense band around the fornix in the medial part of the lateral septal nucleus.The Incerto-Hypothalamic Dopaminergic System These axons most probably originate in cell bodies of the diencephalic A11, A13 and A14 cell groups, and are found in the lateral septal nucleus at the level of the anterior commissure.     

Evidence of sensory neurons in the wall of the small intestine revealed by horseradish peroxidase technique

Sensory neurons in the wall of the small intestine were studied by means of retrograde transport of horseradish peroxidase (HRP). After HRP injection into the mesenteric nerve trunks, peroxidase positive nerve cells were observed in the myenteric and submucous plexuses. Labeled cells of different shape and size were compared with neurones impregnated by silver nitrate. On the basis of HRP-labeled neurons it is concluded that some of the myenteric and submucous nerve cells send processes towards the celiac ganglia; these may correspond to afferent neurons in the wall of the small intestine.