Retrograde intraaxonal transport of horseradish peroxidase by neurons in octopus

While retrograde axonal transport is the basis of a widely used neuroanatomical method, it has been rigorously demonstrated in vivo only in a few vertebrate species and not yet in an invertebrate. Evidence is presented that motor neurons of the octopus stellate ganglion are capable of retrograde intraaxonal transport of horeseradish peroxidase. This demonstration shows that retrograde transport occurs in widely divergent groups of animals, and may be a general property of neurons.     

The role of nerve-growth factor (NGF) in the central nervous system

NGF is a protein that stimulates growth and differentiation of sympathetic and sensory components of the peripheral nervous system. The purpose of this review is to examine the evidence that NGF has similar activity in the central nervous system. First, the primary mode of interaction of NGF with the nerve cell will be discussed, and the possibility that such an interaction takes place in the brain will be examined. Recent studies have demonstrated that NGF promotes regenerative sprouting of damaged catecholamine-containing neurons in the brain. The next part of the paper reviews this literature, and other findings that indicate or contraindicate a role of NGF in brain maturation or maintenance. The final part of this paper suggests specific avenues for future research in this area, and presents conclusions regarding the literature on brain activity of NGF to date.     

Target-derived BDNF (brain-derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo-optic nucleus

Neurons in the peripheral nervous system depend on single neurotrophic factors, whereas those in the brain are thought to utilize many different trophic factors. This study examined whether some neurons in the brain critically depend on a single trophic factor during development. Neurons in the isthmo-optic nucleus (ION) of chick embryos respond to exogenous brain-derived neurotrophic factor (BDNF). Relatively high concentrations of endogenous BDNF were present in the ION of 14-18-day-old chick embryos. ION target cells in the retina were immunolabeled for BDNF but showed surprisingly low levels of BDNF mRNA. These data suggest that ION target cells derive some BDNF from other retinal sources. No BDNF mRNA was detected in the ION itself. ION neurons had a very efficient retrograde transport system for BDNF and exogenous BDNF arrived in the ION intact. When the ION was deprived of endogenous trkB ligands by injection of trkB fusion proteins in the eye, cell death of ION neurons was enhanced, and this effect was mimicked by BDNF-specific blocking antibodies in the eye. TrkB fusion proteins in the retina induced cell death of ION neurons prior to visible effects on ION target cells in the retina. Immunolabel for endogenous BDNF was sparse in pyknotic ION neurons, suggesting that ION neurons with low BDNF content were eliminated by apoptosis. These data show that BDNF is an essential target-derived trophic factor for developing ION neurons and thereby validate the neurotrophic hypothesis for at least one neuronal population in the brain.     

Binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons

Newborn rat dissociated sympathetic neurons were grown in a chamber culture system, where a Teflon ring sealed with silicon grease separated the axonal plexus from the corresponding nerve cell bodies. The binding of 35S-labeled herpes virus suis (HVS) to the neurites was partially inhibited by an excess of unlabeled HVS as well as by concanavalin A, indicating the presence of specific binding sites for the virus. Specific binding was a prerequisite for the subsequent uptake and retrograde transport of HVS to the nerve cell bodies. Predominantly free nucleocapsids were detected by electron microscopy in the axons at the time of retrograde transport, both in culture and in vivo, suggesting the possibility that nucleocapsids without lipid membrane and not contained in cellular membrane compartments can be transported by retrograde axonal transport.     

Receptor-mediated retrograde transport in CNS neurons after intraventricular administration of NGF and growth factors.

Radiolabel tracer techniques were used to follow the distribution of nerve growth factor (NGF) and other neuromodulatory factors after intraventricular injection. Autoradiography showed that shortly after intraventricular injection of radio-iodinated NGF (125I-NGF), substantial amounts of radioactivity had penetrated the ventricular wall surfaces; this binding was transient and nonspecific. The 125I-NGF was progressively cleared from the central nervous system (CNS), presumably via the flow of cerebrospinal fluid (CSF) into the blood. A relatively small proportion of the injected 125I-NGF was taken up by NGF receptor-positive neurons in the CNS. Retrograde accumulation of radiolabel was observed within the basal forebrain cholinergic neurons at 5 hours after intraventricular injection. Labeling intensity was maximal at 18 hours and much reduced by 30 hours. This labeling was blocked by co-injection of an excess of unlabeled NGF. Specific and saturable retrograde labeling was also observed within other NGF receptor-bearing neurons, including the prepositus hypoglossal nucleus and the raphe obscurus nucleus. When epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-I), or IGF-II was radiolabeled and injected intraventricularly, specific labeling of neurons was observed for 125I-IGF-II and 125I-LIF within separate subpopulations of the dorsal and medial raphe. No retrograde accumulation within neurons was observed for EGF, TGF-beta 1, PDGF-AA, PDGF-BB, or IGF-I. This study describes an in vivo method for identifying putative neuromodulatory factors and their responsive neurons.     

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red–nerve growth factor (DTR–NGF) conjugate. DTR–NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR–NGF labelling in SCG neurons was shown to be dose-dependent with an EC₅₀ of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR–NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85–93%) reduction in numbers of DTR–NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.     

Effects of sympathetic histamine on vasomotor responses of blood vessels in rabbit ear to electrical stimulation

Objective

To investigate the effects of histamine receptor antagonists on vasoconstriction induced by electrical stimulation (ES) on posterior auricular nerve, and to explore the pre- and post-synaptic effects of sympathetic histamine on the vasomotor responses of vascular smooth muscle in rabbit ear.

Methods

ES was applied to posterior auricular nerves of the whole rabbit ear at 10 Hz, 20 Hz and 40 Hz, respectively. Besides, the whole ear was perfused with different histamine receptor antagonists under constant perfusion pressure, and the changes in the flow rate of perfusate were observed.

Results

The flow rate of venous outflow was decreased by ES at all the 3 frequencies. The ES-induced vasoconstriction at 20 Hz and 40 Hz could be partly inhibited by H₁ receptor antagonist chlorpheniramine (P < 0.05). After exhaustion of histamine in mast cells by pretreatment with specific mast cell degranulator compound 48/80, chlorpheniramine could still inhibit the ES-induced flow rate reduction. In contrast, H₂ receptor antagonist cimetidine could enhance the 40-Hz ES-induced flow rate reduction (P < 0.05). Moreover, ES-induced vasoconstriction at the 3 frequencies could all be enhanced by H₃ receptor antagonist thioperamide (P < 0.05).

Conclusion

Stimulation on the auricular nerve may evoke histamine release from sympathetic nerves rather than from mast cells. Moreover, the functions of sympathetic histamine vary from pre-synaptic modulation to post-synaptic vasoconstriction or vasodilatation, via activation of different histamine receptors.     

Thalamostriatal neurons with collateral projection onto the rostral reticular thalamic nucleus: anatomical study in the rat by retrograde axonal transport of iron-dextran and horseradish peroxidase

Injection of horseradish peroxidase (HRP) into the head of the reticular thalamic nucleus (RT) of rats having undergone large cortical and striatal lesions, led to the labeling of thalamic neurons in medial thalamic nuclei. After injection of iron-dextran into the corpus striatum and HRP into the rostral RT of intact rats, double-labeled neurons were observed in the medial thalamus, mainly in the central lateral nucleus.     

Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys

There is controversy about whether axotomized neurons undergo death or only severe atrophy after spinal cord injury (SCI) in mammals. Lampreys recover from complete spinal transection, but only about half of the severed spinal-projecting axons regenerate through the site of injury. The fates of the unregenerated neurons remain unknown, and until now death of axotomized spinal-projecting neurons has not been described in the lamprey brain. We now report that in animals allowed to survive for 12 or more weeks after spinal cord transection, several identified reticulospinal (RS) neurons were missing in Nissl-stained or neurofilament-immunostained brain whole mounts. At earlier times, these neurons were swollen and pale in Nissl-stained preparations. Retrograde fluorescent labeling from the site of transection combined with TUNEL histochemistry suggested that neuronal death, including that of the identified RS neurons, began in animals 4 weeks posttransection, reaching a peak at 12-16 weeks. This was not seen in untransected animals. The TUNEL positivity suggests that some cells were dying by apoptosis. Of special interest, among the identified neurons, this delayed cell death was restricted to neurons that at earlier posttransection times have a low probability of regeneration. These data show that SCI induces delayed cell death in lamprey spinal-projecting neurons and suggest that the reason why some neurons are "bad regenerators" is that they are already undergoing apoptotic cell death. Thus protection from apoptosis may be necessary in order to enhance axonal regeneration after SCI.     

Reduction by reserpine of the accumulation of retrogradely transported [I]nerve growth factor in sympathetic neurons

Experiments were carried out to determine if stimuli which augment preganglionic nerve activity to sympathetic neurons, and thereby cause trans-synaptic induction, increase the retrograde transport of nerve growth factor (NGF). It was found that nerve activity had no effect on retrograde transport of [¹²⁵I]NGF. It was found, however, that reserpine decreased retrograde transport of [¹²⁵I]NGF and this inhibition was characterized. Reserpine decreased the maximal accumulation of intravenously administered[¹²⁵I]NGF in superior cervical ganglia (SCG) by about 60%. It also caused a distinct shift in the time course of accumulation so that maximal accumulation was seen 12 h after [¹²⁵I]NGF injection rather than at 9 h as in control animals. Reserpine had no effect on retrograde transport in sensory neurons. Dose-response curves showed that maximal inhibition occurred with doses of reserpine of 2.5 mg/kg i.p. and that reserpine was not able to completely block transport at any dose.The maximal inhibition of retrograde transport was achieved within 30 min of reserpine administration and inhibitory activity was unchanged for 36 h. The ability of sympathetic neurons to transport [¹²⁵I]NGF subsequently recovered and was normal 96 h after reserpine administration. The inhibitory effect of reserpine appears to be due to an action at or very near to the nerve terminal since it was effective at reducing NGF transport at very low doses (0.33 μg) when co-administered directly into the eye with [¹²⁵I]NGF. An action of reserpine at the nerve terminal was further suggested by the inability of reserpine to affect transport if the drug was given 4 h after [¹²⁵I]NGF administration. Based upon these data, it is suggested that there may be two pools of retrogradely transported NGF and that only more rapidly turning over pool is reserpine-sensitive. This pool may represent the retrogradely moving synaptic vesicles or some derivative of the vesicles.     

Regulation of Rat Sympathetic Nerve Density by Target Tissues and NGF in Maturity and Old Age

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of -50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density -50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target-derived neurotrophic factors.     

Retrograde transport of dopamine β-hydroxylase antibodies in sympathetic neurons: Effects of drugs modifying noradrenergic transmission

Antibodies to dopamine beta-hydroxylase (anti-D beta H) were taken up by noradrenergic nerve terminals in the iris following attachment to D beta H, and were transported back to, and accumulated in, the superior cervical ganglion (SCG). Concurrent, or prior destruction of noradrenergic terminals with 6-hydroxydopamine, injected intraocularly, blocked the retrograde transport of anti-D beta H. However, recovery was rapid, reaching 50% of control values within 1 day. Such transport was characterized by a shorter time period before accumulation could be detected in the SCG and by a slower rate of accumulation. These results suggest that noradrenergic neurons recover their ability to turn over synaptic vesicles by exocytosis and transport these back to the ganglion early during the period of axonal regeneration when the axonal length is shorter than normal. The uptake and transport of anti-D beta H was regulated by alpha-adrenergic agents administered locally in the vicinity of noradrenergic nerve terminals. Thus intraocular injection of phentolamine resulted in an increased accumulation of anti-D beta H in the SCG, while amphetamine and the postsynaptic alpha-receptor antagonist, phenylephrine, decreased accumulation. Clonidine and desipramine, which have a predominant presynaptic action, failed to influence the transport of anti-D beta H. These results suggest that in vivo the uptake of anti-D beta H can be increased more by local postsynaptic reflex actions than by a mechanism depending on the inhibition of presynaptic alpha-receptors.     

Neurotrophin receptors and selective loss of cholinergic neurons in Alzheimer disease

The most consistent neuropathological finding in Alzheimer disease (AD) is the loss of cholinergic neurons of the nucleus basalis of Meynert (NbM). Using immunohistochemistry, we have previously shown that cholinergic neurons located in the ventral striatum were affected, whereas those of the caudate nucleus, putamen, and mesencephalon were spared. Since cholinergic neurons that degenerate in AD are sensitive to NGF and those that are spared are not, it has been hypothesized that the loss of neurotrophins receptors may play a role in the death of cholinergic neuronsin AD. Using immunohistochemistry, we have detected the presence of TrkA on most cholinergic neurons from the NbM, on some from those of the striatum, but not on those of the mesencephalon in the human brain. In AD patients, the number of neurons that expressed TrkA was markedly decreased in the NbM very likely as a consequence of cholinergic neuronal loss. In the striatum, despite the loss of high-affinity NGF binding prevously reported, no loss of TrkA was observed. Taken together, these results suggest a decreased expression of NGF receptors on the striatal cholinergic neurons in AD. This loss may contribute, when it reaches a crucial threshold, to the death of cholinergic neurons occurring in AD.     

The T1 domain of Kv1.3 mediates intracellular targeting to axons

Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [Gu, C., Jan, Y.N. & Jan, L.Y. (2003) Science,301, 646-649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment-specific endocytosis or compartment-specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein-labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild-type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting.     

Somatostatinergic neurons in the insular cortex project to the spinal cord: combined retrograde axonal transport and immunohistochemical study

A double-labeling method combining immunohistochemistry and a retrograde tracer technique using biotin-horseradish peroxidase (B-HRP) was employed to identify a descending somatostatinergic fiber system from the insular cortex to the spinal cord. Injection of B-HRP into the spinal cord at cervical or lumbar levels resulted in the labeling of a number of neurons in the insular cortex. Simultaneous immunostaining revealed the existence of double-labeled neurons in the insular cortex. The result provides direct evidence for the presence of a descending somatostatinergic pathway from the insular cortex to lumbar levels of the spinal cord.     

Synapses on axons of sympathetic preganglionic neurons in rat and rabbit thoracic spinal cord

Axosomatic and axodendritic synapses occur on sympathetic preganglionic neurons, but it is not yet known whether their axons receive synaptic input, which could be particularly effective at regulating sympathetic outflow. Here, we examined retrogradely labelled sympathetic preganglionic axons to see if they received synapses. Cholera toxin B subunit (CTB) or CTB conjugated to horseradish peroxidase (CTB-HRP) was used to label neurons projecting to the rat or rabbit superior cervical ganglion, the rat adrenal medulla, or the rabbit stellate ganglion. At the light microscopic level, small groups of CTB-immunoreactive axons travelled through the ventral horn near its lateral boundary, with occasional axons taking a more medial course. The axons passed through the ventrolateral funiculus to exit at the ventral roots. In parasagittal section, a few axons branched within the ventral horn, sending processes rostrally and caudally for short distances before they turned ventrally to exit the spinal cord. At the ultrastructural level, CTB-immunoreactive rat and rabbit sympathetic preganglionic axons were almost exclusively unmyelinated. In contrast, labelling with CTB-HRP revealed both myelinated and unmyelinated axons in the ventral horn, the ventrolateral white matter, and the ventral roots. CTB-HRP also allowed the detection of the initial segment of a sympathetic preganglionic axon. Synapses, with vesicles clustered presynaptically and membrane specializations postsynaptically, were found on some unmyelinated CTB-immunoreactive axons. Occasional axons received several synapses. Synapses were most common on CTB-containing axons just ventral to the intermediolateral cell column. One synapse was found on an axon within 2 μm of its origin from a proximal dendrite. Rare synapses were found several hundred micrometers ventral to the intermediolateral cell column. One branching axon had synapses just below the branch point on both the main axon and the axonal branch. These findings indicate an extensive synaptic input to the axons of at least some sympathetic preganglionic neurons. These axoaxonic synapses could have a profound effect on sympathetic activity. © 1995 Wiley-Liss, Inc.     

Basal forebrain nitric oxide synthase (NOS)-containing neurons project to microvessels and NOS neurons in the rat neocortex: cellular basis for cortical blood flow regulation

Stimulation of basal forebrain neurons results in local increases in cortical cerebral blood flow that are dependent upon cholinergic and nitrergic mechanisms. In the present study, we investigated the possibility that basal forebrain nitric oxide synthase (NOS)-containing neurons project to microvessels and NOS interneurons in the rat cerebral cortex. We performed quisqualic (QUIS) acid lesions of the basal forebrain and evaluated their effects on cortical NOS immunostained nerve terminals, with emphasis on those associated with microvessels and NOS interneurons, both at the light and/or electron microscopic levels. The results show that basal forebrain NOS neurons provide about one third of the overall cortical NOS innervation. Further, the data indicate that basalocortical NOS fibres establish privileged associations with microvessels and NOS neurons, as respective denervations of 60 and 45% were observed following lesion. At the electron microscopic level, most perivascular NOS neuronal elements corresponded to nerve terminals and a majority ( approximately 25%) of these were located in the immediate vicinity of the blood vessels, similar to the perivascular distribution reported previously for classic neurotransmitters/neuromediators. NOS terminals abutting on cortical NOS neurons were primarily nonjunctional. Altogether, these results raise the possibility that not only cholinergic but also nitrergic basal forebrain neurons are involved in the flow response observed following stimulation of the basal forebrain. Further, they suggest interactions between basalocortical and intracortical NOS neurons. We conclude that these interactions are involved in the spatial and temporal regulation of cortical perfusion following basal forebrain activation, and that they may become dysfunctional in pathologies such as Alzheimer's disease which affects both the basal forebrain and the cortical NOS neurons.