Cytochemistry of undamaged neurons transporting exogenous protein in vivo.

The uptake, transport and fate of exogenous protein in undamaged cranial motor and neurosecretory neurons in mice have been investigated using enzyme cytochemical techniques for horseradish peroxidase (HRP) and lysosomal acid hydrolases. Labeling of these neuronal perikarya with HRP is accomplished by retrograde axoplasmic transport from their axon terminals following vascular injection of the protein or by direct somal-dendritic uptake subsequent to cerebral ventriculo-cisternal perfusion of peroxidase. Axon terminals of neurosecretory neurons innervating the posterior pituitary gland and those of cranial motoneurons can remain unexposed to HRP administered intraventricularly and, therefore, cannot directly incorporate the protein. Perikaryal organelles containing HRP reaction product include multivesicular bodies, small vacuoles, membrane-delimited cisterns and dense bodies. The concentrations of these different types of peroxidase-positive organelles are consistently greater in retrogradely labeled cell bodies than in perikarya which have pinocytosed the protein from the perisomal clefts, even though the amount of HRP bathing the cell bodies is much greater than the amount exposed to the axon terminals. Since the same groups of dense bodies contain lysosomal acid hydrolases as well as exogenous peroxidase, the HRP-labeled dense bodies are indeed lysosomes. Exogenous peroxidase does not stimulate lysosome proliferation; the concentration of acid hydrolase-positive lysosomes appears to remain the same whether or not the cells are labeled with HRP. Despite peroxidase labeling of cranial motor and neurosecretory cell bodies after intraventricular injection, the anterograde axonal transport of peroxidase in these neurons is negligible. This transport can be demonstrably increased in the neurosecretory system, but not in cranial motor nerves, in response to hyperosmolarity induced in the animals by salt loading. Under such a stimulus many axons and Herring bodies in the posterior pituitary gland contain HRP-labeled cisterns and 1,200–2,000 Å wide dense bodies. Both types of organelles also contain acid hydrolase activity and are confluent with each other. When compared to normal controls, the concentration of acid hydrolase-positive organelles in pituitary stalk axons and Herring bodies from osmotically stressed mice is noticeably increased. Axonal cisterns with acid hydrolase activity and those transporting peroxidase in an anterograde or a retrograde direction appear similar morphologically. Our results indicate that peroxidase pinocytosed by the neuron is eventually sequestered within perikaryal lysosomes for enzymatic degradation. Under normal conditions acid hydrolase activity and the majority of lysosomes are confined largely to dendrites and the cell body, and anterograde axonal transport of HRP is minor. In the osmotically stressed neurosecretory system, HRP appears to be carried coincidentally and anterogradely along with the movement of acid hydrolases, most likely from secondary lysosomes in the cell body. The movement of acid hydrolases down the axon is presumably increased for degradative purposes in the posterior pituitary gland. A part of the cisternal network conveying acid hydrolases and transporting peroxidase in anterograde and retrograde directions may represent a special compartment of agranular reticulum specifically involved with the lysosomal system of organelles and important in the segregation of exogenous macromolecules within the neuron for transport to or from lysosomes.     

UPTAKE AND RETROGRADE AXONAL TRANSPORT OF HORSERADISH PEROXIDASE IN NORMAL AND AXOTOMIZED MOTOR NEURONS DURING POSTNATAL DEVELOPMENT

Olsson T. & Kristensson K. (1979) Neuropathology and Applied Neurobiology 5 , 377–387
Abstract Uptake and retrograde axonal transport of horseradish peroxidase in normal and axotomized motor neurons during postnatal development
The axonal uptake and somatopetal transport of horseradish peroxidase (HRP) was studied during early postnatal development of facial neurons in mice and rats. HRP injected systemically or locally into the muscles of the vibrissae, diffused into the region of the immature neuromuscular junction and was incorporated into vesicles in the axon terminals on the first and third postnatal days, at a time when synaptic vesicles were already present. HRP later was found in the nerve cell bodies of the facial nucleus in the brain stem indicating a somatopetal transport of the tracer in axons. The response of facial neurons to nerve transection changed from rapid neuronal death to prolonged survival between the 6th and 10th postnatal day. HRP was transferred to nerve cell bodies after topical application to the proximal stump of transected facial nerves in rats 3 days-of-age. In the perikaryon it was localized to vesicles and vacuoles with no signs of leakage into the cytoplasm. In the light of our findings different hypotheses for the mechanism of the neuronal death in the immature animals are discussed.     

Quantitative analysis of multivesicular bodies (MVBs) in the hypoglossal nerve: Evidence that neurotrophic factors do not use MVBs for retrograde axonal transport

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density, and size of internal vesicles. Although target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain‐ and glial‐cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot‐labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions. J. Comp. Neurol. 514:641–657, 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructure of exogenous peroxidase in cerebral cortex

Protein uptake and transport and the intercellular spaces were studied electron microscopically in normal rabbit, cat and monkey cerebral cortex using horseradish peroxidase (HRP) as a cytochemical tracer. Extensive endocytosis and accumulation of tracer were seen as a result of delivering high concentrations of HRP into the intercellular space followed by long observation experiments, up to 120 h. Incorporation of HRP into neurons and their processes after a single application was studied by following the progress of neuron organelle labeling; the disappearance of tracer from these structures gave new information on the optimal times when HRP could be seen as a cellular tracer in cortical neurons.Neurons and their processes demonstrated a remarkable facility to take up peroxidase in coated invaginations and vesicles and accumulate it in multivesicular and other lysosomal bodies. The first structures labeled were coated vesicles, synaptic vesicles, sacs and tubules. HRP disappeared from the intercellular space after 24 h, and coated vesicles and synaptic vesicles no longer contained it at this time. Sacs and tubules were free of tracer by 48 h. Multivesicular bodies contained HRP after 30 min and lost all label between 72–96 h. Dense bodies became labeled after 1.5 h, and remained labeled until 96–120 h. Evidence of axon transport of the tracer was added to that extant on this pathway.     

Presumptive GABAergic centrifugal input to the lamprey retina: a double-labeling study with axonal tracing and GABA immunocytochemistry

The distribution of GABA-like immunoreactivity (GABA-LI) was performed in the lamprey retinopetal system which was previously identified by either anterograde or retrograde axonal tracing methods. This study was carried out at the ultrastructural level for the retina and under both the light and electron microscope for the mesencephalic retinopetal centers (M5 and RMA). The GABA-LI was distributed in about 40% of anterogradely HRP-labeled axon terminals in the inner retina. These made synaptic contacts upon either HRP-labeled ganglion cell dendrites or mostly on GABA-LI or on immunonegative amacrine cell dendrites and somata. The other immunonegative HRP-labeled axon terminals also established synaptic contacts on amacrine cell dendrites and somata. The mesencephalic retinopetal neurons, retrogradely labeled with HRP or [³H]proline, were GABA-LI in 65% of M5 somata and only in 15% of RMA neurons. M5 and RMA retinopetal neurons and dendrites, either GABA-LI or immunonegative, were contacted: (1) asymmetrically by HRP-labeled or unlabeled axon terminals containing rounded synaptic vesicles, always immunonegative and (2) symmetrically by HRP-unlabeled axon terminals containing pleiomorphic synaptic vesicles, which were either GABA-LI or immunonegative. The role of GABA as a putative neurotransmitter in the centrifugal visual system is discussed.     

The morphology of optic tract axons arborizing in the superior colliculus of the hamster

The axonal endoplasmic reticulum (ER) and synaptic-like (micro)vesicles within axon terminals of the neurohypophysis and their contribution to the secretory process in hypothalamo-neurohypophysial neurons have been investigated cytochemically in normal mice and in mice given 2% salt water to drink for stimulation of hormone synthesis in and release from these neurons. Cytochemical techniques included the peroxidase-antiperoxidase (PAP) immunocytochemical method for localization of neurophysin, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) as a tracer for the anterograde axonal transport of membrane from within the perikaryon, and blood-borne native horseradish peroxidase (HRP) as a tracer for internalized axon terminal membrane. The primary antiserum employed was directed against neurophysins I and II, the carrier proteins for the peptide hormones oxytocin and vasopressin, respectively. PAP reaction product was observed over neurosecretory granules but never over the endoplasmic reticulum, microvesicles or other organelles in axons and terminals of the neurohypophysis. WGA-HRP was delivered extracellularly to cell bodies of paraventricular neurons by cerebral ventriculocisternal perfusion. Internalized perikaryal surface membrane tagged with WGA-HRP was recycled through the innermost Golgi saccule (GERL) from which neurosecretory granules were formed. The anterograde axonal transport of membrane-bound WGA-HRP was manifested within the neurosecretory granules; WGA-HRP did not label the axonal reticulum or terminal microvesicles in the neurohypophysis. Blood-borne native HRP endocytosed into neurohypophysial terminals was associated with a plethora of microvesicles measuring 40-70 nm in diameter and vacuoles similar in size to the 100-300-nm-wide neurosecretory granules. The microvesicles contributed to the formation of numerous vacuoles. The internalization of axon terminal membrane as microvesicles incorporating HRP was quantitatively greater than vacuoles in both salt-stressed and control mice. The results suggest that in the hypothalamo-neurohypophysial system of the mouse the axonal ER and terminal microvesicles are not involved in the transport, storage, and exocytosis of neurosecretory material and perhaps other molecules processed through the innermost Golgi saccule. Nevertheless, a prominent population of the microvesicles within axon terminals of the neurohypophysis does participate in the secretory process. These vesicles are involved directly in the internalization of the terminal surface membrane subsequent to release of secretory granule content.(ABSTRACT TRUNCATED AT 400 WORDS)     

The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study

Retrograde transport of horseradish peroxidase (HRP) from the region of retinal genglion cell axon terminals back to the cell bodies has been studied by light and electron microscopy. After injection of HRP into the chick optic tectum, it was taken up by axon terminals and unmyelinated axons as well as by other processes and cell bodies of the outer tectal layers. Subsequently the HRP was obseved in vesicles, multivesicular bodies, cup-shaped organelles and small tubules within axons in the stratum opticum, optic tract, optic nerve and optic fiber layer of the retina with accumulation in the retinal ganglion cell bodies. Pinocytosis of extracellular HRP along the axon shaft was rare. After a short postinjection interval, HRP was found in organelles within the axons of the optic nerve but not in the extracellular spaces. After larger injections or longer postinjection intervals, extracellular HRP diffused from the injection site to the back of the eye, but none was found in the extracellular spaces of the retina; ganglion cells were the only cells of the retina which contained HRP product. HRP disappeared from the cell bodies 3–4 days after transport. These findings suport the concept of intraaxonal retrograde movement of HRP. Within axons the vesicles carrying HRP frequently were partially or completely surrounded by a regualr array of microtubules. Doses of colchicine greater than 5–10 µ/eye administered 4 days before tectal injection of HRP interfered with the uptake and/or transport of HRP. HRP also moved in an anterograde direction in membrane-bound vesicles within the ganglion cell axons, although apparently more slowly and in smaller quantities than in the retrograde direction. The localization of HRP in neurons of the isthmo-optic nucleus following intravitreal injections has also been studied. The marker enzyme was found in neuronal cell bodies 4 hours after injection, indicating a rate of retrograde transport of at least 84 mm/day in these neurons.     

Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localization in perikaryon.

Horseradish peroxidase (HRP) applied to crushed mouse sciatic nerves diffused through the damaged perineurium into the endoneurium. In the injured area, HRP passed into damaged myelinated and unmyelinated axons forming columns of reaction product, which extended for several millimeters proximally to the lesion. Ultrastructurally, HRP adhered to the inner surface of the axoplasm and to the surfaces of neurotubules and neurofilaments in such columns. At more proximal levels axons contained HRP in vesicular and tubular organelles and, later, nerve cell bodies of the corresponding spinal ganglia showed HRP, accumulation in cytoplasmic vesicles, cup-shaped bodies, multivesicular bodies and tubules of agranular endoplasmic reticulum. Markedly less HRP reached neurons in the spinal ganglia when applied to the nerve 30 or 60 min after the crush. After such time intervals solid HRP containing axons were also less frequently observed. Conceivably, HRP enters crushed axons momentarily after a crush as an injured cell reaction. Subsequently it is incorporated into organelles higher up in the axons, from where retrograde transport to the perikaryon will fellow. This phenomenon of a sudden non-specific influx of exogenous macromolecules into axotomized neurons and their subsequent transport to the perikaryon might be relevant for development of certain biochemical and morphological responses, e.g. lysosomal alterations, of the neuron to an axonal injury.     

Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane----endocytic structures----endosomes (presecondary lysosomes)----transfer vesicles----transmost Golgi saccule----vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of axonally transported label in chick retinal ganglion cell axons after intravitreal injections of wheat germ agglutinin conjugated to horseradish peroxidase

We have studied the subcellular localization of peroxidase-labeled organelles after anterograde axonal transport by chick retinal ganglion cells that had been exposed 23-25 h earlier to wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After intravitreal injection of WGA-HRP, we found in the optic tectum that 82% of labeled organelles were located within axons and axon terminals. The organelles included: tubules and cisternae of the smooth endoplasmic reticulum, hypolemmal cisternae, vesicles, dense bodies and multivesticular bodies. We also measured the distances between the centers of the labeled organelles and the plasma membrane of these profiles. The density of organelles (number of organelles/micron 2) was plotted as a function of distance from the plasma membrane. Irrespective of the dose of lectin-peroxidase injected, labeled organelles were most densely concentrated in a 30 nm wide annular zone centered 75 nm in from the plasma membrane. In axon terminals the labeled organelles were most concentrated 75-90 nm in from the plasma membrane. Assuming that the peroxidase label indicates the presence of WGA-HRP, we conclude that after anterograde axonal transport the lectin accumulates in lysosomal organelles and elements of the smooth endoplasmic reticulum. Therefore, in contrast to the more restricted localization of [125I]WGA as inferred from electron microscopic autoradiography after uptake and transport by the same cell type, WGA-HRP-labeled organelles are found more diffusely within the axoplasm, particularly in axon terminals. Furthermore, peroxidase-labeled organelles in dendritic, glial or neuronal cell bodies in the tectum were seen less frequently than expected based on evidence of frequent transfer to second cells after intravitreal injections of [125I]WGA. Thus, we infer that at these concentrations WGA labeled with HRP may not be transferred intercellularly as efficiently as even lower concentrations of iodinated WGA are apparently transferred.     

Uptake of horseradish peroxidase by sensory terminals of lamellated corpuscles in mouse foot pads

Summary Uptake of horseradish peroxidase (HRP) by sensory axon terminals was studied in lamellated corpuscles of mouse plantar foot pads after i.v. and local s.c. HRP injections. The corpuscles, considered to be rapidly adapting mechanoreceptors, are localized in dermal papillae and have discoid axon terminals enclosed in 1–5 lamellae of lamellar cells. The injected HRP diffused into interlamellar and adaxonal clefts and was taken up by sensory terminals, being incorporated in coated pits and coated vesicles formed by the axolemma. The tracer further appeared in the axoplasm in smooth vesicles, multivesicular bodies, and vacuoles. Similar incorporation of HRP into vesicles, inclusion bodies, and vacuoles was observed in the lamellae and perinuclear cytoplasm of lamellar cells. Vibratory stimulation considered to be adequate for this type of mechanoreceptors did not result in any perceptible increase in HRP uptake by sensory terminals. It is suggested that endocytosis by sensory terminals may serve for incorporation of regulatory macromolecules that presumably mediate information to the perikaryon about conditions of the distant terminals and their microenvironment.     

Retrograde axonal transport of horseradish peroxidase

Summary Eye muscles were examined histochemically for the presence of horseradish peroxidase (HRP) activity after intravenous injections of large doses of this tracer protein. HRP had penetrated through the blood vessels and diffused into the areas of motor end plates, where it outlined axon terminals at the synaptic clefts. HRP was incorporated into pinocytotic vesicles in the axons from where an intraaxonal transport in the retrograde direction to the nerve cell bodies in the brainstem followed. Accumulation of HRP in perikarya of motor neurons can therefore be the result of a physiological process of pinocytosis at the axon terminals. In this way exogenous macromolecules in the blood can by-pass the blood-brain barrier and reach the lower motor neurons in the CNS.     

Uptake of horseradish peroxidase in sensory nerve terminals of mouse trigeminal nerve

Summary Uptake of horseradish peroxidase (HRP) in sensory nerve terminals in sinus hair in the nose of the mice was studied after local (s.c.) or i.v. injection. HRP spread into the sinus hair follicles and surrounded the nerve terminals of the Merkel disc endings and lanceolate terminals. It was incorporated into vacuoles and vesicles of these terminals. Subsequently, HRP was demonstrated in nerve cell bodies in the ophthalmomaxillary part of the trigeminal ganglion, indicating a somatopetal axonal transport of the tracer. In this way the sensory neurons may be subjected to various influences from the periphery     

Intraaxonal transport of horseradish peroxidase in the sympathetic nervous system

Following a single injection of horseradish peroxidase (HRP) into the superior cervical ganglion (SCG) of the rabbit, the uptake and anterograde transport of this label was confirmed in the ganglion cell bodies, postganglionic axons, and preterminal and terminal ending axons in the ciliary processes of the eye. From the same injection site the intraaxonal HRP reaction product was demonstrated in myelinated axons, presumably by retrograde transport.

Intracytoplasmic HRP was identified in large, single membrane-bound, dense vesicles predominantly in perinuclear orientation. Intraaxonal HRP appeared throughout, either within single membrane-bound round or oblong vesicles of variable sizes and densities. Frequently, the HRP vesicles in the axons revealed elaborate membranous subunits. A limited number of whole axons or axon fascicles were diffusely stained with HRP reaction product at or near the injection site. This phenomenon may be the result of membrane injury to neurons. The HRP label was found in small amounts in axons and terminals in the ciliary processes of the eye as early as 4 h following injection into the SCG, indicating a rapid anterograde transport of HRP from a single extracellular source. Likewise the HRP label disappeared from the ganglion cell bodies and processes by the 6th day following injection. The presence of numerous HRP-labeled myelinated and non-myelinated axons in the SCG confirms the bidirectional transport of HRP in the sympathetic nervous system.     

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F_F-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F_P-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.     

Morphological features of spiking and nonspiking cells in the paratracheal ganglion of the ferret

The present series of experiments was designed to study details of the morphology and connectivity of functionally identified cells located in the paratracheal ganglia of the ferret. The morphology of 11 spiking (AH cells) and seven nonspiking (type B cells) ganglion cells was examined. Intra-axonally injected horseradish peroxidase (HRP) was used as the label. Each spiking and nonspiking cell was identified by intracellular recording prior to the HRP injection. "Whole mount preparations" were processed for HRP histochemistry with diaminobenzidine as the chromogen. HRP-labeled cell bodies of both the spiking AH and nonspiking type B neurons demonstrated similar morphological features. Both types of ganglion cells showed axons arising from a small, ill-defined axon hillock which exited from the cell as single or multiple branches of equal diameter and coursed unidirectionally through the interganglionic nerve trunk to an adjacent ganglion; short, fine, tapering processes (presumptive dendrites) in the immediate vicinity of the injected cell; and processes extending out of the ganglion cell perpendicular to the interganglionic nerve trunk which could be followed into the smooth muscle. Extraperikaryal injections of HRP into a ganglion retrogradely labeled perikarya in the adjacent ganglia. These results demonstrate that in airway ganglia the morphology of spiking and nonspiking neurons is remarkably similar despite electrophysiological differences. In addition it appears that ganglion cells project to adjacent ganglia and to smooth muscle by means of independent axonal processes. These morphological features of the ganglion cells in airways and the trajectories of their axons correspond to known features of their physiology: i.e., the axon of a ganglion cell travels unidirectionally toward the adjacent ganglion and arborizes there, providing anatomical evidence of communication between ganglia via the interganglionic nerve trunk; and the spiking and nonspiking neurons possess similar morphological features that are typical of ganglion cells described in other systems, such as in the myenteric plexus.     

The neuronal endoplasmic reticulum: its cytochemistry and contribution to the endomembrane system. II. Axons and terminals

The morphology and cytochemistry of the endoplasmic reticulum (ER) in axons and terminals of a number of different types of neurons in brains from mice were investigated ultrastructurally. The neurohypophysis received particular attention because the morphology and enzyme cytochemical activities of many of the preterminal swellings of hypothalamo-neurohypophysial axons are altered by chronic salt-stress. Membrane contrast and enzyme cytochemical staining techniques were employed to characterize the axonal reticulum and to determine if organelles representing the lysosomal system in the axon and the tubular profiles participating in the anterograde axonal transport of native horseradish peroxidase (HRP) are associated with the ER. Potential enzyme cytochemical markers for the axonal ER included glucose-6-phosphatase (G6Pase), thiamine pyrophosphatase, nucleoside diphosphatase, and acid hydroxylase activities. The anterograde transport of HRP was analyzed in undamaged hypothalamo-neurohypophysial neurons and in facial and hypoglossal motoneurons of mice receiving the protein in the lateral cerebral ventricle. The ER pervaded the axon and appeared as parallel, 20-40-nm-wide tubules interconnected by oblique anastomoses. Membrane thickness of the axonal reticulum measured 60-100 A, which is similar to that of the perikaryal ER. Enzyme cytochemical activities associated with the ER or lysosomes were not conspicuous in axons and terminals under normal conditions but became prominent in some axons and preterminal swellings manifesting an autophagic appearance within neurohypophyses from salt-stressed mice. Only G6Pase activity was a marker for the ER in these axons and preterminals. Many ER profiles in non-incubated sections and in G6Pase cytochemical preparations of salt-stressed neurohypophyses were wrapped around or interspersed among secretory granules, multilamellar bodies, and vacuoles that may represent forms of lysosomes involved in autophagy and crinophagy. Acid hydrolase activities were localized within the vacuoles as well as within 80-130-nm-wide, blunt-ended tubules in pituitary stalk axons; similar reactive tubules were confluent with large secondary lysosomes in neurosecretory cell bodies and may be derived from these lysosomes. Morphologically identical tubules transporting HRP in the anterograde direction were observed only in the salt-stressed hypothalamo-neurohypophysial neuron. The HRP-positive tubules very likely are affiliated with the lysosomal system.     

Synaptic connections between spinal motoneurons and dorsal root ganglion cells in the cat

Light- and electron-microscopical horseradish peroxidase (HRP) studies have been employed in conjunction with a degeneration study in order to clarify the origin and axonal passage of afferent synaptic terminals in cat dorsal root ganglia. After injection of HRP into ganglia (C3) without involvement of the ventral roots and spinal nerves, a few ipsilateral spinal ventral horn neurons (C3) were retrogradely labeled with HRP. The labeled neurons were localized in the dorsomedial and the ventromedial nuclei. Following ventral rhizotomy of C3, the afferent terminals in the ganglia (C3) anterogradely degenerated and contained accumulated and disintegrated neurofilaments, depleted, aggregated and enlarged synaptic vesicles. Subsequent to an HRP and wheat germ agglutinin (WGA)-HRP-mixture injection into the dorsal neck or suboccipital muscles, many spinal motoneurons (C3) were labeled retrogradely with an HRP mixture. On the other hand, the afferent synaptic terminals in ganglia contained the membrane-bound and electron-dense bodies which were anterogradely labeled with an HRP mixture in addition to the normal synaptic elements. The present findings strongly suggest that some spinal motoneurons send their axon collaterals to the dorsal root ganglia, in which the terminals of the axon collaterals directly synapse with the dorsal root ganglion cells.     

Neuronal transport of acid hydrolases and peroxidase within the lysosomal system of organelles: Involvement of agranular reticulum-like cisterns

Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5–8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400–1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cupshaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for degradation in the cell body. These observations extend our previously reported findings in the axon to the cell body and suggest that agranular reticulum-like cisterns in the perikaryon, like those in the axon, may be part of the lysosomal system rather than associated with the agranular reticulum. A diagram summarizing the lysosomal system of organelles and proposed transport of acid hydrolases and peroxidase in neurosecretory neurons specifically and in neurons in general is provided.     

Horseradish peroxidase localization of the sympathetic postganglionic neurons innervating the cat heart

The localization of the sympathetic postganglionic neurons innervating the cat heart has been investigated by using retrograde axonal transport of horseradish peroxidase (HRP). HRP was injected into the subepicardial layers of 4 different cardiac regions. The animals were sacrificed 72-96 h later and fixed by perfusion via the left ventricle. The paravertebral sympathetic ganglia from the superior cervical, middle cervical and stellate ganglia to T10 ganglia were removed and processed for HRP identification. Following injections of HRP into the apex of the heart, the sinoatrial (SA) nodal region and the ventral wall of the right ventricle, we observed that HRP-labeled sympathetic neurons were localized predominantly in the right stellate ganglia, and to a lesser extent, in the right superior and middle cervical ganglia, and left stellate ganglia. Fewer labeled cells were found in the right T4-T6. T8 and T9. After HRP injection into the dorsal wall of the left ventricle, HRP-labeled cells were present mainly in the left stellate ganglia.