Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP

Horseradish peroxidase (HRP) conjugates of 8 different lectins (wheat germ agglutinin, ricinus communis I and II, peanut agglutinin, lens culinaris, soybean agglutinin, limulus polyhemus, ulex europaeus I) and cholera toxin (CT) or free HRP (FHRP) were individually injected into the submandibular gland (SMG) or anterior chamber (AC) of the eye and the retrogradely labeled neurons in the superior cervical ganglion (SCG) were quantitated. The effect of using 3 different cross-linking reagents (glutaraldehyde, p-benzoquinone and periodic acid) on the results obtained with HRP conjugates of wheat germ agglutinin (WG) was also examined. The results in 100 rats demonstrated the superior sensitivity of ligand-HRP conjugates over FHRP as retrogradely transported markers. After SMG injections, HRP conjugates of CT, WG and soybean agglutinin were 20-50 times more sensitive than FHRP. After AC injections, HRP conjugates of CT and WG consistently yielded labeled SCG neurons while FHRP failed to do so even when the amount of FHRP injected was increased 10-fold. The sensitivity of HRP conjugates of WG was similar after SMG injections using each of the 3 cross-linking reagents, but AC injections of conjugates produced with p-benzoquinone yielded twice as many labeled SCG neurons as the other WG conjugates. Mixtures of conjugates with and without FHRP were no more sensitive than the most sensitive individual ligand-HRP conjugates, except after SMG injections of a conjugate mixture with FHRP. Additional experiments demonstrated the specificity of the ligand-"receptor" interaction of WG and CT and that the superior sensitivity of these ligand-HRP conjugates does not depend on the tissue destruction produced by the injection procedure.     

Immunoblot analysis of horseradish peroxidase conjugates of wheat germ agglutinin before and after retrograde transport in the rat peripheral nervous system

Immunoblot studies were performed on tissue extracts of rats injected with horseradish peroxidase (HRP) conjugates of wheat germ agglutinin (WGA) using anti-HRP and anti-WGA antisera. Immunoblots of uninjected, purified conjugates (WGA-HRP) demonstrated a 62-kilodalton (kd) conjugate (monomeric WGA conjugated to HRP). Other prominent immunobands included HRP (40 kd), monomeric WGA (22 kd), and 15- to 30-kd HRP breakdown products. Following injections of WGA-HRP into the submandibular gland of rats which survived 16 hr to 8 days, immunoblots were performed on homogenates of injected submandibular glands and of superior cervical ganglia containing neurons retrogradely labeled with WGA-HRP. The anti-HRP antiserum detected unconjugated, 40-kd HRP and a 62-kd immunoband corresponding to WGA-HRP in the superior cervical ganglia and submandibular glands. No immunobands were detected with the antiserum to WGA in superior cervical ganglia homogenates. Blots of submandibular gland homogenates harvested 24 hr after a WGA-HRP injection, but not at later time points, contained an immunoband reactive with the anti-WGA antiserum; it corresponded to monomeric WGA. These studies analyze for the first time the molecular composition of WGA-HRP conjugates before and after retrograde transport; they provide a novel approach for probing the intraneuronal transport and degradation of proteins. We conclude that morphologically defined endocytic pathways using protein markers reflect the endocytosis and transport of immunochemically altered and unaltered forms of the markers.     

The superior cervical ganglion: Origin of sympathetic fibers in the facial and hypoglossal nerves in the cat

Location of superior cervical ganglion (SCG) neurons, sending axons into the facial and hypoglossal nerves, was investigated in the cat by means of retrograde axonal transport of horseradish peroxidase (HRP). After wheat germ agglutinin conjugated HRP (WGA-HRP) was injected into these nerves, many retrogradely labeled neurons were found widely in the ipsilateral SCG, particularly around the caudal half of the SCG. These neurons were round or oval in shape and 70-80% of these were medium in size. In fluorescent experiments, fast blue (FB) was used in combination with diamidino yellow (DY). After injections of FB into the facial nerve and DY into the hypoglossal nerve, a few FB-DY double-labeled neurons occurred in the SCG ipsilaterally.     

Transcellular transfer of HRP in the amphibian visual system

Unilateral intraocular injections of horseradish peroxidase (HRP) were made in the green tree frog, Hyla cinerea. Survival times ranged from 1 to 28 days. Control injections were placed in the orbit, peritoneum, or third ventricle. By 1 day after ocular injection anterogradely transported HRP was observed in the optic nerve and tract and in thalamic and midbrain retinal recipient zones. Retrograde filling of motor neurons was also observed by 1 day. At 3 days, HRP-positive magnocellular preoptic neurons became apparent. Finally, at 3-5 days post-injection, ependymal cells radially adjacent to HRP-positive neuropils, but not retrogradely filled cells, contained a small amount of reaction product. By 7 days these ependymal cells were densely filled and processes could be seen extending toward the neuropils. There was never evidence of HRP uptake by neurons in or adjacent to these HRP-positive neuropils. Neither retinal fibers nor ependymal cells were HRP-positive after any control injection or after processing uninjected material by the HRP histochemistry protocols. In contrast, motor cells were filled following orbital and peritoneal, but not ventricular, injections, suggesting blood-borne HRP reaching motor endplates could account for some of the motor neuron filling. Preoptic cells were filled after all control injections, demonstrating that they too could take up circulating HRP. The specificity of ependymal cell filling, however, suggests that anterogradely transported HRP can be released at the axon terminals and taken up specifically by ependymoglial cells.     

Immunocytochemical localization of GABA in neurons projecting to the ventrolateral nucleus of the solitary tract

To determine the origin of gamma-aminobutyric acidergic (GABAergic) input to the ventrolateral solitary tract nucleus (vlnTS), we used a double-labeling procedure for retrogradely transported horseradish peroxidase (HRP) and the immunocytochemical localization of GABA. Following HRP injections into the vlnTS, double-labeled neurons were found within the B?tzinger Complex. We conclude that these double-labeled cells are the inhibitory B?tzinger neurons and that GABA is a likely transmitter in this respiratory nucleus.     

Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistochemistry

The distribution of cholinergic neurons in the midbrain and pons which project directly to the thalamus was investigated in the rat using a procedure which allows the simultaneous detection of retrogradely transported horseradish peroxidase (HRP) and immunohistochemical demonstration of choline acetyltransferase (ChAT) in the same neurons. HRP injections were placed in the dorsal half of the anterior third of the thalamus on one side which included the anteroventral nucleus as well as portions of the rostral intralaminar and reticular nuclei. These thalamic nuclei showed the highest density of immunohistochemically detectable cholinergic fibers. Neurons containing both HRP and ChAT, which represented cholinergic neurons projecting directly to the thalamus, were found in the midbrain and pons in the lateral tegmental reticular formation, parabrachial region and lateral dorsal tegmental nucleus. Ipsilateral to the injection site over 91% of the HRP labeled neurons in all of these regions were cholinergic, while an average of 60% of the cholinergic neurons had transported HRP. Contralateral to the injection site 5-6% of the cholinergic neurons in these regions were also retrogradely labeled. These findings demonstrate direct cholinergic projections to the thalamus from neurons in several regions in the tegmentum and suggest that tegmental projections to the thalamus are predominantly cholinergic.     

Secondary vestibular cholinergic projection to the cerebellum of rabbit and rat as revealed by choline acetyltransferase immunohistochemistry, retrograde and orthograde tracers.

Previously we have shown that four regions of the cerebellum, the uvula-nodulus, flocculus, ventral paraflocculus, and anterior lobe 1, receive extensive, but not exclusive, cholinergic mossy fiber projections. In the present experiment we have studied the origin of three of these projections in the rat and rabbit (uvula-nodulus, flocculus, ventral paraflocculus), using choline acetyltransferase (ChAT) immunohistochemistry in combination with a double label, retrogradely transported horseradish peroxidase (HRP). We have demonstrated that in both the rat and rabbit the caudal medial vestibular nucleus (MVN) and to a lesser extent the nucleus prepositus hypoglossus (NPH) contain ChAT-positive neurons. Neurons of the caudal MVN are double-labeled following HRP injections into the uvula-nodulus. HRP injections into the uvula-nodulus also labeled less than 5% of the neurons in the cholinergic vestibular efferent complex. Fewer ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the flocculus. Almost no ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the ventral paraflocculus. Injections of Phaseolus leucoagglutinin (PHA-L) into the caudal MVN of both the rat and rabbit demonstrated projection patterns to the uvula-nodulus and flocculus that were qualitatively similar to those observed using ChAT immunohistochemistry. We conclude that the cholinergic mossy fiber pathway to the cerebellum in general and the uvula-nodulus in particular is likely to mediate secondary vestibular information related to postural adjustments.     

Serotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique

The retrograde transport of horseradish peroxidase (HRP) has been used in combination with peroxidase antiperoxidase (PAP) immunocytochemistry in order to investigate serotonin-containing projections to the thalamus of the rat. Sections were histochemically stained to reveal retrogradely transported HRP and then PAP immunostained using a monoclonal anti-serotonin (5-HT) antibody. Following HRP injections into the ventral thalamus, retrogradely labelled cells were observed in a number of sites in the brainstem and including areas known to be rich in 5-HT-containing neurons. At rostral levels of the dorsal raphe nucleus, retrogradely labelled cells were observed both on the midline and in a distinct lateral group extending diffusely into the periaqueductal gray (PAG). In both of these areas many 5-HT-immunoreactive HRP retrogradely labelled neurons were observed. However, except for the most rostral levels of the dorsal raphe nucleus, such double-labelled cells represented only a small proportion of the total population of 5-HT-immunoreactive neurons. In the lateral group, the retrograde labelling was mainly unilateral to the injection site but some contralateral labelling was also seen. At caudal levels of the dorsal raphe nucleus, retrogradely labelled cells were observed predominantly in the lateral group. At the level of the dorsolateral tegmental nucleus, few 5-HT of 5-HT/HRP labelled cells were observed in the lateral group, although HRP retrogradely labelled neurons were present. Double-stained cells were detected also in the medial raphe nucleus (corresponding to the B8 cell group according to the nomenclature of Dahlström and Fuxe¹³), among the fibres of the medial lemniscus (B9), and in nucleus raphe pontis (B5).     

Involvement of the plasminogen enzymatic cascade in the reaction to axotomy of rat sympathetic neurons

Axotomy of superior cervical ganglion (SCG) neurons is characterized by peripheral regeneration of injured axons and temporary disassembly of the intraganglionic synapses, necessary for synaptic silencing. Both events require remodeling of the extracellular matrix achieved through controlled proteolysis of its components by different enzymatic systems. In this study, we investigate the involvement of the plasminogen enzymatic cascade in the response to axotomy of rat SCG neurons. All components of this proteolytic pathway, tissue plasminogen activator (tPA), plasminogen, membrane receptor annexin II and tPA inhibitor (PAI-1), are constitutively expressed in uninjured SCG and increase significantly after SCG neuron axotomy. Immunolocalization of plasminogen, the key protein converted into the enzymatically active plasmin by tPA, in both neurons and non-neuronal cells indicates that all cell types are involved in the response to axotomy. The time course of activation of tPA/plasmin enzymatic pathway suggests its involvement in both intraganglionic synapse remodeling and axonal regeneration.     

Trigeminal projections to supratentorial pial and dural blood vessels in cats demonstrated by horseradish peroxidase histochemistry

Anatomical and clinical observations suggest that supratentorial vascular structures contain afferent projections from the trigeminal ganglia. To characterize this innervation, horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin were applied to the pial and dural arteries and sinuses of 33 cats. HRP was restricted to the site of interest by applying it dissolved in a viscous polymer, polyvinyl alcohol (PVA), to achieve slow release and minimize diffusion. The ganglia of cranial nerves V, VII, IX, and X and the superior cervical ganglia (SCGs) were examined bilaterally for the presence of retrogradely transported protein. Horseradish peroxidase applied to the proximal middle cerebral artery was located in cell bodies occupying the portion of the ipsilateral trigeminal ganglion corresponding to the ophthalmic division and throughout both SCGs. When the tracer was applied to the right anterior or posterior superior sagittal sinus, HRP-positive cells were present as above, predominantly in the ipsilateral trigeminal ganglia corresponding to the ophthalmic division and throughout both SCG. When applied to the right middle meningeal artery, HRP was observed within neurons of ipsilateral SCG and in the ophthalmic division of trigeminal ganglia; a few enzyme-containing cells were present in ipsilateral regions corresponding to the second and third divisions. These observations support the concept that supratentorial vascular structures receive afferent nervous projections from trigeminal neurons.     

Direct amygdaloid projections to the superior salivatory nucleus: a light and electron microscopic study in the cat.

Amygdaloid projections to the superior salivatory nucleus (SSN) were investigated in the cat by using the anterograde and retrograde tracing techniques of horseradish peroxidase (HRP). After HRP injections were made into the lingual nerve, retrogradely labeled SSN neurons were located in the lateral tegmental field medial to the spinal trigeminal nucleus from the middle level of the superior olivary nucleus to the caudal level of the facial nucleus. These labeled neurons, triangular, oval or polygonal in shape, were small to medium-sized (12-29 microns) and formed loosely packed clusters. In further HRP studies, HRP injections were made into the amygdala and in the reticular formation containing the SSN neurons. The results suggested that the SSN receives direct afferents from the central nucleus of the amygdala with ipsilateral predominance. Final proof of such direct connections from amygdala to the SSN can be obtained only by electron microscopic study. Therefore, HRP injections were made into the lingual nerve and in the amygdala in the same animal and electron microscopic observations were carried out on the SSN. It appeared that anterogradely labeled amygdalo-tegmental fibers formed axosomatic and axodendritic synaptic contacts with retrogradely labeled SSN neurons.     

Ultrastructural identification of labeled neurons in the dorsal motor nucleus of the vagus nerve following injections of horseradish peroxidase into the vagus nerve and brainstem

The efferent connections of two types of neurons in the dorsal motor nucleus of the vagus nerve (DMV) were studied in the cat by light and electron microscopy following horseradish peroxidase (HRP) injections into the cervical vagus nerve or brainstem. After injections of HRP into the vagus nerve, up to 80% of medium-sized neruons averaging 26 × 20 μm in 1-μm-thick sections were retrogradely labeled while no small neurons were labeled in the DMV. Incubation with either diaminobenzidene (DAB) or p-phenylenediamine-pyrocatechol (PPD-PC) chromogens yielded electron-dense reaction products localized mainly in lysosomes. Identification of label at the ultrastructural level was facilitated by omitting lead citrate staining and by counting numbers of lysosomes, which were higher in labeled neurons. Quantitative comparisons of the dimensions of labeled and unlabeled somata demonstrated that retrograde transport and incorporation of HRP had no effect on cell size within the 2–3-day survival times used in this study. In order to determine whether neurons in the DMV project to higher levels of the brain stem, large injections of HRP (1–3 μl) were made into the pons, mesencephalon, hypothalamus, and amygdala. After injections of HRP into the brainstem, only small neurons, measuring 17 × 10 μm, were retrogradely labeled. Approximately 90% of the small neurons remained unlabeled following the HRP injections. The ultrastructrual features of the labeled small neurons included an invaginated nucleus, low cytoplasmic/nuclear ratio, and relatively fewer organelles than the medium-sized neurons. A quantitative analysis of labeled and unlabeled small neurons demonstrated that the labeled neurons were significantly larger than the unlabeled small neurons. Thus, two populations of small neurons may exist in the DMV. One population appears to have ascending projections to higher levels of the brainstem while the other more numerous population may be interneurons or project for only short distances.     

Colocalization of fixative-modified glutamate and glutaminase but not GAD in rubrospinal neurons

In an attempt to identify putative neurotransmitters of rubrospinal neurons, immunocytochemical procedures were utilized in combination with retrograde tracing techniques in 15 adult male rats. Following injections of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) into the spinal cord, midbrain sections were processed with a combined procedure that allowed visualization of both the retrograde tracer and one or more antigens including glutamate, glutaminase, and glutamatic acid decarboxylase (GAD). Initial colocalization studies demonstrated that glutamatelike and glutaminaselike immunoreactivities were cocontained within the same neurons. Following injections of HRP or WGA-HRP into the spinal cord approximately 53% of retrogradely labeled neurons contained glutamate immunoreactivity. Triple-labeling experiments indicated that glutamatelike immunoreactivity was colocalized with glutaminase immunoreactivity in retrogradely labeled rubrospinal neurons. Retrogradely labeled neurons did not contain GAD immunoreactivity. Moreover, triple labeling experiments verified that glutamatelike immunoreactive retrogradely labeled cells did not cocontain GAD immunoreactivity. These studies demonstrate that glutamate and its synthesizing enzyme, glutaminase, are present in some rubrospinal neurons and raise the possibility that a component of the rubrospinal projection may be glutamatergic. GAD, on the other hand, is not present in rubrospinal neurons. This finding supports the hypothesis that GABAergic neurons play a role as interneurons in the red nucleus.     

Projection neurons of the basolateral amygdala: A correlative Golgi and retrograde tract tracing study

This study analyzed the projection neurons of the anterior subdivision of the rat basolateral amygdaloid nucleus (BLa) by correlating the morphology of Golgi-stained neurons with the morphology of neurons that were retrogradely labeled by injections into the main terminal fields of BLa. In each animal multiple injections of horseradish peroxidase (HRP) and wheat germ agglutinin-conjugated HRP were made into the prefrontal cortex and rostral striatum. These injections labeled approximately 85% of BLa neurons. The great majority of labeled neurons were the same shape and relative size as the pyramidal (class I) neurons described in previous Golgi studies. The unlabeled neurons appeared to correspond to the nonpyramidal (class II and class III) neurons described in Golgi studies. Thus this investigation provides experimental evidence that the pyramidal neurons are the main projection neurons of BL, whereas most of the nonpyramidal cells are local circuit neurons.     

Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: Their uptake and clearance, transganglionic and retrograde transport and sensitivity

Horseradish peroxidase (HRP) conjugates of 6 different lectins and cholera toxin (CTHRP) were quantitatively compared with respect to: (a) their behavior at the injection site and (b) their ability to label, by means of transganglionic and retrograde axonal transport, axon terminals and neurons in the medulla of the rat subsequent to injections of each probe into the anterior two thirds of the tongue. HRP conjugates of wheat germ agglutinin (WGHRP) and CTHRP were more sensitive than any of the other lectin-HRP conjugates. Both were far superior to free-HRP (FHRP) in demonstrating these projections and CTHRP was the most sensitive transganglionic and retrograde probe. Additional experiments demonstrated that this superiority was not an artifact of the volume of material injected into the tongue nor of the injection site area or survival time selection. These experiments demonstrated further that CTHRP and WGHRP remain at the injection site approximately twice as long as FHRP and that their removal from or degradation in retrogradely labeled neurons requires approximately twice as much time as that required for FHRP. These observations, together with earlier studies from this laboratory, suggest the following conclusions: (1) CTHRP and WGHRP are superior in sensitivity to FHRP for studies of neuronal connectivity; and (2) HRP conjugates of ligands such as CTHRP and WGHRP are internalized, transported and/or degraded by mammalian neurons in a manner which differs from that of FHRP, a macromolecule for which neuronal plasma membrane 'receptors' are lacking.     

Brainstem cells of origin of the cervical vagus and cardiopulmonary nerves in the neonatal pig (Sus scrofa)

The distribution of the cells of origin of the cervical vagus and cardiopulmonary nerves has been studied in neonatal piglets (Sus scrofa) ranging in age from 1 to 60 days. Cardiopulmonary nerves were identified physiologically and anatomically prior to injection of horseradish peroxidase (HRP) into the nerves. Following injection of HRP into the cervical vagus nerve retrogradely labeled neurons were present in the dorsal motor nucleus of the vagus nerve (DMV), the nucleus of the solitary tract, the nucleus ambiguus (NA), ventrolateral to the NA and in an intermediate zone between the DMV and the NA. Two unique clusters of neurons were also retrogradely labeled after injections into the vagus nerve. One group was located lateral to the most caudal levels of the DMV and extended as far caudally as the C1 spinal segment. The second distinctive group was located ventrolateral to the nucleus ambiguus in a cell column identified as the ventrolateral nucleus ambiguus (VLNA). After injections of HRP into cardiopulmonary nerves, the majority of neurons were found in the VLNA and the distinct clusters of neurons in this cell column were particularly heavily labeled. Small numbers of cells were labeled in the DMV and NA and none were labeled in the solitary nucleus after cardiopulmonary nerve injections. There were no apparent age-related differences in the degree or distribution of retrograde labeling.The distribution of neurons in the medulla oblongata projecting into cardiopulmonary nerves in the piglet is similar to that described in other species, i.e., the nucleus ambiguus, particularly its ventrolateral cell column, is the primary site of cardiomotor neurons. In addition, in the piglet there is a morphologically distinct cluster of cells related to the heart, and possibly the lungs, which does not appear to be present in other species.     

Cortical projection of giant neostriatal neurons in the cat. Light and electron microscopic horseradish peroxidase study

Following voluminous injections of horseradish peroxidase (HRP) in various neocortical fields, a small number of labeled large neurons are observed ipsilaterally in the putamen, striatal ponticuli, caudate nucleus, and nucleus accumbens septi. The bulk of the corticopetal cells are found in the putamen and in the striatal ponticuli. A more significant number of labeled neurons is encountered following injections in auditory and sensorimotor cortex, followed by the prefrontal and premotor cortex; very few cells project to the visual cortex. Ultrastructurally, the large HRP-labeled neurons display an eccentrically located, indented nucleus, abundant granular endoplasmic reticulum forming Nissl bodies, well developed Golgi zones, and numerous dense bodies. The simultaneous demonstration of retrogradely transported HRP and acetylcholinesterase (AChE) suggest that the large neurons are presumably cholinergic. These results provide evidence that at least some of the giant striatal neurons are efferent cells. The coincidence of cytological, histochemical, and hodological criteria invite the speculation that the giant corticopetal neostriatal neurons might be related to the magnocellular cholinergic groups of the basal forebrain (especially the Ch4 group).     

The organization of projections to selected points of somatosensory cortex from the cat ventrobasal complex

The organization of neurons in the cat ventrobasal complex (VB) which project to somatosensory cortex (SI) was investigated by the use of the retrograde transport of the enzyme horseradish peroxidase (HRP). Two histochemical procedures were used to visualize retrogradely transported HRP. Injections of HRP in electrophysiologically characterized points of SI cortex labeled distinctive zones of neurons in VB ipsilateral to the injections. Injections placed in the forelimb or hindlimb cortical areas labeled laminar-like aggregates of neurons have long axis corresponded to the long axis of VB. Injections of the SI trigeminal representation resulted in very compact aggregates of HRP positive neurons which were less clearly laminar. The density of projection from VB to various portions of SI paralleled the general innervation density of the peripheral skin. Injections of the cortical vibrissal, face and forepaw representations labeled a greater number of neurons in VB per unit area of cortex injected than did injections of the hindpaw or trunk representations. For a given somatotopic area, the number of labeled neurons in VB increased linearly as the area of the cortical HRP injection increased. Differences in the sensitivity of each histochemical procedure and the relationship of differing sensitivities to the observed results are also discussed.     

Study of the neurons of the supraspinal systems of the cat brain using the method of retrograde axonal transport of horseradish peroxidase]

Horseradish peroxidase (HRP) was injected into cervical, thoracic, lumbar and sacral segments of 18 cats. In all the cases HRP was transported retrogradely to the brain stem neurons in the medial reticular formation, vestibular complex and the red nucleus. The present findings demonstrate that, in addition to the above mentioned groups, there exist several other groups of neurons. Many labelled neurons are present in the locus coeruleus and the nucleus subcoeruleus, ipsilaterally. These neurons seem to be located in the same position as the monoaminergic (catecholamine-containing) neurons. Labelled HPR-positive neurons are present in the retroambiguous nucleus and in the upper pontine tegmentum adjoining the rubrospinal tract, mainly contralaterally, in the bulbar raphe, the mesencephalic central gray and the hypothalamus, mainly ipsilaterally. The occurrence of retrograde-labelled neurons in the locus coeruleus and the dorsal hypothalamus after HRP injections as far caudally as the lumbar segments, indicates that these cell groups give rise to descending fibres which pass almost the entire length of the spinal cord.     

Withdrawal of collaterals of sympathetic axons to the rat eye during postnatal development: the role of function

Neurones in the superior cervical ganglion (SCG) of the rat can be retrogradely labelled by injection of Fast blue (FB) into the anterior eye chamber with the maximum number being labelled during the second postnatal week. In adult rats, however, many fewer neurones can be so labelled. In the present study, we have investigated whether this reduction may be due to the withdrawal of collaterals of neurones which project to the posterior eye compartments. For these experiments, we have used either one (FB) or two (FB and horseradish peroxidase (HRP)) retrogradely transported markers injected with a micropipette into the anterior or posterior (vitreous) parts of the eye in rats aged 14, 31 and greater than 50 days. Using FB, we have shown that in 14-day-old rats, 40% of the neurones which project to the eye have collaterals in anterior and posterior compartments while only 1% of neurones maintain dual projections in animals aged more than 7 weeks. Furthermore, there is a reduction in the total number of neurones projecting to the eye between 14 and 31 days postbirth. The presence of significantly greater numbers of cells with collaterals to both eye compartments in the young versus the adult rats has been confirmed using both FB and HRP as retrograde markers. The importance of function on the rearrangement of these projections was investigated by either rearing animals in the dark from the time of birth, or by sectioning the preganglionic nerve trunk to the SCG, soon after birth.(ABSTRACT TRUNCATED AT 250 WORDS)