Glutamatergic and cholinergic projections to the pontine inhibitory area identified with horseradish peroxidase retrograde transport and immunohistochemistry

Previous studies in our laboratory have shown that microinjection of acetylcholine and non-N-methyl-D-aspartate (NMDA) glutamate agonists into the pontine inhibitory area (PIA) induce muscle atonia. The present experiment was designed to identify the PIA afferents that could be responsible for these effects, by use of retrograde transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), glutamate immunohistochemistry and NADPH-diaphorase staining techniques. Experiments were performed in both decerebrate and intact cats. Dense retrograde WGA-HRP labelling was found in neurons in the periaqueductal gray (PAG) and mesencephalic reticular formation (MRF) at the red nucleus (RN) level, ventral portion of paralemniscal tegmental field (_VFTP), retrorubral nucleus (RRN), contralateral side of PIA (_CPIA), pontis reticularis centralis caudalis (PoC), and most rostral portion of the nucleus parvicellularis (NPV) and nucleus praepositus hypoglossi (PH) at the level of the pontomedullary junction; moderate labelling was seen in pedunculopontine nucleus, pars compacta (PPNc), laterodorsal tegmental nucleus (LDT), superior colliculus (SC), MRF and PAG at the level caudal to RN, medial and superior vestibular nuclei, and principle sensory trigeminal nucleus (5P); and light labelling was seen in dorsal raphe (DR) and locus coeruleus complex (LCC). The projection neurons were predominantly ipsilateral to the injection site, except for both vFTP and RRN, which had more projection cells on the contralateral side. Double labelled WGA-HRP/NADPH-d neurons could be found in PPNc and LDT. Double labelled WGA-HRP/glutamatergic neurons could be seen at high densities in MRF, RRN, vFTP, and cPIA, moderate densities in SC, LDT, PPNc, PoC, and NPV, and low densities in PH, 5P, DR, LCC, and PAG. No cells in LDT and PPNc were triple labelled with NADPH-d, glutamate antibody and WGA-HRP. The mesopontine efferents identified here may mediate the suppression of muscle tone in REM sleep and coordinate muscle tone during head and neck movements. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase

The distribution and morphology of preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the cat have been studied with the horseradish peroxidase (HRP) tracing technique. HRP applied to the cut pelvic nerve was identified in cells located ipsilaterally, primarily in the intermediate gray matter. They formed a column approximately 10 mm long, usually contained within two, but occasionally three, sacral segments. S₂ contained a majority of cells. In transverse sections the SPN had the appearance of an inverted “L.” Cells were medium-sized, oval or spindle-shaped, and transversely oriented. They were distributed among two major components and one minor one: (1) dorsal band (34%) located mainly in lamina V beneath the dorsal horn (cells and dendrites horizontally oriented), (2) lateral band (64%) along the lateral edge of the gray matter in laminae VII through V (cells oriented dorsoventrally with dendrites extending within the nucleus and into the dorsolateral funiculus), and (3) a small group (2%) of cells at the rostral end of the SPN in lamina VII in the middle of the ventral horn. These data coupled with the results of other investigations indicate that the SPN has a viscerotopic organization wherein the colon is innervated primarily by cells in the dorsal band and the urinary bladder is innervated primarily by cells in the lateral band.     

Morphology of sympathetic preganglionic neurons in the neonatal rat spinal cord: an intracellular horseradish peroxidase study

Understanding the central neural control of autonomic functions requires a knowledge of the morphology of the preganglionic neurons, for the location of the dendritic arborizations of these neurons will indicate which central pathways may have access to them. In the present study, individual sympathetic preganglionic neurons in the neonatal rat spinal cord have been examined by the intracellular injection of horseradish peroxidase (HRP) in an in vitro preparation. Seventeen HRP-labeled preganglionic neurons in thoracic segments T1-T3 were examined in detail; of these, 12 somata were located in the intermediolateral cell column (IML), one in the lateral funiculus (LF), two in the intercalated nucleus (IC), and two at the border between IML and IC. All of the neurons had extensive dendritic arborizations arising from an average of six primary dendrites; the average total dendritic length for these cells was 2,343 microns. The morphology of preganglionic neurons differed depending on the location of their cell bodies. Preganglionic neurons located in the IML were essentially two-dimensional: the cells had some dendrites that coursed rostrocaudally for 300-500 microns within the IML and others that coursed mediolaterally, extending to the lateral surface of the cord and close to the central canal. Axons of these cells coursed ventrally from the cell body and exited from the spinal cord at the first ventral root caudal to the cell body. No intraspinal axon branches were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonidet P40 and the retrograde transport of horseradish peroxidase in undamaged visceral nerves

The cell bodies of origin of peripheral nerves, in particular visceral nerves, are often difficult to identify using standard horseradish peroxidase (HRP) methods. The non-ionic surfactant Nonidet-P40, when applied to intact peripheral nerve along with HRP, allows the investigator to examine the neurons of origin of the nerve without cutting the fibers or injecting label into its peripheral terminal field.     

Separate populations of lumbar preganglionic neurons identified with the retrograde transport of horseradish peroxidase (HRP) and 4,6-diamidino-2-phenylindole (DAPI)

Horseradish peroxidase (HRP) was applied to the central cut end of the intermesenteric trunk or the caudal lumbar sympathetic trunk in golden hamsters. Preganglionic cells labeled from the intermesenteric trunk were located in the intermediolateral cell column, bilaterally, and in the dorsal commissural nucleus. The segmental distributions of the cells in these two nuclear groups were similar. Preganglionic neurons labeled from the lumbar sympathetic trunk were located mainly in the ipsilateral intermediolateral cell column. The simultaneous application of HRP to one trunk and 4,6-diamidino-2-phenylindole (DAPI) to the other delincated two separate preganglionic cell populations. Cells labeled from the two trunks were distributed as above. HRP-filled cells and DAPI-filled cells were intermixed in the intermediolateral cell column ipsilateral to the backfilled sympathetic trunk. No double labeled cells were present. The results show that the dorsal commissural nucleus in the lumbar cord preferentially innervates prevertebral ganglia, that lumbar paravertebral ganglia are innervated by cells located mainly in the ipsilateral intermediolateral cell column, and that paravertebral and prevertebral ganglia are innervated by non-overlapping sets of lumbar preganglionic neurons.     

Morphology of sympathetic preganglionic neurons in the thoracic spinal cord of the cat: an intracellular horseradish peroxidase study

Horseradish peroxidase was intracellularly injected into sympathetic preganglionic neurons (SPN) of the third thoracic segment in cats. Seven neurons were reconstructed from serial horizontal or parasagittal sections of the spinal cord. The cell bodies of all neurons were located in the n. intermediolateralis pars principalis (ILp). They were spindle-shaped with the long axis in craniocaudal direction or large and multipolar or small and oval in shape. Preferentially on the cranial and caudal pole of the cell body, five to eight primary dendrites arose from the cell body. Dendritic branches were traced to their terminations at distances up to 1,330 microns from the cell body. The dendritic fields of all SPNs were strictly oriented in the longitudinal direction with a total length of 1,500-2,540 microns. The cranial and caudal dendritic fields were about equal in length but, with one exception, the degree of branching was always greater in the cranial than in the caudal dendritic field. The dendritic fields of all SPNs were primarily restricted to the ILp. In the mediolateral direction it extended from 130 to 360 microns and in the dorsoventral direction from 50 to 180 microns. Only rarely, a higher-order dendrite left the boundaries of the ILp and projected dorsolaterally or laterally into the white matter or ventromedially or medially into the adjacent n. intercalatus. All dendrites showed various forms of spines. At a distance of 132-437 microns from the cell body the axon arose as a direct extension of a process which closely resembled a primary or second-order dendrite. The axons projected ventrally and mostly caudally along the lateral border of the gray matter until they turned laterally at the end of the ventral horn. No axon collaterals were observed.     

Environmental co-regulation of substance P, somatostatin and neurotransmitter synthesizing enzymes in cultured sympathetic neurons

Mechanisms regulating peptide neurotransmitter metabolism were examined in dissociated cell cultures of the neonatal rat superior cervical ganglion (SCG). The pineal gland, a target of the SCG, produced a soluble factor (PCM) which increased substance P (SP) levels more than 15-fold in sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Elimination of the non-neuronal cells decreased SP to negligible levels and abolished the stimulatory effects of PCM on SP expression. These observations suggest that ganglion non-neuronal cells stimulate sympathetic expression of SP, and that the pineal influences neuronal SP by acting on, or in concert with, ganglion support cells. PCM also influenced other neurotransmitter systems. In the presence of ganglion non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) and decreased tyrosine hydroxylase (TOH) and somatostatin (SO). By contrast, PCM treatment of pure neuronal cultures resulted in negligibleCHAC and SP levels and a doubling of SO with a small increase in TOH. In sympathetic neurons, SP expression may be associated with cholinergic development, whereas SO may be associated with noradrenergic phenotypic expression. Moreover, there is a reciprocal relationship between SP and SS expression by sympathetic neurons analogous toe previously described relationship between noradrenergic and cholinergic expression^17–19.     

Olivocerebellar connections in sheep studied with the retrograde transport of horseradish peroxidase

We describe here the morphology of the inferior olive and the localization of labeled cells after HRP injections into various lobules of vermis and hemisphere of the cerebellum of the sheep. The medial part of the caudal half of the medial accessory olive projects to a medial zone in the anterior lobe, the simple lobule, and the lobules VII and VIII. The lateral part of the medial accessory olive projects to more lateral parts of these lobules with the exception of lobule VII. The group beta projects in a differential manner to the lateral parts of the lobules VII and VIII and the medial parts of the lobules IX and X. The dorsomedial cell column projects to lobules VIII, IX, and X; the connections of the dorsal cap are restricted to lobule X. Fibers from the caudal limb of the dorsal accessory olive terminate in the B zone, the simple lobule, and in lobule VIII. The rostral half of the medial accessory olive projects to lobule IX and to the hemisphere. The other projections of the accessory olives and the principal olive to the hemisphere are similar to those reported for the cat. An accessory cell group in the sheep, located between the principal and the dorsal accessory olive, has connections with the caudal vermis and the hemisphere.     

Projections of lamprey spinal neurons determined by the retrograde axonal transport of horseradish peroxidase.

The spinal cords of larval sea lampreys (Petromyzon marinus) and adult river lampreys (Ichthyomyzon unicuspis) were injected with horseradish peroxidase through a transection 1 cm caudal to the last gill. Some animals also had a spinal hemisection 1 cm caudal to the injection. After recovery periods of 1 to 52 days, the spinal cords were treated with diaminobenzidene and hydrogen peroxide, and the projections of various cell types determined in wholemount slides. From these observations the following conclusions were drawn. Most dorsal cells (primary sensory cells) are bipolar with a long rostral projection and a short caudal projection of no more than 5-10 mm. Both processes travel in the ipsilateral dorsal column. Their peripheral processes enter the dorsal roots as branches of their central axons. Some dorsal cells send processes out three or more dorsal roots both rostral and caudal to the cell body. Myotomal motoneurons have characteristic locations in the medial gray column and send prominent transversely oriented dendrites into the lateral columns. A few motoneurons are unusually large. In addition to giant interneurons the majority of smaller rostrally projecting interneurons also have decussating axons. A recently described cell type, the oblique bipolar cell, appears to have an exclusively crossed rostral projection. Although most edge cells project rostrally, as many as 20% may have a caudal projection or both rostral and caudal projections. Edge cells project equally to the ipsilateral and contralateral spinal hemicord, but their processes do not extend more than about 18 mm in sea lamprey larvae and 37 mm in adult river lampreys. Lateral cells project exclusively to the ipsilateral caudal hemicord. A few cells which resemble lateral cells in location and in possessing large lateral dendrites, project rostrally. However, these have atypical morphologic features which probably distinguish them from true lateral cells. Thus far, regardless of cell type, all decussating axons seem to pass ventral to the central canal, while decussating medial dendrites pass dorsally.     

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.     

A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons.

The phenomenon of retrograde intraaxonal transport of extracellular markers introduced at the level of the axon terminal has been suggested as a possible mechanism of communication between the axon terminal and the neuron cell body. We tested the hypothesis that communication after axotomy might consist of a change in the rate of uptake or of transport of material by injured neurons. Small lesions were made with a needle in one retinal quadrant of chicks and immediately afterwards horseradish peroxidase (HRP) was injected into the vitreous body of the eye. The amount of HRP accumulated by some of the neurons of the isthmo-optic nucleus (ION) which project to the damaged area was clearly different from that of nearby cells which project to the non-damaged portions of the retina. The uninjured cells accumulated enzyme marker beginning at 3.5 h after injection. The injured neurons did not accumulate significant amounts of HRP until between 4 and 6 h after injection. Between 6.75 h and 18 h the injured cells in the ION accumulated greater amounts of HRP than cells in other regions, but by 24 h the cells of the ION in the region of injury contained distinctly less label. This pattern of enzyme accumulation was confirmed by counts of the number of HRP-positive granules within cells of chicks fixed 4, 11.75, 12.25, 27.6 and 72 h after injury. In another series of experiments, the axon terminals of the ION were first exposed to HRP, and 1 h later some of the axons were damaged with a needle. In these cases, there was no difference between the injured and control neurons in the time of first appearance of labeled cells in the ION within the first 4 h after injection of HRP. These findings suggest that injury initially results in a decrease in the uptake of the marker rather than a decrease in the rate of retrograde transport. The amount of marker found in the injured neurons later is greater than that found in the control neurons. This subsequent difference may represent an increase in the rate of uptake, transport, or both or a decrease in the rate of degradation of HRP within the cell body as a response to injury of the axon.     

A sensitive method for histochemical demonstration of horseradish peroxidase in neurons following retrograde axonal transport

A study was made on the effects of various fixatives and some other histochemical parameters used in the procedure for demonstrating labeled neurons following retrograde axonal transport of horseradish peroxidase (HRP). The enzyme was injected into the tongue of adult mice and the results were obtained by counting labeled hypoglossal neurons following certain variations in the procedure.Paraformaldehyde in the fixative should be avoided since it reduces the number of labeled neurons as compared to glutaraldehyde in a concentration of 1.5–2.5% Fixation for about 4 h is recommended followed by a wash in 5% sucrose buffer overnight.Variables in the histochemical procedure were systematically studied in order to determine optimal pH, buffer type, buffer concentration and substrate concentration. The effect of using a ‘preincubation’ in buffer containing only diaminobenzidine tetrahydrochloride (DAB) was also examined. These results were used to develop a modified histochemical procedure which produced a substantial increase in the number of detectable HRP-labeled neurons as compared to equivalent sections that were reacted in the incubation medium described by Graham and Karnovsky⁵. The modified histochemical procedure involves incubation (no preincubation with DAB only) of sections in the dark for 30 min in a solution consisting of 10 ml cacodylate buffer (pH5.1; 0.1 M), 20 mg DAB and 0.1 ml of 1% hydrogen peroxide. The Kodak Wratten no. 46 filter is recommended for light-microscopical identification of labeled neurons since it is closely matched to the absorption spectrum of the DAB reaction product and consequently greatly increases the contrast of HRP-labeled neurons.     

A cholinergic projection to the rat superior colliculus demonstrated by retrograde transport of horseradish peroxidase and choline acetyltransferase immunohistochemistry

Acetylcholinesterase (AChE) has been localized by histochemistry in the superior colliculus and in the tegmentum of the caudal midbrain and rostral pons of the rat. The pattern of AChE localization in the superior colliculus was characterized by homogeneous staining in the superficial layers and patchlike staining in the intermediate gray layer. In the tegmentum, AChE was localized in the pedunculopontine nucleus (PPN), beginning rostrally at the caudal pole of the substantia nigra and extending caudally to the level of the parabrachial nuclei, and in the lateral dorsal tegmental nucleus (LDTN) of the central gray. The localization of AChE in these nuclei overlapped the distribution of neurons stained by immunohistochemistry using an antibody to choline acetyltransferase (ChAT), the synthesizing enzyme of the neurotransmitter acetylcholine. Other neighboring areas that were stained with AChE, but that did not contain ChAT-immunoreactive neurons, included the microcellular tegmental nucleus and the ventral tegmental nucleus. Neurons in the PPN and LDTN were determined to be potential sources of the cholinergic projection to the intermediate gray layer of the rat superior colliculus by double labelling with retrograde transport of horseradish peroxidase (HRP) combined with the immunohistochemical localization of ChAT. Three populations of neurons were identified. A predominantly ipsilateral ChAT-immunoreactive population was located in the pars compacta subdivision of PPN (PPNpc). Retrograde HRP-labelled neurons in the pars dissipata subdivision of the PPN (PPNpd), located ventral to the superior cerebellar peduncle (SCP) at the level of the inferior colliculus, composed a second population that was predominantly contralateral but was not ChAT immunoreactive. A third population of retrogradely labelled neurons was predominantly ipsilateral and ChAT immunoreactive and was located in the LDTN. These findings compared favorably with the full extent of the projection from this tegmental region revealed by retrograde transport of HRP from the superior colliculus when more compatible fixation and chromogen procedures were used. The results suggest that the PPN and the LDTN are two sources of the cholinergic input to the superior colliculus. Since the PPN also has extensive efferent, and afferent, connections with basal-ganglia-related structures, this cholinergic excitatory input to the superior colliculus, like the GABA-ergic inhibitory input from the substantia nigra pars reticulata, may provide the basis for an additional influence of the basal ganglia on visuomotor behavior.     

Muscle sensory neurons in the spinal ganglia in the rat determined by the retrograde transport of horseradish peroxidase

The method of retrograde transport of horseradish peroxidase (HRP) was used to identify muscle sensory neurons in the spinal ganglia in the rat. Experiments were conducted on 25 albino rats. Injections of 0.06 to 0.08 ml 2 to 20% Sigma type VIHRP were made unilaterally into anterior tibial muscle. Cells of origin of muscle receptors and motor endings in the same area where HRP was administered were demonstrated. The labeled cells, medium to large, were found in fourth and fifth lumbar ganglia ipsilateral to the site of injection. Simultaneously, labeled neurons were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia.     

Identification of the trochlear motoneurons by retrograde transport of horseradish peroxidase

A common technique for demonstrating projections in the brain is to electrically stimulate one part of the brain and record mass or field potentials from another part. We showed in the visual system of the cat, where connections between retina, lateral geniculate nucleus, and superior colliculus are very well known, that the recording of field potentials is not at all sufficient to demonstrate connections. The most prominent potential after electrical stimulation of the optic tract is the field potential created by the Y-ganglion cell fibers of the optic nerve. We recorded this potential in the optic nerve head of the eye, in the lateral geniculate nucleus, and in the superior colliculus. To our surprise, we also could record this potential 7 mm in front of the retina, with the electrode in the vitreous, and 5 mm above the lateral geniculate nucleus and the superior colliculus, where there are no direct inputs from the optic tract. These results show quite clearly that field potentials can “stray” much farther than the underlying anatomical structure projects.     

Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the lateral reticular nucleus (LRN). Labeled cells were found at all spinal levels and in particular large numbers in cervical and lumbar segments. Various spinal areas gave rise to cells of origin of this tract, which appears to be more prominent than any other tract previously studied with a similar approach. Labeling common to all spinal segments was observed in (1) ventromedial parts of both intermediate zone and ventral horn (laminae VII, VIII and X), mainly contralaterally; (2) the reticular extension of the neck of the dorsal horn, partly bilateral; and (3) superficial layers of the dorsal horn and nucleus of the dorsolateral funiculus (NDLF), mainly contralateral and projecting essentially to the lateral zone of the LRN. Additional labeling was observed at cervical and lumbar levels, each with specific qualities: (1) the cervical enlargement, which displayed labeling in the central part of the ipsilateral intermediate zone (lamina VII); (2) the rostral lumbar levels, which had projections from the contralateral median portion of the neck of the dorsal horn. These latter projections appear to be specific to pathways reaching the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive (except lamina I and NDLF projections) and the differences between these afferents and those projecting to the dorsal reticular formation, i.e., the nucleus reticularis ventralis.     

Identification of abducens motoneurons, accessory abducens motoneurons, and abducens internuclear neurons in the chick by retrograde transport of horseradish peroxidase

The location of the motoneurons innervating the lateral rectus, pyramidalis, and quadratus muscles of the chick has been determined by application of horseradish peroxidase (HRP) to these muscles and their nerve branches, and internuclear neurons in the abducens nucleus have been identified by injection of HRP into the oculomotor nucleus. Quantitative results were obtained by means of a semiautomatic image analyzer. Lateral rectus motoneurons were observed only in the ipsilateral principal abducens nucleus, where they numbered 500-550, and quadratus and pyramidalis motoneurons only in the ipsilateral accessory abducens nucleus. The 325-375 internuclear neurons that appeared in the principal abducens nucleus contralateral to the oculomotor nucleus injected with HRP were practically confined to the rostral two thirds of the nucleus, where they tended to surround the lateral rectus motoneurons in dorsal or lateral positions, though a minority of interneurons also mingled with the motoneurons in the center or at the medial face of the nucleus. Most interneurons were small and elongated, but a minority of larger interneurons morphologically similar to the lateral rectus motoneurons were also distinguishable. The 100-110 quadratus motoneurons and the 45-55 pyramidalis motoneurons mingled in the accessory abducens nucleus were larger than the lateral rectus motoneurons and sent their axons into the ipsilateral abducens nerve.