Corticotrigeminal motor pathway in the rat--I. Antidromic activation

1. The rat corticotrigeminal motor pathway was electrophysiologically investigated. 2. Fifty-one cortical neurons were antidromically activated by stimulation of the contralateral motor trigeminal nucleus (MTN). 3. Twenty-eight of the neurons were examined to see whether they were pyramidal tract (PT) neurons and seven were the PT neurons. 4. Forty peduncular axons were antidromically activated by stimulation of the contralateral MTN and eight of them were the PT axons. 5. Most MTN projecting axons showed slower conduction velocities than their stem anons.     

3H]-GABA selective retrograde labeling of neurons in the pigeon thalamo-Wulst pathway

Evidence is presented which is consistent with a specific retrograde labeling of GABAergic neurons following [3H]-GABA application in their zone of termination. [3H]-GABA injection in the pigeon Wulst leads to perikaryal retrograde labeling in the ipsilateral thalamic visual relay, n. dorsolateralis anterior thalami, pars lateralis (DLLv). This result gives further support to the biochemical evidence of the existence, in the pigeon, of a GABAergic projection from DLLv to the ipsilateral visual Wulst.     

A novel method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal.

Examination of repaired spinal cord tracts has usually required separate groups of animals for anterograde and retrograde tracing owing to the incompatibility of techniques such as tissue fixation. However, anterograde and retrograde labeling of different animals subjected to the same repair may not allow accurate examination of that repair strategy because widely variable results can occur in animals subjected to the same strategy. We have developed a reliable method of labeling spinal cord motor tracts bidirectionally in the same animal using DiI, a lipophilic dye, to anterogradely label the corticospinal tract and Fluoro-Gold (FG) to retrogradely label cortical and brainstem neurons of several spinal cord motor tracts in normal and injured adult rats. Other tracer combinations (lipophilic dyes or fluorescent dextrans) were also investigated but were less effective. We also developed methods to minimize autofluorescence with the DiI/FG technique, and found that the DiI/FG technique is compatible with decalcification and immunohistochemistry for several markers relevant for studies of spinal cord regeneration. Thus, the use of anterograde DiI and retrograde FG is a novel technique for bidirectional labeling of the motor tracts of the adult spinal cord with fluorescent tracers and should be useful for demonstrating neurite regeneration in studies of spinal cord repair.(J Histochem Cytochem 49:1111-1122, 2001)     

Endocytosis and retrograde axonal traffic in motor neurons

Spinal cord motor neurons control voluntary movement by relaying messages that arrive from upper brain centres to the innervated muscles. Despite the importance of motor neurons in human health and disease, the precise control of their membrane dynamics and its effect on motor neuron homoeostasis and survival are poorly understood. In particular, the molecular basis of the co-ordination of specific endocytic events with the axonal retrograde transport pathway is largely unknown. To study these important vesicular trafficking events, we pioneered the use of atoxic fragments of tetanus and botulinum neurotoxins to follow endocytosis and retrograde axonal transport in motor neurons. These neurotoxins bind specifically to pre-synaptic nerve terminals, where they are internalized. Whereas botulinum neurotoxins remain at the neuromuscular junction, tetanus toxin is retrogradely transported along the axon to the cell body, where it is released into the intersynaptic space and is internalized by adjacent inhibitory interneurons. The high neurospecificity and the differential intracellular sorting make tetanus and botulinum neurotoxins ideal tools to study neuronal physiology. In the present review, we discuss recent developments in our understanding of the internalization and trafficking of these molecules in spinal cord motor neurons. Furthermore, we describe the development of a reliable transfection method for motor neurons based on microinjection, which will be extremely useful for dissecting further the molecular basis of membrane dynamics and axonal transport in these cells.     

Innervation of the knee joint in the rabbit and the Formosan rock-monkey (Macaca cyclopis): a retrograde HRP study.

The number and segmental distribution of cell bodies of sensory afferents and sympathetic efferent innervating to the knee joint of the rabbit and the Formosan rock-monkey were investigated using retrograde transport with horseradish peroxidase (HRP). After injecting HRP into the articular knee joint capsule of the rabbits, labeled neurons were found in the ipsilateral L4-S2 dorsal root ganglia (DRG). However, following injection of HRP into the articular cavity of the knee joint in the rabbit and the monkey, labeled neurons were found in both the ipsilateral DRG (L5-S2 and L4-S1 of the rabbit and monkey, respectively) and in the ipsilateral sympathetic ganglia (SG) (L4-S3 (rabbit) and L3-S1 (monkey)). The majority of labeled neurons within the DRG and the SG were composed of medium and large neurons in the monkey and the rabbit, respectively. The present findings suggest that the sensory projections from and sympathetic projection to the knee joint in rabbits and monkeys are similar, but that both projections of monkeys were "shifted" one segment cranially compared to the rabbit on both projections.     

A method for measuring the amount of retrogradely transported HRP in the rat masseteric motoneuron using Mesulam's HRP histochemical protocol and an image processing system. An investigation of the effect of dopamine receptor antagonists on the retrograde transport of HRP

We developed a method for a determination of the amount of retrogradely transported HRP in the rat masseteric motoneuron using a modification of Mesulam's HRP histochemical protocol and an image processing system combined with a light microscope and a television camera. To test the validity and reproducibility of the new method, a quantitative comparison of the amount of dark blue granules of HRP-product in the cell body of masseteric motor neurons was performed between the right and left trigeminal motor nuclei of 70 rats, which resulted in no significant difference. An additional study used the method was made of the effects of administration of five dopamine receptor antagonists with different biochemical and pharmacologic properties on retrograde transport of HRP in the rat masseteric motoneuron. As a result, chlorpromazine, haloperidol, SCH 23390, and sulpiride significantly enhanced retrograde transport of HRP as against domperidone which showed no significant change in the transport. A possible regulatory system for retrograde transport of HRP in the masseteric motoneuron was discussed in relation to the action of the dopamine receptor.     

Cells of origin of the branches of the facial nerve: a retrograde HRP study in the rabbit

The origin of different branches of the facial nerve in the rabbit was determined by using retrograde transport of HRP. Either the proximal stump of specific nerves was exposed to HRP after transection, or an injection of the tracer was made into particular muscles innervated by a branch of the facial nerve. A clear somatotopic pattern was observed. Those branches which innervate the rostral facial musculature arise from cells located in the lateral and intermediate portions of the nuclear complex. Orbital musculature is supplied by neurons in the dorsal portion of the complex, with the more rostral orbital muscles receiving input from more laterally located cells while the caudal orbital region receives innervation from more medial regions of the dorsal facial nucleus. The rostral portion of the ear also receives innervation from cells located in the dorsomedial part of the nucleus, but the caudal aspect of the ear is supplied exclusively by cells located in medial regions. The cervical platysma, the platysma of the lower jaw, and the deep muscles (i.e., digastric and stylohyoid) receive input from cells topographically arranged in the middle and ventral portions of the nuclear complex. It is proposed that the topographic relationship between the facial nucleus and branches of the facial nerve reflects the embryological derivation of the facial muscles. Those muscles that develop from the embryonic sphincter colli profundus layer are innervated by lateral and dorsomedial portions of the nuclear complex. The muscles derived from the embryonic platysma layer, including the deep musculature, receive their input from mid to ventral regions of the nuclear complex.     

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle,as revealed by the retrograde transport of the cholera toxin B subunit

Summary Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L₃–L₆. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.     

Role of lipids in the retrograde pathway of ricin intoxication

The plant toxin ricin binds to both glycosphingolipids and glycoproteins with terminal galactose and is transported to the Golgi apparatus in a cholesterol-dependent manner. To explore the question of whether glycosphingolipid binding of ricin or glycosphingolipid synthesis is essential for transport of ricin from the plasma membrane to the Golgi apparatus, retrogradely to the endoplasmic reticulum or for translocation of the toxin to the cytosol, we have investigated the effect of ricin and the intracellular transport of this toxin in a glycosphingolipid-deficient mouse melanoma cell line (GM95), in the same cell line transfected with ceramide glucosyltransferase to restore glycosphingolipid synthesis (GM95-CGlcT-KKVK) and in the parental cell line (MEB4). Ricin transport to the Golgi apparatus was monitored by quantifying sulfation of a modified ricin molecule, and toxicity was studied by measuring protein synthesis. The data reveal that ricin is transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum and translocated to the cytosol equally well and apparently at the same rate in cells with and without glycosphingolipids. Importantly cholesterol depletion reduced endosome to Golgi transport of ricin even in cells without glycosphingolipids, demonstrating that cholesterol is required for Golgi transport of ricin bound to glycoproteins. The rate of retrograde transport of ricin was increased strongly by monensin and the lag time for intoxication was reduced both in cells with and in those without glycosphingolipids. In conclusion, neither glycosphingolipid synthesis nor binding of ricin to glycosphingolipids is essential for cholesterol-dependent retrograde transport of ricin. Binding of ricin to glycoproteins is sufficient for all transport steps required for ricin intoxication.     

The neurons of origin of non-cortical afferent connections to the oculomotor nucleus. A retrograde labeling study in the rabbit.

The cellular origin of the brainstem projections to the oculomotor nucleus in the rabbit has been investigated by using free (HRP) and lectin-conjugated horseradish peroxidase (WGA-HRP). Following injections of these tracers into the somatic oculomotor nucleus (OMC), retrogradely labeled cells have been observed in numerous brainstem structures. In particular, bilateral labeling has been found in the four main subdivisions of the vestibular complex, predominantly in the superior and medial vestibular nuclei and the interstitial nucleus of Cajal, while ipsilateral labeling was found in the rostral interstitial nucleus of the medial longitudinal fascicle (Ri-MLF), the Darkschewitsch and the praepositus nuclei. Neurons labeled only contralaterally have been identified in the following structures: mesencephalic reticular formation dorsolateral to the red nucleus, abducens internuclear neurons, group Y, several areas of the lateral and medial regions of the pontine and medullary reticular formation, ventral region of the lateral cerebellar nucleus and caudal anterior interpositus nucleus. This study provides also information regarding differential projections of some centers to rostral and caudal portions of the OMC. Thus, the rostral one-third appears to receive predominant afferents from the superior and medial vestibular nuclei, while the caudal two-thirds receive afferents from all the four vestibular nuclei. Finally, the group Y sends afferents to the middle and caudal, but not to the rostral OMC.     

Topographical representation of the jaw muscles within the trigeminal motor nucleus. An HRP study in the mallard, Anas platyrhynchos

The location of the trigeminal motoneurons of the jaw muscles has been determined in the brainstem of the mallard utilizing retrograde axonal transport of horseradish peroxidase (HRP). Injections with HRP into the jaw muscles or application of HRP to the mandibular nerve showed that the trigeminal motor nucleus can be subdivided into five subnuclei, mV1-mV5. Three functional groups of jaw muscles are represented in separate subnuclei. The most lateral subnucleus mV2 innervates all but one adductor muscles, the intermediate mV1 innervates the pterygoid muscles + one adductor and the medial mV4 the two protractor muscles. The most ventral subnucleus mV3 contains the neurons innervating two extrinsic tongue muscles as well as some perikarya of adductor muscles. Subnucleus mV5 lies dorsomedial to mV4 and contains the motoneurons of the depressor muscle of the lower eye lid. Elements of the proprioceptive system, viz. presumptive gamma-neurons and mesencephalic trigeminal nucleus cells, could also be visualized. The topological and functional aspects of the subdivision of the motor nucleus are discussed.     

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

Identification of the teleost Edinger-Westphal nucleus by retrograde horseradish peroxidase labeling and by electrophysiological criteria

Summary A homolog of the Edinger-Westphal nucleus of other vertebrates is described in two species of serranid basses of the genusParalabrax, a group possessing a wide range of ocular accommodation but lacking a pupillary reflex to light. The nucleus was found by retrograde labeling from the ciliary ganglion and lies dorsolateral to the ipsilateral oculomotor nucleus. The nucleus consists of 60 to 100 neurons with an average soma diameter of about 20 m in animals weighing 70 to 150 g. Electrophysiological experiments support the identification. Microstimulation of the nucleus evokes contraction of the ipsilateral lens retractor muscle and slight constriction of the caudal ipsilateral iris. Multi- and single-unit recordings in the nucleus reveal spontaneous firing (about 30 spikes/s in single units), the rate of which decreases during visually-evoked lens retractor relaxations (accommodation to near stimuli). Recordings of muscle fiber activity in the lens retractor show essentially the same behavior, which suggests that the ciliary ganglion and neuromuscular junctions simply relay impulses with little if any synaptic integration. The existence of a discrete Edinger-Westphal nucleus devoted largely to accommodation makesParalabrax a good model system for the further tracing of central accommodation control pathways.Abbreviations CNS central nervous system - EW Edinger-Westphal nucleus - HRP horseradish peroxidase - WGA wheat germ agglutinin     

The somatotopy of the masticatory neurons in the rat trigeminal motor nucleus as revealed by HRP study

Young adult albino rats of Wistar strain were used for the present study. 0.5 to 15 microliters of 20-50% of horseradish peroxidase (HRP) were injected into each individual muscle of mastication to label neurons in the trigeminal motor nucleus (TMON) for light microscopic study. The results reveal that: (1) Many HRP-labeled, multipolar neurons are observed in the motor nucleus in each jaw-closing muscle (JCM) with less in each the jaw-opening muscle (JOM). (2) The motor neurons innervating each masticatory muscle in the motor nucleus show a somatotopic arrangement: (a) those innervating the temporalis muscle are located in the medial and dorsomedial parts; (b) those innervating the masseter muscle are located in the intermediate and lateral; (c) those innervating the medial and lateral pterygoid muscles are located in the lateral, ventrolateral and ventromedial parts, respectively; and (d) those innervating the mylohyoid and the anterior belly of the digastric muscles are located in the most ventromedial part of the caudal one-third of the nucleus. Axons of most masticatory motor neurons run ventrolaterally in between the motor and the chief sensory nuclei of the trigeminal nerve. However, those of the mylohyoid and anterior belly of the digastric muscles ascend dorsally to the dorsal aspect of the caudal nucleus and then turn ventrolaterally to join the motor root of the trigeminal nerve. Furthermore, the dendrites of the motor neuron of JCM converge dorsocaudally to the supratrigeminal region. The diameters of neurons of each JCM display a bimodal distribution. However, an unimodal distribution is present in the motor neurons from each JCM. It is suggested that the motor nucleus innervating the JCM is comprised of comprised of alpha- and gamma-motor neurons. It, thus, may provide a neural basis for the regulation of the muscle tone and biting force.     

Identification of sensory neurons supplying receptors in lingual muscles of the rat: histochemical and retrograde labeling study with horseradish peroxidase.

Sensory innervation of lingual musculature was studied in young adult Wistar rats using retrograde labeling by horseradish peroxidase (HRP) and combined silver impregnation and acetylcholinesterase (AchE) methods. Intra-lingual injection of HRP resulted in labeling of neuronal somata in the trigeminal, superior vagal, and second cervical spinal (C2) ganglia. When HRP was directly applied to the proximal stump of severed hypoglossal nerve, labeling occurred only in the cervical and superior vagal ganglia. Morphometric analysis revealed that the labeled neurons were of the small-sized category in all ganglia. However, in the trigeminal and C2 ganglia, labeling occurred also among the medium-sized neurons. Combined silver and AchE preparations from lingual muscles revealed the absence of typical muscle spindles. Instead, there were free and spiral nerve terminals in the interstitium, and epilemmal knob-like or bouton-like endings surrounding non-encapsulated muscle fibers. These terminals showed AchE -ve reaction in contrast to the motor ones. Few ganglionic cells were scattered along the hypoglossal nerve with uniform AchE +ve reaction in their perikarya. This indicates that medium-sized neurons in the trigeminal and C2 ganglia, and probably sensory neurons along the hypoglossal nerve mediate lingual muscle sensibility perceived by atypical sensory terminals.     

Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway

3-Ethynylbenzoate (3EB) functions as a novel, activity-dependent, fluorogenic, and chromogenic probe for bacterial strains expressing the TOL pathway, which degrade toluene via conversion to benzoate, followed by meta ring fission of the intermediate catechol. This direct physiological analysis allows the fluorescent labeling of cells whose toluene-degrading enzymes have been induced by an aromatic substrate.     

Organization of the cortico-ponto-cerebellar pathway to the dorsal paraflocculus. An experimental study with anterograde and retrograde transport of WGA-HRP in the cat.

To reveal the organization and relative magnitude of connections from various parts of the cerebral cortex to the dorsal paraflocculus via the pontine nuclei, WGA-HRP was injected in the dorsal paraflocculus in conjunction with injection of the same tracer in various parts of the cerebral cortex in 17 cats. Termination areas of cortical fibres (anterogradely labelled) and pontine neurons projecting to the dorsal paraflocculus (retrogradely labelled) were carefully plotted in serial transverse sections. As an average of countings in ten cats, 90% of the labelled cells were found in the pontine nuclei contralateral to the injection, and the majority (70%) were located in the rostral half of the nuclei. The highest degree of overlap between anterograde and retrograde labelling was found after injections of the parietal association cortex (areas 5 and 7). In an experiment with double anterograde tracing, it was shown that both area 5 and 7 contribute substantially to the cerebral inputs to the dorsal paraflocculus. High degree of overlap also occurred after injections of several visual cortical areas (areas 17, 18, 19, 20 and the posteromedial lateral suprasylvian visual area, PMLS). Cases with injections restricted to individual visual areas indicate that they all contribute to the parafloccular input. Considerably less overlap occurred after injections of the primary sensorimotor region (SI, MI) and second somatosensory area (SII), while the supplementary motor area, the auditory cortex and gyrus cinguli probably have no or very restricted access to the dorsal paraflocculus. It is concluded that the dorsal paraflocculus has its major cortical input from the parietal association cortex and the visual cortical areas. Since all the various cortical regions studied project to largely different parts of the pontine nuclei, and overlap with neurons projecting to the dorsal paraflocculus takes place at numerous places, it follows that the pontine neurons projecting to the dorsal paraflocculus must consist of many subgroups differing with regard to their cortical input.