Viruses as transneuronal tracers.

Tracing chains of neurones requires the use of transneuronal tracers, which are transferred between connected neurones. The conventional transneuronal tracers used so far produce weak labelling of recipient neurones, probably because only a small amount of tracer is transferred. Live neurotropic viruses are beginning to be used as transneuronal tracers. The viruses are replicated in recipient neurones after transneuronal transfer. This replication, which is a unique characteristic of viruses, produces strong transneuronal labelling. The findings indicate that herpes-viruses in particular represent powerful tools for demonstrating neuronal connections across synapses, for example between peripheral nerves and neurones in the brain.

Propriospinal neurons with ascending collaterals to the dorsal medulla, the thalamus and the tectum: a retrograde fluorescent double-labeling study of the cervical cord of the rat.

Branching neurons with descending propriospinal collaterals and ascending collaterals to the dorsal medulla, the thalamus and the tectum were studied in the rat's cervical spinal cord (C1-C8), using the retrograde fluorescent double-labeling technique: Diamidino Yellow Dihydrochloride (DY) was injected in the cord at T2, True Blue (TB) was injected in the brain stem. DY-labeled descending propriospinal neurons were present in all laminae, except lamina IX. They were concentrated in lamina I, laminae IV to VIII, and in the lateral spinal nucleus, LSN. TB-labeled neurons projecting to the dorsal medulla were concentrated in lamina IV and the medial parts of laminae V and VI (probably representing postsynaptic dorsal column--PSDC--neurons), but were also present in lamina I, the LSN, the lateral dorsal horn, and in laminae VII and VIII. DY-TB double-labeled neurons giving rise to both a descending propriospinal collateral and an ascending collateral to the dorsal medulla were intermingled with the TB single-labeled neurons. About 4% of the descending propriospinal neurons gave rise to an ascending collateral to the dorsal column nuclei; these double-labeled cells constitute a sizable fraction (10%) of the PSDC neurons. TB-labeled spinothalamic and spinotectal neurons were located in lamina I, the lateral cervical nucleus (LCN), and LSN, the lateral lamina V, lamina VII and VIII, lamina X and in the spinal extensions of the dorsal column nuclei, predominantly contralateral to the TB injections. DY-TB double-labeled neurons were present throughout C1-C8 in the LSN, lateral lamina V, lamina VIII, ventromedial lamina VII, and lamina X. Only very few were observed in lamina I and the LCN, and none in the spinal extensions of the dorsal column nuclei. The double-labeled neurons constituted only a minor fraction of all labeled neurons; 3-5% of the spinothalamic neurons and about 1-7% of the spinotectal neurons were double-labeled. Conversely, only about 1% of the labeled descending propriospinal neurons gave rise to an ascending spinothalamic collateral, and even fewer (0.1 to 0.6%) to a collateral to the dorsal midbrain. The LSN displayed the highest relative content of branching neurons. Up to 20% of its ascending spinothalamic and spinotectal neurons and up to 8% of its descending propriospinal neurons were found to be branching neurons, indicating that the LSN constitutes an unique cell-group in the rat spinal cord.

Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem.

The transneuronal transfer of neurotropic viruses may represent an effective tool for tracing chains of connected neurons because replication of virus in the recipient neurons after transfer amplifies the "tracer signal." Herpes simplex virus type 1 was transferred transneuronally from forelimb and hindlimb nerves of rats to the cortical and brainstem neurons that project to the spinal enlargements to which the nerves receiving injections are connected. This transneuronal transfer of herpes simplex virus type 1 from peripheral nerves has the potential to be used to identify neurons in the brain that are related transsynaptically to different nerves and muscles.

Spinocerebellar neurons and propriospinal neurons in the cervical spinal cord: a fluorescent double-labeling study in the rat and the cat.

In the cervical spinal cord of the rat and the cat, the distributions of spinocerebellar and of descending propriospinal neurons were investigated using the retrograde fluorescent double-labeling technique. Moreover, a search was made for the presence of neurons with both ascending spinocerebellar and descending propriospinal axoncollaterals. Diamidino Yellow Dihydrochloride (DY) was injected at T2, while True Blue (TB) (in rats) or Fast Blue (FB) (in cats) was injected in the cerebellum. The distributions of labeled neurons were very similar in the rat and the cat. DY-labeled propriospinal neurons, projecting to T2 or below, were most numerous in lamina I and laminae IV to VIII. In the rat, such neurons were also present in the lateral spinal nucleus (LSN). TB- or FB-labeled spinocerebellar neurons were concentrated in the central cervical nucleus (CCN) at C1-C4, in the central part of lamina VII at C5-T1, in the medial part of lamina VI and the adjoining dorsomedial part of lamina VII at C2/C3-T1, and in Clarke's column. They were also found in lamina V at C1 and C7-T1, and in lamina VIII at all levels. In both species only very few DY-TB/FB double-labeled neurons, representing neurons with branching axons, were observed; in C1-T1, only about 0.5% of all TB/FB-labeled spinocerebellar neurons and about 0.05% of all DY-labeled descending propriospinal neurons were double-labeled. The double-labeled neurons were all located centrally in lamina VII at C5-T1, but even in that area they constituted not more than 1.5% (rat) and 4% (cat) of the labeled spinocerebellar neurons. These findings indicate that, in the cervical cord of the rat and the cat, descending propriospinal neurons and spinocerebellar neurons are to a large extent separate populations.

Distribution of corticospinal neurons with collaterals to the lower brain stem reticular formation in monkey (Macaca fascicularis).

An earlier retrograde double-labeling study in cat showed that up to 30% of the corticospinal neurons in the medial and anterior parts of the precruciate motor area represent branching neurons which project to both the spinal cord and the reticular formation of the lower brain stem. These neurons were found to be concentrated in the rostral portion of the motor cortex, from where axial and proximal limb movements can be elicited. In the present study the findings in the macaque monkey are reported. The fluorescent retrograde tracer DY was injected unilaterally in the spinal cord at C2 and the fluorescent tracer FB was injected ipsilaterally in the medial tegmentum of the medulla oblongata. In the contralateral hemisphere large numbers of single DY-labeled corticospinal neurons and single FB-labeled corticobulbar neurons were present. A substantial number of DY-FB double-labeled corticospinal neurons were also found, which must represent branching neurons projecting to both the spinal cord and the bulbar reticular formation. These neurons were present in: 1. The anterior portion of the "cingulate corticospinal area" in the lower bank of the cingulate sulcus; 2. The supplementary motor area (SMA); 3. The rostral part of precentral corticospinal area; 4. The upper portion of the precentral face representation area; 5. The caudal bank of the inferior limb of the arcuate sulcus; 6. The posterior part of the insula. In these areas 10% to 30% of the labeled neurons were double-labeled. The functional implications of the presence of branching corticospinal neurons in these areas is discussed.

Brainstem projections to spinal motoneurons: an update.

1. The existence of direct projections to spinal motoneurons and interneurons from the raphe pallidus and obscurus, the adjoining ventral medial reticular formation and the locus coeruleus and subcoeruleus is now well substantiated by various anatomical techniques. 2. The spinal projections from the raphe nuclei and the adjoining medial reticular formation contain serotonergic and non-serotonergic fibres. These projections also contain various peptides, several of which are contained within the serotonergic fibres. Whether still other transmitter substances (e.g. acetylcholine) are present in the various descending brainstem projections to motoneurons remains to be determined. 3. The spinal projections from the locus coeruleus and subcoeruleus are mainly noradrenergic, but there also exists a non-noradrenergic spinal projection. 4. Pharmacological, physiological and behavioural studies indicate an overall facilitatory action of noradrenaline and serotonin (including several peptides) on motoneurons. This may lead to an enhanced susceptibility for excitatory inputs from other sources. 5. The brainstem areas in question receive an important projection from several components of the limbic system. This suggests that the emotional brain can exert a powerful influence on all regions of the spinal cord and may thus control both its sensory input and motor output.

Retrograde transneuronal transfer of herpes simplex virus type 1 (HSV 1) from motoneurones.

The use of Herpes simplex virus (HSV) as a retrograde transneuronal tracer would have the unique advantage that the virus would be replicated in the second order neurones, resulting in strong labelling. HSV was injected in the XII nerve (mice). The virus was detected immunohistochemically. Four stages in the brainstem distribution of HSV-positive neurones were distinguished. These stages were correlated with injected amounts/survival time. In stage 1, positive neurones were restricted to the XII nucleus; glial cells were present around the intramedullary XII rootlets. In stages 2-4, positive neurones and glial cells were also present outside the XII nucleus: (a) in the lateral reticular formation, Kölliker-Fuse nucleus, raphe and nucleus coeruleus; and (b) in the area around the XII rootlets, including parts of the inferior olive. In view of their distribution, many of the neurones in (a) must have received the virus by retrograde transneuronal transfer from XII motoneurones. The neurones in (b) were probably infected through a different route, i.e. local transfer of virus from XII axons via glial cells. This local transfer does not lead to extensive spread of the infection, yet, when using HSV for retrograde transneuronal tracing it may represent a source of error.

Brainstem projections to lumbar motoneurons in rat--I. An ultrastructural study using autoradiography and the combination of autoradiography and horseradish peroxidase histochemistry.

In 11 rats the descending projections from the ventrolateral medullary medial reticular formation, the medullary raphe nuclei and the area of the nucleus coeruleus and subcoeruleus to lumbar motoneuronal cell groups were studied by means of electron microscopical autoradiography after [3H]leucine injections in the respective brainstem areas. The distribution of the transported radioactivity in the autoradiographs was determined using the circle method [Williams (1977), in Practical Methods in Electron Microscopy, Vol. 6, pp. 85-173] which showed that the vast majority of the silver grains was located over terminal profiles. In the motoneuronal cell groups six different types of terminals were distinguished. After injections in the ventrolateral medial reticular formation the majority of the silver grains was located over F-type terminal profiles while many fewer silver grains were found over S- and G-types. After injections in the raphe nuclei and the adjoining medial reticular formation approximately equal numbers of silver grains were found over F- and G-type terminals while fewer were found over S-type. A small proportion of silver grains was present over C-type terminals and only after injections in the ventrolateral medial reticular formation. After [3H]leucine injections in the area of the nucleus coeruleus and subcoeruleus the majority of silver grains were located over E- and S-type terminals whereas relatively few were located over F-type terminals. The E-type terminal, which has not been described before in the motoneuronal cell groups, is characterized by the fact that it contains relatively small vesicles and occasionally elongated or canaliculi-like structures. In the three groups of experiments approximately 40-50% of the labelled S- and F-type terminal profiles established synaptic contacts, but only approximately 10% of the labelled E- and G-type terminal profiles did so. In all cases these synaptic contacts were established mainly with proximal dendrites. In the autoradiographs some of the silver grains were concentrated into clusters. The vast majority of these clusters, consisting of six or more silver grains, were centred over terminal profiles. The differential distribution of these clusters over the different types of terminal profiles in the various experiments was roughly the same as found by means of the circle method. In two rats [3H]leucine injections in the ventrolateral medial reticular formation were combined with horseradish peroxidase injections in the ipsilateral hindleg muscles, resulting in retrograde labelling of the corresponding motoneurons as visualized by means of the tetramethyl benzidine incubation method.(ABSTRACT TRUNCATED AT 400 WORDS)

Branching neurons in the cervical spinal cord: a retrograde fluorescent double-labeling study in the rat.

Branching neurons giving rise to ascending and descending collaterals were studied in the cervical spinal cord of the rat. After unilateral injection of two retrograde fluorescent tracers, i.e. DY.2HCl at T2 or more caudal levels and TB at C1 or more rostral levels, many DY-TB double-labeled neurons were found in C3 to C8. These neurons were located bilaterally throughout the spinal grey matter, as well as in the lateral spinal nucleus (LSN). However, no double-labeled neurons could be detected in the laminae I and II on either side. The double-labeled neurons must represent branching neurons giving rise to a collateral ascending to the rostral injection-site or above, and another collateral descending to the caudal injection-site or below. The descending collaterals were found to extend to various spinal levels, including the lumbosacral cord. However, most of them terminated at shorter distances from their parent cell bodies; thus 20% of the C3-C8 neurons projecting to C1 or above had a descending collateral reaching T2, 8% had a collateral reaching T9, and 3% a collateral reaching L2/L3. The ascending collaterals of the majority of the branching neurons passed into the most caudal part of the medulla oblongata, and about half of these collaterals reached the level of the rostral part of the inferior olive. In regard to the neurons located in the segments C5-C8, about 13% of those projecting to T2 or below distribute an ascending collateral restricted to C2-C4, while 29% of those had an ascending collateral to C1 or above.

Branching cortical neurons in cat which project to the colliculi and to the pons: a retrograde fluorescent double-labeling study.

The fluorescent double-labeling technique has been used to determine whether the corticopontine and the corticotectal fibers in the cat are derived from two different sets of neurons or whether they are derived from branching neurons which distribute collaterals to the pontine grey and the colliculi. After unilateral DY.2HCl injections in the pontine grey and FB injections in the ipsilateral colliculi, large numbers of FB-DY.2HCl double-labeled neurons were present in the cortex of the ipsilateral hemisphere. However, the labeled neurons in its rostral part may have represented pyramidal tract neurons which were labeled retrogradely because their fibers descended through the DY.2HCl injection area. Therefore, also DY.2HCl injections were made in the pyramid (i.e. caudal to the pons) and the cortical pyramidal tract area, containing the retrograde DY.2HCl-labeled neurons, was delineated. In the rest of the experiments only the DY.2HCl-labeled neurons in the caudal two thirds of the hemisphere (outside the pyramidal tract area) were taken into account because only these neurons could, with confidence, be regarded as corticopontine neurons. In some anterograde HRP transport experiments the trajectories of the corticotectal and the corticopontine fibers were visualized. On the basis of the findings the DY.2HCl injections in the pontine grey were placed such that they could not involve any of the corticotectal fibers passing from the cerebral peduncle to the colliculi. Thus artifactual double-labeling of cortical neurons was avoided. However, also under these circumstances many double-labeled neurons were present in the caudal two thirds of the hemisphere. This led to the conclusion that in the cat a large proportion of the corticopontine neurons in the caudal two thirds of the hemisphere represent branching neurons which also distribute collaterals to the colliculi. The parietal (anterior part of the lateral gyrus, middle and posterior suprasylvian gyri) and the cingulate areas together contained three quarters of all labeled corticopontine neurons outside the pyramidal tract area. In the parietal areas roughly 25% of them were double-labeled and in the cingulate area 14%. However, in the visual areas 18 and 19 a much larger percentage (30-60%) was double-labeled.(ABSTRACT TRUNCATED AT 400 WORDS)

Some aspects of the organization of the output of the motor cortex.

The precentral motor cortex in the macaque is defined here as that portion of the precentral motor-sensory areas which projects to the intermediate zone and motor neuronal cell groups in the spinal cord and their bulbar counterparts, i.e. the lateral reticular formation and motor nuclei of the lower brainstem. In this respect the precentral motor cortical areas differ from postcentral areas such that the descending projections from the latter are focused on the spinal dorsal horn and the spinal V complex. Differences in the distribution of the corticospinal fibres in different species are mentioned and differences in findings obtained by means of different tracing techniques are discussed. The projections from the precentral motor cortex to various brain-stem cell groups are also discussed and the areas of origin of these projections are delineated. The presence of branching neurons distributing collaterals to several of these areas is considered.

Collaterals of corticospinal and pyramidal fibres to the pontine grey demonstrated by a new application of the fluorescent fibre labelling technique.

Selective visualization of collaterals of corticospinal and pyramidal fibres to the pons in cat was obtained by retrograde transport of the fluorescent tracer fast blue (FB) through the stem fibres. Unilateral FB injections in the cervical cord and the pyramidal tract respectively produced soft blue fluorescent labelling of pyramidal fibres and of fibres and structures resembling 'terminals' in the pontine grey: contralateral to the spinal injections and ipsilateral to the pyramidal injections. These labelled elements were concluded to represent collaterals of corticospinal and pyramidal fibres because (a) their distribution corresponded to that of the pericruciate corticopontine fibres, (b) their labelling was prevented when the FB injections were preceded by a transection of either the cerebral peduncle or the pyramidal tract which lesions also prevented the FB labelling of the distal parts of the transected axons. Similar findings were obtained when using wheat germ agglutinin-horseradish peroxidase. In other experiments FB-labelling of pyramidal collaterals was combined with retrograde labelling of pontine neurones projecting to the contralateral anterior lobe of the cerebellum using diamidino yellow dihydrochloride as the second tracer. The distributions of the retrogradely labelled neurones and of the pyramidal collaterals in the pontine grey showed an almost complete overlap indicating that these collaterals mainly establish connections with the cerebellar anterior lobe.

The involvement of monkey premotor cortex neurones in preparation of visually cued arm movements.

Some neurones in macaque postarcuate premotor area modulate their firing frequency in relation to motor tasks which require visual information. We previously reported that a large proportion of these neurones modulate during execution of a detour reaching task in which the movement phase was separated in time from the phase in which the monkey received a visual cue for the movement required to retrieve a food reward. A large proportion of task-related neurones (75%) modulated during this 'visual' phase, in which no task-related movements were made. This modulation was related to the position of the food reward, which served as the visual cue. Most of these neurones were located in cortical area 6, close to the arcuate curvature and its spur, but also more caudally in area 4 and rostrally in area 8. In the present chronic recording experiments in monkeys, several variations of the original task were used in order to test whether the 'visual'-related neuronal modulation could be involved in preparation of the upcoming movement. This modulation is unlikely to be related to any eye or arm movements occurring during the visual phase or to changes in environmental illumination. Neither can it be related to the presence of the visual cue in a particular part of the visual field, since the pattern of neuronal modulation was similar when a cue with a fixed position was used. This modulation was, however, contingent upon the occurrence of food retrieval during the subsequent 'movement phase', since it was abolished or diminished during presentation of a 'food-reward' which the monkey did not retrieve. For several neurones, modulation pattern during the visual phase depended on whether the food reward was to be retrieved with a gross hand movement or with relatively independent finger movements. It is likely, therefore, that neurones in the postarcuate premotor cortex are involved in preparation for arm movements with the help of visual cues. The results are discussed in view of corticocortical pathways which might be involved in transmission of visual information from visual areas through parietal association areas and premotor cortex to the primary motor cortex.

Differential corticospinal projections in the cat. An autoradiographic tracing study.

An autoradiographic study of the corticospinal projections from different parts of the cat sensorimotor cortex produced the following findings. The lateral part of area 4 projects contralaterally to the lateral intermediate zone of the cervical enlargement only. The intermediate part of area 4 projects throughout the spinal cord, contralaterally to the lateral part of the intermediate zone and bilaterally to its ventromedial part. The lateral and medial part of area 3 project contralaterally to the cervical and lumbosacral dorsal horn (including laminae I and II), respectively.

Large layer VI cells in macaque striate cortex (Meynert cells) project to both superior colliculus and prestriate visual area V5.

Layer VI of macaque striate cortex contains a number of large solitary neurones called Meynert cells. It has been shown earlier that these Meynert cells project to the posterior bank of the superior temporal sulcus (area V5), but it has also been shown that they project to the superior colliculus. In retrograde fluorescent double-labelling experiments, it was found that Meynert cells represent a class of neurones which distribute divergent axon collaterals to the posterior bank of the superior temporal sulcus and to the superior colliculus, i.e. to a distant cortical and a subcortical structure. This feature appears to be unique among projecting neurones in monkey visual cortex.

A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing.

In the present article a method is described which allows the delineation of the projections of a single neuron as well as the identification of one or more of its chemical components. The technique is a combination of retrograde tracing and fluorescent dyes based on the work of Kuypers and collaborators and indirect immunofluorescence histochemistry as originally described by Coons and collaborators. The crucial parameters including the selection of the dyes, the injection technique and tissue processing as well as the appropriate immunohistochemical fluorescent markers and filter combinations are discussed. The method of choice involves the use of the retrogradely transported dyes Fast Blue, True Blue or Propidium Iodide, and in addition, for double labeling experiments, Diamidino Yellow or Primuline. They are combined with FITC (Propidium Iodide) or TRITC (Fast Blue, True Blue, Diamidino Yellow, Primuline) as immunofluorescence markers.

Retrograde double labeling of neurons: the combined use of horseradish peroxidase and diamidino yellow dihydrochloride (DY X 2HCl) compared with true blue and DY X 2HCl in rat descending brainstem pathways.

Three series of double-labeling experiments were carried out in a study of the collateralization of brainstem nuclei which project to the spinal cord in the rat. The fluorescent tracer Diamidino Yellow Dihydrochloride (DY X 2HCl) was injected in one half of the spinal gray and white matter at T7-T8 or T13-L1. Subsequently, either True Blue (TB), wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or free HRP were injected ipsilaterally in the gray matter at C5-C8. The distributions of single and double retrogradely labeled neurons were studied in the following cell groups: red nucleus, interstitial nucleus of Cajal, ventrolateral pontine tegmentum, nuclei coeruleus and subcoeruleus and nuclei raphe magnus and raphe pallidus including the adjoining ventral reticular formation. The numbers of TB- or HRP-labeled neurons present in those nuclei were counted and the percentages of double-labeled neurons were calculated when TB or HRP had been used in combination with DY X 2HCl. The results indicate: The HRP-TMB reaction product and the DY X 2HCl fluorescence can be visualized simultaneously in retrogradely single- or double-labeled neurons. The distributions of single- and double-labeled neurons in the various brainstem nuclei were entirely comparable when using TB with DY X 2HCl or HRP with DY X 2HCl. The percentages of double-labeled neurons obtained with HRP and DY X 2HCl were consistent over a series of cases, and were comparable to those obtained with TB and DY X 2HCl in several structures. However, in the red nucleus slightly lower percentages of double-labeled neurons were obtained using HRP and DY X 2HCl as compared with the percentages obtained using TB and DY X 2HCl.

Collateralization of brainstem pathways in the spinal ventral horn in rat as demonstrated with the retrograde fluorescent double-labeling technique.

The collateralization of brainstem pathways to the spinal ventral horn was studied in rat by means of injections of True Blue (TB) and Diamidino Yellow Dihydrochloride (DY .2HCl) at different levels in the spinal cord. TB (or DY .2HCl) was injected in the cervical gray and DY .2HCl (or TB) was injected ipsilaterally either at mid-thoracic or at lumbar levels. The retrogradely single- and double-labeled neurons were studied in the interstitial nucleus of Cajal, the lateral vestibular nucleus of Deiters, the nucleus (sub) coeruleus and the nucleus raphe pallidus, including the adjoining ventral medullary reticular formation. In all those brainstem nuclei many double-labeled neurons were present after both mid-thoracic and lumbar injections. This indicates that these brainstem spinal pathways to the ventral horn probably give off many collaterals along their trajectory in the spinal cord.

Cortical afferents and efferents of monkey postarcuate area: an anatomical and electrophysiological study.

A study has been made of the corticocortical efferent and afferent connections of the posterior bank of the arcuate sulcus in the macaque monkey. The distribution of efferent projections to the primary motor cortex (MI) was studied by injecting three different fluorescent retrograde tracers into separate regions of MI. The resultant labeling showed a discrete and topographically organized projection: neurons lying below the inferior limb of the arcuate sulcus project into the MI face area, while neurons located in the posterior bank of the inferior limb of the arcuate sulcus and in the arcuate spur region project into the MI hand area. These findings were confirmed electrophysiologically by demonstrating that postarcuate neurons could only be activated antidromically by stimulation within restricted regions of MI. HRP injections within postarcuate cortex indicated that afferents to this region arise from a number of cortical areas. However, the largest numbers of labeled neurons were found in the posterior parietal cortex (area 7b; PF) and in the secondary somatosensory region (SII). Neurons in both 7b (PF) and SII could be antidromically activated by postarcuate stimulation. It was further shown that stimulation of area 7b (PF) gives rise to short-latency synaptic responses in postarcuate neurons, including some neurons with identified projections to MI. The results are discussed in relation to the possible function of the postarcuate region of the premotor cortex in the sensory guidance of movement.

Distribution of corticospinal neurons with collaterals to lower brain stem reticular formation in cat.

The fluorescent retrograde double-labeling technique has been used to determine whether corticospinal neurons in the cat sensorimotor cortex distribute collaterals to the lower brain stem reticular formation. In this study the fluorescent tracers Nuclear Yellow and Diamidino Yellow 2HCl were used in combination with Fast Blue. One tracer was injected unilaterally in the spinal cord and the other was injected ipsilaterally in the bulbar medial reticular formation. The distribution of the retrogradely labeled neurons was studied in the contralateral hemisphere. In the sensorimotor cortex a large population of neurons was found which were labeled from the spinal cord and were double-labeled from the brain stem. These branching neurons were concentrated in the rostromedial part of the area 4 and the adjoining lateral part of area 6. In this region the percentages of corticospinal neurons which were double-labeled from the brain stem ranged from 5% laterally to 30% medially. In two cats it was demonstrated by means of the anterograde transport of HRP that the corticobulbar fibers from this region which must include the corticospinal collaterals are distributed to the reticular formation of the lower brain stem. In view of the fact that the double-labeled neurons are concentrated in the anterior part of the motor cortex, those branching neurons are in all likelihood involved in the control of neck, back and shoulder movements. This control is probably exerted by way of two routes i.e. by way of the direct corticospinal connections to spinal interneurons, and by way of the indirect cortico-reticulospinal connections established by the cortical fibers to the bulbar reticular formation. The present findings suggest that this dual control may be exerted by one and the same cell.