Basal forebrain and hypothalamic connection to frontal and parietal cortex in the Rhesus monkey.

Horseradish peroxidase was injected in different parts of the frontal and parietal cortex in 17 rhesus monkeys. In all cases the enzyme was transported retrogradely to neurons in the substantia innominata and hypothalamus as well as in the thalamus. These new findings demonstrate that these cortical areas receive direct afferent fibers from limbic basal forebrain areas concerned with emotion and motivation.

Premotor cortical ablations in monkeys: contralateral changes in visually guided reaching behavior.

In rhesus monkeys (Macaca mulatta), ablation of the premotor and supplementary motor areas and the adjoining rostral half of the precentral gyrus impairs the capacity of the contralateral arm to reach around a transparent obstacle to a visible food reward, and results in a tendency of this arm to reach around a transparent obstacle to a visible food reward, and results in a tendency of this arm to reach straight to where the food is visible. This may reflect a disinhibition of brainstem pathways which steer the arm and hand straight ot a visual target.

Splitbrain monkeys: cerebral control of ipsilateral and contralateral arm, hand, and finger movements.

The connections of the descending pathways in the monkey suggest that each hemisphere controls independent arm, hand, and finger movements contralaterally, but mainly arm movements ipsilaterally. This difference could be observed in splitbrain monkeys executing a visuomotor task with one eye covered, provided tactile guidance of the movements was largely prevented.

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.

Fluorescent retrograde double labeling: axonal branching in the ascending raphe and nigral projections.

Red fluorescent Evans blue and blue fluorescent DAPI-primuline were injected into the anterior-medial and lateral-caudal forebrains, respectively, of the same rats. Separate clusters of cells labeled by retrograde transport were observed in the substantia nigra, while in the dorsal raphe many cells were double-labeled. Thus, single raphe cells send divergent axon collaterals to widespread forebrain areas.

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.

The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods.

Experiments using two retrogradely transported fluorescent dyes (bisbenzimide-true blue, and Evans blue-granular blue) were performed in order to determine whether the same or different populations of neurons of the paraventricular nucleus of the hypothalamus (PVH) project to the pituitary gland, dorsal vagal complex, and spinal cord in the rat. The results suggest that cells projecting to the pituitary gland are concentrated in the magnocellular core of the nucleus, while the descending connections arise primarily from the surrounding parvocellular division. The occurrence of neurons double-labeled with both dyes further indicate that at lease 10-15% of the labeled cells in the parvocellular division send divergent axon collaterals to the dorsal vagal complex and to the spinal cord. Cell counts suggest that at least 1,500 cells in the PVH project to the medulla and/or spinal cord. These results, combined with a cytoarchitectonic analysis, show that the PVH consists of eight distinct subdivisions, three magnocellular and five parvocellular. The lateral hypothalamic area and zona incerta also contain a large number of cells projecting to the dorsomedial medulla and spinal cord; approximately 15% of such cells are the double-labeled following injections of separate tracers into these two regions of the same animal.

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)

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.

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.

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.

Quantitative EM study of projection terminal in the rats AV thalamic nucleus. Autoradiographic and degeneration techniques compared.

An attempt was made to determine whether the labeled amino acid tracing technique combined with electron microscope (EM) autoradiography can be used to identify reliably the terminals of different to a cell group in the brain, using the rat's antero-ventral thalamic (AVTh) nucleus as a model. For this purpose in one group of rats [3H]leucine was injected in cingulate cortex, hippocampus and mammilary bodies, respectively, and in another group lesions were made in these structures. With one month autoradiographic exposure time it could be demonstrated that after cortical and hippocampal injections the radioactivity was transported to small terminals on distal dendrites, while after mammillary body injections it was transported to large terminals on proximal dendrites. After 4 months exposure time altogether 12% of the small terminals and 80% of the large ones had been labelled; A comparison with the degeneration findings showed that the EM autoradiography is in many respects more efficient in identifying the various terminals than the EM degeneration technique.

Morphology of rats AV thalamic nucleus in light and electron microscopy.

The structure of the rat's antero-ventral thalamic (AVTh) nucleus has been investigated in order to provide background information for the accompanying study in which an attempt was made to identify the synaptic terminals of the different afferent fiber systems to this nucleus by means of both EM autoradiography and the EM degeneration techniques. Nissl stained sections showed that the rat's AVTh nucleus contains mainly relatively light staining neurons which in Golgi material were found to possess tufted dendrites. In EM material three types of synaptic terminals were found which showed a topical distribution over the neuronal surface. Soma and stem dendrites carry a limited number of terminal with symmetrical synapses and flattened vesiclesmproximal dendrites carry mainly large asymmetrical synaptic terminals while distal dendrites are crowded with small asymmetrical synaptic terminals.

Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels. A HRP study in cat and rhesus monkey.

In the spinal cord of cat and rhesus monkey the cells of origin of long and short propriospinal fibers and those of the spinal fibers ascending to supraspinal levels were identified by means of retrograde HRP labeling after large unilateral HRP-injections, i.e. in the spinal white and grey matter at different levels, in the pons and in the dorsal column nuclei. The findings indicate the existence of the following arrangement. Long ascending supraspinal fibers arise mainly from neurons in the dorsal grey (laminae I-IV and the medial parts of laminae V and VI) as well as from neurons in the medial part of the ventral grey (lamina VIII), in Clarke's column and in the spinal border cell area. Some neurons in the dorsal grey projects to the dorsal column nuclei, which in turn distribute fibers back to the spinal cord. Long propriospinal fibers mainly derived from neurons in the medial part of the ventral grey (lamina VIII), while short propriospinal fibers are characteristically derived from neurons in the intermediate zone (lateral halves of laminae V and VI and lamina VII). The neurons located laterally in laminae V-VII distribute fibers mainly ipsilaterally, while those located medially in lamina VII distribute them to some degree bilaterally. The findings in cats with transections of either the dorsal or the ventral halves of the spinal white matter (both above and below the injected segment), show that the fibers from the dorsal grey and the lateral parts of laminae V-VII travel mainly through the dorsal half of the white matter, while those from the medial part of lamina VII and from lamina VIII travel mainly through the ventral half.

Retrograde neuronal labeling of cells of origin of descending brainstem pathways in rat using SITS as a retrograde tracer.

Recently, the retrograde tracer SITS has been introduced, a cytoplasmic label, which seems to be taken up by terminals only. In order to test the transport of SITS from terminals of brainstem pathways to he spinal cord SITS was injected in different rats at different spinal levels. It could be concluded that SITS is taken up primarily from axon terminals.

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.

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.

A search for corticospinal collaterals to thalamus and mesencephalon by means of multiple retrograde fluorescent tracers in cat and rat.

An attempt has been made to determine anatomically whether in rat and cat cortical projections to ventrolateral nucleus of thalamus and to mesencephalon are in part composed of corticospinal collaterals. For this purpose two different fluorescent tracers were injected: one in the spinal cord and the other contralaterally in the lateral thalamus and in the mesencephalon respectively. In these experiments Fast Blue and True Blue were used in combination with Nuclear Yellow. Evans Blue was used in combination with Granular Blue. After injections of the tracers into the thalamus and spinal cord two different populations of single retrogradely labeled cortical neurons were found, while after injections in mesencephalon and spinal cord double-labeled cortical neurons occurred. This has lead to the conclusion that in cat and rat corticospinal neurons do not distribute collaterals to specific thalamic nuclei, but do distribute collaterals to mesencephalon. Moreover, the preferential distribution of the double-labeled corticospinal neurons in cat suggest that the corticospinal neurons distributing collaterals to the mesencephalon in part are concentrated in those cortical areas which subserve the steering of movements of the head, neck and trunk.

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.

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.