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We examined the location and spatial distribution of prefrontal cortical (PF) cells projecting to the supplementary eye field (SEF) and presupplementary motor area (pre-SMA) using a double retrograde-labeling technique in monkeys (Macaca fuscata). The SEF and pre-SMA were physiologically identified based on the findings of intracortical microstimulation and single cell recordings. Two fluorescent tracers, diamidino yellow and fast blue, were injected into the SEF and pre-SMA of each monkey. Retrogradely labeled cells in the PF were plotted with an automated plotting system. The cells projecting to the SEF and pre-SMA were mainly distributed in the upper and lower banks of the principal sulcus (area 46), with little overlap. Cells projecting to the SEF, but not to the pre-SMA, were observed in areas 8a, 8b, 9, 12, and 45. These findings suggest that the SEF and pre-SMA receive different sets of information from the PF cells.

Anesthetized cats were injected with 2% Fast Blue and 0.5% Nuclear Yellow into the intermediate and deep layers of the left and right superior colliculus, respectively. In the right caudal part of the cerebellar fastigial nucleus (cFN), double-labeling was found in 38.5% of the neurons labeled with Fast Blue, and in 11.5% of the neurons labeled with Nuclear Yellow. In the left cFN, 52.2% of the neurons labeled with Fast Blue and 11.0% of the neurons labeled with Nuclear Yellow were double-labeled. The results suggest a role of bifurcating fastigial fibers in cerebellar visual control.

Summary The aim of this study was to investigate the chemical coding of mammary gland-projecting SChG neurons using double-labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland-projecting neurons were found in Th1-3, Th9-14 and L1-4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1-4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary-projecting neurons contained immunoreactivities to TH (96.97%) and/or DsH (95.92%). Many TH/DsH-positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.

A double-labeling fluorescence microscopic study was performed on the mesencephalic and thalamic distribution of fastigial efferents. Anesthetized cats were injected with 2% fast blue into the suprageniculate nucleus and with 0.5% nuclear yellow into the superior colliculus. Analysis of serial sections through the cerebellar fastigial nucleus revealed that 25% of the neurons projecting to the superior colliculus and 10% of those projecting to the thalamus were double labeled. The results suggest that bifurcating fastigial fibers to the mesencephalon and to the visual thalamus may play a role in cerebellar visual control.

The distribution of neurons innervating the uterus of the pig was studied with the use of fluorescent retrograde tracer Fast Blue. Tracer injections were made into the uterine cervix, pericervical, middle and perioviductal part of the right uterine horn. After tracer injection into the uterine cervix tracer-positive neurons were found bilaterally in the inferior mesenteric ganglia, paracervical ganglia, paravertebral ganglia L1–S3 and dorsal root ganglia Th10–L4 and S2–S3. After tracer injection into the pericervical part of the right uterine horn the distribution of tracer-positive neurons resembled the one described earlier, except the tracer-positive neurons were absent in the left dorsal root ganglia. After tracer injection into the middle part of the right uterine horn tracer-positive neurons were found in the ipsilateral inferior mesenteric ganglion, bilaterally in the paracervical ganglia and in the ipsilateral paravertebral ganglia (L2–L6). Injection of the tracer into the perioviductal part of the right uterine horn revealed tracer-positive neurons bilaterally in the inferior mesenteric ganglia and paracervical ganglia and single cells in the ipsilateral paravertebral ganglia.

The retrograde axonal transport method was used to compare the topography and organization of the visual zone of the claustrum in rat, guinea pig, rabbit and cat. First, massive Fluoro-Gold injections were placed into the primary visual cortex and the secondary areas. Experiments showed differences in the location of the visual zone among the animals under study. In rat, the visual zone occupied the posteroventral part of the claustrum and spread to its anterior pole. In guinea pig, neurons projecting to the visual cortex were located dorsally in the posterior half of the claustrum. In rabbit, similarly to the rat, they were localized in the posteroventral part; however, they did not reach the anterior pole. In cat, neurons that project to the visual cortex were concentrated dorsally in the posterior fourth of the claustrum. In double-injection experiments, Fast Blue and Diamidino Yellow were placed into the primary and secondary visual areas in various combinations. The experiments showed that in the rat and the rabbit claustral neurons project to primary visual cortex (area 17) as well as to both secondary visual areas (areas 18a and b). Populations of neurons sending axons to the primary and secondary areas showed full overlap. The presence of double-labeled neurons indicates that some claustral neurons project both to the primary and secondary fields. In cat, neurons that project to the primary visual cortex appear to be clearly separated from those connected with the secondary visual area, as no double-labeled neurons were found. In all studied species, the double injections placed into the visual and primary somatosensory cortex did not result in any double-labeling neurons. Our results indicate that the location of the visual zone in the posterior part of the claustrum is a phylogenetically stable feature, whereas its dorsoventral shift as well as the extent toward the anterior pole is related to the particular species. The overlap of neurons projecting to the primary and secondary visual areas in the rat and rabbit as well as the separation of both projections in cat appear to reflect the higher degree of complexity of the visual system in the latter.

Methods of retrograde axonal transport were employed to evaluate the topography and overlap of claustroneocortical connections in the rat. Fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) were injected simultaneously in various combinations into the motor, somatosensory, auditory and visual cortical areas. Experiments showed that claustroneocortical projections are organized in two main cortico-related zones: sensorimotor and visuoauditory. The sensorimotor zone occupies the anterodorsal part whereas the visuoauditory occupies the posteroventral part of the claustrum. Between these two main zones only a scanty overlap was observed. In the sensorimotor zone a large overlap between neurons projecting to the motor and somatosensory cortical areas exists. The visuoauditory zone is characterized by a full overlap of neuronal populations projecting to the visual and auditory areas.

To investigate if Ca2+ currents of acutely isolated superior cervical ganglion (SCG) neurons are altered after dye labeling or ultraviolet (UV) exposure, SCG neurons were labeled by multiple injections of 2% fast blue (FB) (5-microliters total) into the submaxillary gland. Ca2+ currents of both labeled and unlabeled neurons were significantly depressed by 2-min exposure to UV, compared to labeled and unlabeled neurons not exposed to UV. The I-V curve of labeled neurons was shifted hyperpolarized after UV exposure. Tail-current activation curves of both labeled and unlabeled neurons were fitted to a double Boltzmann equation. In labeled neurons, 2-min exposure to UV produced a significant reduction of the amplitude of the more depolarized component as well as a hyperpolarizing shift of the half activation potential of both components of the activation curve. However, the short time (10 s) required to identify a target-specific neuron had no significant effect on the biophysical properties of Ca2+ currents of FB-labeled SCG neurons. Thus, use of this label in combination with the patch-clamp technique comprises a powerful approach to study membrane currents of target-specific neurons.

Despite the contribution of nodose ganglia neurons to the innervation of the heart being the subject of several studies, specific neuronal subpopulations innervating the four different chambers of the heart have not been distinguished. In our study, the application of Fast Blue-loaded patch to the epicardial surface of different chambers of the rat heart (the right or left atrium or the right or left ventricle) resulted in labeling of discrete populations of immunohistochemically diverse neurons. About one half (55%) of these neurons showed immunoreactivity for the 200-kDa neurofilament protein (marker of myelinated neurons), with a higher proportion of positive staining among neurons projecting to the left than to the right ventricle. Isolectin B4 immunoreactivity (characteristic for a subset of nonmyelinated non-peptidergic neurons) was more abundant among neurons projecting to the right side of the heart (right atria and right ventricles) compared to the left side (23% vs. 16%). Calretinin immunoreactivity (possible marker of mechanosensitive neurons) was significantly higher among neurons projecting to the ventricles than among those projecting to atria (36% vs. 11%). These findings reveal that chambers of the rat heart are innervated with immunohistochemically different subpopulations of neurons from the nodose ganglia.

To provide endpoints for developmental studies of rubrospinal plasticity in the North American opossum, we have attempted to determine the degree to which rubrospinal neurons survive axotomy in the adult animal. Bilateral or unilateral injections of the long-lasting fluorescent marker fast blue were made into the T-10 or the T-11 segment of the spinal cord to label rubrospinal neurons, and 7 days later, the rubrospinal tract was cut unilaterally four segments rostral to the injection(s). In cases with unilateral injections, the lesion was made ipsilateral to the injection. The animals were allowed to survive for 30–60 days before being sacrificed and perfused so that sections through the red nuclei could be examined for labeled neurons. The results show that most axotomized neurons survived the lesion, suggesting that lesion-dependent cell death is not a major factor in the failure of the rubrospinal tract to regenerate in the adult animal.

Summary The organization of projections from the inferior olive (IO) to the uvula of the vermis and the paramedian lobule (PML) of the same hemisphere in the rabbit cerebellar cortex was studied using retrograde tracers: Diamidino Yellow (DY) and Fast Blue (FB) dyes were injected unilaterally into these two targets in the same animals. The distribution patterns of DY- and FB-labeled neurons consistently detected in spatially separate regions of the contralateral IO subnuclei show that the IO-uvular and IO-PML projections display a distinct topography. The absence of double labeling indicates that no neurons project divergently to both the uvula and PML. The lateral and medial regions of the medial accessory olive and of the ventral lamina of the caudal principal olive project to the uvula and PML, respectively. Several neuronal groups at more rostral levels of the principal olive send fibers solely to the PML. The medial and lateral regions of the β nucleus and the dorsomedial cell column supply the uvula and PML, respectively. Neurons in the dorsal cap and the ventrolateral outgrowth also tend to display this type of localization. Moreover, the caudal part of the dorsal accessory olive and the dorsal lamina of the principal olive project exclusively to the PML. The most caudal and rostral aspects of the IO do not participate in projection. Topographical relationships in the IO-uvular and IO-PML projections have not been previously demonstrated in the rabbit, and they are discussed in the context of inter-animal differences and cerebellar zonal organization. The possible functional significance of the connections is also discussed.

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12-14 cm oral to the ileo-caecal junction. Over 80% of the descending and ascending DiI-labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12-14 cm) FB-labeled MP neurons. Nitric oxide synthase (NOS)-IR, peripheral choline acetyltransferase (pChAT)-IR, and substance P (SP)-IR occurred in both descending and ascending myenteric neurons. NOS-IR was in approximately 60% of FB-labeled descending and ascending neurons, whereas those expressing pChAT-IR were 67 +/- 15% and 60 +/- 14%, respectively. Descending neurons expressing SP-IR were 48 +/- 15% and ascending were 56 +/- 12%. NOS-IR and pChAT-IR, and SP-IR and pChAT-IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP-IR neurons were also pChAT-IR (98 +/- 3%) and NOS-IR (99 +/- 2 NOS(+)/SP(+)/pChAT(-)). Many FB-labeled descending neurons showed both NOS- and pChAT-IR. Descending neurons may represent inhibitory motor neurons (NOS(+)/SP(+)/pChAT(-)) and two classes of interneurons (pChAT(+)/NOS(-), and pChAT(+)/NOS(+)/SP(+)). In ascending pathways, most neurons are pChAT(+)/NOS(+)/SP(+). Thus, in sheep, ascending interneurons and ascending excitatory motor neurons both have the same phenotype, and other markers are needed to distinguish them.

The use of frozen sectioning is a convenient and rapid means of observing the results obtained using fluorescent retrograde tracers. Quantitation of these results using the biased stereological methods currently available can be associated with large errors. A recently developed stereological tool, the optical dissector, provides unbiased and efficient results, however, the requirements for its use in frozen sections has not previously been established. In this study, a comparison was made of neuron numbers, estimated using the optical dissector method, in the motoneuron pool retrogradely labelled from the rat sciatic nerve with either Fast Blue or Tetramethylrhodamine dextran (fluoro-ruby) in methacrylate embedded and frozen spinal cord specimens. Despite over 50% shrinkage in the frozen sections, compared with virtually no shrinkage in the methacrylate sections, no significant difference in labelled motoneuron numbers was observed, provided this shrinkage was taken into account. Correction for section shrinkage is therefore essential in order to use the optical dissector with confidence to count fluorescent labelled neurons in frozen tissue.

Programmed cell death has been demonstrated in several specific neuronal populations as a mechanism for modulating the population size following differentiation, but its applicability to all neuronal types is unclear. Evidence for programmed cell death in some populations such as the numerous spinal interneurons has been lacking. We have studied the incidence of apoptosis in the rat spinal cord with three different methods and found a previously undocumented wave of apoptosis occurring in spinal grey matter shortly after birth. The apoptotic morphology was confirmed ultrastructurally. Dying cells were identified as neurons by immunocytochemical labelling for neuronal markers and had an anatomical distribution which indicated that most of the apoptotic cells were interneurons not motoneurons. This wave of apoptosis has the characteristics of a discrete developmental process and occurs later than that of either ventral horn motoneurons or dorsal root ganglion cells, to which most spinal interneurons are connected. These findings indicate that interneurons do undergo programmed cell death, and we suggest that this occurs in response to the earlier reduction in size of their main synaptic targets.

Although there have been reports of sparse projections from temporal areas TE, TF, and even TH to area V1, it is generally believed that cortical afferents to V1 originate exclusively from prestriate areas. Injections of anterograde tracers in anterior occipital and temporal areas, however, consistently produce labeled terminals in area V1. In order to confirm these results and display the full range of foci projecting to V1, we injected V1 in two monkeys with the retrograde tracer fast blue. Feedback connections were found, as expected, from several prestriate areas (V2, V3, V4, and MT). These originate from neurons in layers 3A and 6. Connections were also found from several more distal regions, namely, areas TEO, TE, TF, TH, and from cortex in the occipitotemporal and superior temporal (STS) sulci. Filled neurons occurred in two small foci in the caudal intraparietal sulcus. These more distal feedback connections tend to originate only from layer 6. An additional injection of the retrograde tracer diamidino yellow in area V2 of one animal revealed a similarly widespread network of feedback connections. In some areas (in the STS and in TEO), 10-15% of fluorescent neurons were double-labeled. These results indicate that feedback connections to early visual cortex derive from a widespread network of areas, including limbic-associated cortices. These connectional patterns testify to the massive recursiveness of anatomical pathways. As there are no reports of projections from V1 to anterior temporal cortices, our results also indicate that some cortical feedback connections may not be strictly reciprocal.

The effect of prenatal X-irradiation on the ontogenesis of corticospinal tract (CST) neurons was examined in rats using retrograde labeling with Fast blue and intracellular Lucifer yellow staining. In prenatally irradiated rats, the cortical laminar architecture of the CST neurons was confused and many cells demonstrated migratory disturbances. Migratory-disordered CST neurons at deeper cortical levels resembled pyramidal cells, but their apical dendrites were oriented in various directions and the development of their dendrites was poor. Migratory-disordered CST neurons near the ependymal layer demonstrated round somata and many thin dendrites with spokewise radiation, suggesting a maturation disturbance. These results suggested that prenatal X-irradiation impeded the migration and maturation of CST neurons. These findings may form the basis for analyzing the mechanisms of radiation-induced mental retardation and behavioral changes.

Neurons in the medial tegmental field project directly to spinal somatic motoneurons and to cranial motoneuron pools such as the hypoglossal nucleus. The axons of these neurons may be highly collateralized, projecting to multiple levels of the spinal cord and to many diverse regions at different levels of the neuraxis. We employed a double fluorescent retrograde tracer technique to examine whether medial tegmental neurons that project to the spinal cord also project to the hypoglossal nucleus. Injections of Diamidino Yellow into the hypoglossal nucleus and Fast Blue into the spinal cord produced large numbers of double labeled neurons in the medial tegmental field, particularly in the caudal raphe nuclei and adjacent ventromedial reticular formation. In these structures the number of neurons projecting to both the hypoglossal nucleus and the spinal cord was equivalent to the number of neurons projecting to multiple levels of the spinal cord observed in control animals. Fewer neurons projecting to both the hypoglossal nucleus and the spinal cord were observed in several other nuclei and subregions of the medial tegmental field, while almost no such neurons were observed in the lateral tegmental field or other pontomedullary structures. These results demonstrate that neurons of the caudal raphe nuclei and adjacent ventromedial reticular formation project to both the spinal cord and the hypoglossal nucleus, and support the concept that the diffuse projections to motoneuron pools from the medial tegmental field globally modulate both spinal and cranial somatic motoneuron excitability.

The organization of corticocortical connections in the representation of the forepaw in cat primary somatosensory cortex (SI) was studied following injections of various tracers into different cortical cytoarchitectonic areas. Small injections of horseradish peroxidase, wheat germ agglutinin-conjugated HRP, Phaseolus vulgaris leukoagglutinin, or fast blue were placed into the representation of the forepaw in areas 3b, 1, or 2. The positions of labeled neurons in SI and the surrounding cortical areas were plotted on flattened surface reconstructions to determine the organization of the corticocortical connections within SI. A strong, reciprocal projection linked the two forepaw representations which have been described in area 3b and the part of area 2 which lies in the anterior bank of the lateral ansate sulcus (see Iwamura and Tanaka 1978a, b). Dense projections also linked these areas with SII, as previously reported (Burton and Kopf 1984a). Additional projections to area 3b arose primarily from areas 3a and 1. Projections to area 2 were more widespread than those to area 3b, and arose from all other areas of SI as well as from areas 4 and 5a. All injections into SI tended to label groups of neurons which lay in mediolateral strips. Corticocortical projection neurons which were most heavily labeled by SI injections were pyramidal cells in layer III. Additional projections from area 2 to 3b, area 5a to 2, and SII to areas 2 and 3b arose from layer VI as well. Although neurons of layers III and VI were always the most densely labeled, large injections into SI labeled neurons in layers II and V as well.

We have combined injections of Fast Blue with lesions of the rubrospinal tract rostral and contralateral to them to determine if an ipsilateral rubrospinal projection exists in adult or developing opossums and, if so, to characterize the neurons giving rise to it. Although the results indicate that some rubral neurons project ipsilaterally, they are very few in number. Using quantitative and image analysis techniques, we have shown that 0.6% of the rubral neurons that project to the lumbar cord in adult opossums do so ipsilaterally and that such neurons are comparable in location and size to those that project contralaterally. Similar results were obtained in developing opossums. Our results are discussed in light of rubrospinal development and ongoing experiments related to rubrospinal plasticity.

Intracochlear bilateral injection of the fluorescent retrograde markers fast blue and diamidino yellow was used to identify the brainstem location of the olivocochlear efferents in the domestic chicken. The overall distribution pattern of neurones was similar to that of recent studies using horseradish peroxidase as the retrograde tracer, although the number of labelled neurones was significantly greater than previously reported. The average number of labelled neurones projecting to any one cochlea was 242, with roughly equal numbers located ipsilaterally or contralaterally. After bilateral injection of the two tracers, no double-labelled neurones were detected.

The distributions of thalamic neurons projecting to the motor cortex and cortical area 3a were studied in cat by means of the retrograde double-labeling technique using Nuclear Yellow (NY) and Fast Blue (FB) as tracers. Following injection of NY and FB into the motor cortex and area 3a respectively, the NY-labeled neurons were found to be mainly located in ventrolateral (VL) nucleus and FB-labeled neurons in ventro-posterolateral nucleus (VPL). However, these two kinds of neurons were intermingled with each other in the border area between VL and VPL. A small number of neurons were double-labeled by both NY and FB. They were also distributed in the border area. Some of them could often be found in centromedian and parafascicular nuclei.

The postnatal maturation of the projection from the lateral geniculate nucleus to the posteromedial lateral suprasylvian visual cortex (PMLS) was studied with injections of fluorescent dyes into the PMLS at various postnatal ages. Labeled neurons projecting to the PMLS were present in all laminae of the ipsilateral lateral geniculate on the day of birth. However, there was a conspicuous change in the distribution of labeled geniculo-PMLS neurons by 11 days of age: now very few labeled neurons were present in lamina A, indicating a loss of geniculo-PMLS connections. The loss of connections began at the peripheral margins of lamina A and proceeded through other laminae toward laminae C1-3. By adulthood, labeled geniculo-PMLS neurons were largely confined to laminae C1-3; they were never observed in lamina A or A1 and were rarely observed in lamina C. To determine whether the lateral geniculate neurons survived after their projections to PMLS were lost, injections of fast blue were made at 1 or 2 days postnatally and the animals were allowed long postinjection survival times. Labeled neurons were found in all lateral geniculate laminae, thereby indicating that for many neurons the loss of connections could be attributed to a loss of their axon collaterals rather than to the death of the neurons themselves. After injections of fast blue into the PMLS and diamidino yellow dihydrochloride into area 17 shortly after birth, many double-labeled neurons were present in all laminae, indicating that they have collaterals to both targets. Thus, the survival of many of the geniculo-PMLS neurons contributing to the transient geniculo-PMLS projection seems to be due to sustaining collateral projections to area 17 or other cortical targets.

When Fast blue is injected into the rabbit spinal cord it is retrogradely transported into nerve cell bodies located in the medial and the descending vestibular nuclei. Approximately 50% of the Fast blue-positive cells also contain glutamic acid decarboxylase-like immunoreactivity. These neurons presumably synthesize γ-aminobutyric acid (GABA) and the rabbit vestibulospinal pathway therefore contains a substantial inhibitory component.

The double-labeling technique based on the retrograde axonal transport of fluorescent tracers (Evans blue, EB; Fast blue, FB; Nuclear yellow, NY) was used in the cat in order to investigate the occurence of axonal branching in the periaqueductal gray (PAG) projections to some thalamic nuclei (n. ventralis postero-lateralis, VPL; n. ventralis postero-medialis, VPM; n. parafascicularis, Pf). In a first group of cats, FB and EB were injected, respectively, within the right and left VPM. In another two groups of cats, FB injections into Pf were combined with either EB or NY injections within VPL or VPM. Double-labeled neurons were found within the PAG only in the animals of the first group. The present results show that some PAG neurons project bilaterally to VPM by means of axons collaterals.

The vast majority of neurons of the rat medial terminal nucleus (MTN) project to the nucleus of the optic tract (NOT), but the MTN also projects to a lesser degree upon a number of other brainstem nuclei controlling optokinetic nystagmus. Because of the diversity of targets of the MTN, it is possible that individual neurons have branched axons that project to two or more brainstem nuclei. The possibility that axons of MTN-NOT neurons collateralize to innervate other MTN targets is examined in the rat with the fluorescent, double-labeling, retrograde tracer technique. Fluoro-Gold was injected into the NOT while Fast Blue was simultaneously injected into each of five other known targets of the MTN: the supraoculomotor-periaqueductal gray; the dorsal cap of the inferior olive; the visual tegmental relay zone; the dorsolateral nucleus of the basal pons; and the superior/lateral vestibular nuclei. Brainstem sections were processed for fluorescence microscopy and the MTN was examined for single- and double-labeled neurons. Results show that virtually all neurons of the MTN (>97.5%), together with neurons in the visual tegmental relay zone immediately surrounding the MTNd, are single-labeled in all paired injections involving the NOT and the other target nuclei. It was found that about 69% of MTN neurons project exclusively to the NOT, 5–10% project to each one of the other nuclei, and 3% of MTN neurons project to more than one target. Based upon cell counts from the fluorescent material, and previous analysis of Nissl-stained serial sections, the findings show that virtually all MTN neurons are projection neurons. It was concluded that the MTN is comprised of independent projection systems, possibly involved in different aspects of generating optokinetic nystagmus.

The efferent projections of the deep cerebellar nuclei which terminate bilaterally in the thalamus were investigated in the rat by means of the retrograde fluorescent double-labeling technique. After injection of True Blue or Fast Blue in the thalamus on one side and Nuclear Yellow on the other, the deep cerebellar nuclei on both sides contained many cells single-labeled from the contralateral thalamic injection and some cells double-labeled from both thalamic injections. Double-labeled cells were more numerous in the caudal part of the medial nucleus than in the other cerebellar nuclei. The present results indicate that some cerebellar cells give origin to bilateral thalamic projections by means of axon collaterals.

To study the retrograde labeling of intact and axotomized retinal ganglion cells (RGCs) over long periods of time, we applied the carbocyanine dye diI to the superior colliculi (SC) and dorsal lateral geniculate nuclei (dLGN) in adult albino rats and examined the retinas by fluorescence microscopy after different periods of survival. Retrogradely labeled RGCs, which were observed in the retinas as early as 3 days after application of the dye, gradually increased in density so that by 7 days more than 80% of the RGCs were labeled and by 30 days diI-labeled cell densities were similar to those observed after short applications of other tracers. Using short-term retrograde labeling with fast blue (FB) as an independent marker of RGCs, it was determined that these neurons remained labeled with diI for periods of up to 9 months without apparent leakage of the tracer to other retinal cells. In addition, diI labeling persisted in the somata of more than 80% of axotomized RGCs whose contact with the source of label had been interrupted for 3 months. Thus, we propose that retrogradely transported diI is a useful label for quantitative studies of neuronal populations, even after axotomy.

In this study we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections of subcoeruleus area neurons to both ‘medial preoptic area (MPA)’ and ‘cervical (C1–C3) spinal cord’. Following microinjections of fluorescent tracers (Fast blue (FB) and Diamidino yellow (DY)) within MPA and C1–C3, substantial numbers of FB and DY single-labeled neurons as well as FB-DY double-labeled branched neurons have been found within subcoeruleus area.

The distribution of visual callosal neurons was identified in tangential sections from flattened hemispheres of normal rats and animals unilaterally enucleated at various postnatal ages with the retrogradely transported fluorescent label, Fast Blue. Following enucleation on or after postnatal day 10, callosal neurons along the 17/18a border appear adult-like in their configuration. Enucleation prior to this age stabilizes callosal development in the hemisphere contralateral to the enucleated eye.

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.

The projections from the cat's superior colliculus to the extrageniculate visual thalamus were examined by the anterograde and retrograde transport of WGA-HRP. An acetylthiocholinesterase (ATChE) stain was employed to facilitate the differentiation of regions within the posterior thalamus. On the basis of the distribution of terminal label as well as the laminar origin of projection neurons, four pathways were delineated. Cells in the stratum griseum superficiale (primarily sublaminae II and III) innervate two regions within the nucleus lateralis posterior (LP): the medial zone, which stains darkly for ATChE, and a restricted portion of the lateral zone, adjacent to the pulvinar. Both of these pathways were found to be topographically organized. By using the fluorescent retrograde tracers, fast blue and rhodamine labeled microspheres, it was determined that the inputs to the medial and lateral zones of LP originate primarily from separate cell populations since very few neurons were found to be double-labeled. A third pathway originates principally from cells in the stratum opticum and terminates in an area just below the cholinesterase-rich region of the LP, designated as the ventral division of the LP. The fourth projection is primarily from the stratum griseum intermedium to the suprageniculate complex. Each of these four pathways arises from a population of neurons with heterogeneous morphological characteristics, and for the most part, each pathway comprises morphologically similar cells. These results suggest that visual information from the superior colliculus is conveyed to the extrageniculate thalamus via multiple pathways that may subserve diverse functions.

After injecting a solution of a fluorescent retrograde neuronal tracer (Fast blue, Diamidino compound 253/50) into the perilymphatic space of the frog labyrinth (Rana esculenta), labeled cells were found in the ventral and dorsal nuclei of the VIIIth nerve and in the nucleus reticularis medius. We consider these labeled cells to be the origin of the afferent innervation of the frog labyrinth. No evidence was found for the existence of a direct cerebello-labyrinthine connection.

The fluorescent dyes Nuclear yellow, a nuclear marker, and Fast blue, a cytoplasmic marker, were utilized in a double-labeling procedure. One or the other of these dyes was injected sequentially into the cervical spinal cords and ciliary ganglia of 6 cynomolgus monkeys. No double labeling of cells was noted in the anterior median nucleus. Edinger-Westphal nucleus, or nucleus of Perlia in the midbrain.

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

Two new fluorescent retrograde neuronal tracers are reported: Nuclear Yellow (Hoechst S 769121), which mainly labels the neuronal nucleus; and Fast Blue (diamidino compound 253/50), which mainly labels the neuronal cytoplasm. Both tracers appear to be transported effectively over long distances in rat and cat.

Thalamo-hyperstriatal projections were studied in the pigeon by retrograde thalamic labeling following bilateral injections of different combinations of fluorescent substances (Fast Blue, Evans Blue, Nuclear Yellow) into the hyperstriatum. The distribution of single and double-labeled neurons within the thalamic nuclei dorsolateralis anterior and superficialis parvocellularis indicated the presence of distinct functional sub-regions containing neuronal populations providing ascending projections which were either ipsilateral, contralateral or bilateral via collateral axon branching.