The relationship between MI and SMA afferents and cerebellar and pallidal efferents in the macaque monkey

The purpose of the present study was to determine the interrelationship between the thalamic afferents arising from the cerebellum (Cb) and the internal segment of the globus pallidus (GPi) with the neurons projecting to the primary motor cortex (MI) and to the supplementary motor area (SMA). We combined fluorescent retrograde tracers with a double anterograde labeling technique. Multiple injections of a combination of Diamidino Yellow and Fast Blue were made into either the MI or SMA hand/arm representation as determined by intracortical microstimulation. In the same animal, biotinylated dextran amine was injected into the GPi and horseradish peroxidase conjugated to wheat germ agglutinin was injected into the contralateral cerebellar nuclei. The results revealed that the cerebellar and pallidal thalamic territories are largely separate. The ventral anterior nucleus (VA) and the ventral lateral nucleus pars oralis (VLo) contained a greater density of pallidal labeling while a greater density of cerebellar label was observed more caudally in the ventral posterior lateral nucleus pars oralis (VPLo) as well as in nucleus X (X). Moreover, we observed that the greatest coincidence of retrograde cell labeling was within the pallidal thalamic territory following the SMA injections and within the cerebellar thalamic territory following the MI injections. However, interdigitating foci of pallidal and cerebellar label were also observed particularly in the ventral lateral nucleus pars oralis (VLo) and the ventral lateral nucleus pars caudalis (VLc). In both VLo and VLc, we additionally observed coincidence between the cerebellar labeling and SMA projection neurons as well as between pallidal labeling and MI projection neurons. These data suggest that while MI primarily receives inputs originating from Cb and SMA primarily receives inputs originating from GPi, it also appears that MI and SMA receive secondary afferents arising from GPi and Cb, respectively.     

Trigeminal Projections to Contralateral Dorsal Horn Originate in Midline Hairy Skin

The present study tested the hypothesis that the trigeminal (V) primary afferent projection to the contralateral dorsal horn originates in midline hairy skin. A prior study (Jacquin et al., 1990) showed that this crossed projection is heaviest to ophthalmic regions of medullary and cervical dorsal horns, and that it does not arise from V ganglion cells that innervate cornea, nasal mucosa, or cerebral dura mater. Here, retrograde double-labeling methods were used to show that many ophthalmic ganglion cells that innervate midline hairy skin via the supraorbital nerve project to the contralateral medullary and upper cervical dorsal horns. Diamidino yellow injections into the right dorsal horn labeled an average of 104 cells in the left V ganglion. Of these contralaterally projecting ganglion cells, an average of 45% were also labeled by horseradish peroxidase (HRP) injections into the left supraorbital nerve, and 25% were also labeled by HRP injections into the midline opthalmic hairy skin. However, only 2% were labeled by HRP injections restricted to left supraorbital vibrissae follicle nerves. Almost all of the double-labeled cells were located in the dorsal one-half of the V ganglion, and they did not differ in size from single-labeled cells.

On the basis of these and prior data, we conclude that a high percentage of contralaterally projecting V ganglion cells originate in midline hairy skin. It is also likely that the contralaterally projecting V ganglion cells serve a low-threshold mechanoreceptive function, given the relatively large ganglion cells and axons giving rise to this pathway and their central terminations in dorsal horn laminae III-V.     

Trigeminal projections to contralateral dorsal horn originate in midline hairy skin

The present study tested the hypothesis that the trigeminal (V) primary afferent projection to the contralateral dorsal horn originates in midline hairy skin. A prior study (Jacquin et al., 1990) showed that this crossed projection is heaviest to ophthalmic regions of medullary and cervical dorsal horns, and that it does not arise from V ganglion cells that innervate cornea, nasal mucosa, or cerebral dura mater. Here, retrograde double-labeling methods were used to show that many ophthalmic ganglion cells that innervate midline hairy skin via the supraorbital nerve project to the contralateral medullary and upper cervical dorsal horns. Diamidino yellow injections into the right dorsal horn labeled an average of 104 cells in the left V ganglion. Of these contralaterally projecting ganglion cells, an average of 45% were also labeled by horseradish peroxidase (HRP) injections into the left supraorbital nerve, and 25% were also labeled by HRP injections into the midline opthalmic hairy skin. However, only 2% were labeled by HRP injections restricted to left supraorbital vibrissae follicle nerves. Almost all of the double-labeled cells were located in the dorsal one-half of the V ganglion, and they did not differ in size from single-labeled cells. On the basis of these and prior data, we conclude that a high percentage of contralaterally projecting V ganglion cells originate in midline hairy skin. It is also likely that the contralaterally projecting V ganglion cells serve a low-threshold mechanoreceptive function, given the relatively large ganglion cells and axons giving rise to this pathway and their central terminations in dorsal horn laminae III-V.     

Characterization of retrograde axonal transport of antibodies in central and peripheral neurons

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.     

Diverging projection of the trigeminocerebellar fibers in the rabbit paramedian lobule

The present study has been attempted to investigate the issue of intralobular branching of cerebellar afferent axons arising from neurons in TSN and terminating in rPML and cPML sublobules, known to be the face-forelimb and hindlimb receiving areas, respectively. In this aim the double fluorescent retrograde technique was employed in the rabbit. No other reports have addressed this question. Non-overlapping unilateral injections of the cytoplasmic tracers FB and the nuclear dye DY into rPML and cPML, respectively, resulted in numerous single FB or DY labeled neurons and small number of double FB + DY ones in Vp, Vo, Vir and Vic bilaterally, with a very clear ipsilateral predominance. No evidence has been disclosed for projection from Vmes and Vc. Distribution pattern of single labeling allows to assume that projection exhibits some degree of topographical organization. Thus, there are populations of TSN neurons projecting independently to rPML and cPML and, to a larger extent, populations of neurons whose projection areas more or less overlap. Profuse projection arises from Vir and less numerous fibers originate from Vp and the rostral part of Vic. Neurons in Vo, mainly in the caudal regions, participate in a relatively moderate degree to this projection. Double labeled neurons recognized herein indicate that TSN projections reaching the two non-homologous PML regions may be collaterals of the same axons. The cells of origin for such projections are distributed in defined regions of Vir (n = 214), Vic (n = 107), Vp (n = 73) and Vo (n = 25). Considering small percent of neurons with divergent axons (about 3% in Vic and Vo, and 2% in Vir and Vp) it can be concluded that trigeminal inputs to rPML and cPML correspond to a larger extent to separate rather than collateral projection. In spite of this the findings indicate that functionally different PML regions are linked. The present results are discussed with those of earlier studies and commented on possible functional meaning of the projection by way of axonal branchings.     

Observations on the projection from the perihypoglossal nuclei onto the cerebellum in the macaque monkey

The occurrence and distribution of retrogradely labeled cells in the perihypoglossal nuclei of the monkey were mapped after injections of horseradish peroxidase in various cerebellar cortical regions. In general the findings are in accord with those made in the cat. The flocculus receives a heavy bilateral projection from the nucleus prepositus, particularly from its caudoventral part, and from the nucleus of Roller. There is an apparently scanty projection from the nucleus intercalatus. The uvula receives a rather similar projection, but in the prepositus the cells projecting to the uvula are on the whole situated more dorsally and rostrally than those supplying the flocculus. The projection to lobules VII-VIII is distinct. More scanty projections have been found to the paramedian lobule and the anterior lobe. The different but partially overlapping sites of origin in the prepositus of fibers to the flocculus and uvula indicate the presence of a topical pattern within the perihypoglosso-cerebellar projection, as in the cat (34). In the monkey the two regions of origin appear to coincide with two particular cell collections in the prepositus (12). Both small and middle sized cells project to the cerebellum, as they do in the cat (9, 48). The nucleus supragenualis nervi facialis in the macaque is morphologically different from the corresponding nucleus in most other mammalian species (12), but it contains labeled cells after injections in the flocculus, uvula and other cerebellar regions. A considerable number of cells in the abducent nucleus are labeled after injections in the flocculus and the posterior vermis.     

Collateral projections from trigeminal sensory nuclei to ventrobasal thalamus and cerebellar cortex in rats

The retrograde fluorescent labeling technique reveals that trigeminal projections to the ventroposteromedial nucleus of the thalamus (VPM) of the rat originate from the main sensory nucleus (MSN) of the trigeminal and subnuclei interpolaris (V1) and caudalis (Vc) of the spinal trigeminal nucleus. These projections are predominantly contralateral; however, the presence of a few ipsilateral labeled cells in MSN suggests an uncrossed trigeminothalamic pathway. Trigeminocerebellar fibers projecting to the paramedian lobule (PML) of the cerebellar cortex are located in Vi and caudal subnucleus oralis (Vo). This is principally an ipsilateral pathway, but several bisbenzimide-labeled cells are present in contralateral Vi. The most notable finding occurred after paired injections of Evans Blue into VPM and bisbenzimide into PML, demonstrating neurons in Vi with divergent projections to both structures. The presence of this type of projection was not found in mice (Steindler: J. Comp. Neurol. 237:155-175, 1985) and has not been reported in other species.     

Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons

The freely diffusible radical, nitric oxide (NO), has been assumed to act as a retrograde signaling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work we have examined the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord. In our experiments we have used histochemical methods for the visualization of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) which label neurons that express nitric oxide synthase (NOS). Both histochemical methods were performed separately or together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were seen mostly in superficial and deep layers of the dorsal horn, preganglionic autonomic neurons and pericentral area. The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in the ventral horns and in neurons of the pericentral and intermediate zone. Besides the above mentioned neurons, also double-labeled neurons were found which contained both the yellow and dark blue histochemical product. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the co-existence of NADPH-d and AChE occurred in the location of interneurons. Our observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission.     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces apoptosis in mouse nigrostriatal glia. Relevance to nigral neuronal death and striatal neurochemical changes

Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed 1 day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta (SNc). Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.     

Innervation of the superficial pineal of the rat using retrograde tracing methods

Fast blue (FB), rhodamine microspheres (RH), horseradish peroxidase (HRP), and wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were used as retrograde tracers to study the innervation of the rat superficial pineal gland (SP). One of the tracers was injected into the gland of each animal. All four retrograde tracers injected into the gland always labeled neurons in the superior cervical ganglia (SCG). No retrograde labeling was ever seen in the suprachiasmatic nuclei, paraventricular hypothalamic nuclei, lateral hypothalamus, habenular nuclei, amygdalar nuclei, or superior salivatory nuclei. Retrograde labeling was seen in the anterior hypothalamic nuclei, anterior thalamic nuclei, lateral geniculate bodies, and midbrain tectal structures when a tracer spread from the injection site to the overlying cortex, tectum, or commissures. Control studies included injection of tracer into the subarachnoid space around the SP or into structures adjacent to the SP. Only the injection of FB or WGA-HRP into the subarachnoid space labeled neurons in the SCG. This labeling was probably due to the spread of tracer to the choroid plexus. These results agree with recent work confirming the existence of a direct projection of the SCG into the interstitium around pinealocytes. The evidence does not substantiate an innervation originating in the habenular nuclei; the superior salivatory nuclei; or any other diencephalic, midbrain, pontine, or medullary structure.     

Expression of Trefoil Factor 1 in the Developing and Adult Rat Ventral Mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson’s disease.     

Target-derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT-4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time-course of degeneration occurs after selective removal of target cells or depletion of target-derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT-4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT-4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release.     

Divergent axon collaterals originate in the estrogen receptive ventromedial nucleus of hypothalamus in the rat

The ventromedial nucleus of the hypothalamus (VMH) plays a crucial role in the mediation of lordosis by integrating predominantly inhibitory limbic signals with cyclic variation of ovarian steroids and sending a stimulatory output to the midbrain, especially the periaqueductal gray (PAG). Tract-tracing studies have established projections of the VMH and Golgi studies have shown these neurons to frequently give rise to axon collaterals, but the anatomical pattern of shared projections has not been explored. We have used a combination of retrograde tracers to map VMH projections to the medial division of the medial preoptic nucleus (MPNm), posterodorsal division of the medial nucleus of the amygdala (MeApd), and the PAG. Neurons with dual projections were mainly confined to the VMHvl and represented 31%–37% of each projection subset. Neurons simultaneously projecting to the MPNm, MeApd, and PAG represented 7%–9% of each projection subset. By combining tract-tracing with steroid autoradiography, we found that approximately one-quarter of each projection subset in the VMHvl concentrated ³H-estradiol. Thus, some of the VMHvl neurons that communicate a facilitatory signal to the PAG may also act to stimulate lordosis through a feedback suppression of the net inhibition formed by efferent signals from the forebrain. The even distribution of estrogen binding among projection subsets suggests a lack of compartmentalization of estrogen-regulated processes that are relevant to lordosis. 1994 John Wiley & Sons, Inc.     

Neurons of visual thalamic nuclei projecting to telencephalon express different types of calcium-binding proteins: A combined immunocytochemical and tracer study

In turtles (Testudo horsfieldi, Emys orbicularis), immunoreactivity to calbindin (CB), parvalbumin (PV), calretinin (CR) and co-localization of CB and PV were studied in neurons of the visual thalamic nuclei (Rot, GLd) projecting to the telencephalon using a combination of immunohistochemical and tracer methods. The prevalence of CB-immunoreactive (-ir) neurons in Rot, CB-ir and CR-ir neurons in GLd, and a smaller number of PV-ir neurons in both nuclei was shown. Double immunofluorescent labeling revealed that within both nuclei PV and CB are colocalized in most PV-ir and fewer CB-ir neurons. After injection of horseradish peroxidase into the Rot and GLs telencephalic projection fields, retrograde labeling was found in corresponding thalamic projection neurons immunoreactive to all the three proteins. After introduction of the fluorescent tracer Fluo-gold into the same telencephalic regions, retrograde labeling was detected in Rot and GLd neurons immunoreactive only to PV and CB as well as in neurons with colocalization of both proteins. These findings provide further evidence that in turtles the CB component prevails in the rotundo-telencephalic pathway while the CB/CR component is dominant in the geniculotelencephalic pathway. The role of functional specialization in segregation of neurons expressing distinct types of calcium-binding proteins is postulated.     

Spatially Reciprocal Inhibition of Inhibition within a Stimulus Selection Network in the Avian Midbrain

Reciprocal inhibition between inhibitory projection neurons has been proposed as the most efficient circuit motif to achieve the flexible selection of one stimulus among competing alternatives. However, whether such a motif exists in networks that mediate selection is unclear. Here, we study the connectivity within the nucleus isthmi pars magnocellularis (Imc), a GABAergic nucleus that mediates competitive selection in the midbrain stimulus selection network. Using laser photostimulation of caged glutamate, we find that feedback inhibitory connectivity is global within the Imc. Unlike typical lateral inhibition in other circuits, intra-Imc inhibition remains functionally powerful over long distances. Anatomically, we observed long-range axonal projections and retrograde somatic labeling from focal injections of bi-directional tracers in the Imc, consistent with spatial reciprocity of intra-Imc inhibition. Together, the data indicate that spatially reciprocal inhibition of inhibition occurs throughout the Imc. Thus, the midbrain selection circuit possesses the most efficient circuit motif possible for fast, reliable, and flexible selection.     

A light microscope-based double retrograde tracer strategy to chart central neuronal connections

This protocol describes a double retrograde tracing method to chart divergent projections in the CNS using light microscope techniques. It is based on immunohistochemical visualization of retrograde transport of cholera toxin b-subunit (CTb) and silver enhancement of a gold-lectin conjugate. Production of the gold-lectin is explained in detail, and a technique is offered to record through the injection pipettes, to help guide accurate placement of injections. Visualization of the two tracers results in light brown staining of CTb-labeled neurons and labeling by black particles of gold-lectin-containing neurons. Both types of label are easily recognized in the same neuron. The labeling is permanent and is well suited for studies in which large areas of the brain need to be surveyed. The whole procedure (excluding survival time) takes approximately 5-7 d to complete.     

Does the reticular thalamic nucleus project to the midbrain?

In this study, we investigated whether the reticular thalamic nucleus has a projection to major centres of the midbrain in rats, rabbits and cats. Various tracers (biotinylated dextran, cholera toxin B subunit, fluorescent latex beads) were injected either into the midbrain tectum (deep layers of the superior colliculus) or tegmentum (midbrain reticular and pedunculopontine nuclei). In other experiments, different coloured latex beads (red and green) were injected into the deep layers of the superior colliculus and into the midbrain reticular nucleus of the same animal (rabbits). Our major finding is that in rats, rabbits and cats, there are no retrogradely labelled cells in the reticular thalamic nucleus after tracer injections into the abovementioned midbrain centres. In rabbits and cats, however, there are retrogradely labelled cells lying close to the ventromedial edge of the reticular thalamic nucleus after such injections. We show, by means of immunocytochemical double-labelling, that these retrogradely labelled cells do not lie in the reticular thalamic nucleus as suggested by previous studies, but in the inner small-celled region, a group of small cells that forms part of the zona incerta. Although there appears to be no clear topography of projection of the inner small-celled region, our tracer double-labelling experiments show that separate cells in the inner small-celled region project to individual centres of the midbrain (i.e., there are very few double-labelled cells after double injections). In rats, unlike in rabbits and cats, there is no clearly defined inner small-celled region and there are no retrogradely labelled cells seen along the ventromedial edge of the reticular thalamic nucleus. Our results suggest that in rats, rabbits and cats, there is no projection of the reticular thalamic nucleus to major centres of the midbrain, suggesting that the nucleus may not have a very strong influence on midbrain function, as it does on dorsal thalamic function.     

Target‐derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT‐4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time‐course of degeneration occurs after selective removal of target cells or depletion of target‐derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT‐4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT‐4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 319–327, 2004     

Intersubnuclear connections within the rat trigeminal brainstem complex

Prior intracellular recording and labeling experiments have documented local-circuit and projection neurons in the spinal trigeminal (V) nucleus with axons that arborize in more rostral and caudal spinal trigeminal subnuclei and nucleus principalis. Anterograde tracing studies were therefore carried out to assess the origin, extent, distribution, and morphology of such intersubnuclear axons in the rat trigeminal brainstem nuclear complex (TBNC). Phaseolus vulgaris leucoagglutinin (PHA-L) was used as the anterograde marker because of its high sensitivity and the morphological detail provided. Injections restricted to TBNC subnucleus caudalis resulted in dense terminal labeling in each of the more rostral ipsilateral subnuclei. Subnucleus interpolaris projected ipsilaterally and heavily to magnocellular portions of subnucleus caudalis, as well as subnucleus oralis and nucleus principalis. Nucleus principalis, on the other hand, had only a sparse projection to each of the caudal ipsilateral subnuclei. Intersubnuclear axons most frequently traveled in the deep bundles within the TBNC, the V spinal tract, and the reticular formation. They gave rise to a number of circumscribed, highly branched arbors with many boutons of the terminal and en passant types. Retrograde single- or multiple-labeling experiments assessed the cells giving rise to TBNC intersubnuclear collaterals. Horseradish peroxidase (HRP) and/or fluorescent tracer injections into the thalamus, colliculus, cerebellum, nucleus principalis, and/or subnucleus caudalis revealed large numbers of neurons in subnuclei caudalis, interpolaris, and oralis projecting to the region of nucleus principalis. Cells projecting to more caudal spinal trigeminal regions were most numerous in subnuclei interpolaris and oralis. Some cells in lamina V of subnucleus caudalis and in subnuclei interpolaris and oralis projected to thalamus and/or colliculus, as well as other TBNC subnuclei. Such collateral projections were rare in nucleus principalis and more superficial laminae of subnucleus caudalis. TBNC cells labeled by cerebellar injections were not double-labeled by tracer injections into the thalamus, colliculus, or TBNC. These findings lend generality to currently available data obtained with intracellular recording and HRP labeling methods, and suggest that most intersubnuclear axons originate in TBNC local-circuit neurons, though some originate in cells that project to midbrain and/or diencephalon.     

Expression and subcellular localization of a novel nuclear acetylcholinesterase protein

Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.