Selective Retrograde Labeling Research Articles

In vivo visualisation of nanoparticle entry into central nervous system tissue

Because the potential neurotoxicity of nanoparticles is a significant issue, characterisation of nanoparticle entry into the brain is essential. Here, we describe an in vivo confocal neuroimaging method (ICON) of visualising the entry of fluorescent particles into the parenchyma of the central nervous system (CNS) in live animals using the retina as a model. Rats received intravenous injections of fluorescence-labelled polybutyl cyanoacrylate nanoparticles that had been synthesised by a standard miniemulsion polymerisation process. We performed live recording with ICON from before and up to 9 days after particle injection and took photomicrographs of the retina. In addition, selective retrograde labelling of the retinal ganglion cells was achieved by stereotaxic injection of a fluorescent dye into the superior colliculus. Using ICON, we observed vascular kinetics of nanoparticles (wash-in within seconds), their passage to the retina parenchyma (within minutes) and their distribution (mainly cellular) under in vivo conditions. For the detection of cell loss--which is important for the evaluation of toxic effects--in another experiment, we semi-quantitatively analysed the selectively labelled retinal neurons. Our results suggest that the dye per se does not lead to neuronal death. With ICON, it is possible to study nanoparticle kinetics in the retina as a model of the blood-brain barrier. Imaging data can be acquired within seconds after the injection, and the long-term fate of cellular uptake can be followed for many days to study the cellular/extracellular distribution of the nanoparticles. ICON is thus an effective and meaningful tool to investigate nanoparticle/CNS interactions.

A Hox Regulatory Network Establishes Motor Neuron Pool Identity and Target-Muscle Connectivity

Spinal motor neurons acquire specialized "pool" identities that determine their ability to form selective connections with target muscles in the limb, but the molecular basis of this striking example of neuronal specificity has remained unclear. We show here that a Hox transcriptional regulatory network specifies motor neuron pool identity and connectivity. Two interdependent sets of Hox regulatory interactions operate within motor neurons, one assigning rostrocaudal motor pool position and a second directing motor pool diversity at a single segmental level. This Hox regulatory network directs the downstream transcriptional identity of motor neuron pools and defines the pattern of target-muscle connectivity.

Embryonic development of connections in Turtle Pallium

We are interested in similarities and conserved mechanisms in early development of the reptilian and mammalian thalamocortical connections. We set out to analyse connectivity in embryonic turtle brains (Pseudemys scripta elegans, between stages 17 and 25), by using carbocyanine dye tracing. From the earliest stages studied, labelling from dorsal and ventral thalamus revealed backlabelled cells among developing thalamic fibres within the lateral forebrain bundle and striatum, which had similar morphology to backlabelled internal capsule cells in embryonic rat (Molnár and Cordery, 1999). However, thalamic crystal placements did not label cells in the dorsal ventricular ridge (DVR) at any stage examined. Crystal placements into both dorsal and lateral cortex labelled cells in the DVR and, reciprocally, DVR crystal placements labelled cells in the dorsal and lateral cortices. Retrograde labelling revealed that thalamic fibres arrive in the DVR and dorsal cortex by stage 19. The DVR received projections from the nucleus rotundus and the dorsal cortex exclusively from the perirotundal complex (including lateral geniculate nucleus). Thalamic fibres show this remarkable degree of specificity from the earliest stage we could examine with selective retrograde labelling (stage 19). Our study demonstrates that axons of similar cells are among the first to reach dorsal and ventral thalamus in mammals and reptiles. Our connectional analysis in turtle suggests that some cells of the mammalian primitive internal capsule are homologous to a cell group within the reptilian lateral forebrain bundle and striatum and that diverse vertebrate brains might use a highly conserved pattern of early thalamocortical development.

The homeodomain transcription factors Islet 1 and HB9 are expressed in adult alpha and gamma motoneurons identified by selective retrograde tracing.

To study gene expression in differentiated adult motoneuron subtypes, we used fluorescent dextrans for both anterograde and retrograde axonal tracing in adult rat and mouse. Application of these dyes to the cut distal and proximal ends of small extramuscular nerve branches revealed both the peripheral ramifications and the cell bodies of subsets of motoneurons. We show that the soleus muscle is innervated by two nerve branches, one of which contains gamma motor and sensory axons but no alpha motor axons. By retrograde tracing of this branch, we selectively labelled gamma motoneurons. In adult rat, the nerves innervating the soleus and extensor digitorum longus muscles contain almost exclusively axons innervating slow (type I) and fast (type 2) muscle fibres, respectively. We selectively labelled slow and fast type motoneurons by retrograde tracing of these nerves. With immunocytochemistry we show that adult motoneurons express several homeodomain genes that are associated with motoneuron differentiation during early embryonic development. Combining selective retrograde labelling with immunocytochemistry we compared the expression patterns in alpha and gamma motoneurons. The homeodomain transcription factors Islet 1 and HB9 were expressed in slow and fast alpha motoneurons and in soleus gamma motoneurons. Motoneurons in each population varied in their intensity of the immunostaining, but no factor or combination of factors was unique to any one population.

Origins of excitatory drive within the respiratory network: anatomical localization.

This study identified several sources of excitatory drive within the rat brain stem respiratory network. Excitatory neuronal projections to the ventral respiratory group (the largest brain stem respiratory neuron population in the rat) were identified by selective retrograde labeling with tritiated aspartate. Neurons were labeled within portions of the nucleus of the tractus solitarius, the region of the ventral respiratory group, superficial to the facial nucleus, the medullary raphe nuclei and the parabrachial/Kölliker-Fuse nuclei. These findings are the first anatomical demonstration of sources of excitatory drive within the respiratory network, and are consistent with electrophysiological data localizing a source of excitatory drive within the pre-Bötzinger complex subdivision of the ventral respiratory group.

Neurotransmitter profile of saccadic omnipause neurons in nucleus raphe interpositus

Saccadic omnipause neurons (OPNs) are essential for the generation of saccadic eye movements. In primates OPNs are located near the midline within the nucleus raphe interpositus (rip). In the present study we used several different neuroanatomical methods to investigate the transmitters associated with OPNs in the monkey. Immunolabeling for the calcium-binding protein parvalbumin was employed to mark OPNs in the monkey and define the homologous cell group in cat and human. The use of antibodies against GABA, glycine (GLY), glutamate (GLU), serotonin (5-HT), and tyrosine hydroxylase revealed that the somata of OPNs are GLY immunoreactive, but they are devoid of GABA and 5-HT immunostaining. In situ hybridization with the GAD67 mRNA probe confirmed the negative GABA immunostaining of OPNs. 3H-GLY was injected into a projection field of OPNs, the rostral interstitial nucleus of the medial longitudinal fascicle (riMLF)--the vertical saccadic burst neuron area. This resulted in selective retrograde labeling of the OPNs in rip, while no labeling was found in the superior colliculus, which sends an excitatory projection to the riMLF. The somata and dendrites of putative burst neurons in the riMLF were contacted by numerous GLY-immunoreactive terminals. The quantitative analysis of immunoreactive terminal-like structures contacting OPNs revealed a strong input from GLY- and GABA-positive terminals on somata and dendrites, whereas GLU-positive puncta were mainly confined to the dendrites. Very few 5-HT and catecholaminergic terminals contacted OPN somata. Our findings suggest that OPNs use GLY as a neurotransmitter, and they receive numerous contacts from GABAergic, glycinergic, and glutaminergic afferents, and significantly fewer from monoaminergic inputs.

Selective retrograde labeling of primary vagal afferent cell-bodies after injection of [ 3H] d-aspartate into the rat nucleus tractus solitarii

A selective retrograde labeling study was performed using [ 3H] d-aspartate to identify putative glutamatergic and/or aspartatergic primary vagal afferent fibers. Unilateral microinjection of [ 3H] d-aspartate into the nucleus tractus solitarii resulted in clear visible labeling of a fraction of the neuronal cell-bodies in the nodose ganglia. The labeled cell-bodies were randomly distributed in the ganglion and more numerous labeled neurons were detected in the ipsilateral than in the contralateral ganglion (4.93% and 0.98% of the neurons sampled within the ipsi- and contralateral ganglia, respectively). These results strongly suggest that primary vagal afferent fibers may utilize excitatory amino acids as transmitters.

Tracing specific transmitter pathways in the rat CNS: combination of [ 3H]serotonin retrograde labelling with immunocytochemical detection of endogenous transmitters

Selective retrograde labelling with [ 3H]serotonin ([ 3H]5-HT) can be used to identify serotonergic cell bodies after specific [ 3H]5-HT uptake by the corresponding nerve terminals. In the present study, we demonstrate that autoradiography of this [ 3H]5-HT radiolabelling can be combined with immunocytochemical detection of endogenous serotonin, GABA or substance P on the same tissue section. The midbrain raphe serotonergic projections to the olfactory bulb and the spinal projections of medullary serotonergic nuclei were investigated. The specificity of retrograde labelling with [ 3H]5-HT was confirmed by immunoreactivity of the radiolabelled cells for serotonin, using an antiserum specific for formaldehyde-fixed serotonin. After spinal injections of [ 3H]5-HT, many retrogradely labelled cells in the medullary raphe were immunopositive for substance P, and a few for GABA. These results are in agreement with the available information on the co-existence of putative transmitters in the spinal projections of caudal raphe neurons. Therefore, autoradiography of [ 3H]5-HT retrograde labelling combined with immunocytochemistry offers a possibility to test the specificity of transmitter-selective retrograde labelling, to identify transmitter-defined neuronal interactions and to investigate the projection fields of multitransmitter containing neurons.

Further evidence for excitatory amino acid transmission in lamprey reticulospinal neurons: Selective retrograde labeling with (3H)D‐aspartate

The distribution of radiolabeled neurons in the brain stem of Lampetra fluviatilis was studied following unilateral injections of (3H)D-aspartate in the rostral spinal cord. After survival periods of 1-3 days, labeled perikarya were present within and nearby the posterior, middle, and anterior rhombencephalic reticular nuclei and in the mesencephalic reticular nucleus. The highest number of (3H)D-aspartate labeled cell bodies were present in the posterior rhombencephalic reticular nucleus. The labeled reticulospinal neurons were distributed mainly ipsilateral to the injection site and included the giant Müller cells as well as medium-sized and small neurons. Contralateral labeling occurred in cell bodies scattered along the lateral margin of the rhombencephalic reticular formation, the most rostral of these contralaterally projecting neurons being the Mauthner cell. The (3H)D-aspartate labeling correlates with previous electrophysiological studies showing that lamprey reticulospinal neurons utilize excitatory amino acid transmission.

Autoradiographic and electrophysiological evidence for excitatory amino acid transmission in the periaqueductal gray projection to nucleus raphe magnus in the rat

Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25–50 nl 10 −2 or 10 −3 M d-[ 3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2–12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and γ- d-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N- methyl- d-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.

Selective retrograde labeling of lateral olivocochlear neurons in the brainstem based on preferential uptake of 3H-D-aspartic acid in the cochlea.

We have previously shown that perfusion of the gerbil cochlea with probe concentrations of 3H-D-aspartic acid (D-ASP) results in immediate, selective labeling of 50-60% of the efferent terminals under the inner hair cells, presumably by high-affinity uptake. The present study was undertaken to determine the origin of these endings. Twenty-four hours after cochlear perfusion with D-ASP, labeled neurons were observed in the ipsilateral, and to a much lesser extent in the contralateral, lateral superior olivary nucleus (LSO). The cells were small, primarily fusiform, and showed fewer synaptic contacts than other LSO cells. Combined transport of D-ASP and horseradish peroxidase indicated that all olivocochlear neurons within the LSO that projected to the injected cochlea were labeled by D-ASP. Labeled fibers coursed dorsally from the LSO, joined contralateral fibers that had passed under the floor of the fourth ventricle, and entered the VIIIth nerve root at its ventromedial edge. Adjacent to the ventral cochlear nucleus (VCN), densely labeled collateral fibers crossed the nerve root to enter the VCN. Labeled fibers and terminals were prominent in the central VCN. Neither retrograde transport of D-ASP by medial olivocochlear and vestibular efferents nor anterograde transport by VIIIth nerve afferents was observed. The D-ASP-labeled cells and fibers are clearly lateral olivocochlear efferents. Retrograde transport of D-ASP thus allows the cells, axons, and collaterals of the lateral olivocochlear system to be studied, morphologically, in isolation from other cells that project to the cochlea. Since the olivocochlear neurons are almost certainly cholinergic, retrograde amino acid transport does not necessarily identify the primary neurotransmitter of a neuron. Rather, it indicates the presence of selective uptake by the processes of that neuron at the site of amino acid injection. Retrograde labeling appears to be markedly enhanced by the use of metabolically inert compounds such as d-isomer amino acids.

Selective retrograde labeling with D‐[3H]‐aspartate in afferents to the mammalian superior colliculus

The selectivity previously reported for retrograde labeling patterns obtained following D-[3H]-aspartate injections and proposed to be related to the transmitter specificity of the labeled pathways was tested in afferents to the superior colliculus (SC) of rats and rabbits. In rats selectivity was assessed by comparing retrograde perikaryal labeling patterns observed in D-[3H]-aspartate experiments with those found after administration of a nonselective tracer, horseradish-peroxidase-labeled wheat germ agglutinin (HRP-WGA), and of the tritiated neurotrasmitter gamma-aminobutyric acid (GABA). Following D-[3H]-aspartate injections into the SC labeling was intense in a large number of cortical and hypothalamic neurons. Other afferents to the SC, however, such as those originating from the ventrolateral geniculate nucleus, the pars reticulata of the substantia nigra, the locus coeruleus, the pontine nuclei, or the retinal ganglion cells, were not labeled. Similar results were obtained in rabbits. In cats, the analysis was focused on the cerebral cortex, since in an earlier investigation no retrograde labeling had been detected in the visual cortex following D-[3H]-aspartate injections in the SC. In the present work, however, retrogradely labeled neurons were observed in various cortical areas including a few in visual cortex. This report shows selective retrograde labeling for a part of the afferents to the SC. This selectivity does not display major differences among the mammalian species studied. Moreover, according to the information available about the distribution of neurotransmitters in the brain, the findings reported here favour the idea that D-[3H]-aspartate is a retrograde tracer selective for glutamatergic and/or aspartatergic pathways.

Selective retrograde labeling in some afferents to the rabbit lateral geniculate nucleus following injections of tritiated neurotransmitter-related compounds

Retrogradely labeled neurons were found in the visual cortex and superior colliculus following d-[ 3H]aspartate injections in the lateral geniculate nucleus (LGN). In these experiments labeling was also observed over the optic tract. [ 3H]dopamine and [ 3H]serotonin injections in the LGN caused weak labeling in a small number of superior colliculus neurons. Furthermore, in [ 3H]serotonin cases, labeled neurons were also found in the dorsal raphé nucleus. In contrast, when other amino acids or monoamines were injected, no retrograde labeling occurred in any of the afferents to the LGN. These results are largely consistent with the idea of d-[ 3H]aspartate being a useful marker for pathways using excitatory amino acids as neurotransmitters. The findings in [ 3H]dopamine and [ 3H]serotonin experiments indicate that these substances may induce retrograde labeling patterns, which are not related to the transmitter specificity of the pathways concerned.

Retrograde Labeling of Dorsal Root Ganglion Neurons after Injection of Tritiated Amino Acids in the Spinal Cord of Rats and Cats

The present experiments are based upon evidence that neurons may selectively take up at their terminals, and retrogradely transport, the same chemical they use as a neurotransmitter or its analogues. In an attempt to identify dorsal root ganglion (DRG) neurons that use glutamic acid as a neurotransmitter, [3H]D-aspartate ([3H]D-Asp) was chosen as a marker, since it is a metabolically inert amino acid known to be taken up by the same affinity mechanism as L-aspartate and L-glutamate. Adult rats and cats received injections of 50 nl to 1.5 microliter of [3H]D-Asp (500 microCi/microliter) in the dorsal horn of cervical segments (C3 to C6). At 9 to 48 hr after injection, all animals were perfused with 5% glutaraldehyde. After sections were processed for autoradiography, the DRG neurons situated most closely to the injection site were chosen from representative cases, and the number and cross-sectional area of labeled and unlabeled perikarya with a nucleolus in the plane of the section were calculated. In rats, about 4% of the sampled DRG neurons were autoradiographically labeled, and the mean perikaryal area of these neurons was about twice that of unlabeled perikarya. In cats, the percentage of labeled perikarya ranged between 6.5% and 13.27% of the sampled population. The ratio of the mean perikaryal area of labeled neurons to that of unlabeled neurons ranged between 1.6 and 2.5. In a control cat injected with [3H]proline at C7, all perikarya in the C7 DRG were autoradiographically labeled. However, with injection of [3H]gamma-aminobutyric acid ([3H]GABA) selective retrograde labeling was observed. Quantitative data in rat showed that perikarya labeled at the C6 level after injection of this amino acid constituted about 8% of the sample population in C6 DRG. The ratio of the size of labeled to unlabeled perikarya was 2.02. In one cat injected with [3H]GABA at caudal C3, the largest number of labeled perikarya were in C4 DRG and comprised up to 5.32% of the sampled population. The ratio of the size of labeled to unlabeled perikarya was 1.57. The results in cases of injection with [3H]D-Asp may be interpreted as consistent with the idea that a fraction of DRG neurons use glutamate and/or aspartate as neurotransmitter(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Selective retrograde labeling in some afferents to the rabbit lateral geniculate nucleus following injections of tritiated neurotransmitter-related compounds

Selective retrograde labeling in some afferents to the rabbit lateral geniculate nucleus following injections of tritiated neurotransmitter-related compounds

Selective retrograde labelling of the rat olivocerebellar climbing fiber system with d-[ 3h]aspartate

Selective retrograde labelling of the olivocerebellar climbing fiber system with d-[ 3H]aspartate has been observed in the rat, and the results have implications for the identification of a transmitter candidate as well as the neuroanatomical understanding of these cerebellar afferents. Microinjections of d-[ 3H]aspartate (50 nl, ca 10 -2 M) were made into various parts of cerebellar cortex. Survival times were 6,12 or 24 h. Pronounced diffusion of the tracer resulted in large injection sites. Within the zone of injection, glial elements were labelled over background. Most granule cells exposed to the tracer were unlabelled; the small numbers demonstrating labelling were believed to have been injured by the micropipette penetration. Beneath injection sites, large numbers of well-labelled nerve fibers appeared in the white matter and could be followed through the brainstem to the contralateral inferior olive, where labelled perikarya were found. After the inferior olivary neurons had been effectively destroyed with 3-acetylpyridine, evidence of cerebellar afferent labelling with d-[ 3H]aspartate was missing. Retrograde labelling of the olivocerebellar system was also observed after superfusion of the verrais with d-[ 3H] aspartate at concentrations in the range of K m for high affinity uptake (10 -5 or 10 -4 M, for 2 h). Mossy fiber or monoaminergic afferents to the cerebellum were never labelled with d-[ 3H]aspartate. The distribution of labelled cells in the olivary subnuclei after injections in different cerebellar areas was in line with the olivocerebellar organization previously described in the cat. Moreover, it was demonstrated that fibers from the different subnuclei follow different routes through the brainstem towards the cerebellum. Labelling of climbing fiber collaterals in uninjected parts of cerebellum indicated that some of the retrogradely migrating d-[ 3H]aspartate was directed in anterograde direction at axonal branching points. Collaterals were demonstrated in all deep cerebellar and Deiters' nuclei, and the results of intranuclear injections suggested that virtually every olivary neuron sends collaterals to these nuclei. Intracortical collaterals were organized in sagittal zones. Midline injections into the anterior lobe and VI lobule labelled collaterals in several zones of the posterior lobe spinal area and uninjected parts of the anterior lobe vermis. Hemispheral injection into copula pyramidis labelled collaterals in two prominent bundles in the anterior lobe. Injection into lobulus paramedianus and crus II gave massive collateral labelling in lobulus simplex, crus I, and laterally in lobuli IV–V, while fewer fibers ran to paraflocculus. Injection into lobulus simplex labelled collaterals in two or three zones of crus II and lobulus paramedianus. Injection into paraflocculus labelled collaterals in crus I, crus II and lobulus paramedianus. These results suggest that all olivocerebellar neurons share a selective affinity for d-[ 3H]aspartate, which is in line with proposals that aspartate may be their transmitter. Further, the study demonstrated the organization of the olivocerebellar pathway through the brainstem, and the complex pattern of climbing fiber collateralization to deep cerebellar nuclei and different regions of cerebellar cortex.

Selective retrograde labeling of neurons of the cat vestibular ganglion with [ 3H] d-aspartate

d-[2,3- 3H]Aspartate ([ 3H] d-Asp) was injected in the cat vestibular nuclei. Labeling patterns resulting from retrograde axonal transport by the vestibular nerve fibers were observed in the vestibular ganglion neurons and also in the nerve fibers. The selectivity of such labeling, related to the neurotransmitter's specificity, is strongly indicated.

Selective retrograde labelling of vestibular efferent neurons with [ 3H]choline

Following administration of [ 3H]choline in the lateral semicircular canal of the cat labyrinth, bidirectional axoplasmic transport of [ 3H]choline and its derivatives was shown by radioautography in the vestibular system. Light-microscopic radioautographs exhibited various patterns of radioautographic labelling. First, a diffuse reaction was observed in vestibular nuclei representing anterograde-labelled, vestibular nerve endings. Second, a heavy labelling limited to perikarya was detected in efferent vestibular neurons and corresponded to retrograde transport. The anterograde migration of [ 3H]choline is known to be non-selective and is related to synthesis of phospholipids, non-diffusable molecules. In contrast, the retrograde perikaryal labelling seems highly selective and related to the cholinergic specificity of the transmitter. The selectivity of such labelling offers a further possibility of identifying cholinergic neurons and is additional evidence that cholinergic mechanisms are involved in the efferent vestibular control.

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

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

Selective retrograde labeling of cholinergic neurons with [3H]choline

Evidence is presented which is consistent with a specific retrograde labeling of cholinergic neurons following [3H]choline application in their zone of termination. [3H]Choline injection in the rat hippocampus leads to perikaryal retrograde labeling in the ipsilateral medial septal nuclease and nucleus of the diagonal band, thus delineating an established cholinergic pathway, while only diffuse presumably anterograde labeling was observed in the lateral septum, the entorhinal cortex, and the opposite hippocampus. After [3H]choline injection in the pigeon visual Wulst, only the ipsilateral thalamic relay, of all inputs, showed similar perikaryal retrograde labeling, an observation supporting the suggestion that at least some thalamo-Wulst neurons are cholinergic.