Rhodamine-labeled Latex Microspheres Research Articles

Substance P-containing medullary projections to the intermediolateral cell column: identification with retrogradely transported rhodamine-labeled latex microspheres and immunohistochemistry

Substance P (SP)-containing medullary neurons that project to the intermediolateral cell column (IML) of the rat were studied. Neurons were retrogradely labeled with rhodamine-labeled latex microspheres (RITC-M) injected into the T-3 IML, and SP-immunoreactive neurons were identified with immunocytochemistry. RITC-M labeled cells occurred in the nucleus reticularis paragiganto-cellular lateralis (RPgcl), adjacent and lateral to the pyramidal tract at the level of the rostral inferior olivary nucleus and extended to the mid-facial nucleus in the medulla. Cells were also labeled caudal to the RPgcl, in the nucleus reticularis ventralis, pars alpha (RVa), rostral tp the RVa, in the nucleus reticularis gigantocellularis (RGc), and in the raphe nuclei. SP immunoreactivity (SP-IR) was seen in cells that were also retrogradely labeled. These double-labeled cells were observed in the RPgcl, RVa and the raphe pallidus. These data show that the IML receives SP-neuronal projections from multiple locations in the medulla. The SP-neuronal projections from the RPgcl of the ventral medulla to the IML likely represent one component of the ventral medullary region that influences cardiovascular functions.

The spinal and commissural projections from the superior colliculus in rat and hamster arise from distinct neuronal populations

Multiple retrograde labelling techniques were used to determine whether the superior collicular (SC) cells that project to the spinal cord in hamster and rat also innervate the contralateral colliculus. In 25 rats and 13 hamsters, various combinations of the tracers horseradish peroxidase, True blue, Diamidino yellow, fluorogold and rhodamine-labelled latex microspheres were used to label tectospinal and SC commissural neurons. No double-labelled cells were observed in any of these experiments. Analysis of neurons that were retrogradely labelled with horseradish peroxidase showed further that SC commissural neurons were much smaller than tectospinal cells. The average soma area for tectospinal cells in hamster was 225.9 μm 2 and that for such neurons in rat was 214.4 μm 2. The mean soma areas for SC commissural neurons in hamster and rat were 85.4 μm 2 and 91.5 μm 2, respectively. In additional experiments (6 rats and 6 hamsters), True blue was injected into the left predorsal bundle and Diamidino yellow was deposited into the left SC. In both hamsters and rats, these injections invariably produced a small number of double-labelled cells in the deep layers of the right SC. In a final group of animals (7 rats and 2 hamsters), large thalamic deposits of Diamidino yellow were combined with bilateral injections of True blue into the spinal cord. This combination also produced small numbers of double-labelled neurons in both colliculi. These results indicate that the tectospinal and SC commissural pathways arise from distinct neuronal populations, but that a small number of cells that send axons into the predorsal bundle also have commissural collaterals. They demonstrate further that some tectospinal cells also send axon collaterals to the thalamus.

A combination of Golgi impregnation and fluorescent retrograde labeling

Central nervous system structures containing neurons labeled by the fluorescent tracers Fast blue (FB), Diamidino yellow dihydrochloride (DY), Rhodamine B isothiocyanate (RITC) and Rhodamine-labeled latex microspheres (RLM) were processed with the Golgi method. The goal was to improve the visualization of the fluorescent labeled neurons and to allow their ultrastructural examination. While the fluorescence of FB and RITC is greatly attenuated by the Golgi method, RLM and DY are still visible in Golgi-impregnated neurons. However, it is usually necessary to remove the silver precipitate by gold-toning.

The topographical organization of neurons in the rat medial frontal, insular and olfactory cortex projecting to the solitary nucleus, olfactory bulb, periaqueductal gray and superior colliculus

In 19 rats two different retrograde tracers (Fast Blue, Diamidino Yellow, Rhodamine-labeled latex microspheres, or wheat germ agglutinin conjugated with HRP) were injected into the solitary nucleus (NTS) and either the olfactory bulb (OB), periaqueductal gray (PAG) or superior colliculus (SC). The pattern of retrogradely labeled neurons in the medial frontal, insular and olfactory cortices was examined to determine the topographical organization of the cell populations projecting to these subcortical targets and the extent to which they overlapped. In the medial frontal cortex (MFC) SC projections originated most dorsally, while NTS and OB projections originated most ventrally and exhibited slight overlap. PAG projections originated from virtually the entire MFC and overlapped with cells projecting to the OB, NTS and SC. These results are consistent with the role of dorsal MFC as the rat's frontal eye field and the ventral MFC as a visceral motor area. Laterally, in the insular cortex there was virtually complete overlap between cells projecting to the NTS and PAG. The extensive overlap of PAG projections with NTS projections medially and laterally and with SC projections medially suggests the PAG is involved in a variety of brain visceral and somatic functions. In the piriform cortex there was overlap between cells projecting to the OB and cells projecting to the SC; the cells projecting to the SC were located in the endopiriform nucleus, and may provide a substrate for orienting responses to odors.

Divergent axon collaterals to cerebellum and amygdala from neurons in the parabrachial nucleus, the nucleus locus coeruleus and some adjacent nuclei. A fluorescent double labelling study using rhodamine labelled latex microspheres and fast blue as retrograde tracers.

After injections in the cat of Rhodamine labelled latex microspheres in the amygdala and of Fast Blue in the cerebellum neurons labelled with one of these tracers as well as some double labelled neurons were found in the parabrachial nucleus, the nucleus locus coeruleus and some adjacent nuclei (the nucleus subcoeruleus and the pontine tegmental reticular formation). All double labelled cells were located on the ipsilateral side. A few double labelled neurons were also found bilaterally in the dorsal raphe nucleus. It therefore appears that a certain number of cerebellar projecting neurons in these brain stem nuclei by means of divergent axon collaterals also project to the amygdala. The location of the double labelled cells found in this study suggests that at least some of the neurons are catecholaminergic. The findings are related to previous reports on the distribution of catecholaminergic neurons and on the amygdaloid and cerebellar projections from this part of the brain stem, and the possible involvement of these connections in cerebellar non-somatic responses are discussed. Some comments are made concerning the use of fluorescent latex microspheres for double labelling studies in combination with another fluorescent tracer.