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PubMed1994

Localization of AMPA-selective excitatory amino acid receptor subunits in identified populations of striatal neurons.

Two-color immunofluorescence histochemistry and immunohistochemistry in combination with retrograde tract-tracing techniques were used to examine the relationship of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-selective glutamate receptor subunits (GluR1, GluR2/3/4c and GluR4) to identified populations of striatal projection neurons and interneurons. The majority of striatonigral and striatopallidal neurons were double-labeled for GluR2/3/4c. These findings were confirmed using calbindin to label matrix projection neurons. In contrast, immunostaining of the GluR1 subunit was not observed to co-localize with any striatal projection neurons. Striatal interneurons immunostained for parvalbumin were also labeled by antibodies directed against the GluR1 subunit. Approximately 50% of parvalbumin neurons also contained GluR2/3/4c. Somatostatin immunoreactivity did not co-localize with either the GluR1 or GluR2/3/4c subunits. GluR4-immunoreactive neurons were not observed in striatum. This study demonstrates that AMPA-selective glutamate receptors are differentially localized on subpopulations of striatal neurons and interneurons. These findings suggest that discrete striatal neuron populations may express different AMPA receptor subunit combinations which may account for their functional specificity.

Animals↗
PubMed1994

Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat.

In order to develop a rodent model displaying a progressive degeneration of the dopamine neurons of the substantia nigra, we bilaterally injected the tracer substance FluoroGold into the terminal field of the nigrostriatal projection, i.e. the striatum. One week later, rats received unilateral injections of 20 micrograms 6-hydroxydopamine into one of the two striatal tracer deposits. Groups of animals were killed one, two, four, eight and 16 weeks later. Ipsilateral to the lesion there was a progressive loss of FluoroGold-labelled nigral cells, with cell counts dropping from 96% of the contralateral side at one week to 59% at two weeks, 35% at four weeks, 23% at eight weeks and down to 15% at 16 weeks. Labelled nigral neurons ipsilateral to the lesion showed a moderate to marked atrophy at all investigated time points. The number of tyrosine hydroxylase-immunoreactive cells was decreased to 83% of contralateral at one week, 39% at two weeks, 44% at four weeks, 34% at eight weeks and 52% at 16 weeks postlesion. Rhodamine fluorescence immunocytochemistry showed that the proportion of surviving ipsilateral fluorogold-labelled cells displaying immunoreactivity for tyrosine hydroxylase was 69% at one week postlesion, 51% at two weeks, 63% at four weeks, 69% at eight weeks and 76% at 16 weeks. We conclude that injection of 6-hydroxydopamine into the terminal field of nigral dopaminergic neurons causes a progressive degeneration of these cells, starting between one and two weeks after lesion and continuing over eight to 16 weeks. This degeneration is preceded, and accompanied by, cellular atrophy and a partial loss of marker enzyme expression, thus yielding an animal model which mimics the degenerative processes in Parkinson's disease more closely than the animal models available so far. The present model may be helpful in investigating the in vivo effects of putative neuroprotective agents and neurotrophic factors.

Animals↗
PubMed1995

Contacts between serotoninergic fibres and dorsal horn spinocerebellar tract neurons in the cat and rat: a confocal microscopic study.

Contacts between serotoninergic nerve fibres and dorsal horn dorsal spinocerebellar tract neurons were analysed in order to investigate the morphological basis of actions of serotonin upon dorsal spinocerebellar tract neurons. In a series of experiments dorsal spinocerebellar tract neurons were labelled with intracellularly injected rhodamine-dextran in the cat. The neurons were monosynaptically excited by group II muscle afferents and cutaneous afferents and were identified by antidromic activation following stimuli applied in the cerebellum. In the second series of experiments dorsal spinocerebellar tract neurons were labelled by retrograde transport of Fluorogold injected into the cerebellum in the rat. In both series, serotoninergic fibres were labelled by using a specific anti-serotonin antiserum and were revealed by immunofluorescence. Appositions between the serotoninergic fibres and the cells were inspected with a dual channel confocal microscope. The merged images obtained with the two channels of the microscope were viewed in single optical planes 2 microns apart and in rotated three-dimensional reconstructions. Serotoninergic nerve fibres were found in apposition to cell bodies of all feline dorsal spinocerebellar tract neurons (n = 7) and of 75% of rat dorsal spinocerebellar tract neurons (n = 90). The numbers of putative contacts on cell bodies varied between less than 100 and nearly 300 (mean 160) in the cat and between about five and 30 in the rat. Contacts with dendrites of feline neurons were seen on 96% of 72 dendrites within 300 microns from soma and on 91% of 23 dendrites at distances of 300-500 microns. The number of such contacts varied from less than five to 150 on a single dendrite within these ranges of distances. Their total number within 100 microns from the soma was comparable or exceeded the number of contacts on the soma.

Animals↗
PubMed1995

The development of striatal patch/matrix organization after prenatal methylazoxymethanol: a combined immunocytochemical and bromo-deoxy-uridine birthdating study.

The antimitotic drug methylazoxymethanol was used to destroy striatal patch neurons during their three-day-period of neurogenesis in the rat. Single or multiple injections of methylazoxymethanol were given during embryonic days 13-15, the period when patch neurons are known to undergo their final cell division. Methylazoxymethanol treatments produced a dramatic reduction in striatal volume. Immunocytochemical analysis revealed the continued presence of patches of neurons that were substance P-immunoreactive and devoid of calbindin and enkephalin immunoreactivity. Both the number of patches and relative volume occupied by patches was reduced in methylazoxymethanol-treated striata. Patch neurons could also be labelled by an intrastriatal injection of FluoroGold during the first postnatal week. The early ingrowth of nigrostriatal dopamine afferents was less noticeably patchy in the methylazoxymethanol-treated animals, in part owing to an overall increase in density. Large reductions in the number of neurons immunoreactive for choline acetyltransferase were observed, whereas NADPH diaphorase-stained neurons were not reduced unless methylazoxymethanol was given on embryonic day 15. Injections of bromo-deoxy-uridine, either during or after the 24 h that each methylazoxymethanol injection was considered to be effective, revealed that (i) some patch neurons continued to be generated in the 24-h period following methylazoxymethanol administration, and (ii) many patch neurons were generated after the effects of methylazoxymethanol had worn off. These findings demonstrate that it was impossible to completely eliminate the patches using methylazoxymethanol injections during the period of patch neurogenesis. However, methylazoxymethanol treatment during this time did produce a dramatic loss of cells and a relatively greater reduction in patch volume. Despite this disruption, the appropriate compartmentalization of neuroactive substances appeared to be maintained.

Acetylcholinesterase↗
PubMed1995

Galanin expression in carotid body afferent neurons.

The present study examined expression and plasticity of the neuropeptide, galanin, in carotid body afferent neurons in the petrosal ganglion of the adult rat. The pattern of galanin expression was compared with that of tyrosine hydroxylase, a selective marker of dopaminergic carotid body afferents in the petrosal ganglion. In normal animals, only 3% of tyrosine hydroxylase-containing petrosal ganglion neurons co-expressed galanin. Retrograde labeling studies, in which FluoroGold was injected into the vascularly isolated carotid body, demonstrated that all tyrosine hydroxylase-positive-galanin-positive cells in the petrosal ganglion project to this target. In addition, however, we unexpectedly found that galanin expression was markedly increased in the petrosal ganglion following FluoroGold injection into the carotid body. On the other hand, tyrosine hydroxylase expression was unchanged, indicating that monoaminergic and peptidergic traits can be differentially regulated in these cells. In summary, these data demonstrate that monoaminergic chemoafferent neurons can co-express a peptidergic trait, similar to catecholaminergic neurons within the central and autonomic nervous systems, and that these cells retain the potential for phenotypic plasticity in adulthood.

Animals↗
PubMed1995

Differential distribution of calbindin-D28k and parvalbumin in somatic and visceral sensory neurons.

The purpose of the present investigation was to determine whether calbindin-D28k and parvalbumin are distributed to different subpopulations of somatic and visceral sensory neurons. Immunofluorescent and retrograde techniques were combined to examine the distribution of calbindin- and parvalbumin-like immunoreactivity in the cell bodies of somatic and visceral primary afferent neurons in dorsal root ganglia L1-S1 of rats. Calbindin and parvalbumin were differentially distributed to essentially non-overlapping subpopulations of primary sensory neurons that could be distinguished by their segmental and size distributions, as well as by their innervation of somatic and visceral structures. Calbindin-like immunoreactivity was found in a population of smaller-sized cell bodies comprising approximately 14%of all dorsal root ganglion cells examined, with the proportions being greatest in L6 and S1. In contrast, parvalbumin was found in a population of larger-sized cells that made up about 11% of dorsal root ganglion cells and that were most concentrated in L4 and L5. Sensory neurons were further characterized by retrograde transport following the application of the neuroanatomical tracer FluoroGold to somatic (sural and gastrocnemius) and visceral (hypogastric and pelvic) nerves. Somatic tissues were innervated by a population of calbindin-containing as well as a separate population of parvalbumin-containing sensory neurons. In contrast, afferent neurons innervating visceral structures contained only a subpopulation of calbindin-containing neurons and very few parvalbumin-positive cells.

Animals↗
PubMed1996

Enkephalin terminals form inhibitory-type synapses on neurons in the rat nucleus locus coeruleus that project to the medial prefrontal cortex.

Norepinephrine-containing fibres in the medial prefrontal cortex derive from the locus coeruleus, a brainstem nucleus which also receives a dense innervation of enkephalin-immunoreactive axon terminals. We combined immunogold-silver labelling of retrogradely transported FluoroGold from the medial prefrontal cortex with immunoperoxidase detection of leucine5-enkephalin in the same section of tissue through the locus coeruleus of adult rats. This dual-labelling experiment was conducted to determine whether axon terminals containing lecuine5-enkephalin target neurons in the locus coeruleus that project to the frontal cortex and, if so, what are their morphological characteristics. By light microscopy, enkephalin-labelled processes overlapped FluoroGold retrogradely labelled neurons in the locus coeruleus. By electron microscopy, retrogradely labelled perikarya and dendrites were commonly enveloped by astrocytic processes and received few afferents in the plane of section examined. However, at sites unoccupied by glial processes, abundant afferent input could be identified. In addition, some FluoroGold-labelled perikarya and dendrites lacked this glial ensheathment but were more frequently apposed by axon terminals. Of 163 FluoroGold-labelled perikarya and dendrites examined where enkephalin immunoreactivity was present in the neuropil, 42% were contacted by enkephalin-immunoreactive axon terminals. The peroxidase-labelled enkephalin terminals as well as the unlabelled terminals often contained both small, clear and large dense core vesicles. Both labelled and unlabelled terminals also formed primary symmetric synapses characteristic of inhibitory transmitters with retrogradely labelled perikarya and proximal dendrites. At times, more than one enkephalin-labelled terminal was found to converge on a common retrogradely labelled perikarya or dendrite. These results demonstrate cellular sites where enkephalin-containing afferents may directly modulate and most likely inhibit the activity of cortically projecting neurons in the locus coeruleus.

Animals↗
PubMed1996

Extensive projections from the midbrain periaqueductal gray to the caudal ventrolateral medulla: a retrograde and anterograde tracing study in the rat.

We investigated the innervation of the caudal ventrolateral medulla by the midbrain periaqueductal gray in the rat using retrograde and anterograde tract-tracing. Iontophoretic injection of Fluoro-Gold or cholera toxin B subunit into the caudal ventrolateral medulla resulted in retrogradely labeled neurons in discrete regions of the periaqueductal gray. These labeled cells were observed throughout the rostrocaudal extent of the periaqueductal gray and were distributed (as percentage of total labeled cells) in its lateral (53-67%), ventrolateral (14-28%), ventromedial (7-16%) and dorsomedial aspects (7-10%). About 70-72% of labeled cells were found in the caudal half of the periaqueductal gray and 28-30% in the rostral half. In the ventromedial periaqueductal gray, more labeled cells were seen in the contralateral side (5-13%) than the ipsilateral side (2-3%), whereas for other periaqueductal gray areas labeling was preferentially ipsilateral. Phaseolus vulgaris leucoagglutinin anterograde tracing was used to confirm the retrograde labeling results. Following iontophoretic injection into the periaqueductal gray, labeled fibers and terminals were observed throughout the rostrocaudal extent of the caudal ventrolateral medulla. Injections in the lateral and/or ventrolateral aspect of the periaqueductal gray yielded more anterograde labeling in the ipsilateral than the contralateral caudal ventrolateral medulla, while injections in the ventromedial aspect of the periaqueductal gray produced labeling preferentially in the contralateral caudal ventrolateral medulla. The present study indicates that specific regions of the periaqueductal gray project to the caudal ventrolateral medulla and may regulate cardiovascular and respiratory functions through these connections.

Animals↗
PubMed1996

Functional and anatomical reconstruction of the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat.

In an attempt to reconstruct the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat we have combined homotopic grafting of embryonic ventral mesencephalon suspensions with the implantation of long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells stretching from the site of the neuronal grafts to the striatum. At seven weeks after receiving both grafts, animals were killed and processed for immunohistochemistry against tyrosine hydroxylase. Tyrosine hydroxylase-immunoreactive axons were seen to extend from the nigral grafts, along the bridge graft to the striatum where terminal arborizations could be seen. The retrograde tracer Fluoro-gold was injected intrastriatally in some of the experimental animals and was taken up by grafted neurons confirming their projection to the striatum. In parallel to the anatomical reconstruction of the system, a decrease in amphetamine-induced rotation was demonstrated in those animals receiving both grafts which had received > 98% complete lesions. This decrease was greatest in those animals with the most tyrosine hydroxylase-immunoreactive axons in their bridge grafts. The presence of the bridge graft also led to an increase in neuronal graft survival with twice as many tyrosine hydroxylase-immunoreactive neurons being found in the grafts of those animals that had received both grafts compared to those that had received a neuronal graft but no bridge graft.

Amphetamine↗
PubMed1996

Morphologically distinct subpopulations of neurotensin-immunoreactive striatal neurons observed in rat following dopamine depletions and D2 receptor blockade project to the globus pallidus.

It has been reported in previous studies that perikaryal neurotensin immunoreactivity is largely absent in the rat striatum except following striatal dopamine depletion or blockade of dopamine D2 receptors, after which, however, neurotensin immunoreactivity is elicited in at least two distinct subpopulations of striatal neurons [Zahm D.S. (1992) Neuroscience 46, 335-350]. One subpopulation of such cells (type I), prominent following D2 receptor blockade, is located mainly in the matrix compartment in the rostral, dorsomedial and ventrolateral parts of the striatum, and comprises neurons at the large end of the medium-sized spectrum that exhibit intense neurotensin immunoreactivity in perikarya and proximal dendrites, but rarely display Fos immunoreactivity [Senger B. et al. (1993) Neuroscience 57, 649-660]. A second subpopulation (type II) resides predominantly in the patch (striosome) and matrix compartments in the dorsolateral quadrant of the striatum, and is prominent following administration of reserpine. These neurons are at the small end of the medium size range and exhibit very light neurotensin immunoreactivity, with little staining of dendrites. Fos immunoreactivity is frequently co-localized in striatal neurons that exhibit a type II striatal neurotensin response [Brog J.S. and Zahm D.S. (1995) Neuroscience 65, 71-86]. In the current study, neurotensin immunoreactivity was elicited in striatal neurons by ventral mesencephalic 6-hydroxydopamine lesions or administration of reserpine or haloperidol. Irrespective of which drug was given, retrogradely transported Fluoro-Gold was prominently co-localized with neurotensin-like immunofluorescence in the perikarya of striatal neurons following injections of the retrograde tracer into the globus pallidus. Few double-labeled neurons were observed following administration of any of these drugs and injections of Fluoro-Gold into the substantia nigra. It is concluded that two subpopulations of neurotensin-immunoreactive striatal neurons project predominantly to the globus pallidus and minimally to the substantia nigra.

Animals↗
PubMed1996

Bridge grafts of fibroblast growth factor-4-secreting schwannoma cells promote functional axonal regeneration in the nigrostriatal pathway of the adult rat.

Axons damaged in the adult mammalian central nervous system are able to regenerate when their inhibitory glial environment is replaced with a more permissive substrate. Here, we have used long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells to allow mechanically lesioned nigrostriatal axons to regenerate back to their original target in the adult rat brain. Regenerated axons were able to leave the bridge graft to form terminal arborizations and increase the density of tyrosine hydroxylase-immunoreactive fibres within the striatum. Bridge grafting also resulted in an increase in the number of neurons within the substantia nigra pars compacta taking up the fluorescent retrograde tracer Fluoro-Gold from the striatum. Animals which had received RN-22 bridge grafts showed lower rates of amphetamine-induced rotation 10 weeks after a mechanical lesion of the nigrostriatal tract compared to lesioned controls, the magnitude of the behavioural effect being related to the number of regenerated axons, and this comparative reduction was reversed by mechanical section of the bridge graft. It is concluded that our bridge grafting strategy allowed the partial anatomical and functional regeneration of the mechanically lesioned nigrostriatal tract, an unmyelinated central axon bundle, and that bridge grafting therefore represents a realistic approach to the repair of central nervous system lesions involving axon tract damage.

Amphetamine↗
PubMed1996

Reduced retrograde labelling with fluorescent tracer accompanies neuronal atrophy of basal forebrain cholinergic neurons in aged rats.

During ageing, basal forebrain cholinergic neurons are prone to degeneration for unknown reasons. In this study we morphometrically evaluated the retrograde labelling of basal forebrain neurons obtained after injection of FluoroGold into multiple sites in the cerebral neocortex in aged (24-33 months) as compared with young adult (four to six months) male Sprague-Dawley rats. In addition, we looked for differences in the distribution of degenerative changes in topographic subdivisions of the basal forebrain cholinergic complex of neurons identified by immunohistochemical detection of the cholinergic markers choline acetyltransferase or low-affinity neurotrophin receptor. After injection of FluoroGold into the cerebral neocortex, the number of retrogradely labelled neurons in the horizontal diagonal band/ substantia innominata and basal nucleus was significantly lower in aged rats, by 41% and 48%, respectively. In aged rats injected with FluoroGold as well as in non-injected aged rats, the numbers of neurons immunoreactive for choline acetyltransferase and low-affinity neurotrophin receptor were significantly lower, by 23-27% in the basal forebrain system as a whole, with no significant difference in the degree of decline amongst different subdivisions (i.e. medial septum, diagonal band, substantia innominata and basal nucleus). The ratios of the number of neurons labelled with FluoroGold as compared with the number of neurons immunoreactive for either cholinergic marker were significantly lower in aged rats, by 32-37%, indicating that the decline in the number of neurons retrogradely transporting tracer was greater than the decline in the number of immunoreactive neurons in aged animals. Immunoreactive as well as retrogradely labelled neurons showed a significant shrinkage of cell surface area of 6-13% in different subdivisions of the basal forebrain cholinergic system in aged rats. These findings confirm significant loss and atrophy of basal forebrain cholinergic neurons in aged rats, and demonstrate significantly reduced retrograde labelling of these neurons with fluorescent tracer applied to their target cortex. This reduced retrograde labelling suggests an impairment of either uptake or retrograde transport mechanisms in these neurons in aged rats. Such an impairment may contribute to the degenerative changes of basal forebrain cholinergic neurons observed in ageing and age-related degenerative conditions such as Alzheimer's disease.

Acetylcholine↗
PubMed1989

Localization of spinal cord preganglionic neurons innervating the superior cervical ganglion in the golden hamster.

The retrograde neuronal tracer Fluoro-Gold (FG) was used to determine the location and distribution of preganglionic sympathetic neurons in the spinal cord of the golden hamster. FG was injected unilaterally into the superior cervical ganglia. Labeled neurons were found only ipsilateral to the injection site in segments C8 to T5 of which the segments T1 to T3 contained about 98% of the labeled cells. Neurons were found in four regions of the spinal cord: the intermediolateral nucleus (43%), the lateral funiculus (55%), the central autonomic area (1%) and the intercalated region (less than 1%). In the intermediolateral nucleus, cells often were arranged in clusters of which several were seen in each spinal segment.

Animals↗
PubMed1990

Collateral input to the paraventricular and supraoptic nuclei in rat. I. Afferents from the subfornical organ and the anteroventral third ventricle region.

Injections of two fluorescent retrograde tracers were used to investigate the existence of collateral branching of input to the hypothalamic magnocellular neuroendocrine neurons. Injection of one tracer (either Fluoro-Gold or rhodamine-labeled microspheres) into the supraoptic nucleus and the other tracer into the ipsilateral paraventricular nucleus produced labeled neurons within the subfornical organ and the anteroventral third ventricle area. Some labeled cells were found to contain both fluorescent tracers (double-labeled cells), suggesting that they project to both the paraventricular and supraoptic nuclei via branching axons. Most double-labeled cells were found within the subfornical organ. Fewer of these cells were located within the nucleus medianus preopticus, and still fewer were distributed in the organum vasculosum lamina terminalis, the bed nucleus of the stria terminalis, and the medial and the lateral preoptic areas. These data present the first direct evidence that single cells may provide input to more than one magnocellular neuroendocrine nucleus. Hypothetically, hormonal release would require coordinated firing of many magnocellular cells. Thus, the branched input to these neurons may assist in the organization and the timely activation of this system in response to physiological stimuli.

Animals↗
PubMed1990

Fate of septohippocampal neurons following fimbria-fornix transection: a time course analysis.

Neurons in the medial septum (MS) and vertical limb of the diagonal band (vDB) undergo degenerative changes following transection of their axons. These changes have been well studied by histological techniques such as Nissl stains and immunocytochemistry. A dramatic loss of stained neurons occurs following axotomy and this has been interpreted as indicative of neuronal death. However, since the staining intensity and the size of affected neurons may be reduced by axotomy, it is possible that the apparent neuronal death may actually be due to a decrease in somal size or the ability to detect neurons by routine histological methods. The present study describes the effects of axotomy on MS and vDB neurons which have been labeled by hippocampal injections of the retrograde tracer, Fluoro-Gold (FG), prior to transection of the fimbria-fornix and supracallosal stria. The number of FG-labeled neurons in the MS decreased by 21% at three weeks, 36% at six weeks, and 31% at ten weeks after fimbria-fornix transection. The reduction was statistically significant at 6 and 10 weeks. The number of FG-labeled neurons in the vDB showed no reduction at three weeks but was decreased by 31% and 37% at six and ten weeks, respectively. This was statistically significant only at 10 weeks. By comparison, the number of neurons immunoreactive for choline acetyltransferase (ChAT) was reduced by 75-80% at these time points. The size (area and diameter) of FG-labeled somata decreased in both the MS and vDB within three weeks following fimbria-fornix transection and remained relatively constant at the six- and ten-week time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗
PubMed1990

Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.

We have examined five conjugated 10,000 mol. wt. dextrans as potential anterograde tract tracers: Lucifer Yellow, Texas Red, fluorescein, Cascade Blue and tetramethylrhodamine. Pressure injections were made into the brain, dorsal root ganglia or footpads of adult rats. The retrograde tracer Fluoro-Gold was injected alone or mixed with the dextrans before injection. Three-14 days after injection, animals were perfused and sections cut with a freezing microtome. Texas Red-, fluorescein- and tetramethylrhodamine-conjugated dextrans produced intense labeling of neuronal cell bodies, axons and dendritic processes at the injection site and were transported by neurons predominantly in an anterograde direction to yield terminal and preterminal labeling. Relative to the fluorescein and tetramethylrhodamine conjugates, the quality and intensity of the anterograde labeling produced by Texas Red was variable. Results with Lucifer Yellow and Cascade Blue conjugates were negative. Optimal results were produced by slow-pressure injections via glass micropipettes. In comparison with Fluoro-Gold, retrograde transport by the dextran conjugates was present, but limited in its extent. Injections of the tetramethylrhodamine conjugate into dorsal root ganglia produced anterograde labeling of afferent fibers in visceral organs and injections into the nucleus ambiguous labeled motor fibers in the esophagus. Double/triple labeling was observed in the brain and spinal cord following multiple injections of fluorescein, tetramethylrhodamine and Fluoro-Gold. Also, Fluoro-Gold could be mixed with one of the dextrans in order to produce specific retrograde and anterograde labeling from the same injection site. The conjugates were compatible with fluorescent immunocytochemical procedures, but proved unsuitable for peripheral injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A double-labeling method for AChE and fluorescent retrograde tracers.

Staining for the degradative enzyme acetylcholinesterase (AChE) is an important tool in studying central cholinergic/cholinoceptive systems. AChE staining has also been useful in identifying the projections of AChE-containing neurons and codistribution of AChE with other neurotransmitters. The intensity and opacity of conventional AChE histochemical reaction products, however, pose problems for such double-labeling studies. Here, we have successfully combined a modified version (37) of the Koelle-Friedenwald AChE reaction with retrograde transport of the fluorescent tracer, Fluoro-Gold (FG). By omitting the final intensification steps of the Koelle-Friedenwald reaction, a translucent, light-stable reaction product is created. Viewed under darkfield illumination, this precipitate is of similar intensity and sensitivity to that produced by conventional AChE histochemical processing. Prior administration of an AChE-inhibitor yields preferential staining of AChE-positive neuronal somata. This nonintensified darkfield AChE (NIDA) histochemical method was compatible with visualization of retrogradely transported FG in AChE-positive neurons, allowing unambiguous identification of the projections of AChE-containing neurons.

Acetylcholinesterase↗
PubMed1992

Supraoptic nucleus afferents from the accessory olfactory bulb: evidence from anterograde and retrograde tract tracing in the rat.

Our earlier electrophysiological work provided evidence of a direct input to the supraoptic nucleus (SON) from the olfactory bulbs; however, these experiments could not determine if the input originated in the main and/or accessory portions of the olfactory bulb. Here, a connection between the accessory olfactory bulb (AOB) and the SON of the rat was examined using a combination of anatomic techniques. We employed neurophysin immunocytochemistry to delineate the morphological boundaries of the SON and the proximal arborizations of supraoptic dendrites. Accessory olfactory bulb efferents to the SON were studied by injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the AOB. The distribution of retrogradely labeled cells within the AOB was also determined after injection of either rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro-Gold (FG) into the SON. Neurophysin immunocytochemistry revealed that SON dendrites extended beyond the generally accepted boundaries of the nucleus, coursing ventrolaterally along the surface of the periamygdaloid cortex. Anterograde tract tracing with WGA-HRP labeled AOB efferents including a dense plexus of terminals and fibers around the ipsilateral SON along the path of the ventrally projecting dendrites. Injections of retrograde tracers into the SON resulted in rhodamine bead or FG labeling of mitral cells throughout the ipsilateral AOB. Taken together, these anatomic studies suggest a direct projection from the accessory olfactory bulb to the SON of the rat and thus a vomeronasal organ to SON pathway.

Afferent Pathways↗