Injections Of Biotinylated Dextran Amine Research Articles

Relationships between striatin-containing neurons and cortical or thalamic afferent fibres in the rat striatum. An ultrastructural study by dual labelling

Striatin, a recently isolated rat brain calmodulin-binding protein belonging to the WD-repeat protein family, is thought to be part of a calcium signal transduction pathway presumably specific to excitatory synapses, at least in the striatum. This study was aimed to specify the cellular and subcellular localization of striatin, and to determine the possible synaptic relationships between the two main excitatory afferent pathways, arising from the cerebral cortex and the thalamus, and the striatin-containing elements, in the rat striatum. Anterograde tract-tracing by means of biotinylated dextran amine injection in the frontoparietal cerebral cortex or the parafascicular nucleus of the thalamus was combined with immunogold detection of striatin. Striatin-immunoreactivity was confined to the neuronal somatodendritic compartment, including spines. Whereas 90–95% of the striatal neurons were striatin-positive, only about 50% of the sections of dendritic spines engaged in asymmetrical synaptic contacts exhibited striatin labelling. Among the sections of striatin-immunopositive dendritic spines, the number of immunogold particles ranged from one to more than seven, indicating an heterogeneity of the spine labelling. Moreover, within each class of spines presenting at least two silver–gold particles, the distribution of the particles varied from a clear-cut alignment under the postsynaptic densities (24–33% of spines) to a location distant from the synaptic area. In the cell bodies and dendrites, striatin labelling was usually not associated with the cytoplasmic membrane nor with the postsynaptic densities. In the striatum ipsilateral to the tracer injections, only 34.8% of the synaptic contacts formed by corticostriatal afferents involved striatin-positive elements (slightly labelled dendritic spines), whereas 56.7% of the synaptic contacts formed by thalamostriatal boutons were made on striatin-positive targets (mostly dendrites). In both cases, striatin labelling was usually not associated with the postsynaptic density. Most of the immunoreactive dendritic spines were in contact with unidentified afferents. These data reveal that striatin is expressed in the vast majority of the cell bodies of striatal spiny neurons, but is heterogeneously distributed among the dendritic spines of those neurons. Data also indicate a preferential relationship between striatin-containing structures and afferents from the parafascicular thalamic nucleus with respect to the frontoparietal cerebral cortex. But, at the dendritic spine level, striatin may be involved in signal transduction mechanisms involving as yet unidentified excitatory afferents to striatal neurons.

Projections from the accessory optic system and pretectum to the dorsolateral thalamus in the pigeon ( Columbia livia ): A study using both anterograde and retrograde tracers

In birds, optic flow is analyzed by two retinal-recipient nuclei: the nucleus of the basal optic root (nBOR) of the accessory optic system (AOS), and the pretectal nucleus, lentiformis mesencephali (LM). Previous anatomical studies have shown that both of these nuclei have descending projections to structures involved in oculomotor, head movement, and postural control. In this report, using biotinylated dextran amine (BDA) and cholera toxin subunit B (CTB) for anterograde and retrograde labelling, respectively, we investigated projections from the nBOR and LM to the dorsal thalamus. After injections of BDA into the nBOR and LM, terminals were consistently found in the nucleus dorsolateralis anterior pars lateralis and pars medialis, and the nucleus dorsalis intermedius ventralis anterior of the thalamus. Some terminals were also found in the nucleus dorsolateralis anterior, nucleus dorsomedialis anterior pars magnocellularis, nucleus dorsolateralis posterior, nucleus superficialis parvocellularis, and the ventrointermediate area. Injections of CTB into the dorsal thalamus resulted in retrogradely labelled cells in the pretectal region, including LM. Numerous cells were also seen in the nBOR pars lateralis and pars dorsalis, but fewer were seen in the nBOR proper. We suggest that the AOS is providing input to a thalamotelencephalic system that may be involved in several functions including: (1) multi-sensory analysis of self-motion, (2) perception of self-motion, (3) perception of the three-dimensional layout of the environment, (4) distinguishing object-motion from self-motion, and (5) spatial cognition.

Direct projections from the periaqueductal gray to pontine micturition center neurons projecting to the lumbosacral cord segments: an electron microscopic study in the rat

Direct projections from the periaqueductal gray (PAG) to the pontine micturition center neurons directly projecting to the lumbosacral cord segments were observed electron microscopically in the rat by a double labeling method. Biotinylated dextran amine (BDA) was injected into the PAG and horseradish peroxidase (HRP) was injected into the lumbosacral cord segments. After injection of BDA into the ventrolateral part of the PAG, many BDA-labeled axons were seen light microscopically in Barrington's nucleus; a moderate number of them were found in the pontine tegmental region just ventral to Barrington's nucleus (D-region [Ding, Y-Q., Takada, M., Tokuno, H. and Mizuno, N., J. Comp. Neurol., 357 (1996) 318–330]). On the other hand, after injection of BDA into the dorsolateral part of the PAG, only a few BDA-labeled axons were seen in Barrington's nucleus or the D-region. BDA-labeled axon terminals were electron microscopically confirmed to be in synaptic contact with HRP-labeled dendrites and somata in Barrington's nucleus and the D-region. The results indicate that the ventrolateral part of the PAG is implicated in regulation of the micturition reflex.

Synaptic targets of pyramidal neurons providing intrinsic horizontal connections in monkey prefrontal cortex.

xũ I sLxxJ In monkey prefrontal cortex, the intrinsic axon collaterals of supragranular pyramidal neurons extend horizontally for considerable distances through the gray matter and give rise to stripe-like clusters of axon terminals (Levitt et al. [1993] J. Comp. Neurol. 338:360-376). Because understanding the functional role of these connections requires knowledge of their synaptic targets, we made injections of biotinylated dextran amine (BDA) into layer 3 of macaque prefrontal area 9 and examined the labeled intrinsic axon collaterals by electron microscopy. Labeled axon terminals formed exclusively asymmetric synapses, and 95.6% of the postsynaptic structures were dendritic spines, presumably belonging to other pyramidal neurons. The remaining postsynaptic structures were dendritic shafts, many of which had the morphological characteristics of local circuit neurons. The prefrontal injections also labeled associational projections that traveled through the white matter to terminate in other areas of prefrontal cortex. All of the synapses formed by these associational axons were asymmetric, and 91.9% were onto dendritic spines. The similarities in synaptic targets of the prefrontal intrinsic and associational axon terminals suggested that these projections might arise from the same neurons, an interpretation confirmed in dual label, retrograde tracing studies. To determine the specificity of the synaptic targets of these prefrontal connections, two additional comparisons were made. In the posterior parietal cortex (area 7a), 94.2% of the synapses furnished by BDA-labeled intrinsic collaterals of supragranular pyramidal neurons were also with dendritic spines. In contrast, only 75.6% of unlabeled asymmetric synapses in the prefrontal cortex were onto dendritic spines. These comparisons suggest that the axons of supragranular pyramidal neurons in primate association cortices are preferentially directed to specific targets. Finally, after injections of BDA, a small number of retrogradely labeled pyramidal neurons were observed within the anterogradely labeled clusters of intrinsic axon terminals. At the ultrastructural level, synapses between anterogradely labeled axon terminals and retrogradely labeled dendritic spines were identified. These findings suggest that reciprocal, monosynaptic connections may exist between pyramidal neurons located in different stripe-like clusters, providing a potential anatomical substrate for reverberating excitatory circuits within the primate association cortices.

Direct projections from the lumbosacral spinal cord to Barrington's nucleus in the rat: a special reference to micturition reflex.

In the present study, direct projections from the lumbosacral cord to Barrington's nucleus in the rat were investigated by using retrograde and anterograde tracing techniques. After injection of cholera toxin B subunit (CTb) into Barrington's nucleus, a number of moderately CTb-labeled neurons were observed in the lumbosacral cord, with a slight ipsilateral dominance; most were located in the spinal parasympathetic and dorsal commissural nuclei of the lumbosacral cord. In addition, some retrogradely labeled neurons were found in the periaqueductal gray (PAG). These findings were confirmed by an anterograde labeling experiment. After biotinylated dextran amine (BDA) was injected into the lumbosacral cord, dense BDA-labeled axon terminals were found in Barrington's nucleus as well as in the PAG. Injection of BDA into the PAG resulted in many BDA-labeled terminals in Barrington's nucleus. The present results provided clear evidence for a direct projection from the spinal parasympathetic and dorsal commissural nuclei to Barrington's nucleus that could subserve conveying bladder-filling information from the lumbosacral cord to Barrington's nucleus in the micturition reflex of the rat.

Organization of the nigro-tecto-bulbar pathway to the parvicellular reticular formation: a light- and electron-microscopic study in the rat.

We examined a nigro-tecto-bulbar pathway to the parvicellular reticular formation (RFp), where many premotor neurons for orofacial motor nuclei are known to be distributed, by using a combined anterograde and retrograde tracing method. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the substantia nigra (SNr) and cholera toxin B subunit (CTb) into the RFp, overlapping distributions of BDA-labeled terminals and CTb-labeled neuronal cell bodies were found in the lateralmost part of the superior colliculus (SC) ipsilateral or contralateral to the site of BDA injection or CTb injection, respectively. After contralateral injections of BDA into the SNr and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) injection into the RFp, ipsilateral labeled axon terminals with BDA were found to make symmetrical synaptic contacts with the somata and dendrites of contralateral labeled neurons with WGA-HRP in the lateralmost part of the SC. Furthermore, we demonstrated that BDA-labeled axon terminals were immunoreactive for GABA, by using the anterograde tracing method combined with immunohistochemistry for GABA. Thus, GABA-like immunoreactive fibers originating from the dorsolateral part of the SNr make monosynaptic contacts with the tectal neurons sending their axons to the RFp.

Demonstration of the corticotectobulbar pathway from the orofacial motor cortex to the parvicellular reticular formation in the rat

We examined a corticotectobulbar pathway from the orofacial motor cortex (OfM) to the parvicellular reticular formation (RFp), where numerous premotor neurons for the orofacial motor nuclei were known to be distributed, light and electron microscopically by using a combination of anterograde and retrograde tracing techniques. After contralateral injections of biotinylated dextranamine (BDA) into the OfM and cholera toxin B subunit (CTb) into the RFp, the overlapping distribution of ipsilateral axon terminals labeled with BDA and contralateral neurons labeled with CTb was found in the lateralmost part of the superior colliculus (SC). Furthermore, contralateral injections of BDA into the OfM and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the RFp resulted in that ipsilateral axons labeled with BDA made asymmetrical synaptic contacts with the dendrites of contralateral SC neurons labeled with WGA-HRP.

Neuronal and transneuronal degeneration of auditory axons in the brainstem after cochlear lesions in the chinchilla: cochleotopic and non-cochleotopic patterns

Terminal axonal degeneration in the brain following cochlear lesions was studied with the Nauta-Rasmussen method. Losses of hair cells and myelinated cochlear fibers were assessed. The cochleotopic map projected, from apex to base, on the ventral-to-dorsal axes of the cochlear nuclei. The cochleotopic correspondence was better for loss of cochlear nerve fibers and inner hair cells, than for outer hair cells. Cochlear fibers were traced to all parts of the cochlear nucleus, including the small-cell shell, also to cell-group Y and the flocculus. Terminal axonal degeneration in nuclei of the superior olivary complex, lateral lemniscus, and inferior colliculus was interpreted as transynaptic, since degenerated axons could not be traced to these locations from the cochlear nerve or trapezoid body. Moreover, biotinylated dextran amine injection in the basal turn of scala media of a normal cochlea labeled cochlear nerve fibers projecting to the high-frequency regions of the cochlear nuclei and to the flocculus, but not to more central auditory nuclei. This is the first detailed account of transynaptic degeneration in the ascending auditory pathway resulting from cochlear damage in an adult mammal. These findings are consistent with a dystrophic process depending on hair-cell loss and/or direct damage to cochlear nerve fibers.

Corticostriatal innervation of the patch and matrix in the rat neostriatum

The distribution of rat corticostrial axons in the patch (striosome) and matrix compartments of the neostriatum was studied by using axonal labeling with biotinylated dextran amine (BDA) and identifying patch and matrix in the same section with calbindin immunocytochemistry. Small injections of BDA were made in the anterior cingulate, medial agranular, lateral agranular, or somatosensory cortex. Each area projected to both the patch and matrix compartments, except for the somatosensory cortex, which had only matrix projections. Within the remaining cortical areas, injections in layers Vb and VI preferentially labeled axons in patches whereas injections in layers III-Va preferentially labeled matrix axons. Axons from these injections formed varicosities preferentially, but not exclusively, in one compartment. There was a population of axons that crossed compartmental boundaries and arborized in both patch and matrix. Two distinct patterns of corticostriatal axonal arborizations were observed. Small, discrete foci of innervation were seen in the patch compartment and in some regions of the matrix. The focal arborizations in the matrix were observed through the rostrocaudal extent of the neostriatum but were most obvious in the caudal one-third. They resembled the matrisomes observed in cat and primate corticostriatal projections. The second pattern of innervation consisted of extended axonal arborizations that covered large regions of the rostral neostriatal matrix. These results support the concept of multiple classes of corticostriatal neurons having different targets within the neostriatum, following different topographical rules, and having different but overlapping distributions across cortical areas. © 1996 Wiley-Liss, Inc.

Corticostriatal innervation of the patch and matrix in the rat neostriatum.

The distribution of rat corticostrial axons in the patch (striosome) and matrix compartments of the neostriatum was studied by using axonal labeling with biotinylated dextran amine (BDA) and identifying patch and matrix in the same section with calbindin immunocytochemistry. Small injections of BDA were made in the anterior cingulate, medial agranular, lateral agranular, or somatosensory cortex. Each area projected to both the patch and matrix compartments, except for the somatosensory cortex, which had only matrix projections. Within the remaining cortical areas, injections in layers Vb and VI preferentially labeled axons in patches whereas injections in layers III-Va preferentially labeled matrix axons. Axons from these injections formed varicosities preferentially, but not exclusively, in one compartment. There was a population of axons that crossed compartmental boundaries and arborized in both patch and matrix. Two distinct patterns of corticostriatal axonal arborizations were observed. Small, discrete foci of innervation were seen in the patch compartment and in some regions of the matrix. The focal arborizations in the matrix were observed through the rostrocaudal extent of the neostriatum but were most obvious in the caudal one-third. They resembled the matrisomes observed in cat and primate corticostriatal projections. The second pattern of innervation consisted of extended axonal arborizations that covered large regions of the rostral neostriatal matrix. These results support the concept of multiple classes of corticostriatal neurons having different targets within the neostriatum, following different topographical rules, and having different but overlapping distributions across cortical areas.

Thalamic collaterals of corticostriatal axons: their termination field and synaptic targets in cats.

Branched cortical projections to the thalamus and striatum were investigated in cats by injecting the retrograde-anterograde tracer biotinylated-dextran amine (BDA) into the caudate nucleus. These injections gave rise to plexuses of labeled fibers and varicosities in widespread thalamic territories. For instance, the lateroposterior nucleus and pulvinar (LP-PUL) mostly contained thick axons that contributed clusters of large-sized varicosities, each forming multiple asymmetric synapses, usually with vesicle-filled dendrites. In contrast, the intralaminar nuclei mostly contained thin axonal segments that emitted small en passant varicosities that formed single asymmetric synapses with spines. Because the caudate nucleus does not project to the thalamus, this labeling had to arise from a neuronal population with branching axons to both structures. Previous findings pointed to three possible sources: brainstem monoaminergic cells, intralaminar thalamic neurons, and corticostriatal cells. The first candidate could be ruled out because monoaminergic neurons contribute small-sized terminals that usually lack membrane specializations. The second possibility was discarded because retrograde tracer injections into the LP-PUL did not give rise to retrograde labeling in the intralaminar nuclear complex but to massive retrograde labeling in deep layers of cortical areas 5 and 7. Therefore, we concluded that the thalamic anterograde labeling originated from corticostriatal neurons, with axons branching to the thalamus. In keeping with this conclusion, Phaseolus vulgaris-leucoagglutinin (PHA-L) injections into cortical areas 5-7 labeled a group of thick corticothalamic fibers that ended in clusters of large boutons in the LP-PUL. These PHA-L-positive terminals were indistinguishable from those labeled after injections of BDA into the caudate nucleus, but they were easy to distinguish from the typical corticothalamic fibers. These findings indicate that the cerebral cortex could coordinate the activity of the striatum and the thalamus via a rich axonal network that collateralizes to both structures. The extent and synaptic organization of this branched projection impose a revision of the traditional scheme of thalamic connectivity.

Anatomy of the ventral nucleus of the lateral lemniscus in rats: A nucleus with a concentric laminar organization

The lateral lemniscus contains relay nuclei of the auditory pathway in which the neurons have been grouped into dorsal and ventral (VNLL) nuclei. The data about the cytoarchitecture of the VNLL are controversial and no agreement exists concerning its tonotopical organization. In this paper, the cytoarchitecture of VNLL and the spatial distribution of its neurons projecting to the central nucleus of the inferior colliculus (CNIC) have been studied by using different tracers. Rats were iontophoretically injected in the CNIC and grouped in three sets. Group 1 rats received large injections of biotinylated dextran amine (BDA). Group 2 animals received restricted single injections of BDA in the low-, medium-, or high-frequency regions of the CNIC. Group 3 rats were double injected, with horseradish peroxidase placed in the high-frequency region of the CNIC, and with biocytin in the low-frequency one. The distribution of retrogradely labeled neurons in the ipsilateral VNLL was three-dimensionally reconstructed by use of a computer microscope. The analysis of labeled neurons and Nissl material suggests that the VNLL contains flat stellate neurons. Labeled flat stellate neurons and fibers are oriented in parallel and form fibrodendritic laminae. The projection from the VNLL to the CNIC is topographically organized: neurons in peripheral laminae project to dorsolateral, low-frequency regions of the CNIC, and those of central laminae project to ventromedial, high-frequency regions. Each VNLL lamina forms a continuous ventrodorsal structure which resembles a helicoid. © 1996 Wiley-Liss, Inc.

Anatomy of the ventral nucleus of the lateral lemniscus in rats: a nucleus with a concentric laminar organization.

The lateral lemniscus contains relay nuclei of the auditory pathway in which the neurons have been grouped into dorsal and ventral (VNLL) nuclei. The data about the cytoarchitecture of the VNLL are controversial and no agreement exists concerning its tonotopical organization. In this paper, the cytoarchitecture of VNLL and the spatial distribution of its neurons projecting to the central nucleus of the inferior colliculus (CNIC) have been studied by using different tracers. Rats were iontophoretically injected in the CNIC and grouped in three sets. Group 1 rats received large injections of biotinylated dextran amine (BDA). Group 2 animals received restricted single injections of BDA in the low-, medium-, or high-frequency regions of the CNIC. Group 3 rats were double injected, with horseradish peroxidase placed in the high-frequency region of the CNIC, and with biocytin in the low-frequency one. The distribution of retrogradely labeled neurons in the ipsilateral VNLL was three-dimensionally reconstructed by use of a computer microscope. The analysis of labeled neurons and Nissl material suggests that the VNLL contains flat stellate neurons. Labeled flat stellate neurons and fibers are oriented in parallel and form fibrodendritic laminae. The projection from the VNLL to the CNIC is topographically organized: neurons in peripheral laminae project to dorsolateral, low-frequency regions of the CNIC, and those of central laminae project to ventromedial, high-frequency regions. Each VNLL lamina forms a continuous ventrodorsal structure which resembles a helicoid.

A nigro-rubro-bulbar pathway to the parvicellular reticular formation in the rat.

A possible pathway from the substantia nigra pars reticulata (SNr) to the parvicellular reticular formation (RFp) via the red nucleus (RN) was examined light and electron microscopically by combining anterograde and retrograde tracing techniques. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the SNr and cholera toxin B subunit (CTb) into the RFp, many CTb-labelled neurones were distributed contralaterally in the dorsal part of the RN, where numerous BDA-labelled axon terminals originating from the ipsilateral SNr were found. After contralateral injections of BDA into the dorsolateral part of the SNr and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the RFp, ipsilateral axons labelled with BDA were found to make synaptic contacts with the somata and dendrites of contralateral neurones labelled with WGA-HRP in the dorsal part of the RN.

1647 A cortico-tecto-bulbar pathway to the parvicellular reticular formation in the rat: combined anterograde and retrograde tracing study

This study has revealed direct projections from the dorsal peduncular cortex (DP) in the medial prefrontal cortex (mPfC) to the trigeminal brainstem sensory nuclear complex and other lower brainstem areas in rats. We first examined the distribution of mPfC neurons projecting directly to the medullary dorsal horn (trigeminal subnucleus caudalis [Vc]) and trigeminal subnucleus oralis (Vo) which are known to receive direct projections from the lateral prefrontal cortex (insular cortex). After injections of the retrograde tracer Fluorogold (FG) into the rostro-dorsomedial part of laminae I/II of Vc (rdm-I/II-Vc), many neurons were labeled bilaterally (with an ipsilateral predominance) in the rostrocaudal middle level of DP (mid-DP) and not in other mPfC areas. After FG injections into the lateral and caudal parts of laminae I/II of Vc, or the Vo, no neurons were labeled in the mPfC. We then examined projections from the mid-DP by using the anterograde tracer biotinylated dextranamine (BDA). After BDA injections into the mid-DP, many axons and terminals were labeled bilaterally (with an ipsilateral predominance) in the rdm-I/II-Vc, periaqueductal gray and solitary tract nucleus, and ipsilaterally in the parabrachial nucleus and trigeminal mesencephalic nucleus. In addition, the connections of the mid-DP with the insular cortex were examined. Many BDA-labeled axons and terminals from the mid-DP were also found ipsilaterally in the caudalmost level of the granular and dysgranular insular cortex (GI/DI). After BDA injections into the caudalmost GI/DI, many axons and terminals were labeled ipsilaterally in the mid-DP. The projections from the mid-DP to the rdm-I/II-Vc and other brainstem nuclei suggest that mid-DP neurons may regulate intraoral and perioral sensory processing (including nociceptive processing) of rdm-I/II-Vc neurons directly or indirectly through the brainstem nuclei. The reciprocal connections between the mid-DP and caudalmost GI/DI suggest that this regulation may involve mid-DP interactions with the caudalmost GI/DI neurons.

Connections of the Olfactory Bulb in the Chinook Salmon <i>(Oncorhynchus tshawytscha)</i>

This report describes the connections of the olfactory bulb in juvenile chinook salmon (Oncorhynchus tshawytscha) as revealed by the retrograde and anterograde transport of biotinylated dextran amine (BDA). Pressure injections of BDA to discrete locations within the bulb reveal that the olfactory bulb projects via medial and lateral tracts to various regions of the dorsal and ventral telencephalon as well as to the diencephalon. Terminal-like boutons are present in the ventral, dorsal, lateral and supracommissural nuclei of the ventral telencephalon and in the lateral-ventral and posterior zones of the dorsal telencephalon. There is also a projection to the nucleus taenia and to the contralateral olfactory bulb. A diencephalic terminal field occurs in the posterior tuberal region of the ventral diencephalon. Olfactory bulb fibers in the preoptic region have axonal varicosities near neurons containing releasing hormones. A synaptic connection is suggested allowing for the possibility that output from the olfactory bulb may directly modulate the activity of these neurons. Telencephalic injections of BDA are used to determine the origins of olfactory bulb efferents. Neurons are retrogradely labeled by BDA in a Golgi-like manner, allowing one to visualize the morphology of olfactory bulb efferents. Three types of neurons in the bulb are filled by injections into the posterior zone of the dorsal telencephalon: mitral cells and ruffed cells in the external cellular layer, and neurons in the internal cellular layer. Although it has been known that both mitral cells and neurons in the internal cellular layer project out of the bulb in teleosts, the use of BDA allowed for an accurate description of the morphology of these extrinsically projecting neurons. This is the first evidence that the ruffed cells in salmon project out of the bulb. The results of this study suggest that the secondary olfactory projections in chinook salmon are consistent with those in other teleosts with minor variations. The data also show that olfactory bulb efferents originate not solely from mitral cells in the external cellular layer but also from ruffed cells in the external cellular layer and neurons in the internal cellular layer. It is proposed that the extrinsically projecting neurons in the internal cellular layer constitute an anterior olfactory nucleus in salmon.

Nucleus reticularis thalami connections with the mediodorsal thalamic nucleus: A light and electron microscopic study in the monkey

Wheat germ agglutinin conjugated horseradish peroxidese (WGA-HRP) and biotinylated dextran amine (BDA) were used as tracers to study nucleus reticularis (NRT) connections with the mediodorsal nucleus (MD). Injections of WGA-HRP in the MO resulted in retrograde labeling of cells in the anteromedial segment of the NRT, the so-called rostral NRT pole. Injections of WGA-HRP and BDA in this NRT region resulted in dense anterograde labeling in the MD. Labeled NRT fibers gave off several collaterals to different MD regions ending with terminal plexuses of thin varicose fibers. In the neuropil, the varicosities were distributed at random, and no tendency to form pericellular baskets was noted. Postembedding immunocytochemistry for GABA was performed on the tissue containing anterograde WGA-HRP label for identification of NRT boutons under electron microscope. The double-labeled boutons were of small to medium size, contained a large number of pleomorphic vesicles, few mitochondria, and formed multiple symmetric synaptic contacts. The number of contacts established by one bouton ranged from 1 to 4 with an average of 1.8 per bouton. About 60% of these boutons made synapses on distal dendrites of GABAergic local circuit neurons; 33% of synaptic contacts were on distal dendrites of thalamocortical neurons, and the rest on their proximal dendrites and soma. NRT boutons were also found in serial synapses and triads. The results demonstrate that the NRT input to the MD is organized so that a single fiber innervates different MD regions and its terminals form numerous synaptic contacts mostly on the distal dendrites of a large number of local circuit neurons and projection neurons.

The cortico-pallidal projection in the rat: an anterograde tracing study with biotinylated dextran amine

The projections from the frontal cortex to the pallidum (e.g. the globus pallidus and the entopenducular nucleus) were studied in the rat using the biotinylated dextran amine (BDA) anterograde tracing method. Injections of BDA into the precentral medial and precentral lateral cortices consistently yielded labeling of thin fibers with small boutons in the ipsilateral pallidium although not in the contralateral pallidum. Electron microscopic observation revealed that BDA-labeled boutons form asymmetric synapses mainly with small dendrites and dendritic spines. When the injection sites included the insular, orbital, and prelimbic cortices, the labeled fibers and boutons were also seen in the ventral pallidum, the basal nucleus of Meynert, and the substantia innominata. Injections of BDA in the parietal and occipital cortices resulted in either very few labeling or no labeled boutons in the pallidum. The cortico-pallidal projections were topographically organized. The density of labeled boutons in the globus pallidus was found to be approximately 10% of the density of cortical terminals in the neostriatum.

The cortico-nigral projection in the rat: an anterograde tracing study with biotinylated dextran amine

The projection from the cortex to the substantia nigra (SN) in the rat was studied using the biotinylated dextran amine (BDA) anterograde tracing method. Injections of BDA into the prefrontal cortex consistently yielded labeling of the thin nerve fibers with small boutons in the SN pars compacta (SNc) and the pars reticulata (SNr). The cortico-nigral projection had a loose medio-lateral and rostro-caudal arrangement. Injections of BDA into the precentral lateral, the parietal, the temporal, and the occipital cortices resulted in sparse or no labeling of boutons in the SN. The density of boutons in the most heavily labeled region of the SN was 0.23/10 3 μm 3. For comparison, similar measurement was performed in the cortico-striatal projection. The density of the boutons in the neostriatum was 4.9/10 3 μm 3. This study indicates that the entire prefrontal cortex projects to the SNc and SNr and that the terminal density of this projection is much less than the cortico-striatal projection.

Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies

Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.