Anterograde Axonal Tracing Research Articles

The organization of the thalamocortical connections of the mediodorsal thalamic nucleus in the rat, related to the ventral forebrain–prefrontal cortex topography

The medial and central segments of the mediodorsal nucleus of the thalamus (MD) receive afferents from the ventral forebrain, including the piriform cortex, the ventral pallidum, and the amygdaloid complex. Because MD is reciprocally interconnected with prefrontal and agranular insular cortical areas, it provides a relay of ventral forebrain activity to these cortical areas. However, there are also direct projections from the piriform cortex and the amygdala to the prefrontal and agranular insular cortices. This study addresses whether this system has a "triangular" organization, such that structures in the ventral forebrain project to interconnected areas in MD and the prefrontal/insular cortex. The thalamocortical projections of MD have been studied in experiments with injections of retrograde tracers into prefrontal or agranular insular cortical areas. In many of the same experiments, projections from the ventral forebrain to MD and to the prefrontal/insular cortex have been demonstrated with anterograde axonal tracers. The connections of the piriform cortex (PC) with MD and the prefrontal/insular cortex form an organized triangular system. The PC projections to the central and medial segments of MD and to the lateral orbital cortex (LO) and the ventral and posterior agranular insular cortices (AIv and AIp) are topographically organized, such that more caudal parts of PC tend to project more medially in MD and more caudally within the orbital/insular cortex. The central and medial portions of MD also send matching, topographically organized projections to LO, AIv and AIp, with more medial parts of MD projecting further caudally. The anterior cortical nucleus of the amygdala (COa) also projects to the dorsal part of the medial segment of MD and to its cortical targets, the medial orbital area (MO) and AIp. The projections of the basal/accessory basal amygdaloid nuclei to MD and to prefrontal cortex, and from MD to amygdaloceptive parts of prefrontal cortex, are not as tightly organized. Amygdalothalamic afferents in MD are concentrated in the dorsal half of the medial segment. Cells in this part of the nucleus project to the amygdaloceptive prelimbic area (PL) and AIp. However, other amygdaloceptive prefrontal areas are connected to parts of MD that do not receive fibers from the amygdala. Ventral pallidal afferents are distributed to all parts of the central and medial segments of MD, overlapping with the fibers from the amygdala and piriform cortex. Fibers from other parts of the pallidum, or related areas such as the substantia nigra, pars reticulata, terminate in the lateral and ventral parts of MD, where they overlap with inputs from the superior colliculus and other brainstem structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Efferent connections of the centromedian and parafascicular thalamic nuclei in the squirrel monkey: a light and electron microscopic study of the thalamostriatal projection in relation to striatal heterogeneity.

The organization of the thalamostriatal projections arising from the centromedian (CM) and parafascicular (Pf) thalamic nuclei in the squirrel monkey (Saimiri sciureus) was studied at both light and electron microscopic levels. Following selective injections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the CM or Pf, patterns of terminal arborization within the striatum were compared to the biochemical heterogeneity of the striatum as revealed by immunohistochemical staining for the calcium-binding protein calbindin D-28k (CaBP), and histochemical staining for the enzymes acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase). The PHA-L-labeled axon terminals within the striatum were further analyzed at the ultrastructural level to characterize their pattern of synaptic organization. Dense and heterogeneous terminal fields occur in the "sensorimotor" territory of the striatum after CM injections, or in the "associative" striatal territory following Pf injections. In the associative territory labeled axons arborize in a diffuse manner predominantly within areas enriched with CaBP, AChE, or NADPH-diaphorase, representing the matrix compartment, and tend to avoid areas poor in these substances, corresponding to the patch/striosome compartment. In the sensorimotor territory labeled axons form bands that occupy a subregion of the NADPH-diaphorase-rich zone in the putamen. The terminal pattern of the CM-striatal projection suggests the existence of a more complex mosaic organization within the sensorimotor territory. Ultrastructural analysis of PHA-L-labeled elements within the striatum reveals that both CM and Pf projections form asymmetric synapses upon dendrites and spines of striatal cells. A total of 339 PHA-L-labeled boutons were examined after CM injections and compared to 293 boutons following Pf injections. After CM injections, 29% of PHA-L-labeled terminals form synapses on dendritic spines and 66% on dendritic shafts, whereas after Pf injections only 12% of synapses occur on dendritic spines compared to 81% on dendritic shafts. Labeled terminals forming axosomatic or axoaxonic synapses were not seen within the striatum following either CM or Pf injections. It is concluded that in the squirrel monkey: 1) Pf-striatal fibers profusely arborize within the matrix compartment of the associative territory, 2) CM-striatal fibers form bands that occupy a subregion of the NADPH-diaphorase-rich zone within the sensorimotor territory, and 3) that both Pf- and CM-striatal projections establish asymmetric synapses with dendrites and spines of medium-sized spiny cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct projections from the globus pallidus to the midbrain and pons in the cat

Employing the anterograde and retrograde axonal tracing techniques with Phaseolus vulgaris leucoagglutinin and cholera toxin B subunit, we demonstrated direct projections from the globus pallidus (GP) to the midbrain and pons in the cat. Cells of origin of these projections were localized in the caudal 2 3 of the GP, and their major target sites included the peripeduncular region, nucleus of the brachium of the inferior colliculus, para-lateral lemniscal zone, nucleus sagulum, external and pericentral nuclei of the inferior colliculus, and cuneiform nucleus. A combination of retrograde axonal tracing and immunohistochemistry for choline acetyltransferase revealed that GP neurons giving rise to such descending projections were primarily non-cholinergic.

Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat.

The periaqueductal or midbrain central gray matter (CG) in the rat contains a dense network of adrenergic and noradrenergic fibers. We examined the origin of this innervation by using retrograde and anterograde axonal tracers combined with immunohistochemistry for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). Following injections of the fluorescent tracers Fast Blue or Fluorogold into the CG, double-labeled neurons in the medulla were identified mainly in the noradrenergic A1 group in the caudal ventrolateral medulla (VLM) and A2 group in the medial part of the nucleus of the solitary tract (NTS); and in the adrenergic C1 group in the rostral ventrolateral medulla and C3 group in the rostral dorsomedial medulla. Injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into these cell groups resulted in a distinct pattern of axonal labeling in various subdivisions of the CG. Anterogradely labeled fibers originating in the medial NTS were predominantly found in the lateral portion of the dorsal raphe nucleus and in the adjacent part of the lateroventral CG (CGlv). Following PHA-L injections into the C3 region the anterogradely labeled fibers were diffusely distributed in the CGlv and the dorsal raphe nucleus at caudal levels, but rostrally tended to be located laterally in the CGlv. In contrast, ascending fibers from the caudal and rostral VLM terminated in the rostral dorsal part of the CGlv and in the dorsal nucleus of the CG, whereas ventral parts of the CG, including the dorsal raphe nucleus, contained few afferent fibers. Double-label studies with antisera against DBH and PNMT confirmed that noradrenergic neurons in the A1 and A2 groups and adrenergic neurons in the C1 and C3 groups contributed to these innervation patterns in the CGlv. Noradrenergic and adrenergic projections from the medulla to the CG may play an important role in a variety of autonomic, sensory and behavioral processes.

Evaluation of projection patterns in the primary olfactory system of rainbow trout

Topographic projections are important for coding sensory information in the visual, auditory, and somatosensory systems but are of uncertain importance in the coding of olfactory information. We searched for topographic projections between olfactory receptor cells and the olfactory bulb of the rainbow trout Oncorhynchus mykiss. Anterograde axonal tracing with HRP revealed that the olfactory axons arising from discrete regions of the olfactory epithelium travel together within the olfactory nerve. The abrupt resorting and redistribution of these axons at the interface between the olfactory nerve and olfactory bulb imply that local cues control and organize axonal projections. The sites of termination of HRP-labeled axons in the glomerular layer could not be predicted from the location of their cell bodies in the periphery. Retrograde tracing with fluorescently labeled latex beads, injected into glomerular subregions as small as 1% of the total glomerular volume, labeled receptor cells dispersed throughout the olfactory epithelium. The distributions of labeled receptor cells were uncorrelated with the bulbar injection sites. Double-labeling experiments revealed that even widely separated sites in the glomerular layer receive axons from comingled populations of receptor cells. Hence, the evidence indicates that the spatial arrangement of olfactory receptor cells in the epithelium is not preserved in the termination of their axons in the olfactory bulb. We conclude that the primary olfactory in trout lacks point-to-point or regionally topographic organization and that the entire extent of the olfactory epithelium contributes axons to each region of the glomerular layer.

Olfactory projections to the hypothalamus

Electrophysiological recording, together with anterograde and retrograde axonal tracers, was used to provide a comprehensive description of the origin and distribution of the olfactory input to the lateral hypothalamus. This input was much more substantial to the caudal part of the hypothalamus than to the rostral part and originates from several different areas of the olfactory cortex. Positive responses to electrical stimulation of the olfactory bulb were found consistently in the postero-lateral hypothalamus, but only occasionally at more rostral levels. In agreement with this, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in the posterior half of the lateral hypothalamus labeled cells in four cortical areas that receive input from the olfactory bulb: the anterior olfactory nucleus, the piriform cortex (in the deepest layer or ventral endopiriform nucleus), the olfactory tubercle (in the deep polymorphic layer), and the anterior cortical nucleus of the amygdala. Injections of WGA-HRP in the anterolateral hypothalamus labeled cells only in the anterior cortical nucleus of the amygdala. Anterograde axonal tracing confirmed these projections. Injections of 3H-leucine in the anterior olfactory nucleus, the piriform cortex, and the olfactory tubercle produced axonal label that was light and confined to the medial forebrain bundle in the rostral hypothalamus but was more substantial and extended throughout the lateral hypothalamic area caudally. Injections in the anterior cortical amygdaloid nucleus labeled axons in the anterior hypothalamus and in the premammillary nuclei as well as in the posterolateral hypothalamic area. In addition, a projection was demonstrated to the nuclei gemini from the polymorphic zone deep to the olfactory tubercle. Injections of two fluorescent retrograde tracers into the mediodorsal nucleus of the thalamus and the posterolateral hypothalamus showed that cells projecting to both diencephalic sites were intermingled in all of the olfactory cortical areas except the anterior olfactory nucleus, where cells were labeled only from the hypothalamus. In the deep layer of the piriform cortex and in the anterior cortical amygdaloid nucleus cells were also double labeled, indicating that they send collateral axons to both parts of the diencephalon.

Projections from the periaqueductal gray to the rostromedial pericoerulear region and nucleus locus coeruleus: Anatomic and physiologic studies

Previous studies showed that the nucleus locus coeruleus (LC) receives two major afferent inputs from 1) nucleus paragigantocellularis and 2) nucleus prepositus hypoglossi, both in the rostral medulla. Recent reports suggested that the midbrain periaqueductal gray (PAG) projects to the rostromedial pericoerulear area and LC. Since the PAG is a major site for control of central antinociception, and since descending noradrenergic fibers have been implicated in pain modulation, we have investigated in detail the functional anatomy of projections from PAG to the dorsolateral pontine tegmentum. A combined anatomical and electrophysiological approach was used to assess the organization and synaptic influence of PAG on neurons in the rostromedial pericoerulear region and in LC proper. Injections of the tracer wheatgerm agglutinin conjugated to horseradish peroxidase encompassing LC proper and the rostromedial pericoerulear area retrogradely labeled neurons in PAG located lateral and ventrolateral to the cerebral aqueduct; injections restricted to LC proper did not consistently label PAG neurons. Deposits of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into this same region of PAG labeled axons that robustly innervated the zone rostral and medial to LC. Only sparse fibers were observed in LC proper. Consistent with these results, focal electrical stimulation of LC antidromically activated only a few PAG neurons (6 of 100); all of these driven cells were located lateral and ventrolateral to the cerebral aqueduct. The majority of neurons in the rostromedial pericoerulear area were robustly activated by single pulse stimulation of PAG. In contrast, single pulse electrical stimulation of lateral PAG produced weak to moderate synaptic activation of some LC neurons; stimulation of ventrolateral PAG produced predominant inhibition of LC discharge, perhaps through recurrent collaterals subsequent to antidromic activation of neighboring LC cells. Taken together, these results indicate that PAG strongly innervates the region rostral and medial to LC, including Barrington's nucleus, but only weakly innervates LC proper. Although recent studies indicate that the dendrites of LC neurons ramify heavily and selectively in the rostromedial pericoerulear region, the results of the present physiological studies suggest that PAG preferentially targets rostromedial pericoerulear neurons rather than LC dendrites.

Autonomic responses and efferent pathways from the insular cortex in the rat

The anatomical distribution of autonomic, particularly cardiovascular, responses originating in the insular cortex was examined by using systematic electrical microstimulation. The localization of these responses to cell bodies in the insular cortex was demonstrated by using microinjection of the excitatory amino acid, D,L-homocysteic acid. The efferents from the cardiovascular responsive sites were traced by iontophoretic injection of the anterograde axonal tracer Phaseoleus vulgaris leucoagglutinin (PHA-L). Two distinct patterns of cardiovascular response were elicited from the insular cortex: an increase in arterial pressure accompanied by tachycardia or a decrease in arterial pressure with bradycardia. The pressor responses were obtained by stimulation of the rostral half of the posterior insular cortex while depressor sites were located in the caudal part of the posterior insular area. Both types of site were primarily located in the dysgranular and agranular insular cortex. Gastric motility changes originated from a separate but adjacent region immediately rostral to the cardiovascular responsive sites in the anterior insular cortex. Tracing of efferents with PHA-L indicated a number of differences in connectivity between the pressor and depressor sites. Pressor sites had substantially more intense connections with other limbic regions including the infralimbic cortex, the amygdala, the bed nucleus of the stria terminalis and the medial dorsal and intralaminar nuclei of the thalamus. Alternatively, the depressor region of the insular cortex more heavily innervated sensory areas of the brain including layer I of the primary somatosensory cortex, a peripheral region of the sensory relay nuclei of the thalamus and the caudal spinal trigeminal nucleus. In addition, there were topographical differences in the projection to the lateral hypothalamic area, the primary site of autonomic outflow for these responses from the insular cortex. These differences in connectivity may provide the anatomic substrate for the specific cardiovascular responses and behaviors integrated in the insular cortex.

Sources of presumptive glutamatergic/aspartatergic afferents to the magnocellular basal forebrain in the rat

The distribution of presumptive glutamatergic and/or aspartatergic neurons retrogradely labeled following injections of [3H]-D-aspartate into the magnocellular basal forebrain of the rat was compared with the distribution of neurons labeled by comparable injections of the nonspecific retrograde axonal tracer wheat germ agglutinin conjugated to horseradish peroxidase. Cells retrogradely labeled by wheat germ agglutinin-horseradish peroxidase were found in a wide range of limbic and limbic-related structures in the forebrain and brainstem. In the telencephalon, labeled neurons were seen in the orbital, medial prefrontal, and agranular insular cortical areas, the amygdaloid complex, and the hippocampal formation. Labeled cells were also seen in the olfactory cortex, the lateral septum, the ventral striatopallidal region, and the magnocellular basal forebrain itself. In the diencephalon, neurons were labeled in the midline nuclear complex of the thalamus, the lateral habenular nucleus, and the hypothalamus. In the brainstem, labeled cells were found bilaterally in the ventral midbrain, the central gray, the reticular formation, the parabrachial nuclei, the raphe nuclei, the laterodorsal tegmental nucleus, and the locus coeruleus. A significant fraction of the afferents to the magnocellular basal forebrain appear to be glutamatergic and/or aspartatergic. Only a few of the regions labeled with wheat germ agglutinin-horseradish peroxidase were not also labeled with [3H]-D-aspartate in the comparable experiments. Most prominent among the non-glutamatergic/aspartatergic projections were those from fields CA1 and CA3 of the hippocampus, the hilus of the dentate gyrus, the dorsal subiculum, the tuberomammillary nucleus, and the ventral pallidum. In addition, most of the lateral hypothalamic and brainstem projections to the magnocellular basal forebrain were not significantly labeled with [3H]-D-aspartate. In addition to these inputs, a commissural projection from the region of the contralateral nucleus of the horizontal limb of the diagonal band was confirmed with both wheat germ agglutinin-horseradish peroxidase and the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin. This projection did not label with [3H]-D-aspartate or [3H]-GABA, suggesting that it is not glutamatergic/aspartatergic or GABAergic. Furthermore, double labeling experiments with the fluorescent retrograde tracer True Blue and antibodies against choline acetyltransferase indicate that the projection is not cholinergic.

Segregation of afferent projections in the central nervous system of the leech Hirudo medicinalis

Sensory axons originating in peripheral tissues converge onto each segmental ganglion in the central nervous system (CNS) of the leech, where they segregate into well-defined regions of the synaptic neuropil. Here we report on several aspects of the molecular and anatomical organizations of these afferent projections that bear upon the hypothesis that surface markers are involved in organizing these axons as they grow into the CNS. First, we show that the distribution of some surface markers in the adult is restricted to axons of peripheral origin and is not present on the neighboring axons of central neurons. Second, we demonstrate that the number of afferents increases postembryonically as the leech increases in size, suggesting that at least some of the cues employed by afferent axons to grow to appropriate central targets must be present throughout the life of the animal. We then show, using anterograde axonal tracing and immunohistochemistry, that there is both convergence and divergence of afferent axons into highly specific regions of the neuropil. Lastly, we examine the distribution of surface markers present on different subsets of afferents and show that axons having one type of marker segregate from those having the second type. Our results, considered together with previous observations in this system, provide new clues about the organization of afferent projections in the nervous system of the leech. They also suggest how a relatively small number of molecular markers might mediate fiber-fiber interactions to organize afferent axons as they grow into the CNS.

Pluripotentiality of the 2-day-old avian germinative neuroepithelium

In a previous study using chick/quail chimeric embryos with homotopic transplants ( Martinez & Alvarado-Mallart, 1989b), we have delimited in the 2-day-old avian embryo the areas of the neural tube giving rise to optic tectum and mesencephalic grissea as well as to isthmic grissea and cerebellum: respectively, “mesencephalic” and “metencephalic” alar plates. To investigate the determination or the competence of these areas, portions of these germinative neuroepithelia from a quail embryo were transplanted in substitution for other areas of the chick neural tube. The analysis of the chimeric brains was done by comparing alternating transverse sections stained for cytoarchitecture and with two different techniques to recognize transplanted versus host cells: either the Feulgen and Rossenbeck DNA histochemical reaction and/or immunohistochemical methods with a monoclonal antibody recognizing quail but not chick cells. The eventual visual innervation of the quail graft was analyzed in many cases by injecting anterograde axonal tracers in the eye contralateral to the graft. The results are as follows: (1) caudal metencephalon transferred to mesencephalon maintained in all cases its presumptive cerebellar phenotype, whereas (2) rostral metencephalon transferred to mesencephalon changed its fate to a tectal phenotype but maintained its cerebellar fate when transferred to diencephalon; (3) caudal mesencephalon maintained its tectal fate in 65% of the cases when transferred to diencephalon, whereas (4) rostral mesencephalon transferred to a cerebellar domain changed its fate and became influenced by the surrounding structures in all cases, but only in 85% of the cases when it was transplanted to diencephalon; (5) the in situ host diencephalon, isolated from its normal environment by a mesencephalic graft, is competent to change its fate and express a mesencephalic phenotype. These results demonstrate that at least some regions of the germinative neuroepithelium from either metencephalon, mesencephalon, and diencephalon are still pluripotent in the 2-day-old avian embryo and that their fate seems to be under the influence of the surrounding structures. Rostral mesencephalon and rostral metencephalon have been more easily influenced by environmental factors than their caudal counterparts, suggesting that regions providing instructive positional factors exist within the 2-day-old germinative neuroepithelium. These regions might play an important role in the determination of the various segments of the neural tube.

The topographical and laminar organization of a commissural-associational entorhino-entorhinal projection in the guinea pig

A commissural-associational entorhino-entorhinal projection in the guinea pig was analyzed using anterograde and retrograde axonal tracing techniques. The projection originated in layer II and terminated in layer Ia of both the medial entorhinal area (MEA) and the lateral entorhinal area (LEA). A few cells in other layers, especially layer III, also contributed to the commissural system. The protection was largely homotopic with the exception of the most medial MEA, which projected ventrally like previously described projections from the para- and presubiculum to the superficial layers of the entorhinal area. The commissural fibers crossed the midline in the dorsal hippocampal commissure. The antero-posterior position of the fibers within the commissure reflected the ventrodorsal position of their origin in the entorhinal area.

Intrinsic Organization and Connectivity of Intrastriatal Striatal Transplants in Rats as Revealed by DARPP-32 Immunohistochemistry: Specificity of Connections with the Lesioned Host Brain.

Intrastriatal grafts of tissue obtained from the striatal or neocortical primordia of rat fetuses have been studied with respect to their intrinsic organization and connectivity using antibodies to DARPP-32 in combination with acetylcholinesterase (AChE) histochemistry, tyrosine hydroxylase (TH) immunocytochemistry, and anterograde and retrograde axonal tracing techniques. The striatal grafts were characterized by distinct patches of DARPP-32-immunoreactive neurons, which were identical to the densely AChE-positive patches stained in adjacent sections from the same specimens. The non-patch areas possessed only few DARPP-32-positive neurons and contained only sparse AChE-positive fibres. The cortical grafts, by contrast, contained no neurons with clear-cut DARPP-32-positivity and they exhibited a sparse, evenly distributed AChE fibre network, similar to that seen in the non-patch areas of the striatal grafts. The host dopaminergic afferents, as revealed by TH immunostaining, had grown selectively into the DARPP-32-positive patches in the striatal grafts, where they formed a dense terminal network around the DARPP-32-positive cell bodies. The non-patch areas, as well as the cortical grafts, received only sparse TH innervation. By contrast, the host cortical afferents, labelled by Phaseolus vulgaris leucoagglutinin from the host frontal cortex, were seen to extend into both the patch and non-patch areas of the striatal grafts. Transplant neurons projecting into the host brain were labelled by Fluoro-Gold injections into the ipsilateral host globus pallidus. These injections labelled large numbers of medium-sized neurons within the striatal grafts and the vast majority of them (over 85%) were confined to the DARPP-32-positive patches. Similar Fluoro-Gold injections labelled only few graft neurons in the cortical grafts. The results indicate that the striatal grafts are composed of a mixture of striatal and non-striatal tissue, and that the striatal graft compartment selectively establishes afferent and efferent connections with the host nigro-pallidal system. These graft connections demonstrate a remarkable specificity in the formation of graft - host connectivity. The results, moreover, suggest that developmental properties of the grafted striatal primordium are retained and expressed in the implanted cell suspension, and that the neuronal systems of the lesioned adult host brain, at least to some extent, remain responsive to growth regulating mechanisms normally operating during ontogenetic development.

The neostriatal mosaic: striatal patch-matrix organization is related to cortical lamination.

The basal ganglia, of which the striatum is the major component, process inputs from virtually all cerebral cortical areas to affect motor, emotional, and cognitive behaviors. Insights into how these seemingly disparate functions may be integrated have emerged from studies that have demonstrated that the mammalian striatum is composed of two compartments arranged as a mosaic, the patches and the matrix, which differ in their neurochemical and neuroanatomical properties. In this study, projections from prefrontal, cingulate, and motor cortical areas to the striatal compartments were examined with the Phaseolus vulgaris-leucoagglutinin (PHA-L) anterograde axonal tracer in rats. Each cortical area projects to both the patches and the matrix of the striatum; however, deep layer V and layer VI corticostriatal neurons project principally to the patches, whereas superficial layer V and layer III and II corticostriatal neurons project principally to the matrix. The relative contribution of patch and matrix corticostriatal projections varies among the cortical areas examined such that allocortical areas provide a greater number of inputs to the patches than to the matrix, whereas the reverse obtains for neocortical areas. These results demonstrate that the compartmental organization of corticostriatal inputs is related to their laminar origin and secondarily to the cytoarchitectonic area of origin.

Proximal trajectory of the brachium conjunctivum in rat fetuses and its early association with the parabrachial nucleus. A study combining in vitro HRP anterograde axonal tracing and immunocytochemistry

The proximal course of the developing brachium conjunctivum (BC) in the rat is described from embryonic day 16 (E16) to one day postnatal (P1). Axons of the cerebellar deep nuclear neurons entering this bundle were identified by anterograde axonal tracing after in vitro horseradish peroxidase (HRP) injections in the cerebellar plate. At all ages, the main ascending limb of the BC can be followed from its emergence, dorsal to the cerebellar plate where it assumes an almost vertical course, up to its decussation. Close to the ventricle at E16, the decussating fibers are progressively displaced ventrally probably because of the fusion, on the midline, of bilaterally produced raphe neurons. In E16 and E17 embryos, labeled BC fibers extend beyond the decussation in the caudal part of the red nucleus. Decussating BC axons, in some E16 early embryos, end with large and complicated growth cones, as described previously in ‘decision regions’ for chick embryo motoneurons. Growth cones were never observed in this region in older embryos. In addition to the main ascending limb of the BC, we also traced its ipsilateral descending limb and the cerebello-olivary projections. In parallel, the development of a nucleus immunoreactive for the vitamin D-dependent calcium-binding protein (CaBP) is reported. By E16, its neurons migrate rostrally and settle in the region where the BC is demonstrated by tracing experiments. At E17 and thereafter this isthmic nucleus is composed of a shell of CaBP-immunoreactive neurons ensheathing an immunonegative cylinder. Between E17 and birth, in spite of the profound modifications of the isthmic region, this CaBP-immunoreactive nucleus remains in close proximity to the BC. This nucleus is identified as the marginal nucleus of the BC or parabrachial nucleus, by double-labeling experiments combining the visualization of the retrogradely labeled axons and neurons of the deep cerebellar nuclei inside the CaBP immunofluorescently labeled parabrachial nucleus. Subsequently the deep cerebellar neurons translocate caudoventrally moving away from the parabrachial nucleus inside which their axons become visible. This pattern of migration could indicate that a few neurons of the deep nuclei remain ectopic, wedged between the restiform body and the BC while receiving an appropriate Purkinje cell (PC) projection.

Synaptology of the cerebello-olivary pathway. Double labelling with anterograde axonal tracing and GABA immunocytochemistry in the rat

The dentato-olivary projection has been ultrastructurally studied in rats that received a wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injection in the nucleus lateralis. Ultrathin sections containing the inferior olive have been double-labelled with the GABA-immunogold method. About 97% of the WGA-HRP labelled axon terminals are GABA-immunopositive. Most of them belong to a single consisting of small boutons establishing symmetrical synapses on dendritis. Nevertheless, there is some morphological and neurochemical diversity among the labelled terminals, and particularly, a small contingent are GABA-immunonegative. Of the GABAergic dentato-olivary boutons, 4% occupy a privileged position, with synaptic contacts straddling two dendritic profiles linked by gap junctions. The strategic location of these inhibitory dentato-olivary synapses suggests that they can modulate the electronic coupling rate between sets of inferior olivary neurons.

Pattern formation in the striatum: developmental changes in the distribution of striatonigral projections

The mammalian striatum (the major subcortical structure in the telencephalon) can be divided into two compartments, the patch and the matrix, on the basis of various neurochemical and hodological markers expressed in the adult. The primary efferent target of striatal neurons is the substantia nigra. We have previously shown that the patch compartment sends projections to the substantia nigra embryonically; whereas the matrix does not form a similar projection until the early postnatal period (Fishell and van der Kooy, J. Neurosci., 7 (1987) 1969–1978). The projection of patch neurons to the substantia nigra is the earliest developmental marker for the patch compartment. Here we ask about the early distribution of patch projections and their possible relation to striatal compartmentalization. Embryonic anterograde axonal tracing of the striatonigral pathway can take advantage of the temporal separation of patch versus matrix projections to reveal the terminal distribution of patch striatonigral neurons independent of the nigral terminal distribution from the striatal matrix. The anterograde tracer rhodamine isothiocyanate was shown in a model system to persist in labeled neurons for more than a week, but to be available for uptake into these neurons for a few days after injection at the most. These properties of rhodamine isothiocyanate were combined experimentally with short and long term survival periods. This allowed assessment of the changing developmental distribution of nigral fibers from specifically the striatal patch compartment. In all experimental cases the anterogradely labeled sections of the substantia nigra were also stained with antibodies to tyrosine hydroxylase, which permitted differentiation of the dopamine cell rich pars compacta from the dopamine cell poor pars reticulata. The results show that in the adult the majority of patch and matrix striatonigral projections are confined to the substantia nigra pars reticulata. Furthermore, their fiber distribution within the pars reticulata is overlapping rather than complementary. Most interestingly, in the late embryonic period (most noticeably at embryonic day 19) there is a marked overlap between patch striatonigral fibers and nigral dopamine perikarya. By early postnatal times, when the matrix compartment begins to form its striatonigral projection, the overlap of patch striatonigral fibers and dopamine cells has largely disappeared. The results suggest that a transient interaction between patch striatonigral fibers and dopamine neurons (which is concomitant with the formation of striatal compartments), may be an important developmental event in the phenotypic maturation of striatal patch/matrix organization.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum—III. Efferent projecting graft neurons and their relation to host afferents within the grafts

Efferent projections of intrastriatally implanted striatal neurons have been studied using a combination of anterograde and retrograde axonal tracers. Adult rats subjected to a unilateral ibotenic acid lesion of the head of the caudate putamen received cell suspension grafts obtained from 14–15-day-old striatal primordia. Three and a half to 20 months after transplantation the rats received either intra-transplant injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin or injections of fluorescent retrograde tracers, Fluoro-Gold and rhodamine-labelled latex beads, into the host globus pallidus and substantia nigra. Injections of Phaseolus vulgaris leucoagglutinin located entirely within the grafts labelled axons that ramified extensively within the tissue itself, as well as axons that extended caudally, across the graft-host border, along the myelinated fascicles of the internal capsule to arborize in the medial parts of the host globus pallidus. A few axons also reached the entopeduncular nucleus. Injections of Fluoro-Gold into the host globus pallidus labelled large numbers of graft neurons, which had a prominent patchy distribution and were most abundant in the caudal portions of the grafts. Clear retro-grade labelling was also seen after injection of Fluoro-Gold or rhodamine beads into the host substantia nigra, although the number of labelled graft neurons was 30–50 times lower than that seen after pallidal injections. Combined injections of Fluoro-Gold into the pallidus and rhodamine beads into the nigra showed that the vast majority of cells labelled from the nigra were also labelled by Fluoro-Gold from the pallidus. In some of the grafted and Fluoro-Gold-injected animals, the fetal donor tissue had been labelled with [ 3H]thymidine prior to transplantation. Many examples of neurons labelled with both [ 3H]thymidine and Fluoro-Gold were found after tracer injections into the host globus pallidus, and double-labelled neurons were identified also after Fluoro-Gold injections into the host substantia nigra. In several animals retrograde tracing was combined with labelling of host dopaminergic afferents (by tyrosine hydroxylase immunohistochemistry) and cortical afferents (by injections of Phaseolus vulgaris leucoagglutinin into the host frontal cortex). Comparison of adjacent sections revealed a striking overlap between the patches of Fluoro-Gold-labelled graft neurons (labelled from the host pallidum) and the dense patches of tyrosine hydroxylase-positive terminals. In addition, many of the Fluoro-Gold-labelled cell patches received a high density of cortical afferents labelled by Phaseolus vulgaris leucoagglutinin. The results thus demonstrate that the intrastriatal striatal grafts are capable of establishing efferent projections to the host globus pallidus and to a lesser extent also to the entopeduncular nucleus and the substantia nigra. The close association between the efferent projecting graft nerons and the dopaminergic and cortical afferent inputs from the host suggests that the grafted striatal neurons become anatomically and functionally integrated into the neuron-depleted host striatal circuitry.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum—II. Cortical afferents

Grafts of fetal striatal tissue, implanted into the ibotenic acid-lesioned caudate-putamen in adult recipient rats, have previously been shown to receive extensive afferent inputs from various subcortical areas in the host. In the present study, the formation of graft afferents from the host neocortex has been analysed, 3–12 months after transplantation, by means of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin, iontophoretically injected at multiple sites into the host frontal cortex, in combination with intra-transplant injections of the fluorescent retrograde tracer, Fluoro-Gold. From the cortical injection sites, fibres labelled with Phaseolus vulgaris leucoagglutinin could be traced through the corpus callosum, along the myelinated fascicles of the internal capsule, into the host caudate-putamen. The labelled axons passed in large numbers across the host-graft border zone to form a rich terminal plexus that covered large parts of the grafted tissue, in close association with the grafted neurons. The labelled corticostriatal terminal network was dense, particularly in the peripheral portions of the grafts, although it did not reach the density seen in the spared portions of the host caudate-putamen. Injections of Fluoro-Gold into the depth of the striatal grafts labelled large numbers of neurons in the frontoparietal cortical regions. Consistent with previous findings, labelled neurons were also found, ipsi- lateral to the graft, in the intralaminar nuclear complex of the thalamus, basolateral amygdala, substantia nigra and dorsal raphe. The host neocortical neurons labelled by Fluoro-Gold injections into the graft had the same laminar and regional distribution as the neurons labelled from the homotopic area of the intact striatum, indicating that the cortical afferents to the graft were derived from those corticostriatal neurons which projected to the area of the ibotenic acid-lesioned caudate-putamen into which the graft had been placed. The number of Fluoro-Gold-labelled cortical neurons ipsilateral to the graft reached in the best cases about 1/3 of the number labelled by identical Fluoro-Gold injections into the intact caudate-putamen. Labelled neurons occurred, with a similar distribution, also in the contralateral frontoparietal cortex, but they were considerably fewer in number. The results demonstrate that fetal striatal tissue, implanted into the previously neuron-depleted adult caudate-putamen, receives innervations from all normal principal cortical and subcortical striatal afferent systems, thus pointing to a remarkable capacity of the implanted fetal striatal neurons to become anatomically integrated into the lesioned host striatal circuitry.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum—I. Subcortical afferents

Subcortical afferents to transplants of fetal striatal tissue, implanted into the excitotoxically lesioned striatum of adult recipient rats, were studied with retrograde and anterograde axonal tracers and immunohistochemistry. One week after a striatal ibotenic acid lesion, involving most of the head of the caudate-putamen, a suspension of fetal striatal tissue (embryonic day 14–15) was injected into the lesioned area. In one group of rats, the ibotenic acid lesion was preceded (10 days) by large intrastriatal injections of True Blue, with injection sites matching the area to be lesioned. This was done to retrogradely pre-label the host brain afferents to the area of the striatum later to be lesioned and grafted. At 3 or 6 months post-transplantation, small injections (50 nl) of rhodamine-labelled latex beads were made into the striatal grafts. In animals where the injections were confined to the graft, retrogradely labelled host brain neurons were found in the thalamus, the substantia nigra, amygdala and dorsal raphe nucleus. Double-labelling analysis revealed that the vast majority of the rhodamine bead-labelled neurons also contained True Blue, which indicates that the host afferents to the graft, to a large extent, were derived from the neurons which normally project to the area of the caudate-putamen which was lesioned by the ibotenic acid injection. To further substantiate these observations a second group of lesioned and grafted animals received unilateral wheatgerm agglutinin-horseradish peroxidase injections into the ipsilateral host thalamus at 4 months post-transplantation in order to anterogradely label the host thalamostriatal axons. In a third group of animals serotonin immunocytochemistry was performed in order to detect possible afferents from the raphe nuclei. In contrast to the serotonin-containing fibers, which were fairly evenly distributed throughout the graft tissue, the peroxidase-labelled thalamic afferents were most prominent in the peripheral zones of the grafts and they were densely aggregated at the graft-host interface. The combined results provide evidence that the intrastriatal grafts receive afferents from the host substantia nigra, thalamus, amygdala and dorsal raphe nucleus, but with different distributions. The afferents from the substantia nigra, amygdala and raphe nuclei seem to distribute throughout the grafted tissue, although they are most dense in the peripheral parts, whereas the thalamic afferents are largely confined to the peripheral areas of the transplants and to the graft-host interface.