Injections Of Wheat Germ Agglutinin Conjugated To Horseradish Peroxidase Research Articles

Presence and connections of auditory neurons in the rostrodorsal and rostrolateral parts of the thalamic reticular nucleus

ObjectiveAuditory neurons have been identified in the caudoventral part of the thalamic reticular nucleus (TRN). We examined the acoustic input to single cells in the rostrodorsal part of the TRN.Material and MethodsIn α-chloralose-anesthetized cats, we extracellularly recorded the responses of single neurons in the rostral TRN to acoustic and light stimuli. Next, to examine efferent projections of auditory neurons in the rostral TRN, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into other thalamic nuclei where auditory neurons were detected, including the lateral posterior nucleus (LP), the lateral medial and suprageniculate nuclei and the centromedian nucleus. Finally, intracortical microstimulation of the LP was performed to demonstrate antidromic activation of the auditory neurons in the rostral TRN.ResultsIn the rostral TRN, 2 types of response to auditory stimuli were observed: brief, short-latency bursts (13–20 ms; mean 16.5 ms) and longer bursts with a long latency (38.8–50 ms; mean 44.8 ms). Injection of WGA-HRP into the medial LP labeled cells only in the rostrodorsal TRN, while extending the injection to the other nuclei labeled cells in the rostrodorsal and rostrolateral parts of the nucleus. Auditory neurons in the rostral TRN were activated antidromically by microstimulation of auditory neurons in the LP, with a latency of 1.2 ms.ConclusionsThese results strongly suggest that auditory neurons in the rostrodorsal TRN project to auditory neurons in the LP. The rostral auditory TRN may be involved in transmission of auditory information via the non-specific association system of the thalamus.

Afferent and Efferent Connections of the Nucleus Geniculatus Lateralis Ventralis Demonstrated by WGA‐HRP in the Chick

Fibre connections of the chick nucleus geniculatus lateralis ventralis (GLv) were investigated using the axonal tracing method with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the GLv, many labelled neurons were observed in layer i of the stratum griseum et fibrosum superficiale (SGFS) in the ipsilateral tectum opticum (TO) and in the nucleus lentiformis mesencephali (LM). In the TO-GLv projection, cells of origin were located in the deeper part of layer i of the TO and were topographically distributed along the direction from the rostrodorsal part to the caudoventral part of the TO relating to a rostrocaudal axis of the GLv. In the LM-GLv connection, the dorsal and ventral parts of the LM connected reciprocally with the rostral and caudal halves of the GLv, respectively. In contrast, in the GLv efferent connection, labelled axon terminals spread widely in the ipsilateral area pretectalis without any clear topographical arrangement.

Afferent and efferent connections of the nucleus rotundus demonstrated by WGA-HRP in the chick.

Organization of the fibre connections in the chick nucleus rotundus (Rt) was investigated by an axonal tracing method using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After an injection of WGA-HRP into the Rt, labelled neurones were observed in the striatum griseum centrale (SGC) in both sides of the tectum (TO) and in the ipsilateral nucleus subpretectalis/nucleus interstito-pretecto-subpretectalis (SP/IPS). Labelled fibres and terminals were also found in the ipsilateral ectostriatum (Ect). These fibre connections were topographically organized rostrocaudally. In the TO-Rt projection, the rostral and the dorsocaudal parts of the Rt received afferents from the superficial part of the SGC, the middle part of the Rt received afferents from the intermediate part of the SGC, and the ventrocaudal part of the Rt received mainly fibres from the deep part of the SGC. These topographic projections were accompanied by a considerable number of diffuse projections to the thalamic regions surrounding the Rt. In addition, the rostral and middle caudal parts of the Rt received afferents from the lateral and medial parts of the SP/IPS, respectively, and respective parts of the Rt sent efferents to the lateral and medial parts of the Ect.

Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry.

Current protocols for a combined approach of anterograde tracing with carbocyanine dyes or horseradish peroxidase (HRP) conjugates and immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was investigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGA-HRP into the nodose ganglion of rats. By use of tyramide-biotin amplification, a dense fiber plexus of vagal afferents was visualized centrally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, large- and small-caliber vagal fibers and terminals could be demonstrated comparably to conventional tracing technique using carbocyanine dyes or WGA-HRP and TMB histochemistry. Combination with immunohistochemistry could easily be done, requiring only one more incubation step, and did not result in loss of sensitivity of the tracing. With this method and confocal microscopy, the presence of Ca binding proteins in vagal afferent terminals could be demonstrated. Tyramide amplification allows sensitive anterograde tracing with low background staining in conjunction with immunohistochemistry of a-axonal markers.

Auditory brainstem projections to the ferret superior colliculus: Anatomical contribution to the neural coding of sound azimuth

The mammalian superior colliculus (SC) contains a neural map of auditory space. It is not known whether this topographic representation emerges at the level of the SC or is relayed there from other auditory areas. We have used retrograde labelling techniques in ferrets to examine the sources and pattern of innervation from auditory brainstem nuclei. After multiple injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the SC, the heaviest concentrations of labelled cells were found in the nucleus of the brachium (BIN) and external nucleus of the inferior colliculus, with much weaker labelling in the nucleus sagulum, dorsal, intermediate and ventral nuclei of the lateral lemniscus, paralemniscal regions, and periolivary nuclei. The projections were predominantly ipsilateral, although labelled cells were found on both sides of the brainstem. Single injections of WGA-HRP or discrete injections of red and green latex microspheres revealed that the caudal and lateral regions of the SC receive the heaviest projections, although the majority of the retrogradely labelled neurons in the contralateral BIN project to rostral SC. On the ipsilateral side, neurons in rostral and caudal regions of the BIN were labelled primarily by the tracer injected into rostral and caudal regions of the SC, respectively. However, no clear segregation was apparent in the BIN after injections into the medial and lateral regions or in any of the other nuclei after either injection paradigm. These data suggest that converging inputs from several auditory brainstem nuclei contribute to the construction of the auditory space map in the SC, although information about sound azimuth may be conveyed to this nucleus via a spatially ordered projection from the ipsilateral BIN. J. Comp. Neurol. 390:342–365, 1998. © 1998 Wiley-Liss, Inc.

Auditory brainstem projections to the ferret superior colliculus: Anatomical contribution to the neural coding of sound azimuth

The mammalian superior colliculus (SC) contains a neural map of auditory space. It is not known whether this topographic representation emerges at the level of the SC or is relayed there from other auditory areas. We have used retrograde labelling techniques in ferrets to examine the sources and pattern of innervation from auditory brainstem nuclei. After multiple injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the SC, the heaviest concentrations of labelled cells were found in the nucleus of the brachium (BIN) and external nucleus of the inferior colliculus, with much weaker labelling in the nucleus sagulum, dorsal, intermediate and ventral nuclei of the lateral lemniscus, paralemniscal regions, and periolivary nuclei. The projections were predominantly ipsilateral, although labelled cells were found on both sides of the brainstem. Single injections of WGA-HRP or discrete injections of red and green latex microspheres revealed that the caudal and lateral regions of the SC receive the heaviest projections, although the majority of the retrogradely labelled neurons in the contralateral BIN project to rostral SC. On the ipsilateral side, neurons in rostral and caudal regions of the BIN were labelled primarily by the tracer injected into rostral and caudal regions of the SC, respectively. However, no clear segregation was apparent in the BIN after injections into the medial and lateral regions or in any of the other nuclei after either injection paradigm. These data suggest that converging inputs from several auditory brainstem nuclei contribute to the construction of the auditory space map in the SC, although information about sound azimuth may be conveyed to this nucleus via a spatially ordered projection from the ipsilateral BIN.

Organization in the auditory sector of the cat's thalamic reticular nucleus

This study describes the organization of cells in the thalamic reticular nucleus (TRN) that project to the auditory part of the cat's dorsal thalamus. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and fluorescent dyes were made into the medial geniculate complex (MG). The resultant retrograde labelling in the TRN was analyzed. Injections of WGA-HRP into the ventral (MGv), dorsal (MGd), or medial (MGm) nuclei of the MG label zones of cells that are restricted to a caudoventral sector of the TRN. In reconstructions, these zones resemble "slabs" that are elongated in the dorsoventral and oblique rostrocaudal dimensions of the nucleus. In comparisons of the zones of labelling in the TRN following tracer injections into different nuclei of the MG, inner and caudal cells project to the pars lateralis of the MGv (MGvl) or to the MGd, and outer and rostral cells project to the pars ovoidea of the MGv (MGvo) or to the MGm. Thus, cells projecting to the MGvl or MGd or to the MGvo or MGm occupy overlapping territories. Double injections of different fluorescent dyes into selected pairs of MG nuclei result in reticular cells that are labelled from either both nuclei or only one or the other nucleus in each pair. These results indicate that the projections of cells in the auditory sector of the TRN to the MGvl or MGvo or to the MGd or MGm are topographically organized. Furthermore, projections to more than one MG nucleus can arise from single reticular cells.

Divergent Projections from the Anterior Inferotemporal Area TE to the Perirhinal and Entorhinal Cortices in the Macaque Monkey

Area TE is located at the latter part of the ventral visual cortical pathway, which is essential for visual recognition of objects. TE projects heavily to the perirhinal region, which is important for visual recognition memory of objects. To study the organization of projections from TE to the perirhinal (areas 35 and 36) and entorhinal (area 28) cortices, we made focal injections of Phaseolus vulgaris leucoagglutinin (PHA-L) and large injections of biocytin or wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into anterior levels of TE in macaque monkeys. Injections of PHA-L into the ventral part of anterior TE (TEav) resulted in labeling of terminals distributed widely in area 36 (approximately one-half of its total extent), although the injection sites were limited to 0.7 mm in width. The labeled terminals tended to be denser in the medial part of area 36. There was less dense but definite labeling in area 35 and the lateral part of area 28. After a single injection of PHA-L or WGA-HRP into the dorsal part of anterior TE (TEad), labeled terminals were confined to a small region at the lateral part of area 36 (less than one-tenth of its total extent). The projections to areas 35 and 28 from TEad were much sparser than those from TEav. The different patterns of projections to the perirhinal and entorhinal cortices, together with previously reported differences in their afferent and other efferent connections, suggest the functional differentiation between TEav and TEad. The divergent projection from TEav to the perirhinal cortex may facilitate the association of different visual features in the perirhinal cortex.

The nucleus of the optic tract (NOT) and the dorsal terminal nucleus (DTN) of opossums (Didelphis marsupialis aurita).

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected unilaterally into the pretectocollicular region of opossums (Didelphis marsupialis aurita), primarily to investigate the existence of a commissural subcortical pathway but also to reveal afferents and efferents of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in this species. Labelled cells and terminals were observed in the contralateral NOT-DTN. Furthermore, HRP was injected bilaterally in the region of the inferior olive (IO) to verify if the distribution of labelled cells in the NOT-DTN overlapped the region of commissural labelled cells. The two subpopulations of retrogradely labelled cells coincided, being distributed within the retinal terminal field attributed to the NOT-DTN, as revealed by contralateral eye injections of HRP. The commissural cells were located slightly more ventral than the olivary cells in the optic tract. The pretectocollicular WGA-HRP injections also labelled cells and terminals bilaterally in the lateral terminal nucleus (LTN), interstitial nucleus of the superior fasciculus, posterior fibers (INSFp), ventral lateral geniculate nucleus (vLGN), and superior colliculus (SC) and ipsilaterally in the medial terminal nucleus (MTN). In addition, further caudally, labelled cells and terminals were observed bilaterally in the nuclei prepositus hypoglossi (PH) and in the medial (MVN) and lateral (LVN) vestibular nuclei. Labelled terminals were found in the ipsilateral nucleus reticularis tegmenti pontis (NRTP) and in the IO with ipsilateral predominance. This study allowed an anatomical delimitation of the NOT-DTN in this opossum species, as defined by the olivary and commissural subpopulations, as well as a hodological evaluation of this region. The existence of some common anatomical aspects with other mammalian species is discussed.

GABAergic projection from the intercalated cell masses of the amygdala to the basal forebrain in cats.

The intercalated cell masses (ICMs) are dense clusters of small GABAergic cells interposed between the basolateral and centromedial nuclear groups of the amygdala. Until now, the ICMs have been largely ignored in anatomical studies of the amygdaloid complex. Thus, this study was undertaken to identify some of their targets by means of tract-tracing methods combined with immunohistochemical techniques. Wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into numerous cortical areas and dorsal thalamic nuclei, in the anterior commissure and/or stria terminalis nuclei, and in the caudate nucleus, as well as into lateral and preoptic hypothalamic areas. Very few retrogradely labeled cells were seen in the ICMs following these injections. In contrast, massive retrograde labeling was found in the rostral groups of ICMs after WGA-HRP injections involving the substantia innominata and horizontal limb of the diagonal band. Furthermore, these retrogradely labeled intercalated cells were also GABA-immunoreactive. Results of iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the rostral ICMs confirmed that they contribute a massive projection to the entire extent of the substantia innominata and horizontal limb of the diagonal band. Electron microscopic observations of ultrathin sections prepared for postembedding GABA or glutamate immunocytochemistry revealed that the ICM terminals labeled with PHA-L displayed GABA, but not glutamate immunoreactivity, and formed symmetric synapses with dendritic profiles. The present findings constitute the first direct demonstration of an amygdalofugal GABAergic projection to the basal forebrain. Considering that the basal forebrain contains a group of cholinergic and GABAergic neurons collectively projecting to the entire cortical mantle, this GABAergic projection of the ICMs could allow the amygdaloid complex to influence the activity of widespread cortical regions to which it is not directly connected, at least in the cat.

Auditory brainstem of the ferret: long survival following cochlear removal progressively changes projections from the cochlear nucleus to the inferior colliculus.

Some effects on auditory brainstem connections of long (1-2.3 years) survival following unilateral cochlear removal in infant and adolescent ferrets were examined by making multiple injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in either the left or the right inferior colliculus (IC). Previous studies have shown that, in normal adult ferrets, about 50 times as many cochlear nucleus (CN) neurons project to the contralateral as to the ipsilateral IC. Right cochlear removal at P25 increased, within 30 days, the number of retrogradely labeled left CN neurons projecting to the left ipsilateral IC by 17% (from n = 235 to n = 275), relative to normals. In this study, longer survival (3 months to 1 year) after right cochlear removal at P25 resulted in larger increases (38-47%; n = 100) in the number of neurons labeled in the left CN after injections of WGA-HRP in the left IC. No change occurred in the number of neurons labeled in the right CN. Taken together, the results of these experiments show that the ratio of the number of labeled neurons in the left CN to that in the right CN increases progressively with survival time out to the maximum time tested (1 year). In contrast to these results, we have previously reported that right cochlear removal at P90 did not change the number of neurons projecting from the left CN to the left IC after 90 days of survival. However, in this study, very long survival (2.3 years) following right cochlear removal at P90 resulted in an increased (51%, from n = 235 to n = 355) number of left CN neurons labeled by WGA-HRP injections into the left IC, relative to normals. The increased number of labeled neurons included neurons throughout each division of the CN and all of the principal morphological types. In a separate series of experiments involving long survival (1-2 years), right cochlear removal at P25 or P40 did not significantly change the number of neurons in either CN retrogradely labeled by injections of WGA-HRP in the right IC, or the ratio between the number of neurons labeled in each CN. Long survival following cochlear removal at P25-P90 did not result in any loss of neurons in the ipsilateral CN or in any shrinkage of CN neurons further than the 10-20% seen at a shorter survival time (90 days).(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of cerebellothalamic and nigrothalamic projections in the dog: a double anterograde tracing study.

The distribution of nigrothalamic and cerebellothalamic projections was investigated in the dog by a double labeling strategy combining the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and tritiated amino acids. Following tritiated amino acid injections into the substantia nigra pars reticulata (SNr) and WGA-HRP injections into the contralateral cerebellar nuclei, we found that the nigrothalamic and cerebellothalamic afferents distribute to three main targets: the central portion of the ventral anterior nucleus (VA) and the ventral lateral nucleus (VL), the internal medullary lamina (IML) region, which includes the paralaminar VA, the mediodorsal nucleus (MD) and the central lateral nucleus (CL), and finally the ventromedial nucleus (VM). We observed three distribution patterns of labeled fibers: a) Dense single label was observed in the central portion of VA following the SNr injections and in VL following the cerebellar nuclei injections. b) A complementary pattern consisting of alternating foci of nigral and cerebellar label was found in the IML region. This pattern was also observed in the caudal intralaminar nuclei where cerebellar label predominated in the centrum medianum (CM), while the parafascicular nucleus (Pf) primarily contained nigral label. c) An overlapping pattern of autoradiographic and WGA-HRP label was found in the lateral half of the VM. Overall, the distribution of nigrothalamic and cerebellothalamic projections was widespread throughout much of rostrocaudal thalamus. However, the pattern of projections varied along a continuum from lateral to medial thalamus. In lateral thalamus, nigral and cerebellar projections distributed to separate nuclei while in medial thalamus, the projection pattern changed to focal and complementary in the IML and overlapping in VM. Taken together, these thalamic projections may constitute crucial links in different functional channels involved in alerting and orienting mechanisms associated with motor behavior. Our findings also suggest that the organization of motor thalamic afferents in the dog shares similarities with the segregated and parallel circuitry characteristic of primates as well as with the overlapping and converging circuits of rodents and other carnivores.

Laminar Termination Patterns of Thalamic, Callosal, and Association Afferents in the Primary Auditory Area of the Rhesus Monkey

Thalamic, callosal, and association afferents to the primary auditory koniocortex, area KA, were investigated in rhesus monkeys with the aid of anterograde tract tracing techniques (radioactively labeled amino acids, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), and the Fink-Heimer silver impregnation method). Different patterns of anterograde label in KA were produced by radioactively labeled amino acid injections in the contralateral primary auditory area, the ipsilateral medial geniculate body (MGB), or auditory association cortex of the superior temporal gyrus (STG). Thalamic afferents from the MGB were found throughout KA in layers IV and III where the density waxed and waned, forming regular patches of higher density label separated by areas of less dense label. Callosal afferents from the contralateral auditory cortices were found in layers IV, III, and II in larger, more spatially separated, and irregular patches. In contrast, association afferents from the auditory association cortex of the STG were found in a continuous band in layer I. To determine the extent to which the patches of callosal afferents overlap with or interdigitate between the high-density thalamic clusters, two types of double anterograde labeling experiments were undertaken. In one set of experiments isotope injections of the MGB were made in conjunction with transection of the corpus callosum in the same animal. In another set of experiments isotope injections of the MGB were made in one hemisphere and an injection of WGA-HRP was made in the opposite supratemporal plane, including KA, in the same animal. Both approaches demonstrate that the patches of callosal afferents in KA mainly occur between the high-density patches of thalamic afferents, although there is a variable degree of overlap at their periphery. Thus it seems that thalamic and callosal inputs to KA are organized in a largely complementary fashion while input from the lateral association cortex is continuous and overlaps both. When considered in light of pertinent anatomical and physiological studies in a variety of species, it appears that the areas mainly occupied by callosal or thalamic inputs may be differentially involved in frequency analysis and sound localization, respectively.

Trigeminocerebellar projections to the posterior lobe in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase.

Cerebellar projections of the nucleus interpolaris and oralis of the spinal trigeminal nucleus were studied in the cat by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into these nuclei labeled many mossy fiber terminals mainly ipsilaterally in the rostral folium of lobule IX (IXa or IXa + b), the simple lobule, the anterior part (sublobule A) of the paramedian lobule and the posterior part of crus II. Labeled terminals were also seen in the anterior lobe, lobules VI and VII, the anterior part of crus I, and the paraflocculus dorsalis. Projection fields in the horizontal plane of lobules were reconstructed from a series of transverse sections through each folium of lobule IX, the paramedian lobule, and the posterior part of crus II on the ipsilateral side. In sublobule IXa + b, labeled terminals were distributed in five longitudinal areas extending along the apicobasal axis of the sublobule. These five areas were located in the apical two-thirds of the ipsilateral half of the sublobule. Labeled terminals were distributed in five longitudinal areas in sublobule A (the rostral part) of the paramedian lobule. In the posterior part of crus II, four aggregations of labeled terminals were present in cross sections through a lobule. They were distributed in the apicobasal extent of the lobules. The present study indicates that the projection fields of trigeminocerebellar fibers are longitudinally arranged along the apicobasal axis of the cerebellar lobules.

Retinal projections in the house musk shrew, Suncus murinus, as determined by anterograde transport of WGA-HRP.

Retinal projections in the house musk shrew (Suncus murinus) were determined by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Unilateral injection of WGA-HRP into the vitreous body resulted in the terminal labeling of the optic projections in the suprachiasmatic nucleus (SCH), the ventral (CGLv) and dorsal (CGLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the pretectum, the superficial layers of the superior colliculus (CS), and the dorsal terminal nucleus (DTN) of the accessory optic system (AOS). Labeling of the SCH was bilateral, with ipsilateral predominance, and covered the whole dorsoventral extent of the nucleus. Immunohistochemical studies revealed that VIP-like immunoreactive neurons and fibers were present in almost all parts of the SCH. No hypothalamic regions other than the SCH received the optic fibers. The ipsilateral projections to the CGLv, CGLd, and IGL were sparse, and a considerable number of uncrossed retinal fibers were found in the pretectal olivary nucleus. No retinal projections to the lateral posterior thalamic nucleus (LP) were found. Ipsilateral optic fibers projected sparsely to the medial part of the CS. The AOS consisted of a small DTN with a very few crossed retinal projections but no lateral and medial terminal nuclei. In addition, the AOS had no inferior fascicle.

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.

Synaptic organization of projections from basal forebrain structures to the mediodorsal thalamic nucleus of the rat

The synaptic organization of the mediodorsal thalamic nucleus (MD) in the rat was studied with the electron microscope, and correlated with the termination of afferent fibers labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Presynaptic axon terminals were classified into four categories in MD on the basis of the size, synaptic vesicle morphology, and synaptic membrane specializations: 1) small axon terminals with round synaptic vesicles (SR), which made asymmetrical synaptic contacts predominantly with small dendritic shafts; 2) large axon terminals with round vesicles (LR), which established asymmetrical synaptic junctions mainly with large dendritic shafts; 3) small to medium axon terminals with pleomorphic vesicles (SMP), which formed symmetrical synaptic contacts with somata and small-diameter dendrites; 4) large axon terminals with pleomorphic vesicles (LP), which made symmetrical synaptic contacts with large dendritic shafts. Synaptic glomeruli were also identified in MD that contained either LR or LP terminals as the central presynaptic components. No presynaptic dendrites were identified. In order to identify terminals arising from different sources, injections of WGA-HRP were made into cortical and subcortical structures known to project to MD, including the prefrontal cortex, piriform cortex, amygdala, ventral pallidum and thalamic reticular nucleus. Axons from the amygdala formed LR terminals, while those from the prefrontal and insular cortex ended exclusively in SR terminals. Fibers labeled from the piriform cortex formed both LR and SR endings. Based on their morphology, all of these are presumed to be excitatory. In contrast, the axons from the ventral pallidum ended as LP terminals, and those from the thalamic reticular nucleus formed SMP terminals. Both are presumed to be inhibitory. At least some terminals from these sources have also been identified as GABAergic, based on double labeling with anterogradely transported WGA-HRP and glutamic acid decarboxylase (GAD) immunocytochemistry.

Extracellular labeling of unmyelinated dorsal root terminals after WGA-HRP injections in spinal ganglia

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) is a widely used neuroanatomical tracer. When compared with other tracers, WGA-HRP may preferentially label unmyelinated fibers. In agreement with this hypothesis, injections of WGA-HRP in cervical and lumbar dorsal root ganglia resulted in more prominent light microscopical labeling in superficial than deep laminae of the dorsal horn. However, ultrastructural examination of these laminae reveals a paucity of terminal labeling in contrast to the abundance of extracellular tracer in the space surrounding unmyelinated fibers and their terminals, and to the widespread occurrence of transneuronal labeling. These results bear upon the mechanism of preferential labeling in the spinal cord and have implications for the interpretation of the labeling obtained when using WGA-HRP.

Spinocerebellar projections from spinal border cells in the cat as studied by anterograde transport of wheat germ agglutinin‐horseradish peroxidase

The cerebellar projections of spinal border cells, which give rise to crossed ascending axons, were studied in the lower lumbar segments by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in the cat. Prior to the injections, the spinal cord was lesioned rostral and ipsilateral to the WGA-HRP injections to eliminate labeling of the ipsilaterally ascending axons. Following injections of WGA-HRP into the L4-L6 segments, labeled terminals were seen in sublobules Ib-Vf of the anterior lobe, sublobules VIf-VIe, lobule VIII, the paramedian lobule, and crus II. More than 90% of the total number of labeled terminals were in the anterior lobe; the projections were predominantly ipsilateral to their cells of origin (about 90% or more labeled terminals of the total number in each of sublobules IIb-Va). Many labeled terminals were seen ipsilaterally in sublobules IIb (11.1-17.5% of the total number), IIIa (11.7-12.9%), IIIb (16.1-17.6%), IVa (8.8-10.8%), IVb (6.2-10.5%), and Va (9.7-10.3%). In the posterior lobe, labeled terminals were numerous only in sublobule C of the ipsilateral paramedian lobule (2.7-4.9%). The projection fields in the horizontal plane of the lobules were reconstructed from a series of cross-sections through each lobule. In the anterior lobe, labeled terminals were distributed in four major areas. In sublobules IIa-IVa, area 1 was located between 1.0 and 2.0 mm lateral to the midline in zones B-C1 of Voogd; area 2, between 2.0 and 3.0 mm lateral to the midline in zones C1-C2; area 3, between 3.0 and 4.0 mm in zones C2-C3; and area 4, lateral to 4.0 mm from the midline in zone C3. These areas extended apicobasally in the apical to middle parts of the lobules. On the rostral side of sublobule C1 of the ipsilateral paramedian lobule, the projection areas extended in the entire apicobasal and the mediolateral axis of the sublobule, except in the most lateral part. The present results suggest that the SBCs project to specific areas in the cerebellar lobules.

Cholinergic innervation of the superior colliculus in the cat

The superficial and intermediate gray layers of the superior colliculus are heavily innervated by fibers that utilize the neurotransmitter acetylcholine. The distribution, ultrastructure, and sources of the cholinergic innervation of these layers have been examined in the cat by using a combination of immuno-cytochemical and axonal transport methods. Putative cholinergic fibers and cells were localized by means of a monoclonal antibody to choline acetyltransferase (ChAT). ChAT immunoreactive fibers are distributed throughout the depth of the superior colliculus, with particularly dense zones of innervation in the upper part of the superficial grey layer and in the intermediate grey layer. Within the superficial grey layer, the fibers form a continuous, dense band, whereas within the intermediate grey layer the fibers are arranged in clusters or patches. Although the patches are present throughout the rostrocaudal extent of the superior colliculus, they are most prominent in middle to caudal sections. The structure of the ChAT immunoreactive terminals was examined electron microscopically. The appearance of the terminals is similar in the superficial and intermediate grey layers. They contain closely packed, mostly round vesicles, and form contacts with medium-sized dendrites that exhibit small, but prominent postsynaptic densities; a few of the terminals contact vesicle-containing profiles. To identify the sources of the cholinergic input to the superior colliculus, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made in the superior colliculus and the sections were processed to demonstrate both the retrograde transport of WGA-HRP and ChAT immunoreactivity. Neurons containing both labels were found in the parabigeminal nucleus, and in the lateral dorsal and pedunculopontine tegmental nuclei of the pontomesencephalic reticular formation. Almost every cell in these nuclei that contained retrograde label was also immunoreactive for ChAT. The similarities between the laminar distributions of the ChAT terminals and the terminations of the pathway from the parabigeminal nucleus (Graybiel: Brain Res. 145:365-374, '78) support the view that the latter nucleus is a source of the cholinergic fibers in the superficial grey layer. The possibility that the pedunculopontine tegmental nucleus is a source of cholinergic fibers in the deep layers was tested by examining the distribution of labeled fibers following injections of WGA-HRP into this region of the tegmentum. Patches of labeled terminals were found in the intermediate grey layer that resemble in distribution the patches of ChAT immunoreactive fibers in this layer.(ABSTRACT TRUNCATED AT 400 WORDS)