Injections Of Phaseolus Vulgaris Leucoagglutinin Research Articles

Amygdala afferents monosynaptically innervate corticospinal neurons in rat medial prefrontal cortex

The amygdala provides the medial prefrontal cortex (mPFC; areas 25, 32, and 24b) with salient emotional information. This study investigated the synaptic connectivity of identified amygdalocortical boutons (ACBs; labeled anterogradely following injections of Phaseolus vulgaris leucoagglutinin into the basolateral nucleus of the amygdala), with the dendritic processes of identified layer 5 corticospinal neurons in the rat mPFC. The corticospinal (CS) neurons in the mPFC had been retrogradely labeled with rhodamine fluorescent latex microspheres and subsequently intracellularly filled with biotinylated lucifer yellow to visualize their basal and apical dendrites. Two main classes of mPFC CS neurons were identified. Type 1 cells had apical dendrites bearing numerous dendritic spines with radiate basal dendritic arbors. Type 2 cells possessed apical dendrites with greatly reduced spine densities and a broad range of basal dendritic tree morphologies. Identified ACBs made asymmetric synaptic junctions with labeled dendritic spines and the labeled apical and basal dendritic shafts of identified CS neurons. On average, eight ACBs were closely associated with the labeled basal dendritic arbors of type 1 CS neurons and five ACBs with type 2 CS basal dendrites. The mean Scholl distance of ACBs from CS somata (for both types 1 and 2 cells) was 66 μm-coinciding with a region containing the highest length density of CS neuron basal dendrites. These results indicate that neurons in the BLA can monosynaptically influence CS neurons in the mPFC that project to autonomic regions of the thoracic spinal cord and probably to other additional subcortical target regions, such as the lateral hypothalamus.

Projections from the inferior colliculus to the tectal longitudinal column in the rat

We have used the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) to study with albino rats the projections from the inferior colliculus (IC) to the tectal longitudinal column (TLC), a newly discovered nucleus that spans the midbrain tectum longitudinally, on each side of the midbrain, immediately above the periaqueductal gray matter. We studied the projections of the medial IC, which includes the classical central nucleus (CNIC) and the dorsal cortex (DCIC), and those of the lateral IC, equivalent to the classical external cortex (ECIC). Following unilateral injections of PHA-L into the medial IC, numerous terminal fibers are labeled bilaterally in the TLC. The ipsilateral projection is denser and targets the entire nucleus, whereas the contralateral projection targets significantly only the caudal half or two-thirds of the TLC. Fibers from the medial IC reach the TLC by two routes: as collaterals of axons that travel in the commissure of the IC and as collaterals of thick ipsilateral colliculogeniculate axons; the latter travel through the deep superior colliculus on their way to the TLC. Within the TLC, individual IC fibers tend to run longitudinally. The injection of PHA-L into the lateral IC indicates that this subdivision sends a weak, bilateral projection to the TLC whose trajectory, morphology and distribution are similar to those of the projection from the medial IC. These results demonstrate that all subdivisions of the IC send projections to the TLC, suggesting that the IC may be one of the main sources of auditory input to this tectal nucleus.

Cytoarchitectonics and afferent/efferent reorganization of neurons in layers II and III of the lateral entorhinal cortex in the mouse pilocarpine model of temporal lobe epilepsy

With the mouse pilocarpine model of temporal lobe epilepsy (TLE), we showed a progressive loss of both principal cells and calbindin (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunopositive interneurons in layers II-III of lateral entorhinal cortex (LEnt) from 2 months to 1 year after pilocarpine-induced status epilepticus (PISE). In the efferent pathway of LEnt, more Phaseolus vulgaris leucoagglutinin (PHA-L)-labelled en passant and terminal boutons with larger diameters were shown in the hippocampus and subiculum; in the prefrontal, piriform, and perirhinal cortices; and in the amygdaloid complex in experimental mice at the two time points compared with the control after iontophoretical injection of an anterograde tracer PHA-L into the LEnt. Furthermore, the numbers of CB- or CR-immunopositive neurons contacted by PHA-L-labelled en passant and terminal boutons decreased in most of these areas at 2 months or 1 year after PISE. In the afferent pathway of LEnt, the numbers of retrogradely labelled neurons were reduced significantly in the ipsilateral piriform cortex and endopiriform nucleus at 2 months and 1 year and in the reuniens thalamic nucleus only at 1 year after injection of a retrograde tracer cholera toxin B subunit (CTB) into the LEnt. The percentages of the number of CTB and CB or CR double-labelled neurons of all the retrogradely labelled neurons were also decreased in the reunions thalamic nucleus at 1 year after PISE. It is concluded that both cytoarchitectonic change and reorganization of afferent and efferent pathways in LEnt may be involved in the occurrence of TLE.

Synaptologic and fine structural features distinguishing a subset of basal forebrain cholinergic neurons embedded in the dense intrinsic fiber network of the caudal extended amygdala

Cholinergic basal forebrain neurons confined within the intrinsic connections of the extended amygdala in the caudal sublenticular region and anterior amygdaloid area (cSLR/AAA) differ from other basal forebrain cholinergic neurons in several morphological and neurochemical respects. These cSLR/AAA cholinergic neurons have been subjected to additional investigations described in this report. First, fibers traced anterogradely following injections of Phaseolus vulgaris-leucoagglutinin in the central amygdaloid nucleus were shown to contact cSLR/AAA cholinergic neurons and dendrites. Second, these neurons were shown to be contacted by numerous GABAergic boutons with symmetric synaptic specializations. Third, the numbers of synaptic densities of morphologically characterized symmetric contacts on the somata and proximal dendrites of cSLR/AAA cholinergic neurons were shown to significantly exceed those of extra-cSLR/AAA cholinergic neurons. Fourth, fine structural features distinguishing cSLR/AAA cholinergic neurons from other basal forebrain cholinergic neurons were revealed. Specifically, cSLR/AAA cholinergic neurons have less abundant cytoplasm and a less well-organized system of rough endoplasmic reticulum than their counterparts in other parts of the basal forebrain. Thus, morphologically and neurochemically distinct cSLR/AAA cholinergic neurons exhibit robust proximal inhibitory inputs, of which a significant number originate in the extended amygdala, while cholinergic neurons outside this region lack a substrate for strong proximal inhibitory input. The implications of these findings for interaction of fear, anxiety, and attention are considered.

Ultrastructural organization of medial prefrontal inputs to the rhinal cortices

Accumulating evidence suggests that the medial prefrontal cortex (mPFC) plays a critical role in the formation, retrieval and long-term storage of hippocampal-dependent memories. Consistent with this, there are direct hippocampal projections to the mPFC. Moreover, the mPFC sends robust projections to the perirhinal and entorhinal cortices, two interconnected cortical fields that funnel information into and out of the hippocampus. However, the significance of the latter projection remains unclear because no data are available regarding the rhinal targets of mPFC axons. This question was examined in the present study using a combination of anterograde tracing with Phaseolus vulgaris leucoagglutinin and pre-embedding gamma-aminobutyric acid (GABA) immunocytochemistry in guinea pigs. Following Phaseolus vulgaris leucoagglutinin injections in the mPFC, anterogradely labeled axons were seen in the perirhinal (mainly superficial layers) and lateral entorhinal (mainly deep layers) cortices. In the electron microscope, the synaptic articulation of anterogradely labeled mPFC axon terminals with perirhinal and entorhinal neurons was found to be nearly identical. In these two rhinal fields, mPFC axon terminals only formed asymmetric synapses, typically with GABA-immunonegative spines ( approximately 70%) but occasionally with dendritic profiles ( approximately 30%), half of which were GABA immunopositive. In the light of earlier observations, these findings indicate that mPFC inputs exert mainly excitatory effects in the rhinal cortices, prevalently on principal neurons. Thus, these results suggest that the mPFC may affect hippocampal-dependent memories by enhancing impulse traffic into and out of the hippocampus at the level of the rhinal cortices.

Calcium binding protein containing neurons in the gliotic mouse hippocampus with special reference to their afferents from the medial septum and the entorhinal cortex

In CA1 area and the hilus of the dentate gyrus of the mouse hippocampus, drastic reduction of NeuN, calbindin, calretinin, or parvalbumin immunopositive neurons was shown at 3, 7 and 60 days after pilocarpine-induced status epilepticus. In gliotic CA1 area at 60 days, few dendritic branches of calcium binding protein immunopositive neurons could be found suggesting reorganization of the afferents of surviving calcium binding protein immunopositive neurons. Calbindin, calretinin, or parvalbumin and 5-bromo-2′-deoxyuridine (BrdU) double labeling showed that calcium binding protein immunopositive neurons in gliotic CA1 area at 60 days were surviving instead of newly generated neurons. Iontophoretic injection of Phaseolus vulgaris leucoagglutinin into the medial septum and the nucleus of the diagonal band of Broca or the lateral entorhinal cortex showed contacts between Phaseolus vulgaris leucoagglutinin immunopositive en passant and terminal boutons and surviving calcium binding protein immunopositive neurons in the hippocampus. The presence in the gliotic hippocampus of enlarged and/or aggregated bouton-like structures 60 days after pilocarpine-induced status epilepticus is indicative for the reorganization of connections between the hippocampal afferents and surviving hippocampal neurons. This reconstruction could be a factor in the ongoing epileptic activity in this model of mesial temporal lobe epilepsy.

Direct projections from caudal ventrolateral medullary depressor sites to the subfornical organ

Experiments were performed in the male Wistar rat to investigate the projections from cardiovascular responsive sites in the ventrolateral medulla (VLM) to the subfornical organ (SFO). Unilateral iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHA-L) were made into either caudal VLM (CVLM) sites at which microinjection of l-glutamate (10 nl; 0.25 M) elicited decreases in mean arterial pressure or into rostral VLM (RVLM) sites at which l-glutamate microinjection elicited increases in arterial pressure. After a survival period of 7–10 days, transverse sections of the forebrain and brainstem were processed for PHA-L immunoreactivity. After injections of PHA-L into the CVLM, axonal and presumptive terminal labeling was found bilaterally throughout the rostrocaudal extent of the SFO, although most of the projections were observed within the rostral half of the nucleus. Within the SFO, labeling was found primarily in the lateral aspects of the nucleus, often in close proximity to blood vessels. In addition, CVLM injections resulted in labeling within the organum vasculosum of the laminae terminalis (OVLT) and within the ventral and dorsal components of the median preoptic nucleus (MnPO) bilaterally, but with an ipsilateral predominance. In contrast, PHA-L injections into the RVLM did not result in axonal labeling in the SFO or OVLT, although a few labeled axons were found to course through the region of the ventral component of MnPO. These data have demonstrated that neurons within the cardiovascular responsive region of the CVLM send direct axonal projections to the SFO and other structures of the laminae terminalis, and suggest that the CVLM may function in the modulation of the activity of neurons of circumventricular organs to intra- and extracellular signals of body fluid balance.

Projections from the raphe nuclei to the suprachiasmatic nucleus of the rat

The presence of serotonergic afferents in the hypothalamic suprachiasmatic nucleus (SCN) is well documented and several functional roles of serotonin (5-HT) in circadian function are well established. However, there is some controversy about the precise location of the serotonergic neurones from where this input arises. Discrete injection of the tracer Cholera toxin, subunit B, (ChB) was centred in the rat SCN, and a few retrograde labelled neurones were distributed in the dorsal and median raphe nuclei (MnR) and in the rostral part of the raphe magnus (RMg), but no neurones were found in the raphe pallidus or raphe obscurus. In addition, a group of neurones was consistently found in the medial part of the pontine supra lemniscal nucleus but not including the serotonergic B 9 region. A combination of retrograde tracing with Fluoro-Gold together with 5-HT-immunolabelling, showed few double-labelled neurones in the dorsal, MnR and B 9. However, the majority of projecting neurones were not co-storing 5-HT immunoreactivity. Phaseolus vulgaris-leucoagglutinin (PHA-L) injections in the dorsal raphe resulted in faint labelling, whereas the MnR gave rise to several labelled fibres in the SCN. Individual delicate PHA-L nerve fibres were found in all compartments of the SCN both in terms of rostrocaudal, ventromedial and dorsomedial extent, without any sign of a topographical organisation of the MnR input to the SCN. PHA-L injections into RMg gave rise to labelling of a few processes within the SCN. In conclusion, the main serotonergic input to the rat SCN originates from a few neurones in the MnR.

Central projection of unmyelinated (C) primary afferent fibers from gastrocnemius muscle in the guinea pig.

We have demonstrated the central projections of muscle C or group IV afferent fibers in the guinea pig by tracing arborizations in the spinal cord. C afferent fibers from the gastrocnemius muscle (GCM) were electrophysiologically identified by conduction velocity (less than 1 m/second). A single neuron in the lumbar 5 dorsal root ganglion (L5 DRG) was intracellularly labeled with Phaseolus vulgaris leucoagglutinin (PHA-L). After iontophoretic injection of PHA-L, we processed the lumbar cord and L5 DRG for PHA-L immunohistochemistry. Six muscle C afferent fibers from 40 animals were labeled, and whole trajectories were recovered. Labeled fibers were reconstructed by tracing of the arbor in serial parasagittal sections. The GCM C afferents projected rostrocaudally for two or three segments and ran at the surface of the dorsal funiculus, giving off collaterals into laminae I and II and sometimes into parts of lamina III. We determined, based on the branching pattern and form of the terminal plexus, that the branching of muscle C afferent fibers showed an intermediate pattern that fell morphologically between the terminal patterns of somatic and visceral afferents. The numbers and sizes of fiber swellings and terminal swellings were measured on all collateral branches. We found that the area of distribution of the terminal swellings of muscle C afferent fibers is larger than that of somatic terminals but that the density of terminal swellings in the terminal area was lower than that of the somatic terminals.

The efferent connections of the pupillary constriction area in the rat medial frontal cortex

This study investigated, in the rat, the efferent projections of the pupillary constriction area, which is located within the medial frontal cortex. In order to identify the location of the pupillary constriction area, in preliminary experiments the medial frontal cortex was microstimulated. Intracortical microstimulation elicited pupillary constriction in a thin strip of cortex near the interhemispheric fissure and bordering the frontal eye field and vibrissae area of the somatomotor cortex. Seven animals received a single iontophoretic injection of Phaseolus vulgaris leucoagglutinin in the pupillary constriction area. In these cases, anterogradely labelled fibres and terminal-like elements were found in both hemispheres. The densest labeling was seen in several areas of the injected hemisphere, where labeled fibers prevailed in the secondary visual cortex. Dense labeled fibers were also found in the retrosplenial and cingulate cortex. In the thalamus, labeled fibers were seen in the intralaminar nuclei and posterior nuclear group. In the midbrain and pons, labeled fibers were located in the anterior pretectal area, superior colliculus and in the dorsolateral portion of the central gray. Contralaterally to the injection site, labeled fibers were distributed in the homotopic region. These findings led us to assume that, in the medial frontal cortex of the rat, besides controlling pupillary constriction, the pupillary constriction area may also be involved in controlling orientation and exploring behavior.

Synaptic organization of GABAergic projections from the substantia nigra pars reticulata and the reticular thalamic nucleus to the parafascicular thalamic nucleus in the rat

The ventrolateral part of the parafascicular thalamic nucleus (PF), which is considered to take part in the control mechanism of orofacial motor functions, receives projection fibers not only from the dorsolateral part of the substantia nigra pars reticulata (SNr) but also from the ventral part of the reticular thalamic nucleus (RT) [Tsumori et al., Brain Res. 858 (2000) 429]. In order to better understand the influence of these fibers upon the PF projection neurons, the morphology, synaptology and chemical nature of them were examined in the present study. After ipsilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the dorsolateral part of the SNr and biotinylated dextran amine (BDA) into the ventral part of the RT, overlapping distributions of PHA-L-labeled SNr fibers and BDA-labeled RT fibers were seen in the ventrolateral part of the PF. At the electron microscopic level, the SNr terminals made synapses predominantly with the medium to small dendrites and far less frequently with the somata and large dendrites, whereas approximately half of the RT terminals made synapses with the somata and large dendrites and the rest did with the medium to small dendrites of PF neurons. Some of single dendritic as well as single somatic profiles received convergent synaptic inputs from both sets of terminals. These terminals were packed with pleomorphic synaptic vesicles and formed symmetrical synapses. After combined injections of PHA-L into the dorsolateral part of the SNr, BDA into the ventral part of the RT and wheat germ agglutinin–horseradish peroxidase (WGA-HRP) into the ventrolateral part of the striatum or into the rostroventral part of the lateral agranular cortex, WGA-HRP-labeled neurons were embedded in the plexus of PHA-L- and BDA-labeled axon terminals within the ventrolateral part of the PF, where the PHA-L- and/or BDA-labeled terminals were in synaptic contact with single somatic and dendritic profiles of the WGA-HRP-labeled neurons. Furthermore, the SNr and RT axon terminals were revealed to be immunoreactive for γ-aminobutyric acid (GABA), by using the anterograde BDA tracing technique combined with immunohistochemistry for GABA. The present data suggest that GABAergic SNr and RT fibers may exert different inhibitory influences on the PF neurons for regulating the thalamic outflow from the PF to the cerebral cortex and/or striatum in the control of orofacial movements.

Evidence for a direct projection from the paraventricular nucleus of the hypothalamus to putative serotoninergic neurons of the nucleus paragigantocellularis involved in the control of erection in rats.

In the male rat, serotoninergic neurons of the ventrolateral medulla send direct projections onto spinal preganglionic neurons that innervate the penis. The role of the paraventricular nucleus of the hypothalamus in the control of penile erection is well recognized. Our aim was to demonstrate anatomical relation between paraventricular neurons and medullary serotoninergic neurons innervating the penis. In adult male rats, stereotaxic iontophoretic injections of Phaseolus vulgaris leuco-agglutinin were performed in the paraventricular nucleus. Neurons in the ventrolateral medulla were retrogradely labelled using transneuronal retrograde transport of pseudorabies virus injected in the corpus cavernosum. Sections of the ventro-lateral medulla were processed for double immunofluorescence to reveal both Phaseolus vulgaris leuco-agglutinin and pseudorabies virus using specific antibodies. Sections were also processed for the simultaneous detection of pseudorabies virus and serotonin. Pseudorabies virus-infected neurons in the ventrolateral medulla were present in the nucleus paragigantocellularis, reticular formation of the medulla, raphe pallidus and raphe magnus. In the nucleus paragigantocellularis, all pseudorabies virus-infected-neurons were immunoreactive for serotonin. Some of them received Phaseolus vulgaris leuco-agglutinin-labelled varicose fibres that ran along the soma of pseudorabies virus-infected neurons. Confocal microscopy suggested the presence of several close appositions between them, which were demonstrated using three-dimensional reconstruction of serial optical sections. Our results show that paraventricular neurons send direct projections in the nucleus paragigantocellularis onto neurons that innervate the penis. They suggest a possible role of the paraventricular nucleus in penile erection through the control of descending serotoninergic raphe-spinal neurons. The neurotransmitter used in this pathway remains to be determined.

Long-term cortical atrophy after excitotoxic striatal lesion: effects of intrastriatal fetal-striatum grafts and implications for Huntington disease.

It is not currently clear whether the cortical atrophy observed in Huntington disease (HD) is entirely a direct consequence of the disease or at least partially a secondary consequence of striatal atrophy. This is of major importance for evaluating the possible therapeutic value of intrastriatal fetal-striatum grafts in HD. Cresyl violet-stained sections from rats that had received striatal excitotoxic lesions 1 wk or 4 wk previously showed small and statistically nonsignificant decreases in the thickness of cortical layers V and VI, while series from rats lesioned 12 months previously showed marked decreases in the thickness of the whole cortex (approximately 35% decrease), layer V (approximately 45%-50%) and layer VI (approximately 45%-50%), together with marked neuron loss in these layers. In deep layer V and layer VI, Fluoro-Jade staining showed labeled neurons in animals lesioned 1 wk previously, labeled neurons and astrocytes in animals lesioned 4 wk previously, and practically no labeling in animals lesioned 12 months previously. Intracortical injection of Phaseolus vulgaris leucoagglutinin revealed that corticostriatal fibers were practically absent from the lesioned area of striata lesioned 12 months previously. However, rats that received intrastriatal fetal-striatum grafts shortly after the lesion and were killed 12 months later showed a significant reduction in cortical atrophy, and a large number of labeled corticostriatal fibers surrounding and innervating the graft. In addition, a reduction in the number of Fluoro-Jade-labeled cells in the cortex was already apparent at 3 wk post-grafting. Regardless of whether HD has a primary effect on the cortex, the present results suggest that the striatal degeneration caused by HD contributes markedly to the cortical atrophy, and that intrastriatal grafts may ameliorate this secondary component of the cortical degeneration.

Characterization of pretectal-nuclear-complex afferents to the pulvinar in the cat.

We investigated anatomical and physiological properties of the projection from the pretectal nuclear complex (PNC) to the ipsilateral lateral posterior-pulvinar complex in the cat. After Phaseolus vulgaris leucoagglutinin injections into the PNC, the majority (70%) of anterogradely labeled terminals was localized in the pulvinar proper, the remaining 30% were scattered in the lateral and medial portions of the LP. No PNC neuron retrogradely labeled from the pulvinar was found to also express glutamic acid decarboxylase (GAD) mRNA, although a large number of neurons carrying the GAD label were found in close vicinity. In contrast, 69% of retrogradely labeled PNC cells also displayed glutamate-like immunoreactivity. Twenty-six out of 96 (27%) visually responsive pulvinar neurons were orthodromically activated by electrical stimulation of the ipsilateral PNC at latencies between 1 and 10 ms (median 1.9 ms). All orthodromically activated neurons responded well to the onset and offset of large visual stimuli and to sudden stimulus shifts. Whenever a saccadic eye movement was executed, these neurons were also activated, except during saccades in darkness. The comparison of saccade-evoked response with responses to visual stimuli that elicit similar retinal image shifts revealed that pretectorecipient pulvinar neurons also seem to receive a saccade-related non-visual input. All response properties correspond to those of a specific class of pulvinar neurons that have been termed "SV" neurons because they respond to visual stimulation as well as during saccades. They also closely resemble response properties of PNC neurons that project to the ipsilateral pulvinar. The results support the proposal that PNC cells not only directly activate their postsynaptic target neurons in the pulvinar, but that they also provide a visual input to these neurons that greatly contributes to their response characteristics.

Hypothalamic dorsomedial nucleus neurons innervate thyrotropin-releasing hormone-synthesizing neurons in the paraventricular nucleus

To determine whether the hypothalamic dorsomedial nucleus (DMN) may serve as a relay center for the central actions of leptin on thyrotropin-releasing hormone (TRH)-synthesizing neurons in the paraventricular nucleus (PVN), axonal projections from the DMN to TRH-containing neurons in the PVN were studied using the anterogradely transported marker substance, Phaseolus vulgaris-leucoagglutinin (PHA-L). Stereotaxic injections of PHA-L were targeted to the mid-dorsal and mid-ventral portions of the DMN. After 10–14-day survival, the brains were prepared for immunohistochemistry and immunostained with an antibody directed against PHA-L. Focal injections confined to the DMN were identified in 14 animals and gave rise to a fiber bundle that entered the PVN at the caudal pole of the nucleus, densely innervating all parvocellular subdivisions of the PVN. In double-labeled preparations using antisera to PHA-L and preproTRH 178-199, the latter as a marker for TRH-containing neurons in the PVN, proTRH-IR neurons were observed to be enmeshed in a network of PHA-L-containing fibers. When the injection site covered the entire DMN or the mid-dorsal part of the DMN, PHA-L-containing axon varicosities were juxtaposed to ∼97 and 90% of proTRH neurons, respectively, in all parvocellular subdivisions of the PVN, and by ultrastructural analysis were shown to be synaptic. In contrast, when the injection site was centered primarily in the mid-ventral part of the DMN, only ∼52% of proTRH-synthesizing neurons appeared to be innervated by PHA-L-containing axons. These data demonstrate that a major projection pathway exists from the DMN, specifically to TRH-producing neurons in the PVN, and suggest that the DMN is anatomically situated to exert a regulatory effect on TRH-synthesizing neurons in the PVN.

Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity

In order to investigate converging projections originating from adjacent populations of cortical neurons, injections of two different anterograde tracers, biotinylated dextranamine (BDA) and Phaseolus vulgaris leucoagglutinin (PHA-L), were made in close proximity. When the two injection sites were separated by around 500 μm and the time between injections was 1–4 h, BDA-labeling of neuronal elements was found not only at the BDA injection site but also at the PHA-L injection site. This false-positive BDA labeling of neurons at the PHA-L injection site was so intense that labeled axons could be traced, both into the neighboring cortical gray matter and into white matter. Increasing the separation distance to 1000 μm resulted in much fewer falsely positive labeled neurons at the PHA-L injection site. Even more effective was extending the time interval between the two injections. Thus, if the BDA injection preceded the PHA-L injection by more than 12 h, virtually no false-positive labeling was associated with the PHA-L injection site. These procedures may be applied to other combinations of anterograde tracers, such as BDA with tetramethylrhodamine-conjugated dextran amine.

The efferent connections to the thalamus and brainstem of the physiologically defined eye field in the rat medial frontal cortex

The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into sites of the rat frontal eye field (FEF) located in the medial frontal cortex. After a single iontophoretic injection of PHA-L into a FEF site where intracortical microstimulation elicited eye movements, anterogradely labelled fibres and terminal-like elements were found in the thalamus in the anterior nuclei, intralaminar nuclei, lateral portion of the mediodorsal nucleus and posterior nuclear group. In the midbrain and pons, labelled fibres were located in the anterior pretectal area, Darkschewitsch nucleus, superior colliculus and dorsolateral portion of the central gray. When the tracer was injected at the FEF periphery, at a site the stimulation of which evoked both eye and whisker movements, labelling distribution in the thalamus differed from that observed after FEF injections, while a similar distribution was observed in the brainstem. In the thalamus, anterograde labelling was observed in these latter cases in the anterior nuclei, ventral nuclei, medial portion of the laterodorsal nucleus. The present findings point out that the FEF and FEF periphery are connected with numerous subcortical structures of the thalamus and brainstem. In addition, the connections of FEF and FEF periphery with the thalamus differ, whereas the midbrain and pons connections of the two subdivisions share common targets.

Divergent backward projections from the anterior part of the inferotemporal cortex (area TE) in the macaque.

The organization of backward projections from the anterior part of the inferotemporal cortex (area TE) to the posterior part of the inferotemporal cortex (area TEO) was studied in the macaque monkey by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). The objectives of the study were to investigate this backward projection and to compare it with 1) the backward projections that have been described previously in the early sensory areas and 2) the forward projection from area TEO to area TE. After a single iontophoretic injection of PHA-L into area TE in three monkeys, a dense distribution of labeled terminals was observed in area TEO and in the ventral bank of the superior temporal sulcus (area PITd) that adjoined area TEO. A less dense distribution was observed in areas V4, V2, and V1. Clusters of labeled terminals in areas TEO and PITd extended more than 4 mm along the cortical surface. The forward projections from area TEO to area TE also were studied for comparison by reanalyzing two previous cases (Saleem et al. ¿1993 Cerebral Cortex 3:454-464). These projections (from area TEO to area TE) were more focal than the terminations that occurred in area TEO after injections into area TE. Nine single axons projecting from area TE to areas TEO/PITd were reconstructed through serial sections. These showed variable, complex branching patterns with multiple arbors (1-12). Arbors were localized in layers 1-3 for four axons, in layer 1 for one axon, layers 5 and 6 for two axons, and in both layers 1-3 and layers 5-6 for two axons. Axons with horizontally elongated arbors confined to layer 1 were not predominant. The size of the individual arbors of these axons along their long axes tended to be larger (1.56 +/- 1.24 mm) than those of TEO-to-TE forward axons (<0.6 mm). Thus, the authors conclude that, like other backward systems described to date, those from area TE to areas TEO/PITd are divergent. However, single axons have more variable laminar patterns of terminal distribution than those in the other backward systems.

Development, neurochemical properties, and axonal projections of a population of last-order premotor interneurons in the white matter of the chick lumbosacral spinal cord.

There is general agreement that last-order premotor interneurons-a set of neurons that integrate activities generated by the spinal motor apparatus, sensory information and volleys arising from higher motor centres, and transmit the integrated signals to motoneurons through monosynaptic contacts-play crucial roles in the initiation and maintenance of spinal motor activities. Here, we demonstrate the development, neurochemical properties, and axonal projections of a unique group of last-order premotor interneurons within the ventrolateral aspect of the lateral funiculus of the chick lumbosacral spinal cord. Neurons expressing immunoreactivity for neuron-specific enolase were first detected in the ventrolateral white matter at embryonic day 9 (E9). The numbers of immunoreactive neurons were significantly increased at E10-E12, while most of them were gradually concentrated in small segmentally arranged nuclei (referred to as major nuclei of Hofmann) protruding from the white matter in a necklace like fashion dorsal to the ventral roots. The major nuclei of Hofmann became more prominent at E12-E16, but substantial numbers of cells were still located within the ventrolateral white matter (referred to as minor nucleus of Hofmann). The distribution of immunoreactive neurons achieved by E16 was maintained during later developmental stages and was also characteristic of adult animals. After injection of Phaseolus vulgaris-leucoagglutinin unilaterally into the minor nucleus of Hofmann, labeled fibres were detected in the ventrolateral white matter ipsilateral to the injection site. Ascending and descending fibres were revealed throughout the entire rostro-caudal length of the lumbosacral spinal cord. Axon terminals were predominantly found within the lateral motor column and the ventral regions of lamina VII ipsilateral to the injection site. Several axon varicosities made close appositions with somata and dendrites of motoneurons, which were identified as synaptic contacts in a consecutive electron microscopic study. With the postembedding immunogold method, 21 of 97 labeled terminals investigated were immunoreactive for glycine and 2 of them showed immunoreactivity for gamma-aminobutyric acid (GABA). The axon trajectories of neurons within the minor nucleus of Hofmann suggest that some of these cells might represent a population of last-order premotor interneurons. J. Exp. Zool. 286:157-172, 2000.

Ascending projections from the area around the spinal cord central canal: A Phaseolus vulgaris leucoagglutinin study in rats.

A single small iontophoretic injection of Phaseolus vulgaris leucoagglutinin labels projections from the area surrounding the spinal cord central canal at midthoracic (T6-T9) or lumbosacral (L6-S1) segments of the spinal cord. The projections from the midthoracic or lumbosacral level of the medial spinal cord are found: 1) ascending ipsilaterally in the dorsal column near the dorsal intermediate septum or the midline of the gracile fasciculus, respectively; 2) terminating primarily in the dorsal, lateral rim of the gracile nucleus and the medial rim of the cuneate nucleus or the dorsomedial rim of the gracile nucleus, respectively; and 3) ascending bilaterally with slight contralateral predominance in the ventrolateral quadrant of the spinal cord and terminating in the ventral and medial medullary reticular formation. Other less dense projections are to the pons, midbrain, thalamus, hypothalamus, and other forebrain structures. Projections arising from the lumbosacral level are also found in Barrington's nucleus. The results of the present study support previous retrograde tract tracing and physiological studies from our group demonstrating that the neurons in the area adjacent to the central canal of the midthoracic or lumbosacral level of the spinal cord send long ascending projections to the dorsal column nucleus that are important in the transmission of second-order afferent information for visceral nociception. Thus, the axonal projections through both the dorsal and the ventrolateral white matter from the CC region terminate in many regions of the brain providing spinal input for sensory integration, autonomic regulation, motor and emotional responses, and limbic activation.