Injections Of Anterograde Tracers Research Articles

Pathways for emotions and memory: I. Input and output zones linking the anterior thalamic nuclei with prefrontal cortices in the rhesus monkey

The anterior thalamic nuclei occupy a central position in pathways associated with emotions and memory [AMA Arch. Neurol. Psychiatry 38 (1937) 725]. The goal of this study was to determine the anatomic interaction of the anterior nuclei with distinct prefrontal cortices that have been implicated in emotion and specific aspects of memory. To address this issue, we investigated the relationship of input and output zones in the anterior thalamic nuclei linking them with functionally distinct orbitofrontal, medial, and lateral prefrontal cortices. We identified input zones by mapping the pattern and topography of terminations of prefrontal axons, and the output zones by mapping projection neurons in the anterior nuclei, after injection of anterograde and bidirectional tracers in distinct prefrontal cortices. The results showed that the anterior nuclei were preferentially connected with some orbitofrontal and medial prefrontal areas. In contrast, the anterior nuclei had comparatively sparse connections with most lateral prefrontal cortices, with the notable exception of frontal polar cortex, which had moderate but consistent connections with the anterior nuclei. Prefrontal cortices were connected mostly with the anterior medial nucleus, though medial areas 32 and 25 as well as the frontal polar cortex were also connected with the anterior ventral nucleus. The zones of axonal terminations were more expansive than the sites with projection neurons in the anterior nuclei, suggesting extensive influence of feedback projections from prefrontal cortices. The results suggest that the anterior thalamic nuclei may act in concert with orbitofrontal and medial prefrontal cortices in processes underlying emotions and long-term memory, and with the frontal polar cortex in prospective aspects of working memory.

Thalamic Relay Nuclei of the Basal Ganglia Form Both Reciprocal and Nonreciprocal Cortical Connections, Linking Multiple Frontal Cortical Areas

Thalamic relay nuclei transmit basal ganglia output to the frontal cortex, forming the last link in corticobasal ganglia circuitry. The thalamus regulates cortical activity through differential laminar connections, providing not only feedback, but also initiating "feedforward" loops, via nonreciprocal projections, that influence higher cortical areas. This study examines the organization of thalamic connections with cortex from basal ganglia relay nuclei, including ventral anterior (VA), ventral lateral (VL), and mediodorsal (MD) nuclei, in the Macaque monkey. Anterograde and bidirectional tracer injections ([3H]-amino acids, dextran conjugates of Fluorescein, Lucifer Yellow or FluoroRuby, or wheat germ agglutinin) into discrete VA/VL, MD, and frontal cortical sites demonstrate specific thalamocortical connections. VL projections target caudal motor areas (primary, supplementary, and caudal premotor areas), whereas VA projections target more rostral premotor areas (including cingulate and presupplementary motor areas) and MD projects to dorsolateral and orbital prefrontal cortices. Thalamocortical projections innervate cortical layers I and III, and to a lesser extent, layer V. In motor areas layer I projections are more extensive than those to layer III (and V). The complex laminar organization of projections from specific thalamic sites suggests differential regulation of cortical function. Injections of bidirectional tracers into thalamic and frontal cortical sites also show that in comparison to thalamocortical projections, corticothalamic projections to VA-VL and MD are more widespread. These findings demonstrate both reciprocal and nonreciprocal components to the thalamo-cortico-thalamic relay. Together, these experiments indicate a dual role for VA-VL and MD nuclei: (1) to relay basal ganglia output within specific cortical circuits and (2) to mediate information flow between cortical circuits.

The sensory trigeminal complex projects contralaterally to the facial motor and the accessory abducens nuclei in the rat

Anterograde tracer injections in the rat sensory trigeminal complex are shown here to demonstrate projections to the contralateral facial motor (VII) and accessory abducens (VIacc) nuclei. Most of the trigeminal fibres originated within the pars oralis (5o) and contacted neurones in the medial and intermediate VII. Moderate projections from the pars caudalis (5c) and interpolaris (5i) reached the lateral and dorsolateral VII. Rare projections from the principal nucleus (5P) were found. Trigeminal projections to the contralateral VIacc originated mainly from the 5P and 5o. Few projections from the 5i and 5c to the contralateral VIacc were found. Retrograde tracer injections in the VII showed premotor neurones to the contralateral VII scattered throughout the 5o and in the ventromedial portion of the caudal 5i and the 5c. Double retrograde tracing experiments provide evidence that neurones in the 5o and 5c project to both the ipsi- and contralateral VII. Such collateralization would play a significant role in the co-ordination of the musculature of the face.

Pain pathways and parabrachial circuits in the rat.

This review presents a schematic attempt to classify the major pain pathways, based on the results of recent studies in our laboratory, with a special emphasis on the parabrachial system. Our view is based on results from experiments in the rat, using very small iontophoretic injections of anterograde tracers. As illustrated in this report, we have found a very dramatic difference between ascending projections originating from deep laminae compared with those arising from lamina I of the dorsal horn. We propose three main pain systems and discuss their functional-anatomical relationships. The first system is centred on the projection from deep laminae to three caudal reticular areas - the lateral reticular nucleus (LRN), the subnucleus reticularis dorsalis (SRD) and the gigantocellular lateral paragigantocellular reticular nuclei (NGc) - and the parabrachial internal lateral subnucleus (PBil). The second system is centred on the projection from lamina I to the ventral posterolateral nucleus (VPL), the ventral posteromedial (VPM), the posterior nuclear group (Po) and triangular posterior nucleus (PoT) of the thalamus. The third system is centred on the projection from lamina I to the lateral parabrachial area. We also present the four main projections from the latter area to the extended amygdala, the hypothalamus, the periaqueductal grey matter (PAG), and the ventrolateral medulla (VLM), and their involvement in emotional and autonomic (homeostatic) aspects of pain.

Differential Metabolic Activity in the Striosome and Matrix Compartments of the Rat Striatum during Natural Behaviors

The striosome and matrix compartments of the striatum are clearly identified by their neurochemical expression patterns and anatomical connections. To determine whether these compartments are distinguishable functionally, we used [14C]deoxyglucose metabolic mapping in the rat and tested whether neutral behavioral states (free movement, gentle restraint, and focal tactile stimulation under gentle restraint) were associated with regions of high metabolic activity in the matrix, in striosomes, or in both. We identified metabolic peaks in the striatum by means of image analysis, striosome-matrix boundaries by [3H]naloxone binding, and primary somatosensory corticostriatal input clusters by injections of anterograde tracer into electrophysiologically identified sites in SI. Peak metabolic activity was primarily confined to the matrix compartment under each behavioral condition. These findings show that during relatively neutral behavioral conditions the balance of activity between the two compartments favors the matrix and suggest that this balance is present in the striatum as part of normal behavior and processing of afferent activity.

Properties and interconnections of trigeminal interneurons of the lateral pontine reticular formation in the rat.

Numerous evidence suggests that interneurons located in the lateral tegmentum at the level of the trigeminal motor nucleus contribute importantly to the circuitry involved in mastication. However, the question of whether these neurons participate actively to genesis of the rhythmic motor pattern or simply relay it to trigeminal motoneurons remains open. To answer this question, intracellular recordings were performed in an in vitro slice preparation comprising interneurons of the peritrigeminal area (PeriV) surrounding the trigeminal motor nucleus (NVmt) and the parvocellular reticular formation ventral and caudal to it (PCRt). Intracellular and extracellular injections of anterograde tracers were also used to examine the local connections established by these neurons. In 97% of recordings, electrical stimulation of adjacent areas evoked a postsynaptic potential (PSP). These PSPs were primarily excitatory, but inhibitory and biphasic responses were also induced. Most occurred at latencies longer than those required for monosynaptic transmission and were considered to involve oligosynaptic pathways. Both the anatomical and physiological findings show that all divisions of PeriV and PCRt are extensively interconnected. Most responses followed high-frequency stimulation (50 Hz) and showed little variability in latency indicating that the network reliably distributes inputs across all areas. In all neurons but one, excitatory postsynaptic potentials (EPSPs) or inhibitory postsynaptic potentials (IPSPs) were also elicited by stimulation of NVmt, suggesting the existence of excitatory and inhibitory interneurons within the motor nucleus. In a number of cases, these PSPs were reproduced by local injection of glutamate in lieu of the electrical stimulation. All EPSPs induced by stimulation of PeriV, PCRt, or NVmt were sensitive to ionotropic glutamate receptor antagonists 6-cyano-7-dinitroquinoxaline and D,L-2-amino-5-phosphonovaleric acid, while IPSPs were blocked by bicuculline and strychnine, antagonists of GABA(A) and glycine receptors. Examination of PeriV and PCRt intrinsic properties indicate that they form a fairly uniform network. Three types of neurons were identified on the basis of their firing adaptation properties. These types were not associated with particular regions. Only 5% of all neurons showed bursting behavior. Our results do not support the hypothesis that neurons of PeriV and PCRt participate actively to rhythm generation, but suggest instead that they are driven by rhythmical synaptic inputs. The organization of the network allows for rapid distribution of this rhythmic input across premotoneuron groups.

Axon collaterals of Meynert cells diverge over large portions of area V1 in the macaque monkey.

Patchy intrinsic connections, originating mainly from horizontal collaterals of pyramidal neurons, have been demonstrated in area V1 and many other cortical areas. In this article, we identify a network of intrinsic connections concentrated in layer 6 of area V1. These are visualized by extracellular injections of anterograde tracers in V1, which label small clusters of large terminal boutons in layer 6, in conjunction with thick axon segments. These segments can be traced back to infragranular Meynert cells (n = 10), which are retrogradely labeled from the injections. By using serial section analysis, we identified the following features of this distinctive system of Meynert cell collaterals: (1) terminal clusters are relatively small (<100 microm); (2) each cluster has a small number of rather large boutons (up to 3.0 microm); (3) there is typically a termination-free zone in the immediate vicinity (0.5-2.0 mm) of the cell body; (4) a single neuron has multiple branches that can extend up to 8.0 mm from the soma; and (5) the collaterals are concentrated in layer 6. These features are different from those of horizontal intrinsic connections in the supragranular layers of area V1. They are consistent with fast dynamics and a possible role in wide-field motion processing, such as has been associated with Meynert cells from other studies.

Projections of the pontine nuclei to the cochlear nucleus in rats

In the cochlear nucleus, there is a magnocellular core of neurons whose axons form the ascending auditory pathways. Surrounding this core is a thin shell of microneurons called the granule cell domain (GCD). The GCD receives auditory and nonauditory inputs and projects in turn to the dorsal cochlear nucleus, thus appearing to serve as a central locus for integrating polysensory information and descending feedback. Nevertheless, the source of many of these inputs and the nature of the synaptic connections are relatively unknown. We used the retrograde tracer Fast Blue to demonstrate that a major projection arises from the contralateral pontine nuclei (PN) to the GCD. The projecting cells are more densely located in the ventral and rostral parts of the PN. They also are clustered into a lateral and a medial group. Injections of anterograde tracers into the PN labeled mossy fibers in the contralateral GCD. The terminals are confined to those parts of the GCD immediately surrounding the ventral cochlear nucleus. There is no PN projection to the dorsal cochlear nucleus. These endings have the form of bouton and mossy fiber endings as revealed by light and electron microscopy. The PN represent a key station between the cerebral and cerebellar cortices, so the pontocochlear nucleus projection emerges as a significant source of highly processed information that is introduced into the early stages of the auditory pathway. The cerebropontocerebellar pathway may impart coordination and timing cues to the motor system. In an analogous way, perhaps the cerebropontocochlear nucleus projection endows the auditory system with a timing mechanism for extracting temporal information.

Origin of the noradrenergic innervation of the superior olivary complex in the rat

In the rat, the superior olivary complex contains lateral and medial olivocochlear neurones, which respectively innervate two separate targets within the cochlea; the auditory afferents contacting the inner hair cells and the outer hair cells themselves. Previous double label immunohistochemical studies have shown that both lateral and medial olivocochlear neurones are contacted by noradrenergic nerve endings, and electrophysiological studies on in-vitro rat brain slices have demonstrated that noradrenaline exerts a direct, predominantly excitatory effect on medial olivocochlear neurones. In this paper, we have investigated the origin of the noradrenergic input to the superior olivary complex (SOC). A retrograde tracer, Fluorogold, was used to map the inputs to the SOC, and this was combined with immunofluorescent staining for dopamine-beta-hydroxylase (DβH) to identify which of the afferent inputs was noradrenergic. These experiments showed small numbers of neurones double-stained for both Fluorogold and DβH in the A6 cell group (the locus coeruleus). In the A7 cell group, within and medial to the lateral lemniscus, numerous Fluorogold labelled and DβH positive neurones were found, but no neurones were seen that were double-labelled. In none of the other major noradrenergic cell groups were labelled Fluorogold neurones ever detected. To confirm the results obtained by retrograde tracer injections, anterograde tracer injections with biotinylated dextran amine were made in the locus coeruleus. This resulted in labelled fibres within all subdivisions of the superior olivary complex. These experiments indicate that the noradrenergic input to the olivocochlear neurones originates solely from the locus coeruleus. The small numbers of double-labelled neurones found in the locus coeruleus indicate a very divergent non-selective noradrenergic input to the SOC.

Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat.

Chewing, swallowing, breathing, and vocalization in mammals require precise coordination of tongue movements with concomitant activities of the mimetic muscles. The neuroanatomic basis for this oro-facial coordination is not yet fully understood. After the stereotaxic microinjection of retrograde and anterograde neuronal tracers (biotin-dextran, Fluoro-Ruby, Fluoro-Emerald, and Fluoro-Gold) into the facial and hypoglossal nuclei of the rat, we report here a direct bilateral projection of hypoglossal internuclear interneurons onto facial motoneurons. We also confirm the existence of a small pool of neurons in the dorsal part of the brainstem reticular formation that project ipsilaterally to both facial and hypoglossal nuclei. For precise tracer injections, both motor nuclei were located and identified by the electrical antidromic activation of their constituent motoneurons. Injections of retrograde tracers into the facial nucleus consistently labeled neurons in the hypoglossal nucleus. These neurons prevalently lay in the ipsilateral side, were small in size, and, like classic intrinsic hypoglossal local-circuit interneurons, had several thin dendrites. Reverse experiments - injections of anterograde tracers into the hypoglossal nucleus - labeled fine varicose nerve fiber terminals in the facial nucleus. These fiber terminals were concentrated in the intermediate subdivision of the facial nucleus, with a strong ipsilateral prevalence. Double injections of different tracers into the facial and the hypoglossal nuclei revealed a small, but constant, number of double-labeled neurons located predominantly ipsilateral in the caudal brainstem reticular formation. Hypoglossal internuclear interneurons projecting to the facial nucleus, as well as those neurons of the parvocellular reticular formation that project to both facial and hypoglossal nuclei, could be involved in oro-facial coordination.

Orbitomedial prefrontal cortical projections to hypothalamus in the rat.

A previous study in the rat revealed that distinct orbital and medial prefrontal cortical (OMPFC) areas projected to specific columns of the midbrain periaqueductal gray region (PAG). This study used anterograde tracing techniques to define projections to the hypothalamus arising from the same OMPFC regions. In addition, injections of anterograde and retrograde tracers were made into different PAG columns to examine connections between hypothalamic regions and PAG columns projected upon by the same OMPFC regions. The most extensive patterns of hypothalamic termination were seen after injection of anterograde tracer in prelimbic and infralimbic (PL/IL) and the ventral and medial orbital (VO/MO) cortices. Projections from rostral PL/IL and VO/MO targeted the rostrocaudal extent of the lateral hypothalamus, as well as lateral perifornical, and dorsal and posterior hypothalamic areas. Projections arising from caudal PL/IL terminated within the dorsal hypothalamus, including the dorsomedial nucleus and dorsal and posterior hypothalamic areas. There were also projections to medial perifornical and lateral hypothalamic areas. In contrast, it was found that anterior cingulate (AC), dorsolateral orbital (DLO), and agranular insular (AId) cortices projected to distinct and restricted hypothalamic regions. Projections arising from AC terminated within dorsal and posterior hypothalamic areas, whereas DLO and AId projected to the lateral hypothalamus. The same OMPFC regions also projected indirectly, by means of specific PAG columns, to many of the same hypothalamic fields. In the context of our previous findings, these data indicate that, in both rat and macaque, parallel but distinct circuits interconnect OMPFC areas with specific hypothalamic regions, as well as PAG columns.

Evidence for a direct projection from the postrhinal cortex to the subiculum in the rat.

Behavioral data indicate that three of the areas which form the parahippocampal region in the rat, i.e., the entorhinal, perirhinal, and postrhinal cortices, have different, although related functions that also differ from those of the hippocampal formation. These functional differences might be related to differences in connectivity, on the one hand with parts of the association cortex, and on the other with the hippocampal formation. In a previous study, we showed the existence of both a direct and an indirect projection from the perirhinal cortex to areas CA1 and subiculum of the hippocampus. Here we present the result of a second study, demonstrating a similarly organized projection from the postrhinal cortex to the subiculum, comprising both a direct and an indirect route. Electrical stimulation of the postrhinal cortex in vivo evoked field potentials throughout the subiculum and the dentate gyrus. Current source density analysis in both the subiculum and dentate gyrus revealed the presence of sink-source pairs, indicative of a synaptic termination. Based on comparison with the sink-source pairs found after stimulation of the medial entorhinal cortex, we conclude that the connection between the postrhinal cortex and the dentate gyrus most likely is formed by a polysynaptic pathway mediated via the medial entorhinal cortex, while the pathway from the postrhinal cortex to the subiculum is likely monosynaptic. In order to substantiate these findings, we carried out several tracer experiments. Anterograde tracer injections in the postrhinal cortex resulted in labeled fibers in limited parts of the subiculum, but no anatomical evidence for a projection of the postrhinal cortex to the dentate gyrus was found. Additional retrograde tracer injections in the subiculum also showed evidence for a direct postrhinal-to-subiculum projection with a strong topological organization. Based on these combined anatomical and electrophysiological data, we conclude that the postrhinal cortex indeed can reach the subiculum via both a direct and an indirect pathway.

Multiple zonal projections of the basilar pontine nuclei to the cerebellar cortex of the rat.

This study revealed a sagittal zonal pattern of projections to the cerebellar cortex after hydraulic or iontophoretic injections of anterograde tracers (tritiated leucine, wheat germ agglutinin-horseradish peroxidase, or biotinylated dextrane amine) in the basilar pontine nuclei of Wistar rats. The zonal pattern of projection was observed only after injections of small size, whereas large injections labeled diffusely wide areas of the cerebellar cortex, masking the zonal projection because the fusion of contiguous stripes. Diverging projections to discrete sets of sagittal stripes in the two sides of the cerebellar cortex arose from single injections. The stripes of fiber terminals were sharply delimited on both sides by areas, interstripes, either virtually void of labeling or with a much lower density of labeling. Thus, the areas of the cerebellar cortex were parceled in sets of sagittal compartments, stripes and interstripes, by the pontine projections. Up to five compartments (three stripes and two interstripes) were observed in the paraflocculus, in the copula pyramidis, and in vermal lobule IX. Up to nine compartments (five stripes and four interstripes) were found in the crus I, the lobulus simplex, the paramedian lobule, and vermal lobules VI-VIII. Up to seven compartments (four stripes and three interstripes) were found in the crus II. Single injections into the basilar pontine nuclei usually labeled symmetric areas of the cerebellar cortex, which, in some cases, showed similar number of stripes. When this was not the case, the stripes were usually more numerous in the contralateral than in the ipsilateral side. All areas of the cerebellar cortex were projected upon, with zonation patterns from different regions of the basilar pontine nuclei. The projections of the basilar pontine nuclei to the cerebellar cortex were arranged according to a fixed pattern specific for each cortical area, independently of the number of stripes labeled within. The mean width of the stripes visualized in the single cortical areas of different rats was similar, despite the different size of the injections. The length of the stripes ranged widely in the various areas of different rats. The data collected in this study are consistent with the idea that all the mossy afferents to the cerebellar cortex are arranged with a zonal pattern.

Morphological connections between the Hypoglassal and facial nerve in the brain stem of the rat

The perfect coordination and synchronization of hypoglossal and facial muscles during chewing, swallowing, breathing, and vocalization requires particular concomitant activities of the facial muscles. In contrast, no direct connection between the facial and hypoglossal nucleus on the level of the brain stem has been detected until now. Facial and hypoglossal nuclei of rats were identified on the basis of their antidromic field potential recorded after peripheral stimulation of the corresponding nerves. Stereotactically single or double fluorescence tracer injections (Biotin-Dextran, Fluorescine-Dextran, Rhodamine-Dextran, Fluoro Gold) were placed into the nuclei. Retrograde tracer injections into the facial nucleus consistently labeled small neurons in the hypoglossal nucleus. In reverse experiments the injection of anterograde tracers into the hypoglossal nucleus labeled fine caliber varicose nerve fibers, but no somata in the facial nucleus. Synchronous injections of different tracers into the facial and hypoglossal nucleus produced a small, but constant number of double-labeled cells in the parvocellular reticular formation. Both, hypoglossal interneurons projecting to the facial nucleus and neurons of the parvocellular reticular formation double-projecting to the facial and hypoglossal nucleus might play an important role in coordinated orofacial movements. Moreover, both populations of neurons might be responsible for the excellent postoperative results after hypoglossal-facial anastomosis.

Cross-modal plasticity of the corticothalamic circuits in rats enucleated on the first postnatal day.

Reorganization of the reciprocal corticothalamic connections was studied as a possible anatomical substrate of the cross-modal compensation of the missing visual input of the visual cortex by somatosensory-evoked activities in neonatally enucleated rats. The use of quantitative retrograde tract-tracing techniques revealed that the contribution of the lateral posterior thalamic nucleus (LP) is significantly increased following enucleation, while that of the dorsolateral geniculate and the lateral dorsal nuclei is decreased in the thalamocortical afferentation of a region in visual cortical area 17. In contrast with the control rats, a dense terminal arborization of afferents was labelled in the LP after the injection of anterograde tracer into the barrel cortex of the enucleated rats. The injection of anterograde tracer into the visual cortex also demonstrated a massive afferentation into the LP of the enucleated rats. Visual and somatosensory corticothalamic afferents exhibited similar ultrastructural features in the LP after enucleation, but their synaptic organizations differed as regards the diameter of the postsynaptic dendrites. Taken together with the previous observations, these results suggest a central role for the LP in the transmission of the somatosensory-evoked activities to the visual cortex after early blindness.

Localization of area prostriata and its projection to the cingulate motor cortex in the rhesus monkey.

Area prostriata is a poorly understood cortical area located in the anterior portion of the calcarine sulcus. It has attracted interest as a separate visual area and progenitor for the cortex of this modality. In this report we describe a direct projection from area prostriata to the rostral cingulate motor cortex (M3) that forms the fundus and lower bank of the anterior part of the cingulate sulcus. Injections of retrograde tracers in M3 resulted in labeled neurons in layers III, V and VI of prostriate cortex. However, injections of anterograde tracers in M3 did not demonstrate axon terminals in area prostriata. This connection was organized topographically such that the rostral part of M3 received input from the dorsal region of prostriate cortex, whereas middle and caudal levels of M3 received input from more ventral locations. Injections of retrograde and anterograde tracers in the caudal cingulate motor cortex (M4) did not produce labeling in prostriate cortex. Cytoarchitectural analysis confirmed the identity of area prostriata and further clarified its extent and borders with the parasubiculum of the hippocampal formation rostrally, and V1 of the visual cortex caudally. This linkage between cortex bordering V1 and cortex giving rise to a component of the corticofacial and corticospinal pathways demonstrates a more direct visuomotor route than visual association projections coursing laterally.

Organization of thalamostriatal terminals from the ventral motor nuclei in the macaque.

This study examines the organization of thalamostriatal projections from ventral tier nuclei that relay basal ganglia output to the frontal cortex. Although previous thalamostriatal studies emphasize projections from the intralaminar nuclei, studies in primates show a substantial projection from the ventral anterior (VA) and ventral lateral (VL) nuclei. These nuclei make up the main efferent projection from the basal ganglia to frontal cortical areas, including primary motor, supplementary, premotor, and cingulate motor areas. Functionally related motor areas of the frontal cortex and VA/VL have convergent projections to specific regions of the dorsal striatum. The distribution of VA/VL terminals within the striatum is crucial to understanding their relationship to motor cortical afferents. We placed anterograde tracer injections into discrete VA/VL thalamic areas. VA/VL thalamostriatal projections terminate in broad, rostrocaudal regions of the dorsal striatum, corresponding to regions innervated by functionally related cortical motor areas. The pars oralis division of VL projects primarily to the dorsolateral, postcommissural putamen, whereas the parvicellular VA targets more medial and rostral putamen regions, and the magnocellular division of VA targets the dorsal head of the caudate nucleus. Whereas these results demonstrate a general functional topography, specific VA/VL projections overlap extensively, suggesting that functionally distinct VA/VL projections may also converge in dorsal striatal areas. Within striatal territories, VA/VL projections terminate in a patchy, nonhomogeneous manner, indicating another level of complexity. Moreover, terminal fields contain both terminal clusters and scattered, long, unbranched fibers with many varicosities. These fiber morphologies resemble those from the cortex and raise the possibility that VA/VL thalamostriatal projections neurons have divergent connectional features.

A method for measuring colocalization of presynaptic markers with anatomically labeled axons using double label immunofluorescence and confocal microscopy

Information concerning the location and distribution of presynaptic neurotransmitter release sites within anatomically labeled axons would be of value for a large number of studies in functional anatomy, development, and plasticity. Here we report a method for localizing presynaptic sites within identified arbors of interest using anterograde anatomical tracer injections to label axonal projections and synaptic vesicle protein (SVP) antibodies to label presumptive presynaptic terminals. The axons and presynaptic sites are independently visualized with double label immunofluorescence and confocal microscopy. Stacks of images representing adjacent focal planes are collected, and image processing techniques are applied to identify the location of each axonal branch segment and each cluster of SVP label in three-dimensional space. Segmentation of the SVP label into distinct pixel clusters in three-dimensional space, followed by colocalization of these clusters with the labeled axons (object-based analysis), yields much more reliable and sensitive measures of colocalization than a simple determination of the number (or summed intensities) of colocalized pixels in a single optical section (pixel-based analysis). The method has been extended to measure the colocalization of antigens that are not located at the presynaptic terminal with a labeled population of axons.

Spinal location of ascending lamina i axons in the macaque monkey

Ascending axons of lamina I neurons were identified at upper cervical and tracic levels in 10 macaque monkeys after confined anterograde tracer injections in the superficial cervical or lumbar dorsal horn. Lamina I axons in the contralateral spinal white matter were of particular interest, because these include spinothalamic tract (STT) axons. These were clearly concentrated in the middle of the lateral funiculus, that is, in the location of the classically described lateral STT of humans. At the C1-2 level, they were more ventrally located. There was evidence of a general topography, in that lumbar axons were located superficially and cervical axons were located medially. There was some dispersion, in that a few axons were also found more dorsally and more ventrally (especially at the midthoracic level), and there was some variability between cases. In addition, ascending axons were more widely distributed in the ipsilateral white matter. These observations are consistent with prior anterograde findings in the cat. Together with clinical and other evidence, these observations indicate that lamina 1 neurons have an integral role in the effects of spinal lesions on pain, temperature, and itch sensations.

Perirhinal cortex input to the hippocampus in the rat: evidence for parallel pathways, both direct and indirect. A combined physiological and anatomical study.

The possibility of a direct projection from the perirhinal cortex (PER) to areas CA1 and subiculum (SUB) in the hippocampus has been suggested on the basis of tracer studies, but this projection has not unequivocally been supported by physiological studies. The demonstration of such a functional pathway might be important to understand the functioning of the hippocampal memory system. Here we present physiological and further anatomical evidence for such a connection between PER and the hippocampus. Electrical stimulation of PER in vivo evoked field potentials (EFPs) at the border area of CA1/SUB, consisting of a short latency and a longer latency component. Current source density analysis revealed that the sink of the short latency component was situated in the molecular layer of area CA1/SUB, while the longer latency component had its sink in the outer molecular layer of the dentate gyrus (DG). Anterograde tracer injections in PER showed labelled fibres in the border area of CA1/SUB, but anatomical evidence for a projection of PER to DG was not found. When synaptic transmission in the entorhinal cortex was partly blocked, the amplitude of the longer latency component of the recorded EFPs in the hippocampus was decreased while the short latency component was not affected, which suggests that the indirect pathway originating in PER is mediated through a synaptic relay in the entorhinal cortex. From the present results we conclude that information originating in PER reaches area CA1/SUB by parallel, direct and indirect, routes. The existence of this parallel organization appears to form an essential feature for the proper function of the medial temporal lobe memory system.