Retrograde Transport Of Fluorescent Tracers Research Articles

Experience-Dependent Plasticity and Vision Restoration in Rats after Optic Nerve Crush

Within the first weeks after brain damage the visual system can spontaneously recover, but it is not known if visual experience plays a role in this process. Therefore, we studied the role of visual experience during recovery by exposing rats to normal, enriched, and impoverished visual conditions. Adult rats, which had learned a six-choice brightness discrimination task, received bilateral partial optic nerve crush (ONC), and were then exposed daily for 34 days to either (1) total darkness, (2) a standard 12-h light:12-h dark cycle, or (3) a 2-h selective visual enrichment. The percentage of correct choices and the maximal performance levels reached were used as recovery end-points. Retinal ganglion cell (RGC) morphology was evaluated following retrograde transport of fluorescent tracer with in vivo confocal neuroimaging (ICON) before and after ONC. Whereas rats kept under normal daylight conditions after ONC recovered their visual functions rather well, rats housed in complete darkness did not recover at all. However, only 2 h of daily exposure to visual enrichment induced significantly enhanced recovery, which was even faster than that seen in rats housed under normal daylight conditions. RGC soma size changes were observed after ONC, but they did not correlate with any measures of behavioral recovery. We conclude that visual experience, even if provided for short daily periods, is a critical factor determining the dynamics of early phases of recovery.

Morphology of brachial segments in mudpuppy (Necturus maculosus) spinal cord studied with confocal and electron microscopy.

The isolated brachial spinal cord of Necturus maculosus is useful for studies of neural networks underlying forelimb locomotion, but information about its cellular morphology is scarce. We addressed this issue by using confocal and electron microscopy. Remarkably, the central region of gray matter was aneural and consisted exclusively of a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled by retrograde transport of fluorescent tracers from ventral roots and axons in the ventrolateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, whereas their dendrites projected widely throughout the white matter. About one-third of labeled INs were found contralaterally, with axons crossing ventral to a thick layer of ependyma surrounding the central canal. Lateral MN dendrites proliferated under the pial surface to form a dense, thin (1-2 microm) plexus immediately beneath a thin layer of glial fibrillary acidic protein-positive glia limitans. The latter contained arrays of unusual tubular structures (diameter 200-400 nm, length 3 microm) that resembled mitochondria but lacked double membranes or cristae. Dorsal roots (DRs) produced dense presynaptic arbors within a wedge-shaped afferent termination zone medial to the dorsal root entry, within which dendrites of MNs and INs mingled with dense collections of synaptic boutons. Our data suggest that a major fraction of synaptic interactions takes place within the white matter. This study provides a detailed foundation for designing electrophysiological experiments to study the neural circuits involved in locomotor pattern generation.

Efferent connections of the caudal part of the globus pallidus in the rat.

The efferent connections of the caudal pole of the globus pallidus (GP) were examined in the rat by employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L), and the retrograde transport of fluorescent tracers combined with choline acetyltransferase (ChAT) or parvalbumin (PV) immunofluorescence histochemistry. Labeled fibers from the caudal GP distribute to the caudate-putamen, nucleus of the ansa lenticularis, reuniens, reticular thalamic nucleus (mainly its posterior extent), and along a thin strip of the zona incerta adjacent to the cerebral peduncle. The entopeduncular and subthalamic nuclei do not appear to receive input from the caudal GP. Descending fibers from the caudal GP course in the cerebral peduncle and project to posterior thalamic nuclei (the subparafascicular and suprageniculate nuclei, medial division of the medial geniculate nucleus, and posterior intralaminar nucleus/peripeduncular area) and to extensive brainstem territories, including the pars lateralis of the substantia nigra, lateral terminal nucleus of the accessory optic system, nucleus of the brachium of the inferior colliculus, nucleus sagulum, external cortical nucleus of the inferior colliculus, cuneiform nucleus, and periaqueductal gray. In cases with deposits of PHA-L in the ventral part of the caudal GP, labeled fibers in addition distribute to the lateral amygdaloid nucleus, amygdalostriatal transition area, cerebral cortex (mainly perirhinal, temporal, and somatosensory areas) and rostroventral part of the lateral hypothalamus. Following injections of fluorescent tracer centered in the lateral hypothalamus, posterior intralaminar nucleus, substantia nigra, pars lateralis, or lateral terminal nucleus, a substantial number of retrogradely labeled cells is observed in the caudal GP. None of these cells express ChAT immunoreactivity, but, except for the ones projecting to the lateral hypothalamus, a significant proportion is immunoreactive to PV. Our results indicate that caudal GP efferents differ from those of the rostral GP in that they project to extensive brainstem territories and appear to be less intimately related to intrinsic basal ganglia circuits. Moreover, our data suggest a possible participation of the caudal GP in feedback loops involving posterior cortical areas, posterior striatopallidal districts, and posterior thalamic nuclei. Taken as a whole, the projections of the caudal GP suggest a potential role of this pallidal district in visuomotor and auditory processes.

Functional organization of corticocortical projections from area 17 to area 18 in the cat's visual cortex

We used anatomical and physiological methods to study the functional organization of the association projection from area 17 to area 18 in the cat's visual cortex. Neurons in area 17 projecting to area 18 (revealed by retrograde transport of fluorescent tracer) tend to be clustered over regions of layer 4 receiving input from the ipsilateral eye (visualized by anterograde transneuronal tracing). Since the contralateral input overlaps these ipsilateral patches, the association cells lie preferentially in regions that are likely to be binocularly innervated. Indeed, almost all cells recorded electrophysiologically within the association clusters were strongly binocular, whereas between the clusters, many neurons were dominated by the contralateral eye. There is sufficient jitter in the retinotopic organization of area 17 for the discontinuous distribution of association cells to provide a continuous representation of the visual field. Cells in each association cluster in the rostral part of area 17 project divergently to innervate a zone extending up to 3 mm wide, anteroposteriorly, in the superficial layers of area 18. The receptive fields of cells at any point in area 18 are larger than for the corresponding point in area 17. Neurons recorded at two points in area 18, separated by a distance equal to the limit of anatomical divergence of the projection from area 17, have receptive fields that overlap by an amount similar to the region of visual field covered by the receptive fields of cells in a single association cluster in area 17 at a similar retinotopic position. Thus, area 18 receives a full and strongly binocular representation of the visual field not only from the lateral geniculate nucleus but also from area 17. The divergence of the area 17 to 18 projection compensates for the difference in receptive field size by ensuring that the receptive fields of each cluster of projecting neurons overlap fairly precisely those of the recipient neurons in area 18.

Neurokinin A coexists with substance P and serotonin in ventral medullary spinally projecting neurons of the rat

The coexistence of neurokinin A (NKA) with substance P (SP) and serotonin (5-HT) in ventral medullary neurons of the parapyramidal region and nucleus raphe pallidus of the rat was studied using multiple immunofluorescence labeling. Nearly all of the NKA-immunoreactive (IR) cells in the parapyramidal region and raphe pallidus were SP-IR and 5-HT-IR, whereas about 70% of the SP-IR neurons and about 60% of the 5-HT-IR neurons contained NKA-IR. There were no apparent differences in the patterns of coexistence between parapyramidal and raphe pallidus neurons. NKA-IR neurons, which colocalized SP-IR and 5-HT-IR, were studied for projections to the lumbar and thoracic spinal cord by use of retrograde transport of fluorescent tracer. Whereas about 50% of the retrogradely labeled neurons of the parapyramidal region and raphe pallidus contained NKA-IR, nearly all of the NKA-IR neurons projected to the thoracic and lumbar spinal cord. In addition, some NKA-IR neurons in the ventral medulla were retrogradely labeled with tracer from localized injections into the thoracic intermediolateral cell column. In summary, this study demonstrated that NKA-IR is colocalized with SP-IR in bulbospinal serotonergic neurons of the parapyramidal region and raphe pallidus, which are known to regulate sensory, motor, and autonomic activities of the spinal cord.

Areal and laminar organization of corticocortical projections in the rat somatosensory cortex

The present study examines patterns of connectivity between the primary somatosensory cortex of the rat (SI) and surrounding cortical areas also implicated in the processing of somatosensory information. The impetus for the study was the recent reports of major differences in the organization of cortex lateral and caudal to the SI in two other rodent species; the mouse (Carvell and Simons, '86: Somatosens. Res. 3:213-237; '87: J. Comp. Neurol. 265:409-427) and the grey squirrel (Krubitzer et al., '86: J. Comp. Neurol 250: 403-430). Corticocortical connections between the somatosensory areas of the rat parietal cortex were examined by using the combined retrograde and anterograde transport of horseradish peroxidase as well as the retrograde transport of fluorescent tracers. Tracer injections were made into different locations within SI and dysgranular cortex as well as into more lateral regions of parietal cortex. The tangential patterns of distribution both of callosal connections and of cytochrome oxidase activity together provided points of reference in determining the relation between injection sites and the resultant patterns of label. The results indicate that two distinct somatosensory areas, SI and the dysgranular cortex, are interconnected with a further lateral somatosensory area referred to as the second somatosensory area (SII). These projections are organized in a topographic fashion, which we interpret as evidence for a single representation of the body surface in SII. The three somatosensory areas each exhibit unique laminar patterns of ipsilateral corticocortical projection neurons and terminations. In SI, projection neurons are found mainly in layers II, III, and Va, and terminations are largely restricted to the infragranular layers. In the dysgranular cortex, projection neurons and terminations are found in all layers except layer I in which only terminal label is detectable and layer Vb in which notably fewer neurons are labelled. In SII, projection neurons and terminations are found in all layers except layer I and are particularly dense in lower layer III and layer IV. Further, whereas the laminar and areal distributions of ipsilateral and contralateral corticocortical projections largely overlap in both SI and the dysgranular cortex, in SII they tend to be areally segregated. Neurons projecting bilaterally to both ipsilateral and contralateral somatosensory cortex were equally rare in all three somatosensory areas. These results are discussed in relation to the organization of SII in other rodent species, and it is concluded that in the rat, like the mouse, cortex lateral and caudal to SI contains a single representation of the body surface.

Cholecystokinin‐, galanin‐, and corticotropin‐releasing factor‐like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat

The parabrachial nucleus (PB) is the main relay for ascending visceral afferent information from the nucleus of the solitary tract (NTS) to the forebrain. We examined the chemical organization of solitary-parabrachial afferents by using combined retrograde transport of fluorescent tracers and immunohistochemistry for galanin (GAL), cholecystokinin (CCK), and corticotropin-releasing factor (CRF). Each peptide demonstrated a unique pattern of immunoreactive staining. GAL-like immunoreactive (-ir) fibers were most prominent in the "waist" area, the inner portion of external lateral PB, and the central and dorsal lateral PB subnuclei. Additional GAL-ir innervation was seen in the medial and external medial PB subnuclei. GAL-ir perikarya were observed mainly rostrally in the dorsal lateral, superior lateral, and extreme lateral PB. CCK-ir fibers and terminals were most prominent in the outer portion of the external lateral PB; some weaker labeling was also present in the central lateral PB. CCK-ir cell bodies were almost exclusively confined to the superior lateral PB and the "waist" area, although a few cells were seen in the Kölliker-Fuse nucleus. The distribution of CRF-ir terminal fibers in general resembled that of GAL, but showed considerably less terminal labeling in the lateral parts of the dorsal and central lateral PB, and the external medial and Kölliker-Fuse subnuclei. The CRF-ir cells were most numerous in the dorsal lateral PB and the outer portion of the external lateral PB; rostrally, scattered CRF-ir neurons were seen mainly in the central lateral PB. After injecting the fluorescent tracer Fast Blue into the PB, the distribution of double-labeled neurons in the NTS was mapped. GAL-ir cells were mainly located in the medial NTS subnucleus; 34% of GAL-ir cells were double-labeled ipsilaterally and 7% contralaterally. Conversely, 17% of the retrogradely labeled cells ipsilaterally and 16% contralaterally were GAL-ir. CCK-ir neurons were most numerous in the dorsomedial subnucleus of the NTS and the outer rim of the area postrema. Of the CCK-ir cells, 68% in the ipsilateral and 10% in the contralateral NTS were double-labeled, whereas 15% and 10%, respectively, of retrogradely labeled cells were CCK-ir. In the area postrema, 36% of the CCK-ir cells and 9% of the Fast Blue cells were double-labeled. CRF-ir neurons were more widely distributed in the medial, dorsomedial, and ventrolateral NTS subnuclei, but double-labeled cells were mainly seen in the medial NTS.(ABSTRACT TRUNCATED AT 400 WORDS)

A study of NADPH-diaphorase positive septohippocampal neurons in rat

Retrograde transport of fluorescent tracers and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical techniques were combined in a study of septohippocampal projections in the rat. The dorsal (DH) and ventral (VH) hippocampus were simultaneously injected with different tracers (Fast Blue or Fluoro-Gold). Histochemical procedures revealed many NADPH-d positive cells located in the medial septum and the horizontal limb of the diagonal band. In the medial septum, NADPH-d positive neurons were mostly located lateral to the midline region and some of these were double-labeled by the tracer injected into the VH. Also, NADPH-d positive cells were found in the horizontal diagonal band and some of these were double-labeled following injections into the DH. No fluorescence/NADPH-d double-labeled neurons were observed in other structures known to project to the hippocampus.

Peripheral inflammation is associated with increased dynorphin immunoreactivity in both projection and local circuit neurons in the superficial dorsal horn of the rat lumbar spinal cord

The present study combined the retrograde transport of fluorescent tracers with the immunocytochemical identification of dynorphin A(1–8) in superficial dorsal horn neurons to examine whether peripheral inflammation-induced dynorphin increases are found in local circuit neurons only or also in neurons projecting at least to the caudal mesencephalon. Evidence is presented that complete Freund's adjuvant-induced inflammation produces a large increase in the number of lamina I dynorphin-containing projection and non-projection neurons, and in the number of lamina II dynorphin local circuit neurons.

Neurons containing calcitonin gene-related peptide in the parabrachial nucleus project to the central nucleus of the amygdala.

The source, distribution, and morphology of axons displaying calcitonin gene-related peptide (CGRP) immunoreactivity in the central amygdaloid nucleus of the adult rat were investigated with immunohistochemical techniques, both alone and in combination with retrograde transport of fluorescent tracers. An extremely dense plexus of CGRP-immunoreactive axons is differentially concentrated within the lateral capsular and lateral central subdivisions of the central nucleus, and much lighter concentrations of labeled fibers are present in the rostral part of the medial subdivision. No immunoreactive neurons were observed in the central nucleus in any of the experimental animals. The immunoreactive axons characteristically form prominent pericellular terminal arborizations surrounding unlabeled neurons. The number of cells receiving this dense input increases at caudal levels of the central nucleus. Retrograde label of central nucleus neurons by dye transport from injections into the pontine parabrachial nucleus and the nucleus of the tractus solitarius combined with CGRP immunohistochemistry established that many neurons in the central nucleus which receive dense pericellular innervation from CGRP-immunoreactive axons are projecting caudally to the parabrachial nucleus or, to a lesser extent, to the nucleus tractus solitarii. Central amygdaloid injections of rhodamine-labeled microspheres or fluorogold followed by immunohistochemical localization of cellular CGRP immunoreactivity revealed that the central amygdaloid CGRP fiber plexus originates bilaterally from the parabrachial nucleus. These multipolar CGRP-containing neurons are preferentially concentrated in the external medial and external lateral subnuclei, in the ventral aspect of the parabrachial nucleus. These results relating central amygdaloid CGRP to ascending and descending brainstem pathways, taken together with the extreme density of the fiber plexus, strongly suggest the relevance of the CGRP input to central nucleus function in cardiovascular and other autonomic regulation.

Cortico-cortical connections of the limbic cortex of the rat.

By means of the retrograde transport of fluorescent tracers (Fast Blue, True Blue, Fluorogold and Diamidino Yellow), the cortico-cortical connections of prelimbic, insular, anterior and posterior cingulate (retrosplenial) areas have been studied. Our results demonstrate that there are, in the cortex of the adult rat, a few cells which have branched axons with connections in the ipsilateral hemisphere (associational neurons) or in the contralateral hemisphere (callosal neurons). The callosal neurons could be separated into two categories: "double callosal" neurons which project both axon collaterals to two cortical areas of the contralateral hemisphere, and "associational-callosal" neurons which send axon collaterals to both hemispheres.

Tracing of lateral pterygoid muscle-related neurons in the trigeminal brainstem nuclei in the rat.

Retrograde transport of fluorescent tracers (diamidino yellow and true blue) was used to study the arrangement of brainstem neurons innervating the lateral pterygoid muscle in the rat. The lateral pterygoid motoneurons were located in the dorsolateral (jaw-closing) part of the trigeminal motor nucleus with clear somatotopy in the caudal part of the nucleus. No muscle-related neurons were present in the mesencephalic trigeminal nucleus. Histological examination of serial sections of lateral pterygoid muscles confirms the notion that, at least in the rat, this muscle is devoid of muscle spindles.

Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord.

We studied the organization of projections from the C1 adrenergic and A1 noradrenergic cell groups in the ventrolateral medulla (VLM) to the hypothalamus and the spinal cord by using a combination of retrograde transport of fluorescent tracers and immunocytochemistry. Three issues were addressed. Neurons in the VLM that stain immunohistochemically for phenylethanolamine N-methyltransferase (PNMT) have been assumed to be adrenergic. However, the presence of PNMT-immunoreactive neurons in the hypothalamus that do not stain for tyrosine hydroxylase (TH) prompted us to re-evaluate the VLM by an elution-restaining immunohistochemical procedure. We confirmed that nearly all of the rostral medullary PNMT-immunoreactive neurons also stained for TH. By contrast, in the caudal medulla, very few TH-positive neurons stained for PNMT. Neurons of the C1 group in the rostral VLM project both to the thoracic spinal cord and to the hypothalamus. To determine whether individual C1 neurons send collaterals to the hypothalamus and spinal cord, we injected different-colored fluorescent dyes (diamidino yellow or fast blue) into the thoracic spinal gray matter and either the median preoptic (MnPO) or paraventricular (PVH) nuclei of the hypothalamus. Very few double-labeled neurons were found in the VLM, indicating that hypothalamic and spinal cord projections arise from almost completely independent populations of cells. Approximately half of the neurons projecting to the spinal cord from rostral VLM were not immunoreactive for TH or PNMT, indicating that a substantial part of this projection is noncatecholaminergic. The MnPO and the PVH both receive extensive catecholaminergic inputs from the VLM. We also used fluorescent retrograde tracers to determine whether individual VLM neurons send collaterals to both hypothalamic sites. Approximately 20% of neurons projecting to the MnPO in the rostral two thirds of the VLM also sent collaterials to the PVH, nearly all of these neurons being TH-positive. The collateralization of the VLM catecholaminergic projection to the hypothalamus may provide an anatomical substrate for integration of fore-brain participation in cardiovascular regulation. In contrast, the adrenergic projection from the VLM to the intermediolateral column of the spinal cord arises from a separate population of neurons.

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.

Topographic organization of peripheral trigeminal ganglionic projections in newborn rats

Retrograde transport of fluorescent tracers (true blue and diamidino yellow) was employed to delineate the topography of the peripheral projections of trigeminal ganglion cells in newborn (<12 h of age) rats. Identical injections were made in adult animals for comparison. In neonates, both inter- and intradivisional topography of ganglionic projections were adult-like. Neurons which innervated mandibular fields were located posterolaterally while cells with ophthalmic or maxillary projections were restricted to the anteromedial and central parts of the ganglion, respectively. An adult-like topographic representation of the mystacial vibrissae follicles was also evident in the neonates.

Quantification of branched neuronal projections labelled by retrograde fluorescent tracing. A study of olivo-cerebellar projections

A method for estimating the number of branched and unbranched neurones projecting from a nucleus to two target sites is presented, based on the retrograde transport of fluorescent tracers. The method initially involves stereological corrections for the size of cytoplasm and nuclei respectively labelled by the two tracers. A second correction is applied to account for doubly labelled cells whose cytoplasm, but not nuclei, are in the plane of section. Finally, the detection rates of the two tracers are determined and appropriate corrections are applied. The projection from the medial accessory olive to the cerebellar vermis was studied using true blue and diamidino yellow to illustrate the method. Application of the method increased the number of branched neurones detected by 18.5%. Of the total increase, 48.4% was due to the correction for size, 9.2% to the correction for doubly labelled cells with nuclei outside the plane of the section and 42.4% to the correction for detection rates. There was no significant masking of one tracer by another, but true blue enhanced the fluorescence of diamidino yellow.

Specificity of spinal projections from hypothalamic and brainstem areas which innervate sympathetic preganglionic neurons

The specificity and topographic organization of afferent projections to the intermediolateral column (IML) were examined using retrograde transport of fluorescent tracers injected into pairs of thoracic spinal segments. Neurons within the hypothalamus (parvocellular paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus), pons (Kolliker-Fuse and A5 nuclei) and medulla (ventrolateral nucleus of the solitary tract and rostral ventrolateral medulla) each appeared to innervate only a single spinal segment. Neurons in each cell group projecting to different spinal segments were intermixed and showed no evidence of topographic organization. These results provide a potential anatomical substrate for organ-specific autonomic responses to physiological and psychological stimuli.

Transnuclear transport and axon collateral projections of the mamillary nuclei in the rat

Transnuclear transport of horseradish peroxidase (HRP) and wheat germ agglutinin-HRP conjugate (WGA-HRP) and the retrograde transport of fluorescent tracers were used to study axon collaterals of neurons in the mamillary nuclei. Tracers were injected into the thalamus or brain stem and after 18 hour-5 day survival periods, the brains were processed for fluorescence microscopy or for light and electron microscopic HRP histochemistry. Neurons in all divisions of the ipsilateral mamillary nuclei projected to both the thalamus and tegmentum. After HRP and WGA-HRP injections, anterogradely labeled axon terminals were observed in the known projection fields of the mamillary nuclei. Mamillary neurons were characterized by deeply invaginated, eccentrically located nuclei. Most labeled terminals of axon collaterals in the contralateral anterodorsal thalamic nucleus and dorsal and ventral tegmental nuclei contained round vesicles and formed asymmetrical synapses with somata and dendrites. The present results demonstrate that transnuclear transport of HRP and WGA-HRP can be used to study the connectivity and ultrastructure of axon collaterals and their cells of origin in the central nervous system in a manner comparable to that of transganglionic transport in the peripheral nervous system.

The distribution and orientation of serotonin fibers in the entorhinal and other retrohippocampal areas. An immunohistochemical study with anti-serotonin antibodies in the rats brain.

The serotonin (5-hydroxytryptamine; 5-HT) innervation of the retrohippocampal region (subiculum, pre- and parasubiculum, area 29e, medial and lateral entorhinal area) in the rat brain has been examined with antibodies against 5-HT used in combination with fluorescence histochemistry. Analysis of consecutive sections cut in the coronal, sagittal, and horizontal planes revealed a widespread distribution of 5-HT immunoreactive fibers throughout the retrohippocampal region. This innervation was with regard to the morphological characteristics of the 5-HT fibers, their density and their spatial orientation. On the basis of morphological criteria, four different types of 5-HT positive processes were distinguished: (a) fine, convoluted fibers with small (approximately 0.5-0.8 micron), round and evenly spaced varicosities: (b) fine fibers with small elongated, irregularly distributed varicosities; (c) thick, possibly myelinated fibers, and (d) a terminal plexus with large (5-10 micron), irregularly spaced varicosities. Analysis of the laminar distribution of the 5-HT fibers showed that whereas all layers contain 5-HT positive fibers, the molecular layer was the most densely innervated. The 5-HT fibers were found to be oriented both parallel and transverse to the longitudinal axis of medial and lateral entorhinal area. This grid-like arrangement was less pronounced in the presubiculum. Although the 5-HT innervation of the retrohippocampal region was found to be dominated by a widespread and apparently diffuse pattern, several areas contained dense clusters of preterminal 5-HT processes: area 29e, dorsal presubiculum (layer II), lateral entorhinal area (layer III and ventral layer II) and the transitional zone of the ventral entorhinal area. The 5-HT fibers were found to enter the retrohippocampal region primarily by three different routes; from the ventral and dorsal aspects and from the piriform and lateral neocortex (via the perirhinal area). Most of the fibers enter the region by the ventral route and these were found to ascend in all layers but predominantly in layer I. The location of the 5-HT cells giving rise to the innervation of the entorhinal area was studied by combining retrograde transport of fluorescent tracers with immunohistochemistry on the same tissue section. Both ipsi- and contralaterally located cells in the dorsal and median raphe nuclei were found to project to the entorhinal area. Most, but not all, of these retrogradely labeled cell bodies also contain 5-HT immunoreactivity.