Cytoarchitecture and efferent projections of the nucleus incertus of the rat

The nucleus incertus is located caudal to the dorsal raphe and medial to the dorsal tegmentum. It is composed of a pars compacta and a pars dissipata and contains acetylcholinesterase, glutamic acid decarboxylase, and cholecystokinin-positive somata. In the present study, anterograde tracer injections in the nucleus incertus resulted in terminal-like labeling in the perirhinal cortex and the dorsal endopyriform nucleus, the hippocampus, the medial septum diagonal band complex, lateral and triangular septum medial amygdala, the intralaminar thalamic nuclei, and the lateral habenula. The hypothalamus contained dense plexuses of fibers in the medial forebrain bundle that spread in nearly all nuclei. Labeling in the suprachiasmatic nucleus filled specifically the ventral half. In the midbrain, labeled fibers were observed in the interpeduncular nuclei, ventral tegmental area, periaqueductal gray, superior colliculus, pericentral inferior colliculus, pretectal area, the raphe nuclei, and the nucleus reticularis pontis oralis. Retrograde tracer injections were made in areas reached by anterogradely labeled fibers including the medial prefrontal cortex, hippocampus, amygdala, habenula, nucleus reuniens, superior colliculus, periaqueductal gray, and interpeduncular nuclei. All these injections gave rise to retrograde labeling in the nucleus incertus but not in the dorsal tegmental nucleus. These data led us to conclude that there is a system of ascending projections arising from the nucleus incertus to the median raphe, mammillary complex, hypothalamus, lateral habenula, nucleus reuniens, amygdala, entorhinal cortex, medial septum, and hippocampus. Many of the targets of the nucleus incertus were involved in arousal mechanisms including the synchronization and desynchronization of the theta rhythm.     

Afferent projections to nucleus reuniens of the thalamus

The nucleus reuniens (RE) is the largest of the midline nuclei of the thalamus and the major source of thalamic afferents to the hippocampus and parahippocampal structures. Nucleus reuniens has recently been shown to exert powerful excitatory actions on CA1 of the hippocampus. Few reports on any species have examined afferent projections to nucleus reuniens. By using the retrograde anatomical tracer Fluorogold, we examined patterns of afferent projections to RE in the rat. We showed that RE receives a diverse and widely distributed set of afferents projections. The main sources of input to nucleus reuniens were from the orbitomedial, insular, ectorhinal, perirhinal, and retrosplenial cortices; CA1/subiculum of hippocampus; claustrum, tania tecta, lateral septum, substantia innominata, and medial and lateral preoptic nuclei of the basal forebrain; medial nucleus of amygdala; paraventricular and lateral geniculate nuclei of the thalamus; zona incerta; anterior, ventromedial, lateral, posterior, supramammillary, and dorsal premammillary nuclei of the hypothalamus; and ventral tegmental area, periaqueductal gray, medial and posterior pretectal nuclei, superior colliculus, precommissural/commissural nuclei, nucleus of the posterior commissure, parabrachial nucleus, laterodorsal and pedunculopontine tegmental nuclei, nucleus incertus, and dorsal and median raphe nuclei of the brainstem. The present findings of widespread projections to RE, mainly from limbic/limbic-associated structures, suggest that nucleus reuniens represents a critical relay in the transfer of limbic information (emotional/cognitive) from RE to its major targets, namely, to the hippocampus and orbitomedial prefrontal cortex. RE appears to be a major link in the two-way exchange of information between the hippocampus and the medial prefrontal cortex.     

Prefrontal cortical projections to the cholinergic neurons in the basal forebrain

The prefrontal cortex (PFC) projections to the basal forebrain cholinergic cell groups in the medial septum (MS), vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB), and the magnocellular basal nucleus (MBN) in the rat were investigated by anterograde transport of Phaseolus vulgaris leuco-agglutinin (PHA-L) combined with acetylcholinesterase (AChE) histochemistry or choline acetyltransferase (ChAT) immunocytochemistry. The experiments revealed rich PHA-L-labeled projections to discrete parts of the basal forebrain cholinergic system (BFChS) essentially originating from all prefrontal areas investigated. The PFC afferents to the BFChS display a topographic organization, such that medial prefrontal areas project to the MS, VDB, and the medial part of the HDB, whereas the orbital and agranular insular areas predominantly innervate the HDB and MBN, respectively. Since the recurrent BFChS projection to the prefrontal cortex is arranged according to a similar topography, the relationship between the BFChS and the prefrontal cortex is characterized by reciprocal connections. Furthermore, tracer injections in the PFC resulted in anterograde labeling of numerous "en passant" and terminal boutons apposing perikarya and proximal dendrites of neurons in the basal forebrain, which were stained for the cholinergic marker enzymes. These results indicate that prefrontal cortical afferents make direct synaptic contacts upon the cholinergic neurons in the basal forebrain, although further analysis at the electron microscopic level will be needed to provide conclusive evidence.     

Amygdaloid projections to the frontal cortex and the striatum in the rat.

Projections from the basolateral nucleus of the amygdala (BLA) to the frontal cortex and the striatum were studied by using Phaseolus vulgaris-leucoagglutinin (PHA-L) anterograde tracing technique in the rat. PHA-L injections into the rostral part of the BLA resulted in a dense labeling of fibers with boutons in the dorsal bank of the rhinal fissure and in the lateral and the medial agranular cortex. PHA-L injections into the caudal part of the BLA produced a dense labeling of fibers in the medial surface of the frontal cortex. In most of the cortical regions, labeled fibers were predominantly distributed in two bands: one in the deep part of layers I and II and the other, heavier band, in layers V and VI. PHA-L injections into the rostral BLA resulted in a dense labeling of fibers with boutons in the olfactory tubercle, the rostral and caudolateral portion of the nucleus accumbens, and a large region of the caudate-putamen. The labeled area of the caudate-putamen included the rostroventral area, the central area, and the area caudal to the anterior commissure and dorsal and lateral to the globus pallidus. PHA-L injections into the caudal BLA produced fiber labeling in the most rostromedial area of the caudate-putamen facing the lateral ventricle, the medial portion of the nucleus accumbens, and the lateral septum. In the rostroventral striatum, PHA-L-labeled fibers selectively innervated the matrix compartment that contains abundant somatostatin-immunoreactive fibers. Compartmental segregation was less clear in the caudodorsolateral caudate-putamen and in the nucleus accumbens. Electron microscopy revealed that PHA-L-labeled boutons in the striatum contained abundant, small, round vesicles. These boutons formed asymmetrical synapses with dendritic spines of striatal neurons.     

Afferent fibers to the cingular vocalization region in the squirrel monkey

Three squirrel monkeys (Saimiri sciureus) received horseradish peroxidase injections in the anterior cingulate cortex at the level of the genu of the corpus callosum, a region yielding vocalization when electrically stimulated. Retrogradely labeled neurons were found at the cortical level within the dorsomedial and lateral prefrontal cortex (areas 9 and 10), orbital cortex (area 11), premotor cortex (areas 44, 6b, and 8), frontoparietal operculum, insula, cortex of the superior temporal sulcus, piriform cortex, subiculum, posterior cingulate, and retrosplenial cortex. Subcortical telencephalic projections came from the the claustrum, diagonal band of Broca, nucleus basalis Meynert, nuclei basalis lateralis and accessorius amygdalae, and cells at the periphery of globus pallidus. Diencephalic structures projecting to the anterior cingulate cortex were the thalamic nuclei anterior medialis, anterior ventralis, ventralis anterior, ventralis lateralis pars medialis, medialis dorsalis, pulvinaris medialis, centralis superior lateralis and limitans; the intralaminar nuclei paracentralis, centralis lateralis and parafascicularis; and the midline nuclei periventricularis, parataenialis, centralis superior, centralis inferior, centralis medialis, and reuniens. In the hypothalamus, projections came from the periventricular, lateral and posterior part, as well as the supramamillary nucleus. Midbrain afferent fibers came from the ventral tegmental area of Tsai, medial substantia nigra, reticular formation, area praerubralis, nucleus peripeduncularis, and periaqueductal gray. The most posterior labeled neurons were found in the locus ceruleus, dorsal tegmental nucleus of Gudden, nucleus annularis, nucleus centralis superior Bechterew, nucleus dorsalis raphae and the most dorsomedial part of the nucleus reticularis tegmenti pontis. Some of those projections have functional significance in the light of the hypothesis that the cingular cortex is involved in the volitional control of emotional reactions on the one hand and the influence of primary emotional reactions on intentional behavior on the other.     

Cortical and thalamic afferent connections of the insular and adjacent cortex of the cat

The thalamo-cortical and cortico-cortical afferents of the cat's insular cortex were investigated with the retrograde horseradish peroxidase technique. The most prominent loci of thalamic labeling were the suprageniculate nucleus and parts of the posterolateral nucleus. Injections into the anterior part of the insular cortex also resulted in labeled cells in the ventromedial posterior nucleus and in the intralaminar nuclei, while injections into posterior parts revealed projections from the medial and dorsal parts of the medial geniculate nucleus. Only the anterior and most ventral parts of the insular cortex overlying the anterior rhinal sulcus were connected with the mediodorsal nucleus of the thalamus. All injections into the gyrus sylvius anterior showed a specific pattern of cortical afferents: With the exception of the labeling in the prefrontal cortex and the inferotemporal region, the labeled cells were very narrowly restricted to the presylvian, the suprasylvian, and the splenial sulcus. The thalamic neurons projecting to the cortex were generally organized in a bandlike pattern which crossed nuclear borders. The majority of the cortico-cortical connections originated from sulcal areas next to the prefrontal, parietal, and cingulate cortex, that is, next to so-called association cortices. In the light of the present results the role of the insular cortex as a multifunctional association area is discussed, as well as its relation to other cortical centers.     

Projections from the lateral nucleus to the basal nucleus of the amygdala: a light and electron microscopic PHA-L study in the rat.

A recent study, carried out in the monkey brain demonstrated a hitherto undescribed projection from the lateral to the basal nucleus of the amygdaloid complex. In the present study, we used light and electron microscopic techniques to determine whether a similar connection exists in the rat brain and to define what type(s) of synaptic contacts are produced by fibers of this projection. Injections of the lectin tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were placed into several levels of the lateral nucleus and the distribution of fibers in the basal (basolateral) nucleus was evaluated. All lateral nucleus injections resulted in labeled fibers in the basal nucleus, though the density and distribution of labeled fibers depended on the position of the injection site within the lateral nucleus. In general, the heaviest labeling of the basal nucleus was observed after injections at midrostrocaudal levels of the lateral nucleus, especially when the injection was located ventrally. Fibers originating from cells labeled by these injections were observed throughout much of the rostrocaudal extent of the basal nucleus. Rostrally situated injections resulted in substantially lower levels of labeled fibers in the basal nucleus. Injections placed caudally in the lateral nucleus resulted in light to medium levels of labeled fibers in the basal nucleus; the terminal field in these cases did not extend as far rostrally as after the rostral and midlevel injections. Electron microscopic analysis of PHA-L labeled fibers revealed that they contributed synapses to the basal nucleus. The majority of PHA-L labeled terminals formed asymmetric contacts on dendritic spines or shafts; a smaller number of PHA-L labeled terminals formed symmetrical synapses.     

Afferents to the entorhinal cortex of the rat studied by the method of retrograde transport of horseradish peroxidase

The afferents to the parahippocampal area of the rat were studied with retrograde transport of horseradish peroxidase injected into the medial entorhinal cortex, lateral entorhinal cortex, parasubiculum, presubiculum, or a large injection which stained all these structures as well as the ventral hippocampus. Control rats were injected with horseradish peroxidase into the overlying visual cortex. Labeled neurons in brains with injections into the medial entorhinal cortex and the adjacent parasubicular region were found in the ipsilateral and contralateral presubicular region, the medial septal nucleus, the thalamic nucleus reuniens, the dorsal part of the lateral nucleus of thalamus, the anterior periventricular nucleus of the thalamus, and the dorsal raphe nucleus. Brains with injections into the lateral entorhinal cortex yielded labeled neurons in the medial septal nucleus, nucleus reuniens, dorsal raphe nucleus, and nucleus locus ceruleus. Injections into the presubiculum resulted, in addition, in labeling of neurons in the lateral nucleus of the thalamus. Control injections aimed at the sensory cortex overlying the parahippocampal area yielded labeled neurons in the medial septal nucleus, the dorsal lateral geniculate nucleus, and the nucleus locus ceruleus.     

Topography of descending projections from anterior insular and medial prefrontal regions to the lateral habenula of the epithalamus in the rat

The epithalamic lateral nucleus of the habenula (LHb) plays a key role in regulating firing of dopamine and serotonin neurons in the midbrain and is thereby involved in various cognitive and affective behaviors. It is not yet clear, however, from where the LHb receives cognitive and affective information relevant to its regulation of the midbrain monoaminergic systems. The prefrontal cortex would be among the ideal sources. Here, using anterograde and retrograde tracer injections in the rat brain, we characterized the topography of the corticohabenular projections. Following injections of cholera toxin subunit B into the LHb, retrogradely labeled neurons were produced in the anterior insular, cingulate, prelimbic and infralimbic cortices. Consistent with this retrograde tracing, injections of biotinylated dextran amine (BDA) into these cortical regions labeled robust terminals in the LHb. Our quantification of the BDA-impregnated varicosities revealed that projections from the anterior insula terminated mainly in the intersection regions of the lateral and ventral two-thirds of the LHb, while projections from the cingulate cortex terminated mainly in the lateral two-thirds of the LHb. By comparison, BDA-labeled terminals originating from the medial prefrontal regions were contained mainly in the medial plus ventral one-third of LHb. Based on these data, we hypothesize that LHb provides a link for conveying cognitive and affective information from prefrontal and insular regions to the midbrain monoaminergic centers.