Observations on the development of certain ascending inputs to the thalamus in rats. I. Postnatal development.

We have studied the postnatal development of the major ascending afferents to the thalamus in postnatal rats using tetramethylbenzidine histochemistry following wheat germ agglutinin-conjugated horseradish peroxidase injections into either the dorsal column nuclei, the deep cerebellar nuclei, or the inferior colliculus. By the day of birth, the efferents from each of these regions have already entered, and arborized extensively within, their appropriate thalamic relay nuclei. However, the overall distribution of each of these ascending afferent systems differs dramatically from that seen in mature rats. In neonatal rats, a substantial proportion of the ascending axons extend beyond the thalamus and often enter the internal capsule, some bypassing the thalamus altogether. In addition, some of the axons which enter and arborize within the thalamus extend beyond their appropriate terminal field into adjoining thalamic nuclei. Retrograde tracing experiments utilizing Fast blue indicate that the cells of origin of these overshooting axons are distributed similarly to the cells of origin of the definitive thalamic afferents. These early erroneous projections are all subsequently eliminated and the characteristically restricted adult distribution of each afferent system is evident by P30. These results indicate that developmental overgrowths and targeting errors of thalamic afferent fibers are not unique to the visual system (where they have been documented previously), but may be a general feature in the development of these pathways.

The entorhinal cortex of the monkey: I. Cytoarchitectonic organization.

As an essential preliminary to a series of experimental studies of the afferent and efferent connections of the monkey entorhinal cortex, we have carried out a detailed analysis of its cytoarchitectonic organization. Primarily on the basis of features observed in Nissl- and fiber-stained preparations, supplemented with Golgi-stained material and preparations stained for heavy metals by Timm's method and histochemically for acetylcholinesterase, the entorhinal cortex has been divided into seven fields that are named according to their rostrocaudal and mediolateral positions except for one rostrally located field that is named for the prominent input that it receives from the olfactory bulb. At rostral levels, the entorhinal cortex is marked by a number of morphological inhomogeneities. The neurons tend to be organized in patches that are surrounded by large, thick, radially oriented bundles of fibers. At caudal levels, the entorhinal cortex has a more distinctly laminated appearance, reminiscent of that in the neocortex, and most of the neurons and fiber fascicles are arranged in discrete radial columns. The cortical region adjoining the entorhinal cortex laterally, which is commonly known as the "perirhinal cortex," is in fact composed of two separate fields corresponding to areas 35 and 36 of Brodmann. Area 35 occupies the fundus and part of the lateral aspect of the rhinal sulcus. Area 36 extends from the lateral bank of the rhinal sulcus into the inferior temporal gyrus, where it borders fields TA and TE rostrally, and field TF of the parahippocampal gyrus caudally. The surface extents of each of the entorhinal fields have been determined by making "unfolded" two-dimensional maps of the region and measuring the areas with a computerized digitizing system.

An EM autoradiographic study of the hypothalamo-hippocampal projection.

Previous studies have shown that in many different mammals there is a small but distinct projection from the supramammillary region in the caudal hypothalamus to the junctional region between the regio superior and regio inferior of the hippocampus. We have analyzed the mode of termination of this hypothalamo-hippocampal projection in the rat by electron microscopic (EM) autoradiography following injections of [3H]proline into the caudal hypothalamus. The projection is confined to the regio inferior where it is centered over the subicular end of field CA3, but also spans the adjoining region, field CA2. In our material the highest densities of labeling have been seen over the deeper part of the pyramidal cell layer and in the adjacent stratum oriens but, in addition, above background levels of labeling have been found superficial to the pyramidal cell layer in the stratum lucidum and the deeper part of the stratum radiatum. Most of the labeled synapses appear to be on the perikarya and primary dendrites of the hippocampal pyramidal cells, but some axo-spinous contacts have also been seen. All the labeled boutons contained clear, spheroidal synaptic vesicles and made asymmetric, Type I, contacts with their targets.

The mode of termination of the hypothalamic projection to the dentate gyrus: an EM autoradiographic study.

It has been suggested, on electrophysiological grounds, that the projection from the hypothalamus to the dentate gyrus constitutes a long-axon, monosynaptic, inhibitory pathway. To clarify the mode of termination of this projection we have examined in EM autoradiographs the distribution and form of labeled synapses in the rat dentate gyrus following the injection of [3H]proline of high specific activity into the supramammillary region of the hypothalamus. As suggested by previous light microscopic studies the hypothalamo-dentate projection has been found to terminate in a narrow zone that extends from about the superficial half of the layer of granule cell somata to the inner one-fifth of the overlying molecular layer. Within this zone more than 80% of the silver grains observed were associated with vesicle-containing profiles, most of which could be identified as forming Type I, asymmetric synapses upon large dendritic shafts. A smaller number of labeled synapses was found upon granule cell somata or upon the sessile dendritic spines that occur on the proximal parts of the granule cell dendrites. Since all of the labeled synapses showed distinct asymmetric membrane specializations and contained spheroidal vesicles it is difficult to reconcile these morphological findings with the view that the hypothalamic afferents to the dentate granule cells are inhibitory.

Some observations on hypothalamo-amygdaloid connections in the monkey.

The projections of the hypothalamus to the amygdala have been studied autoradiographically in a series of eleven cynomolgus monkeys (Macaca fascicularis) in which injections of [3H]amino acids had been made in different regions of the caudal two-thirds of the hypothalamus. The most prominent projection arises from the ventromedial nucleus of the hypothalamus and terminates most heavily in the medial, magnocellular division of the central nucleus. Injections confined to the ventromedial nucleus also result in labeling of the piriform cortex, the periamygdaloid cortex, the anterior amygdaloid area, the medial amygdaloid nucleus and the parvocellular divisions of both the basal and basal accessory nuclei. All these projections are bilateral (although the contralateral component is much smaller) and show evidence of a rostro-caudal topographic organization. Isotope injections that involve the caudal part of the lateral hypothalamic area label projections to the medial division of the central amygdaloid nucleus, to the medial and cortical nuclei and to the anterior amygdaloid area. When such caudally placed injections also involved the lateral mamillary nucleus, the lateral division of the central amygdaloid nucleus was additionally labeled. Although the medial mamillary nucleus does not project to the amygdala, there is evidence for a minor projection from the supramamillary region to the medial amygdaloid nucleus. The ventral tegmental area appears to project to the lateral division of the central nucleus and the medial portion of the substantia nigra has a small projection to both divisions of the central nucleus. All of these projections reach the amygdala by way of the so-called ventral amygdalofugal pathway, but at least some of the fibers that arise in the ventromedial nucleus run in the stria terminalis.

The morphology and connections of the posterior hypothalamus in the cynomolgus monkey (Macaca fascicularis). I. Cytoarchitectonic organization.

The cytoarchitectonic organization of the posterior hypothalamus of the cynomolgus monkey (Macaca fascicularis) was analyzed in Nissl, Golgi, acetylcholinesterase, and reduced silver preparations. The region consists of a number of cell masses that differ considerably in their discreteness and in the homogeneity of their neuronal populations. The nuclei identified include: the medial mamillary nucleus (in which at least three distinct subdivisions can be recognized--a pars medialis, a pars lateralis, and a pars basalis); the small-celled nucleus intercalatus; the large-celled lateral mamillary nucleus; a single premamillary nucleus; the tuberomamillary nucleus; the posterior hypothalamic nucleus; the caudal extension of the lateral hypothalamic area; the supramamillary area; and the paramamillary nucleus (which appears to correspond to the nucleus of the ansa lenticularis of other workers). As a basis for the subsequent experimental study of the efferent connections of the posterior hypothalamus, the location of each of these cell masses is described and illustrated in a series of low-power photomicrographs, as are the form and distribution of the resident neuronal populations of the various components of the mamillary complex as seen in Golgi preparations.

The morphology and connections of the posterior hypothalamus in the cynomolgus monkey (Macaca fascicularis). II. Efferent connections.

The efferent connections of the posterior hypothalamus have been analyzed autoradiographically in a series of eight cynomolgus monkey (Macaca fascicularis) brains with injections of 3H-amino acids in different regions of the mamillary complex and the surrounding areas. The medial mamillary nucleus was found to project through the mamillothalamic tract to the ipsilateral anteroventral, anteromedial, and interanteromedial nuclei, and by way of the mamillotegmental tract principally to the deep tegmental nucleus (of Gudden). It also appears to contribute fibers to the medial forebrain bundle, some of which reach as far rostrally as the medial septal nucleus. The lateral mamillary nucleus projects through the mamillothalamic tract bilaterally upon the anterodorsal nuclei of the thalamus, and through the mamillotegmental system to the dorsal tegmental nucleus; it also appears to contribute fibers to the medial forebrain bundle. The supramamillary area has extensive ascending and descending connections that are distributed with the medial forebrain bundle to the hypothalamus and rostral midbrain; in addition, it gives rise to an unusually well-defined projection to field CA2 of the hippocampus and to a narrow zone overlying the outer part of the granule cell layer and the adjoining part of the molecular layer of the dentate gyrus. We have not been able to distinguish the connections of the posterior hypothalamic nucleus from those of the caudal part of the lateral hypothalamic area: they both appear to contribute substantially to the ascending components of the medial forebrain bundle, and through its descending projection to the tegmental fields of the midbrain, the nucleus centralis superior of the raphe complex, the locus coeruleus, and the central gray as far caudally as the facial nerve. Their further projections to the spinal cord were not examined. Viewed broadly, and in the light of previous work, our observations confirm, once again, the constancy of the connections of the hypothalamus in the mammalian brain, and the pivotal position that the posterior hypothalamus occupies in the elaborate system of connections that links the limbic areas of the forebrain with the complex of structures that Nauta has aptly designated the "midbrain limbic region."

The medial forebrain bundle of the rat. II. An autoradiographic study of the topography of the major descending and ascending components.

The medial forebrain bundle (MFB) is a complex fiber system that courses through and partly arises and partly terminates within the lateral preoptic and lateral hypothalamic areas. It consists mainly of thin fibers and may be comprised of as many as 50 descending and ascending components of varying lengths and of different origins and/or destinations (Nieuwenhuys et al., '82). With the aid of an an atlas of the MFB and the surrounding brain areas in the rat presented in the preceding paper (Nieuwenhuys et al., '82), the position and topographic relationships of some 21 components of the bundle have been analyzed in detail, in brains that had been prepared for autoradiography following injections of tritiated amino acids into a number of structures that are known to contribute fibers to the MFB. From this analysis it is clear that most of the labeled components occupy specific and rather constant positions within the MFB. For example, the ascending components are largely confined to the dorsal half of the bundle; those arising from the medial preoptic area and the various hypothalamic nuclei are distributed rather diffusely over much of the MFB; and the descending components that arise from the olfactory tubercle and the magnocellular preoptic nucleus are confined to restricted parts of the bundle. These findings indicate that the neurons which occupy different parts of the lateral hypothalamic area probably receive distinctive inputs, and to a first approximation these are likely to be determined principally by their position within the MFB.

Subcortical afferents to the hippocampal formation in the monkey.

The distribution of neurons in the basal telencephalon, the diencephalon, and the brainstem that project to the hippocampal formation has been analyzed in mature cynomolgus monkeys (Macaca fascicularis) by the injection of horseradish peroxidase into different rostro-caudal levels of the hippocampal formation. After injections which involve Ammon's horn, the dentate gyrus, and the subicular complex, retrogradely labeled neurons are found in the following regions: in the amygdala (specifically in the anterior amygdaloid area, the basolateral nucleus, and the periamygdaloid cortex); in the medial septal nucleus and the nucleus of the diagonal band; in the ventral part of the claustrum; in the substantia innominata and the basal nucleus of Meynert; in the rostral thalamus (specifically in the anterior nuclear complex, the laterodorsal nucleus, the paraventricular and parataenial nuclei, the nucleus reuniens, and the nucleus centralis medialis); in the lateral preoptic and lateral hypothalamic areas, and especially in the supramammillary and retromammillary regions; in the ventral tegmental area, the tegmental reticular fields, the raphe nuclei (specifically in nucleus centralis superior and the dorsal raphe nucleus), in the nucleus reticularis tegmenti pontis, the central gray, the dorsal tegmental nucleus, and in the locus coeruleus.

Evidence for an input to the molecular layer and the stratum granulosum of the dentate gyrus from the supramammillary region of the hypothalamus.

Injections of a mixture of tritiated amino acids were made into the posterior hypothalamus in a series of rats and cats. In every case in which the injection involved a significant proportion of the cells in the supramammillary region, labeled fibers could be followed to the dentate gyrus, the anterior hippocampal rudiment and the induseum griseum of both sides. In the dentate gyrus the hypothalamic afferents terminate in a narrow band in the outer half of the stratum gramulosum and the inner 20 micron or so, of the stratum moleculare, immediately deep to the zone of termination of the associational and commissural afferents. As judged by silver grain counts across the width of the zone of labeled terminals, the projection to the ipsilateral side is several times as heavy as that to the contralateral side, and although it involves the entire septo-temporal (=rostro-caudal) extent of the gyrus on both sides, the projection to the suprapyramidal (inner) blade of the dentate gyrus is approximately twice as heavy as that to the infrapyramidal (outer) blade.

An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat.

The efferent connections of the lateral hypothalamic area (LHA) have been analyzed in a series of 30 rat brains with injections of 3H-amino acids into different parts of the area and the surrounding regions. Our findings indicate that all parts of the LHA contribute ascending and descending fibers to the medial forebrain bundle, and also project medially to certain of the adjoining hypothalamic nuclei. All levels of the LHA appear to send some fibers to a continuous group of structures that extends from the medial septal-diagonal band complex rostrally, through the lateral preoptic and lateral hypothalamic areas to the mammillary complex and the ventral tegmental area caudally. In addition, it is evident that cells at different levels within the LHA may have differential projections. Thus, the anterior and lateral parts of the LHA also appear to project substantially to the anterior hypothalamic area, the ventromedial and dorsomedial hypothalamic nuclei, the parataenial and paraventricular nuclei of the thalamus, and the medial part of the lateral habenular nucleus. Similarly, cells in the tuberal and posterior parts of the LHA project to the central gray, the longest projections from the posterior region reaching as far caudally as the central tegmental field, the parabrachial nucleus, the locus coeruleus, and the superior central and dorsal nuclei of the raphe. Viewed as a whole, the LHA is therefore well-suited to integrate inputs from the limbic system and brainstem and to relay them on the one hand to the medial zone of the hypothalamus and on the other to virtually every structure closely associated with the medial forebrain bundle and to the nuclei of origin of the major ascending monoaminergic systems.

The efferent connections of the anterior hypothalamic area of the rat, cat and monkey.

The general morphology and topographic relations of the anterior hypothalamic area (AHA) in the rat, cat and squirrel monkey have been described, and its efferent connections analyzed autoradiographically, after small injections of 3H-labeled amino acids into, or around, the area. In all three species the AHA is rather poorly separated from the surrounding preoptic and hypothalamic areas and nuclei but shows three distinct cellular condensations, located rostrally, centrally, and posterodorsally. Closely associated with the AHA are the retrochiasmatic area, the anterior periventricular nucleus and the scattered neurons usually referred to as the accessory supraoptic nucleus. The AHA has primarily short connections to the adjoining medial preoptic area, the lateral hypothalamic area, the periventricular nucleus, the dorsomedial nucleus, and to the "capsule" of the ventromedial nucleus. However, it also has certain more distant projections, rostrally to a narrow zone centered in the ventral part of the lateral septal nucleus, and caudally to the dorsal premammillary nuclei, the posterior hypothalamic area and the central gray. There is some evidence to suggest that the various subdivisions of the AHA have different efferent connections. Thus the posterodorsal cell condensation appears to give rise to the bilateral projection to the dorsal premammillary nuclei, while the projections to the septum, the posterior hypothalamic area and the central gray seem to have their origin in the central condensation. Similarly, the retrochiasmatic area sends its efferents through the ventral supraoptic commissure to the amygdala, the anterior periventricular nucleus contributes to the periventricular fiber system and to the external lamina of the median eminence, and the accessory supraoptic neurons project to the internal lamina of the median eminence.

The efferent connections of the ventromedial nucleus of the hypothalamus of the rat.

The efferent connections of the ventromedial nucleus of the hypothalamus (VMH) of the rat have been examined using the autoradiographic method. Following injections of small amounts (0.4-2.0 muCi) of tritium labeled amino acids, fibers from the VMH can be traced forward through the periventricular region, the medial hypothalamus and the medial forebrain bundle to the preoptic and thalamic periventricular nuclei, to the medial and lateral preoptic areas, to the bed nucleus of the stria terminalis and to the ventral part of the lateral septum. Some labeled axons continue through the bed nucleus of the stria terminalis into the stria itself, and hence to the amygdala, where they join other fibers which follow a ventral amygdalopetal route from the lateral hypothalamic area and ventral supraoptic commissure. These fibers terminate in the dorsal part of the medial amygdaloid nucleus and in the capsule of the central nucleus. A lesser number of rostrally directed fibers from the VMH crosses the midline in the ventral supraoptic commissure and contributes a sparse projection to the contralateral amygdala. Descending fibers from the VMH take three routes: (i) through the medial hypothalamus and medial forebrain bundle; (ii) through the periventricular region; and (iii) bilaterally through the ventral supraoptic commissure. These three pathways are interconnected by labeled fibers so that it is not possible to precisely identify their respective terminations. However, the periventricular fibers seem to project primarily to the posterior hypothalamic area and central gray, as far caudally as the anterior pole of the locus coeruleus, while the medial hypothalamic and medial forebrain bundle fibers apparently terminate mainly in the capsule of the mammillary complex, in the supramammillary nucleus and in the ventral tegmental area. The ventral supraoptic commissure fibers leave the hypothalamus closely applied to the medial edges of the two optic tracts. After giving off their contributions to the amygdala, they continue caudally until they cross the dorsal edge of the cerebral peduncle to enter the zona incerta. Some fibers probably terminate here, but others continue caudally to end in the dentral tegmental fields, and particularly in the peripeduncular nucleus. Within the hypothalamus, the VMH appears to project extensively to the surrounding nuclei. However, we have not been able to find evidence for a projection from the VMH to the median eminence. Isotope injections which differentially label the dorsomedial or the ventrolateral parts of the VMH have shown that most of the long connections (to the septum, amygdala, central tegmental fields and locus coeruleus) originate in the ventrolateral VMH, and there is also some evidence for a topographic organization within the projections of this subdivision of the nucleus.

Hippocampo-hypothalamic connections: origin in subicular cortex, not ammon's horn.

An autoradiographic study of the subcortical projections of the rat hippocampal formation shows that the efferent fibers of the hippocampus proper (fields CA1-4 OF Ammon's horn) do not project to the hypothalamus but are confined to the precommissural fornix, ending primarily in the septum. The fibers that are distributed by way of the fornix system to the hypothalamus (principally the arcuate-ventromedial region and the mammillary nuclei) and the anterior thalamus arise from the subicular region of the cerebral cortex (that is, the subiculum, presubiculum, and parasubiculum).

The efferent connections of the suprachiasmatic nucleus of the hypothalamus.

The efferent connections of the suprachiasmatic nucleus of the hypothalamus have been studied in the rat by the injection of 3H-proline into the nucleus and the surrounding regions of the rostral hypothalamus, and by the injection of the enzyme marker, horseradish peroxidase, into the region of the ventromedial hypothalamic nucleus. After an injection of 3H-proline confined to the ventral portion of the suprachiasmatic nucleus, transported label can be followed, in the autoradiographs, dorsally and caudally in the periventricular area as far as the caudal end of the ventromedial nucleus, into the triangular area between this nucleus and the arcuate nucleus, and along the ventral aspect of the tuberal region, just lateral to the ventromedial nucleus. A small number of silver grains are also seen over the internal lamina of the median eminence. No label can be followed rostrally or immediately lateral to the nucleus. Comparable injections into adjoining regions of the hypothalamus (especially the anterior hypothalamic area, the medial preoptic area, and the retrochiasmatic region) show transported label over the same regions, but with a somewhat different pattern of grain distribution; in addition, the anterior hypothalamic area shows an extensive projection through the medial forebrain bundle to the mammillary and supramammillary nuclei, the midbrain tegmentum, and certain of the midline thalamic nuclei. Although it is difficult in our autoradiographs to distinguish between the course of the efferent fibers from the suprachiasmatic nucleus and the zones in which they terminate, our evidence favors a termination among the cells of the periventricular area, and upon dendrites of the cells in the ventromedial, dorsomedial and arcuate nuclei, which extend beyond the limits of the nuclei into the periventricular area and to the area beneath the ventromedial nucleus.