The organization of afferent projections to the midbrain periaqueductal gray of the rat

The retrograde transport technique was utilized in the present study to investigate the afferent projections to the periaqueductal gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductal gray of 22 experimental animals and into regions adjacent to the periaqueductal gray in 6 control animals. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductal gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled cells occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornical nucleus and the area of the tuber cinereum. The largest mesencephalic input to the periaqueductal gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis, the ventral tegmental area, the locus coeruleus and the parabrachial nuclei. The medullary and pontine reticular formation provide the largest input to the periaqueductal gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain.The sources of afferent projections to the periaqueductal gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.     

The distribution of the projection from the hippocampal formation to the nucleus accumbens in the rat: an anterograde- and retrograde-horseradish peroxidase study

We have investigated the distribution and organization of the projection from the hippocampal formation to the nucleus accumbens in the rat. In the first experiments, horseradish peroxidase was injected into the fimbria fornicis. This procedure resulted in massive anterograde labelling of fornix fibers, and enabled the hippocampo--accumbens projection to be charted in detail. Labelled fornix fibers are distributed to the entire length of the nucleus accumbens and do not spread lateral to the anterior limb of the anterior commissure, that is, the projection is limited to the medial nucleus accumbens. Terminal labelling is particularly dense in a caudal dorsomedial sector of accumbens immediately adjacent to the septum. In the second part of the study, horseradish peroxidase was microelectrophoretically injected into the nucleus accumbens, in order to confirm the findings from the anterograde experiments. When the deposit was localized within the fornix distribution field, as indicated by the first experiment, retrogradely-labelled cells were observed in the subiculum, prosubiculum, and to a lesser extent, in hippocampal field CAI. When the deposit was located outside of the fornix distribution field, no hippocampal labelling was noted. The topography of the projection, suggested by the retrograde labelling experiments, is discussed. The findings confirm earlier reports of such a projection from the hippocampal formation to the nucleus accumbens, and with the use of a relatively novel anterograde-horseradish peroxidase technique, provide a picture of fiber labelling representing the entire field of origin of the fornix system. The striatal projection field of this pathway is discussed in relation to other striatal afferents, with particular emphasis on limbic afferentation of the striatum. Finally, the so-called 'hippocampal district' of striatum is discussed with respect to its neurotransmitter composition, as well as its functional importance regarding limbic system influence on central motor mechanisms.     

The amygdalostriatal projection in the rat—an anatomical study by anterograde and retrograde tracing methods

Tritiated leucine and proline injected into the amygdaloid complex was found to label a voluminous amygdalostriatal fiber system which is distributed to all parts of the striatum except an antero-dorsolateral striatal sector. The connection is established by way of the longitudinal association bundle as well as the stria terminalis, and includes a modest (10–15%), symmetrically distributed contralateral component conveyed by the anterior commissure. Both autoradiographic findings and subsequent observations in retrograde cell-labelling (horseradish peroxidase) material indicate that the amygdalostriatal projection originates mainly from the nucleus basalis lateralis amygdalae, in much lesser volume from the nucleus basalis medialis, and minimally from the nucleus lateralis amygdalae; no other contributing amygdaloid cell group could be identified.A comparison of the present findings with earlier reports indicates that the amygdalostriatal projection widely overlaps the striatal projections from the ventral tegmental area, the mesencephalic raphe nuclei and the prefrontal cortex. Like the amygdalostriatal projection, these striatal afferents largely or entirely avoid the antero-dorsolateral striatal quadrant, which thus appears to be the striatal region most sparsely innervated by afferents originating from structures within the circuitry of the limbic system. Findings in additional autoradiographic material identify this relatively non-limbic striatal quadrant as the main region of distribution of the corticostriatal projection from the sensorimotor cortex.     

Subcortical afferents of the nucleus accumbens septi in the cat, studied with retrograde axonal transport of horseradish peroxidase and bisbenzimid

The afferent connections of the nucleus accumbens septi from subcortical centers in the cat were studied with the aid of two different retrograde tracer substances. In most experiments horseradish peroxidase was injected in the nucleus accumbens, either mechanically or by microiontophoresis. In a few cats injections of bisbenzimid were placed and subsequently the retrogradely labelled cells were visualized with fluorescence microscopy. In a number of experiments neighbouring nuclei of the nucleus accumbens were involved in the injection site. Control injections were placed in the nucleus caudatus.With both tracers the same topography of labelled cells could be demonstrated following injections into the nucleus accumbens. Labelled cells were found in the basolateral nucleus of the amygdaloid complex. In the thalamus the nucleus paraventricularis and the nucleus parataenialis were most heavily labelled. Other midline nuclei, which showed fewer labelled cells, included the nucleus interanteromedialis, the nucleus rhomboidalis, the nucleus centralis medialis and the nucleus reuniens. Some projections were also found to originate from the medial part of the parafascicular nucleus. In the mesencephalic tegmentum the ventral tegmental area of Tsai, the interfascicular nucleus and the retrorubral nucleus contained labelled cells. A smaller number of cells was found to be labelled in the substantia nigra proper. In addition, the rostral linear nucleus, the central linear nucleus and the dorsal raphe nucleus, all belonging to the raphe nuclei, showed labelling of cells.A comparison with the distribution of labelled cells following injections into the caudate nucleus showed that the accumbens and caudate nuclei share many projections from the mesencephalon and the thalamus. However, the accumbens can be distinguished from the caudate because it receives afferents from the amygdala.     

The rapid anterograde transport of horseradish peroxidase

Intravitreal injections of horseradish peroxidase in the rat consistently resulted in the labelling of contralateral and ipsilateral efferents of the retina. Simultaneous intravitreal injections of horseradish peroxidase and tritiated amino acids yielded identical projection patterns. Intravitreal injections of colchicine and pentobarbital reversibly blocked this transport of horseradish peroxidase from the eye to the brain. Furthermore, silver impregnation procedures indicated that this transport occurs in the absence of significant neurol damage. The anterograde transport of horseradish peroxidase along the optic pathways occurs at a rate between 288 and 432 mm per day.These observations show that the anterograde migration of horseradish peroxidase is subserved by fast axonal transport and that its occurrence does not depend on neurol injury or passive diffusion. The anterograde transport of horseradish peroxidase thus offers a valid and reliable anatomical method for tracing efferent connections in the nervous system.     

Claustral efferents to the cat's limbic cortex studied with retrograde and anterograde tracing techniques

The claustral projections to the cat's limbic cortex were investigated with horseradish peroxidase retrograde tracing technique and with autoradiography. Autoradiographic injections covered small portions of either the dorsal anterior claustrum or intermediate to posterior regions of the claustrum. Injections of horseradish peroxidase were made into the subicular, insular, entorhinal, prepiriform, cingulate, retrosplenial and prefrontal cortex. Both methods revealed fully consistent data for substantial claustral efferents to the cingulate, retrosplenial, entorhinal and subicular cortex. For the prepiriform cortex claustral efferents could be established unequivocally only with the horseradish peroxidase technique. Only a rather minor projection could be traced for the claustro-insular projection. Unilateral injections of horseradish peroxidase revealed the existence of a minor number of labeled claustral cells in the contralateral hemisphere for all loci except insular and prepiriform ones. Our data show that claustral cells reach the majority of the allocortical areas of the brain. They thereby confirm the view that the claustrum projects to most regions of the cortex and furthermore that a certain kind of topography exists in the claustro-cortical afferents with a minor number of claustral cells sending afferents to the contralateral cortical hemisphere. In addition, our data reveal that the distribution of claustro-cortical afferents is uneven and that the ventral claustrum (or nucleus endopiriformis) sends fibers to more cortical regions than previously assumed. It is suggested that the claustrum participates in the integration of sensory, motivational, emotional and mnemonic information via its reciprocal claustro-neocortical and its claustro-limbic connections.     

Technical considerations on the use of horseradish peroxidase as a neuronal marker

Techniques are described for increasing the reliability and sensitivity of retrograde labeling of neurons with horseradish peroxidase. Labeling is increased by injecting peroxidase where axons are severed. Exclusive use of glutaraldehyde as a fixative and shortening the time used to infiltrate tissue with cryoprotectives are recommended to reduce degradation of injected peroxidase. Localization of the reaction product is facilitated by gallocyanin counterstaining, by use of phase-contrast optics, and by preincubation treatment of tissue in cobalt chloride. The use of diaminobenzidine and its safe disposal are considered.     

A study of the rat septohippocampal pathway using anterograde transport of horseradish peroxidase

The projection of the septohippocampal pathway in the rat was studied using anterograde transport of horseradish peroxidase. This technique provides a number of advantages over other methods including the ability to differentiate between terminal and preterminal axon labeling, a very high ‘signal to noise’ ratio, and a short delay in obtaining results. As applied to the septohippocampal projection, anterograde transport of horseradish peroxidase reveals a dense septal input to the dentate hilus and stratum oriens of CA3, a modest input to the dentate molecular layer and stratum radiatum of CA3, and a very sparse input to stratum oriens and stratum lacunosum-moleculare of CA1 with most labeling in this field confined to axons passing through it. In addition, our results suggest that a septal projection to the supragranular region of the dentate is present only within the rostral pole of the hippocampal formation. Potential artifacts such as labeling of fibers-of-passage and ‘collateral’ filling do not appear to interfere with the results but transneuronal transport of horseradish peroxidase may occur when large amounts of the protein are injected.     

Basal ganglia projections to the brain stem in the lizard Varanus exanthematicus as demonstrated by retrograde transport of horseradish peroxidase

In the present study the cells of origin of basal ganglia projections to the brain stem have been studied with the horseradish peroxidase technique in the lizard, Varanus exanthematicus. Injections of horseradish peroxidase were made at various levels of the brain stem from the mesodiencephalic border to the obex as well as in the tectum mesencephali. Efferent libers from the telencephalon to the diencephalon and the brain stem were found to arise predominantly from the striatum. From the present data it seems likely that the basal ganglia in Varanus exanthematicus as in other reptiles consist of two parts, a rostral ‘striatal’ part with projections mainly to the diencephalon and mesencephalon including the substantia nigra and a caudal ‘pallidal’ part with projections to the intercollicular nucleus and the rhombencephalic reticular formation.Injections of horseradish peroxidase into various parts of the rhombencephalic reticular formation have shown rather extensive projections from diencephalic and mesencephalic structures which receive afferents from the striatum: the posterior entopeduncular nucleus, the intercollicular nucleus and the substantia nigra were found to project as far caudal as the nucleus reticularis inferior. The substantia nigra shows, as regards its fiber connections, striking similarities to the mammalian substantia nigra, whereas the intercollicular nucleus possibly represents the reptilian homologue of the mammalian pedunculopontine nucleus.Injections of horseradish peroxidase into the tectum mesencephali have shown labeled cells in the nucleus of the posterior commissure, the posterior entopeduncular nucleus and the substantia nigra, all centers which are known to receive afferents from the striatum. Thus, the striatum can influence bisynaptically the reptilian homologue of the mammalian superior colliculus.It can be concluded that the striatum of the lizard, Varanus exanthematicus, has extensive direct as well as indirect projections to centers which influence the motor apparatus of the brain stem and spinal cord. Thus in reptiles it seems likely that the striatum exerts its influence on motor activity mainly via descending projections, in contrast to mammals where both descending and ascending striatal efferent pathways occur.     

Neocortical and basal telencephalic origins of the anterior commissure of the cat

The neocortical and basal telencephalic origins of the anterior commissure of the cat have not been described in earlier studies of the great cerebral commissures. In this anatomical study, all cerebral commissures, except the anterior commissure, of twelve cats were first transected. Subsequent unilateral injections of large quantities of horseradish peroxidase throughout the right hemisphere revealed the entire origins of the three branches of the anterior commissure in the left hemisphere. Since the anterior commissure was the only interhemispheric fibre system remaining, only the cells, fibres and anterogradely-labelled terminals of the anterior commissure were labelled by horseradish peroxidase in the uninjected hemisphere.Approximately three-quarters of the neurons of the anterior commissure are in the neocortex, mostly in layers V and VI. These neocortical cells occupy an extensive field stretching from gyrus proreus to the posterior ectosylvian gyrus and from the rhinal sulcus to the suprasylvian sulcus. Other fibres of the anterior commissure, however, were found to have their cell bodies in regions not considered part of the neocortex, and these included the anterior olfactory nucleus, the pyriform cortex, olfactory tubercles, nucleus of the lateral olfactory tract, part of the amygdaloid nuclei, the periamygdaloid nucleus and the lateral entorhinal area.Finally, it was found that the fibres of the anterior commissure do not have an exclusive neocortical territory from which cells of other commissural fibres are excluded. Rather, there appears to be a substantial overlap between the field of origin of fibres of the anterior commissure and those of the largest cerebral commissure, the corpus callosum. The disposition of this field may help to explain why visual information fails to transfer between the hemispheres in cats whose corpus callosum has been cut, in contrast to the success of such transfer in primates.     

The septohippocampal projection in the rat: an electron microscopic horseradish peroxidase study

In order to identify the specific targets of the septohippocampal projection in the rat, horseradish peroxidase localization at the electron microscopic level was used. Following injections of free horseradish peroxidase into the medial septum, sections of the dorsal hippocampal formation were reacted with diaminobenzidine and processed for electron microscopy by routine methods. Sections were viewed unstained. Horseradish peroxidase labeling in the dentate gyrus was predominantly in the supra- and infragranular layers. All postsynaptic elements were neuronal. They included granule cell somata and somata and dendrites of hilar cells; these may include pyramidal basket cells. No synaptic contacts with vascular or glial elements were found. These results provide a basis for comparing the specific targets of the septohippocampal projection with those of the sympathohippocampal pathway, which innervates the dentate following lesions of the septohippocampal projection.     

Periaqueductal grey projection to the ventrobasal complex in the cat: An horseradish peroxidase study

Horseradish peroxidase (HRP) was injected within the thalamic ventrobasal complex of 14 cats. The aim was to ascertain whether the periaqueductal grey matter (PAG) sends fibres to this complex. Retrogradely labelled cells were found within the PAG following HRP delivery either in the nucleus ventralis posterolateralis (VPL) or ventralis posteromedialis (VPM). PAG-VPL projection is only ipsilateral and arises mainly from lateral PAG. PAG-VPM projection is bilateral and originates from latero-ventral regions of the central grey. The hypothesis that PAG might control the activity of ventrobasal nociceptive neurones is proposed.     

Endocytosis in glial cells (pituicytes) of the rat neurohypophysis demonstrated by incorporation of horseradish peroxidase.

The uptake and intracellular disposition of an extracellular marker, horseradish peroxidase, was traced at various time intervals in glial cells of the neurohypophysis, the pituicytes. Glands from normal rats and rats in which neurohypophysial secretion was activated by various stimuli were studied.Following an intravenous injection of the enzyme, reaction product rapidly appeared in the pituicyte cytoplasm, within numerous membrane-bound vacuoles of various size and morphology. The vacuoles presumably arose from large bristle coated invaginations of the plasmalemma that were marked by the tracer as soon as 5 min after injection. By 24 h, the reaction product was sequestered mainly in lysosomal multivesicular bodies and dense bodies.The observations suggest that endocytosis normally occurs in pituicytes. Furthermore, this endocytotic activity appears to be related to neurohypophysial secretion: morphometric analysis indicated that at each time period studied, the relative volume occupied by the peroxidase-labelled structures in cells from stimulated preparations was significantly greater than their corresponding controls. The possible role of uptake of extracellular fluid and accompanying plasma membrane that result from endocytosis is discussed.     

Cortical projections to the periaqueductal grey in the cat: a retrograde horseradish peroxidase study

Stereotaxic fluid microinjections of horseradish peroxidase into different parts of the rostral and caudal periaqueductal grey (PAG) in cats have provided substantial retrograde evidence that the somatosensory cortex (I and II), frontal cortex, insular and cingular cortex are the principal sources of cortical-PAG projections. The somatosensory cortex II projects to all the regions of the rostral and caudal PAG. The frontal cortex projects to dorso-lateral quadrant of the PAG. The same projections were determined from insular and cingular cortex to PAG. The findings revealed a morphological substratum of corticofugal effects on PAG.     

Afferent connections of the mesencephalic reticular formation: A horseradish peroxidase study in the rat

The afferent connections of the mesencephalic reticular formation were studied experimentally in the rat by the aid of the retrograde horseradish peroxidase tracer technique. The results suggest that the rostral portion of the mesencephalic reticular formation receives its main input from the cerebral cortex, the zona incerta and the fields of Forel, the central gray substance, the nuclei reticularis pontis oralis and caudalis, and the deep cerebellar nuclei. Substantial input to the same territory of the mesencephalic reticular formation appears to come from the superior colliculus, the substantia nigra, the parabrachial area, the spinal trigeminal nucleus, and the nucleus reticularis gigantocellularis, whereas several other brain structures, among which the locus coeruleus and the raphe complex, seem to represent modest but consistent additional input sources. The afferentation of more caudal portions of the mesencephalic reticular formation appears to conform to the general pattern outlined above with only three exceptions: the cerebral cortex, the deep cerebellar nuclei and the spinal trigeminal nucleus seem to be relatively modest sources of projections to these levels.Considering that the mesencephalic reticular formation is a critical structure in the “ascending activating systems”, the present results, confirming and extending those of many other investigators, characterize a set of pathways that seem to be an important part of the anatomical substrate of the sleep-waking cycle.     

The cells of origin of the incertofugal projections to the tectum,thalamus, tegmentum and spinal cord in the rat: A study using the autoradiographic and horseradish peroxidase methods

Efferent projections of the zona incerta were examined in the rat using the autoradiographic and horseradish peroxidase methods, with special reference to the cytoarchitectonic structure of the zona incerta.Autoradiographic experiments showed that the incertofugal fiber systems reach ipsilaterally to the thalamus (lateral dorsal, central lateral, ventral lateral geniculate, parafascicular, subparafascicular and reuniens nuclei, and posterior nuclear complex), to the hypothalamus (dorsal, lateral and posterior hypothalamic areas), to the tectum (medial pretectal area, deep pretectal and pretectal nuclei, superior colliculus and periaqueductal gray) and to the midbrain tegmentum, pons and medulla oblongata (subcuneiform, cuneiform and red nuclei, nuclei of the posterior commissure and Darkschewitsch, interstitial nucleus of Cajal, pedunculopontine tegmental nucleus, oral and caudal pontine reticular nuclei, nucleus raphe magnus, gigantocellular reticular nucleus, pontine gray and inferior olivary complex). Contralaterally, incertal efferent fibers reach to the zona incerta.Cells of origin of the incertofugal fiber systems to the tectum, thalamus, tegmentum and spinal cord were examined using the retrograde horseradish peroxidase method. Cells of origin of the incertotectal pathway are located mainly in the ventral and caudal parts of the zona incerta and partly in the antero-polar, dorsal and postero-polar parts. Cells projecting to the thalamus (at least to the lateral dorsal and central lateral nuclei) are situated in the ventral and caudal parts of the zona incerta, but they are rare in the other incertal structures. Cells of origin of the incertotegmental system are located mainly in the dorsal, magnocellular and caudal parts and partly in the antero- and postero-polar parts, but they are not situated in the ventral part. Cells of the magnocellular part project more caudally to the medulla oblongata and spinal cord than those of the other parts of the zona incerta. Forel's field contains many cells projecting to the tegmentum.The results provide good evidence that the cells of origin of efferent projections are topographically organized and are related to cytoarchitectonic areas within the zona incerta.     

The cerebellar projections to the superior colliculus and pretectum in the cat: An autoradiographic and horseradish peroxidase study

Efferent projections from the cerebellar nuclei to the superior colliculus and the pretectum have been studied using both retrograde and orthograde labeling techniques in the cat. In order to identify what parts of the cerebellar nuclei project to the superior colliculus and the pretectum, the retrograde horseradish labeling technique was employed. In another set of experiments, tritiated amino acids were injected into each of the cerebellar regions from which the cerebello-tectal and cerebellopretectal projections arise, and the laminar and spatial distributions of orthograde labeling in the superior colliculus and the pretectum were compared.The results showed that the cerebello-tectal projections arise from two different regions of the cerebellar nuclei: the caudal half of the medial nucleus and the ventrolateral part of the posterior interposed nucleus. Fibers arising from the medial nucleus distribute bilaterally in the superficial zone of the intermediate gray layer in the superior colliculus, while those originating from the posterior interposed nucleus terminate contralaterally in the deeper aspect of the intermediate gray layer and in the deep gray and white layers. Although the lateral nucleus does not contribute to the cerebello-tectal projection, it projects profusely to the pretectum contralaterally. The origin of the cerebello-pretectal projection lies in the parvicellular part of the lateral nucleus. Among several pretectal nuclei, the posterior pretectal, the medial pretectal nucleus and the reticular part of the anterior pretectal nucleus receive the cerebellar afferents.The findings of the differential projections from the cerebellum to the superior colliculus and the pretectum suggest that the cerebellum exerts a regulatory influence on visuo-motor and somato-motor transfer in these midbrain structures by differential circuits.     

Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: a horseradish peroxidase study

To detect the cell bodies of neurones which project to the area of the hypothalamic para-ventricular nucleus, 10–40 nl of a solution containing horseradish peroxidase and poly-l-ornithine were pressure-injected into one paraventricular nucleus of the rat. After 24 or 48 h, the enzyme remaining at the site of injection was detected by the diaminobenzidine procedure. Retrogradely transported horse-radish peroxidase was visualized by using o-dianisidine as the chromogen substrate.The extent and the intensity of labelling correlated with the apparent volume of the injection site. Labelled cell bodies were observed, ipsilateral to the injection, in the mediobasal hypothalamus, in the limbic system (lateral septum, posteromedial amygdala, ventral subiculum) and in several cell clusters in the brain stem (dorsal raphe nucleus, locus coeruleus, parabrachial nucleus, nucleus of the solitary tract and lateral reticular nucleus). In some animals, light labelling in the organum vasculosum laminae terminalis and in the subfornical organ was observed. No labelled neurones could be detected in the spinal cord.