The ascending reticular activating system (ARAS) mediates arousal, an essential component of human consciousness. Lesions of the ARAS cause coma, the most severe disorder of consciousness. Because of current methodological limitations, including of postmortem tissue analysis, the neuroanatomic connectivity of the human ARAS is poorly understood. We applied the advanced imaging technique of high angular resolution diffusion imaging (HARDI) to elucidate the structural connectivity of the ARAS in 3 adult human brains, 2 of which were imaged postmortem. High angular resolution diffusion imaging tractography identified the ARAS connectivity previously described in animals and also revealed novel human pathways connecting the brainstem to the thalamus, the hypothalamus, and the basal forebrain. Each pathway contained different distributions of fiber tracts from known neurotransmitter-specific ARAS nuclei in the brainstem. The histologically guided tractography findings reported here provide initialevidence for human-specific pathways of the ARAS. The unique composition of neurotransmitter-specific fiber tracts within each ARAS pathway suggests structural specializations that subserve the different functional characteristics of human arousal. This ARAS connectivity analysis provides proof of principle that HARDI tractography may affect the study of human consciousness and its disorders, including in neuropathologic studies of patients dying in coma and the persistent vegetative state.

The hypothesis that leptin (OB protein) acts in the hypothalamus to reduce food intake and body weight is based primarily on evidence from leptin-deficient, ob/ob mice. To investigate whether leptin exerts similar effects in normal animals, we administered leptin intracerebroventricularly (icv) to Long-Evans rats. Leptin administration (3.5 microg icv) at the onset of nocturnal feeding reduced food intake by 50% at 1 h and by 42% at 4 h, as compared with vehicle-treated controls (both P < 0.05). To investigate the basis for this effect, we used in situ hybridization (ISH) to determine whether leptin alters expression of hypothalamic neuropeptides involved in energy homeostasis. Two injections of leptin (3.5 microg icv) during a 40 h fast significantly decreased levels of mRNA for neuropeptide Y (NPY, which stimulates food intake) in the arcuate nucleus (-24%) and increased levels of mRNA for corticotrophin releasing hormone (CRH, an inhibitor of food intake) in the paraventricular nucleus (by 38%)(both P < 0.05 vs. vehicle-treated controls). To investigate the anatomic basis for these effects, we measured leptin receptor gene expression in rat brain by ISH using a probe complementary to mRNA for all leptin receptor splice variants. Leptin receptor mRNA was densely concentrated in the arcuate nucleus, with lower levels present in the ventromedial and dorsomedial hypothalamic nuclei and other brain areas involved in energy balance. These findings suggest that leptin action in rat hypothalamus involves altered expression of key neuropeptide genes, and implicate leptin in the hypothalamic response to fasting.

A sensitive immunofluorescence technique was used to describe systematically the distrubution of dopamine-beta-hydroxylase (DBH)-containing cell bodies, non-terminal fiber pathways, and terminal fields in the brain of the male albino rat. DBH is the enzyme that catalyzes the conversion of dopamine to noradrenaline, and as such is useful as an anatomical marker for noradrenaline and possibly adrenaline neurons. The enzyme is not present in dopamine- or indolamine-containing neurons. Ten micron frozen sections (1-in 20 series) were prepared in the frontal, sagittal, and horizontal planes from the olfactory bulb to the upper cervical segments of the spinal cord; adjacent sections in each plane were stained for DBH and for cells (toluidine blue=azure II). An atlas consisting of 40 projection drawings of selected frontal sections illustrates the results of the investigation. DBH perikarya are confined to three groups in the pons and medulla: the well defined locus coeruleus, a more diffuse but continuous subcoeruleus group that arches through the pons and ventral medulla, and a third dorsal medullary group centered in the dorsal motor nucleus of the vagus. A single principal adrenergic fiber system distributes a great many of the axons from these neuron groups to a majority of nuclear areas in the brain. In the pons and medulla two components of the fiber system may be distinguished. A medullary branch may be followed from the posterior aspect of the subcoeruleus group dorsally and then anteriorly through the lateral tegmental field and ventral aspect of the vestibular complex to a position subjacent to the locus coeruleus, where it is joined by a subcoeruleus branch consisting of a large number of fibers coursing among cells along the length of the subcoeruleus group, and by fibers arising from the locus coeruleus. Anterior to the locus coeruleus the principal adrenergic bundle courses as a single fiber tract immediately ventrolateral to the central gray in the mesencephalon and in the zona incerta and substantia innominata in the diencephalon. At the level of the septal area separate bundles reach the cortex dorsally over the genu of the corpus calosum via the medial septal-diagonal band nuclei and the lateral septum and ventrally between the olfactory tubercle and caudate-putamen. In the medulla and pons adrenergic fibers undoubtedly course in both directions. Anterior to the most rostral pontine cell bodies, however, all fibers presumably ascend. Along the course of the bundle distinct branches emerge to innervate circumscribed terminal fields. In addition, certain regions of the brain such as the reticular formation and pontine gray receive diffuse DBH innervation derived from less clearly defined pathways. A small number of areas in the brain contain little or no detectable DBH. These include the caudate-putamen, nucleus accumbens, globus pallidus, olfactory tubercle, subthalamic nucleus, substantia nigra, pretectal area, third, fourth and sixth cranial verve nuclei, and the trapezoid body nucleus.

Small injections of tritiated leucine and proline confined to the ventral tegmental area (AVT) were found to label fibers ascending:(a) to the entire ventromedial half of the striatum, but most massively to the ventral striatal zone that includes the nucleus accumbens;(b) to the thalamus: lateral habenular nucleus, nuclei reuniens and centralis medius, and the most medial zone of the mediodorsal nucleus;(c) to the posterior hypothalamic nucleus and possibly the lateral hypothalamic and preoptic region;(d) to the nuclei amygdalae centralis, lateralis and medialis;(e) to the bed nucleus of the stria terminalis, the nucleus of the diagonal band, and the medial half of the lateral septal nucleus;(f) to the anteromedial (frontocingulate) cortex; and (g) to the entorhinal area. Further AVT efferents descend to the medial half of the midbrain tegmentum including an anterior region of the median raphe nucleus, to the ventral half of the central grey substance including the dorsal raphe nucleus, to the parabrachial nuclei, and to the locus coeruleus. Similar injections centered in the pars compacta of the substantia nigra (SNC) label fibers that are distributed in the striatum in an orderly medial-to-lateral arrangement, and almost entirely avoid the nucleus accumbens and olfactory tubercle. With the exception of the lateral quarter of the substantia nigra, which apparently does not project to the extreme rostral pole of the striatum, each small SNC locus, regardless of its anteroposterior localization, distributes nigrostriatal fibers throughout the length of the striatum. Descending SNC efferents are distributed to the same general regions that receive descending AVT projections, except that no SNC fibers appear to enter the locus coeruleus. Isotope injections confined to the pars reticulata (SNR) label sparse nigrostriatal fibers, and numerous nigrothalamic fibers ascending mainly to the nucleus ventromedialis and in lesser number to the parafascicular nucleus and the paralamellar zone of the nucleus mediodorsalis. Descending SNR fibers leave the nigra as a voluminous fiber bundle that bifurcates into a large nigrotectal and a smaller nigrotegmental component, the latter terminating largely in the pedunculopontine nucleus of the pontomesencephalic tegmentum.

Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Ch1 and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5-Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5-Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation. These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.

The efferent connections of the parabrachial nucleus have been analyzed in the rat using the anterograde autoradiographic method. Fibers originating from the lateral parabrachial nucleus (PBl) ascend in the periventricular system, the dorsal tegmental bundle and the central tegmental tract. The PBl projects to the dorsal raphe nucleus, the superior central raphe nucleus, and the Edinger-Westphal nucleus. It also innervates the intralaminar (centromedian, centrolateral, paracentral, parafascicular), the midline (paraventricular, reuniens), and the ventromedial basal (VMb) thalamic nuclei as well as much of the hypothalamus, including the dorsomedial, the ventromedial, the arcuate and the paraventricular nuclei, the lateral hypothalamic and the lateral preoptic areas. The PBl sends fibers via the ansa peduncularis into the amygdala, innervating the anterior, the central, the medial, the basomedial, and the posterior basolateral nuclei. In addition, it projects to the lateral part of the bed nucleus of the stria terminalis. Descending PBl fibers travel mainly through the ventrolateral medulla, passing through the region of the A1 and A5 catecholamine cell groups, the ventrolateral reticular formation and the region that contains parasympathetic preganglionic neurons. A small component travels in Probst's bundle to the ventral part of the nucleus of the solitary tract. Only a few PBl axons continue caudally into the lateral funiculus of the spinal cord, but these could not be followed beyond the first few cervical segments. The projections of the medial parabrachial nucleus (PBm) are similar to those of PBl, but two major differences have been noted. One difference is that the PBm provides a direct input to 4 regions of cerebral cortex:(1) the granular insular cortex;(2) the deep layers of the frontal cortex;(3) the septo-olfactory area; and (4) the infralimbic cortex. The other difference is that unlike the PBl, the PBm appears to provide almost no input to the medial hypothalamic nuclei (dorsomedial, ventromedial, arcuate nuclei) nor to the medial amygdaloid nucleus. The PBm projects heavily to the nucleus ambiguus and there was no evidence for an input to the nucleus of the solitary tract. The projections of the Kölliker-Fuse nucleus (KF) are distinct from those of either PBm or PBl. The KF projects via the central tegmental tract to the lateral hypothalamic area, the lateral preoptic area, and the central nucleus of the amygdala. The contralateral projection to the zona incerta, the lateral hypothalamic area, and the lateral preoptic areas is more prominent than the ipsilateral projections. Descending KF fibers travel mainly through the ventrolateral medullary reticular formation passing through regions which give rise to parasympathetic preganglionic fibers of the VIIth, IXth and Xth cranial nerves and the A1 and A5 catecholamine cell groups. In one experiment, fibers could be followed to the intermediolateral cell column of the upper thoracic spinal cord.

Ascending projections from the caudal (general-visceroceptive) part of the nucleus of the solitary tract (NTS) were studied experimentally in the rat by the aid of the anterograde autoradiographic and the retrograde horseradish peroxidase (HRP) tracer techniques. Microelectrophoretic deposits of tritiated proline and leucine which involved the caudal part of the NTS, the dorsal motor nucleus of the vagus (dmX), and portions of the hypoglossal nucleus, nucleus intercalatus and/or nucleus gracilis were found to label ascending fibers that, besides going to numerous brain stem territories that included prominently the parabrachial area, could also be traced to serveral forebrain structures, namely, the bed nucleus of the stria terminalis (BST), the paraventricular (PA), dorsomedial (HDM) and arcuate (ARC) nuclei of the hypothalamus, the central nucleus of the amygdaloid complex (AC), the medial preoptic area (PM) and the periventricular nucleus of the thalamus (TPV). Smaller isotope injections almost completely confined to the NTS and dmX resulted in lighter labeling of a similar set of parabrachial and forebrain projections, whereas in another case, in which the deposit was almost exclusively limited to the nucleus gracilis, no label was seen in the aforementioned structures. In another series of experiments, aimed at further localizing the neurons of origin of the prosencephalic projections under consideration, small microelectrophoretic HRP injections confined almost totally to BST, PA, HDM, AC, PM or TPV, as well as both small and large injections involving ARC, resulted in labeled neurons situated in the dorsal medullary region, mainly in the medial portion of the NTS at the level of and caudal to the area postrema. Taken together, these observations indicate for the first time the existence of relatively direct conduction lines by which interoceptive information might be conveyed to limbic forebrain structures; some of the possible physiological correlates of these anatomical findings are discussed.

A method that allows the concurrent localization of an antigen and a retrogradely transported fluorescent dye (true blue) was used to identify, immunohistochemically, cells in the paraventricular nucleus of the hypothalamus (PVH) that project to autonomic centers in the brainstem or in the spinal cord of the adult albino rat. After placing injections of true blue in the dorsal vagal complex or in upper thoracic segments of the spinal cord, series of evenly spaced sections through the PVH were stained with antisera directed against oxytocin, vasopressin, somatostatin, methionine-enkephalin, or leucine-encephalin. The results indicate that both oxytocin- and vasopressin-stained cells in the PVH project to the spinal cord and (or) to the dorsal vagal complex, although about three times as many oxytocin-stained cells were doubly labeled after injections centered in either terminal field. The oxytocin- and vasopressin-stained cells that give rise to these long descending projections were found primarily in caudal part of the parvocellular division of the PVH, where immunoreactive cells were shown to be significantly smaller than oxytocin- and vasopressin-stained cells in parts of the nucleus that project to the posterior pituitary. Small populations of cells in the PVH that cross-react with antisera against somatostatin, leucine-enkephalin, or methionine-enkephalin were also shown to project directly to the region of the dorsal vagal complex and to the spinal cord, and to have a unique distribution within the PVH. Collectively, the total number of doubly labeled cells that were identified in these experiments accounts for only about one-fourth of the total number of PVH neurons with long descending projections, thus suggesting that additional neuroactive substances are contained within these pathways.