Arthur D. Loewy

A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections.

Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.

Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response.

During stress, the activity of the sympathetic nervous system is changed in a global fashion, leading to an increase in cardiovascular function and a release of adrenal catecholamines. This response is thought to be regulated by a common set of brain neurons that provide a dual input to the sympathetic preganglionic neurons regulating cardiac and adrenal medullary functions. By using a double-virus transneuronal labeling technique, the existence of such a set of central autonomic neurons in the hypothalamus and brainstem was demonstrated. These neurons innervate both of the sympathetic outflow systems and likely function in circumstances where parallel sympathetic processing occurs, such as in the fight-or-flight response.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labeling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T4-T13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactive phenylethanolamine-N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

Spinal origin of sympathetic preganglionic neurons in the rat

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000-2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100-400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (approximately 800 cells) and dorsal root input (approximately 300 cells); it has a widespread segmental input (T4-T12) with the T8 segment being the major source.

Projections of the pedunculopontine tegmental nucleus in the rat: evidence for additional extrapyramidal circuitry.

The projections of the pedunculopontine tegmental nucleus (PPT) were studied in the rat using anterograde and retrograde transport methods. Ascending fibers to the substantia nigra, the subthalamic nucleus, the globus pallidus, the entopeduncular nucleus, the neostriatum, the ventral thalamus, and the medial and sulcal frontal cortical areas were identified. PPT has been reported to receive afferents from the substantia nigra, the subthalamic nucleus, the entopeduncular nucleus and the neostriatum. The connections of PPT provide an additional limb to extrapyramidal circuitry.

Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat

The descending spinal projections from the raphe pallidus and raphe obscurus nuclei were studied by the anterograde autoradiographic technique in the rat. Both regions project to the intermediolateral cell columns. These pathways were destroyed by intraventricular injections of 5,7-dihydroxytryptamine, a neurotoxin relatively specific for serotonergic cells.

Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat.

Serotonergic neurons of the ventral medulla (B1 and B3 cell groups are retrogradely labeled after HRP injection in the T1-T2 levels of the spinal cord. Following stereotaxic injections of 3H-labeled amino acids in this region, a direct projection to th intermediolateral cell column and ventral horn can be demonstrated.

Brainstem projections to midline and intralaminar thalamic nuclei of the rat

The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin β ‐subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nuclei—the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental field. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem‐thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions. J. Comp. Neurol. 448:53–101, 2002.

CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat.

In the rat, approximately 20% of total body heat-loss occurs by sympathetically mediated increases in blood flow through an elaborate system of arteriovenous anastomoses in the skin of its tail. In this study, the CNS cell groups that regulate this sympathetic outflow were identified by the viral transneuronal labeling method. Pseudorabies virus was injected into the wall of the ventral tail artery in rats that had their cauda equina transected to eliminate the somatic innervation of the tail. After 4-7 days survival, the pattern of CNS transneuronal labeling was studied. Sympathetic preganglionic neurons in the T11-L2 (mainly L1) levels of the intermediolateral cell column (IML) were labeled by 4 days. After 5 days, sympathetic pre-motor neurons (i.e., supraspinal neurons that project to the IML) were identified near the ventral medullary surface; some of these contained serotonin immunoreactivity. Additional groups of the sympathetic premotor areas were labeled by 6 days post-injection, including the rostral ventrolateral medulla (C1 adrenergic neurons), rostral ventromedial medulla, caudal raphe nuclei (serotonin neurons in the raphe pallidus and magnus nuclei), A5 noradrenergic cell group, lateral hypothalamic area and paraventricular hypothalamic area (oxytocin-immunoreactive neurons). Seven days after the PRV injections, additional cell groups in the telencephalon (viz., bed nucleus of the stria terminalis, medial and lateral preoptic areas and medial preoptic nucleus), diencephalon (viz., subincertal nucleus, zona incerta as well as dorsal, dorsomedial, parafascicular, posterior and ventromedial hypothalamic nuclei) and midbrain (viz., periaqueductal gray matter, precommissural nucleus, Edinger-Westphal nucleus and ventral tegmental area) were labeled. The discussion is focused on the CNS cell groups involved in the control of body temperature and fever.

Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion

The viral transneuronal labeling method was used in combination with immunohistochemical procedures to identify CNS neuropeptide and monoamine neurons that innervate the sympathetic preganglionic neurons (SPNs) which project to the stellate ganglion--the principal source of the sympathetic supply to the heart. Transneuronal labeling was found at three CNS levels: spinal cord, brainstem, and hypothalamus. In the thoracic spinal cord, apart from the pseudorabies virus (PRV)-labeled stellate SPNs, PRV-labeled neurons were localized in laminae I/II, IV, V, VII, and X as well as in the lateral spinal nucleus and lateral funiculus. In the C1-C4 spinal segments, labeled neurons were found in the lateral funiculus as well as laminae V and VII of the spinal gray matter. PRV-labeled cells were identified in lamina V and the dorsolateral funiculus of the lumbar spinal cord. Three medullary areas were consistently labeled: rostral ventromedial medulla (RVMM), rostral ventrolateral medulla (RVLM), and caudal raphe nuclei. The greatest concentration of labeling was found in the RVMM. This projection arose from adrenergic, serotonergic (5-HT), thyrotropin releasing hormone (TRH), substance P, somatostatin, enkephalin, and vasoactive intestinal peptide (VIP) immunoreactive neurons. The RVLM projection originated mainly from C1 adrenergic neurons, some of which contained immunoreactive neuropeptide Y (NPY). C3 adrenergic-NPY neurons lying near the floor of the 4th ventricle were also labeled. Enkephalin-, somatostatin- and VIP-immunoreactive RVLM neurons also contributed to this projection. 5-HT neurons of the caudal raphe nuclei (raphe pallidus, raphe obscurus, and raphe magnus) were labeled; some of these contained substance P or TRH-immunoreactivity with an occasional neuron staining for all three putative neurotransmitters. In the pons, catecholamine neurons in the A5 cell group, subcoeruleus and Kolliker-Fuse nuclei were labeled. The midbrain contained relatively few infected cells, but some were present in the Edinger-Westphal and precommissural nuclei. Forebrain labeling was concentrated in the paraventricular hypothalamic nucleus (PVN) with lesser amounts in the lateral hypothalamic area (LHA) and the perifornical region. In the PVN, oxytocin-immunoreactive neurons accounted for the greatest chemically-defined projection while corticotrophin releasing factor (CRF), vasopressin-, and angiotensin II-immunoreactive neurons provided successively lesser inputs. In the LHA, angiotensin II-immunoreactive neurons were labeled. In summary, this study provides the first detailed map of the chemically-coded CNS neurons involved in the control of the cardiosympathetic outflow.