Cynthia J. Forehand

Expression and regulation of the retinoic acid synthetic enzyme RALDH‐2 in the embryonic chicken wing

Retinaldehyde dehydrogenase type 2 (RALDH‐2) is a major retinoic acid (RA) generating enzyme in the embryo. Here, we report immunolocalization of this enzyme (RALDH‐2‐IR) in the developing wings of stage 17–30 chicken embryos. RALDH‐2‐IR is located in the area of the presumptive muscle masses, although it is not colocalized with developing muscle cells. RALDH‐2‐IR is located in tendon precursor cells and may be present in muscular connective tissue. We show that motor neurons and blood vessels, tissues showing RALDH‐2‐IR as they enter the limb, are capable of synthesizing and releasing RA in culture. RALDH‐2‐IR in the limb mesenchyme is under the control of both the vasculature and the motor innervation; it is decreased with denervation and increased with hypervascularization. RALDH‐2‐IR is present in the motor neuron pool of the brachial spinal cord, but this expression pattern is apparently not under the control of limb target tissues, RA in the periphery, or somitic factors. RA is known to be a potent inducer of cellular differentiation; we propose that locally synthesized RA may be involved in aspects of wing tissue specification, including cartilage condensation and outgrowth, skeletal muscle differentiation, and recruitment of smooth muscle cells to the vasculature.

Enhanced Vascular Neuropeptide Y– Immunoreactive Innervation in Two Hypertensive Rat Strains

Considerable evidence indicates an enhanced sympathetic innervation of resistance arterial smooth muscle in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) control. In addition to sympathetic hyperinnervation, an increased vascular innervation by neuropeptide Y-containing fibers, which are known to exert a vasoconstrictive and trophic action in vascular smooth muscle, has also been described. In addition to genetic hypertension, the SHR expresses hyperactive behavior and hyperreactivity to stress. To determine whether the enhanced neuropeptide Y-immunoreactive vascular innervation is specifically associated with hypertension and/or these behavioral abnormalities, four genetically related, inbred rat strains were used: SHR, which are hypertensive and hyperactive; WKY rats, which are neither hypertensive nor hyperactive; WKHA, which are hyperactive but normotensive; and WKHT, which are hypertensive but not hyperactive. The present study demonstrated that whereas the hypertensive strains (SHR and WKHT) exhibited smooth muscle hypertrophy in both superior mesenteric and caudal arteries in adulthood (10 months) but not at a prehypertensive age (1 month), both arteries exhibited significantly increased neuropeptide Y-immunoreactive innervation at both ages. It was further observed that the mesenteric artery in WKHA, a normotensive strain, had significant smooth muscle hypertrophy at 10 months; however, neuropeptide Y innervation in this artery was no different from that of WKY controls. The findings indicate that there is a cosegregation of neuropeptide Y hyperinnervation of the vasculature with the hypertensive phenotype, evident as early as 1 month of life in the hypertensive strains, and this should be considered further as a contributory factor in genetic hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of differential preganglionic projections to pre‐ and paravertebral sympathetic ganglia

Sympathetic preganglionic axons project to spatially distinct targets in the periphery. A precise topographic pattern exists within the thoracic preganglionic cell column relative to the direction of axonal projections within the sympathetic chain. In this study, the time course and pattern of axonal outgrowth from different populations of preganglionic neurons in the chicken embryo is examined in detail to clarify the origin of the topography in this system. Projections to prevertebral targets are established by development of the splanchnic nerves by stage 25, well after the earliest somatic motor projections at stage 19 but at least two stages before the reported onset of paravertebral projections. Further, preganglionic axons that project rostrally into the sympathetic chain may do so earlier than those that project caudally in the chain. The separation of preganglionic axons into prevertebral, rostral paravertebral or caudal paravertebral directions occurs at a common site in the ventral mesenchyme, established by the initial ventromedial projection of the splanchnic nerves. Analysis of the axonal trajectories of rostrally and caudally projecting cells reveals that preganglionic axons are not selectively fasciculated before their point of separation at the sympathetic chain. The patterning of the preganglionic cell column is specified before the establishment of functional connections within the chain, indicating that target contact is not a determinant of the segmental pattern. We suggest that the differential outgrowth of preganglionic axons to peripheral targets is determined by the unique identities of underlying subpopulations of preganglionic axons. J. Comp. Neurol. 382:1‐18, 1997.

Segmental restriction and target specificity of bullfrog preganglionic neurons that exhibit galanin-like immunoreactivity

These experiments were designed to examine the distribution of galanin-like peptide immunoreactivity (GAL-IR) in bullfrog sympathetic preganglionic neurons and to identify the peripheral target organs affected by these neurons. Cells expressing GAL-IR were observed in the intermediolateral column of segments 7 and 8 only. Apparent GAL-IR innervation is present, but rare, in sympathetic chain ganglia. Double-labelling with retrogradely transported fast blue and galanin antiserum demonstrated that most GAL-IR neurons project via splanchnic nerves to innervate the adrenal gland, which receives a dense plexus of GAL-IR fibers surrounding chromaffin cells. The adrenal gland is also innervated by preganglionic neurons in segments 5 and 6 that do not express GAL-IR. Because nitric oxide is expressed in sympathoadrenal preganglionic neurons in mammals (Anderson, C.R., Neurosci. Lett., 139 (1992) 280), we examined whether it is expressed in bullfrog preganglionic neurons. Nicotinamide adenine dinucleotide phosphate-diaphorase positive neurons are present in bullfrog spinal grey at segments 5 through 8. These neurons were not double-labelled with fast blue retrogradely transported from the sympathetic chain, celiac ganglion, or adrenal gland; nor were they double-labelled with GAL-antiserum. Thus nitric oxide is apparently not expressed in bullfrog sympathetic preganglionic neurons.

Mutation of low affinity nerve growth factor receptor gene is associated with the hypertensive phenotype in spontaneously hypertensive inbred rat strains

We previously reported a missense mutation in the low affinity nerve growth factor receptor (LNGFR) gene of spontaneously hypertensive rats (SHR), proposing this gene as a promising candidate in genetic hypertension. In this study we provide further support for implicating this gene in genetic hypertension using two new inbred strains, WKHT and WKHA rats. These strains originated from crossbreeding SHR rats with normotensive Wistar-Kyoto rats (WKY): WKHT rats are hypertensive but not hyperactive, and WKHA rats are hyperactive but not hypertensive. Nucleotide sequence analysis of the LNGFR gene revealed that WKHT has the same mutation as SHR, whereas WKHA has the normal sequence, as seen in WKY. These results support our original hypothesis that the mutated LNGFR gene is linked to hypertension, since the mutation had co-segregated with the hypertensive trait, and not hyperactivity trait of SHR.

Development and segmental organization of rostrocaudal dendrites of rat sympathetic preganglionic neurons

The longitudinal organization of preganglionic sympathetic neurons in the adult mammalian spinal cord takes the general form of a ladder. In the rat, the preganglionic neurons of the intermediolateral cell column (IML) have extensive dendritic arborizations in both rostrocaudal and mediolateral directions. We have studied the development of the rostrocaudal dendritic projection by retrogradely labeling single sympathetic preganglionic spinal segments with the membrane label DiI. The rostrocaudal dendrites of sympathetic preganglionic neurons in the IML begin to develop prenatally on embryonic day 19-20 (E19/20), several days after these neurons develop mediolaterally oriented dendrites. Between E19 and postnatal day 1 (P1), the rostrocaudal dendrites attain a length of approx. 200 microns. As the rostrocaudal dendrites elongate, the preganglionic neurons form distinct clusters between which dendritic bundles are seen. Following a growth spurt from E19 to P1, the average rostrocaudal dendritic length approximates twice the distance between clusters from P1 to P9.

The effect of cAMP signaling on the longitudinal extension of spinal sensory neurons in the chicken embryo.

Developing sensory axons grow into the spinal cord in a three‐step process: the axons extend toward and into the cord, then branch rostrally and caudally to establish a longitudinal pathway, and finally grow into the grey matter. This study investigated regulation by cAMP of the longitudinal extension of this pathway within the spinal cord. The cAMP pathway was pharmacologically altered in chicken embryos to determine its effects on the establishment of the longitudinal extension of the dorsal funiculus. A forskolin‐induced increase in cAMP in ovo inhibited longitudinal growth by sensory afferents. Furthermore, blocking cAMP activation of protein kinase A (PKA) in ovo with H‐89 substantially increased longitudinal extension. These results demonstrate a specific role for the cAMP/PKA pathway in the initial longitudinal spinal afferent growth in the chicken embryo.

Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus

Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.