Story C. Landis

A call for transparent reporting to optimize the predictive value of preclinical research

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase

Abstract Horseradish peroxidase (HRP) injected into rat hippocampus was transported to the perikarya of neurons which project to the hippocampus. HRP-labeled cells were present in both medial and lateral entorhinal cortex; cells of the medial entorhinal cortex appeared to be topographically organized. The mediaal septal nucleus contained stained cells; its mediaal aspect was labeled after dorsal hippocampal injections, while ventral hippocampal injections resulted in the labeling of more laterally located cells. Stained cells were also observed in the ipsilateral nucleus locus coeruleus, dorsal and median raphe nuclei and areas CA3–4 of the contralateral hippocampus. In additions, cells in the supramammillary region, an area not previously recognized to project to the hippocampus, were labeled. Finally, the mossy fiber terminal zone and the CA3–4 terminal zone in the dentate molecular layer of the ipsilateral hippocampus demonstrated HRP activity, presumably the result of orthograde axonal transport from the injection site.

Evidence for neurotransmitter plasticity in vivo: Developmental changes in properties of cholinergic sympathetic neurons

We have examined the cholinergic sympathetic innervation of sweat glands in footpads of adult and developing rats. Acetylcholinesterase staining reveals a plexus of heavily stained fibers in the sweat glands of adult rats. Reaction product appears among and around bundles of axons that lie at a considerable distance from the cells of the secretory tubule. Each bundle contains 8-12 axons that possess numerous varicosities and contain small clear and large dense core vesicles. The glands of the hindpaws and their innervation develop during the first three weeks after birth. Catecholamine-containing axons were associated with the forming glands. At 7 and 10 days, intensely fluorescent fibers surrounded the tubules, and all of the axon profiles associated with the glands contained small granular vesicles (SGV) after permanganate fixation to reveal vesicular stores of norepinephrine. At 14 days the sweat gland plexus was less intensely fluorescent than at earlier ages and relatively few SGV were present. By 21 days, no endogenous catecholamine fluorescence and no SGV were detectable. However, following exposure to exogenous catecholamine, fluorescent fibers were present in the sweat glands of mature rats and they corresponded in position and density to the plexus localized with acetylcholinesterase staining. Catecholamine uptake was blocked by incubation in the cold and by desmethylimipramine and was not observed in cholinergic parasympathetic fibers in the iris or salivary glands. After intraperitoneal administration of 5-hydroxydopamine and permanganate fixation, all the axons in the sweat glands contained a few SGV. Thus, the developing sweat glands appear to be innervated by noradrenergic axons that lose their stores of endogenous catecholamines but not their capacity for uptake and storage as they elaborate an axonal plexus in the maturing glands. These observations support the hypothesis that cholinergic sympathetic neurons appear to undergo a transition from noradrenergic to cholinergic function during development in vivo similar to that previously described in cell culture.

Small intensely fluorescent cells in culture: role of glucocorticoids and growth factors in their development and interconversions with other neural crest derivatives

The neural crest gives rise to a number of adrenergic derivatives, including sympathetic neurons and adrenal chromaffin cells, which contain catecholamines (CAs) but differ in other morphological and functional characteristics. Small intensely fluorescent (SIF) cells, which exist primarily as a minority cell population in autonomic ganglia, are a third cell type in the sympathoadrenal branch of the neural crest lineage. In some respects these cells appear intermediate in phenotype between sympathetic neurons and adrenal chromaffin cells. We established pure dissociated cell cultures of SIF cells from rat superior cervical ganglia (SCG) and used these to study the role of environmental factors in SIF cell development and the relationship of these cells to the other cell types of the sympathoadrenal lineage. When cells from neonatal rat SCG were grown for 3 weeks in the presence of glucocorticoid and in the absence of nerve growth factor (NGF), pure cultures of SIF cells developed. The properties of the cells included (i) small cell size and the occasional presence of short neurites, (ii) intense CA histofluorescence and immunoreactivity for CA synthetic enzymes, (iii) synthesis and storage of CA from radioactive precursors, and (iv) characteristic ultrastructure. The concentration of the glucocorticoid and the presence or absence of non-neuronal cell factors influenced which types of SIF cells developed. In micromolar glucocorticoid most of the cells resembled adrenal chromaffin or type II SIF cells: they displayed immunohistochemically detectable phenylethanolamine-N-methyltransferase (PNMT), synthesized and stored epinephrine, and contained large granular vesicles (100 to 300 nm). When SCG cells were grown in 10(-8) M hormone, many fewer SIF cells developed and a higher percentage of these lacked PNMT immunoreactivity, had neurites, and contained vesicles of smaller mean diameter (70 to 130 nm), similar to those of type I SIF cells in vivo. In the presence of conditioned medium (medium conditioned by non- neuronal cells) as well as glucocorticoid, virtually all of the cells morphologically resembled type I SIF cells. In the absence of glucocorticoid, no SIF cells were ever observed after 3 weeks in culture. By following the development of CA histofluorescence and SIF cell ultrastructure in the cultures over time, we demonstrated that SIF cells were not present in large numbers in these cultures immediately after plating, but were induced from an apparently undifferentiated precursor by the hormonal environment, whereas most of the principal neurons died.

The NIH BRAIN Initiative

The NIH BRAIN Initiative will build on recent successes in neuroscience to create and apply new tools for understanding brain activity. On 2 April 2013, President Barack Obama announced the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. In front of some 200 scientists in the East Room of the White House, the President declared, “…there is this enormous mystery waiting to be unlocked, and the BRAIN Initiative will change that by giving scientists the tools they need to get a dynamic picture of the brain in action and better understand how we think and how we learn and how we remember. And that knowledge could be—will be—transformative” (1).

Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been localized with indirect immunofluorescence techniques in the cholinergic sympathetic fibers that innervate eccrine sweat glands in the rat. This innervation also contains vasoactive intestinal peptide-like immunoreactivity (VIP-IR). A small proportion of principal neurons in stellate and lumbar sympathetic ganglia which provide innervation to the sweat glands contain detectable CGRP-immunoreactivity. The CGRP-IR neurons are immunoreactive for VIP; however, many VIP-IR neurons in these ganglia do not contain detectable levels of CGRP-IR.

Neural crest cell lineages

Abstract A number of derivatives of the neural crest can be interconverted by manipulation of environmental cues. Developmental lineages are proposed for cells in the peripheral nervous system based on these experimental interconversions.

Catecholamine Synthesis is Mediated by Tyrosinase in the Absence of Tyrosine Hydroxylase

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to l-dihydroxyphenylalanine (l-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to l-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

The p75 neurotrophin receptor influences NT-3 responsiveness of sympathetic neurons in vivo.

To determine the role of the p75 neurotrophin receptor (p75NTR) in sympathetic neuron development, we crossed transgenic mice with mutations in p75NTR, nerve growth factor (NGF) and neurotrophin-3 (NT-3). Neuron number is normal in sympathetic ganglia of adult p75NTR–/– mice. Mice heterozygous for a NGF deletion (NGF+/–) have 50% fewer sympathetic neurons. In the absence of p75NTR (p75NTR–/– NGF+/–), however, neuron number is restored to wild-type levels. When NT-3 levels are reduced (p75NTR–/– NGF+/– NT3+/–), neuron number decreases compared to p75NTR–/– NGF+/– NT3+/+. Thus, without p75NTR, NT3 substitutes for NGF, suggesting that p75 alters the neurotrophin specificity of TrkA in vivo.

Developmental changes in the neurotransmitter properties of dissociated sympathetic neurons: a cytochemical study of the effects of medium☆

Abstract Sympathetic principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in several culture conditions shown previously to affect the transmitter status of the neurons. In three of these conditions the neurons are known to develop adrenergic functions over a 3- to 4-week period; in a fourth condition, they develop predominantly cholinergic functions. In this ultrastructural study, the transmitter status of the neurons during development in the several different media was examined after permanganate fixation which causes a granular precipitate in synaptic vesicles containing norepinephrine (small granular vesicles or SGV). It was found that as early as 4 days after plating, synapses and varicosities were present. In all four conditions, all of the terminals contained numerous SGV, indicating that the neurons both synthesize and store norepinephrine. Under “adrenergic” growth conditions, the terminals remained adrenergic in appearance during further development. Under “cholinergic” conditions, terminals of cholinergic appearance were present as early as 7 days and their incidence increased with time. Although the cholinergic terminals contained little or no endogenous norepinephrine, many were initially able to take up and store exogenous catecholamine. These results indicate that the dissociated sympathetic neurons of newborn rats which survive in culture acquired adrenergic transmitter functions early. Under “cholinergic” culture conditions, the neurons lose the ability to synthesize detectable quantities of norepinephrine; the ability to take up and store detectable quantities of exogenous catecholamines disappears more slowly.