Nana Sunn

The sensory circumventricular organs of the mammalian brain.

The brains three sensory circumventricular organs, the subfornical organ, organum vasculosum of the lamina terminalis and the area postrema lack a blood brain barrier and are the only regions in the brain in which neurons are exposed to the chemical environment of the systemic circulation. Therefore they are ideally placed to monitor the changes in osmotic, ionic and hormonal composition of the blood. This book describes their. General structure and relationship to the cerebral ventricles Regional subdivisions Vasculature and barrier properties Neurons, glia and ependymal cells Receptors, neurotransmitters, neuropeptides and enzymes Neuroanatomical connections Functions.

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 μg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10−7 M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10−7 M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood–brain barrier may have regulatory influences on fluid balance during pregnancy in rats.

Nuclear Factor I Gene Expression in the Developing Forebrain

Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al. [ 1999 ] Proc Natl Acad Sci U S A 96:11946–11951; Shu et al. [ 2003 ] J Neurosci 23:203–212; Steele‐Perkins et al. [ 2005 ] Mol Cell Biol 25:685–698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell‐autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell‐autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell‐autonomous roles in cortical development. J. Comp. Neurol. 508:385–401, 2008.

Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib -deficient mice

BackgroundAgenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation.ResultsOur investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant.ConclusionThe formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.

Identification of efferent neural pathways from the lamina terminalis activated by blood-borne relaxin.

The ovarian hormone relaxin, in addition to its role in pregnancy, exerts an action on the brain to influence oxytocin and vasopressin secretion, water drinking, and cardiovascular function. Intravenous (i.v.) infusion of relaxin causes an acute water drinking response, confirming its role as a dipsogenic hormone. The aim of this study was to determine whether neurones in the lamina terminalis, which project to the hypothalamic paraventricular and supraoptic nuclei, are activated by elevated levels of circulating relaxin in conscious rats. Immunocytochemistry combined with retrograde neuronal tracing with cholera toxin B subunit conjugated to cholera toxin B (CTB‐gold) was used to identify populations of neurones responding with elevated cells of Fos protein to i.v. relaxin administration and which project to these specific hypothalamic sites. Neurones exhibiting Fos were present in the outer parts of the subfornical organ (SFO), the dorsal part of the organum vasculosum (OVLT), the supraoptic nucleus and the paraventricular nucleus. These did not occur in control rats with i.v. infusions of isotonic saline. Approximately 90% of neurones concentrated in the outer parts of the SFO and in the dorsal OVLT showed both retrogradely transported CTB‐gold and Fos in response to i.v. infusion of relaxin. These data support a role for relaxin acting on the brain to regulate body fluid and electrolyte homeostasis by activating neural pathways subserving water drinking, vasopressin and oxytocin secretion.

Circulating angiotensin II activates neurones in circumventricular organs of the lamina terminalis that project to the bed nucleus of the stria terminalis

The aim of this study was to determine, in conscious rats, whether elevated concentrations of circulating angiotensin II activate neurones in both the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) that project to the bed nucleus of the stria terminalis (BNST). The strategy employed was to colocalize retrogradely transported cholera toxin B subunit (CTB) from the BNST, with elevated levels of Fos protein in response to angiotensin II. Circulating angiotensin II concentrations were increased by either intravenous infusion of angiotensin II or subcutaneous injection of isoproterenol. Neurones exhibiting Fos in response to angiotensin II were present in the subfornical organ, predominantly in its central core but with some also seen in its peripheral aspect, the dorsal and lateral margins of the OVLT, the supraoptic nucleus and the parvo‐ and magnocellular divisions of the paraventricular nucleus. Fos‐labelling was not apparent in control rats infused with isotonic saline intravenously or injected with either CTB or CTB conjugated to gold particles (CTB‐gold) only. Of the neurones in the subfornical organ that were shown by retrograde labelling to project to BNST, approximately 50% expressed Fos in response to isoproterenol. This stimulus also increased Fos in 33% of neurones in the OVLT that project to BNST. Double‐labelled neurones were concentrated in the central core of the subfornical organ and lateral margins of the OVLT in response to increased circulating angiotensin II resulting from isoproterenol treatment. These data support a role for circulating angiotensin II acting either directly or indirectly on neurones in subfornical organ and OVLT that project to the BNST and provide further evidence of functional regionalization within the subfornical organ and the OVLT. The function of these pathways is yet to be determined; however, a role in body fluid homeostasis is possible.

Neuregulin-1 potentiates agrin-induced acetylcholine receptor clustering through muscle-specific kinase phosphorylation

At neuromuscular synapses, neural agrin (n-agrin) stabilizes embryonic postsynaptic acetylcholine receptor (AChR) clusters by signalling through the muscle-specific kinase (MuSK) complex. Live imaging of cultured myotubes showed that the formation and disassembly of primitive AChR clusters is a dynamic and reversible process favoured by n-agrin, and possibly other synaptic signals. Neuregulin-1 is a growth factor that can act through muscle ErbB receptor kinases to enhance synaptic gene transcription. Recent studies suggest that neuregulin-1–ErbB signalling can modulate n-agrin-induced AChR clustering independently of its effects on transcription. Here we report that neuregulin-1 increased the size of developing AChR clusters when injected into muscles of embryonic mice. We investigated this phenomenon using cultured myotubes, and found that in the ongoing presence of n-agrin, neuregulin-1 potentiates AChR clustering by increasing the tyrosine phosphorylation of MuSK. This potentiation could be blocked by inhibiting Shp2, a postsynaptic tyrosine phosphatase known to modulate the activity of MuSK. Our results provide new evidence that neuregulin-1 modulates the signaling activity of MuSK and hence might function as a second-order regulator of postsynaptic AChR clustering at the neuromuscular synapse. Thus two classic synaptic signalling systems (neuregulin-1 and n-agrin) converge upon MuSK to regulate postsynaptic differentiation.

High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

BackgroundIn utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure.ResultsIn 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was similar in sham experiments, 54% (33/61), for which procedures were identical but no microinjection was performed, suggesting that surgery and manipulation of the uterus were the main causes of embryonic death.ConclusionUltrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of ≥60%. Target accuracy of these sites and of the exocoelomic cavity at E7.5 was ≥51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development.

Collecting Duct-Derived Cells Display Mesenchymal Stem Cell Properties and Retain Selective In Vitro and In Vivo Epithelial Capacity

We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.

Developmental regulation of cardiovascular function is dependent on both genotype and environment

Adverse developmental environments can increase the risk of adult cardiovascular disease, but not all individuals are affected, suggesting the importance of genotype. Genetically distinct mouse strains allow the genetic dissection of complex traits; however, they have not been used to evaluate the developmental origins of adult cardiovascular disease. Our objective was to determine the effect of prenatal nutrient restriction (R) on adult cardiovascular function in A/J (AJ) and C57BL/6J (B6) mice and whether a postnatal high-fat (HF) diet exacerbates these effects. Pregnant AJ and B6 mice underwent a 30% R or ad libitum diet, and their offspring underwent a HF or control diet. Hypertension (+17 mmHg; P<0.001) was observed in B6R mice at 9 wk, and their arterial pressure tended to remain high at 25 wk (+13 mmHg; not significant). In AJR mice, the normal decrement in arterial pressure over this age range in this strain was abolished. Heart rate prematurely increased in B6R and decreased in AJR (all; P<0.05) mice from 9 to 25 wk. There was no effect of postnatal HF diet on these relationships. The Tei index (from a 26-wk microultrasound) was increased in both AJR and B6R mice (all; P<0.05), suggesting an improved global myocardial performance. Neither R nor HF alone changed diastolic (ratio of E wave to A wave) or systolic (%fractional shortening) function in either strain; however, R and HE combined improved diastolic function in B6 (P<0.05) but not in AJ mice. Therefore, there are strain-dependent alterations in adult cardiovascular function in response to prenatal nutrient restriction. Unexpectedly, a postnatal HF diet did not exacerbate the effects of prenatal nutrient restriction on postnatal cardiovascular outcomes.