Persistent expression of activated notch in the developing hypothalamus affects survival of pituitary progenitors and alters pituitary structure.

BACKGROUND: As the pituitary gland develops, signals from the hypothalamus are necessary for pituitary induction and expansion. Little is known about the control of cues that regulate early signaling between the two structures. Ligands and receptors of the Notch signaling pathway are found in both the hypothalamus and Rathke's pouch. The downstream Notch effector gene Hes1 is required for proper pituitary formation; however, these effects could be due to the action of Hes1 in the hypothalamus, Rathke's pouch, or both. To determine the contribution of hypothalamic Notch signaling to pituitary organogenesis, we used mice with loss and gain of Notch function within the developing hypothalamus. RESULTS: We demonstrate that loss of Notch signaling by conditional deletion of Rbpj in the hypothalamus does not affect expression of Hes1 within the posterior hypothalamus or expression of Hes5. In contrast, expression of activated Notch within the hypothalamus results in ectopic Hes5 expression and increased Hes1 expression, which is sufficient to disrupt pituitary development and postnatal expansion. CONCLUSIONS: Taken together, our results indicate that Rbpj-dependent Notch signaling within the developing hypothalamus is not necessary for pituitary development, but persistent Notch signaling and ectopic Hes5 expression in hypothalamic progenitors affects pituitary induction and expansion.

Early postnatal expression and localization of matrix metalloproteinases-2 and -9 during establishment of rat hippocampal synaptic circuitry.

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes that contribute to pericellular remodeling in a variety of tissues, including brain, where they function in adult hippocampal synaptic structural and functional plasticity. Synaptic plasticity and remodeling are also important for development of connectivity, but it is unclear whether MMPs--particularly MMP-2 and -9, the major MMPs operative in brain--contribute at these stages. Here, we use a combination of biochemical and anatomical methods to characterize expression and localization of MMP-2 and MMP-9 in early postnatal and adult rat hippocampus. Gene and protein expression of these MMPs were evident throughout hippocampus at all ages examined, but expression levels were highest during the first postnatal week. MMP-2 and MMP-9 immunolocalized to punctate structures within the neuropil that codistributed with foci of proteolytic activity, as well as with markers of growing axons and synapses. Taken together, discrete foci of MMP proteolysis are likely important for actively shaping and remodeling cellular and connectional architecture as hippocampal circuitry is becoming established during early postnatal life.

Notch/Rbpjκ signaling regulates progenitor maintenance and differentiation of hypothalamic arcuate neurons.

The hypothalamic arcuate nucleus (Arc), containing pro-opoiomelanocortin (POMC), neuropeptide Y (NPY) and growth hormone releasing hormone (GHRH) neurons, regulates feeding, energy balance and body size. Dysregulation of this homeostatic mediator underlies diseases ranging from growth failure to obesity. Despite considerable investigation regarding the function of Arc neurons, mechanisms governing their development remain unclear. Notch signaling factors such as Hes1 and Mash1 are present in hypothalamic progenitors that give rise to Arc neurons. However, how Notch signaling controls these progenitor populations is unknown. To elucidate the role of Notch signaling in Arc development, we analyzed conditional loss-of-function mice lacking a necessary Notch co-factor, Rbpjκ, in Nkx2.1-cre-expressing cells (Rbpjκ cKO), as well as mice with expression of the constitutively active Notch1 intracellular domain (NICD) in Nkx2.1-cre-expressing cells (NICD Tg). We found that loss of Rbpjκ results in absence of Hes1 but not of Hes5 within the primordial Arc at E13.5. Additionally, Mash1 expression is increased, coincident with increased proliferation and accumulation of Arc neurons at E13.5. At E18.5, Rbpjκ cKO mice have few progenitors and show increased numbers of differentiated Pomc, NPY and Ghrh neurons. By contrast, NICD Tg mice have increased hypothalamic progenitors, show an absence of differentiated Arc neurons and aberrant glial differentiation at E18.5. Subsequently, both Rbpjκ cKO and NICD Tg mice have changes in growth and body size during postnatal development. Taken together, our results demonstrate that Notch/Rbpjκ signaling regulates the generation and differentiation of Arc neurons, which contribute to homeostatic regulation of body size.

Persistent expression of activated Notch inhibits corticotrope and melanotrope differentiation and results in dysfunction of the HPA axis.

The hypothalamic-pituitary-adrenal (HPA) axis is an important regulator of energy balance, immune function and the body's response to stress. Signaling networks governing the initial specification of corticotropes, a major component of this axis, are not fully understood. Loss of function studies indicate that Notch signaling may be necessary to repress premature differentiation of corticotropes and to promote proliferation of pituitary progenitors. To elucidate whether Notch signaling must be suppressed in order for corticotrope differentiation to proceed and whether Notch signaling is sufficient to promote corticotrope proliferation, we examined the effects of persistent Notch expression in Pomc lineage cells. We show that constitutive activation of the Notch cascade inhibits the differentiation of both corticotropes and melanotropes and results in the suppression of transcription factors required for Pomc expression. Furthermore, persistent Notch signaling traps cells in the intermediate lobe of the pituitary in a progenitor state, but has no effect on pituitary proliferation. Undifferentiated cells are eliminated in the first two postnatal weeks in these mice, resulting in a modest increase in CRH expression in the paraventricular nucleus, hypoplastic adrenal glands and decreased stress-induced corticosterone levels. Taken together, these findings show that Notch signaling is sufficient to prevent corticotrope and melanotrope differentiation, resulting in dysregulation of the HPA axis.

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary.

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.

Talampanel suppresses the acute and chronic effects of seizures in a rodent neonatal seizure model.

PURPOSE: To test the efficacy of the novel candidate anticonvulsant talampanel (GYKI 53773) in a rodent model of hypoxic neonatal seizures. Talampanel is a noncompetitive antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid subtype of the glutamate receptor (AMPAR). We have previously shown that AMPARs play a critical role in the generation of acute seizures and later-life seizure susceptibility in this model of neonatal seizures. METHODS: Seizures were induced in postnatal day (P) 10 Long-Evans rat pups by a 15 min exposure to global hypoxia. Acute seizure activity at P10 and subsequent susceptibility to seizure-induced neuronal injury with a "second-hit" kainate-induced seizure at P30-31 were compared between animals receiving talampanel (1, 5, 7.5, or 10 mg/kg) intraperitoneally (i.p.) versus saline vehicle treatment. RESULTS: Talampanel treatment suppressed seizures in a dose-dependent manner, with maximal effect at 7.5 and 10 mg/kg. In addition, talampanel treatment 30 min before hypoxia prevented later-life increases in seizure-induced neuronal injury as assessed by in situ DNA nick end-labeling (ISEL). DISCUSSION: We have previously demonstrated efficacy of other AMPAR antagonists such as NBQX and topiramate in this model. The present finding shows that the novel agent talampanel, under evaluation as an antiepileptic drug in children and adults, may have clinical potential in the treatment of neonatal seizures, particularly those occurring in the context of hypoxic encephalopathy.

Early alterations of AMPA receptors mediate synaptic potentiation induced by neonatal seizures.

The highest incidence of seizures during lifetime is found in the neonatal period and neonatal seizures lead to a propensity for epilepsy and long-term cognitive deficits. Here, we identify potential mechanisms that elucidate a critical role for AMPA receptors (AMPARs) in epileptogenesis during this critical period in the developing brain. In a rodent model of neonatal seizures, we have shown previously that administration of antagonists of the AMPARs during the 48 h after seizures prevents long-term increases in seizure susceptibility and seizure-induced neuronal injury. Hypoxia-induced seizures in postnatal day 10 rats induce rapid and reversible alterations in AMPAR signaling resembling changes implicated previously in models of synaptic potentiation in vitro. Hippocampal slices removed after hypoxic seizures exhibited potentiation of AMPAR-mediated synaptic currents, including an increase in the amplitude and frequency of spontaneous and miniature EPSCs as well as increased synaptic potency. This increased excitability was temporally associated with a rapid increase in phosphorylation at GluR1 S845/S831 and GluR2 S880 sites and increased activity of the protein kinases CaMKII (calcium/calmodulin-dependent protein kinase II), PKA, and PKC, which mediate the phosphorylation of these AMPAR subunits. Postseizure administration of AMPAR antagonists NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline), topiramate, or GYKI-53773 [(1)-1-(4-aminophenyl)-3-acetyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] attenuated the AMPAR potentiation, phosphorylation, and kinase activation and prevented the concurrent increase in in vivo seizure susceptibility. Thus, the potentiation of AMPAR-containing synapses is a reversible, early step in epileptogenesis that offers a novel therapeutic target in the highly seizure-prone developing brain.