Shirley B. House

An immunochemical analysis of oxytocin and vasopressin prohormone processing in vivo.

Antisera against partially processed, unamidated forms of AVP and OT were raised and characterized by radioimmunoassay and immunocytochemistry. These antibodies, and antibodies that recognize fully processed, amidated forms of AVP and OT, were used together with various fractionation methods to study the content of prohormones, partially processed and fully processed forms of AVP and OT in the hypothalamo-neurohypophysial system of adult and fetal (E21) rats. The levels of cleaved AVP and OT in the fetus were lower than those of the adult (1 to 3 orders of magnitude for brain and pituitary, respectively), and the detection of cleaved OT in brain and pituitary was delayed compared to that of AVP. Pro-AVP cleavage efficiency in the adult and the fetus was high (99 and 95% cleavage, respectively) resulting in formation of fully processed amidated forms of AVP, with no detectable partially processed peptides. Pro-OT processing in the adult was very similar (over 99% cleavage) resulting in formation of fully processed amidated OT. However, Pro-OT processing efficiency in the fetus was very low and incomplete, resulting in 40% unprocessed precursor and the accumulation of C-terminally extended unamidated intermediate forms (OT-Gly, OT-Gly-Lys, and OT-Gly-Lys-Arg).

Regulatory Domains in the Intergenic Region of the Oxytocin and Vasopressin Genes that Control their Hypothalamus-Specific Expression In Vitro

Previous studies of oxytocin (OT) and vasopressin (VP) cell-specific gene expression in the hypothalamus using transgenic mouse and rat models focused attention on the intergenic region (IGR) as the site of critical enhancer elements. In this study, we used organotypic slice-explant cultures of rat hypothalamus as in vitro models, and particle-mediated gene transfer (biolistics) transfection methods to identify critical DNA sequences in the IGR between the OT and VP genes responsible for hypothalamic-specific gene expression. Reducing the 5′ flanking region in the mouse VP gene from 3.5 kbp to 288 bp did not alter the efficacy of its expression in hypothalamic slices. All subsequent VP constructs were based on this 288 bp VP gene construct with changes made only to the IGR. These studies, which used various constructs with OT and VP promoters driving enhanced green fluorescent protein reporter gene expression, demonstrated that the IGR is necessary for OT and VP gene expression in hypothalamic slices in vitro. The DNA sequences in the IGR responsible for both OT and VP gene expression were located in a 178 bp domain immediately downstream of exon 3 of the VP gene. In addition, another domain in the IGR, 430 bp immediately downstream of exon 3 of the OT gene, contained a positive regulatory element for OT gene expression in the hypothalamus. Alignment of the DNA sequences in the 178 and 430 bp domains reveals four common sequences (motifs) that may be candidates for the putative enhancers in the IGR that regulate OT and VP gene hypothalamic-specific expression.

Neural Activity Protects Hypothalamic Magnocellular Neurons against Axotomy-Induced Programmed Cell Death

Axotomy typically leads to retrograde neuronal degeneration in the CNS. Studies in the hypothalamo-neurohypophysial system (HNS) have suggested that neural activity is supportive of magnocellular neuronal (MCN) survival after axotomy. In this study, we directly test this hypothesis by inhibiting neural activity in the HNS, both in vivo and in vitro, by the use of tetrodotoxin (TTX). After median eminence compression to produce axonal injury, unilateral superfusion of 3 μM TTX into the rat supraoptic nucleus (SON), delivered with the use of a miniature osmotic pump for 2 weeks in vivo, produced a decrease in the number of surviving MCNs in the TTX-treated SON, compared with the contralateral untreated side of the SON. In vitro application of 2.5 μM TTX for 2 weeks to the SON in organotypic culture produced a 73% decrease in the surviving MCNs, compared with untreated control cultures. Raising the extracellular KCl in the culture medium to 25 mM rescued the MCNs from the axotomy- and TTX-induced cell death. These data support the proposal that after axotomy, neural activity is neuroprotective in the HNS.

The magnocellular neuronal phenotype: cell-specific gene expression in the hypothalamo-neurohypophysial system.

The magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system are exceptional cell biological models to study mechanisms of cell-specific gene expression and neurosecretion of neuropeptides in the central nervous system. Single cell differential gene expression experiments have further defined these phenotypes by identifying novel and distinct regulatory molecules in these neurons. Transgenic mouse studies have led to the intergenic region (IGR) hypothesis, which states that the DNA sequences between the OT- and VP-genes contain critical enhancer sites for their cell-specific expression. The recent cloning and sequencing of the human IGR, and its comparison with the mouse IGR sequence has identified conserved sequences as putative, cell-specific enhancer sites which are now being evaluated by biolistic transfections of organotypic hypothalamic cultures. With these data, it is possible to target the gene expression of specific molecules to magnocellular neurons both in vivo and in vitro, in order to perturb and/or visualize neurosecretory and other processes.

Gene transfer into hypothalamic organotypic cultures using an adeno-associated virus vector.

Organotypic cultures of rat hypothalamic slice cultures were successfully transduced using adeno-associated viral vectors. Using nuclear-targeted Lac-Z as the reporter gene, transduction was found to be very effective, occurring in as high as 89% of a specific cell type, the oxytocin neurons, present in the cultured explants. These transduction levels were not accompanied by any deleterious effects in the cultured cells 7 days after transduction. Such an in vitro approach should be valuable for the study of cell-specific gene expression in neurons in the central nervous system for which there are no homologous (surrogate) cell lines.

Interactions of cyclic adenosine monophosphate, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor treatment on the survival and growth of postnatal mesencephalic dopamine neurons in vitro

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 microM db-cAMP or 10 microM Forskolin plus 100 microM IBMX in the culture medium. Neither 10-50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 microM H89 or 20 microM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro.

Long‐Term Effects of Ciliary Neurotrophic Factor on the Survival of Vasopressin Magnocellular Neurones in the Rat Supraoptic Nucleus In Vitro

The use of hypothalamic organotypic cultures for the long‐term study of mechanisms in magnocellular neurones (MCNs) of the hypothalamic‐neurohypophysial system has been limited by the relatively poor maintenance of the vasopressin MCNs in vitro. Recent studies have shown that addition of ciliary neurotrophic factor (CNTF) to the media significantly reduced the apoptosis of both oxytocin and vasopressin MCNs. Here, we studied various temporal factors in the CNTF treatment that can influence the efficacy of MCN survival. Immunohistochemistry was used to identify and count surviving vasopressin and oxytocin MCNs in the supraoptic nucleus (SON) in hypothalamic slices cultured in the presence of CNTF (10 ng/ml media) for various time intervals, and in situ hybridization for vasopressin mRNA was used to evaluate the vasopressin mRNA gene expression in the SON under the same conditions. The presence of CNTF in the medium for 10 days produced a maximal increase in the survival of vasopressin MCNs (by 11‐fold) and in the survival of oxytocin‐MCNs (by approximately four‐fold) over controls. These effects persisted for an additional 7–10 days even in the absence of CNTF. The ability of CNTF to increase survival of the MCNs or increase vasopressin mRNA levels in the SON required that the CNTF be present during the initial 7–10 days of culture. CNTF failed to rescue vasopressin or oxytocin MCNs when added to the media only for the last 7 days of a total of 14 days in vitro. Similar results were observed when SON vasopressin mRNA levels were measured. These results indicate that the presence of CNTF is required at the outset to rescue the vasopressin and oxytocin MCN from axotomy induced apoptosis, and that, after 10 days in CNTF, the MCNs no longer require the CNTF for survival.

Depolarization and Neurotransmitter Regulation of Vasopressin Gene Expression in the Rat Suprachiasmatic Nucleus In Vitro

Vasopressin (VP) transcription in the rat suprachiasmatic nucleus (SCN) in organotypic culture was studied by in situ hybridization histochemistry using an intron-specific VP heteronuclear RNA probe. The circadian peak of VP gene transcription in the SCN in vitro is completely blocked by a 2 h exposure to tetrodotoxin (TTX) in the culture medium, and this TTX inhibition of VP gene transcription is reversed by exposure of the SCN to either forskolin or potassium depolarization. This suggests that an intrinsic, spontaneously active neuronal mechanism in the SCN is responsible for the cAMP- and depolarization-dependent pathways involved in maintaining peak VP gene transcription. In this paper, we evaluate a variety of neurotransmitter candidates, membrane receptors, and signal-transduction cascades that might constitute the mechanisms responsible for the peak of VP gene transcription. We find that vasoactive intestinal peptide (VIP) and a VPAC2 (VIP receptor subtype 2) receptor-specific agonist, Ro-25-1553, are the most effective ligands tested in evoking a cAMP–mitogen-activated protein kinase signal transduction cascade leading to an increase in VP gene transcription in the SCN. In addition, a second independent pathway involving depolarization activating L-type voltage-gated calcium channels and a Ca-dependent kinase pathway [inhibited by KN62 (1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine)] rescues VP gene transcription in the presence of TTX. In the absence of TTX, these independent pathways appear to act in a cooperative manner to generate the circadian peak of VP gene transcription in the SCN.

Bcl-xL and caspase inhibition increase the survival of rat oxytocin and vasopressin magnocellular neurons in organotypic culture

Hypothalamic magnocellular neurons (MCNs) are highly vulnerable to axotomy-induced cell death in vivo and in vitro. In this study, we determined whether the anti-apoptotic agent Bcl-xL, a member of the Bcl-2 family which prevents programmed cell death in the central nervous system, can rescue oxytocin (OT) and vasopressin (VP) MCNs in the supraoptic nucleus (SON) in organotypic culture. We found that the novel, membrane permeant form of Bcl-xL that we employed in these studies protected both OT and VP MCNs from degeneration as long as the Bcl-xL was present in the medium. In contrast, z-VAD-fmk, an inhibitor of caspases that are involved in apoptosis, was less effective in that it significantly rescued OT MCNs (P < 0.01) but not VP MCNs (P > 0.09). Unlike the Bcl-xL, Z-VAD-fmks effectiveness in reducing MCN cell death was not sustained for the full 15 days in vitro.

Effects of ciliary neurotrophic factor and leukemia inhibiting factor on oxytocin and vasopressin magnocellular neuron survival in rat and mouse hypothalamic organotypic cultures

Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) have served as important experimental models for the molecular and physiological study of this neuronal phenotype. However, it has been difficult to maintain significant numbers of the MCNs, particularly vasopressin MCNs, in these cultures for long periods. In this paper, we describe the use of the neurotrophic factors, leukemia inhibiting factor (LIF) and ciliary neurotrophic factor (CNTF) to rescue rat vasopressin (Avp)- and oxytocin (Oxt)-MCNs from axotomy-induced, programmed cell death in vitro. Quantitative data are presented for the efficacy of the LIF family of neurotrophic factors on the survival of MCNs in three nuclei, the paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei in the mouse and rat hypothalamus.