The -216- to -100-bp Sequence in the 5'-Flanking Region of the Oxytocin Gene Contains a Cell-Type Specific Regulatory Element for its Selective Expression in Oxytocin Magnocellular Neurones.

The oxytocin (OXT) gene is abundantly and highly selectively expressed in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system. Previous DNA sequence deletion studies in vivo have shown that the -216- to -100-bp sequence in the 5'-flanking region of the oxytocin gene was required for its cell-type specific expression in the rat supraoptic nucleus. In the present study, we test the coupled hypotheses that this -216- to -100-bp sequence is responsible for (i) the selective expression of the OXT gene in OXT-MNCs and (ii) its selective repression in vasopressin (AVP)-MCNs. We show that, consistent with hypothesis 1, removal of the -216- to -100-bp sequence from the OXT gene completely eliminates its expression in OXT-MCNs in vivo but, in contrast to the prediction of hypothesis 2, there was no appearance of OXT gene expression in AVP-MCNs. Taken together, these and other data demonstrate that the -216- to -100-bp sequence in the 5'-flanking region of the oxytocin gene contains only an activator of transcription operating in the OXT-MCNs.

Cell-type specific expression of oxytocin and vasopressin genes: an experimental odyssey.

The supraoptic nucleus (SON) is a particularly good model for the study of cell-type specific gene expression because it contains two distinct neuronal phenotypes, the oxytocin (OT) and vasopressin (AVP) synthesising magnocellular neurones (MCNs). The MCNs are found in approximately equal numbers and selectively express either the OT or the AVP gene in approximately 97% of the MCN population in the SON. An unresolved issue has been to determine what mechanisms are responsible for the highly selective regulation of the cell-type specific expression of OT and AVP genes in the MCNs. Previous attempts to address this question have used various bioinformatic and molecular approaches, which included using heterologous cell lines to study the putative cis-elements in the OT and AVP genes, and the use of OT and/or AVP transgenes in transgenic rodents. The data from all of the above studies identified a region < 0.6 kbp upstream of OT exon I and approximately 3 kb upstream of AVP exon I as being sufficient to produce cell-specific expression of the OT and AVP genes, respectively, although they failed to identify the specific cis-domains responsible for the MCN-specific gene expression. An alternative experimental approach to perform promoter deletion analysis in vivo (i.e. to use stereotaxic viral vector gene transfer into the SON to further dissect the cis-elements in the OT and AVP genes) will be described here. This in vivo method uses adeno-associated viral (AAV) vectors expressing OT-promoter deletion constructs and utilises the enhanced green fluorescent protein (EGFP) as the reporter. The AAV constructs are stereotaxically injected into the rat brain above the SON and, 2 weeks post injection, the rats are sacrificed and assayed for EGFP expression. Using this method, it has been possible to identify specific regions upstream of the transcription start site in the OT and AVP gene promoters that are responsible for conferring the cell-type specificity of the OT and AVP gene expression in the SON.

Differential kinetics of oxytocin and vasopressin heteronuclear RNA expression in the rat supraoptic nucleus in response to chronic salt loading in vivo.

Previous studies have shown that the secretion of oxytocin and vasopressin from the posterior pituitary always accompanies systemic hyperosmotic stimuli in rats, and that oxytocin and vasopressin mRNAs consistently increase in response to prolonged hyperosmotic stimuli. Hence, it has been widely interpreted that oxytocin and vasopressin secretion and gene expression are closely coupled. In the present study, we used both vasopressin and oxytocin intron- specific probes to measure vasopressin and oxytocin heteronuclear RNA (hnRNA) levels, respectively, by in situ hybridisation in the rat supraoptic nucleus (SON) in conjunction with radioimmunoassays of vasopressin and oxytocin peptide levels in plasma and in the posterior pituitary in normally hydrated rats and after 1-5 days of salt loading. Increased oxytocin secretion in response to hyperosmotic stimuli exceeded vasopressin secretion at every time point studied. Vasopressin hnRNA in the SON increased to near maximal levels within minutes after the hyperosmotic stimulus, and was maintained throughout all 5 days of salt loading. By contrast, oxytocin hnRNA did not significantly change from control levels until approximately 2 days after hyperosmotic stimulation, and was not maximal until 3 days. In summary, increases in oxytocin gene transcription in response to osmotic stimuli are dramatically delayed compared to increases in vasopressin gene transcription under the same conditions. These data indicate that oxytocin gene transcription is not as closely correlated with pituitary peptide secretion as is vasopressin gene transcription, and suggests that there is a fundamental difference in excitation-secretion-transcription coupling mechanisms that regulate these two closely related genes in the rat magnocellular neurones in the SON.

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.

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.

Targeting of green fluorescent protein to secretory granules in oxytocin magnocellular neurons and its secretion from neurohypophysial nerve terminals in transgenic mice.

Oxytocin (OT) is a hypothalamic nonapeptide that is synthesized as part of a larger precursor protein that also contains an approximately 10-kDa protein called neurophysin at its C-terminus. This precursor protein is trafficked through the regulated secretory pathway into secretory granules and then axonally transported to and secreted from nerve terminals in the neural lobe of the pituitary. In this paper, we show that the AI-03 transgene that contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence undergoes depolarization-induced calcium-dependent secretion. The endogenous fluorescence in the neural lobes is sufficiently intense to image secretory events in individual OT nerve terminals (neurosecretosomes) isolated from the posterior pituitaries in these transgenic mice.

Direct stimulation of arginine vasopressin gene transcription by cAMP in parvocellular neurons of the paraventricular nucleus in organotypic cultures.

The regulation of arginine vasopressin (AVP) gene transcription in the paraventricular nucleus (PVN) was studied in rat hypothalamic organotypic cultures using intronic in situ hybridization. While AVP heteronuclear (hn) RNA was not detected in the PVN under basal conditions, a marked induction of AVP hnRNA was observed after 2 and 3 h incubation of slices with forskolin. In contrast to the stimulatory effects of forskolin, phorbol 12-myristate 13-acetate (PMA) was completely ineffective in inducing AVP hnRNA in the PVN at any time examined (1-3 h). Forskolin-induced AVP hnRNA expression was unaffected by blockage of neurotransmission by the sodium channel inhibitor, tetrodotoxin, indicating that forskolin acts directly on AVP cells in the PVN. Dual staining in situ hybridization of forskolin-stimulated hypothalamic sections using both radio labeled AVP hnRNA and digoxigenin-labeled CRH mRNA probes revealed colocalization of both transcripts, indicating AVP hnRNA is expressed in the parvocellular neurons. The data demonstrate that cAMP directly activates AVP gene transcription in parvocellular neurons of the PVN.

Vasopressin gene expression: experimental models and strategies.

The intergenic region (IGR) separating the genes for vasopressin (VP) and oxytocin (OT) has been shown to be critical for the cell-specific expression of these peptide genes in hypothalamic neurons. To date, the most relevant information about the putative cis-elements in the IGR that might determine cell-specific gene expression has come from studies in transgenic models. As a first step toward increasing the efficiency of the IGR sequence deletion studies in transgenic animals, a comparative genomics approach comparing the IGR sequence in humans versus mice was used to identify conserved sequences that might be candidate regulatory elements. The nucleotide sequence of the IGR between the human VP and OT genes was determined and compared to the mouse IGR, and 26 conserved sequences in three distinct clusters were found. These conserved sequences and motifs may be important for the cell-specific expression of the VP and OT genes. However, before further significant progress can be made, a "high-throughput" method for the analysis of deletion constructs in relevant cell types in vitro is needed. It is proposed here that organotypic culture models combined with the use of particle-mediated gene transfer methods may provide an effective, general strategy for the study of cell-specific expression in the central nervous system.

Cell-specific expression and subcellular localization of neurophysin-CAT-fusion proteins expressed from oxytocin and vasopressin gene promoter-driven constructs in transgenic mice.

The cell-specific expression of both the oxytocin (OT) and vasopressin (VP) genes in magnocellular neurons (MCNs) of the hypothalamus has been proposed to be under the control of cis-elements in an intergenic region downstream of the VP gene. We examined this hypothesis using transgenic mice containing mouse genomic DNA-derived constructs linked to chloramphenicol acetyltransferase (CAT) reporters. VP gene expression was studied using constructs containing 3.8 kbp of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to a CAT reporter. The two VP-transgene constructs differed by the lengths of their VP gene 3' flanking regions (2.1 versus 3.6 kbp). A similar construct for the oxytocin CAT transgene was used which contained the full-length (3.6 kbp) downstream intergenic region between the mouse genes. All three transgenic constructs produced cell-specific expression of the CAT-reporter in the magnocellular neurons as determined by CAT-immunoreactivity. Oxytocin transgene expression was restricted to OT cells in two founders, and the two VP transgenes to VP cells in five founders. Electron microscopic immunocytochemistry showed that the CAT fusion proteins produced from the OT- and VP-transgenes were efficiently trafficked through the regulated secretory pathways in their respective magnocellular neurons, packaged into large dense core vesicles, and transported to nerve terminals in the posterior pituitary.

Gene regulation in the magnocellular hypothalamo-neurohypophysial system.

The hypothalamo-neurohypophysial system (HNS) is the major peptidergic neurosecretory system through which the brain controls peripheral physiology. The hormones vasopressin and oxytocin released from the HNS at the neurohypophysis serve homeostatic functions of water balance and reproduction. From a physiological viewpoint, the core question on the HNS has always been, "How is the rate of hormone production controlled?" Despite a clear description of the physiology, anatomy, cell biology, and biochemistry of the HNS gained over the last 100 years, this question has remained largely unanswered. However, recently, significant progress has been made through studies of gene identity and gene expression in the magnocellular neurons (MCNs) that constitute the HNS. These are keys to mechanisms and events that exist in the HNS. This review is an inventory of what we know about genes expressed in the HNS, about the regulation of their expression in response to physiological stimuli, and about their function. Genes relevant to the central question include receptors and signal transduction components that receive and process the message that the organism is in demand of a neurohypophysial hormone. The key players in gene regulatory events, the transcription factors, deserve special attention. They do not only control rates of hormone production at the level of the gene, but also determine the molecular make-up of the cell essential for appropriate development and physiological functioning. Finally, the HNS neurons are equipped with a machinery to produce and secrete hormones in a regulated manner. With the availability of several gene transfer approaches applicable to the HNS, it is anticipated that new insights will be obtained on how the HNS is able to respond to the physiological demands for its hormones.

Chronic hypoosmolality induces a selective decrease in magnocellular neurone soma and nuclear size in the rat hypothalamic supraoptic nucleus.

The magnocellular neurones of the hypothalamo-neurohypophysial system (HNS) play a vital role in the maintenance of body homeostasis by regulating oxytocin (OT) and vasopressin (VP) secretion from the posterior pituitary. During hyperosmolality, OT and VP mRNA levels are known to increase by approximately two-fold, whereas during chronic hypoosmolality, OT and VP mRNA levels decrease to approximately 10-20% of basal levels. In these studies, we evaluated changes in cell size associated with these physiological conditions. Cell and nuclear sizes of neurones in the supraoptic nucleus (SON), the nucleus of the lateral olfactory tract (LOT) and the medial habenular nucleus (MHB) were measured from neurones identified by in situ hybridization histochemistry for beta(III)-tubulin mRNA, and measurements were made from OT and AVP magnocellular neurones in the SON after phenotypic identification by immunohistochemistry. Under hypoosmolar conditions, the cell and nuclear sizes of OT and VP magnocellular neurones decreased to approximately 60% of basal values, whereas cell and nuclear sizes of OT and VP neurones in hyperosmolar rats increased to approximately 170% of basal values. In contrast, neither hyperosmolality, nor hypoosmolality significantly affected cell and nuclear sizes in the LOT and MHB. These results confirm previous studies that showed that magnocellular neurones increase cell size in response to hyperosmolar conditions and, for the first time, demonstrate a marked decrease in cell size in the SON in response to chronic hypoosmolar conditions. These dramatic changes in cell and nuclear size directly parallel changes in OT and VP gene expression in the magnocellular neurones of the SON and, consequently, are consistent with the pronounced bidirectional changes in gene expression and cellular activity found during these osmotic perturbations. Our results therefore support the concept of global alterations in the synthetic activity of magnocellular OT and AVP neurones in response to extracellular osmolality.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality.

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10-20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na(+)-K(+)-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide's messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage-activated calcium channel subunit genes, alpha1A-D, alpha2, and beta1-4 are expressed in virtually all MNCs. However, the alpha1E subunit gene is not expressed at detectable levels in these cells. The expression of all of the beta-subunit genes in each MNC may account for the variations in physiological and pharmacological properties of the high voltage-activated channels found in these neurons. (Endocrinology 140: 5391-5401, 1999)

Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus.

Oxytocin (OT) and vasopressin (VP) are peptide hormones that are derived from genes predominantly expressed in distinct magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Recent evidence suggests that some magnocellular neurons coexpress both peptides. Our qualitative RT-PCR experiments on single cells show that the majority of magnocellular neurons coexpress both peptide messenger RNAs (mRNAs) in varying amounts. Using a competitive RT-PCR method combined with a standard calibration curve, we quantitatively determined OT and VP mRNA in single magnocellular neurons from the normal female rat SON, with a detection sensitivity of less than 30 mRNA molecules/cell. We defined the phenotypes of the single magnocellular neurons according to their ratios of these two peptide mRNAs. Using this approach, we identified three major phenotypes: oxytocin neurons, where the average OT to VP mRNA ratio is about 256; vasopressin neurons, where the average VP to OT mRNA ratio is about 182; and one oxytocin/vasopressin coexisting neuron, where the OT/VP mRNA ratio is 2. Thus, there is some OT and VP mRNA coexpression in virtually all of the magnocellular neurons in supraoptic nuclei of hypothalamus. However, clear phenotypes are identifiable by considering quantitative as opposed to qualitative differences.

Analysis of synaptotagmin I-IV messenger RNA expression and developmental regulation in the rat hypothalamus and pituitary.

Synaptotagmins are a large family of synaptic vesicle membrane proteins, that appear to be involved in neurotransmitter secretion from small secretory vesicles. We have quantitatively analysed the messenger RNA levels of synaptotagmin I-IV isoforms in adult hypothalamic and pituitary tissues in order to determine which of these isoforms dominate in these tissues which mainly secrete peptides from large dense core vesicles. We also studied the expression of these isoforms during prenatal (E15, and E17) and postnatal (P1, P7, P14 and P21) rat hypothalamic development. In order to assay small individual samples (e.g., pituitary and embryonic tissues), we employed quantitative reverse transcription-polymerase chain reaction methods. Our results show that synaptotagmin I messenger RNA is the most abundant isoform in all tissues, and is about 5.4- or 38-fold higher in hypothalamus than in neurointermediate and anterior pituitary lobe, respectively. Synaptotagmin II, which is very abundant in cerebellum, is relatively low in hypothalamus (5% of cerebellum) and virtually absent from the pituitary. Synaptotagmin III is about 10 times greater in the neural tissues versus the pituitary, and synaptotagmin IV was the least abundant isoform in all the tissues. Developmental analyses of the synaptotagmin isoforms in rat hypothalamus shows that all isoforms are at low levels during embryonic stages and increase postnatally. Synaptotagmin I and II have similar patterns and rise to maximum (adult) levels around P14, whereas synaptotagmin III and IV reach their maximum levels considerably earlier, at P1. These data show that synaptotagmin I is the dominant isoform in both predominantly peptide secreting systems (e.g., in pituitary tissues) and in neurotransmitter secreting systems (e.g., in cerebellum). While the developmental expression patterns of synaptotagmin I and II parallels the temporal development of synaptogenesis in the nervous system, the early maximal expression of synaptotagmin III and IV suggests that these isoforms may have other functions during early postnatal development.

Effects of osmotic pressure and brain-derived neurotrophic factor on the survival of postnatal hypothalamic oxytocinergic and vasopressinergic neurons in dissociated cell culture.

Neurons from hypothalamic paraventricular nuclei (PVN) and supraoptic nuclei (SON) from postnatal day 6-8 rats were enzymatically dissociated and separately maintained in monolayer cultures for 14 days. The osmotic pressure of the culture medium, based on Neurobasal medium (Life Technologies), was varied (255, 300 and 330 mOsm/l) by adjustment using mannitol. The survival of oxytocin (OT), vasopressin (VP) and oxytocin-vasopressin (OT/VP) coexpressing neurons were studied under these varied conditions, and the identification of the cell phenotypes in the cultures was carried out by using double-label immunofluorescence. Under control osmolar conditions (300 mOsm/l) equivalent numbers of OT and VP neurons were found in the SON (P = 0.8398) and PVN (P = 0.4721) cultures. The OT neurons' survival did not change in 255 or 330 mOsm media in the SON cultures, but the VP neurons in the SON cultures were significantly increased in 255 mOsm/l medium as compared to control (300 mOsm/l) medium (P = 0.0088). No significant changes were found in VP neuron survival in SON cultures between the 300-330 mOsm/l media (P = 0.2372). Similar data were obtained for the VP neurons in PVN-derived cultures, but the OT neurons in these cultures survived significantly better at 300 mOs/l than at 255 mOsm/l (P<0.0001), but were not significantly different at 330 mOsm/l (P = 0.1208). In general, the VP neurons were more vulnerable than OT neurons to increases of culture medium osmolarity with respect to their survival. The number of OT/VP coexpressing neurons was greater in SON-derived cell cultures as compared to PVN-derived cell cultures, and their numbers were higher in the lower osmolarity media. The effects of adding brain-derived neurotrophic factor (BDNF) to the culture medium on survival were determined. BDNF significantly increased the numbers of all three types of neurons in both PVN and SON cell cultures (P = 0.0001-0.0060). The phenotypically identified cells, cultured in the 300 mOsm/l medium, responded by depolarization or hyperpolarization when transferred to hypertonic or hypotonic perfusion salines, respectively.

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.

Quantitative analysis of gene expression in organotypic slice-explant cultures by particle-mediated gene transfer.

Biolistics, also known as particle-mediated gene transfer, has been used as an effective, method to transfect primary neurons in cultured slices when all other methods have proven unsuccessful. Most of these uses have provided qualitative or semi-quantitative data based on visual assays such as immunohistochemistry. In this paper, we describe a quantitative method of biolistics to analyze gene expression in organotypic cultures of hippocampus and hypothalamus. The method involves co-transfection of the experimental promoters and standard (cytomegalovirus or Rous sarcoma virus) promoters coupled to different reporters (luciferase or beta-galactosidase), with the standard promoter-reporter construct used to 'normalize' the experimental data. Examples and validations of this technique with various cell specific promoters are given: for example, astrocyte-specific and neuron-specific (alpha-tubulin and N-type calcium channel alpha-1B gene) promoters and various tissues (Neuro 2A cells and hippocampal and hypothalamic organotypic slice-explants). An analysis of deletion constructs of the alpha 1B calcium channel subunit gene is described. This method should provide a new opportunity for the analysis of gene expression in diverse neuronal phenotypes.

Stationary organotypic cultures of oxytocin and vasopressin magnocellular neurones from rat and mouse hypothalamus.

Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long-term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427 +/- 63 OT cells and 217 +/- 27 VP cells per cultured rat hypothalamus, and 380 +/- 72 OT cells and 622 +/- 91 VP cells per cultured mouse hypothalamus. Following a 7-day adaptation period in standard culture medium containing serum, the rat slice-explants survived very well after subsequent transfer to defined, serum- free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612 +/- 147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c-fos gene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c-fos expression in 87-91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long-term physiological and gene expression studies to be done under defined media conditions.

Molecular diversity in neurosecretion: reflections on the hypothalamo-neurohypophysial system.

1. The diversity of molecules involved in various aspects of neurosecretion, such as proprotein processing, axonal transport of large dense core vesicles (LDCVs), and regulated secretion, is discussed in the context of the hypothalamo-neurohypophysial system (HNS). 2. Recent studies have uncovered a family of at least seven processing enzymes known as proprotein convertases (PCs) which are involved in proteolytically cleaving protein precursors at paired basic amino acid motifs to yield biologically active peptides. Three of these, PC1(3), 2, and 5, are found in neurons and are involved in producing regulated secretory peptide products. 3. The axonal transport of LDCVs occurs on microtubule tracks by still unknown mechanisms. There are over 11 distinct kinesin-related molecules that have now been identified as possible microtubule motor candidates. 4. Calcium channels in the nervous system are known to be derived from at least five alpha-subunit and four beta-subunit genes with multiple alternatively spliced isoforms in each case. These could account, in part, for the varied calcium currents found in the HNS. 5. The large number of proteins and isoforms now demonstrated to be involved in regulated secretion are discussed, with a focus on LDCV compositions and the synaptotagmin gene family.