Vasopressin (VP) is an antidiuretic hormone that is synthesized and released by osmosensitive magnocellular neurosecretory cells (MNCs) to regulate water homeostasis in the body. The rate and firing pattern of MNCs determines the amount of VP release, which is elevated during physiological stress particularly dehydration. During acute osmotic changes the MNCs shrink and swell due to hypertonic and hypotonic stimuli, respectively. In contrast to hippocampal neurons, which display regulatory volume increases (RVI) and regulatory volume decreases (RVD) in response to hypertonic and hypotonic stimuli, MNCs do not have compensatory mechanisms. The MNCs undergo hypertrophy as a part of their physiological structural and functional plasticity during chronic dehydration. These changes are thought to be important during long term osmotic changes for the sustained and high level releases of hormone. However, the mechanism of hypertrophy is still unclear and it is difficult to address this issue in vivo. We therefore undertook studies on acutely isolated MNCs to test hypertrophy in MNCs. We observed that acutely isolated MNCs treated with hyperosmolar solution (325 mOsmol kg-1) for 150 minutes in vitro showed hypertrophy (a 9% increase in CSA) and recovered their original size when returned to isotonic solution (295 mOsmol kg-1) for another 60 minutes. Whole cell patch clamp experiments showed a 34% increase in cell membrane capacitance following treatment with hypertonic solution for 90-150 minutes. The osmotically-evoked hypertrophic response was blocked by using a TAT (human immunodeficiency virus transactivator of transcription) peptide (TAT-NSF700) that prevents SNARE-mediated exocytotic fusion by blocking the function of NSF (N-ethylmaleimide-sensitive factor). The hypertrophic response did not appear to be altered by a scrambled version of the peptide, showing that osmotically-evoked hypertrophy depends on SNARE-mediated exocytotic fusion. The VP and OT-MNCs exposed to hyperosmolar solution for two hours showed increased immunofluorescence for L-type Ca²⁺ channels (both Cav1.2 and Cav1.3). Our data suggest that the osmotically-evoked hypertrophy is associated with an increase in the total membrane surface area due to the exocytotic fusion of intracellular granules with the plasma membrane and with increased expression of L-type Ca2+ channels. This study will be helpful in understanding of the adaptation that MNCs undergo during long term dehydration and pathological conditions that lead to increased plasma osmolality.

Visual information is processed by two separate visual pathways. One is the magnocellular visual pathway (M-pathway), which carries high temporal frequency information but low spatial frequency information. The other is the parvocellular visual pathway (P-pathway), which carries low temporal information but high spatial information. Kveraga and colleagues (2007) presented participants with high and low spatial frequency images and found that participants made faster and more accurate categorization responses to the low spatial frequency images. They hypothesized this was due to low spatial frequency “gist” information being rapidly carried by the M-pathway. Using diffuse light and hand posture manipulations, we replicated the advantage for low spatial frequency (LSF) images in both experiments, and also found a larger advantage for LSF information when biasing the M-pathway (using hand posture). We were unable to inhibit the M-pathway using red diffuse light. Thus, it does appear “gist” processing is uniquely carried by the M-pathway.

Visual information is processed by two separate visual pathways. One is the magnocellular visual pathway (M-pathway), which carries high temporal frequency information but low spatial frequency information. The other is the parvocellular visual pathway (P-pathway), which carries low temporal information but high spatial information. Kveraga and colleagues (2007) presented participants with high and low spatial frequency images and found that participants made faster and more accurate categorization responses to the low spatial frequency images. They hypothesized this was due to low spatial frequency “gist” information being rapidly carried by the M-pathway. Using diffuse light and hand posture manipulations, we replicated the advantage for low spatial frequency (LSF) images in both experiments, and also found a larger advantage for LSF information when biasing the M-pathway (using hand posture). We were unable to inhibit the M-pathway using red diffuse light. Thus, it does appear “gist” processing is uniquely carried by the M-pathway.

Vasopressin (VP) is an antidiuretic hormone that is synthesized and released by osmosensitive magnocellular neurosecretory cells (MNCs) to regulate water homeostasis in the body. The rate and firing pattern of MNCs determines the amount of VP release, which is elevated during physiological stress particularly dehydration. During acute osmotic changes the MNCs shrink and swell due to hypertonic and hypotonic stimuli, respectively. In contrast to hippocampal neurons, which display regulatory volume increases (RVI) and regulatory volume decreases (RVD) in response to hypertonic and hypotonic stimuli, MNCs do not have compensatory mechanisms. The MNCs undergo hypertrophy as a part of their physiological structural and functional plasticity during chronic dehydration. These changes are thought to be important during long term osmotic changes for the sustained and high level releases of hormone. However, the mechanism of hypertrophy is still unclear and it is difficult to address this issue in vivo. We therefore undertook studies on acutely isolated MNCs to test hypertrophy in MNCs. We observed that acutely isolated MNCs treated with hyperosmolar solution (325 mOsmol kg-1) for 150 minutes in vitro showed hypertrophy (a 9% increase in CSA) and recovered their original size when returned to isotonic solution (295 mOsmol kg-1) for another 60 minutes. Whole cell patch clamp experiments showed a 34% increase in cell membrane capacitance following treatment with hypertonic solution for 90-150 minutes. The osmotically-evoked hypertrophic response was blocked by using a TAT (human immunodeficiency virus transactivator of transcription) peptide (TAT-NSF700) that prevents SNARE-mediated exocytotic fusion by blocking the function of NSF (N-ethylmaleimide-sensitive factor). The hypertrophic response did not appear to be altered by a scrambled version of the peptide, showing that osmotically-evoked hypertrophy depends on SNARE-mediated exocytotic fusion. The VP and OT-MNCs exposed to hyperosmolar solution for two hours showed increased immunofluorescence for L-type Ca²⁺ channels (both Cav1.2 and Cav1.3). Our data suggest that the osmotically-evoked hypertrophy is associated with an increase in the total membrane surface area due to the exocytotic fusion of intracellular granules with the plasma membrane and with increased expression of L-type Ca2+ channels. This study will be helpful in understanding of the adaptation that MNCs undergo during long term dehydration and pathological conditions that lead to increased plasma osmolality.

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The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

Magnocellular neurones (MCNs) are capable of secreting vasopressin and oxytocin from the somato-dendritic compartment, which can occur independently to secretion from nerve terminals. One hypothesis of the mechanism that regulates this differential release is that dendrites utilise different vesicle pools compared to those found in terminals. Little is known for the function of neuronal dendrites, especially the mechanism for peptide release. One theory is that vesicles stored in dendrites are non-released vesicles ready for recycling or degradation. Immunofluorescent labelling was performed on hypothalamic slices of the transgenic rat where enhanced green fluorescent protein (eGFP) was tagged to vasopressin. Lysosomes were detected by the lysosome-associated membrane protein LAMP1. Correlation analysis of LAMP1 labelling and VP-eGFP had shown that localisation of lysosomes in dendrites is positively correlated to loci of high vasopressin expression. This suggests active degradation of vesicles in dendrites. It is not known whether preferential release of peptides occurs along the profile of dendrites. Experiments were carried out using a temperature block to block exit of vesicles from the Golgi apparatus. Release of the temperature block triggered release of a wave of newly synthesised vesicles from the Golgi apparatus. Measurement of the fluorescent intensity of VP-eGFP showed that preferential release of peptides does not occur along the profile of dendrites. I have also utilised confocal live cell imaging to study the dynamics of dendritic vasopressin release using VP-eGFP slice explants. Experiments using high potassium stimulation showed significant increase in the release of vasopressin after priming with thapsigargin (intracellular calcium mobiliser), in accordance to in vitro release and microdialysis studies. These results demonstrate that live cell imaging can be achieved in magnocellular neurons, providing a robust model system in the study of dendritic peptide release. Large dense core vesicles (LDCVs) in other cell types such as bovine adrenal chromaffin cells were shown to segregate according to vesicle age, suggesting that vesicle age is an important factor in the regulation of peptide release. Whether vesicles of different age groups exist in magnocellular dendrites is not known. Thus, biolistic transfection with exogenous fluorescent proteins for expression under temporal control was carried out. However, low transfection rate in magnocellular neurones and the high background fluorescence caused by scattered gold particles used as bullets for transfection deemed this method inappropriate for the purpose of imaging vesicles. Hence, development of an adenoviral transduction system was employed. By using an inducible adenovirus gene construct coupled with a fluorescent reporter gene, it is possible to visualise vesicle pool segregation under different experimental conditions. Subcloning of a red fluorescent construct tagged to ppANF was tested on PC12 cells to show targeting of fluorescence expression to LDCVs. Successful production of an inducible adenoviral DNA with the red fluorescent construct insert was confirmed by PCR and DNA sequencing. Whilst the generation of viral particles is still to be achieved, successful production of the virus will be an invaluable system for inducible gene expression in neurones.

L'objectif de cette thèse était l'investigation du fonctionnement et des dysfonctionnements des deux systèmes visuels principaux (magnocellulaire et parvocellulaire). Une synthèse théorique sur la dissociation de ces deux systèmes et une revue sur les procédures expérimentales permettant de les dissocier constituaient la première partie de cette thèse. Ensuite, des arguments neuropsychologiques concernant le vieillissement normal et pathologique du système visuel ont été synthétisés et différents résultats de la littérature ont été analysés pour étayer cette dissociation. La quinzaine d’expériences réalisées a mis en évidence deux résultats principaux. Il y a un déficit massif du système parvocellulaire dans le vieillissement normal dès les étapes précoces du traitement et qui perdure aux étapes plus tardives, ainsi qu’un déficit magnocellulaire sous certaines conditions seulement. L'étude des dysfonctionnements visuels dans les pathologies à syndromes extrapyramidaux a montré que ces patients présentaient un déficit spécifiquement magnocellulaire en lien avec la dénervation dopaminergique dans le système central, objectivée à l’aide d’une technique de scintigraphie cérébrale. / The objective was to investigate the normal functioning and the impairment of the two main visual systems, the parvocellular and the magnocellular systems. A theoretical review on the two systems and on the experimental procedures that have been used to dissociate them is proposed in the first chapter. In a second part, a synthesis of the neuropsychological studies focusing on the normal aging of the visual system and on the visual deficits found in various pathologies, that support the parvocellular / magnocellular dissociation is presented. The experimental chapter comprised about fifteen experiments and showed two main results: 1/ a major low-level parvocellular impairment with normal aging that was not compensated at the later stages, and a slighter magnocellular impairment under certain conditions: 2/ a specific deterioration of the magnocellular system in extrapyramidal patients, correlated with dopamine denervation in two structures of the central system.

Magnocellular neurosecretory cells (MNCs) are neuroendocrine cells with somata located in the hypothalamus and nerve terminals in the posterior pituitary. They receive neuronal inputs from the brain and release vasopressin and oxytocin into the blood to regulate many important functions such as water balance, lactation, and parturition. The process of hormone release depends on Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCCs) on the plasma membranes of neuroendocrine cells. To better understand the cellular and molecular components that are involved in regulating secretory vesicle exocytosis, this thesis work was conducted to investigate the expression and function of different subtypes of VGCCs in MNCs and pituicytes (the glial cells surrounding MNC nerve terminals).<p> Molecular biology, immunohistochemistry and cellular biology were used to detect expression and alternative splicing of different VGCC subtypes in MNCs, neurons, and pituicytes. First, the presence of CaV2.2 and CaV2.3 channels were detected on the pituicytes in situ. When the pituicytes were isolated and cultured for 14 days, more VGCC subtypes were expressed including CaV1.2 channels. Regulation of VGCC expression was measured in normal and dehydrated rats, and CaV1.2 channels were found to be selectively up-regulated in pituicytes after 24 hours of dehydration.<p> Second, two splice variants of CaV2.1 channels (CaV2.1Ä1 and Ä2) that lack a large portion of the synprint (synaptic protein interaction) site were detected in the rat brain. To determine whether the splice variants were expressed in MNCs, we did immunocytochemistry using two antibodies (the selective and the inclusive antibody) that recognized the carboxyl-terminus of channels and the synprint site, respectively, in different cell types. We found that vasopressin MNCs, but not the oxytocin MNCs, and one type of endocrine cell (the melanotropes of the pituitary gland) expressed the synprint site deleted variants, whereas the hippocampal neurons mainly expressed the full-length isoform. The splice variants were properly distributed on the plasma membrane of the somata and nerve terminals of the MNCs, suggesting the synprint site is not essential for CaV2.1 channel targeting into the nerve terminals of neuroendocrine cells.<p> Third, expression and distribution of CaV2.2 channels were studied in the MNCs. All CaV2.2 isoforms we detected contained the full-length synprint site. To test the importance of the CASK/Mint1 binding site for CaV2.2 channel targeting, we over-expressed a peptide that inhibits the interaction between CaV2.2 channels and CASK/Mint1 in differentiated PC12 cells (a neuroendocrine cell line). We found that the distribution of CaV2.2 channels in the growth cones of PC12 cells were significantly decreased, suggesting that the CASK/Mint1 interaction is important for CaV2.2 channel targeting into the neuroendocrine terminals.<p> In conclusion, these results provide insights of VGCC expression in neuroendocrine cells, and also give rise to a better understanding of the molecular components that are involved in forming the exocytotic machinery in these cells.