An important issue that we continue to address in the laboratory is how to quantitatively measure gene expression in the CNS. Previous studies of gene expression in the HNS have been performed by in situ hybridization histochemistry (ISHH) using exon-specific probes, and measured the steady-state levels of mRNA, which is determined by both gene transcription and mRNA degradation processes. In contrast, measurements using intron-specific probes measure pre-mRNA or heteronuclear RNA (hnRNA) levels in the neuron which, because of the rapid turnover of the primary transcript and intermediate forms of RNA in the cell nucleus, is believed to primarily reflect the transcription rate of the gene. Since an effective intronic probe for VP hnRNA had previously been developed, we developed an effective intron-specific OT probe de novo, and then used both these intronic probes, together with other well established exonic OT and VP probes to reevaluate OT and VP gene expression in the hypothalamus under three classical physiological conditions, acute and chronic osmotic stimulation and lactation. We found that while there was the expected large increase in VP hnRNa after acute salt loading, there was no change in OT hnRNA, indicating that acute hyperosmotic stimuli produce increased VP but not OT gene transcription which was surprising. Since both neuropeptides are robustly and equivalently secreted from the neurohypophysis following acute salt-loading it had always been assumed that the gene expression responses of the OT and VP MCN phenotypes would be equivalent. It has always been believed that OT and VP gene expression and secretion are closely coupled. We then extended these observations over a wide range of times and found that VP hnRNA levels in the SON increased to near maximal levels after 2 hours following the NaCl injection (p<0.05), and reached maximal levels by 24 hours (p<0.001), which was sustained thereafter. The VP gene transcriptional activity in the SON is as rapid and sensitive to increases of plasma osmolality as is VP secretion. In contrast, the salt-loading stimulus did not produce statistically significant changes in OT hnRNA levels after 2, 24 or 48 hours but did increase the OT hnRNA to a maximal level by 72 hours (p<0.05), which was maintained for 120 and 168 hours (p<0.001). Since both OT and VP are secreted equivalently under these conditions, one possible interpretation is that OT gene expression is not closely correlated with secretion as is VP gene expression. If this is the case, this would further suggest that there are significant differences in excitation-transcription coupling mechanisms that regulate the OT and VP genes in the rat magnocellular neurons in the SON. ? ? Given the above findings that the OT- and VP MCNs gene expression responses to acute osmotic stimuli appear to be dramatically different, we sought to determine whether direct application of the presumed neurotransmitter signals for secretion on to the SONs would also differentiate between the OT and VP MCNs. For this purpose, we used a novel experimental paradigm in which ALZET mini osmotic pumps attached to pre-positioned cannulae position over each SON in male rats were used to infuse control (PBS only) solutions over the left SON and an excitatory cocktail consisting of of a mixture of NMDA, AMPA and bicuculline (NAB) were infused over the right SON. In this way, each animals left SON serves as a control for the experimental right SON, and the hnRNA measurements on the experimental side are expressed as a percentage of the values measured on the control side. We found that there was an increase of VP hnRNA in the NAB-treated SON (right SON) as compared to the PBS-control infused SON, indicating that the NAB was a very effective stimulus to increase VP hnRNA expression in the stimulated SON, but there was no change in OT hnRNA in the NAB-treated SONs as compared to the PBS-control SON. Thus, these data are consistent with the view that the VP hnRNA is regulated by excitatory amino acid input, but that under the same conditions the OT hnRNA is not. In summary, we find that direct excitatory neurotransmitter and acute systemic osmotic stimuli both activate VP gene transcription in the SON, whereas neither stimulus effects OT gene transcription. While NAB activates both MCN phenotypes as measured by an increase in c-fos expression and increased VP gene expression as well, it clearly is an inadequate stimulus for increasing OT gene expression. In addition, the ALZET mini osmotic pumps experimental paradigm used as described above, especially when combined with Laser Capture Microdissection and Microarrays, is a novel approach we will continue to use in our future studies of the regulation of gene expression in the CNS in vivo.? ? With regard to signal-transduction issues we previously reported that the SCN in organotypic culture exhibits a robust circadian rhythm in VP gene transcription and that the daytime peak of VP transcription is completely inhibited to reduced night-time levels by a 2 hour exposure to tetrodotoxin (TTX) in the culture medium. We found that VIP activating a VPAC2-receptor maintains VP gene transcription at peak levels, and in addition, that potassium depolarization was as effective as a stimulus of VP gene transcription in the SCN in TTX as was Forskolin. This suggested that there might also be a depolarization-evoked, presumably calcium ion-dependent pathway that could increase VP gene transcription in the SCN), i.e., a VIP-cAMP-MAPkinase pathway and a depolarization-calcium ion influx (L-channel)-CaCAM kinase pathway, possibly acting co-operatively to phosphorylate CREB, and possible activate other transcription factors. We are currently applying a similar strategy to study the signal transduction of OT and VP in the SON but in vivo, by using stereotaxic injections into and AZET miniosmotic pump infusions into the SON to affect the MNCs as described above.? ? Another major effort in our laboratory has been to establish a viral vector gene transfer methodology in our laboratory based on adeno-associated viral & lentiviral vectors that is being used to transduce MCNs in vivo and in vitro with various wild-type and mutant vectors driven by cell-specific promoters. In general we favor using the lentiviral vector initially because of its larger insert size capacity (8kb), versus the AAV which is significantly smaller (<4.3kb). Both viral vectors have been successfully used as vehicles for gene transfer in the CNS in vivo, and we have found that AAV is more efficient in organotypic culture. We are using the Lentilox 3.7 (pLL3.7) self-inactivating (third generation) lentivirus vector with a highly modified, partial HIV type 1 genome containing a CMV promoter driving an EGFP reporter, and a U6 promoter upstream of cloning sites for shRNA expression. We also have replaced the U6 promoter in this plasmid by a CMV promoter in order to express proteins from this site. In addition, we are presently modifying the LV-cre vector for this purpose. Thus far, we have succeeded in using standard protocols for the production, packaging, purification, and titering of both lentiviral and AAV-2 vectors. In the past year, we have been successful in doing stereotaxic injections of various viral vectors into the SON in vivo. Experiments currently in progress are to test our positive control constructs for OT and VP cell specific gene expression, i.e., OT III.EGFP.IGR 3.6 (insert length 5.9kb) and VPIII.EGFP.IGR 2.1 (insert length 8.3kb), in the lentivirus vector, and to do stereotaxic injections over the SON and PVN in vivo.
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