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NEUROENDOCRINOLOGY |
Laboratory of Neurochemistry (B.-J.Z., K.K., H.G.), and Electron Microscope Facility (P.Z.), National Institute of Neurological Diseases and Stroke, and Section on Neural Gene Expression (A.I., W.S.Y.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Harold Gainer, Ph.D., Laboratory of Neurochemistry, National Institutes of Neurological Diseases and Stroke/National Institutes of Health, Building 36, Room 4D04, Bethesda, Maryland 20892. E-mail: . gainerh{at}ninds.nih.gov
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Introduction |
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Targeted gene expression in the oxytocin (OT) and vasopressin (VP) magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS) has also been achieved (reviewed in Refs. 14, 15, 16). However, the minimal cell-specific enhancer elements in these genes that are responsible for their cell-specific expression are still unclear, and all the successful constructs thus far have contained all the introns and exons of the native genes. Transgenic constructs have been made containing reporters at various exonic sites, and the consensus is that placement of the reporter gene after exon III is the most efficacious site for gene expression (17, 18, 19). Consequently, Young et al. (19) placed an enhanced GFP (EGFP) reporter gene after exon III in the oxytocin gene, as an in-frame fusion with the carboxyl terminus of the OT pre-prohormone. This construct was similar to an OT-VP minilocus configuration (called V1) that had previously been successful in producing robust expression of a rat transgene (but with no reporter) specifically in OT-MCNs (20). The resulting OT-EGFP transgenic mouse line, designated AI-03, showed cell-specific expression of the EGFP reporter only in OT-MCNs (19).
In the absence of a reporter in exon III, the transgenic mice containing the V1 transgene showed, in addition to its cell-specific expression, very efficient targeting of the rat gene to the secretory granules in the OT-MCNs (21). One study on the intracellular trafficking of a vasopressin pre-prohormone construct that had a full length chloramphenicol acetyl transferase (CAT) reporter attached to its C terminus showed that the reporter protein was targeted to secretory granules in vivo (22). This suggested that the similar length EGFP in the OT pre-prohormone of the AI-03 mouse being studied here would exhibit a similar trafficking pattern. Indeed, the initial report that the EGFP in the AI-03 transgenic mouse was detectable in OT containing regions of the posterior pituitary (19) is consistent with this expectation. In this paper, we have examined the specificity and efficiency of the expression of the EGFP protein in OT-MCNs in the AI-03 mouse and have especially focused on its intracellular localization. We find that it is trafficked to secretory granules in OT neurons and transported in these vehicles to OT nerve terminals in the posterior pituitary, where the calcium-dependent secretion of the EGFP can be demonstrated in neurosecretomes by fluorescence imaging techniques.
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Materials and Methods |
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). The transgenic mice used in these experiments were primarily heterozygous, and similar data were obtained for the three homozygous mice that were used. All procedures were carried out in accordance with the NIH guidelines on the Care and Use of Animals and animal study protocols approved by the NINDS and NIMH Animal Care and Use Committees.
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Neurosecretosomes that were attached to poly-L-lysine-coated glass coverslips were fixed with 4% paraformaldehyde in PBS for 1–2 h. After several washes in PBS, the preparations were incubated in PBS containing 0.1% Triton X-100 and 10% normal goat serum overnight. Double immunofluorescence staining was carried out sequentially employing a rabbit polyclonal antibody, THR, or monoclonal antibody PS41 against VP-neurophysin (VP-NP), and the monoclonal antibody, PS38, against OT-neurophysin (OT-NP). The EGFP fluorescence in the neurosecretosomes was sufficiently intense so that immunocytochemical staining for the EGFP was not necessary.
Fluorescence microscopic observations and photographs were made using either a Nikon 5' eclipse E-400 microscope with a SONY Digital photo camera DKC-5000 system or a Carl Zeiss LM 410 confocal microscope (Carl Zeiss, Inc., Thornwood, NY).
Electron microscopic immunocytochemistry
After dissecting the neural lobe tissue free from the anterior lobe of the pituitary, it was immersion fixed in 1.0% glutaraldehyde/formaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4 C. The tissue was processed according to the protocol of Phend et al. (26) with the following modifications: the filipin and digitonin step was removed, no propylene oxide was used in the dehydration, all steps up to the ethanol were done at 4 C, and the tissue was embedded in Epon. The blocs were polymerized at 60 C for 48 h.
Before immunocytochemistry, a bloc was placed into an unmasking solution of 0.1 M sodium citrate (pH 5.8) solution that was microwaved in a Pelco 3450 oven purchased from Ted Pella Inc. (Redding, CA) at full power for three cycles of 5 min each with a 1-min break between each cycle (27). The bloc and the sodium citrate were allowed to equilibrate to room temperature, at which time the bloc was removed and allowed to air dry. Thin sections were made using a Reichert Ultracut microtome (Leica, Solms, Germany) and placed on single slot nickel grids. All remaining steps were done at room temperature. Sections, on the grids, were etched for 30 min with saturated sodium meta-periodate, then floated on a drop of blocking solution, 0.05 M TBS (pH 7.5) with 0.05% Triton X-100, 1% goat serum, and 1% BSA for 1 h. Double-labeling was performed by mixing the two antibodies, the PS38 Mab against oxytocin-neurophysin diluted 1:15 and the EGFP antibody (CLONTECH Laboratories, Inc.) diluted 1:10 in the blocking solution. The grid was placed onto a small drop of the diluted antibodies in a moist chamber for 3 h. The grid was then passed through three drops of blocking solution for 1 min each and then onto the two colloidal golds mixed together; goat antimouse IgG (10 nm gold) diluted 1:15, and goat antirabbit IgG (20 nm gold) diluted 1:20, in the blocking solution for one hour. Both of the colloidal gold IgGs were purchased from Ted Pella, Inc. Controls were performed by omitting the primary antibodies. Grids were dipped twice into distilled water, air dried, and counterstained with uranyl acetate and lead citrate. The grids were viewed and micrographs recorded in the JEOL 100CX.
Preparation of neurosecretosomes for fluorescence imaging
The neural lobe was dissected out from a single animal, placed in 60 µl of an isotonic sucrose solution (270 mM sucrose; 20 mM HEPES; 100 µM EGTA, pH 7.4 (28, 29), and homogenized in a small glass-homogenizer at room temperature. The suspension (
60 µl) was spread on to two 16-mm diameter glass coverslips precoated with poly-L-lysine. After 15-min incubation, the neurosecretosomes were attached to the coverglass surface and the suspension medium was replaced by control saline (in mM: 140 NaC1; 5 KC1; 2 CaC12; 1 MgC12; 10 glucose; 5 HEPES, pH 7.4, which contained 0.15% BSA) and incubated for 2–3 h at 37 C before use in physiological experiments.
Digital fluorescence microscopy and Fura-2 measurements
A glass coverslip containing attached neurosecretosomes was mounted in a bath (700 µl volume) on the stage of upright fluorescence microscope (Carl Zeiss Axioscope, 63x water immersion objective lens) and perfused with control saline at 3 ml/min keeping the bath temperature between 33 and 36 C. Several fluorescent, relatively large secretosomes (>3 µm in diameter) could be found within the illumination field and these were selected for recording EGFP intensity changes. The secretosomes were excited at 488 nm (0.1-sec illumination period at 5-sec cycles) and the emitted light was collected through the 510 nm filter by an intensified charge-coupled device camera (Attofluor Ratiovision, Atto Instruments, Rockville, MD). During recording, the perfusion solution was typically switched from Ca2+-containing normal saline to Ca2+-free medium with secretagogues (cf., 4 ml of 50 and 90 mM K-salines). Each period of the perfusion lasted 100 sec. After recording, data obtained from individual secretosomes was normalized (
F/F), where the F is the change in fluorescence intensity during tests and the F is the average fluorescence intensity before the test. To observe changes in intracellular calcium concentration ([Ca2+]i) of the same secretosomes during the recording of EGFP intensity change, the neurosecretosomes were pretreated with 2 µM Fura-2 acetoxymethylester (Molecular Probes, Inc.) for 15 min at 37 C followed by washing for 20 min with Fura-2-free control saline. These secretosomes were excited alternately at three wavelengths (340 nm and 380 nm for Fura-2, and 488 nm for EGFP; with 0.12-sec switching intervals) using 10 nm band-pass interference filters (340HT15, 380HT15, and DF 490HBW; Omega Optics, Brattleboro, VT). Fluorescence emissions were passed through a dichroic mirror, which had two coatings to reflect both EGFP and Fura-2 signals (Chroma 505DCXRU) and a long pass emission filter that had a sharp cut-off below 500 nm. Thus, EGFP and Fura-2 fluorescence intensity changes (average gray level of pixels within the region of interest) were recorded alternately. To reduce illumination intensities of 380 and 488 nm, neutral density filters (OD = 0.2 and 0.5, respectively) were placed in front of these excitation filters. The ratios (340 vs. 380 signals) were converted by employing two point calibration method to obtain free [Ca2+] from the equation [Ca2+]=Kd[R-R(Lo)/R(H)-R] x Den(Lo)/Den(H) in which Kd is the dissociation constant for the dye and taken to be 224 nM (30, 31). The R is the measured ratio. The R(H) and R(Lo) are ratio values for high concentration and low concentration standards, respectively. The Den(H) and Den(Lo) are respectively denominator wavelength intensity for high and low concentration standards.
Drugs and solutions
Compositions of the homogenization solution for preparing the neurosecretosomes and the perfusion salines are described above. High K-salines (50 and 90 mM) were prepared by replacing equimillimolar NaC1. Ca2+-free saline was prepared by replacing 2 mM CaC12 in the control saline with equimolar MgC12 and adding 0.5 mM EGTA. D-Ala2-dynorphin A was purchased from Peninsula Laboratories, Inc. (San Carlos, CA). Osmolarity of all salines was kept between 302 and 315 milliosmoles.
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), as determined by in situ hybridization histochemistry (19). In this study, we have focused on the extent of expression and localization of the fluorescent protein (Fig. 1B) that was expressed in this transgenic mouse line.
Figure 2 shows a typical pattern of expression of the EGFP in the AI-03 mouse hypothalamus. The EGFP protein was found exclusively in magnocellular oxytocin neurons in the PVN (Fig. 2A) and SON (Fig. 2B) of the hypothalamus, confirming the previous report about the absence of ectopic expression of this transgene in the AI03 transgenic line (19). Further examination of the specificity of this expression was made by double-label immunocytochemistry and by comparing the EGFP-immunoreactivity (ir) with that of the immunoreactivity of the specific neurophysins (i.e. OT- or VP-associated neurophysin) expressed by the MCNs (Fig. 3). By using highly specific monoclonal antibodies directed against the OT-NP and VP-NP in immunohistochemistry (23, 24), it became apparent that many MCNs in the PVN and SON that expressed the EGFP-ir also expressed OT-NP-ir (Figs. 3, A and B). In contrast, few VP-NP-ir MCNs were found to clearly express EGFP-ir (a rare example is shown at the arrow in Fig. 3C). Overall, there is a selective EGFP expression of the transgene in oxytocin as opposed to vasopressin MCNs, and this is supported by the quantitative analysis of the colocalizations of both peptides and the EGFP-ir shown in Table 1A. Approximately 41% of the 35 OT neurons sampled in an average section contained EGFP-ir, whereas only 2% of the VP neuronal population contained EGFP-ir (Table 1A). The latter finding of a 2% incidence of expression of the OT-transgene (i.e. the EGFP-ir) in a VP-MCN is consistent with recent findings that between 1 and 3% of MCNs normally contain comparable levels of both OT and VP mRNA and peptide immunoreactivity (32, 33, 34). From these data, one can conclude that the OT-transgene (Fig. 1A) was being expressed selectively in OT-MCNs.
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shows that the degree of expression of the transgene in the MCNs (as determined from EGFP-ir) ranged from robust to barely detectable. When the MCNs were examined for intrinsic EGFP fluorescence very few OT-MCNs could be visualized without the aid of immunohistochemistry.
EGFP accumulates in OT nerve terminals in the neural lobe of the pituitary and is present in secretory granules
The expected translated product of the AI-03 transgene contained the entire OT pre-prohormone fused at the C-terminal region of its neurophysins to EGFP (Fig. 1B). If this EGFP would not interfere with the OT-precursor’s self-association, which is believed to be involved in its packaging into secretory granules (35, 36), then the green fluorescence should be trafficked to the regulated secretory pathway and transported to nerve terminals in the posterior pituitary. Figure 4 shows cross-sections through the neural lobe, and in contrast to the cell bodies of the MCNs, sufficient EGFP accumulated in the neural lobes so that the intrinsic EGFP fluorescence was relatively robust. The fluorescence in the neural lobe did not require immunohistochemistry to be visualized (Fig. 4A), and was located only in the neural lobe (compare Fig. 4, A and B). Nevertheless, immunohistochemistry, using an antibody to EGFP and a green fluorescent second antibody did greatly enhance the fluorescent signal observed in the neural lobe (Fig. 4C), and showed the same distribution pattern of fluorescence as observed for the intrinsic EGFP fluorescence. The EGFP was primarily located in the periphery of the neural lobe (Figs. 4, A and C), in a pattern similar to the endogenous OT found in the rat neural lobe (37, 38). When double-label immunocytochemistry was performed using a specific OT-NP antibody (in red fluorescence, Fig. 4C), the merged view illustrated in Fig. 4D shows that the principal EGFP-ir and the predominant OT-NP-ir in the neural lobe clearly overlap. This accumulation of high concentrations of EGFP in the neural lobe in regions corresponding to OT nerve fibers and endings indicates that the EGFP synthesized in the MCN was being transported to the pituitary in a manner similar to the endogenous OT for storage and secretion.
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illustrates that when the double-label immunocytochemistry is performed at the electron microscopic level, nerve terminals in the neural lobe contain EGFP (20 nm gold, see arrows in Fig. 5A) in the same secretory granules that also contain OT-NP-ir (seen as small, 10 nm gold). Also shown is a non-OT nerve terminal (presumably containing VP) that contains neither label (arrowhead in Fig. 5A), and an OT-terminal but with no EGFP (asterisk in Fig. 5B). These data indicate that the EGFP is packaged into OT-NP containing secretory granules for axonal transport to the pituitary in these subcellular organelles. Because the secretory granules are also the vehicles for regulated, (calcium-dependent) secretion of OT and OT-NP from nerve terminals in the pituitary, we then examined whether the EGFP fluorescence could be secreted in a calcium-dependent manner.
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) and VP-NP-ir (Fig. 6B) with their intrinsic EGFP fluorescence show that colocalization (yellow) occurs virtually only in OT-NP containing neurosecretosomes (Fig. 6A), and only rarely in the VP-NP containing ones (Fig. 6B). However, as was observed with the cell-bodies of the MCNs (Fig. 3), many OT-NP-containing neurosecretosomes did not contain EGFP. Several smaller sized neurosecretosomes in Fig. 6A appear green because in these terminals the OT-NP-ir was present but weak (not shown) and hence, the merged view is biased in favor of the intrinsic EGFP signal. In the quantitative analysis of colocalization in neurosecretosomes that is shown in Table 1B, the green and red fluorescence signals were recorded independently (to increase the sensitivity of visualization) and compared. These measurements showed that 29% of the OT-NP-ir neurosecretosomes exhibited EGFP fluorescence, whereas only 0.5% of the NP-VP-ir neurosecretosomes contained EGFP fluorescence. These data are consistent with the expression specificities and efficiencies found in the MCNs (Table 1A), indicating a selective but only partial expression of the transgene in the OT-MCNs. The above data were obtained using heterozygous transgenic mice, but similar observations and measurements were made using neurosecretosomes derived from three homozygous (2 females, 1 male) transgenic mice (data not shown).
The intrinsic EGFP fluorescence found in the neurosecretosomes is secreted in response to potassium depolarization in a calcium-dependent manner (Fig. 7). The routine stimulation protocol used (in top abscissa of Fig. 7) involved exposure to high K+ (50 mM or 90 mM) in normal Ca2+- containing saline, followed by perfusion and stimulation in Ca2+-free saline; and after recovery from Ca2+-free solution, by perfusion with normal Ca2+-containing saline; after which the potassium depolarization was repeated in calcium containing medium. The resulting EGFP intensity changes are shown as the average F/F and the SEM of five separate measurements. In these experiments, we found that the percentage reduction of the fluorescence intensity (F/F) found in response to application of 90 mM K-saline and 50 mM K-saline were 9.3 ± 1.3% (n = 23), and 5.7 ± 1.3% (n = 18), respectively. In some preparations, intrasecretosomal [Ca2+] changes were also monitored by Fura-2 signals (see bottom of Fig. 7). The basal [Ca2+] of secretosomes before stimulation was 63 ± 4.3 nM (n = 19). Application of either 50 or 90 mM K+-saline for 100 sec in normal [Ca2+]o-saline raised intrasecretosomal [Ca2+] to 828.8 ± 211.0 nM (n = 11). These values are similar to the basal and stimulated [Ca2+] values observed in rat neurosecretosomes (39, 40, 41, 42). In [Ca2+]o-free saline, neither 50 mM nor 90 mM K+-saline increased intrasecretosome [Ca2+] or decreased the EGFP fluorescence (Fig. 7).
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41%) of the OT MCNs (Table 1A). In contrast, the original V1 construct, which did not contain foreign coding sequence as a reporter, was expressed in over 90% of the OT MCNs in transgenic mice (20). Is the reduced expression that was observed with the EGFP constructs due to the influence of the EGFP reporter? In this regard, it is interesting that a similar cell-specific gene expression pattern with a partial penetrance was also observed when a similar OT transgene construct but with a different reporter, CAT was used to produce transgenic mice (22). Therefore, it is unlikely that the low efficiency of transgene expression observed in the OT MCNs in these mice is due to the presence of the particular reporter (CAT or EGFP) used. Furthermore, because this low penetrance was found in multiple founder lines for the EGFP (four lines) and CAT (two lines) constructs, this suggests that integration site factors were not involved. The principal issue being addressed in this paper is whether the EGFP in the AI-03 transgene would be targeted to the secretory granules in the OT-MCNs, and whether the regulated secretion of the fluorescent proteins in individual neurohypophysial terminals could be visualized by imaging techniques. OT is a nine-amino acid neuropeptide hormone that is synthesized as part of a larger precursor protein (the OT pre-prohormone) in MCNs in the hypothalamus. The OT pre-prohormone contains the N-terminal signal sequence followed by the OT peptide, and an approximately 10-kDa protein called neurophysin at its C terminus. The OT precursor protein is packaged into secretory granules (i.e. large dense core vesicles, LDCVs), in which the prohormone is processed and transported to the posterior pituitary for secretion of the peptide products (reviewed in Refs. 15 , 43, 44, 45). The neurophysin is in a noncovalent association with the neurohypophysial peptides in the secretory granules (35, 46, 47). Based on the biophysical properties of the nonapeptide-neurophysin complex (35) and its crystal structure (48), it was proposed that the self-association properties of this peptide-neurophysin complex as well as the precursor protein might play a critical role in the sorting of the VP precursor to the regulated secretory pathway and the stabilization of the high concentrations of peptides in the LDCVs against disulfide exchange and degradation (35, 49, 50). Several studies supporting the hypothesis that the interaction between the peptide and neurophysin in the protein precursor is necessary for its efficient sorting and packaging to the regulated secretory pathway have been reported (reviewed in Ref. 36). In this report, we tracked the intracellular locations and secretion of the EGFP-tagged, OT-precursor protein to secretory granules in the OT MCNs cells in transgenic mice directly by electron microscopic immunocytochemistry and studied its secretion from individual living cell processes by digital fluorescence imaging techniques.
Very few studies have examined the ultrastructural localization and trafficking of OT- or VP-transgene peptide products. The first was the rat "minilocus" transgene that expressed the wild-type rat OT gene in transgenic mice (20). The rat OT-NP could be discriminated from mouse OT-NP immunocytochemically by using a monoclonal antibody, PS67, which was specific for the rat OT-NP. Both the transgenic rat and endogenous mouse OT-NPs were found to be colocalized in secretory granules in cell bodies and nerve terminals in only the OT MCNs (21). In another study, a rat VP transgene containing a short epitope tag at the end of exon 3 of the VP gene (18) was found to be trafficked to secretory granules in the posterior pituitaries of transgenic rats (17). In the studies by Jeong et al. (22), a full-length CAT reporter was fused to the C termini of either a VP-pre-pro-hormone or OT pre-pro-hormone transgene, and in both cases the reporter was selectively trafficked to secretory granules in the MCNs. In the AI-03 construct being studied here the EGFP moiety would be attached to the end of the OT-NP in the OT prohormone (see Fig. 1B). Because the peptide (OT or VP) and neurophysin interaction in the prohormone is believed to be involved in the sorting process to the regulated secretory pathway (35, 36, 51), it is interesting that the long EGFP adduct attached to the OT-NP did not prevent trafficking of the OT-prohormone-EGFP fusion protein to the secretory granules (Fig. 5). In fact, it is remarkable that the OT prohormone EGFP fusion protein used in this study was effectively sorted to the secretory granules in the MCNs, even though the EGFP reporter was more than two times larger in molecular weight than the entire OT prohormone to which it was fused.
Because in this OT-EGFP transgenic mouse the fluorescent reporter is targeted to secretory granules in the OT magnocellular neurons that are subsequently transported to nerve endings in the neural lobe (Figs. 4 and 5), this provided a unique opportunity to visualize neurosecretion from individual OT nerve terminals. Previous measurements of OT and VP peptide secretion in the HNS, from nerve terminals in the neural lobe (28, 29, 39, 40, 52, 53) or from dendrites in the hypothalamus (54), have been made by RIA of samples, collected either by superfusion in vitro from neural lobe or by microdialysis in vivo in hypothalamus. While the RIA method is extremely sensitive and has provided much valuable information about OT and VP secretion, the topographic (dendrite vs. nerve terminal) and kinetic aspects of the secretion of these peptides cannot be studied by these procedures. In the present study, we have been able to use the AI-03 transgenic mouse and imaging methods by which the secretion process can be directly visualized (55, 56) to demonstrate calcium-dependent secretion from individual OT nerve endings (neurosecretosomes) isolated from their neural lobes (Fig. 7).
Using this paradigm, it should also be relatively easy to perform various pharmacological studies of neurosecretion from individual identified peptidergic nerve terminals that have previously only been done using whole neural lobes or mixed neurosecretosomes and RIA assays (28, 29, 40, 41). For example, 
opioid receptor agonists, such as dynorphin, are known to inhibit depolarization-evoked secretions of OT from rat neural lobes and neurosecretosomes (42, 57, 58, 59, 60). In preliminary experiments, we have confirmed that the treatment of neurosecretosomes (derived from AI-03 transgenic mice) with 2.5 x 10-6 M dynorphin A inhibited the 50 mM K+-induced secretion of EGFP (i.e. the fluorescence intensity decrease) in normal Ca2+-containing saline from individual OT nerve terminals (n = 3). This dynorphin effect was reversible after washing out the dynorphin and replacing it with normal saline (data not shown). Given the availability of AI-03 transgenic mice that contain fluorescent secretory granules, and the various dissociated and organotypic tissue cultured neuron models that can be derived from them (25, 61), it should then be possible, in the future, to compare the pharmacologies of secretion from the dendrites and nerve terminals of intact OT-MCNs in vitro.
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Footnotes |
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Abbreviations: CAT, Chloramphenicol acetyl transferase; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; HNS, hypothalamo-neurohypophysial system; ir, immunoreactivity; LDCVs, large dense core vesicles; MCNs, magnocellular neurons; NP, neurophysin; OT, oxytocin; VP, vasopressin.
Received August 31, 2001.
Accepted for publication November 19, 2001.
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-opioid receptors inhibits depolarization-evoked exocytosis but not the rise in intracellular Ca2+ in secretory nerve terminals of the neurohypophysis. Brain Res 574:138–146[CrossRef][Medline]
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