Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons an
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内分泌学杂志 2005年第1期
Department of Physiology (Y.U., H.F., R.S.), School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan; U-583 Institut National de la Santé et de la Recherche Médicale (G.D.), L’Institut des Neurosciences de Montpellier, Hopital Saint Eloi, Montpellier F-34091, France; Department of Anatomy and Neurobiology (H.O., K.-i.M., M.K.), Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; Department of Biological Information Processing (J.Y., A.F.), Graduate School of Electronic Science and Technology, Shizuoka University, Hamamatsu 432-8011, Japan; Department of Physiology (S.U., A.F.), Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan; and Molecular Neuroendocrinology Research Group (D.M.), The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Bristol BS1 3NY, United Kingdom
Address all correspondence and requests for reprints to: Yoichi Ueta, M.D., Ph.D., Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yoichi@med.uoeh-u.ac.jp.
Abstract
We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.
Introduction
THE HYPOTHALAMO-NEUROHYPOPHYSEAL SYSTEM (HNS) consists of large peptidergic magnocellular neurons (MCNs; 20–40 μm cell body diameter) of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, the axons of which course through the internal zone of the median eminence (ME) and terminate on blood capillaries of the posterior (neural) lobe of the pituitary gland (1). These neurons synthesize the neuropeptide hormone arginine vasopressin (AVP) in abundance. The SON is a homogeneous collection of MCNs, whereas the PVN is divided into a lateral and more medial subdivision of magnocellular and parvocellular neurons, respectively. Parvocellular neurons have smaller cells bodies (10–15 μm) whose axons project to the external zone of the ME (2). The HNS produces other neuropeptides in addition to AVP, for example, the closely related hormone oxytocin (OXT). Single-cell RT-PCR enables vasopressin and OXT transcripts to be detected together in the same MCN (3, 4), but expression levels of each neuropeptide RNA differ by orders of magnitude, and it is not clear whether the minority species is translated. Only 3–5% of MCNs express high, equivalent levels of both peptides (5, 6), although this proportion increases after dehydration (7). AVP is also expressed in the hypothalamic suprachiasmatic nucleus (SCN) (8).
AVP is synthesized as part of a prepropeptide precursor in the cell bodies of hypothalamic neurons (9). After signal peptide removal and disulfide bond formation in the endoplasmic reticulum, the propeptide is sorted (10) and packaged into dense core granules of the regulated secretory pathway in the trans-Golgi network. Subsequent processing events proceed as the granules are transported down axons from the cell body to nerve terminals (9). Here, the mature peptide is stored until mobilized for secretion by electrical activity evoked by physiological cues (11).
The functions of hypothalamic AVP depend upon its anatomical origin. Thus, the hormone made in the magnocellular cell bodies of the SON and PVN is transported to the posterior pituitary from where it is released into the general circulation, acting upon not only the kidney to conserve water in its classical role as antidiuretic hormone but also arterioles to cause vasoconstriction. The AVP made in the parvocellular PVN is known to be involved in stress (12, 13), whereas the SCN-derived material appears to control circadian rhythmicity (14).
Electrophysiological studies have characterized the electrical properties of MCNs in the SON and PVN using both in vivo and in vitro brain preparations (15). Although it has been established that the firing of AVP-secreting MCNs in vivo is inhibited by activation of baroreceptor stimulation (16), AVP-secreting MCNs in the SON and PVN can be identified only by the characteristic phasic firing pattern that shows alternating periods of activity and silence in an in vitro slice preparation. In the isolated SON neurons, a cell-specific increase in intracellular concentration of Ca2+ ([Ca2+]i) induced by either AVP or OXT was also demonstrated (17, 18). The firing of AVP-secreting neurons sometimes shows continuous or irregular patterns. Although it is possible to retrospectively identify AVP-secreting neurons marked by injection of intracellular dye, with subsequently immunohistochemistry using a specific antibody against AVP (19, 20, 21), it is difficult to easily identify the living AVP neurons in in vitro preparations except under certain electrophysiological studies. In addition, using acutely dissociated SON neurons to record electrical activity and/or [Ca2+]i, it is possible to identify AVP or OXT neurons only by immunohistochemistry after [Ca2+]i measurement experiments (18, 22). The ability to directly identify living AVP-secreting neurons or AVP nerve terminals of the neurohypophysis with the naked eye using classical fluorescence microscopy would contribute greatly to progress in AVP physiological studies.
In the present study, we introduced a novel AVP-enhanced green fluorescent protein (eGFP) fusion gene into transgenic rats. GFP is made by the bioluminescent jellyfish (Aequorea victoria). GFP has been cloned and modified as eGFP (23). Expression of eGFP in specific neurons under the control of cell-restricted promoters enables their direct visualization and identification. In our rats, transgene expression faithfully recapitulates that of the endogenous AVP gene, being confined to AVP neurons in the SON, PVN, and SCN. Transgenic eGFP appears to enter the regulated secretory pathway, being present in axons of the hypothalamo-neurohypophyseal tract and nerve terminals in the posterior pituitary gland. Furthermore, like the endogenous AVP, expression of the transgene is up-regulated by the physiological challenge of dehydration. Importantly, eGFP expression can be visualized in live AVP cells in brain slices, isolated SON neurons, and their nerve terminals in the neurohypophysis enabling their direct identification for electrophysiological and [Ca2+]i microspectrofluorimetry/[Ca2+]i imaging studies.
Materials and Methods
Animals
Transgenic Wistar rats were bred and housed under normal laboratory conditions (12-h light, 12-h dark cycle) with free access to food and drinking water. For dehydration, water was deprived for 2 d with free access to dry food. For fluorescent microscopic observation and in situ hybridization histochemistry after dehydration, 8-wk-old AVP-eGFP transgenic male rats and transgene (–) male rats as control were used. All experiments were performed in strict accordance with guidelines on the use and care of laboratory animals as set out by the Physiological Society of Japan and approved by the Ethics Committee of Animal Care and Experimentation of our University.
Constructs for microinjection
The AVP-eGFP transgene was constructed using pRVP[S-E/BD-S]Not (24), which consists of the AVP structural gene flanked by approximately 3 kbp of 5' upstream sequences and 2 kbp of flanking downstream sequences. In exon III, a 66-bp SmaI fragment has been replaced with a NotI site. The eGFP coding sequence of a 732-bp fragment obtained from pLP-IRES2-eGFP (Clontech Laboratories, Inc., Tokyo, Japan) were inserted in the NotI site of the exon III of the AVP gene in pRVP[S-E/BD-S]Not. Thus, the AVP-eGFP transgene encodes a modified AVP precursor with eGFP fused in-frame at the C terminus (Fig. 1A).
FIG. 1. A–C, Structure of the AVP-eGFP transgene (A) and representative autoradiographs of brain sections hybridized to a 35S-labeled oligodeoxynucleotide probe for eGFP mRNA in a transgenic rat (B and C). D and E, Enlargements from the boxed areas in B. F, Enlargement from the boxed area in C. White is the most intense signal, and black is the least intense signal. Scale bars, 1 mm (B and C) and 50 μm (D–F). VP, vasopressin; EX, exon; OT, optic tract.
The AVP-eGFP fusion gene was validated by sequencing. The transgene fragment was microinjected into the pronuclei of fertilized oocytes obtained from Wistar rats. The transgenic founders were identified by Southern blot analysis using genomic tail DNA with a 32P-labeled eGFP probe. The founders were bred, and F1 rats were screened by PCR analysis of genomic DNA extracted from rat tail. The PCR was performed using the oligonucleotide primers (sense sequence, 5'-CAC CAT CTT CTT CAA GGA CGA C-3'; antisense sequence, 5'-ATG ATA TAG ACG TTG TGG CTG TTG T-3').
Furthermore, frozen and/or 4% paraformaldehyde-fixed brain sections obtained from eGFP-positive rats (F1) were observed under the fluorescence microscope (Eclipse E600, Nikon, Tokyo, Japan) with a GFP filter (Nikon) for screening whether eGFP was visually expressed in the hypothalamus and pituitary gland. Fluorescent microphotographs were taken by digital camera (COOLPIX 990, Nikon).
In situ hybridization histochemistry for eGFP
Cryostat sections were cut into 12-μm slices and thaw mounted on gelatin/chrome alum-coated slides. The locations of the hypothalamic areas including the SON, the PVN, and the SCN were determined according to coordinates given by the rat brain atlas (25). 35S 3'-end-labeled deoxyoligonucleotide complementary to transcripts encoding eGFP were used (eGFP probe sequence, 5'-CGG CCA TGA TAT AGA CGT TGT GGC TGT TGT AGT TGT ACT CC-3'). The probe was 3'-end labeled using terminal deoxynucleotidyl transferase and [35S]dATP. The in situ hybridization protocol has been described previously in detail (26). Briefly, sections were fixed in 4% (wt/vol) formaldehyde for 5 min and incubated in saline containing 0.25% (vol/vol) acetic anhydride and 0.1 M triethanolamine for 10 min and then dehydrated, delipidated in chloroform, and partially rehydrated. Hybridization was carried out overnight at 37 C in 45 μl hybridization buffer under a Nescofilm (Bando Kagaku, Osaka, Japan) coverslip. A total count of 1 x 106 cpm for eGFP transcripts per slide was used. After hybridization, sections were washed for 1 h in four changes of 1x standard saline citrate (150 nM NaCl and 15 nM sodium citrate) at 55 C and for an additional 1 h in two changes of 1x standard saline citrate at room temperature. Hybridization sections were exposed to autoradiography (Hyperfilm, Amersham, Little Chalfont, UK) for 2 wk for eGFP probe. The resulting images were analyzed by computerized densitometry using a MCID imaging analyzer (Imaging Research Inc., Ontario, Canada). The mean OD of autoradiographs was measured by comparing it with simultaneously exposed 14C-labeled micro-scale samples (Amersham) and presented in arbitrary units setting the mean OD obtained from control rats.
Immunohistochemistry for AVP and OXT
Deeply anesthetized animals were perfused transcardially with 0.1 M phosphate buffer (PB) (pH 7.4) containing heparin (1000 U/liter) followed by 4% (wt/vol) paraformaldehyde. The brains were then removed and divided into three blocks. The blocks that included the hypothalamus were postfixed with the same fixative for 48 h at 4 C. The tissues were then immersed in 20% (wt/vol) sucrose in 0.1 M PB for 48 h at 4 C for cryoprotection. Then, the brains were quickly frozen using powdered dry ice and sectioned at 30 μm on a cryostat (Leica CM3050, Nussloch, Germany). The sections were rinsed twice with 0.1 M PBS containing 0.3% (vol/vol) Triton X-100. The floating sections were incubated with a primary AVP antibody (Chemicon International, Inc., Temecula, CA) (diluted 1:8000) or OXT antibody (Chemicon) (diluted 1:8000) for 3 d at 4 C. After washing for 20 min in 0.1 M PBS solution containing 0.3% (vol/vol) Triton X-100, the sections were incubated for 2 h with Alexa Fluor 546 conjugated antirabbit IgG (Molecular Probes, Eugene, OR) (diluted 1:1000). After final washing with 0.1 M PB, sections were mounted on the glass slides and coverslipped with hydrophilic mounting media (Tris-HCl containing polyvinyl alcohol and glycerol); subsequently, the sections were observed under a confocal laser scanning microscopy (LSM510, Carl Zeiss Co. Ltd., Jena, Germany).
Electrophysiological recordings from brain slice preparations
AVP-eGFP transgenic female rats (5 wk-old virgins; n = 3) were anesthetized with halothane before decapitation. The brain was placed in ice-cold artificial cerebrospinal fluid containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 20 glucose (298–302 mOsm/liter; pH 7.4) bubbled with 95% (vol/vol) O2:5% (vol/vol) CO2. Coronal slices (350 μm thick) were cut with a vibratome (VT-1000S; Leica) and stored in bubbled artificial cerebrospinal fluid for more than 1 h before recordings. Using a cooled charge-coupled device camera (ORCA-ER C4742-95; Hamamatsu Photonics, Hamamatsu, Japan) fitted to an epifluorescence and infrared-differential-interference-contrast upright microscope (ECLIPSE E600FN; Nikon), the SON area and eGFP-expressing cells in the SON were easily identified. Whole-cell patch-clamp recordings were made at 31–32 C from identified SON MCNs expressing GFP. Patch electrodes were fabricated from borosilicate capillary tubing of 1.5 μm diameter (Garner Glass Co., Claremont, CA) using a Narishige PP-83 vertical puller (Narishige, Tokyo, Japan). The pipette solution contained (in mM) a50 potassium methanesulfonate, 5 KCl, 0.1 K-EGTA, 5 Na-HEPES, 3 Mg-ATP, and 0.4 Na-GTP (pH 7.4) (4–5 M). Membrane potentials were recorded in current-clamp mode, with a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA). Recorded signals were low-pass-filtered at 1–2 kHz, digitized at 2–5 kHz with an A/D converter (Digidata 1322A; Axon Instruments), and analyzed with the pClamp8 software (Axon Instruments). During recordings, the leak current was monitored, and if the parameter changed to greater than 10 pA, the recording data were excluded from further data analysis.
Preparation of dissociated axon terminals and SON neurons
Two neurohypophyses devoid of the pars intermedia and two SON from the same rat were taken for each experiment. Axon terminals were isolated from the neurohypophysis as described previously (27). Briefly, rats were killed by decapitation with a guillotine following the guidelines laid down by the Japanese ethical committee before isolating the pituitary. After removal of the anterior and the intermediate lobe of the pituitary, the neurohypophysis was dissociated in a buffer at 37 C containing (in mM) 270 sucrose, 1 EGTA, and 10 HEPES, buffered to pH 7.25 with Tris. The dissociated neurohypophysis was spun at 100 x g for 1 min with the supernatant then spun at 2400 x g for 4 min. The resulting pellet was then resuspended in normal Locke buffer at 37 C that contained (in mM): 140 NaCl, 5 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.25); the osmolality was between 298 and 302 mOsmol/liter. Although the vast majority of the isolated axon terminals are less than 2 μm in diameter, several hundred nerve terminals and swellings of larger diameter (4–10 μm) can also be found. One hour after, the terminals were observed under the fluorescence microscope (Axiovert 135TV, Carl Zeiss) with a GFP filter (No. 17 for fluorescein isothiocyanate).
The SON were dissected after brain removal after decapitation, and two narrow pieces lateral to the optic chiasma were dissected and transferred to Locke buffer (28). Enzymatic dissociation was carried out by incubating the tissue pieces in oxygenated Locke buffer supplemented with 0.5 mg/ml of deoxyribonuclease I, 1 mg/ml protease X, and 1 mg/ml protease XIV for 30 min at 34 C under constant oxygenation. The tissues pieces were then rinsed with Locke buffer and dissociated mechanically by gentle titration. The cell suspension was plated onto glass-bottom culture dishes (HBSt or GWSt-3522 series; 22 mm diameter, 0.17 mm thickness; WillCo Wells BV, Amsterdam, The Netherlands). One hour after, the cells were also observed under fluorescence microscope (Axiovert 135TV, Carl Zeiss) with a GFP filter (No. 17 for fluorescein isothiocyanate).
Statistical analysis
All data are given as mean ± SEM, calculated from the results of the in situ hybridization histochemistry. Each group within an experiment was compared with the control group. The data were analyzed using a one-way ANOVA followed by a Bonferroni-type adjustment for multiple comparisons. The statistical significance was set at P < 0.05.
Results
Production of transgenic rats
The AVP-eGFP transgene was injected into the pronuclei of 203 fertilized oocytes obtained from Wistar rat donors. A total of 52 pups were obtained. Twelve transgenic founders (nine male and three female) were identified by Southern blot analysis using genomic tail DNA with a 32P-labeled eGFP probe. The copy number of the eGFP gene was between 10 and 100. Twelve founders were bred, and F1 rats were screened by PCR analysis of genomic DNA extracted from rat tail. The PCR product was identified as the band of 171 bp in gel.
Frozen brain sections obtained from eGFP-positive rats (F1) were examined by fluorescent microscopic observation. Two lines (54-9 and 66-1) demonstrating robust expression of eGFP in the SON, the PVN, and the SCN were selected for physiological experiments and breeding. The copy numbers of the eGFP gene in 54-9 and 66-1 were 30 and 100, respectively. In the present study one line (66-1) was used.
Distribution of eGFP mRNA and eGFP in the central nervous system (CNS)
In situ hybridization histochemistry revealed that eGFP mRNA was localized in the SON, the magnocellular and parvocellular divisions of the PVN, and dorsomedial parts of the SCN (Fig. 1). There was no ectopic expression of the eGFP gene in the CNS (Fig. 1). The fluorescence of eGFP was observed throughout the SON (Fig. 2A). There were scattered eGFP-expressing neurons in the parvocellular division of the PVN and abundant fluorescence of eGFP in the magnocellular division of the PVN (Fig. 2B). In the dorsomedial parts of the SCN, eGFP-expressing neurons were of relatively small size, and the fluorescence of eGFP in the fibers was also clearly observed (Fig. 2C). In the hypothalamo-neurohypophyseal tract, intense fluorescence of eGFP was also observed (Fig. 2D). In the posterior pituitary gland, the fluorescence of eGFP was clearly observed in the axon terminals (Fig. 2E).
FIG. 2. Endogenous fluorescence of eGFP in the SON (A), the PVN (B), the SCN (C), the infundibulum (D), and the posterior pituitary (PP) (E). Scale bars, 50 μm. OT, optic tract; 3V, third ventricle; IL, intermediate lobe; AP, anterior pituitary.
Immunostaining for AVP and OXT
In the SON, most eGFP-expressing MCNs also exhibited AVP immunoreactivity (AVP-ir) (Fig. 3, A–C). In the PVN, the majority of both the magnocellular and parvocellular neurons that expressed eGFP fluorescence also exhibited AVP-ir (Fig. 3, D–F). In the SCN, almost all eGFP-expressing neurons exhibited AVP-ir (Fig. 3, G–I). In the SON, a few eGFP-expressing MCNs exhibited OXT-ir (Fig. 4, A–C). In the PVN, no eGFP-expressing neurons exhibited OXT-ir (Fig. 4, D–F).
FIG. 3. Endogenous fluorescence of eGFP in the SON (A), the PVN (D), and the SCN (G). The AVP antibodies were visualized as red fluorescence, using Alexa 546-conjugated secondary antibody (B, E, and H). Merged view of fluorescence of eGFP and specific AVP is seen as yellow (C, F, and I). Scale bars, 50 μm. OT, optic tract; 3V, third ventricle.
FIG. 4. A and D, Endogenous fluorescence of eGFP in the SON (A) and the PVN (D). B and E, OXT antibodies were visualized as red fluorescence, using Alexa 546-conjugated secondary antibody. C and F, Merged view. Scale bars, 50 μm. OT, optic tract.
Effects of dehydration on the expression of the eGFP gene
Water deprivation for 2 d caused a marked increase in the expression of the eGFP transgene in the SON and the PVN but not the SCN as determined by in situ hybridization (Fig. 5, A–E). Water deprivation for 2 d also caused marked increases in the level of fluorescence of eGFP throughout the SON and in majority of the magnocellular cells and a few parvocellular cells of the PVN (Fig. 5, F–I).
FIG. 5. A–D, Effects of water deprivation for 2 d on the expression of the eGFP gene in the SON (A and B), the SCN (A and B), and the PVN (C and D). The changes of eGFP mRNA levels in the SCN, SON, and PVN after water deprivation for 2 d are shown in E. F–I, Effects of water deprivation for 2 d on the endogenous fluorescence of eGFP in the SON (F and G) and the PVN (H and I). A, C, F, and H, Sections obtained from euhydrated rats; B, D, G, and I, sections are obtained from dehydrated rats. Scale bars, 1 mm (A–D) and 100 μm (F–I). OT, optic tract; 3V, third ventricle.
Electrophysiological properties of eGFP-expressing magnocellular neurons in the SON
Whole-cell patch-clamp studies were undertaken to elucidate the physiological properties of eGFP-expressing MCNs in the SON, which were identified microscopically in acutely prepared brain slices (Fig. 6, A–C). Mean resting potential and input resistance was –59.9 ± 4.7 mV and 115 ± 8.5 Mohm, respectively (n = 6). Current-clamp recordings revealed that these eGFP-expressing neurons had spontaneous firing of action potentials (Fig. 6D). Figure 6E shows examples of current-voltage relationships taken at resting membrane potential. All neurons (n = 6) exhibited no rebound depolarization at the offset (Fig. 6 E). In addition, eGFP-expressing neurons in the magnocellular division of the PVN and the SCN were also easily identified and successfully recorded (data not shown).
FIG. 6. Electrophysiological properties of visually identified eGFP-expressing MCNs in the SON and freshly isolated SON neurons and axon terminals. A–C, Photomicrograph of the positioning of a recording electrode (A) approaching an eGFP-expressing cell identified by infrared-differential-interference-contrast (B) and epifluorescence (C) images. In B and C, the dotted line indicates recording electrode. D–F, Typical firing pattern (D) and voltage responses to current injections from –0.4 nA to +0.2 nA (E and F) recorded from the eGFP-positive MCN shown in B and C (arrowheads). Scale bars, 500 μm (A) and 30 μm (B and C). OT, optic tract. G and H, Arrowheads indicate visually identified neurons with high-intensity endogenous fluorescence of eGFP in a freshly isolated SON neuron with a neighboring neuron that is negative (G) or in the freshly isolated axon terminals (H). Note that only few terminals are eGFP positive, although some terminals are negative. Scale bars, 50 μm (G) and 25 μm (H).
Visualization of eGFP expression in isolated SON neurons and axon terminals
Isolated axon terminals and SON neurons were widely used for many physiological studies using electrophysiological, fluorescence photometry and peptide release techniques; and in a few reports, the cells were identified only after the recordings (18, 29, 30, 31, 32). Here, we have succeeded to observe the eGFP expression in only half of the populations of the freshly isolated SON neurons (Fig. 6G) and axon terminals (Fig. 6H). In three different preparations of SON and terminals from the same transgenic rats, we observed approximately 50% (SON, 13 of 28; terminals, 33 of 61) of the cells were eGFP positive, suggesting that they are likely to be AVP neurons and AVP terminals. In control rats that were born from the same female rat but eGFP gene negative, none of the SON neurons (n = 17) or axon terminals (n = 28) showed eGFP positive (image not shown). Additional immunohistochemical identification studies are under investigation.
Discussion
As the first known mammalian neuropeptide secretory system, the HNS has become a model. Studies on the HNS have consistently pointed physiological and neuroscience research toward novel concepts, as evidenced from our present understanding of neuroanatomy (anatomical separation of the synthesis and secretion of neuropeptides), neurosecretion (biosynthesis of neuropeptides and electrophysiology in phenotypically identified neurons), neuroendocrinology (integration of and communication between the brain and periphery), and neuronal plasticity (functional remodeling of neuronal activity and physiological regulation of gene expression). This guiding role has been augmented by the application of transgenic techniques that enable gene function and regulation to be studied within the context of the physiological integrity of the intact organism (33).
Transgenic animals are widely used to understand the physiological role and regulation of neuroendocrine genes (33, 34). Most transgenic studies have been performed on mice. But it is the rat that continues to be the species of choice for studies in neuroscience and physiology, particularly in the field of neuroendocrinology. The mouse is relatively inappropriate; in contrast, the anatomy of the rat brain is well mapped, and the structure, function, and regulation of the rat CNS have been the subject of detailed study for many years and numerous effective behavioral paradigms have been developed. The large size of the rat makes it easily accessible for a whole range of physiological measurement and intervention, particularly in terms of electrophysiological studies, but its reproductive capacity and gestation time are equivalent to the mouse. Rats can be transformed by microinjection of fertilized one-cell eggs (23, 34, 35). With the completion of the sequence of the rat genome (36), the utility of the rat as an experimental model is further enhanced (37, 38).
In the HNS, magnocellular neurosecretory cells divide into two types of cells: AVP- and OXT-secreting cells. On the basis of electrical recording or [Ca2+]i measurements and subsequent immunohistochemistry, these cells can be identified as being either AVP- or OXT-secreting cells in in vitro preparations (19, 20, 21, 22). However, such identification was limited to only a few cells and limited to few laboratories.
To have a simple and easy visualization of a particular type of tissue, a fluorescent marker, eGFP, can be expressed transgenetically in specific neuronal cell types. For example, eGFP has been expressed in GnRH neurons in transgenic mice (39) and rats (40). In GnRH-eGFP transgenic rats, electrophysiological recordings from eGFP-expressing neurons were obtained (40). In mice, Young and colleagues (35, 41) succeeded in the transgenic expression of eGFP in OXT-secreting neurons. eGFP fused to the C terminus of the OXT preprohormone was expressed selectively in OXT-MCNs. The reporter entered the regulated secretory pathway and transported to neurosecretory terminals in the neurohypophysis, where the eGFP fluorescence underwent depolarization-induced calcium-dependent secretion. The fluorescence was sufficient to image secretory events in individual OXT nerve terminals.
We have now succeeded, for the first time, in the transgenic expression of eGFP in AVP-secreting neurons in the SON, the PVN, and the SCN in rats. Expression of eGFP in our rats faithfully recapitulates that of the endogenous gene. In situ hybridization histochemistry, and fluorescence microscopy revealed that the expression of the AVP-eGFP transgene was restricted to the SON, the PVN, and the SCN. It is worth noting that, at least at the levels of sensitivity used in this study, there was no expression of the eGFP gene seen elsewhere in the brain at the level of the SON and the PVN (Fig. 1). A confocal laser scanning microscopy survey of the entire brain from forebrain to brain stem found scattered eGFP-expressing neurons in the septal area and the bed nucleus of stria terminalis, although the intensity of eGFP fluorescence in those nuclei was very weak. Because these regions are known to express AVP (42), these eGFP-expressing neurons in the extrahypothalamus should be compared with hypothalamic eGFP-expressing neurons and be identified by immunohistochemistry for AVP by further study.
We then sought to identify the specific neurochemical phenotype of the eGFP-expressing neurons in the hypothalamus. Using a specific antibody for AVP, we have shown that almost all AVP-containing neurons express eGFP. Conversely, in the PVN, no eGFP-expressing neurons exhibited OXT-ir. In the dorsal part of the SON, a few eGFP-expressing neurons exhibited OXT-ir. These results suggest that almost all eGFP-expressing neurons in the SON, the PVN, and the SCN express AVP, although a few eGFP-expressing neurons in the SON exhibited OXT. This is consistent with previous observations showing that AVP and OXT expression are largely mutually exclusive, with only 3–5% of MCNs expressing high, equivalent levels of both peptides (5, 6).
In the present study we have been successful in visualizing the high intensity of expression of eGFP in the freshly isolated SON neurons and their axon terminals. It should be noted that only approximately 50% of the neurons/terminals were eGFP positive. These neurons and terminals were widely used for their cell-specific physiology (17, 18, 22, 29, 30, 31, 43, 44). The preidentified eGFP-AVP neurons and axon terminals will be the interesting model for further exploration of their physiology.
The AVP-eGFP transgene was based on genomic sequences previously described (24), all of which, when modified by the inclusion of a chloramphenicol acetyltransferase reporter sequence into the third exon, elicited expression in SON and PVN MCNs, but not in PVN parvocellular neuron nor in the SCN (45, 46). Interestingly, the AVP-eGFP transgene is expressed in the AVP parvocellular cells of the PVN and the SCN. It appears that the choice of reporter similarly located in the third exon can greatly influence the expression of AVP transgenes; although a ?-galactosidase reporter failed to express at all in the hypothalamus (46), a chloramphenicol acetyltransferase-based reporter is directed to MCNs of the PVN and the SON, whereas our eGFP reporter allows faithful recapitulation of endogenous AVP expression in the SON and the magnocellular and parvocellular compartments of the PVN and the SCN.
After water deprivation for 2 d, the fluorescence of eGFP in the SON and most magnocellular and a few parvocellular cells of the PVN were markedly increased. However, there was no effect of dehydration on the fluorescence of eGFP in the SCN as has previously been observed (47), suggesting that there may be a different regulatory mechanism in the magnocellular neurosecretory neurons in the SON and the PVN compared with the SCN neurons.
We note that the eGFP moiety, as a C-terminal fusion with the AVP prepropeptide, seems to enter the regulated pathway. The eGFP produced in the cell bodies of the SON and the PVN enters the pituitary stalk and accumulates in the axon terminals of the posterior pituitary lobe. In addition, there were many strong intense fluorescent varicosities in the hypothalamo-pituitary tract in dehydrated transgenic rats.
An electrophysiological study was performed to record neuronal activity from eGFP-expressing neurons in the SON, the PVN, and the SCN in an in vitro slice preparation. There was sufficiently intense fluorescence of eGFP to identify neurons for electrophysiological recording. Our results show that the membrane properties of eGFP-expressing SON neurons are similar to those previously described as being characteristic of AVP SON MCNs (48). All recordings from eGFP-expressing neurons in the SON exhibited no rebound depolarization at the offset. AVP-secreting neurons in the SON lacked a strong sag and exhibited no rebound, although OXT-secreting neurons in the SON have a sag during small hyperpolarization and exhibited a rebound depolarization that produced spikes (49, 50). Although a small sag during hyperpolarization was observed in recorded neurons (Fig. 6), the specific sag observed in OXT-secreting neurons was not seen.
In conclusion, we have described, for the first time, AVP-eGFP transgenic rats that elicit robust fluorescence specifically in AVP-secreting neurons of the SON, the PVN, and the SCN and their fibers and axon terminals in the posterior pituitary gland. These rats will revolutionize research on the HNS. For the first time, living AVPergic neurons can be directly identified by virtue of their inherent fluorescence. We anticipate broad application for these rats in the study of the physiological role of AVP neurons in the CNS and the dynamics of AVP synthesis and secretion in living neurons, either in dispersed culture, organotypic culture, or living slices or, indeed, by using advanced imaging techniques (51) in the intact organism. Particularly, these animals will enable the direct identification of living AVP neurons for electrophysiology experiments. In addition, in vivo fluorescence fiber-optic probes could be used to monitor eGFP levels in the hypothalamus (i.e. synthesis) and in the pituitary (release) and, possibly, to be coupled with in vivo electrophysiological recordings (52, 53).
Acknowledgments
D.M. gratefully acknowledges the support of the Biotechnology and Biological Sciences Research Council (UK) and the British Heart Foundation.
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Address all correspondence and requests for reprints to: Yoichi Ueta, M.D., Ph.D., Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yoichi@med.uoeh-u.ac.jp.
Abstract
We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.
Introduction
THE HYPOTHALAMO-NEUROHYPOPHYSEAL SYSTEM (HNS) consists of large peptidergic magnocellular neurons (MCNs; 20–40 μm cell body diameter) of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, the axons of which course through the internal zone of the median eminence (ME) and terminate on blood capillaries of the posterior (neural) lobe of the pituitary gland (1). These neurons synthesize the neuropeptide hormone arginine vasopressin (AVP) in abundance. The SON is a homogeneous collection of MCNs, whereas the PVN is divided into a lateral and more medial subdivision of magnocellular and parvocellular neurons, respectively. Parvocellular neurons have smaller cells bodies (10–15 μm) whose axons project to the external zone of the ME (2). The HNS produces other neuropeptides in addition to AVP, for example, the closely related hormone oxytocin (OXT). Single-cell RT-PCR enables vasopressin and OXT transcripts to be detected together in the same MCN (3, 4), but expression levels of each neuropeptide RNA differ by orders of magnitude, and it is not clear whether the minority species is translated. Only 3–5% of MCNs express high, equivalent levels of both peptides (5, 6), although this proportion increases after dehydration (7). AVP is also expressed in the hypothalamic suprachiasmatic nucleus (SCN) (8).
AVP is synthesized as part of a prepropeptide precursor in the cell bodies of hypothalamic neurons (9). After signal peptide removal and disulfide bond formation in the endoplasmic reticulum, the propeptide is sorted (10) and packaged into dense core granules of the regulated secretory pathway in the trans-Golgi network. Subsequent processing events proceed as the granules are transported down axons from the cell body to nerve terminals (9). Here, the mature peptide is stored until mobilized for secretion by electrical activity evoked by physiological cues (11).
The functions of hypothalamic AVP depend upon its anatomical origin. Thus, the hormone made in the magnocellular cell bodies of the SON and PVN is transported to the posterior pituitary from where it is released into the general circulation, acting upon not only the kidney to conserve water in its classical role as antidiuretic hormone but also arterioles to cause vasoconstriction. The AVP made in the parvocellular PVN is known to be involved in stress (12, 13), whereas the SCN-derived material appears to control circadian rhythmicity (14).
Electrophysiological studies have characterized the electrical properties of MCNs in the SON and PVN using both in vivo and in vitro brain preparations (15). Although it has been established that the firing of AVP-secreting MCNs in vivo is inhibited by activation of baroreceptor stimulation (16), AVP-secreting MCNs in the SON and PVN can be identified only by the characteristic phasic firing pattern that shows alternating periods of activity and silence in an in vitro slice preparation. In the isolated SON neurons, a cell-specific increase in intracellular concentration of Ca2+ ([Ca2+]i) induced by either AVP or OXT was also demonstrated (17, 18). The firing of AVP-secreting neurons sometimes shows continuous or irregular patterns. Although it is possible to retrospectively identify AVP-secreting neurons marked by injection of intracellular dye, with subsequently immunohistochemistry using a specific antibody against AVP (19, 20, 21), it is difficult to easily identify the living AVP neurons in in vitro preparations except under certain electrophysiological studies. In addition, using acutely dissociated SON neurons to record electrical activity and/or [Ca2+]i, it is possible to identify AVP or OXT neurons only by immunohistochemistry after [Ca2+]i measurement experiments (18, 22). The ability to directly identify living AVP-secreting neurons or AVP nerve terminals of the neurohypophysis with the naked eye using classical fluorescence microscopy would contribute greatly to progress in AVP physiological studies.
In the present study, we introduced a novel AVP-enhanced green fluorescent protein (eGFP) fusion gene into transgenic rats. GFP is made by the bioluminescent jellyfish (Aequorea victoria). GFP has been cloned and modified as eGFP (23). Expression of eGFP in specific neurons under the control of cell-restricted promoters enables their direct visualization and identification. In our rats, transgene expression faithfully recapitulates that of the endogenous AVP gene, being confined to AVP neurons in the SON, PVN, and SCN. Transgenic eGFP appears to enter the regulated secretory pathway, being present in axons of the hypothalamo-neurohypophyseal tract and nerve terminals in the posterior pituitary gland. Furthermore, like the endogenous AVP, expression of the transgene is up-regulated by the physiological challenge of dehydration. Importantly, eGFP expression can be visualized in live AVP cells in brain slices, isolated SON neurons, and their nerve terminals in the neurohypophysis enabling their direct identification for electrophysiological and [Ca2+]i microspectrofluorimetry/[Ca2+]i imaging studies.
Materials and Methods
Animals
Transgenic Wistar rats were bred and housed under normal laboratory conditions (12-h light, 12-h dark cycle) with free access to food and drinking water. For dehydration, water was deprived for 2 d with free access to dry food. For fluorescent microscopic observation and in situ hybridization histochemistry after dehydration, 8-wk-old AVP-eGFP transgenic male rats and transgene (–) male rats as control were used. All experiments were performed in strict accordance with guidelines on the use and care of laboratory animals as set out by the Physiological Society of Japan and approved by the Ethics Committee of Animal Care and Experimentation of our University.
Constructs for microinjection
The AVP-eGFP transgene was constructed using pRVP[S-E/BD-S]Not (24), which consists of the AVP structural gene flanked by approximately 3 kbp of 5' upstream sequences and 2 kbp of flanking downstream sequences. In exon III, a 66-bp SmaI fragment has been replaced with a NotI site. The eGFP coding sequence of a 732-bp fragment obtained from pLP-IRES2-eGFP (Clontech Laboratories, Inc., Tokyo, Japan) were inserted in the NotI site of the exon III of the AVP gene in pRVP[S-E/BD-S]Not. Thus, the AVP-eGFP transgene encodes a modified AVP precursor with eGFP fused in-frame at the C terminus (Fig. 1A).
FIG. 1. A–C, Structure of the AVP-eGFP transgene (A) and representative autoradiographs of brain sections hybridized to a 35S-labeled oligodeoxynucleotide probe for eGFP mRNA in a transgenic rat (B and C). D and E, Enlargements from the boxed areas in B. F, Enlargement from the boxed area in C. White is the most intense signal, and black is the least intense signal. Scale bars, 1 mm (B and C) and 50 μm (D–F). VP, vasopressin; EX, exon; OT, optic tract.
The AVP-eGFP fusion gene was validated by sequencing. The transgene fragment was microinjected into the pronuclei of fertilized oocytes obtained from Wistar rats. The transgenic founders were identified by Southern blot analysis using genomic tail DNA with a 32P-labeled eGFP probe. The founders were bred, and F1 rats were screened by PCR analysis of genomic DNA extracted from rat tail. The PCR was performed using the oligonucleotide primers (sense sequence, 5'-CAC CAT CTT CTT CAA GGA CGA C-3'; antisense sequence, 5'-ATG ATA TAG ACG TTG TGG CTG TTG T-3').
Furthermore, frozen and/or 4% paraformaldehyde-fixed brain sections obtained from eGFP-positive rats (F1) were observed under the fluorescence microscope (Eclipse E600, Nikon, Tokyo, Japan) with a GFP filter (Nikon) for screening whether eGFP was visually expressed in the hypothalamus and pituitary gland. Fluorescent microphotographs were taken by digital camera (COOLPIX 990, Nikon).
In situ hybridization histochemistry for eGFP
Cryostat sections were cut into 12-μm slices and thaw mounted on gelatin/chrome alum-coated slides. The locations of the hypothalamic areas including the SON, the PVN, and the SCN were determined according to coordinates given by the rat brain atlas (25). 35S 3'-end-labeled deoxyoligonucleotide complementary to transcripts encoding eGFP were used (eGFP probe sequence, 5'-CGG CCA TGA TAT AGA CGT TGT GGC TGT TGT AGT TGT ACT CC-3'). The probe was 3'-end labeled using terminal deoxynucleotidyl transferase and [35S]dATP. The in situ hybridization protocol has been described previously in detail (26). Briefly, sections were fixed in 4% (wt/vol) formaldehyde for 5 min and incubated in saline containing 0.25% (vol/vol) acetic anhydride and 0.1 M triethanolamine for 10 min and then dehydrated, delipidated in chloroform, and partially rehydrated. Hybridization was carried out overnight at 37 C in 45 μl hybridization buffer under a Nescofilm (Bando Kagaku, Osaka, Japan) coverslip. A total count of 1 x 106 cpm for eGFP transcripts per slide was used. After hybridization, sections were washed for 1 h in four changes of 1x standard saline citrate (150 nM NaCl and 15 nM sodium citrate) at 55 C and for an additional 1 h in two changes of 1x standard saline citrate at room temperature. Hybridization sections were exposed to autoradiography (Hyperfilm, Amersham, Little Chalfont, UK) for 2 wk for eGFP probe. The resulting images were analyzed by computerized densitometry using a MCID imaging analyzer (Imaging Research Inc., Ontario, Canada). The mean OD of autoradiographs was measured by comparing it with simultaneously exposed 14C-labeled micro-scale samples (Amersham) and presented in arbitrary units setting the mean OD obtained from control rats.
Immunohistochemistry for AVP and OXT
Deeply anesthetized animals were perfused transcardially with 0.1 M phosphate buffer (PB) (pH 7.4) containing heparin (1000 U/liter) followed by 4% (wt/vol) paraformaldehyde. The brains were then removed and divided into three blocks. The blocks that included the hypothalamus were postfixed with the same fixative for 48 h at 4 C. The tissues were then immersed in 20% (wt/vol) sucrose in 0.1 M PB for 48 h at 4 C for cryoprotection. Then, the brains were quickly frozen using powdered dry ice and sectioned at 30 μm on a cryostat (Leica CM3050, Nussloch, Germany). The sections were rinsed twice with 0.1 M PBS containing 0.3% (vol/vol) Triton X-100. The floating sections were incubated with a primary AVP antibody (Chemicon International, Inc., Temecula, CA) (diluted 1:8000) or OXT antibody (Chemicon) (diluted 1:8000) for 3 d at 4 C. After washing for 20 min in 0.1 M PBS solution containing 0.3% (vol/vol) Triton X-100, the sections were incubated for 2 h with Alexa Fluor 546 conjugated antirabbit IgG (Molecular Probes, Eugene, OR) (diluted 1:1000). After final washing with 0.1 M PB, sections were mounted on the glass slides and coverslipped with hydrophilic mounting media (Tris-HCl containing polyvinyl alcohol and glycerol); subsequently, the sections were observed under a confocal laser scanning microscopy (LSM510, Carl Zeiss Co. Ltd., Jena, Germany).
Electrophysiological recordings from brain slice preparations
AVP-eGFP transgenic female rats (5 wk-old virgins; n = 3) were anesthetized with halothane before decapitation. The brain was placed in ice-cold artificial cerebrospinal fluid containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 20 glucose (298–302 mOsm/liter; pH 7.4) bubbled with 95% (vol/vol) O2:5% (vol/vol) CO2. Coronal slices (350 μm thick) were cut with a vibratome (VT-1000S; Leica) and stored in bubbled artificial cerebrospinal fluid for more than 1 h before recordings. Using a cooled charge-coupled device camera (ORCA-ER C4742-95; Hamamatsu Photonics, Hamamatsu, Japan) fitted to an epifluorescence and infrared-differential-interference-contrast upright microscope (ECLIPSE E600FN; Nikon), the SON area and eGFP-expressing cells in the SON were easily identified. Whole-cell patch-clamp recordings were made at 31–32 C from identified SON MCNs expressing GFP. Patch electrodes were fabricated from borosilicate capillary tubing of 1.5 μm diameter (Garner Glass Co., Claremont, CA) using a Narishige PP-83 vertical puller (Narishige, Tokyo, Japan). The pipette solution contained (in mM) a50 potassium methanesulfonate, 5 KCl, 0.1 K-EGTA, 5 Na-HEPES, 3 Mg-ATP, and 0.4 Na-GTP (pH 7.4) (4–5 M). Membrane potentials were recorded in current-clamp mode, with a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA). Recorded signals were low-pass-filtered at 1–2 kHz, digitized at 2–5 kHz with an A/D converter (Digidata 1322A; Axon Instruments), and analyzed with the pClamp8 software (Axon Instruments). During recordings, the leak current was monitored, and if the parameter changed to greater than 10 pA, the recording data were excluded from further data analysis.
Preparation of dissociated axon terminals and SON neurons
Two neurohypophyses devoid of the pars intermedia and two SON from the same rat were taken for each experiment. Axon terminals were isolated from the neurohypophysis as described previously (27). Briefly, rats were killed by decapitation with a guillotine following the guidelines laid down by the Japanese ethical committee before isolating the pituitary. After removal of the anterior and the intermediate lobe of the pituitary, the neurohypophysis was dissociated in a buffer at 37 C containing (in mM) 270 sucrose, 1 EGTA, and 10 HEPES, buffered to pH 7.25 with Tris. The dissociated neurohypophysis was spun at 100 x g for 1 min with the supernatant then spun at 2400 x g for 4 min. The resulting pellet was then resuspended in normal Locke buffer at 37 C that contained (in mM): 140 NaCl, 5 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.25); the osmolality was between 298 and 302 mOsmol/liter. Although the vast majority of the isolated axon terminals are less than 2 μm in diameter, several hundred nerve terminals and swellings of larger diameter (4–10 μm) can also be found. One hour after, the terminals were observed under the fluorescence microscope (Axiovert 135TV, Carl Zeiss) with a GFP filter (No. 17 for fluorescein isothiocyanate).
The SON were dissected after brain removal after decapitation, and two narrow pieces lateral to the optic chiasma were dissected and transferred to Locke buffer (28). Enzymatic dissociation was carried out by incubating the tissue pieces in oxygenated Locke buffer supplemented with 0.5 mg/ml of deoxyribonuclease I, 1 mg/ml protease X, and 1 mg/ml protease XIV for 30 min at 34 C under constant oxygenation. The tissues pieces were then rinsed with Locke buffer and dissociated mechanically by gentle titration. The cell suspension was plated onto glass-bottom culture dishes (HBSt or GWSt-3522 series; 22 mm diameter, 0.17 mm thickness; WillCo Wells BV, Amsterdam, The Netherlands). One hour after, the cells were also observed under fluorescence microscope (Axiovert 135TV, Carl Zeiss) with a GFP filter (No. 17 for fluorescein isothiocyanate).
Statistical analysis
All data are given as mean ± SEM, calculated from the results of the in situ hybridization histochemistry. Each group within an experiment was compared with the control group. The data were analyzed using a one-way ANOVA followed by a Bonferroni-type adjustment for multiple comparisons. The statistical significance was set at P < 0.05.
Results
Production of transgenic rats
The AVP-eGFP transgene was injected into the pronuclei of 203 fertilized oocytes obtained from Wistar rat donors. A total of 52 pups were obtained. Twelve transgenic founders (nine male and three female) were identified by Southern blot analysis using genomic tail DNA with a 32P-labeled eGFP probe. The copy number of the eGFP gene was between 10 and 100. Twelve founders were bred, and F1 rats were screened by PCR analysis of genomic DNA extracted from rat tail. The PCR product was identified as the band of 171 bp in gel.
Frozen brain sections obtained from eGFP-positive rats (F1) were examined by fluorescent microscopic observation. Two lines (54-9 and 66-1) demonstrating robust expression of eGFP in the SON, the PVN, and the SCN were selected for physiological experiments and breeding. The copy numbers of the eGFP gene in 54-9 and 66-1 were 30 and 100, respectively. In the present study one line (66-1) was used.
Distribution of eGFP mRNA and eGFP in the central nervous system (CNS)
In situ hybridization histochemistry revealed that eGFP mRNA was localized in the SON, the magnocellular and parvocellular divisions of the PVN, and dorsomedial parts of the SCN (Fig. 1). There was no ectopic expression of the eGFP gene in the CNS (Fig. 1). The fluorescence of eGFP was observed throughout the SON (Fig. 2A). There were scattered eGFP-expressing neurons in the parvocellular division of the PVN and abundant fluorescence of eGFP in the magnocellular division of the PVN (Fig. 2B). In the dorsomedial parts of the SCN, eGFP-expressing neurons were of relatively small size, and the fluorescence of eGFP in the fibers was also clearly observed (Fig. 2C). In the hypothalamo-neurohypophyseal tract, intense fluorescence of eGFP was also observed (Fig. 2D). In the posterior pituitary gland, the fluorescence of eGFP was clearly observed in the axon terminals (Fig. 2E).
FIG. 2. Endogenous fluorescence of eGFP in the SON (A), the PVN (B), the SCN (C), the infundibulum (D), and the posterior pituitary (PP) (E). Scale bars, 50 μm. OT, optic tract; 3V, third ventricle; IL, intermediate lobe; AP, anterior pituitary.
Immunostaining for AVP and OXT
In the SON, most eGFP-expressing MCNs also exhibited AVP immunoreactivity (AVP-ir) (Fig. 3, A–C). In the PVN, the majority of both the magnocellular and parvocellular neurons that expressed eGFP fluorescence also exhibited AVP-ir (Fig. 3, D–F). In the SCN, almost all eGFP-expressing neurons exhibited AVP-ir (Fig. 3, G–I). In the SON, a few eGFP-expressing MCNs exhibited OXT-ir (Fig. 4, A–C). In the PVN, no eGFP-expressing neurons exhibited OXT-ir (Fig. 4, D–F).
FIG. 3. Endogenous fluorescence of eGFP in the SON (A), the PVN (D), and the SCN (G). The AVP antibodies were visualized as red fluorescence, using Alexa 546-conjugated secondary antibody (B, E, and H). Merged view of fluorescence of eGFP and specific AVP is seen as yellow (C, F, and I). Scale bars, 50 μm. OT, optic tract; 3V, third ventricle.
FIG. 4. A and D, Endogenous fluorescence of eGFP in the SON (A) and the PVN (D). B and E, OXT antibodies were visualized as red fluorescence, using Alexa 546-conjugated secondary antibody. C and F, Merged view. Scale bars, 50 μm. OT, optic tract.
Effects of dehydration on the expression of the eGFP gene
Water deprivation for 2 d caused a marked increase in the expression of the eGFP transgene in the SON and the PVN but not the SCN as determined by in situ hybridization (Fig. 5, A–E). Water deprivation for 2 d also caused marked increases in the level of fluorescence of eGFP throughout the SON and in majority of the magnocellular cells and a few parvocellular cells of the PVN (Fig. 5, F–I).
FIG. 5. A–D, Effects of water deprivation for 2 d on the expression of the eGFP gene in the SON (A and B), the SCN (A and B), and the PVN (C and D). The changes of eGFP mRNA levels in the SCN, SON, and PVN after water deprivation for 2 d are shown in E. F–I, Effects of water deprivation for 2 d on the endogenous fluorescence of eGFP in the SON (F and G) and the PVN (H and I). A, C, F, and H, Sections obtained from euhydrated rats; B, D, G, and I, sections are obtained from dehydrated rats. Scale bars, 1 mm (A–D) and 100 μm (F–I). OT, optic tract; 3V, third ventricle.
Electrophysiological properties of eGFP-expressing magnocellular neurons in the SON
Whole-cell patch-clamp studies were undertaken to elucidate the physiological properties of eGFP-expressing MCNs in the SON, which were identified microscopically in acutely prepared brain slices (Fig. 6, A–C). Mean resting potential and input resistance was –59.9 ± 4.7 mV and 115 ± 8.5 Mohm, respectively (n = 6). Current-clamp recordings revealed that these eGFP-expressing neurons had spontaneous firing of action potentials (Fig. 6D). Figure 6E shows examples of current-voltage relationships taken at resting membrane potential. All neurons (n = 6) exhibited no rebound depolarization at the offset (Fig. 6 E). In addition, eGFP-expressing neurons in the magnocellular division of the PVN and the SCN were also easily identified and successfully recorded (data not shown).
FIG. 6. Electrophysiological properties of visually identified eGFP-expressing MCNs in the SON and freshly isolated SON neurons and axon terminals. A–C, Photomicrograph of the positioning of a recording electrode (A) approaching an eGFP-expressing cell identified by infrared-differential-interference-contrast (B) and epifluorescence (C) images. In B and C, the dotted line indicates recording electrode. D–F, Typical firing pattern (D) and voltage responses to current injections from –0.4 nA to +0.2 nA (E and F) recorded from the eGFP-positive MCN shown in B and C (arrowheads). Scale bars, 500 μm (A) and 30 μm (B and C). OT, optic tract. G and H, Arrowheads indicate visually identified neurons with high-intensity endogenous fluorescence of eGFP in a freshly isolated SON neuron with a neighboring neuron that is negative (G) or in the freshly isolated axon terminals (H). Note that only few terminals are eGFP positive, although some terminals are negative. Scale bars, 50 μm (G) and 25 μm (H).
Visualization of eGFP expression in isolated SON neurons and axon terminals
Isolated axon terminals and SON neurons were widely used for many physiological studies using electrophysiological, fluorescence photometry and peptide release techniques; and in a few reports, the cells were identified only after the recordings (18, 29, 30, 31, 32). Here, we have succeeded to observe the eGFP expression in only half of the populations of the freshly isolated SON neurons (Fig. 6G) and axon terminals (Fig. 6H). In three different preparations of SON and terminals from the same transgenic rats, we observed approximately 50% (SON, 13 of 28; terminals, 33 of 61) of the cells were eGFP positive, suggesting that they are likely to be AVP neurons and AVP terminals. In control rats that were born from the same female rat but eGFP gene negative, none of the SON neurons (n = 17) or axon terminals (n = 28) showed eGFP positive (image not shown). Additional immunohistochemical identification studies are under investigation.
Discussion
As the first known mammalian neuropeptide secretory system, the HNS has become a model. Studies on the HNS have consistently pointed physiological and neuroscience research toward novel concepts, as evidenced from our present understanding of neuroanatomy (anatomical separation of the synthesis and secretion of neuropeptides), neurosecretion (biosynthesis of neuropeptides and electrophysiology in phenotypically identified neurons), neuroendocrinology (integration of and communication between the brain and periphery), and neuronal plasticity (functional remodeling of neuronal activity and physiological regulation of gene expression). This guiding role has been augmented by the application of transgenic techniques that enable gene function and regulation to be studied within the context of the physiological integrity of the intact organism (33).
Transgenic animals are widely used to understand the physiological role and regulation of neuroendocrine genes (33, 34). Most transgenic studies have been performed on mice. But it is the rat that continues to be the species of choice for studies in neuroscience and physiology, particularly in the field of neuroendocrinology. The mouse is relatively inappropriate; in contrast, the anatomy of the rat brain is well mapped, and the structure, function, and regulation of the rat CNS have been the subject of detailed study for many years and numerous effective behavioral paradigms have been developed. The large size of the rat makes it easily accessible for a whole range of physiological measurement and intervention, particularly in terms of electrophysiological studies, but its reproductive capacity and gestation time are equivalent to the mouse. Rats can be transformed by microinjection of fertilized one-cell eggs (23, 34, 35). With the completion of the sequence of the rat genome (36), the utility of the rat as an experimental model is further enhanced (37, 38).
In the HNS, magnocellular neurosecretory cells divide into two types of cells: AVP- and OXT-secreting cells. On the basis of electrical recording or [Ca2+]i measurements and subsequent immunohistochemistry, these cells can be identified as being either AVP- or OXT-secreting cells in in vitro preparations (19, 20, 21, 22). However, such identification was limited to only a few cells and limited to few laboratories.
To have a simple and easy visualization of a particular type of tissue, a fluorescent marker, eGFP, can be expressed transgenetically in specific neuronal cell types. For example, eGFP has been expressed in GnRH neurons in transgenic mice (39) and rats (40). In GnRH-eGFP transgenic rats, electrophysiological recordings from eGFP-expressing neurons were obtained (40). In mice, Young and colleagues (35, 41) succeeded in the transgenic expression of eGFP in OXT-secreting neurons. eGFP fused to the C terminus of the OXT preprohormone was expressed selectively in OXT-MCNs. The reporter entered the regulated secretory pathway and transported to neurosecretory terminals in the neurohypophysis, where the eGFP fluorescence underwent depolarization-induced calcium-dependent secretion. The fluorescence was sufficient to image secretory events in individual OXT nerve terminals.
We have now succeeded, for the first time, in the transgenic expression of eGFP in AVP-secreting neurons in the SON, the PVN, and the SCN in rats. Expression of eGFP in our rats faithfully recapitulates that of the endogenous gene. In situ hybridization histochemistry, and fluorescence microscopy revealed that the expression of the AVP-eGFP transgene was restricted to the SON, the PVN, and the SCN. It is worth noting that, at least at the levels of sensitivity used in this study, there was no expression of the eGFP gene seen elsewhere in the brain at the level of the SON and the PVN (Fig. 1). A confocal laser scanning microscopy survey of the entire brain from forebrain to brain stem found scattered eGFP-expressing neurons in the septal area and the bed nucleus of stria terminalis, although the intensity of eGFP fluorescence in those nuclei was very weak. Because these regions are known to express AVP (42), these eGFP-expressing neurons in the extrahypothalamus should be compared with hypothalamic eGFP-expressing neurons and be identified by immunohistochemistry for AVP by further study.
We then sought to identify the specific neurochemical phenotype of the eGFP-expressing neurons in the hypothalamus. Using a specific antibody for AVP, we have shown that almost all AVP-containing neurons express eGFP. Conversely, in the PVN, no eGFP-expressing neurons exhibited OXT-ir. In the dorsal part of the SON, a few eGFP-expressing neurons exhibited OXT-ir. These results suggest that almost all eGFP-expressing neurons in the SON, the PVN, and the SCN express AVP, although a few eGFP-expressing neurons in the SON exhibited OXT. This is consistent with previous observations showing that AVP and OXT expression are largely mutually exclusive, with only 3–5% of MCNs expressing high, equivalent levels of both peptides (5, 6).
In the present study we have been successful in visualizing the high intensity of expression of eGFP in the freshly isolated SON neurons and their axon terminals. It should be noted that only approximately 50% of the neurons/terminals were eGFP positive. These neurons and terminals were widely used for their cell-specific physiology (17, 18, 22, 29, 30, 31, 43, 44). The preidentified eGFP-AVP neurons and axon terminals will be the interesting model for further exploration of their physiology.
The AVP-eGFP transgene was based on genomic sequences previously described (24), all of which, when modified by the inclusion of a chloramphenicol acetyltransferase reporter sequence into the third exon, elicited expression in SON and PVN MCNs, but not in PVN parvocellular neuron nor in the SCN (45, 46). Interestingly, the AVP-eGFP transgene is expressed in the AVP parvocellular cells of the PVN and the SCN. It appears that the choice of reporter similarly located in the third exon can greatly influence the expression of AVP transgenes; although a ?-galactosidase reporter failed to express at all in the hypothalamus (46), a chloramphenicol acetyltransferase-based reporter is directed to MCNs of the PVN and the SON, whereas our eGFP reporter allows faithful recapitulation of endogenous AVP expression in the SON and the magnocellular and parvocellular compartments of the PVN and the SCN.
After water deprivation for 2 d, the fluorescence of eGFP in the SON and most magnocellular and a few parvocellular cells of the PVN were markedly increased. However, there was no effect of dehydration on the fluorescence of eGFP in the SCN as has previously been observed (47), suggesting that there may be a different regulatory mechanism in the magnocellular neurosecretory neurons in the SON and the PVN compared with the SCN neurons.
We note that the eGFP moiety, as a C-terminal fusion with the AVP prepropeptide, seems to enter the regulated pathway. The eGFP produced in the cell bodies of the SON and the PVN enters the pituitary stalk and accumulates in the axon terminals of the posterior pituitary lobe. In addition, there were many strong intense fluorescent varicosities in the hypothalamo-pituitary tract in dehydrated transgenic rats.
An electrophysiological study was performed to record neuronal activity from eGFP-expressing neurons in the SON, the PVN, and the SCN in an in vitro slice preparation. There was sufficiently intense fluorescence of eGFP to identify neurons for electrophysiological recording. Our results show that the membrane properties of eGFP-expressing SON neurons are similar to those previously described as being characteristic of AVP SON MCNs (48). All recordings from eGFP-expressing neurons in the SON exhibited no rebound depolarization at the offset. AVP-secreting neurons in the SON lacked a strong sag and exhibited no rebound, although OXT-secreting neurons in the SON have a sag during small hyperpolarization and exhibited a rebound depolarization that produced spikes (49, 50). Although a small sag during hyperpolarization was observed in recorded neurons (Fig. 6), the specific sag observed in OXT-secreting neurons was not seen.
In conclusion, we have described, for the first time, AVP-eGFP transgenic rats that elicit robust fluorescence specifically in AVP-secreting neurons of the SON, the PVN, and the SCN and their fibers and axon terminals in the posterior pituitary gland. These rats will revolutionize research on the HNS. For the first time, living AVPergic neurons can be directly identified by virtue of their inherent fluorescence. We anticipate broad application for these rats in the study of the physiological role of AVP neurons in the CNS and the dynamics of AVP synthesis and secretion in living neurons, either in dispersed culture, organotypic culture, or living slices or, indeed, by using advanced imaging techniques (51) in the intact organism. Particularly, these animals will enable the direct identification of living AVP neurons for electrophysiology experiments. In addition, in vivo fluorescence fiber-optic probes could be used to monitor eGFP levels in the hypothalamus (i.e. synthesis) and in the pituitary (release) and, possibly, to be coupled with in vivo electrophysiological recordings (52, 53).
Acknowledgments
D.M. gratefully acknowledges the support of the Biotechnology and Biological Sciences Research Council (UK) and the British Heart Foundation.
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