Local production of angiotensin II in the subfornical organ causes elevated drinking

The mechanism controlling cell-specific Ang II production in the brain remains unclear despite evidence supporting neuron-specific renin and glial- and neuronal-specific angiotensinogen (AGT) expression. We generated double-transgenic mice expressing human renin (hREN) from a neuron-specific promoter and human AGT (hAGT) from its own promoter (SRA mice) to emulate this expression. SRA mice exhibited an increase in water and salt intake and urinary volume, which were significantly reduced after chronic intracerebroventricular delivery of losartan. Ang II–like immunoreactivity was markedly increased in the subfornical organ (SFO). To further evaluate the physiological importance of de novo Ang II production specifically in the SFO, we utilized a transgenic mouse model expressing a floxed version of hAGT (hAGT^flox), so that deletions could be induced with Cre recombinase. We targeted SFO-specific ablation of hAGT^flox by microinjection of an adenovirus encoding Cre recombinase (AdCre). SRA^flox mice exhibited a marked increase in drinking at baseline and a significant decrease in water intake after administration of AdCre/adenovirus encoding enhanced GFP (AdCre/AdEGFP), but not after administration of AdEGFP alone. This decrease only occurred when Cre recombinase correctly targeted the SFO and correlated with a loss of hAGT and angiotensin peptide immunostaining in the SFO. These data provide strong genetic evidence implicating de novo synthesis of Ang II in the SFO as an integral player in fluid homeostasis.

The renin-angiotensin system (RAS) is known to play a critical role in body fluid regulation through its actions within the kidney and central nervous system. Direct intracranial or intracerebroventricular injection of Ang II causes increases in blood pressure and dipsogenic behavior (1–3). Furthermore, centrally administered Ang II receptor antagonists are known to eliminate the cardiovascular effects caused by exogenous Ang II (4–6). Although these experiments have provided important evidence that the central nervous system is a target for the pressor and dipsogenic actions of exogenous Ang II, it remains unclear how endogenous Ang II is formed within the brain, where it is located, and how regional Ang II production correlates with specific cardiovascular responses.

Renin catalyzes the first of 2 steps in the processing of angiotensinogen (AGT) to Ang II and has been reported to be expressed in numerous tissues, including the brain. Indeed, there is now clear evidence that all components of the RAS are expressed within the brain. AGT exhibits a widespread distribution in astroglia throughout the brain but a much more selective regional distribution in neurons (7–9). Renin was previously identified in neurons (10, 11), and using a renin promoter–directed enhanced GFP (EGFP) gene, we reported that renin-expressing cells are located in or near regions controlling cardiovascular function and are primarily neurons (12, 13). Ang II, like other neurotransmitters, has been localized in neuronal processes and in the vicinity of nerve terminals (14). That renin and AGT are located in close proximity to Ang II receptor–containing neurons suggests a model for local production and action of Ang II. Renin released from neurons would initiate the processing of AGT released from glial cells (and perhaps neurons), eventually forming Ang II, which binds locally to Ang II receptor–containing neurons. Other evidence supporting the existence of an alternative form of renin in the brain that is not secreted, coupled with the observation that some neurons appear to coexpress both renin and AGT (and angiotensin converting enzyme), suggests the potential for intracellular generation of Ang II (15–18). Whether this Ang II is packaged into secretory or synaptic vesicles has yet to be determined experimentally.

In the brain, the subfornical organ (SFO) has long been known to be an important cardiovascular control region regulating fluid homeostasis in response to Ang II (19). However, the SFO is one of several circumventricular organs that lie outside the blood-brain barrier and thus have access to circulating and intraventricular Ang II. Indeed, systemic Ang II causes drinking that is mediated by the SFO (19–21). This, coupled with the observation that AGT is abundantly expressed in the SFO, suggests the possibility that SFO Ang II may either be derived (taken up) from the circulation or cerebrospinal fluid (CSF) or synthesized locally from AGT (22). Angiotensinergic connections between the SFO and hypothalamic nuclei that control drinking have been described in detail (23–25). Consequently, the purpose of this study was to examine the physiological consequences on fluid homeostasis of regional production of Ang II derived from local renin and AGT. Genetic techniques that allow for spatiotemporal control of gene expression and ablation in mice provide innovative tools with which to experimentally dissect complex regulatory systems such as the RAS in the brain (26, 27). To test the hypothesis that the SFO plays a role in the dipsogenic actions of Ang II derived from the local synthesis of renin and AGT, we used a combination of cell-specific promoters to drive renin and AGT synthesis (28), an AGT gene modified for cell-specific deletion by Cre recombinase (29–31), and the stereotaxic delivery of adenoviruses encoding Cre recombinase (AdCre) to the SFO (27, 31). We report that targeted expression of hREN and hAGT resulted in a marked increase in water and salt intake, which significantly decreased in response to the blockade of AT1 receptors in the brain. There mice exhibited elevated Ang II specifically in the SFO. SFO-specific ablation of hAGT synthesis by Cre recombinase resulted in a significant decrease in hAGT and Ang II immunostaining in the SFO and correlated with a significant decrease in water intake. These results provide genetic evidence that local de novo synthesis of Ang II in the SFO plays an integral role in regulating fluid hemostasis.

Our recent studies using a dual-reporter transgenic model in which renin- and AGT-expressing cells were identified with EGFP and β-galactosidase, respectively, indicated close localization of renin- and AGT-containing cells in a number of cardiovascular control regions, with renin primarily localized to neurons (12, 13). Therefore, in order to overexpress Ang II in the brain while retaining the normal cellular distribution of Ang II formation, we bred mice expressing human AGT (hAGT) under the control of its own endogenous promoter with mice expressing human renin (hREN) under the control of the neuron-specific synapsin I promoter (SR mice). We previously reported that the source of circulating hAGT in the hAGT mouse is the liver, whereas in the brain, hAGT is expressed in both astrocytes and neurons (22, 32). In SR mice, we confirmed that the expression of hREN in this model is restricted to neurons and is not released into the systemic circulation (28). Because of the species specificity of the renin-AGT enzymatic reaction and the absence of circulating hREN, double-transgenic mice expressing hREN from a neuron-specific promoter and hAGT from its own promoter (SRA mice) would be expected to have increased synthesis of Ang II specifically in the brain.

We first measured arterial pressure in SRA mice by radiotelemetry and observed a significant increase in arterial pressure (121 ± 6 mmHg in SRA mice versus 97 ± 4 mmHg in nontransgenic mice, n = 7; P = 0.004). We then focused entirely on water and electrolyte homeostasis and compared water intake, urinary volume, and salt intake between SRA mice and control littermates. SRA mice exhibited a nearly 3-fold increase in baseline water intake (Figure 1A). Accordingly, there was a marked elevation in the production of urine (Figure 1B), which was osmotically dilute (Table 1). When given a choice of tap water or hypertonic saline, the SRA mice exhibited an increased preference (2.3-fold) for sodium consumption (Figure 1C). To determine whether the increase in drinking was due to an Ang II receptor, type 1–dependent (AT₁-dependent) mechanism, we chronically infused losartan using an icv microinfusion pump in separate groups of SRA and control mice. Chronic icv infusion of losartan significantly reduced water intake, urinary volume, and salt intake in both SRA and control mice (Table 2), but the decrease was significantly greater in double-transgenic SRA mice (Figure 2). Chronic icv infusion of artificial cerebrospinal fluid (aCSF) did not affect water intake (1.6 ml/10 g body weight [BW]/d versus 1.5 ml/10 g BW/d; P = 0.57) in control mice. Importantly, chronic subcutaneous infusion of the same dose of losartan did not affect water intake in SRA mice (5.4 ml/10 g BW/d versus 5.1 ml/10 g BW/d; P = 0.95).

Figure 1

Volume homeostasis in SRA mice. Water intake (A), urine volume (B), and salt intake (C) in SRA mice (n = 14) and control littermates (Con; n = 25). *P < 0.001 compared with control mice.

Figure 2

The effect of chronic icv administration of losartan on volume homeostasis in SRA mice. Change in water intake (A), urine volume (B), and salt intake (C) in SRA mice (n = 9) and nontransgenic mice (n = 6) in response to losartan administered icv. *P = 0.007 (A); **P < 0.001 (B); ^#P = 0.002 (C) compared with control mice.

Table 1

Electrolyte homeostasis

Table 2

Fluid homeostasis in SRA mice before and after icv administration of losartan

One of the first phenotypes we observed was a smaller number of weaned SRA offspring than would be expected based on Mendelian inheritance. From a total 2,097 offspring from 123 breeding females, only 259 (in contrast to the expected 524) offspring were genotyped at weaning as double transgenic (P < 0.0001, χ² test). There was no apparent difference in the number of nontransgenic (SR^–/A^–, n = 653) or single-transgenic mice (SR^–/A⁺ or SR⁺/A^–, n = 1,185) compared with what was expected, and once SRA mice survived to weaning, there was little mortality observed thereafter. Moreover, there was no difference in the litter size of offspring derived from a breeding of SRA mice (5.9 ± 1.9 pups/litter from 86 litters) compared with other double-transgenic strains, in which no mortality was observed (6.0 ± 1.3 pups per litter from 66 litters; P = 0.72) (33). To determine a possible reason for mortality in young SRA mice, we measured sodium and potassium output in the survivors. We observed a significant increase in both sodium and potassium output, which suggested the possibility that newborn SRA mice may have had difficulty concentrating their urine or retaining electrolytes (Table 1).

We next performed immunohistochemistry to assay for the location of angiotensin peptide production in the brain of SRA mice. At the regional level, we observed a dramatic increase in Ang II–like immunoreactivity in the SFO of SRA mice (in Figure 3, compare B with A and E with D). The specificity of the immunohistochemistry was confirmed by preabsorption with the immunizing peptide (data not shown). Interestingly, this was the only region where an obvious increase in Ang II immunostaining was evident. This observation lead us to hypothesize that the increase in drinking was due to increased de novo production of Ang II in the SFO.

Figure 3

Localization of angiotensin peptide in the brain of SRA and SRA^flox mice. Representative photomicrographs of immunostaining for Ang I/II peptide in the SFO by immunofluorescence (A–C) and immunoperoxidase (D and E) in nontransgenic control (A and D), SRA (B and E), and SRA^flox (C) mice. n = 4 or more mice. Scale bars: 0.4 mm (A–C); 0.1 mm (D and E).

To directly test this hypothesis and to distinguish de novo synthesis of Ang II in the SFO from synthesis of circulating or ventricular Ang II detected by the SFO, we examined the drinking response after targeted ablation of Ang II substrate (AGT) synthesis in the SFO. We took advantage of a genetic tool that we believe to be novel in order to accomplish this goal. We employed what we believe is a new double-transgenic strain (SRA^flox), which was bred from a synapsin-hREN mouse (same as that used to generate SRA mice) crossed with a transgenic mouse engineered to express a modified hAGT containing loxP sites surrounding exon II (hAGT^flox) (29–31). The presence of loxP sites surrounding the major coding exon of the gene provides a genetic means of ablating the exon in the presence of Cre recombinase (27). In the absence of Cre recombinase, the hAGT^flox gene is fully functional and expresses hAGT. However, when Cre recombinase is expressed, the gene is functionally crippled because of the deletion of exon II (29). Expression of Cre recombinase was achieved by stereotaxic microinjection of AdCre directly into the SFO (27, 31).

We previously reported that the hAGT^flox and hAGT transgenes exhibit the same tissue-specific and cell-specific pattern of expression (29, 30). However, the level of hAGT expression is higher in the brain of hAGT^flox mice than in the brain of hAGT mice (Figure 4A). An initial evaluation of 23 randomly chosen SRA^flox mice revealed a marked increase in water intake averaging 6.8 ± 0.6 ml/10 g BW/d and a concomitant increase in urine volume (6.7 ± 0.8 ml/10 g BW/d) and salt intake (5.7 ± 0.5 μg/g BW/d). Although the magnitude of the drinking response was less, a highly significant increase in water intake (P < 0.001) was also observed in a second independent study pairing SRA^flox mice with age- and sex-matched littermate controls (Figure 4B). SRA^flox mice also exhibited an increase in angiotensin peptides in the SFO (Figure 3C).

Figure 4

Expression hAGT in the brain and water intake in SRA^flox mice. (A) An RNase protection assay of total RNA purified from independent brain samples removed from 3 SRA and 3 SRA^flox mice. The positions of the hAGT- and 28S-protected bands are shown. (B) Water intake in SRA^flox (n = 4) and age- and sex-matched littermate controls (n = 9). *P < 0.001 compared with control mice.

Next, we directly targeted the SFO by microinjection of either adenovirus encoding EGFP (AdEGFP) alone or both AdEGFP and AdCre together. Seven days after the microinjection, site-specific expression of EGFP on brain sections was confirmed by examination with a fluorescent stereomicroscope (Figure 5A). To confirm Cre-mediated recombination, we obtained punches of brain tissue from EGFP-positive and -negative brain segments and performed RT-PCR to confirm the presence of either the normal hAGT mRNA encoding exons 1–5 or the Cre-deleted hAGT mRNA lacking exon 2 (direct splicing of exons 1 to 3, as previously reported in ref. 29). Cre-mediated recombination was evident in those segments exhibiting EGFP staining but not in control segments (Figure 5B). At the cellular level, we confirmed that the level of hAGT protein in the SFO was unaltered in mice receiving only AdEGFP and that hAGT and EGFP were clearly coexpressed in the same cells (Figure 6, A–C). A few cells neighboring the SFO were also observed to express EGFP but not hAGT (arrows in Figure 6C), indicating transduction of cells both in and just outside the SFO. On the contrary, there was a marked reduction in hAGT staining in the SFO of mice receiving both AdEGFP and AdCre, specifically in those cells expressing the EGFP reporter (Figure 6, D–F). Note that there was no overlap in the staining of hAGT and EGFP (the marker of Cre recombinase) except for perhaps 1 or 2 cells (arrow in Figure 6F).

Figure 5

Site-specific deletion of hAGT mRNA in the SFO. (A) Representative brain slice from an SRA^flox mouse coinfected with AdEGFP and AdCre and photographed under a fluorescence stereomicroscope. Selected from over 12 photos each from independent mice (n = 12). Brain tissue from an EGFP-positive region and from a control region (EGFP–) were carefully dissected for the experiment in B. (B) RT-PCR reaction for hAGT mRNA from brain punches derived from the EGFP-positive and -negative regions indicated in A.

Figure 6

Site-specific deletion of hAGT protein in the SFO. Representative coronal sections through the SFO from mice injected with either AdEGFP alone or both AdEGFP and AdCre directly into the SFO. AGT was localized by immunofluorescence (red) and EGFP by its intrinsic fluorescence (green). A–C show colocalization of AdEGFP and AGT but no deletion or diminution of AGT staining. The arrows in C indicate EGFP-positive, hAGT-negative cells adjacent to the SFO. D–F show loss of AGT in regions where the virus mixture (AdEGFP and AdCre) is located. The arrow in F represents a cell in which hAGT was not ablated, which may represent a cell infected with AdEGFP but not AdCre.

We next measured water intake before and after injection of AdCre. In the control groups, there was no effect on drinking in response to AdEGFP injection into SRA^flox mice or AdCre/AdEGFP injection into SRA mice lacking hAGT^flox (Figure 7A). There was also no decrease in water intake in SRA^flox mice in which we confirmed that AdCre and AdEGFP missed the SFO. On the contrary, AdEGFP/AdCre-transfected SRA^flox mice in which the SFO-specific microinjection was confirmed by EGFP fluorescence and RT-PCR exhibited a significant decrease in water intake that reached a nadir 2 days after injection and remained low for the next 6 days (P < 0.05) (Figure 7A). A comparison of the change in water intake over the 8-day period after AdCre/AdEGFP injection into SRA^flox mice that either missed or hit the SFO clearly demonstrates that local hAGT production in the SFO was required to mediate the elevated drinking (Figure 7B).

Figure 7

Effect of AdCre transfection into the SFO on water intake. (A) Drinking volume of water before and after SFO-directed injection (arrow) of AdCre/AdEGFP into SRA mice lacking loxP sites (filled squares; n = 8); AdEGFP alone into SRA^flox mice (filled triangles; n = 8); AdCre/AdEGFP into SRA^flox mice, where the injection missed the SFO (filled diamonds; n = 21); and AdCre/AdEGFP into SRA^flox mice, where the injection into the SFO was confirmed (filled circles; n = 8). Data were analyzed using 2-way repeated measures ANOVA with the Bonferroni posthoc test. *P < 0.05 versus SRA^flox mice injected with AdEGFP; ^†P < 0.05 versus SRA^flox mice injected with AdCre, which missed the SFO; ^‡P < 0.05 versus SRA mice lacking loxP sites injected with AdCre/AdEGFP. (B) Change in water intake by mice after SFO-directed injection of AdCre/AdEGFP into SRA^flox mice, where the injection missed the SFO (open circles) and of AdCre/AdEGFP into SRA^flox mice, where the injection into the SFO was confirmed (filled circles). *P < 0.003.

Finally, to confirm that the decrease in drinking was due to decreased angiotensin peptides in the SFO, we performed immunocytochemistry for angiotensin I/II in the SFO of SRA^flox mice that were either injected with AdEGFP alone (Figure 8A) or with AdCre/AdEGFP (Figure 8B). These data clearly show a significant decrease in angiotensin peptide staining after AdCre injection that matches the background of the immunocytochemical assay evaluated by eliminating the primary antibody (Figure 8C). There was no decrease in angiotensin peptide in the SFO when the injection of AdCre/AdEGFP was confirmed to have missed the SFO (Figure 8D). Ultrastructural analysis showed strong angiotensin peptide staining in an SFO neuron in the nucleus and cytosol and vesicular structures of uninjected SRA^flox mice (Figure 9, A and B) but a significant decrease in electron-dense material after injection of AdCre/AdEGFP (Figure 9C).

Figure 8

Effect of AdCre on SFO Ang II. Immunocytochemistry for angiotensin I/II in the SFO of SRA^flox mice. (A) SRA^flox mouse injected with AdEGFP alone. (B) Diminished Ang I/II signal in SFO after SFO-directed injection of AdCre/AdEGFP. (C) Control SFO section from SRA^flox mice where the primary antibody directed against Ang I/II was omitted. (D) Retention of Ang I/II immunoreactivity in SFO after an injection of AdCre/AdEGFP that missed the SFO. The SFO is bordered by a dashed line. 3V, third ventricle. Scale bars: 100 μm.

Figure 9

Ultrastructural localization of Ang I/II in the SFO. (A) Strong expression of Ang I/II (arrows) in the nucleus, cytosol, and vesicular structures of SFO neurons of an uninjected SRA^flox mouse. (B) Fragment of cytoplasm of an SFO neuron in A containing immunoreactive vesicular structures (arrows). (C) Note the very low level of immunoreactivity around the neuronal nucleus (arrows) and absence of immunoreactivity in cytoplasm and neuropil after AdCre/AdEGFP was directly injected into the SFO. N, neuron. Scale bars: 2.0 μm (A and C); 1.0 μm (B).

There is now considerable evidence that all components of the RAS exist within the brain. At the cellular level, angiotensin II is located in neuronal somata and axons and at nerve terminals (14). Angiotensin receptors are abundantly expressed on postsynaptic membranes of cell bodies as well as on neuronal fibers (34, 35). The location of Ang II immunoreactive fibers as well as binding sites for Ang II correlate well with nuclei controlling blood pressure, water and electrolyte homeostasis, and baroreceptor reflex pathways (36). Exogenous administration of Ang II into Ang II receptor–containing regions of the brain elicits both blood pressure and drinking responses, and the blockade of endogenous Ang II production or action has effects on both (reviewed in ref. 37). Although the central actions of exogenous Ang II are well documented, the mechanisms controlling its de novo production in the brain remain very incomplete.

The Ang II substrate, AGT, is abundantly expressed and widely distributed in astroglia (8). Other reports suggest that AGT is also expressed in neurons, although in a much more regionally restricted manner (7, 22). Renin, on the other hand, has been difficult to identify because its level of expression is quite low and commonly available antisera directed at renin are only marginally effective for immunohistochemical methods. Using a pair of transgenic mice expressing sensitive reporter genes under the control of the renin and AGT promoters, we recently identified regions of the brain containing renin-expressing neurons and AGT-expressing cells that were both closely localized (adjacent) and colocalized (expressed in the same cell) (13). We therefore developed the SRA and SRA^flox models employed herein to assess the physiological consequences of elevated de novo Ang II production under conditions in which renin and AGT were correctly expressed in a cell-specific manner. In our model, the synapsin I promoter targeted expression of renin to neurons, while the endogenous AGT promoter targeted its expression to glial cells and, importantly, the correct population of AGT-expressing neurons (22).

This cell-appropriate overexpression of Ang II resulted in a striking increase in water intake, salt preference, and urine output. Immunostaining revealed that the SFO was the only nucleus with an obvious increase in immunoreactive angiotensin peptides. Interestingly, inspection of multiple brain sections did not provide convincing evidence for elevated Ang II production in other nuclei implicated in drinking, such as the median preoptic nucleus (MnPO) or the paraventricular nucleus (PVN). Consequently, it is possible that in this model system, the SFO was the only nucleus where hREN and hAGT were sufficiently closely localized to generate Ang II. Because the promoter elements used to target expression of the hREN and hAGT genes emulated their natural cellular pattern of expression, one might hypothesize that the SFO is one of the primary locations for de novo synthesis of Ang II in the brain. Nevertheless, future studies will have to address whether enhanced production of Ang II, perhaps at a lower level than is easily detected by immunocytochemistry, occurred in other nuclei regulating fluid homeostasis. Similarly, as our experiment is admittedly an overexpression study, future studies must address whether local production of Ang II in the SFO is required to mediate drinking responses under normal physiological conditions.

The SFO has long been thought to be a critical integrating center for regulating many of the physiological responses of Ang II, including drinking (19). Neurons in the SFO are rich in AT₁ receptors, and Ang II immunoreactive fibers can be traced from the SFO to other regions implicated in homeostatic control (24). Destruction of these pathways by lesioning the SFO or regions such as the MnPO, which contain angiotensinergic projections from the SFO, or transection of these neural pathways abolishes the drinking response to i.v. or locally administered Ang II (24, 25, 38). That elevated drinking in the SRA model was reduced to baseline when losartan was delivered chronically into the brain strongly supports the dipsogenic response as being AT₁ mediated. Our previous studies suggest the AT_1B subtype of AT₁ receptors may mediate the dipsogenic responses to Ang II (39). Although the drinking response was completely normalized by AT₁ receptor blockade, future experiments will be required to assess the relative contribution of AT_1A and AT_1B receptors or the role, if any, of AT₂ receptors, which have also been implicated in regulating the drinking response to central Ang II (40).

The SFO is among a number of circumventricular organs that have access to the circulation. Several models have been proposed to explain the mechanism of angiotensinergic stimulation of the SFO (reviewed in ref. 41). In one model, an SFO neuron takes up circulating Ang II by a receptor-mediated mechanism and then releases Ang II inside the cell. The internalized Ang II then undergoes axonal transport and is ultimately released as a neurotransmitter at the synapse, where it binds to AT₁ receptors on the postsynaptic membrane of the connecting neuron or interneuron. Despite the implication that Ang II acts as a neurotransmitter between the SFO and its efferent targets, the one criterion that has long been lacking to classify Ang II as a neurotransmitter is de novo synthesis of Ang II in the presynaptic neuron (reviewed in ref. 41). It is interesting to note therefore that the SFO is one of the sites in the central nervous system containing both neuronal AGT and renin, which suggests the potential for de novo local synthesis of Ang II even when circulating levels of Ang II are normal (13, 22). Our data showing that ablation of Ang II substrate synthesis specifically in the SFO markedly diminishes Ang II production in the SFO and attenuates drinking provides compelling evidence for the local production of Ang II in the SFO and its role in signaling the dipsogenic response. The data also complement our previous studies demonstrating that ablation of SFO AGT attenuates the pressor response to an icv infusion of recombinant hREN (31). These data therefore provide an additional criterion implicating Ang II as a neurotransmitter originating from neuron cell bodies located in the SFO. Our data also suggest the possibility that there are “microdomains” of Ang II synthesis in critical regions of the CNS controlling Ang II–dependent neural output and that Ang II production in these microdomains may be under tight physiological regulation. One of these regions, the rostral ventral lateral medulla (RVLM), controls sympathetic outflow and expresses renin and AGT locally (13). A recent, elegant study by Allen et al. showed that targeting a constitutively active AT₁ receptor to glial cells in the RVLM caused hypertension (42). One model posited by the authors is that glial AT₁ receptors may modulate the synthesis, and perhaps release, of glial AGT, thus providing a regulatory circuit controlling the level of Ang II substrate available to interact with neuronal renin.

In interpreting our results, we recognize that the level of drinking was not completely normalized after SFO-specific delivery of Cre recombinase. This would be consistent with either an incomplete ablation of Ang II production in the SFO or local Ang II synthesis in other hypothalamic nuclei controlling drinking (43). Whereas our immunocytochemical data show efficient ablation of hAGT synthesis in EGFP-labeled cells, the extent of EGFP reporter expression in the SFO varied among experimental mice. Consequently, the most likely explanation is incomplete ablation of Ang II production in the SFO. However, although we did not obtain convincing evidence for elevated Ang II production in other nuclei implicated in drinking, the importance of these nuclei cannot be ruled out by the studies performed herein. Consequently, studies must be undertaken to determine whether de novo Ang II production in other circumventricular organs or in nuclei along the neural pathway initiated in the SFO is necessary for regulating angiotensinergic dipsogenic responses initiated in the SFO. Whereas our results indicate that de novo synthesis of Ang II in the SFO is necessary for increased water intake, studies are currently underway to assess whether Ang II production in the SFO and elsewhere is sufficient to mediate increased water intake.

Along these lines, we and others have provided convincing evidence that the Cre-loxP recombinase system offers a unique tool to dissect neural pathways in the brain (31). At the level of the whole brain, cell-specific promoters can be used to target Cre recombinase expression to specific cell types (i.e., neurons or glia). Although our ability to target specific populations of neurons has improved (44, 45), examining regional angiotensinergic pathways has become more difficult, as promoters that have the ability to target specific regions of the brain, such as the SFO or MnPO, have yet to be identified. Certainly will be one of the long-anticipated outcomes of the brain genome and mapping projects (46). In the meantime, viral delivery of Cre recombinase provides an innovative tool for the region-specific ablation or even activation of gene expression (47). Recent technological developments that employ retrograde transport of infected viruses or the cellular tropism of specific viruses have provided a means to target specific cell types in the brain, follow specific neuronal pathways, and ablate expression of genes at sites distant from the sites of infection (27, 48). Although we did not observe evidence for retrograde transport of AdEGFP from the SFO in our experimental system, studies could be performed in which the virus is injected into the PVN or MnPO to assess the relative contribution of the SFO-MnPO and SFO-PVN pathways. The next obvious step will be to use the molecular tools developed herein to dissect the neuronal pathways mediating the drinking response.

This work was supported by grants from the NIH (HL48058, HL61446, and HL55006 to C.D. Sigmund; HL63887 and HL14388 to R.L. Davisson), the American Heart Association (0030017N and 0540114N to R.L. Davisson). K. Sakai was previously funded by the Japan Society for the Promotion of Science. P. Sinnayah was funded by a postdoctoral fellowship from the American Heart Association (0225723Z). K. Agassandian was funded by the Center on Functional Genomics of Hypertension. Transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, supported by the Roy J. and Lucille A. Carver College of Medicine. AdCre and AdEGFP viruses were obtained from the University of Iowa Vector Core, DNA sequencing was performed at the University of Iowa DNA Core, and the dissecting fluorescence microscope was used in the Central Microscopy Facility of the University of Iowa. We gratefully acknowledge the generous research support of the Roy J. Carver Charitable Trust.

Address correspondence to: Curt D. Sigmund, Departments of Internal Medicine and Molecular Physiology and Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. Phone: (319) 335-7604; Fax: (319) 353-5350; E-mail: curt-sigmund@uiowa.edu.

Nonstandard abbreviations used: aCSF, artificial cerebrospinal fluid; AdCre, adenovirus encoding Cre recombinase; AdEGFP, adenovirus encoding EGFP; AGT, angiotensinogen; AT₁, Ang II receptor, type 1; BW, body weight; CSF, cerebrospinal fluid; EGFP, enhanced GFP; hAGT, human AGT; hREN, human renin; MnPO, median preoptic nucleus; PVN, paraventricular nucleus; RAS, renin-angiotensin system; SFO, subfornical organ; SRA mice, double-transgenic mice expressing hREN from a neuron-specific promoter and hAGT from its own promoter.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:1088–1095 (2007). doi:10.1172/JCI31242

See the related article at The renin-angiotensin system: it’s all in your head.

1. Johnson, A.K., Epstein, A.N. 1975. The cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin. Brain Res. 86:399-418.
   View this article via: PubMed Google Scholar
2. Buggy, J., Fisher, A.E., Hoffman, W.E., Johnson, A.L., Phillips, M.I. 1975. Ventricular obstruction: effect on drinking induced by intracranial injection of angiotensin. Science. 190:72-74.
   View this article via: PubMed Google Scholar
3. Buggy, J., Fisher, A.E. 1974. Evidence for a dual central role for angiotensin in water and sodium intake. Nature. 250:733-735.
   View this article via: PubMed Google Scholar
4. Phillips, M.I., et al. 1977. Lowering of hypertension by central saralasin in the absence of plasma renin. Nature. 270:445-447.
   View this article via: PubMed Google Scholar
5. DiNicolantonio, R. 1984. Angiotensin converting enzyme blockade and thirst. Clin. Exp. Hypertens. A. 6:2025-2029.
   View this article via: PubMed Google Scholar
6. Epstein, A.N., Fitzsimons, J.T., Johnson, A.K. 1973. Prevention by angiotensin II antiserum of drinking induced by intracranial angiotensin. J. Physiol. 230:42P-43P.
   View this article via: PubMed Google Scholar
7. Thomas, W.G., Sernia, C. 1988. Immuno­cytochemical localization of angiotensinogen in the rat brain. Neuroscience. 25:319-341.
   View this article via: PubMed Google Scholar
8. Stornetta, R.L., Hawelu-Johnson, C.L., Guyenet, P.G., Lynch, K.R. 1988. Astrocytes synthesize angiotensinogen in brain. Science. 242:1444-1446.
   View this article via: PubMed Google Scholar
9. Imboden, H., Harding, J.W., Hilgenfeldt, U., Celio, M.R., Felix, D. 1987. Localization of angiotensinogen in multiple cell types of rat brain. Brain Res. 410:74-77.
   View this article via: PubMed Google Scholar
10. Fuxe, K., et al. 1980. Renin-like immuno­cyto­chemical activity in the rat and mouse brain. Neurosci. Lett. 18:245-250.
    View this article via: PubMed Google Scholar
11. Slater, E.E., Defendini, R., Zimmerman, E. 1980. Wide distribution of immunoreactive renin in nerve cells of human brain. Proc. Natl. Acad. Sci. U. S. A. 77:5458-5460.
    View this article via: PubMed Google Scholar
12. Lavoie, J.L., Cassell, M.D., Gross, K.W., Sigmund, C.D. 2004. Localization of renin expressing cells in the brain using a REN-eGFP transgenic model. Physiol. Genomics. 16:240-246.
    View this article via: PubMed Google Scholar
13. Lavoie, J.L., Cassell, M.D., Gross, K.W., Sigmund, C.D. 2004. Adjacent expression of renin and angiotensinogen in the rostral ventrolateral medulla using a dual-reporter transgenic model. Hypertension. 43:1116-1119.
    View this article via: PubMed Google Scholar
14. Lind, R.W., Swanson, L.W., Ganten, D. 1985. Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. An immunohistochemical study. Neuroendocrinology. 40:2-24.
    View this article via: PubMed Google Scholar
15. Sinn, P.L., Sigmund, C.D. 2000. Identification of three human renin mRNA isoforms resulting from alternative tissue-specific transcriptional initiation. Physiol. Genomics. 3:25-31.
    View this article via: PubMed Google Scholar
16. Re, R.N. 2003. Intracellular renin and the nature of intracrine enzymes. Hypertension. 42:117-122.
    View this article via: PubMed Google Scholar
17. Lee-Kirsch, M.A., Gaudet, F., Cardoso, M.C., Lindpaintner, K. 1999. Distinct renin isoforms generated by tissue-specific transcription initiation and alternative splicing. Circ. Res. 84:240-246.
    View this article via: PubMed Google Scholar
18. Pickel, V.M., Chan, J., Ganten, D. 1986. Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ. J. Neurosci. 6:2457-2469.
    View this article via: PubMed Google Scholar
19. Simpson, J.B., Routtenberg, A. 1973. Subfornical organ: site of drinking elicitation by angiotensin II. Science. 181:1172-1175.
    View this article via: PubMed Google Scholar
20. Fitzsimons, J.T. 1998. Angiotensin, thirst, and sodium appetite. Physiol. Rev. 78:583-686.
    View this article via: PubMed Google Scholar
21. Simpson, J.B., Routtenberg, A. 1978. Subfornical organ: a dipsogenic site of action of angiotensin II. Science. 201:379-381.
    View this article via: PubMed Google Scholar
22. Yang, G., Gray, T.S., Sigmund, C.D., Cassell, M.D. 1999. The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system. Brain Res. 817:123-131.
    View this article via: PubMed Google Scholar
23. Hoffman, W.E., Phillips, M.I. 1976. The effect of subfornical organ lesions and ventricular blockade on drinking induced by angiotensin II. Brain Res. 108:59-73.
    View this article via: PubMed Google Scholar
24. Eng, R., Miselis, R.R. 1981. Polydipsia and abolition of angiotensin-induced drinking after transections of subfornical organ efferent projections in the rat. Brain Res. 225:200-206.
    View this article via: PubMed Google Scholar
25. Tanaka, J., Nomura, M. 1993. Involvement of neurons sensitive to angiotensin II in the median preoptic nucleus in the drinking response induced by angiotensin II activation of the subfornical organ in rats. Exp. Neurol. 119:235-239.
    View this article via: PubMed Google Scholar
26. Lavoie, J.L., Sigmund, C.D. 2003. Minireview: overview of the renin-angiotensin system—an endocrine and paracrine system. Endocrinology. 144:2179-2183.
    View this article via: PubMed Google Scholar
27. Sinnayah, P., et al. 2004. Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain. Physiol. Genomics. 18:25-32.
    View this article via: PubMed Google Scholar
28. Morimoto, S., Cassell, M.D., Sigmund, C.D. 2002. Glial- and neuronal-specific expression of the renin-angiotensin system in brain alters blood pressure, water intake, and salt preference. J. Biol. Chem. 277:33235-33241.
    View this article via: PubMed Google Scholar
29. Stec, D.E., Davisson, R.L., Haskell, R.E., Davidson, B.L., Sigmund, C.D. 1999. Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of cre-recombinase in vivo. J. Biol. Chem. 274:21285-21290.
    View this article via: PubMed Google Scholar
30. Stec, D.E., Keen, H.L., Sigmund, C.D. 2002. Lower blood pressure in floxed angiotensinogen mice after adenoviral delivery of cre-recombinase. Hypertension. 39:629-633.
    View this article via: PubMed Google Scholar
31. Sinnayah, P., et al. 2006. Genetic ablation of angiotensinogen in the subfornical organ of the brain prevents the central angiotensinergic pressor response. Circ. Res. 99:1125-1131.
    View this article via: PubMed Google Scholar
32. Yang, G., Merrill, D.C., Thompson, M.W., Robillard, J.E., Sigmund, C.D. 1994. Functional expression of the human angiotensinogen gene in transgenic mice. J. Biol. Chem. 269:32497-32502.
    View this article via: PubMed Google Scholar
33. Davisson, R.L., et al. 1998. The brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes. Circ. Res. 83:1047-1058.
    View this article via: PubMed Google Scholar
34. Glass, M.J., Kruzich, P.J., Colago, E.E., Kreek, M.J., Pickel, V.M. 2005. Increased AMPA GluR1 receptor subunit labeling on the plasma membrane of dendrites in the basolateral amygdala of rats self-administering morphine. Synapse. 58:1-12.
    View this article via: PubMed Google Scholar
35. Huang, J., et al. 2003. Angiotensin II subtype 1A (AT1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin. Neuroscience. 122:21-36.
    View this article via: PubMed Google Scholar
36. Bunnemann, B., et al. 1992. The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain. Neurosci. Lett. 142:155-158.
    View this article via: PubMed Google Scholar
37. Printz, M.P., Ganten, D., Unger, T., and Phillips, M.I. 1982. The brain renin-angiotensin system. In The renin-angiotensin system in the brain: a model for the synthesis of peptides in the brain. D. Ganten, M.P. Printz, M.I. Phillips, and B.A. Scholkens, editors. Springer-Verlag. Berlin, Germany. 3–52.
38. Simpson, J.B., Epstein, A.N., Camardo (Jr.), J.S. 1978. Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of rat. J. Comp. Physiol. Psychol. 92:581-601.
    View this article via: PubMed Google Scholar
39. Davisson, R.L., Oliverio, M.I., Coffman, T.M., Sigmund, C.D. 2000. Divergent functions of angiotensin II receptor isoforms in brain. J. Clin. Invest. 106:103-106.
    View this article via: JCI PubMed Google Scholar
40. Camara, A.K., Osborn, J. 2001. Central AT1 and AT2 receptors mediate chronic intracerebroventricular angiotensin II-induced drinking in rats fed high sodium chloride diet from weaning. Acta. Physiol. Scand. 171:195-201.
    View this article via: PubMed Google Scholar
41. Ferguson, A.V., Washburn, D.L., Latchford, K.J. 2001. Hormonal and neurotransmitter roles for angiotensin in the regulation of central autonomic function. Exp. Biol. Med. (Maywood). 226:85-96.
    View this article via: PubMed Google Scholar
42. Allen, A.M., et al. 2006. Expression of constitutively active angiotensin receptors in the rostral ventrolateral medulla increases blood pressure. Hypertension. 47:1054-1061.
    View this article via: PubMed Google Scholar
43. Tanaka, J., et al. 2001. Angiotensinergic and noradrenergic mechanisms in the hypothalamic paraventricular nucleus participate in the drinking response induced by activation of the subfornical organ in rats. Behav. Brain Res. 118:117-122.
    View this article via: PubMed Google Scholar
44. Xu, A.W., Ste-Marie, L., Kaelin, C.B., Barsh, G.S. 2007. Inactivation of signal transducer and activator of transcription 3 in proopiomelanocortin (Pomc) neurons causes decreased pomc expression, mild obesity, and defects in compensatory refeeding. Endocrinology. 148:72-80.
    View this article via: PubMed Google Scholar
45. Kwon, C.H., et al. 2006. Neuron-specific enolase-cre mouse line with cre activity in specific neuronal populations. Genesis. 44:130-135.
    View this article via: PubMed Google Scholar
46. Gray, P.A., et al. 2004. Mouse brain organization revealed through direct genome-scale TF expression analysis. Science. 306:2255-2257.
    View this article via: PubMed Google Scholar
47. Kilby, N.J., Snaith, M.R., Murray, J.A.H. 1994. Site-specific recombinases: tools for genome engineering. Trends Genet. 9:413-421.
    View this article via: PubMed Google Scholar
48. Sinnayah, P., et al. 2002. Selective gene transfer to key cardiovascular regions of the brain: comparison of two viral vector systems. Hypertension. 39:603-608.
    View this article via: PubMed Google Scholar
49. Hatae, T., Takimoto, E., Murakami, K., Fukamizu, A. 1994. Comparative studies on species-specific reactivity between renin and angiotensinogen. Mol. Cell. Biochem. 131:43-47.
    View this article via: PubMed Google Scholar
50. Franklin, K.B.J., and Paxinos, G. 1997. The mouse brain in stereotaxic coordinates. Academic Press. San Diego, California, USA. 216 pp.
51. Morimoto, S., Cassell, M.D., Sigmund, C.D. 2002. Neuron-specific expression of human angiotensinogen in brain causes increased salt appetite. Physiol. Genomics. 9:113.
    View this article via: PubMed PubMed Google Scholar