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The centrally mediated cardiovascular regulatory actions of angiotensin II (Ang II) in normal and hypertensive rats include Ang II type 1 receptor (AT1R)-mediated actions at the paraventricular nucleus (PVN) of the hypothalamus. Since the PVN consists of multiple neuronal populations, it is important to understand which neuronal types in the PVN are influenced by Ang II. Here we have developed a viral vector (AAV2-PAG-eGFP; [PAG, phosphate activated glutaminase promoter]) to drive expression of green fluorescent protein (GFP) primarily within glutamate neurons. At 10 to 14 days following bilateral microinjection (200 nL/side; 1.2 × 1012 genome copies [gc]) of AAV2-PAG-eGFP into adult Sprague Dawley rat PVN, animals were euthanized, brains removed and used for isolation and culture of PVN neurons. Fluorescence microscopy and immunostaining using neuron and PAG-specific antibodies revealed the presence of GFP-containing glutamatergic neurons in these PVN cultures. Whole cell patch clamp recordings demonstrated that Ang II (100 nmol/L) produced a 16% decrease in delayed rectifier potassium current (Ikv) in ~50% of the GFP-containing neurons, an effect that was abolished by the AT1R antagonist losartan (1 μmol/L). Consistently, 9 of 28 GFP/PAG-expressing neurons contained AT1R mRNA, as indicated by single cell RT-PCR. Furthermore, certain of the GFP/PAG-positive neurons in the PVN that project to the rostral ventrolateral medulla (RVLM) of the brainstem express immunoreactive AT1R. In conclusion, we have demonstrated the presence of functional AT1R on PAG-positive (largely glutamate) neurons within rat PVN, certain of which project to the RVLM.
The paraventricular nucleus (PVN) of the hypothalamus has a key role in the regulation of sympathetic outflow, neuroendocrine secretions and behavioral homeostatic responses.1 Activation of PVN neurons contributes to increases in sympathetic tone associated with conditions such as hypertension,2 water deprivation,3, 4 increased plasma osmolality,5 and heart failure.6, 7 The PVN contains heterogeneous populations of neurons that can be differentiated based on either morphological, phenotypic or electrophysiological criteria. For example, the PVN contains glutamate, oxytocin (OT), vasopressin (AVP), corticotropin-releasing hormone (CRH), enkephalin (ENK), and a small number of γ-aminobutyric acid (GABA) neurons. In general, from a morphological standpoint the PVN can be separated into large magnocellular neurons and smaller parvocellular neurons. The magnocellular neurons synthesize and secrete OT or AVP into the circulation from terminals at the posterior pituitary. The smaller parvocellular neurons, which include populations of most of the other transmitters as well as AVP and OT neurons1 have different projection sites. Some parvocellular neurons influence sympathetic outflow by projecting to brainstem cardiovascular regulatory sites such as the rostral ventrolateral medulla (RVLM), or directly to preganglionic sympathetic neurons in the intermediolateral column (IML) of the spinal cord. Other parvocellular neurons (CRH) project to the median eminence and influence cardiovascular regulation particularly during stress. Magnocellular and parvocellular neurons can also be distinguished by different threshold of action potential and A-type K+ current properties.8
Due to its central role in the regulation of sympathetic outflow, it is important to understand the factors that modulate the activity of PVN neurons. One such factor is angiotensin II (Ang II), which acts in the brain via its type 1 receptors (AT1R) to increase sympathetic outflow and blood pressure, 9 and the high densities of AT1R present in the PVN mediate the pressor action of centrally injected Ang II.10 Over activity of CNS AT1R, including those in the PVN, contributes to neurogenic hypertension.11 Evidence from autoradiographic,12 in situ hybridization,13 immunostaining14 and electrophysiological15 analyses indicates that AT1R are localized on parvocellular neurons in the PVN. By contrast there is little evidence that magnocellular neurons contain AT1R.16 Further, while it is known that Ang II elicits AT1R-mediated excitation of PVN neurons including those that innervate the RVLM,17 the location of these AT1R (pre-or postsynaptic, and on which neuronal phenotype) is not established. Electrophysiological data supports a presynaptic location of AT1R in the PVN,18, 19 but evidence for a postsynaptic location of these sites, especially on neurons that innervate brainstem or spinal cord cardiovascular control centers, is lacking.
Since previous studies demonstrated that many of the sympathetic regulatory neurons that project from the PVN to the RVLM are glutamatergic,4 we focused on PVN glutamate neurons as a possible postsynaptic location of AT1R. The present data provide evidence that a population of AT1R are located postsynaptically on PVN neurons that contain the glutamate synthetic enzyme phosphate activated glutaminase (PAG), and that certain of these AT1R bearing neurons innervate the RVLM.
Adult male Sprague Dawley (SD) rats were obtained from Charles River Laboratories (Wilmington, Mass). Rats were kept on a 12-hour light/dark cycle in a climate-controlled room. Rat chow and water were provided ad libitum. Animal use protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida. Details of the chemicals used in this study are available in the online supplement at http://hyper.ahajournals.org.
Neuronal cultures were prepared from a block of hypothalamic tissue containing the PVN or from the parietal cortex of 5–7 -week-old SD rats as detailed in the online supplement at http://hyper.ahajournals.org.
Construction of the recombinant vector AAV2-PAG-eGFP-WPRE was performed as detailed in the online supplement at http://hyper.ahajournals.org. PAG is phosphate activated glutaminase, a key enzyme in glutamate synthesis, catalyzing the conversion of glutamine to glutamate in neurons. The PAG promoter drives gene expression primarily within glutamate neurons, and also in a small percentage of GABA neurons.20 However, the PVN contains very few GABAergic neurons, and those present are within the peripheral margin of the nucleus4,21. Most GABA neurons in this region surround, but are not within, the PVN.4,21 Taking this into account, the strategy that we utilized to microinject AAV2-CBA-eGFP-WPRE into the PVN (see below) minimizes the possibility that GFP expression will be driven in GABA neurons.
AAV2-PAG-eGFP-WPRE (200nL/side/10 mins; 1.2× 1012 gc) was delivered bilaterally into the PVN, and rhodamine-labeled fluorescent microspheres (100nL/side; Red Retrobeads; Lumafluor, Naples, FL) were delivered bilaterally into the RVLM. Details of the procedures used are contained in the online supplement at http://hyper.ahajournals.org.
Recordings of delayed rectifier K+ current (IKv) from PVN neurons in culture were made using whole-cell patch clamp procedures in the voltage clamp mode, as detailed in the online supplement at http://hyper.ahajournals.org.
Levels of mRNA of AT1R, PAG, GAD67 (67kDa glutamic acid decarboxylase), AVP, OT, ENK, CRH and β-actin were analyzed via quantitative real-time RT-PCR using either Taqman or SYBR green kits (Bio-Rad, Hercules, CA), as detailed in the online supplement at http://hyper.ahajournals.org.
RT-PCR analysis of AT1R and β-actin in single PVN neurons in culture was performed as described in the online supplement at http://hyper.ahajournals.org.
PAG, GAD67, AT1R, NeuN (Neuron-specific nuclear protein) and GFAP (glial fibrillary acidic protein) immunostaining was performed as described in the online supplement at http://hyper.ahajournals.org.
Data are expressed as means ± SEM. Statistical significance was evaluated using Student’s t-tests. Differences were considered significant at P < 0.05, and individual P values are noted in the figure legends.
The overall goal of this study was to determine whether functional AT1R are localized postsynaptically to glutamatergic neurons in the PVN. We utilized two experimental approaches, both of which relied on the use of AAV2-PAG-eGFP to target GFP expression to PAG-containing neurons in the PVN. The PAG promoter drives gene expression primarily within glutamate neurons, as explained in the methods. The first approach was to microinject AAV2-PAG-eGFP into the PVN, and after 10–14 days isolate cells from the PVN, and use single cell RT-PCR and electrophysiology to determine whether individual green fluorescing neurons in culture (PAG-positive) contain functional AT1R. In a second complementary approach we again microinjected AAV2-PAG-eGFP into the PVN, and then utilized immunostaining to demonstrate that the green fluorescing neurons in the PVN in situ contain AT1R. Further, in this latter approach we employed retrograde labeling techniques to demonstrate that certain of the GFP expressing/AT1R positive neurons in the PVN project to the RVLM.
It was first essential to establish the procedures for isolation and culture of neurons from adult rat brain tissue containing the PVN, and their characterization with respect to expression of AT1R and PVN-typical neurotransmitters. A block of hypothalamic tissue containing the PVN was dissected from 4 SD rats, tissue pooled, cells isolated and cultured for at least 4 days as detailed in the methods. Approximately 40% of the isolated/cultured cells exhibited neuron-like morphology (Figure S1A), and their presence was confirmed by immunostaining using antibodies against the neuron-specific marker NeuN (Figure S1B). The remaining cells in these cultures were mostly astroglia, as evidenced by the presence of immunoreactive GFAP (Figure S1B). Real time RT-PCR analysis of the hypothalamic brain tissue used for isolation and culture of PVN cells revealed the presence of mRNAs for AVP, OT, ENK, CRH, PAG (used as a glutamate neuron marker) and GAD67 (used as a GABA neuron marker) [Figure S2A]. This indicates that the hypothalamic block contains transmitters that are typical of the PVN (AVP, OT, ENK, CRH, glutamate) or which surround the PVN (GABA).4 Parallel analyses of parietal cerebral cortical tissues revealed the expected high levels of ENK, CRH, PAG and GAD67, but neither OT nor AVP (Figure S2A), validating the RT-PCR procedure. RT-PCR analyses revealed that all of the above neuron types were maintained in the hypothalamic cells in culture (Figure S2B). The PVN contains a high density of AT1R13–18 and AT1R mRNA was present in the hypothalamic tissue used for isolation and culture of neurons, and in the cultures (Figure S2B, C). As expected, the hypothalamic cultures contain greater levels of AT1R mRNA compared with parietal cortex neurons in culture (Figure S2C). Single cell RT-PCR analysis revealed the presence of the AT1R gene in neurons from a representative dish of cells in culture (Figure S2D). The presence of functional AT1R in the hypothalamic cultures was confirmed by electrophysiological experiments. Ang II (100nmol/L) produced a significant decrease in delayed rectifier K+ current (Ikv) of hypothalamic neurons cultured from adult rats (mean ± SEM current density of IKv was 25.70±5.92 pA/pF in control neurons and 17.60±1.55 pA/pF in Ang II-treated neurons; n=7, p<.01), similar to its effects in newborn rat neurons. 22 This effect of Ang II was time-dependent, and was reversed by treatment with the AT1R antagonist losartan (1μmol/L) (Figures S3A,B). The data in Figure S3C demonstrate that Ang II produced respective 14.71±3.02% and 3.02±1.21% decreases in IKv in the absence or presence of losartan (cell capacitance was 20.11±1.58 pF; n=7 neurons; p<0.001). Collectively, these data indicate that we are able to isolate and culture neurons from a block of adult rat hypothalamus containing the PVN, that these cultures contain populations of neurons that are typical of the PVN, and that some of the isolated neurons express functional AT1R.
Based on the above experiments the cells that were isolated and cultured from the block of adult rat hypothalamus containing the PVN include glutamate neurons. To target this neuronal population for further experiments on AT1R localization and Ang II actions, we utilized the vector AAV2-PAG-eGFP to drive GFP expression primarily within glutamate neurons in the PVN. The AAV2-based vector was highly effective in vivo, as bilateral microinjection of AAV2-PAG-eGFP (200nL; 1.2 × 1012 gc/mL) into the PVN of rats elicited significant expression of GFP in this nucleus within 7–10 days, and expression persisted for several months after the injection (see representative fluorescence micrograph taken at 10 days after injection of AAV2-PAG-eGFP; Figure 1A). This PAG-driven GFP expression was largely specific to PVN glutamate neurons, as indicated by co-labeling with immunoreactive NeuN (neuron-specific marker; Figure 1B) and immunoreactive PAG (Figure 1C). AAV2-PAG-eGFP driven expression of GFP within GABA neurons (GAD67 immunopositive) was minimal. GFP expression did not co-localize with GAD67 within the PVN, and any co-localization was restricted to the area surrounding the PVN (Figure 2). Many of the GFP-expressing neurons within the PVN of AAV2-PAG-eGFP injected rats contain immunoreactive AT1R (Figure 3). Further, a number of the GFP-positive cells in the PVN are devoid of immunoreactive AT1R, and that AT1R are also present on GFP-negative cells (Figure 3). To determine if the AT1R on the GFP expressing PVN neurons are functional, neurons were isolated and cultured as above from hypothalamic tissue containing the PVN of rats that were microinjected with AAV2-PAG-eGFP (100nL/side; 1.2 × 1012 gc/mL) into the PVN 10 days previously. This yielded a population of cells that exhibited neuron-like morphology, green fluorescence and immunoreactive PAG (Figure 4A). Next we determined whether the GFP-expressing neurons contain AT1R using both single cell RT-PCR and electrophysiology. Single cell RT-PCR analysis of a random sample of 28 viable (β-actin positive) green fluorescing neurons revealed that 9 cells (~30%) expressed AT1R, based on the presence of a 125bp AT1R DNA fragment on agarose gels (Figure 4B). Ang II (100 nmol/L) produced a significant (~16%) decrease in IKv (current density of 37.51±4.63 pA/pF under control conditions versus 30.73±4.62 pA/pF in the presence of Ang II, P< 0.05; Cm was 18.22±1.93 pF) in 8 of 16 GFP-positive neurons tested (Figure 4C,D). As shown in Figure 4D, 50% of the selected green cells were not responsive to Ang II (IKv current density of 37.93±6.45 pA/pF under control conditions and 36.7±6.39 pA/pF in the presence of Ang II, p> 0.05, Cm was 19.49±1.89 pF). This inhibitory action of Ang II on IKv in the GFP expressing neurons was abolished by 1 μmol/L losartan (Figure 4E). Collectively, the data from Figures 1 through through44 suggest that a population of GFP expressing (PAG positive) neurons within the PVN contain functional AT1R. Considering the locus of the microinjections and the GFP expression, and the fact that few of the GFP-positive neurons are GABAergic, it is likely that the majority of GFP expressing (PAG positive) neurons are glutamatergic.
Many of the PVN neurons that project to the RVLM are glutamatergic.4 Thus, rats were injected into the PVN with AAV2-PAG-eGFP (200nL; 1.2× 1012 gc/mL) as above, and on the same days later received a unilateral injection of rhodamine labeled retrobeads (Red Retrobeads) into the RVLM. After a further 10–14 days, brains were removed and sectioned, and viewed under a fluorescence microscope for detection of neurons containing GFP (PAG-positive neurons) and rhodamine fluorescence (PVN to RVLM projecting neurons). GFP was widely distributed within the PVN, and the rhodamine fluorescence from Red Retrobeads was localized mostly to parvocellular regions (Figure 5A). In addition, there is a high degree of overlap between the GFP and rhodamine fluorescence (Figure 5A) in the parvocellular regions. The micrographs in Figure 5B show a higher power view of a single cell that is co-labeled with GFP and rhodamine. Following the detection of GFP and Red Retrobead fluorescence, brain slices were processed for immunostaining as described in the Methods. This process involved treatment with Target Retrieval Solution (DAKO Cytomation) at 95°C, which resulted in complete elimination of the fluorescence from the GFP and Red Retrobeads. Subsequent immunostaining with anti-GFP and AT1R antibodies revealed that many of the same cells which exhibited GFP and Red Retrobead fluorescence contain AT1R and GFP immunoreactivity. An example is shown in Figure 5C (same cell as shown in Figure 5B). Thus, these data indicate that certain of the neurons that co-express AT1R and GFP (PAG positive) in the PVN project to the RVLM.
The major findings of the present study are that AT1R are located on PAG positive neurons within the PVN, and that certain of these AT1R bearing neurons innervate the RVLM. While we cannot exclude the possibility that some of the neurons identified as AT1R-containing are GABAergic, it is probable that the majority of these cells are glutamatergic, as the AAV2-PAG-eGFP vector appears to drive GFP expression almost entirely in the latter type of neuron when injected into the PVN.
The significance of these results is straightforward. The PVN has a key role in the CNS control of sympathetic outflow and blood pressure, and in regulating the activity of the hypothalamic-pituitary (HPA) axis.1 The PVN contains high densities of AT1R, mostly located within the parvocellular regions of this hypothalamic nucleus,13–15 and the actions of Ang II via these receptors contributes to its CNS-mediated stimulatory effects on sympathetic outflow and blood pressure.9 Data also suggest that Ang II can act via AT1R in the PVN to alter the activity of the HPA axis.13 Furthermore, experimental models of hypertension such as the spontaneously hypertensive rat (SHR) exhibit increased levels and activity of AT1R in the PVN.2, 23 Thus, it is essential to understand the specific location of AT1R within the PVN, and which neuronal pathways are regulated by Ang II within this nucleus. The current findings, which provide the first definitive evidence that AT1R are located on PAG-containing neurons in the PVN, are a significant step forward in this regard. Nonetheless, the results also raise many questions.
One primary issue concerns the discrete location of the AT1R on glutamate neurons, whether they occur pre- or postsynaptically or in both locations. Several previous studies have provided good evidence that Ang II acts at presynaptic AT1R in the PVN, located either on glutamatergic interneurons or on GABAergic neuron terminals.18,19,24 It has also been demonstrated that Ang II elicits AT1R-mediated excitation of PVN neurons that innervate the RVLM,17,25 but these studies did not define the synaptic location of this particular set of AT1R. Data from the present study indicate the presence of immunoreactive AT1R on at least some of the PAG-positive neurons that project from PVN to RVLM (Figure 5). Further, we have recorded electrical responses following AT1R stimulation in PAG-containing neurons isolated from the PVN, have demonstrated AT1R mRNA expression within these neurons and have shown the presence of AT1R immunoreactivity on the cell bodies of GFP expressing (PAG) neurons in brain slices (Figures 3 and and4).4). Collectively, these data suggest a postsynaptic location of a population of functional AT1R on PAG-containing neurons in the PVN, including those that project to the RVLM. While our experiments have focused on the PVN neurons that project to the RVLM, it is established that other PVN neuron populations can influence sympathetic outflow by projecting directly to preganglionic sympathetic neurons in the IML of the spinal cord, and that some PVN-spinal neurons branch to innervate neurons in the RVLM.25, 26 Thus, it will also be interesting to determine whether these populations of PVN neurons are PAG positive and contain AT1R.
Many neurons within the CNS (including the PVN) contain co-transmitters.22 Thus, even though our data indicate the presence of AT1R on PAG positive (mostly glutamate) neurons in the PVN, it is very likely that these neurons also contain at least one other transmitter. One possible candidate is CRH, as previous studies have demonstrated that PVN neurons that express CRH and project to the median eminence also express AT1R mRNA and immunoreactive AT1R.13, 14 With respect to the AT1R-containing PAG-positive neurons that project from the PVN to the RVLM, we do not know whether they contain a co-transmitter, or if so the identity. However, a preliminary single-cell PCR screen of the GFP-expressing (PAG-positive) PVN neurons within the cultures has revealed that many are oxytocinergic. One future direction will be to solidify this data and further assess the phenotypic nature of the AT1R-containing PVN neurons. Such an effort will be made easier if specific promoters that can be used to drive gene expression within OT or CRH neurons become available.
Our ability to drive gene expression within PAG-positive neurons in the PVN, and the present demonstration that certain of these neurons contain AT1R opens up new avenues of investigation concerning the regulation of Ang II actions at this site. Namely, in previous studies we have demonstrated that macrophage migration inhibitory factor (MIF) serves as a negative regulator of the AT1R-mediated actions of Ang II at the PVN.10 The current results and the development of a vector for gene transduction within PAG-positive neurons gives us the rationale and means for determining the effects of over expression of MIF specifically in these neurons in the PVN on Ang II-induced cardiovascular responses.
In summary, the results presented here provide the first evidence that AT1R are located on PAG-positive (likely glutamatergic) neurons in the PVN, and that certain of these AT1R bearing neurons innervate the RVLM. Furthermore, our data provide evidence that a population of AT1R are located postsynaptically. As such, the data are a first step to understanding which specific neuronal pathways in the PVN are modulated by Ang II, and open the door to studies that target each specific pathway and how it contributes to the CNS-mediated cardiovascular actions of this peptide.
Knowledge of which neuronal pathways in the PVN are influenced by Ang II/AT1R interactions, and how these Ang II actions are regulated, is essential to understanding the CNS mechanisms that are involved in blood pressure regulation and neurogenic hypertension. While it is known that the PVN contains AT1R and that these receptors have a role in blood pressure control and hypertension, evidence concerning their discrete localization within this hypothalamic nucleus is lacking. The present study gives a clear demonstration of the presence of functional AT1R on PAG-positive neurons in the PVN, including those that project to the RVLM. As such, this study provides information that is fundamental to our understanding of the CNS control of blood pressure.
Sources of Funding: Supported by NIH grant HL 076803