Phosphate-activated glutaminase-containing neurons in the rat paraventricular nucleus express angiotensin type 1 receptors

Abstract

The centrally mediated cardiovascular regulatory actions of angiotensin II in normal and hypertensive rats include angiotensin II type 1 receptor (AT1R)-mediated actions at the paraventricular nucleus (PVN) of the hypothalamus. Because the PVN consists of multiple neuronal populations, it is important to understand which neuronal types in the PVN are influenced by angiotensin II. Here we have developed a viral vector (Adeno-associated vector 2 [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 after bilateral microinjection (200 nL per side; 1.2 x10(12) genome copies) of AAV2-PAG-eGFP into adult Sprague-Dawley rat PVN, animals were euthanized and 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 angiotensin II (100 nmol/L) produced a 16% decrease in delayed rectifier potassium current in approximately 50% of the GFP-containing neurons, an effect that was abolished by the AT1R antagonist losartan (1 mumol/L). Consistently, 9 of 28 GFP/PAG-expressing neurons contained AT1R mRNA, as indicated by single-cell RT-PCR. Furthermore, specific GFP/PAG-positive neurons in the PVN that project to the rostral ventrolateral medulla of the brain stem 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 rostral ventrolateral medulla.

Figure 1. AAV2-PAG-eGFP produces expression of GFP in PAG-positive PVN neurons

(A) Representative fluorescence micrograph of GFP expression in the PVN 10 days after microinjection of AAV2-PAG-eGFP confirming the injection location. Bar, 50μm. (3V: third cerebroventricle) Inset is lower power view. (B) Neuron-specific localization of GFP in the PVN. Representative fluorescence micrographs from the same field in the PVN show GFP-positive cells (green) and NeuN immunoreactivity (red). G + N, overlap of GFP and NeuN (Bar, 50μm). (C) AAV2-PAG-eGFP induced transduction of GFP into PAG-positive PVN neurons. Representative fluorescence micrographs from the same field in the PVN show GFP-positive cells (green) and PAG immunoreactivity (red). P + G, overlap of PAG and GFP (Bar, 50μm). Data are representative of 4 rats.

Figure 2. Relative distribution of AAV2-PAG-eGFP-induced GFP expression and GABA neurons in and surrounding the PVN

(A) Representative fluorescence micrograph of GFP and GAD67immunoreactivity in the PVN 10 days after microinjection of AAV2-PAG-eGFP. Pink arrow indicates GAD67 positive neurons surrounding the PVN. Cyan arrow is overlap GFP and GAD67. (3V: third cerebroventricle). (B)–(D) Higher power of a different field view showing GFP immunostaining (B), GFP + GAD67 (C) and GFP + GAD67 +DAPI nuclear stain within the PVN. (E)–(G) Higher power of a different field view showing GFP-positive (B), GFP + GAD67 (C) and GFP + GAD67 +DAPI nuclear stain at the margin of the PVN. Pink arrow indicates a GAD67 positive neuron surrounding the PVN. Data are representative of 5 rats. Bar, 50μm.

Figure 3. Immunoreactive AT1R on PAG-positive neurons in the PVN

(A) Representative fluorescence micrograph of GFP expression in the PVN 10 days after microinjection of AAV2-PAG-eGFP. (B) Grayscale view of Panel A indicating the location of the 3^rd cerebroventricle (3V). (C) Localization of AT1R-immunoreactive cells within the same field as in Panel A. (D) G + A, overlap of GFP and AT1R. Data are representative of 4 rats. Bar, 100μm

Figure 4. Functional AT1R on PAG specific PVN Neurons

(A) Representative micrographs of GFP-expressing neurons dissociated from a rat that was previously injected into the PVN with AAV2-PAG-eGFP. Left to right: Phase contrast image; GFP; Immunoreactive PAG (red); P + G, overlap of GFP and PAG. Bar, 100μm. (B) Presence of AT1R mRNA in a population of PAG positive neurons. Ethidium bromide-stained gel showing RT-PCR products from seven single GFP (PAG) expressing PVN neurons in culture. Upper panel, β-actin mRNA. Lower panel, AT1R mRNA is present in cells 1, 3, 5 and 6. L, 100bp DNA ladder; P, positive control, mRNA purified from PVN; N, negative control, RNase-free water instead of the sample mRNA. (C) Top: Representative current tracings showing inhibition of I_Kv by Ang II (100nmol/L) in GFP (PAG) expressing neurons. Control recordings (Con; Black) were made before the application of Ang II (Red), followed by washout of Ang II (Wash; Pink). Bottom: Blockade of the inhibitory effect of Ang II on I_Kv by 1μmol/L Los. Los alone, Black; Los + Ang II, Red. (D) Ang II decreased neuronal I_Kv in 8 of 16 neurons (Responders/Resp). Bar graphs are means±SEM, * P<0.05. (E) The inhibitory effects of Ang II were blocked by Los (1μmol/L). Data are means±SEM from 5 neurons, * P< 0.05.

Figure 5. Immunoreactive AT1R on PAG-positive neurons that project to the RVLM

(A) Left Panel: Representative fluorescence micrograph of GFP expression in the PVN 14 days after microinjection of AAV2-PAG-eGFP; Middle Panel: The same PVN field as shown in the left panel, indicating rhodamine fluorescence derived from Red Retrobeads that were injected into the RVLM 3 days earlier. Inset: Fluorescence micrograph showing the site of Retrobead injection into the RVLM Right Panel: G+B, merged GFP and Retrobead fluorescence images. Colocalization indicates the GFP expressing (PAG) neurons that project to RVLM. (B) Higher power view of the same section as shown in (A). Pink arrows (pink) indicate co-localization of GFP and rhodamine fluorescence in a single neuron. (C) Following elimination of the GFP and rhodamine fluorescence from the PVN as described in the methods and results, brain slices underwent immunostaining using anti-GFP and anti AT1R antibodies. Left to Right Panels: High power fluorescence micrographs showing GFP and AT1R immunoreactivity colocalized in the same PVN cell (pink arrows) as shown in (B). Bar, 50μm.