5-HT1A receptors of the nucleus tractus solitarii facilitate sympathetic recovery following hypotensive hemorrhage in rats

Abstract

The role of serotonin in the hemodynamic response to blood loss remains controversial. Caudal raphe serotonin neurons are activated during hypotensive hemorrhage, and their destruction attenuates sympathetic increases following blood loss in unanesthetized rats. Caudal raphe neurons provide serotonin-positive projections to the nucleus tractus solitarii (NTS), and disruption of serotonin-positive nerve terminals in the NTS attenuates sympathetic recovery following hemorrhage. Administration of 5-HT1A-receptor agonists following hemorrhage augments sympathetic-mediated increases in venous tone and tissue hypoxia. These findings led us to hypothesize that severe blood loss promotes activation of 5-HT1A receptors in the NTS, which facilitates sympathetic recovery and peripheral tissue perfusion. Here, we developed an adeno-associated viral vector encoding an efficacious small hairpin RNA sequence targeting the rat 5-HT1A receptor. Unanesthetized rats subjected to NTS injection of the anti-rat 5-HT1A small hairpin RNA-encoding vector 4 wk prior showed normal blood pressure recovery, but an attenuated recovery of renal sympathetic nerve activity (-6.4 ± 12.9 vs. 42.6 ± 15.6% baseline, P < 0.05) 50 min after 21% estimated blood volume withdrawal. The same rats developed increased tissue hypoxia after hemorrhage, as indicated by prolonged elevations in lactate (2.77 ± 0.5 vs. 1.34 ± 0.2 mmol/l, 60 min after start of hemorrhage, P < 0.05). 5-HT1A mRNA levels in the commissural NTS were directly correlated with renal sympathetic nerve activity (P < 0.01) and inversely correlated with lactate (P < 0.05) 60 min after start of hemorrhage. The data suggest that 5-HT1A receptors in the commissural NTS facilitate tissue perfusion after blood loss likely by increasing sympathetic-mediated venous return.

Keywords: adeno-associated virus; blood pressure; lactate; serotonin; shRNA.

Fig. 1.

A: human embryonic kidney (HEK)-293 cells cotransfected with a mCherry-5-HT_1A receptor fusion protein (mCherry-5-HT_1AR) and an efficacious small hairpin RNA (shRNA) sequence targeting the rat 5-HT_1AR (1ARshRNA#2; top) or a control sequence targeting a protein not expressed by HEK-293 cells, i.e., the rat tryptophan hydroxylase 2 (TPH₂) gene (TPHshRNA; bottom). Left panels show mCherry fluorescence, and right panels show humanized Renilla reniformis green fluorescent protein (hrGFP) fluorescence produced by shRNA vector in same cells. Four additional shRNAs tested were not efficacious and so are not shown. B: PCR performed on cells cotransfected with the mCherry-tagged receptor confirmed reductions in 5-HT_1AR mRNA (5-HT_1AR). The experiment was repeated 6 times on separate occasions. C: Western blot using a custom rabbit anti-rat 5-HT_1AR antibody revealed a 46-kDa band in lysates from cells transfected with untagged 5-HT_1AR (1AR), but not in lysates from cells cotransfected with the untagged receptor and 1ARshRNA#2 (1AR + 1ARshRNA#2) or from those left untransfected (UT). The experiment was repeated 3 times on separate occasions. D: cotransfection of cells with the untagged receptor and 1ARshRNA#2 (1ARshRNA#2) decreased protein expression of the 46-kDa band compared with cells cotransfected with the untagged receptor and shRNA targeting the rat TPH₂ gene (TPHshRNA). Summary data in B and D are means ± SE. *P < 0.05 or **P < 0.01.

Fig. 2.

A: schematic shows a dorsal view of the rat brain stem with location of 4 adeno-associated viral vector (AAV) injection sites shown with numbers indicating order of injection. B: location of 4 injection sites are also demonstrated in schematic of coronal brain stem sections with injection marked by black squares (26). Numbers indicated rostrocaudal plane of coronal section relative to bregma. C: fluorescent images from coronal sections (∼14.3 mm from bregma) demonstrating neuronal transduction (hrGFP expression) in rats injected with AAVs encoding either the scrambled shRNA (ScramshRNA; left) or a shRNA targeting the rat 5-HT_1AR (1ARshRNA#2, right). AP, area postrema; cNTS, commissural nucleus tractus solitarii; DMVX, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus, CC, central canal. D: neurolucida tracings of sequential brain stem sections from rats injected with virus encoding the scrambled sequence (left) or the 1ARshRNA sequence (right). Sections are superimposed with symbols indicating cells with intense (solid squares) or light (shaded triangles) fluorescent label. Approximate rostral-caudal level (vs. bregma) is similar for neighboring sections and is shown for first and last sections on the right stack of sections.

Fig. 3.

Coronal brain sections showing the typical pattern of hrGFP expression (green) around serotonin-positive caudal raphe neurons (red) in individual rats injected with AAV encoding the ScramshRNA (A and C) or 1ARshRNA (B and D). Both the raphe magnus (A and B) and raphe obscurus (C and D) are shown. Scale is the same in all images. Scale bar in D represents 100 μm.

Fig. 4.

Representative quantitative PCR amplification of GAPDH and rat 5-HT_1AR obtained from cDNA isolated from micropunches of cNTS (A) and raphe obscurus (B) of rats injected with AAV encoding the ScramshRNA (solid lines) and 1ARshRNA (dashed lines) sequences. C: relative 5-HT_1AR mRNA levels normalized to GAPDH and presented as fold change from average 5-HT_1AR mRNA levels in corresponding brain regions of rats injected with AAV encoding the ScramshRNA. D: relative α_2A-adrenergic receptor (αAR2A) mRNA levels in commissural and medial NTS normalized to GAPDH. Nos. inside bars, sample numbers for each location. Values are group means ± SE. **P < 0.01. αAR_2A, α-adrenergic 2A receptors; RFU, relative fluorescence units; mNTS, medial NTS; RO, raphe obscurus; DR, dorsal raphe.

Fig. 5.

A: representative sample recordings (2.5 s) of arterial pressure (AP) and raw renal sympathetic nerve activity (RSNA) taken during different phases of hemorrhage. Data are shown for individual rats injected with AAV encoding ScramshRNA (A, top) and 1ARshRNA (A, bottom). Data were sampled before hemorrhage start (baseline); 3, 8, 20, and 60 min after start of hemorrhage; and after administration of the ganglionic blocker hexamethonium chloride (Hex). B: group data showing mean arterial pressure (MAP), heart rate (HR), and RSNA for rats injected with AAV encoding scrambled (Scram) sequence or 1ARshRNA. Group n values are shown in parentheses. RSNA was successfully recorded in 6 rats injected with the 1ARshRNA-encoding virus and from 8 rats injected with AAV encoding the Scram sequence. Group n values for MAP and HR are indicated by high end of range shown in parentheses. *P < 0.05 and **P < 0.01, group difference. BPM, beats/min. C: correlation of the percent change in RSNA 60 min after start of hemorrhage and relative 5-HT_1AR mRNA expression in the cNTS (left) and mNTS (right) in a subset of animals with successful nerve recordings shown in B. The brains of a subset of rats with successful nerve recordings (3 control and 1 1ARshRNA) were reserved for detection of 5-HT_1AR by Western blot or immunohistochemistry and so were not available for PCR. However, the available antibodies for the 5-HT_1AR were not sufficiently selective to identify the receptor in tissue, and as such these data are not included.

Fig. 6.

A: lactate levels at baseline, just before hemorrhage (0), and 10 and 60 min after the start of hemorrhage in rats injected with AAV encoding ScramshRNA or 1ARshRNA. Values are group means ± SE. **P < 0.01 within 1ARshRNA injected group or between groups. B: Spearman correlation between lactate levels 60 min after the start of hemorrhage and relative 5-HT_1AR mRNA levels in the cNTS (left) and the mNTS (right). In some cases, brains were used for attempted Western blot or immunohistochemistry rather than quantitative PCR. Therefore, data shown in B are from a subset of rats used to obtain data in A.