Adult C57BL/6 (WT; n = 20) and transgenic C57BL/TLR4^loxp/loxp (n = 20) were procured from the Jackson Laboratory, United States. C57BL (VGAT)-Venus mice (n = 10), previously characterized by Wang et al. (2009), were obtained from Dr. Janice Nagle at Wesleyan University. All animals used for this experiment weighed between 22 and 25 g. Animals were kept under standard laboratory condition and handled using NIH guidelines for animal care and use in research. All protocols used were reviewed and approved by the Institutional Animal Care and Use Committee of the Louisiana State University.

The projections from the PVN were identified using fluorescent retrograde anatomical tract tracing and immunohistochemistry. Flame pulled glass pipettes were back-filled with mineral oil and mounted on a Nanoject (Drummond Instruments). The tip of the pipette was trimmed with forceps to increase the width to 200 μm and reduce resistance to the injected bolus. Rhodamine B + Amine [10,000 MW] (Life Technologies; Molecular Probe) was diluted (10%) in saline and drawn into tip of the glass pipette. The coordinate of the Nucleus of solitary tract (NTS) was determined using a stereotaxic apparatus (NTS: Anteroposterior −6.96 mm, Mediolateral +1.35 mm; Franklin and Paxinos, 1997). A dental drill was used to remove the bone on the marked AP/ML intersection until the dura became visible. The exposed brain area was kept moist with oxygenated artificial cerebrospinal fluid [aCSF; in mM 125 NaCl, 25 NaHCO₃, 3 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, and 25 Glucose] throughout the duration of the experiment. The pipette (Nanoject) was lowered gradually into the NTS (Dorsoventral: +5.5 mm) following which two boli (5 nL each) were injected at 120 s interval. After a resting period of 3 min, the pipette was lowered further (DV: +6.0 mm) to label the SolL/SolV area. After a 7-day survival period, animals were euthanized following which the whole brain was removed and kept in ice-cold oxygenated aCSF. Vibrotome sliced coronal slices (500 μm thick) exposing the PVN (Bregma −0.58 mm to −1.22 mm) or NTS were fixed in 4% 10 mM phosphate buffered paraformaldehyde (4% PB PFA). In subsequent processing, 60 μm thick cryostat sections were obtained from the fixed slices and labeled further in immunofluorescence methods.

AAV-CMV-eGFP and AAV-CMV-Cre-eGFP were procured from the University of Iowa Vector Core and stored at −80°C. TLR4^loxp/loxp mice were anesthetized using Ketamine/Xylazine (100mg/Kg:10mg/Kg body weight). The PVN was targeted stereotaxically for injections at coordinates relative to the bregma [AP −0.85, ML + 1.5 mm, DV +5.5 mm] (Franklin and Paxinos, 1997). Control (AAV-CMV-eGFP; n = 10 TLR4^loxp/loxp mice) or Cre-expressing AAV (AAV-CMV-Cre-eGFP; n = 10 TLR4^loxp/loxp mice) was injected into the PVN (500 nL) using a low resistance flame pulled glass pipette (200 μm wide; Lee et al., 2017). The pipette was filled with mineral oil and mounted on a Nanoject (Drummond Instruments, USA). After exposing the dura at the designated coordinates, adenoviral cocktail was aspirated into the tip of the pipette and checked for flow before being lowered into the neural tissue. The injections were done in multiple boli of 23 nL each at 60 s interval. After the last injection, the pipette stayed in position for 180 s before it was gradually withdrawn. AAV gene expression (eGFP reporter) was verified 3 weeks after the injection in wet mount brain slices; using an Olympus BX 51 Fluorescence microscope.

Isoproterenol (Sigma, USA) was diluted in normal saline and administered in two separate intraperitoneal injections of 150 mg/kg (per injection) per animal (24 h′ interval; Gupta et al., 2013). ISOP treatment was done in the following groups; WT/ISOP (n = 10), TLR4^loxp/loxp/ISOP (n = 10) and TLR4^Cre/−/ISOP (n = 10). The animals were euthanized 24 h after the last injection to obtain brain (PVN) sections for immunofluorescence. A separate group of n = 10 WT mice were treated with normal saline (i.p).

To assess physiological effects in the brains of some animals, after 48 h of ISOP acute treatment, animals were decapitated following which the whole brain was removed and kept in ice-cold oxygenated artificial cerebrospinal fluid (ACSF; in mM 125 NaCl, 25 NaHCO₃, 3 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, and 25 Glucose). Vibratome sectioned coronal brain sections (400 μm thick) containing the PVN (Bregma −0.58 to −1.22 mm) were prepared in cold oxygenated artificial cerebrospinal fluid (ACSF) and transferred to oxygenated ACSF at 34°C for recovery (1 h). Subsequently, the brain slices were moved to a perfusion chamber mounted on an Olympus BX 51 phase contrast microscope. To record from neurons using the perforated patch technique, freshly aliquoted 3 mg of Amphotericin B was dissolved in 50 μl of dimethyl sulfonate (DMSO) and stored at −20°C. Subsequently, 8 μl of Amphotericin B solution was added to 2 ml of intracellular pipette solution in mM 135 Potassium gluconate, 7 NaCl, 10 4-(2hydroxy ethyl)-1-piperazineethanesulfonic gluconate, 1-2Na₂ATP, 0.3 Guanosine Trisphosphate (GTP) and MgCl_2; the pH was adjusted with KOH and final Osmolality was set at 290 mOsm; Amphotericin B (0.24 mg/ml)] following which it was kept on ice and stored away from light. Flame pulled glass pipette electrode (with resistance 3–5 MΩ) were prepared on a P-97 pipette puller (Stutter Instruments) and filled with the intracellular solution containing Amphotericin B. Using a micromanipulator, the electrode was visually guided to create a perforated patch (12–15 min) to isolate single-cell potentials (mV) in current clamp mode. Only cells with access resistance of 5–20 MΩ were recorded. Frequency (Hz) of evoked membrane depolarization potential was detected using Multiclamp 700B Amplifier and Digidata 1440A digital acquisition system (Molecular Instruments) after a step current was injected (60pA). Analysis of evoked action potentials was done in ClampFit (Axon Instrument, Sunnyvale, CA).

For western blot analyses, 15 μl brain tissue lysate containing 15 μg of protein was processed for SDS-PAGE electrophoresis for n = 7 samples per group. After subsequent western blotting (wet transfer), Polyvinylidene fluoride membrane (PVDF) was incubated in Tris-buffered saline (with 0.01% Tween 20) (TBST) for 15 min in TBST at room temperature. Subsequently, the membrane was blocked in 3% Bovine serum albumin (prepared in TBST) for 50 min at room temperature. The protein of interest, and control (Homer-1 and GAPDH) were detected using the following primary antibodies; Mouse anti-CaMKIIα (Cell Signaling-#50049), Rabbit anti p-CaMKIIα (Cell Signaling- #12716S), Rabbit anti MAPK/ErK (Cell Signaling- #9102S), Rabbit anti p-MAPK/ErK (4370S), Rabbit anti NMDAR1 (abcam-# ab17345), Rabbit anti GABA_BR (abcam-#ab166604), Rabbit anti GAPDH (Cell Signaling-#517S), Rabbit anti Homer1 (Cell Signaling-#8231), Mouse anti TLR4 (abcam-# 76B357.1) and Rabbit anti NeuN-Alexa 488 Conjugate (abcam-#ab190195). All primary antibodies were diluted in the blocking solution at 1:1,000. Subsequently, the primary antibodies were detected using HRP-conjugated secondary antibodies (goat anti-rabbit-#65-6120 and goat anti-mouse-#65-6520; Invitrogen; dilution of 1:5,000 or 1:10,000) following which the reaction was developed using a chemiluminescence substrate (Thermo Fisher-#34579).

Cardiac tissue was stained in Hematoxylin and Eosin (n = 5 per group) to demonstrate the general morphology of the ventricular wall using the methods of Fischer et al. (2008). Images were acquired in Nanozoomer (Hamatsu, USA). The affected area was determined in using Image J (NIH, USA).

WT and transgenic mice (n = 10 per group) were deeply anesthetized in isoflurane and perfused transcardially through the left ventricle using 10 mM phosphate buffered saline. Subsequently, 4% phosphate buffered paraformaldehyde (PB-PFA) was introduced for perfusion fixation. The whole brain was harvested through cranial dissection and fixed in 4% PB-PFA overnight, following which it was transferred to 4% PB-PFA containing 30% Sucrose for cryopreservation. Free-floating cryostat sections (40 μm) were collected into 10 mM PBS stored away from light (4°C). Before the commencement of staining, the sections were washed 3 times (5 min each) in 10 mM PBS (pH 7.4). To block non-specific binding, sections were incubated in normal Goat serum (Vector Labs), prepared in 10 mM PBS (with 0.03% Triton-X 100), at room temperature for 1 h. Subsequently, the sections were washed 3 times in PBS and moved to a primary antibody solution for overnight incubation at 4°C. Primary antibodies [Mouse anti-CaMKIIα (1:100; Cell Signaling-#50049), Guinea pig anti-VGLUT 2 (1:250; EMD Millipore-AB2251), Rabbit anti-GABA (1:200; Abcam-ab8891), Rabbit anti-MAPK/ErK1/ErK2 (1:100; Cell Signaling-#9102)] were prepared in 10 mM PBS, 0.03% Triton X-100 and Normal Goat Serum. In further processing, the sections were washed (as described previously) and incubated in secondary antibody solution [Goat anti-Rabbit Alexa-568, Goat anti-Guinea pig Alexa-568, and Goat anti-Mouse Alexa-568 (1:1,000), 10 mM PBS, 0.03% Triton X-100 and Normal Goat Serum] for 1 h at room temperature in a dark enclosure. Labeled sections were washed and mounted on gelatin coated slides with plain anti-fade mounting medium (Vector Labs).

Imaging of adenoviral (AAV-CMV-eGFP or AAV-CMV-Cre-eGFP) gene reporter expression (eGFP) was performed on serial cryostat coronal brain section (400 μm) using an Olympus BX 51 florescence microscope. The distribution of immunolabeled proteins (MAPK/ErK1/ErK2, CaMKIIα, GABA, and VGLUT 2) in the PVN were determined using confocal microscopy (Olympus FluoView 10i). To demonstrate cellular changes in inhibitory neurons (Venus) of the PVN, whole brain confocal scans and montaged higher magnification images were generated for (VGAT)-Venus mice after ISOP treatment. Immunolabeled cells and proteins were counted in confocal PVN images (n = 5 per group per protein) using Image J (NIH, USA). Furthermore, the average count per unit area was estimated bilaterally relative to the third ventricle. For each PVN on a section, the count was determined for n = 7 areas (per μm²) in different field of views. We determined the limits of the PVN using cellular morphology characterization and region-dependent cell density. This was combined with retrograde tracing (as above) to determine the approximate PVN region projecting to the NTS.

The average values for cell counting per unit area and fluorescence yield were compared for WT vs. WT/ISOP, TLR4^loxP/loxP/ISOP vs. TLR4^Cre/−/ISOP, and (VGAT)-Venus/ISOP vs. (VGAT)-Venus /Saline (control) mice using Student's t-test (GraphPad Prism Version 5). The outcome of statistical analysis was presented in bar chart with error bar representing the mean ± SEM.

To identify the approximate localization of pre-autonomic sympathetic neurons, we employed retrograde anatomical tracing methods to outline of the PVN after a dye was injected into the NTS (Figures 1a,b). The outcome of this experiments showed that retrogradely labeled cells were relatively abundant in the superiomedial aspect of the PVN. As shown in Figures 1c,d, retrograde vesicles were co-localized with neuronal cell bodies labeled with NeuN.

After βARA, in confocal microscopy, we found a significant decrease in total MAPK/ErK expression within the PVN of the WT/ISOP group when compared with the control (Figures 2a,b; p < 0.05). However, the expression of phosphorylated MAPK/ErK increased in the WT-ISOP group when compared to the total MAPK expression in quantitative immunoblotting (Figure 2c; p < 0.05). Overall, the total MAPK expression reduced significantly for the WT/ISOP group when compared with the control (Figures 2a–c). Alterations in MAPK/ErK and p-MAPK/ErK were accompanied by a significant loss of CaMKIIα in the PVN neurons of ISOP treated mice when compared with the control (Figures 2d,e). Interestingly, in quantitative protein analysis, we recorded a significant decrease in PVN CaMKIIα (p < 0.01), and an increase in phosphorylated CaMKIIα (p < 0.01) when the WT/ISOP group was compared with the control (Figures 2f,g). This suggests that a change in systemic sympathetic βARA elicited neural changes in PVN by altering the expression of pattern of kinases involved in the regulation of synaptic function within the PVN (Figure 2h). Specifically, this involved a preferential expression of p-MAPK/ErK and downregulation of CaMKIIα through phosphorylation (increased p-MAPK/ErK).

In subsequent experiments we evaluated the significance of changes in MAPK/CaMKIIα on components of the PVN glutamate/GABA system. Here we showed the distribution of transporters (VGLUT2 and VGAT) and associated receptors (NMDAR and GABA_BR) in the PVN of control and ISOP treated mice (WT/ISOP). In addition to a change in MAPK/ErK/CaMKIIα, ISOP treated mice showed a significant upregulation of VGLUT 2 (Figures 3a,b; p < 0.01), but a decrease in NMDAR expression in the PVN when compared with the control (Figure 3c; p < 0.01). In a separate experiment, transgenic (VGAT)-Venus mice, which express the Venus fluorescent protein (an enhanced form of YFP) in inhibitory GABAergic and glycinergic neurons, were treated with ISOP to determine the effect of βARA on inhibitory neurons in the PVN. A significant decrease in Venus fluorescence intensity was observed in the PVN of ISOP treated (VGAT)-Venus mice when compared with control (VGAT)-Venus group (Figures 3d,e). Furthermore, there was a significant loss of GABA_BR in the PVN (p < 0.01; Figure 3f). Based on these outcomes, we deduced that that acute systemic βAR activation promotes pre-autonomic glutamatergic vesicular activity, while suppressing inhibitory neurotransmission.

Since we have shown that a systemic adrenergic activation may cause certain indirect effects on preautonomic function, we verified this observation by determining the profile of spontaneous EPSCs in acute slices prepared from ISOP treated mice (Figure 4a). Using whole-cell perforated patch recordings from the PVN of WT/ISOP group (n = 5), we found an increase in spontaneous EPSCs when compared with the control (p < 0.01). Additionally, there was an increase in the amplitude of the EPSCs (p < 0.01) vs. the control (Figures 4b–e). In support of this outcome, we quantified the expression of nNOS as an indicator of sympathoexcitation that may be related to both NMDAR and GABA_BR function. Interestingly, we found a decrease in nNOS in the PVN of WT/ISOP groups when compared with the control (p < 0.01; Figures 4f,g). Ultimately, these changes were associated with an increase in PVN TLR4 expression (p < 0.05; Figures 4h,i).

From these findings, we hypothesized that an alteration of synaptic kinases may be responsible for the overall change in synaptic function associated with a systemic βARA. Since the balance between CaMKIIα and MAPK/ErK expression, and phosphorylation, is involved in inflammatory receptor (TLR4) signaling, we asked whether manipulation of TLR4 in the PVN reduces the threshold of MAPK/CaMKIIα imbalance and PVN sympathoexcitation (Figure 5a).

After stereotactic injection of AAV-cocktail in the PVN, successful AAV gene transfection was confirmed through fluorescence detection of the reporter (eGFP) for AAV-CMV-eGFP and AAV-CMV-Cre-eGFP in the PVN after 21 days (Figure 5b). To confirm the knockdown of TLR4 in the PVN of TLR4^loxp/loxp mice, we estimated TLR4 expression using western blotting analysis. Our results showed a significant reduction in TLR4 expression in untreated TLR4^Cre/− when compared with the untreated TLR4^loxp/loxp PVN (Figure 5c).

After knockdown of TLR4, TLR4^loxp/loxp, and TLR4^Cre/− (PVN specific knockdown) mice were treated with ISOP (48 h) following which we evaluated the expression of MAPK/ErK and other associated molecules in the PVN; as described previously in WT. A significant decrease in MAPK/ErK expression was observed in the PVN of TLR4^Cre/− mice, after βARA, when compared with the TLR4^loxp/loxp mice (p < 0.05) in confocal microscopy (Figures 6a,b). In subsequent analysis, using western blotting technique, we showed a significant decrease in p-MAPK/ErK (p < 0.001), but not MAPK/ErK (p < 0.05) in the PVN of TLR4^Cre/− ISOP group; when compared with the TLR4^loxp/loxp PVN (Figure 6c). The significance of p-MAPK/ErK downregulation in TLR4^Cre/− PVN was further verified by quantitative analysis of CaMKIIα in eGFP expressing neurons in TLR4^loxp/loxp and TLR4^Cre/−PVN. Interestingly, the loss of CaMKIIα was significantly attenuated in eGFP positive neurons of TLR4^Cre/− and not in TLR4^loxp/loxp PVN after ISOP treatment (p < 0.05; Figures 6d,e). In protein quantification, CaMKIIα expression was significantly higher in TLR4^Cre/− PVN after ISOP treatment; vs. TLR4^loxp/loxp PVN (p < 0.01). Evidently, the TLR4^loxp/loxp ISOP group recorded a significant increase p-CaMKIIα when compared with TLR4^Cre/− PVN; which showed less p-CaMKIIα (p < 0.001; Figures 6f,g).

In keeping with the role of TLR4 knockout in the attenuation of MAPK/ErK/CaMKIIα imbalance, there was a corresponding reduction of VGLUT2 (Figures 7a,b), and an upregulation of NMDAR in the TLR4^Cre/− ISOP when compared with the TLR4^loxp/loxp PVN (Figure 7c). Additionally, our results showed that a change in PVN MAPK/ErK/CaMKIIα expression in TLR4^loxp/loxp ISOP group was associated with a decrease in GABA (p < 0.001; Figures 7d,e) and GABA_BR (p < 0.01; Figures 7f,g); like the WT/ISOP. However, in the TLR4^Cre/− PVN, after ISOP treatment, GABA and GABA_BR depletion was rescued; vs. the TLR4^loxp/loxp PVN (Figures 7d,g).

Similar to the observation in WT/ISOP PVN, TLR4^loxp/loxp ISOP PVN neurons showed an increase in the frequency and amplitude of spontaneous EPSCs when compared with the WT control (Figures 8a,b). Interestingly, after TLR4 knockout, there was an increase in frequency but not the amplitude of EPSCs in the PVN neuron (p < 0.01, ns; Figures 8c,d). As a confirmation of a reduction in sympathoexcitation see Sharma et al. (2011), we found that the expression of nNOS was significantly higher for the TLR4 ^Cre/− ISOP; when compared with the TLR4^loxp/loxp ISOP group (Figure 8e–g; p < 0.01).

Since TLR4 knockout in the PVN reduced the imbalance between MAPK/ErK and CaMKIIα, we sought to elucidate if this contributes to the overall profile of cardiac tissue morphology (histology). After 48 h of ISOP treatment, we confirmed sympathoexcitation by serologically determining the areas affected systemic βARA in ventricular wall (Figures 9a,b). While WT/ISOP and TLR4^loxp/loxp ISOP showed significant changes in ventricular wall, no prominent change was recorded in the TLR4^Cre/− ISOP (Figure 9c). However, there was an increase in lumen size for the TLR4^Cre/− ISOP group when compared with the control.

In support of this outcome, we observed increase in cardiac thickness after 48 h of isoproterenol treatment in WT mice as reflected by a significant increase in the thickening of interventricular septum in systole, posterior wall thickness in both systole and diastole. In addition, WT mice infused with ISO showed an increase in heart rate. In contrast, treatment of TLR4 knockout mice with ISO did not increase ventricular thickness or heart rate. In agreement with echocardiographic finding, histological finding revealed thickening of the wall without an increase in LV cavity size indicating enhanced contraction in WT mice treated with ISO but not in the TLR4 knockout mice. These finding indicate beta adrenergic stimulation increased cardiac contractility and this effect was blunted in TLR4 knockout mice indicating a role for inflammatory molecule in cardiac dysfunction and exaggerated sympathetic activity in mice (Table 1).

Here we showed that an acute βAR activation was associated with alterations in preautonomic synaptic substrates. This was generally accounted for a change in glutamatergic and GABAergic systems (Figure 2). Although VGLUT2 expression increased in the preautonomic area, there was a significant decrease in NMDAR expression in the PVN of ISOP treated mice. However, a striking decrease in VGAT/GABA and GABA_BR suggests a decreased inhibition of the PVN during an acute systemic ISOP treatment. In support of this proposition, a decrease in GABA and GABA_BR was associated with an increased threshold and frequency for spontaneous EPSCs in the preautonomic area (Figure 4).

TLR4 may alter the expression of nNOS through the activity of MAPK/ErK at synaptic densities (Yao et al., 2012; Wang et al., 2016). Moreover, a reduction in the expression of nNOS, during inflammation, has been described in the loss of NO and GABA activity (Lethbridge et al., 2012; Di Mauro et al., 2013). Ultimately, our results showed that an increase in sympathoexcitation may involve MAPK-mediated glutamatergic activity or nNOS-related GABA loss after βARA. While nNOS is required for NO-mediated GABAergic activation, a reduction in the tissue level of NO and GABA has been described in the PVN after a phase of TLR4-mediated inflammation (Affleck et al., 2012). Furthermore, the activity of downstream pro-inflammatory molecules- such as NF-κB and IL-1β—have been identified in nNOS/GABA inhibition in the PVN (Crowley et al., 2015; Dange et al., 2015). Based on our results, and reports by other groups (Kim et al., 2006; Dange et al., 2015; Crowley et al., 2015), we deduced that βARA caused sympathoexcitation, in part, through TLR4/MAPK/ErK-mediated loss of GABA activity in the PVN. As such, when a tissue—specific TLR4 knockdown was implemented, we observed no significant loss in GABA and nNOS expression in the PVN (TLR4^Cre/−) after ISOP treatment. TLR4 knockdown also had a significant effect on the amplitude, but not the frequency of EPSCs in the PVN. As such, there was no significant change in fEPSC, while the TLR^Cr/e− ISOP responses exhibited a lower threshold vs. TLR4^loxp/loxp ISOP.

It is noteworthy to mention that methods such as RT-PCR can serve as useful tools in adequately measuring the extent of knockdown attained using the AAV cocktail and protein expression. However, western blotting gave us an overall outcome on protein expression pattern. This was ultimately used to determine the extent of the knockdown in PVN lysate. While our study has opened a new premise for assessing the crosstalk between PVN synaptic kinases and systemic cardiovascular activation, there are several gaps that our study does not address. Majorly, since ISOP remains systemic thereafter, the actual mechanism through which these changes may occur remains open for further investigation. A logical explanation may involve a broad up-regulation of p-MAPK (systemic) capable of causing changes in blood vessels, and permeability of the blood brain barrier (González-Mariscal et al., 2008; Pan et al., 2011). Additionally, systemic ISOP treatment may involve neural stress response mechanism—through adrenergic systems—and oxidative stress (Rathore et al., 2000) that is capable of eliciting changes in the PVN. Therefore, there is a need for further characterization of stress response systems, and determining the profile of the blood-brain barrier after acute ISOP treatment.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.