Alcohol abuse plays a crucial role in the prevalence of cardiovascular and neuronal degenerated diseases. However, the central mechanism that mediates these effects are not fully understood. It has been well know that autonomic hypothalamic paraventricular nucleus (PVN) play an important role in regulating sympathetic outflow and arterial blood pressure (ABP). Moreover, it has been demonstrated that the increased glutamatergic activity among PVN neurons contributed to the sympathoexcitation and development of hypertension. Furthermore, we have reported that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in autonomic central nucleus of amygdala. Combing with the fact that autonomic PVN neurons are abundantly expressed NMDA receptors, we hypothesize that acetate, the metabolic products from alcohol, activates the PVN neurons through the increase in glutamatergic activity and contributes to the sympathoexcitation and development of chronic diseases, such as hypertension. In anesthetized rats, the effect of acetate microinjected in the PVN on the renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) was determined. The PVN acetate microinjection increased sympathoexcitatiory and pressor response in a dose dependent dose-dependent (1.5mm, 0.5mm, 2.0mm, 7.5mm) manner, and 2mm of acetate showed a minimum dose evoked a maximal response. To determine the role of acetate-stimulated glutamatergic receptor activation in driving sympathoexcitatory and pressor responses, either 2mm acetate or pre-treatment of Kynurenic acid (KYN, 7.2mm) ionotropic excitatory amino acid receptor blocker, or D-2-amino-5-phosphopentanoate (AP5, 3.0mm), a N-methyl-D-aspartate (NMDA) receptor antagonist, followed by 2mm acetate were microinjected into the PVN of male Sprague Dawley rats (300-500g, n= 6-9/ group). RSNA and ABP responses were compared among vehicle (saline) and 2mm acetate, pre-treatment of KYN and 2mm acetate, or pre-treatment AP5 and 2mm acetate protocols. 2mm acetate significantly increased RSNA (60-70% baseline, p < 0.001) and mean arterial pressure (MAP, 10-12mmHg, p < 0.05). Non-selective glutamatergic receptor antagonist, KYN significantly blocked the sympathoexcitatory (RSNA, p < 0.05) and pressor response (MAP, p < 0.05) evoked by 2mm acetate in the PVN. Furthermore, selective NMDA receptor antagonist, AP5 significantly attenuates the sympathoexcitatory response induced by PVN 2mm acetate (RSNA, P < 0.05 and MAP, p < 0.01). These data indicate acetate can increase glutamatergic activity and excitability of pre-sympathetic PVN neurons via activation of local NMDA receptors. The metabolism of ethanol to acetate following by alcohol consumption may contribute, in part, to the development of neurogenic hypertension and/or other cardiovascular diseases associated with increased sympathetic outflow. R15HL145655, R15HL150703 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Upregulation of neuroinflammatory signaling molecules following ethanol consumption has been identified as a key cellular mechanism to contribute to neuronal excitotoxicity. Evidence indicates that ethanol excitotoxicity occurs during ethanol withdrawal/clearance, times at which acetate concentrations are still elevated. Studies have demonstrated that acetate is a very active and powerful metabolite of ethanol, which may be one of the key components to underlie the mechanisms of neuronal excitotoxicity/apoptosis induced by ethanol consumption. Here, we tested the hypothesis that acetate increases neuroinflammatory cytokines, which may contribute to neuronal excitotoxicity/apoptosis and enhanced cellular death. Whole brain neuronal cultures incubated with acetate (2 mM) for 3 hrs. increased mRNA levels significantly (p < 0.05 vs control) of tumor necrosis factor alpha (TNFα), interleukin‐1β (IL‐1β) and fos related antigen 1 (fosl1); 2 fold, 8 fold and 2 fold respectively. Furthermore, 24 hr. treatment with acetate (2 mM) in whole brain neuronal cultures followed by staining with propidium iodide and analyzed via flow cytometry (1000 neurons, triplicates) showed significant neuronal death (p < 0.05 vs control), acetate (62.3 %) vs control (25.5 %). In a separate treatment, whole brain neuronal cultures were incubated with acetate (2 mM) for 24 hrs. and then stained with Fluo‐4AM (calcium dye) and analyzed via flow cytometry (1000 neurons, triplicates) and showed a significant 5 % increases in cytosolic calcium (p < 0.05 vs control). This data suggests that physiological circulating levels of acetate (2 mM) following ethanol consumption and metabolism lead to increased neuroinflammation which enhances whole brain cellular apoptosis/death and may be the underlying cause of alcohol associated neurodegeneration. Support or Funding Information AHA 16PRE27780121, Andrew Chapp, AHA 11SDG7420029, Zhiying Shan; R15‐HL122952, Qinghui Chen
Control of sympathetic outflow by pre-sympathetic paraventricular nucleus (PVN) neurons is partially mediated through a monosynaptic pathway that innervates the rostral ventrolateral medulla (RVLM). Here we investigated the role of the thapsigargin (TG), the endoplasmic reticulum (ER) Ca 2+ ATPase inhibitor, in regulating the in vitro excitability of PVN neurons projecting to RVLM (PVN-RVLM) under brain slice preparation. In current-clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater ( P < 0.05) in the presence of TG (0.5 μ M) (32 ± 4 Hz, n=5). Next, the effect of Ca 2+ store depletion from the ER in regulating SNA and blood pressure was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased the renal sympathetic nerve activity (SNA) and mean arterial pressure (MAP) in a dose-dependent manner. Maximum increase in renal SNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre-treatment of CyPPA (5 nmol/100 nl), a SK channel activator/opener (n=3), remarkably attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for renal SNA and 4 ± 7 mmHg for MAP). PVN injection of CyPPA did not significantly alter the baseline of renal SNA and MAP. Our data indicate that inhibition of ER function by TG contributes to the enhanced in vitro excitability of PVN-RVLM neurons and increased in vivo renal SNA and MAP. The effect of TG on the excitability of pre-sympathetic PVN neurons and sympathetic outflow through the Ca 2+ store depletion from the ER may involve the down-regulation of SK channel function. Support: NSDP2010008 (JZ) and AHA10SDG2640130 (CQH).
ABSTRACT Binge alcohol consumption elicits robust sympathoexcitation and excitatory neuronal output. However, the central mechanism that mediates these effects remains elusive. We investigated the effects of ethanol metabolism within the central nucleus of the amygdala (CeA) on sympathoexcitation, and elucidated the role of acetate in these excitatory responses. In vivo arterial blood pressure, heart rate and sympathetic nerve activity responses to CeA microinjected ethanol or acetate with appropriate inhibitors/antagonists were tracked. In vitro whole-cell electrophysiology recording responses to acetate in CeA neurons with axon projecting to the rostral ventrolateral medulla (CeA-RVLM) were investigated, and cytosolic calcium responses in primary neuronal cultures were quantified. We demonstrate that in Sprague Dawley rats, local brain metabolism of ethanol in the CeA to acetic acid/acetate elicits sympathoexcitatory responses in vivo through activation of NMDA receptor (NMDAR). Alcohol dehydrogenase or aldehyde dehydrogenase inhibition using fomepizole or cyanamide and NMDAR antagonism using AP5 or memantine blunted these effects. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. NMDAR antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability, and memantine suppressed the direct activation of NMDAR-mediated inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca 2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. These findings suggest that within the CeA, ethanol is sympathoexcitatory through local brain metabolism, which generates acetic acid/acetate leading to activation of NMDAR. NEW AND NOTEWORTHY Brain ethanol metabolism to acetic acid (vinegar)/acetate causes activation of N-methyl-D-aspartate receptors (NMDARs) in the central nucleus of the amygdala and elicits sympathoexcitatory responses. This excitatory mechanism is opposite to the inhibitory effects of ethanol at NMDAR. Understanding the active compounds that arise from ethanol metabolism, and the molecular mechanisms by which they influence alcohol reward and cardiovascular function, may be beneficial in developing targeted intervention strategies for both alcohol use disorder and its cardiovascular sequelae. Graphical Figure Proposed mechanisms for ethanol and acetate induced increases in sympathoexcitation within the central nucleus of the amygdala (CeA). Abbreviations: Acetic acid (HOAc), acetate ( - OAc), ADH (alcohol dehydrogenase), ALDH (aldehyde dehydrogenase), BBB (blood brain barrier), FOM (fomepizole), CYAN (cyanamide), CYP450 (cytochrome P450), IML (intermediolateral nucleus), RVLM (rostral ventrolateral medulla), SNA (sympathetic nerve activity).
We have demonstrated that hypertension induced by high salt diet combined with chronic low‐dose infusion of angiotensin II (AngII‐Salt HTN) involves dysfunction of small conductance calcium‐activated potassium (SK) channels in the hypothalamic paraventricular nucleus (PVN). Diminished current conducted through SK channels augments neuronal excitability, and inhibition of SK channel function in the PVN augments sympathetic outflow and arterial blood pressure (ABP); however, the underlying mechanisms responsible for inhibition of SK channel function among PVN neurons in AngII‐Salt HTN remain unclear. It is well‐known that intracellular calcium is one of the key regulators gating SK channel conductance. Here, we demonstrated the importance of intracellular calcium homeostasis in controlling neuronal excitability in PVN neurons with axon projections to the rostral ventrolateral medulla (PVN‐RVLM) in vitro, and sympathetic nerve activity (SNA) and ABP in vivo. In whole‐cell current‐clamp recordings under brain slice preparation, graded current injections (0, 50, 100, 150, 200, 250 and 300 pA) evoked graded increases in spike frequency in pre‐sympathetic PVN‐RVLM neurons identified by retrograde labelling. Maximum discharge was evoked by +200 pA current injections and averaged 20 ± 2 Hz (n=10) in the presence of a low level of the calcium chelator EGTA (0.1mM). In contrast, neuronal excitability in response to +200pA current injection was significantly greater (P<0.05) in the presence of high EGTA (5.0mM) (31±4 Hz, n=7) or BAPTA (10mM) (30±1 Hz, n=8), a fast calcium chelator, loaded through the intracellular pipette solution. In order to examine the effects of diminished intracellular calcium on sympathetic outflow, in vivo recordings of splanchnic SNA (SSNA), renal SNA (RSNA) and mean arterial pressure (MAP) were assessed via bilateral PVN microinjection of BAPTA‐AM, a cell membrane permeable calcium chelator compared to membrane‐impermeable BAPTA, in anesthetized rats. PVN microinjection of BAPTA‐AM (20nmol/100nl) in the PVN augmented (P<0.05) SSNA (66±11% vs 17±12%, n=3), RSNA (52±13% vs. 11±8%, n=3) and MAP (8±1 vs. 1±2 mmHg, n=3) compared to membrane‐impermeable BAPTA control (20nmol/100nl). Additionally, blockade of calcium channels via PVN microinjection of the non‐selective calcium channel blocker cadmium chloride (CdCl 2 , 10nmol/100nl) significantly (P<0.05) augmented SSNA (61±16%, n=3), RSNA (80±21%, n=3) and MAP (12±1 mmHg, n=3). Collectively, our data indicate that disruption of cytoplasmic calcium significantly augmented excitability of pre‐sympathetic PVN neurons in vitro, and increased sympathetic outflow and ABP in vivo. Normal intracellular calcium homeostasis among PVN neurons plays an important role in limiting neuronal excitability and regulating sympathetic outflow, likely through activation of calcium activated potassium channels including SK channels. Support or Funding Information 11SDG7420029 (ZYS) and R15‐HL122952 (QHC)
The central nucleus of amygdala (CeA) is involved in the sympathoexcitatory and pressor responses to ethanol intake. However, the underlying neural mechanisms have not been determined. Here we investigated the role of acetate, the ethanol metabolite, in regulating the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM) under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +250 pA injections and averaged 18 ± 1 Hz (n=7). Bath application of acetate (0, 7.5, 37.5 and 75 mM) increased the excitability of CeA‐RVLM neurons in a dose‐dependent manner. Maximum spike discharge in the presence of acetate (75 mM) was 53 ± 3 Hz (n=4), which was significantly (p<0.01) higher than vehicle control. Pre‐treatment with AP5 (60 µM, n=7), the NMDA receptor blocker, significantly prevented increase in excitability (37 ± 5 Hz, p<0.05) elicited by acetate. AP5 alone was without effect on baseline excitability. The contribution of acetate in regulating sympathetic nerve activity (SNA) was assessed by microinjecting acetate into the CeA of anesthetized rat. Acetate (0.2 µmol/100 nl) significantly (P<0.05) increased lumber SNA by 90 ± 1 %, splanchnic SNA by 76 ± 18% and MAP by 4 ± 1 mmHg (n=3). Immunohistochemistry study demonstrates expression of NMDA NR1 in CeA‐RVLM neurons. Our data indicate that activation of NMDA receptors by acetate contributes to the increased excitability of CeA‐RVLM neurons. The CeA acetate elicited sympathoexcitatory responses involve the activation of CeA‐RVLM neurons, which may underlie the mechanisms of increased SNA during the development of alcohol associated hypertension. Grant Funding Source : Supported by AHA 10SDG2640130
Small conductance Ca 2+ activated K + (SK) channels act as negative feedback regulators of neuronal excitability. We have reported that SK channels expressed in the paraventricular nucleus (PVN) are involved in controlling neuronal activity, sympathetic nerve activity (SNA) and arterial pressure (AP). Blockade of SK channels in the pre‐sympathetic PVN neurons increases neuronal excitability, SNA and AP. Here we investigated the effect of angiotensin II type 1 receptor (AT1R) blockade on the sympathoexcitatory and presser responses elicited by the inhibition of SK channels in the PVN. In anesthetized rats, PVN microinjection of SK channel blocker with apamin (12.5 pmol, 50nl) (n=7) increased splanchnic SNA (SSNA) (278 ± 50%), renal SNA (RSNA) (193 ± 12%), and mean AP (MAP) (32 ± 4mmHg). Pre‐treatment with PVN injection of losartan (20 nmol), an AT1R antagonist, significantly attenuated (n=6) the elevated SSNA (129 ± 37%; p<.05 vs vehicle control), RSNA (84 ± 40%; p<.05 vs vehicle control), and MAP (10 ± 6mmHg; p<.05 vs vehicle control) after PVN injection of apamin. Pre‐treatment with PVN injection of ZD7155 (1.0 nmol), another AT1R antagonist, significantly attenuated (n=5) the elevated SSNA (112 ± 30%; p<.05 vs vehicle control), RSNA (81 ± 23%; p<.05 vs vehicle control) and MAP (7 ± 2mmHg; p<.05 vs vehicle control) elicited by PVN apamin. No significant difference was found between pretreatment with losartan and ZD7155 for SNA and MAP. Our data indicate that sympathoexcitation and pressor responses induced by inhibition of SK channel activity involve the activation of AT1R in the PVN. Support: AHA7420029 (ZYS) 2640130 (QHC).
Prospective memory (PM), which involves remembering to execute a future action, is impacted by Alzheimer's disease. This study examined the role of PM in instrumental activities of daily living (IADLs) and the use of compensatory aids in mild cognitive impairment (MCI) and healthy aging.Participants included 44 healthy older controls and 49 persons with MCI. Simple PM was measured by asking participants to remember to execute a single action- to request a pill after completing each of a series of neuropsychological tests. Complex PM was then measured by asking participants to request a specific number of pills depending on whether a memory test was just completed or a non-memory test was completed. The Instrumental Activities of Daily Living- Compensation (IADL-C) scale was used to assess IADLs and the use of compensatory aids for completing these activities.Regression analyses revealed that both types of PM together predicted the IADLs of money/self-management skills and home/daily living skills in healthy older controls. Both types of PM also predicted total IADLs in controls. PM did not predict IADLs in MCI or the use of compensatory aids in either group.Results indicated that PM was predictive of IADLs in healthy older adults. When PM skills are poorer, as in MCI, they are not predictive of IADLs.
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