Dietary α‐Linolenic Acid, Marine ω‐3 Fatty Acids, and Mortality in a Population With High Fish Consumption: Findings From the PREvención con DIeta MEDiterránea (PREDIMED) Study

Background: Epidemiological evidence suggests a cardioprotective role of α‐linolenic acid (ALA), a plant‐derived ω‐3 fatty acid. It is unclear whether ALA is beneficial in a background of high marine ω‐3 fatty acids (long‐chain n‐3 polyunsaturated fatty acids) intake. In persons at high cardiovascular risk from Spain, a country in which fish consumption is customarily high, we investigated whether meeting the International Society for the Study of Fatty Acids and Lipids recommendation for dietary ALA (0.7% of total energy) at baseline was related to all‐cause and cardiovascular disease mortality. We also examined the effect of meeting the society's recommendation for long‐chain n‐3 polyunsaturated fatty acids (≥500 mg/day). Methods and Results: We longitudinally evaluated 7202 participants in the PREvención con DIeta MEDiterránea (PREDIMED) trial. Multivariable‐adjusted Cox regression models were fitted to estimate hazard ratios. ALA intake correlated to walnut consumption (r=0.94). During a 5.9‐y follow‐up, 431 deaths occurred (104 cardiovascular disease, 55 coronary heart disease, 32 sudden cardiac death, 25 stroke). The hazard ratios for meeting ALA recommendation (n=1615, 22.4%) were 0.72 (95% CI 0.56–0.92) for all‐cause mortality and 0.95 (95% CI 0.58–1.57) for fatal cardiovascular disease. The hazard ratios for meeting the recommendation for long‐chain n‐3 polyunsaturated fatty acids (n=5452, 75.7%) were 0.84 (95% CI 0.67–1.05) for all‐cause mortality, 0.61 (95% CI 0.39–0.96) for fatal cardiovascular disease, 0.54 (95% CI 0.29–0.99) for fatal coronary heart disease, and 0.49 (95% CI 0.22–1.01) for sudden cardiac death. The highest reduction in all‐cause mortality occurred in participants meeting both recommendations (hazard ratio 0.63 [95% CI 0.45–0.87]). Conclusions: In participants without prior cardiovascular disease and high fish consumption, dietary ALA, supplied mainly by walnuts and olive oil, relates inversely to all‐cause mortality, whereas protection from cardiac mortality is limited to fish‐derived long‐chain n‐3 polyunsaturated fatty acids. Clinical Trial Registration URL: http://www.Controlled-trials.com/. Unique identifier: ISRCTN35739639

Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei

Recent advances in optogenetic methods demonstrate the feasibility of selective photoactivation at the soma of neurons that express channelrhodopsin-2 (ChR2), but a comprehensive evaluation of different methods to selectively evoke transmitter release from distant synapses using optogenetic approaches is needed. Here we compared different lentiviral vectors, with sub-population-specific and strong promoters, and transgenic methods to express and photostimulate ChR2 in the long-range projections of paraventricular nucleus of the hypothalamus (PVN) neurons to brain stem cardiac vagal neurons (CVNs). Using PVN subpopulation-specific promoters for vasopressin and oxytocin, we were able to depolarize the soma of these neurons upon photostimulation, but these promoters were not strong enough to drive sufficient expression for optogenetic stimulation and synaptic release from the distal axons. However, utilizing the synapsin promoter photostimulation of distal PVN axons successfully evoked glutamatergic excitatory post-synaptic currents in CVNs. Employing the Cre/loxP system, using the Sim-1 Cre-driver mouse line, we found that the Rosa-CAG-LSL-ChR2-EYFP Cre-responder mice expressed higher levels of ChR2 than the Rosa-CAG-LSL-ChR2-tdTomato line in the PVN, judged by photo-evoked currents at the soma. However, neither was able to drive sufficient expression to observe and photostimulate the long-range projections to brainstem autonomic regions. We conclude that a viral vector approach with a strong promoter is required for successful optogenetic stimulation of distal axons to evoke transmitter release in pre-autonomic PVN neurons. This approach can be very useful to study important hypothalamus-brainstem connections, and can be easily modified to selectively activate other long-range projections within the brain. © 2012 Elsevier B.V

Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei

Recent advances in optogenetic methods demonstrate the feasibility of selective photoactivation at the soma of neurons that express channelrhodopsin-2 (ChR2), but a comprehensive evaluation of different methods to selectively evoke transmitter release from distant synapses using optogenetic approaches is needed. Here we compared different lentiviral vectors, with sub-population-specific and strong promoters, and transgenic methods to express and photostimulate ChR2 in the long-range projections of paraventricular nucleus of the hypothalamus (PVN) neurons to brain stem cardiac vagal neurons (CVNs). Using PVN subpopulation-specific promoters for vasopressin and oxytocin, we were able to depolarize the soma of these neurons upon photostimulation, but these promoters were not strong enough to drive sufficient expression for optogenetic stimulation and synaptic release from the distal axons. However, utilizing the synapsin promoter photostimulation of distal PVN axons successfully evoked glutamatergic excitatory post-synaptic currents in CVNs. Employing the Cre/loxP system, using the Sim-1 Cre-driver mouse line, we found that the Rosa-CAG-LSL-ChR2-EYFP Cre-responder mice expressed higher levels of ChR2 than the Rosa-CAG-LSL-ChR2-tdTomato line in the PVN, judged by photo-evoked currents at the soma. However, neither was able to drive sufficient expression to observe and photostimulate the long-range projections to brainstem autonomic regions. We conclude that a viral vector approach with a strong promoter is required for successful optogenetic stimulation of distal axons to evoke transmitter release in pre-autonomic PVN neurons. This approach can be very useful to study important hypothalamus-brainstem connections, and can be easily modified to selectively activate other long-range projections within the brain. © 2012 Elsevier B.V

Chronic intermittent hypoxia and hypercapnia inhibit the hypothalamic paraventricular nucleus neurotransmission to parasympathetic cardiac neurons in the brain stem

Obstructive sleep apnea is associated with chronic intermittent hypoxia/hypercapnia (CIHH) episodes during sleep that heighten sympathetic and diminish parasympathetic activity to the heart. Although one population of neurons in the paraventricular nucleus of the hypothalamus strongly influences sympathetic tone and has increased activity after CIHH, little is known about the role of this pathway to parasympathetic neurons and how this network is altered in CIHH. We hypothesized that CIHH inhibits the excitatory pathway from the paraventricular nucleus of the hypothalamus to parasympathetic cardiac vagal neurons in the brain stem. To test this hypothesis, channelrhodopsin was selectively expressed, using viral vectors, in neurons in the paraventricular nucleus of the hypothalamus and channelrhodopsinexpressing fibers were photoactivated to evoke postsynaptic currents in cardiac vagal neurons in brain stem slices. Excitatory postsynaptic currents were diminished in animals exposed to CIHH. The paired-pulse and prolonged facilitation of the postsynaptic current amplitudes and frequencies evoked by paired and bursts of photoactivation of channelrhodopsin fibers, respectively, occurred in unexposed rats but were blunted in CIHH animals. In response to an acute challenge of hypoxia/hypercapnia, the amplitude of postsynaptic events was unchanged during, but increased after hypoxia/hypercapnia in unexposed animals. In contrast, postsynaptic currents were inhibited during hypoxia/hypercapnia in rats exposed to CIHH. In conclusion, the excitatory pathway to cardiac vagal neurons is diminished in response to both acute and chronic exposures to hypoxia/hypercapnia. This could elicit a reduced cardioprotective parasympathetic activity and an enhanced risk of adverse cardiovascular events in episodes of apnea and chronic obstructive sleep apnea. © 2014 American Heart Association, Inc

Optogenetic stimulation of locus ceruleus neurons augments inhibitory transmission to parasympathetic cardiac vagal neurons via activation of brainstem α1 and β1 receptors

Locus ceruleus (LC) noradrenergic neurons are critical in generating alertness. In addition to inducing cortical arousal, the LC also orchestrates changes in accompanying autonomic system function that compliments increased attention, such as during stress, excitation, and/or exposure to averse or novel stimuli. Although the association between arousal and increased heart rate is well accepted, the neurobiological link between the LC and parasympathetic neurons that control heart rate has not been identified. In this study, we test directly whether activation of noradrenergic neurons in the LC influences brainstem parasympathetic cardiac vagal neurons (CVNs). CVNs were identified in transgenic mice that express channel-rhodopsin-2 (ChR2) in LC tyrosine hydroxylase neurons. Photoactivation evoked a rapid depolarization, increased firing, and excitatory inward currents in ChR2-expressing neurons in the LC. Photostimulation of LC neurons did not alter excitatory currents, but increased inhibitory neurotransmission to CVNs. Optogenetic activation of LC neurons increased the frequency of isolated glycinergic IPSCs by 27 ± 8% (p = 0.003, n = 26) and augmented GABAergic IPSCs in CVNs by 21 ± 5% (p = 0.001, n = 26). Inhibiting α1, but not α2, receptors blocked the evoked responses. Inhibiting β1 receptors prevented the increase in glycinergic, but not GABAergic, IPSCs in CVNs. This study demonstrates LC noradrenergic neurons inhibit the brainstem CVNs that generate parasympathetic activity to the heart. This inhibition of CVNs would increase heart rate and risks associated with tachycardia. The receptors activated within this pathway, α1 and/or β1 receptors, are targets for clinically prescribed antagonists that promote slower, cardioprotective heart rates during heightened vigilant states. © 2014 the authors

Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons

© 2014, PLOS ONE. Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection

Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons.

Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection

Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons

© 2014, PLOS ONE. Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection