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Ventromedial medullary pathway mediating cardiac responses evoked from periaqueductal gray

Open AccessPublished:August 12, 2020DOI:https://doi.org/10.1016/j.autneu.2020.102716

      Highlights

      • Periaqueductal gray activation increases cardiac chronotropy and inotropy.
      • Periaqueductal gray sends monosynaptic projections to raphe pallidus neurons.
      • Raphe pallidus neurons control cardiac responses evoked from periaqueductal gray.

      Abstract

      Periaqueductal gray (PAG) is a midbrain region that projects to areas controlling behavioral and autonomic outputs and is involved in the behavioral and physiological components of defense reactions. Since Raphe Pallidus (RPa) is a medial medullary region comprising sympathetic premotor neurons governing heart function, it is worth considering the PAG-RPa path. We assessed: i) whether PAG projects to RPa; ii) the amplitude of cardiac responses evoked from PAG; iii) whether cardiovascular responses evoked from PAG rely on RPa. Experiments conducted in Wistar rats (±300 g) were approved by Ethics Committee CEUA-UFG (092/18). Firstly, (n = 3), monosynaptic retrograde tracer Retrobeads was injected into RPa; PAG slices were analyzed. Other two groups (n = 6 each) were anesthetized with urethane (1.4 g/kg) and chloralose (120 mg/kg) and underwent craniotomy, tracheostomy, catheterization of femoral artery and vein and of cardiac left ventricle. In one group, we injected the GABAA receptor antagonist, bicuculline methiodide (BMI – 40 pmol/100 nL) into lateral/dorsolateral PAG. Another group was injected (100 nL) with the GABAA receptor agonist muscimol (20 mM) into RPa, 20 min before BMI into PAG. The results were: i) retrogradely labelled neurons were found in PAG; ii) PAG activation by BMI caused positive chronotropism and inotropism, which were accompanied by afterload increases; iii) RPa inhibition with Muscimol reduced heart rate, arterial and ventricular pressures; iv) the subsequent PAG activation still increased arterial pressure, cardiac chronotropy and inotropy, but these responses were significantly attenuated. In conclusion, PAG activation increases cardiac chronotropy and inotropy, and these responses seem to rely on a direct pathway reaching ventromedial medullary RPa neurons.

      Keywords

      1. Introduction

      Stressful stimuli give rise to defense reactions that are organized by central nervous system (
      • Steimer T.
      The biology of fear- and anxiety-related behaviors.
      ) (
      • Strigo I.A.
      • Bud Craig A.D.
      Interoception, homeostatic emotions and sympathovagal balance.
      ), which increases heart and respiratory rates, blood pressure, body temperature and produces some behaviors (
      • Carrive P.
      • Bandler R.
      Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study.
      ) (
      • Farkas E.
      • Jansen A.S.P.
      • Loewy A.D.
      Periaqueductal gray matter input to cardiac-related sympathetic premotor neurons.
      ). Many studies that were conducted to unravel neural pathways organizing these responses show that diencephalic and midbrain activities are pivotal (
      • Fontes M.A.P.
      • Filho M.L.
      • Santos Machado N.L.
      • de Paula C.A.
      • Souza Cordeiro L.M.
      • Xavier C.H.
      • Marins F.R.
      • Henderson L.
      • Macefield V.G.
      Asymmetric sympathetic output: the dorsomedial hypothalamus as a potential link between emotional stress and cardiac arrhythmias.
      ,
      • Fontes M.A.P.
      • Xavier C.H.
      • Marins F.R.
      • Limborço-Filho M.
      • Vaz G.C.
      • Müller-Ribeiro F.C.
      • Nalivaiko E.
      Emotional stress and sympathetic activity: contribution of dorsomedial hypothalamus to cardiac arrhythmias.
      ,
      • Fontes M.A.P.
      • Xavier C.H.
      • de Menezes R.C.A.
      • DiMicco J.A.
      The dorsomedial hypothalamus and the central pathways involved in the cardiovascular response to emotional stress.
      ,
      • Fontes M.A.P.
      • Menezes R.C.A.
      • Villela D.C.
      • Da Silva L.G.
      The dorsomedial hypothalamus and the organization of the cardiovascular response to emotional stress: a functional perspective.
      ). Despite providing clear evidence that the autonomic nervous system composes neural circuitries governing physiological responses (
      • Fontes M.A.P.
      • Xavier C.H.
      • de Menezes R.C.A.
      • DiMicco J.A.
      The dorsomedial hypothalamus and the central pathways involved in the cardiovascular response to emotional stress.
      ) (
      • Kober H.
      • Barrett L.F.
      • Joseph J.
      • Bliss-Moreau E.
      • Lindquist K.
      • Wager T.D.
      Functional grouping and cortical-subcortical interactions in emotion: a meta-analysis of neuroimaging studies.
      ), literature has few details on the descending pathways from midbrain that would mediate autonomic efferences recruited during defensive reactions.
      Periaqueductal gray (PAG) is a midbrain region surrounding cerebral aqueduct that is considered a behavioral-autonomic interface (
      • Faull O.K.
      • Subramanian H.H.
      • Ezra M.
      • Pattinson K.T.S.
      The midbrain periaqueductal gray as an integrative and interoceptive neural structure for breathing.
      ), as it connects different brain levels through ascending and descending projections (
      • Vianna D.M.L.
      • Brandão M.L.
      Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear.
      ). PAG also integrates emotional processing systems and motor pathways used for expressing behaviors during defense reactions (
      • Vianna D.M.L.
      • Brandão M.L.
      Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear.
      ). However, PAG does not project to spinal intermediolateral column, the efferent system conducting responses organized by the autonomic sympathetic branch. Therefore, it requires connections with autonomic premotor neurons to produce some responses (
      • Farkas E.
      • Jansen A.S.P.
      • Loewy A.D.
      Periaqueductal gray matter input to cardiac-related sympathetic premotor neurons.
      ;
      • Schenberg L.C.
      • Brandao C.A.L.
      • Vasquez E.C.
      Role of periaqueductal gray matter in hypertension in spontaneously hypertensive rats.
      ) (
      • Bandler R.
      • Carrive P.
      • Zhang S.P.
      Chapter 13 integration of somatic and autonomic reactions within the midbrain periaqueductal grey: viscerotopic, somatotopic and functional organization.
      ) (
      • Behbehani M.M.
      Functional characteristics of the midbrain periaqueductal gray.
      ). In fact, studies show that PAG projects to vagal preganglionic nuclei (
      • Farkas E.
      • Jansen A.S.P.
      • Loewy A.D.
      Periaqueductal gray matter projection to vagal preganglionic neurons and the nucleus tractus solitarius.
      ) and interferes with the neurotransmission within these areas (
      • Haxhiu M.A.
      • Yamamoto B.K.
      • Dreshaj I.A.
      • Ferguson D.G.
      Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.
      ). Also, experiments with retrograde transneuronal tracer injected into the sympathetic stellate ganglion showed the PAG-sympathetic integration (
      • Jansen A.S.P.
      • Wessendorf M.W.
      • Loewy A.D.
      Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion.
      ).
      PAG is subdivided in columns that also project to each other within this midbrain area (
      • Vianna D.M.L.
      • Landeira-Fernandez J.
      • Brandão M.L.
      Dorsolateral and ventral regions of the periaqueductal gray matter are involved in distinct types of fear.
      ) (
      • Ruiz-Torner A.
      • Olucha-Bordonau F.
      • Valverde-Navarro A.A.
      • Martínez-Soriano F.
      The chemical architecture of the rat’s periaqueductal gray based on acetylcholinesterase histochemistry: a quantitative and qualitative study.
      ) (
      • Jansen A.S.P.
      • Farkas E.
      • Mac Sams J.
      • Loewy A.D.
      Local connections between the columns of the periaqueductal gray matter: a case for intrinsic neuromodulation.
      ). The pattern of anatomical connection seems to be related to the function mediated by every part of this columnar system (
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Cliffer K.D.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. I. Ascending projections.
      ,
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. II. Descending projections.
      ) (
      • Depaulis A.
      • Keay K.A.
      • Bandler R.
      Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat.
      ) (
      • Behbehani M.M.
      Functional characteristics of the midbrain periaqueductal gray.
      ) (
      • Farook J.M.
      • Wang Q.
      • Moochhala S.M.
      • Zhu Z.Y.
      • Lee L.
      • Wong P.T.H.
      Distinct regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG rats on the cat-freezing test apparatus.
      ) (
      • Vianna Daniel M.L.
      • Graeff F.G.
      • Brandão M.L.
      • Landeira-Fernandez J.
      Defensive freezing evoked by electrical stimulation of the periaqueductal gray: comparison between dorsolateral and ventrolateral regions.
      ). Stimulation of lateral PAG displays defensive behavioral responses and increases cardiac output and blood pressure (
      • Lovick T.A.
      Inhibitory modulation of the cardiovascular defence response by the ventrolateral periaqueductal grey matter in rats.
      ) (
      • da Silva L.G.
      • Menezes R.C.A.
      • Villela D.C.
      • Fontes M.A.P.
      Excitatory amino acid receptors in the periaqueductal gray mediate the cardiovascular response evoked by activation of dorsomedial hypothalamic neurons.
      ). Previous studies have shown that stimulation of the lateral PAG changes vasomotion, peripheral resistance, heart rate and respiration (
      • Bandler R.
      • Carrive P.
      • Zhang S.P.
      Chapter 13 integration of somatic and autonomic reactions within the midbrain periaqueductal grey: viscerotopic, somatotopic and functional organization.
      ) (
      • Bandler R.
      • Carrive P.
      Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat.
      ), which altogether mimic the responses typically evoked by acute emotional stress (
      • Dampney R.
      Emotion and the cardiovascular system: postulated role of inputs from the medial prefrontal cortex to the dorsolateral periaqueductal gray.
      ). It was demonstrated that lateral and dorsolateral PAG columns control the tachycardia and pressor responses evoked from hypothalamic activation and from acute stress exposure (
      • da Silva L.G.
      • Menezes R.C.A.
      • Villela D.C.
      • Fontes M.A.P.
      Excitatory amino acid receptors in the periaqueductal gray mediate the cardiovascular response evoked by activation of dorsomedial hypothalamic neurons.
      ;
      • Da Silva L.G.
      • Alvim De Menezes R.C.
      • Souza dos Santos R.A.
      • Campagnole-Santos M.J.
      • Peliky Fontes M.A.
      Role of periaqueductal gray on the cardiovascular response evoked by disinhibition of the dorsomedial hypothalamus.
      ;
      • Villela D.C.
      • da Silva Junior L.G.
      • Fontes M.A.P.
      Activation of 5-HT receptors in the periaqueductal gray attenuates the tachycardia evoked from dorsomedial hypothalamus.
      ) (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ). Notwithstanding these important findings, little is known on the descending medullary pathways mediating cardiovascular responses evoked from PAG.
      Anatomic data obtained by using transsynaptic tracing techniques show that PAG connects – directly or indirectly – with medullary sympathetic premotor neurons, such as those comprised within rostral ventrolateral (RVLM) and ventromedial medulla (
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. II. Descending projections.
      ) (
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Cliffer K.D.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. I. Ascending projections.
      ). Additional studies confirmed that the cardiovascular responses caused by the PAG stimulation are mediated by the RVLM premotor neurons that control vasomotion, which may reflect a pattern of sympathetically-mediated pressor responses during conditioned fear and other stressor stimuli (
      • Carrive P.
      The periaqueductal gray and defensive behavior: functional representation and neuronal organization.
      ) (
      • Carrive P.
      • Gorissen M.
      Premotor sympathetic neurons of conditioned fear in the rat.
      ). Although RVLM is a descending pathway recruited by PAG to evoke cardiovascular responses (
      • Carrive P.
      • Bandler R.
      Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study.
      ), there is no data on the role of ventromedial medullary paths and on the amplitude of the cardiac inotropic responses resulting from PAG activation. Since Raphe Pallidus (RPa) is a ventromedial medullary region encircling autonomic premotor neurons connected to adrenergic supplies innervating the heart (
      • Cao W.H.
      • Morrison S.F.
      Disinhibition of rostral raphe pallidus neurons increases cardiac sympathetic nerve activity and heart rate.
      ) (
      • Jansen A.S.P.
      • Wessendorf M.W.
      • Loewy A.D.
      Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion.
      ) (
      • Loewy A.D.
      Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat.
      ) (
      • Ter Horst G.J.
      • Hautvast R.W.M.
      • De Jongste M.J.L.
      • Korf J.
      Neuroanatomy of cardiac activity-regulating circuitry: a transneuronal retrograde viral labelling study in the rat.
      ) (
      • Ter Horst G.J.
      • Van Den Brink A.
      • Homminga S.A.
      • Hautvast R.W.M.
      • Rakhorst G.
      • Mettenleiter T.C.
      • De Jongste M.J.L.
      • Lie K.I.
      • Korf J.
      Transneuronal viral lab elling of rat heart left ventricle controlling pathways.
      ), it is worth considering the PAG-RPa path. We next investigated: i) whether PAG projects directly to RPa; ii) the amplitude of the inotropic and chronotropic responses evoked from PAG; iii) whether cardiovascular responses evoked from PAG rely on RPa.

      2. Material and methods

      The animals were provided by the local animal facility (Biotério Central UFG). Experimental procedures were approved by Ethics Committee on Animal Use of the Federal University of Goiás (Brazil) (CEUA-UFG protocol 092/18). We used male Wistar rats (250–350 g). Our procedures followed the rules established by the Brazilian Committee for Animal Experiment (COBEA) and by United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals needed to complete the experiments.

      2.1 Experimental procedures

      Procedures involving drug injections (see experiments 1–3 ahead) into PAG and RPa were conducted under urethane (1.4 g/kg) and chloralose (120 mg/kg). Its adequacy was verified by the absence of a withdrawal response to a nociceptive stimulation of a hindpaw. Supplemental doses of anesthetics were given when necessary. Polyethylene catheters were placed into the femoral artery and vein for recordings arterial pressure (AP) and for drug injections, respectively. Left ventricular pressure (LVP) was measured using polyethylene catheter inserted into the left ventricle through the right common carotid artery. Peak values of the first derivative from LVP, i.e., LVdP/dT peak (a measure of contractility), were computed online. Subsequently, the animals were positioned on a heating pad in a prone position. Head was placed in a stereotaxic frame (AVS Projetos, Brazil), in ventral decubitus with the head positioned −3.3 mm below the interaural line. The skull was exposed in the midline for visualization of Bregma and Lambda. The coordinates to reach PAG (relative to bregma – anteroposterior: −7.8 mm; dorsoventral: −4.8 mm; and lateral: −0.7 mm) and RPa (relative to lambda – anteroposterior: −3.0 mm; dorsoventral: −9.6 mm; and mediolateral: 0 mm) were in according with Atlas of Paxinos and Watson, (
      • Paxinos G.
      • Watson C.
      The Rat Brain in Stereotaxic Coordinates - The New Coronal Set. English.
      ). After performing a local craniotomy, an ultra-fine tipped glass micropipette was directed to the PAG and/or RPa and drugs were injected. The nanoinjection volume for all drugs, including the Evans Blue dye, was 100 nL. Body temperature was monitored using rectal thermometer and maintained in the range of 37–37.5 °C with a heating pad. In all experiments, the animals underwent anesthesia procedures and surgical procedures described above; after a minimum period of 20 min, for stabilization of the hemodynamic parameters of the animals, injections of the drugs were performed in the brain regions.

      2.1.1 Experiment 1: cardiovascular responses evoked from disinhibition of PAG (n = 6)

      This experiment aimed to reveal the amplitude of the cardiac chronotropic and inotropic responses evoked from disinhibition of lateral/dorsolateral (l/dl) PAG columns. After anesthesia, surgical procedures and cardiovascular parameters stabilization, vehicle was injected in RPa (NaCl 0.9%) 20 min before injecting the GABAA antagonist Bicuculline Methiode (0.4 mM) into l/dl PAG.

      2.1.2 Experiment 2: cardiovascular responses evoked from inhibition of RPa neurons (n = 6)

      This experiment aimed to reveal the amplitude of the chronotropic and inotropic responses evoked by inhibition of RPa neurons. After anesthesia, surgical procedures and cardiovascular parameters stabilization, GABAA agonist muscimol (20 mM) was injected in RPa.

      2.1.3 Experiment 3: cardiovascular responses evoked from PAG following inhibition of RPa neurons (n = 6)

      This experimental series aimed to reveal the amplitude of the chronotropic and inotropic responses evoked from PAG following inhibition of RPa neurons. After anesthesia, surgical procedures and cardiovascular parameters stabilization, GABAA agonist muscimol (20 mM) was injected in RPa 20 min before injecting the GABAA antagonist Bicuculline Methiode (0.4 mM) into l/dl PAG.

      2.2 Histological analysis

      At the end of the experiments 1, 2 and 3 the animals were injected with Evans' blue dye (100 nL) into lateral and dorsolateral columns of PAG, followed by an overdose of anesthetic (urethane 5 g/kg). The brains were removed, kept in paraformaldehyde (PFA) 10% (24 h) and then transferred to sucrose solution 30% for 48 h. Brain slices (40 μm) were cut in cryostat and mounted on previously gelatinized glass slides. Mounted slides with fresh brain slices were set in a microscope and the blue spots marked by the dye were used to confirm injection site, which was compared with the Atlas of Paxinos and Watson (
      • Paxinos G.
      • Watson C.
      The Rat Brain in Stereotaxic Coordinates - The New Coronal Set. English.
      ).

      2.2.1 Experiment 4: monosynaptic projection from PAG to RPa using retrograde neuronal tracer injections (RetroBeads) (n = 3)

      This experiment aimed to reveal whether PAG projects monsynaptically to RPa neurons and was conducted as previously described (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ) (
      • Apps R.
      • Ruigrok T.J.H.
      A fluorescence-based double retrograde tracer strategy for charting central neuronal connections.
      ). The animals were anesthetized with ketamine (10 mg/kg, i.p.) and xylazine (4 mg/kg, i.p.) and fixed in a stereotaxic frame. Undiluted red Retrobeads™ (Luma Fluor) were nanoinjected (100 nL) into the RPa. After the surgery, an antibiotic [Pentabiotic® (Benzathine, Benzilpeniciline, Sodium Benzilpeniciline, Potassium Benzilpeniciline, Procaine, Benzilpeniciline and Estreptomicin) 5 mg/kg; 0.2 mL] and an analgesic solution [Banamine Pet®, (Flunixim Meglumine) 1.1 mg/kg] were injected (i.m.). Seven days later, animals were euthanized by an overdose of anesthetic (urethane 5 g/kg, i.p.), the brains were removed, maintained in 4% PFA for 24 h and then transferred to a 30% sucrose solution for 48 h. Brain slices (40 μm) containing injection spots into RPa and PAG regions were taken using a cryostat and mounted on silanized glass slides. Analyses and depicting of retrogradely labelled neurons within PAG were performed using a fluorescence microscope (excitation max of 530 nm and emission max of 590 nm).

      2.3 Statistical analysis

      The software used for cardiovascular parameters acquisition and analysis was LabChart 7 (ADInstruments, Australia). Mean maximal changes were sampled from 30 second periods during baseline (pre-injection) and maximum responses, obtained following central injections. The statistical test used was two-tailed student t-test (GraphPad Prism 6). The significance level considered was P < 0.05 and all values are presented as mean ± standard error of the mean.

      3. Results

      3.1 Periaqueductal gray projects monosynaptically to Raphe Pallidus neurons

      Fig. 1 shows PAG neurons labelled with retrograde monosynaptic tracer that was injected into Raphe Pallidus seven days before the neurohistological analyses. This is an evidence that PAG projects directly to ventromedial medullary RPa neurons.
      Fig. 1
      Fig. 1Panel A – Photomicrographs of brain slice depicting injection spots from animals injected with red Retrobeads into RPa, at the level of rostral ventromedial medulla. Panel B - Photomicrographs of brain slice depicting retrogradely labelled neurons at the level of PAG, seven days past injection of red Retrobeads into RPa. Panel C - Zoom view of the PAG neurons depicted in Panel B. Aq: aqueduct. Py: pyramids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      3.2 Cardiac responses evoked from periaqueductal gray neurons

      Table 1 shows baseline and mean maximal changes in cardiovascular parameters sampled during the first experimental series. Fig. 2 shows the effect of the GABAA receptors antagonist injection into the unilateral lateral/dorsolateral PAG columns upon cardiac function and arterial pressure. BMI into PAG significantly increased mean arterial pressure (MAP), heart rate (HR), peak values of left ventricular pressure (LVP Peak) and of its first derivative (LVdP/dt peak). These effects were already evident at the initial minute past injection.
      Table 1Basal and peak values obtained in the first experimental series. Mean arterial pressure (MAP), peak values of left ventricular pressure (LVP Peak), heart rate (HR) and peak values of ventricular pressure derivative (LVdP/dt peak). Values expressed as mean ± standard error of the mean.
      Experiment 1
      BasalMaximalDelta
      MAP (mmHg)95 ± 3144 ± 11
      P < 0.05 vs. basal.
      49 ± 13
      LVP peak (mmHg)123 ± 15190 ± 23
      P < 0.05 vs. basal.
      67 ± 23
      LVdP/dt peak (mmHg/s)5772 ± 11528209 ± 1773
      P < 0.05 vs. basal.
      2278 ± 826
      HR (bpm)418 ± 12469 ± 17
      P < 0.05 vs. basal.
      50 ± 5
      low asterisk P < 0.05 vs. basal.
      Fig. 2
      Fig. 2Representative chart records and changes in LVP max, MAP, LVdP/dT peak and heart rate evoked by the nanoinjection of bicuculline methiodide into unilateral PAG (n = 6). *P < 0.05 basal vs. maximal responses (Paired student t-test).

      3.3 Cardiac effects evoked by inhibition of Raphe Pallidus

      Table 2 shows baseline and mean maximal changes in cardiovascular parameters sampled during the second experimental series. Fig. 3 shows the effect of the GABAA receptors agonist injected into the unilateral lateral/dorsolateral PAG columns upon cardiac function and arterial pressure. Inhibition of RPa neurons with muscimol evoked significant reductions in MAP, LVP Peak, HR and LVdP/dt peak. These effects were already evident just after injection and were maintained until the subsequent experimental procedure.
      Table 2Basal and peak values obtained in the second experimental series. Mean arterial pressure (MAP), peak values of left ventricular pressure (LVP Peak), heart rate (HR) and peak values of ventricular pressure derivative (LVdP/dt peak). Values expressed as mean ± standard error of the mean.
      Experiment 2
      BasalMaximalDelta
      MAP (mmHg)126 ± 259 ± 4
      P < 0.05 vs. basal.
      −66 ± 5
      LVP peak (mmHg)138 ± 959 ± 12
      P < 0.05 vs. basal.
      −84 ± 17
      LVdP/dt peak (mmHg/s)5159 ± 14233654 ± 831−1505 ± 654
      HR (bpm)360 ± 8323 ± 9
      P < 0.05 vs. basal.
      −34 ± 11
      low asterisk P < 0.05 vs. basal.
      Fig. 3
      Fig. 3Representative chart records and changes in LVP max, MAP, LVdP/dT peak and heart rate evoked by the nanoinjection of muscimol into RPa neurons (n = 6). *P < 0.05 basal vs. maximal responses (Paired student t-test).

      3.4 Cardiac responses evoked from periaqueductal gray following inhibition of Raphe Pallidus are attenuated

      Table 3 shows baseline and mean maximal changes in cardiovascular parameters sampled during the third experimental series. Fig. 4 shows the effect of the GABAA receptors antagonist injected into the unilateral lateral/dorsolateral PAG columns following RPa inhibition upon cardiac function and arterial pressure. BMI into PAG still evoked significant increases in pressure and contractility (Fig. 4), but the amplitude of these responses was attenuated when compared to those sampled in animals injected with BMI into PAG without RPa inhibition (Fig. 5).
      Table 3Basal and peak values obtained in the third experimental series. Mean arterial pressure (MAP), peak values of left ventricular pressure (LVP Peak), heart rate (HR) and peak values of ventricular pressure derivative (LVdP/dt peak). Values expressed as mean ± standard error of the mean.
      Experiment 3
      BasalMaximalDelta
      MAP (mmHg)56 ± 5126 ± 3
      P < 0.05 vs. basal.
      70 ± 8
      LVP peak (mmHg)53 ± 9138 ± 9
      P < 0.05 vs. basal.
      79 ± 12
      LVdP/dt peak (mmHg/s)3653 ± 8294993 ± 1427
      P < 0.05 vs. basal.
      1473 ± 640
      HR (bpm)345 ± 23400 ± 30
      P < 0.05 vs. basal.
      104 ± 19
      low asterisk P < 0.05 vs. basal.
      Fig. 4
      Fig. 4Representative chart records and changes in LVP max, MAP, LVdP/dT peak and heart rate evoked by the nanoinjection of bicuculline methiodide into unilateral PAG following injection of muscimol into RPa (n = 6). *P < 0.05 basal vs. maximal responses (Paired student t-test).
      Fig. 5
      Fig. 5Mean maximal changes in LVP max, MAP, LVdP/dT peak and heart rate evoked by the nanoinjection of bicuculline methiodide into unilateral PAG (black bars) (n = 6) compared with those evoked by same bicuculine midbrain injection following inhibition of RPa neurons (gray bars). *P < 0.05 (Unpaired student t-test).

      4. Discussion

      The present study revealed that a direct projection from PAG to RPa controls the cardiac responses evoked from this mesenphalic periaqueductal region. Previous studies have described the anatomical communication between PAG and RPa, but these past methodological choices used transsynaptic tracings (
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Cliffer K.D.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. I. Ascending projections.
      ,
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. II. Descending projections.
      ). In the light of this, one question that still remained open was whether there would be a direct projection from PAG to RPa, a medullary region comprising cardiac sympathetic premotor neurons (
      • Jansen A.S.P.
      • Wessendorf M.W.
      • Loewy A.D.
      Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion.
      ) (
      • Morrison S.F.
      • Gebber G.L.
      Classification of raphe neurons with cardiac-related activity.
      ) (
      • Loewy A.
      • Spyer K. Michael
      Anatomy of the autonomic nervous system: an overview.
      ). We found that PAG projects to RPa and these projections probably activate sympathetic premotor neurons, thus provoking the positive cardiac chronotropic and inotropic responses herein reported. Since inhibition of RPa neurons reduced the magnitude of the cardiac contractile responses evoked from PAG stimulation, we believe that descending excitatory projections from PAG stimulate sympathetic premotor neurons of rostral ventromedial medulla. In addition to the autonomic-cardiovascular involvement, this pathway may also be involved in the somatosensory component of defense reactions (
      • Bandler R.
      • Carrive P.
      Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat.
      ) (
      • Abols I.A.
      • Basbaum A.I.
      Afferent connections of the rostral medulla of the cat: a neural substrate for midbrain-medullary interactions in the modulation of pain.
      ) (
      • Holstege G.
      • Tan J.
      Projections from the red nucleus and surrounding areas to the brainstem and spinal cord in the cat. An HRP and autoradiographical tracing study.
      ).
      Plenty of studies have reported on RPa role in the organization of cardiovascular and thermogenic responses, including those evoked from acute stress exposure. Cao and Morrison showed that RPa stimulation increases blood pressure, heart rate and temperature accompanied by augmented outflow to cardiac sympathetic nerves (
      • Cao W.H.
      • Morrison S.F.
      Disinhibition of rostral raphe pallidus neurons increases cardiac sympathetic nerve activity and heart rate.
      ). RPa inhibition reduces the amplitude of sympathetic responses evoked from skin cooling, a model of thermal stress (
      • Nakamura K
      • Morrison SF
      Central efferent pathways mediating skin cooling-evoked sympathetic thermogenesis in brown adipose tissue.
      ). Prior studies showed that stimulation of RPa neurons produces cardiac chronotropic and inotropic responses that rise in an afterload-independent manner (
      • Mcallen R.M.
      • May C.N.
      • Shafton A.D.
      Functional anatomy of sympathetic premotor cell groups in the medulla.
      ) (
      • Salo L.M.
      • Nalivaiko E.
      • Anderson C.R.
      • McAllen R.M.
      Control of cardiac rate, contractility, and atrioventricular conduction by medullary raphe neurons in anesthetized rats.
      ), which supports the idea that cardiac responses evoked from RPa activation are predominantly consequence of augmented sympathetic outflow to the heart. Another study demonstrated that inhibition of RPa neurons attenuates the tachycardia evoked by acute emotional stress (
      • Zaretsky D
      • Zaretskaia M
      • Samuels B
      • Cluxton L
      • Dimicco J
      Microinjection of muscimol into raphe pallidus suppresses tachycardia associated with air stress in conscious rats.
      ), thus confirming that defense reactions depend on increases in excitatory inputs to these sympathetic premotor neurons to produce tachycardia. Our study adds the evidence that projections from PAG may be another direct source of excitatory inputs that activate ventromedial medullary sympathetic premotor neurons.
      The responses evoked by neuronal disinhibition with an antagonist of GABAA receptors indicate that PAG is under a tonic inhibition in baseline conditions. The methodological choice of using a GABAA antagonist to disinhibit PAG neurons has been already reported in the literature (
      • Behbehani M.M.
      • Jiang M.
      • Chandler S.D.
      • Ennis M.
      The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat.
      ). Our findings on cardiovascular responses evoked from injecting a GABAA antagonist into PAG corroborate with the hypothesis of a possible tonic gabaergic inhibition. Although not being per se an excitatory drug (
      • Ueno S.
      • Bracamontes J.
      • Zorumski C.
      • Weiss D.S.
      • Steinbach J.H.
      Bicuculline and gabazine are allosteric inhibitors of channel opening of the GABA(A) receptor.
      ), bicuculline would remove the inhibitory (hyperpolarizing) component that acts over PAG neurons, thus allowing excitatory inputs to predominate. Such excitatory predominance would be responsible for activating (depolarizing) PAG neurons injected with bicuculline (
      • Ueno S.
      • Bracamontes J.
      • Zorumski C.
      • Weiss D.S.
      • Steinbach J.H.
      Bicuculline and gabazine are allosteric inhibitors of channel opening of the GABA(A) receptor.
      ). Data from the literature support this methodological choice [for review, see (
      • Fontes M.A.P.
      • Xavier C.H.
      • Marins F.R.
      • Limborço-Filho M.
      • Vaz G.C.
      • Müller-Ribeiro F.C.
      • Nalivaiko E.
      Emotional stress and sympathetic activity: contribution of dorsomedial hypothalamus to cardiac arrhythmias.
      ) (
      • DiMicco J.A.
      • Samuels B.C.
      • Zaretskaia M.V.
      • Zaretsky D.V.
      The dorsomedial hypothalamus and the response to stress: part renaissance, part revolution.
      )], which was based on the hypothesis that reducing gabaergic activity would result in a predominant excitatory influence upon PAG (
      • Bandler R.J.
      Chemical stimulation of the rat midbrain and aggressive behaviour.
      ) (
      • Behbehani M.M.
      • Jiang M.
      • Chandler S.D.
      • Ennis M.
      The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat.
      ). The predominance of these excitatory inputs upon PAG is further evidenced during stress (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ) (
      • de Menezes R.C.
      • Zaretsky D.V.
      • Sarkar S.
      • Fontes M.A.
      • Dimicco J.A.
      Microinjection of muscimol into the periaqueductal gray suppresses cardiovascular and neuroendocrine response to air jet stress in conscious rats.
      ), as PAG inhibition is able to attenuate the stress-evoked tachycardia (
      • Da Silva L.G.
      • Alvim De Menezes R.C.
      • Souza dos Santos R.A.
      • Campagnole-Santos M.J.
      • Peliky Fontes M.A.
      Role of periaqueductal gray on the cardiovascular response evoked by disinhibition of the dorsomedial hypothalamus.
      ) (
      • da Silva L.G.
      • Menezes R.C.A.
      • Villela D.C.
      • Fontes M.A.P.
      Excitatory amino acid receptors in the periaqueductal gray mediate the cardiovascular response evoked by activation of dorsomedial hypothalamic neurons.
      ) (
      • de Menezes R.C.
      • Zaretsky D.V.
      • Sarkar S.
      • Fontes M.A.
      • Dimicco J.A.
      Microinjection of muscimol into the periaqueductal gray suppresses cardiovascular and neuroendocrine response to air jet stress in conscious rats.
      ) (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ). Pharmacological approaches assessing the balance between neuronal activation/disinhibition and inhibition were also attempted on other central structures associated with the control of cardiovascular function at baseline conditions and during defense reactions (
      • Cao W.H.
      • Morrison S.F.
      Disinhibition of rostral raphe pallidus neurons increases cardiac sympathetic nerve activity and heart rate.
      ) (
      • Mendonça M.M.
      • Santana J.S.
      • da Cruz K.R.
      • Ianzer D.
      • Ghedini P.C.
      • Nalivaiko E.
      • Fontes M.A.P.
      • Ferreira R.N.
      • Pedrino G.R.
      • Colugnati D.B.
      • Xavier C.H.
      Involvement of GABAergic and adrenergic neurotransmissions on paraventricular nucleus of hypothalamus in the control of cardiac function.
      ) (
      • Müller-Ribeiro F.C.F.
      • Goodchild A.K.
      • McMullan S.
      • Fontes M.A.P.
      • Dampney R.A.L.
      Coordinated autonomic and respiratory responses evoked by alerting stimuli: role of the midbrain colliculi.
      ).
      By using the same tracing methodology, we have previously reported a bidirectional projection between PAG and dorsomedial hypothalamus (DMH) (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ). These respective midbrain and diencephalic regions are key in the control of physiological responses to stress (
      • Fontes M.A.P.
      • Xavier C.H.
      • Marins F.R.
      • Limborço-Filho M.
      • Vaz G.C.
      • Müller-Ribeiro F.C.
      • Nalivaiko E.
      Emotional stress and sympathetic activity: contribution of dorsomedial hypothalamus to cardiac arrhythmias.
      ) (
      • Fontes M.A.P.
      • Menezes R.C.A.
      • Villela D.C.
      • Da Silva L.G.
      The dorsomedial hypothalamus and the organization of the cardiovascular response to emotional stress: a functional perspective.
      ) (
      • Xavier C.H.
      • Mendonça M.M.
      • Marins F.R.
      • da Silva E.S.
      • Ianzer D.
      • Colugnati D.B.
      • Pedrino G.R.
      • Fontes M.A.P.
      Stating asymmetry in neural pathways: methodological trends in autonomic neuroscience.
      ). Other studies showed that the cardiovascular and thermogenic responses evoked from DMH are attenuated following PAG inhibition (
      • Dampney R.
      Emotion and the cardiovascular system: postulated role of inputs from the medial prefrontal cortex to the dorsolateral periaqueductal gray.
      ) (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ) (
      • de Menezes R.C.
      • Zaretsky D.V.
      • Sarkar S.
      • Fontes M.A.
      • Dimicco J.A.
      Microinjection of muscimol into the periaqueductal gray suppresses cardiovascular and neuroendocrine response to air jet stress in conscious rats.
      ) (
      • da Silva L.G.
      • Menezes R.C.A.
      • Villela D.C.
      • Fontes M.A.P.
      Excitatory amino acid receptors in the periaqueductal gray mediate the cardiovascular response evoked by activation of dorsomedial hypothalamic neurons.
      ). Therefore, it is possible that ascending projections to hypothalamus would be involved in the organization of responses triggered by PAG stimulation (
      • de Menezes R.C.A.
      • Zaretsky D.V.
      • Fontes M.A.P.
      • DiMicco J.A.
      Cardiovascular and thermal responses evoked from the periaqueductal grey require neuronal activity in the hypothalamus.
      ) (
      • Xavier Carlos Henrique
      • Ianzer D.
      • Lima A.M.
      • Marins F.R.
      • Pedrino G.R.
      • Vaz G.
      • Menezes G.B.
      • Nalivaiko E.
      • Fontes M.A.P.
      Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.
      ). This shall to be supported by evidences showing that other hypothalamic nuclei participate in the generation of PAG-evoked responses: besides receiving projections from PAG, hypothalamic neurons connect with PAG and with different medullary nuclei involved in the control of cardiovascular function (
      • Cao W.H.
      • Fan W.
      • Morrison S.F.
      Medullary pathways mediating specific sympathetic responses to activation of dorsomedial hypothalamus.
      ) (
      • Fontes M.A.P.
      • Tagawa T.
      • Polson J.W.
      • Cavanagh S.J.
      • Dampney R.A.L.
      Descending pathways mediating cardiovascular response from dorsomedial hypothalamic nucleus.
      ). In fact, PAG projects to paraventricular hypothalamus (PVH) (
      • Vianna D.M.L.
      • Brandão M.L.
      Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear.
      ), a region that is strongly involved in the cardiovascular control and governs cardiac function (
      • Mendonça M.M.
      • Santana J.S.
      • da Cruz K.R.
      • Ianzer D.
      • Ghedini P.C.
      • Nalivaiko E.
      • Fontes M.A.P.
      • Ferreira R.N.
      • Pedrino G.R.
      • Colugnati D.B.
      • Xavier C.H.
      Involvement of GABAergic and adrenergic neurotransmissions on paraventricular nucleus of hypothalamus in the control of cardiac function.
      ) (
      • Akine A.
      • Montanaro M.
      • Allen A.M.
      Hypothalamic paraventricular nucleus inhibition decreases renal sympathetic nerve activity in hypertensive and normotensive rats.
      ) (
      • Allen A.M.
      Inhibition of the hypothalamic paraventricular nucleus in spontaneously hypertensive rats dramatically reduces sympathetic vasomotor tone.
      ). PAG activation recruits hypothalamic neurons (
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Cliffer K.D.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. I. Ascending projections.
      ,
      • Cameron A.A.
      • Khan I.A.
      • Westlund K.N.
      • Willis W.D.
      The efferent projections of the periaqueductal gray in the rat: a Phaseolus vulgaris-leucoagglutinin study. II. Descending projections.
      ), which in turn, send direct excitatory projections to sympathetic preganglionic neurons and to RVLM and RPa (
      • Cao W.H.
      • Fan W.
      • Morrison S.F.
      Medullary pathways mediating specific sympathetic responses to activation of dorsomedial hypothalamus.
      ;
      • Samuels B.C.
      • Zaretsky D.V.
      • DiMicco J.A.
      Tachycardia evoked by disinhibition of the dorsomedial hypothalamus in rats is mediated through medullary raphe.
      ). Both hypothalamus and medulla are important areas regulating cardiovascular function and play a leading role in keeping blood pressure and cardiac function. In the light of the seminal viscerotopic data suggesting that RVLM is in charge of controlling PAG-evoked pressor responses (afterload) (
      • Carrive P.
      • Bandler R.
      • Dampney R.A.L.
      Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray.
      ) (
      • Carrive P.
      • Gorissen M.
      Premotor sympathetic neurons of conditioned fear in the rat.
      ) (
      • Carrive P.
      • Bandler R.
      Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study.
      ), we propose that the PAG-RPa pathway is prominently responsible for organizing cardiac components of the cardiovascular responses typically seen during PAG-dependent defense reactions. However, the RPa inhibition did not evoke significant modifications in the amplitude of the positive chronotropism evoked by the subsequent PAG disinhibition, which allows us to suggest that additional mechanisms, such as the activation of cardiac noradrenergic terminals recruited by baroreflex, may be involved (
      • Guyenet P.G.
      The sympathetic control of blood pressure.
      ;
      • Verberne A.J.M.
      • Guyenet P.G.
      Midbrain central gray: influence on medullary sympathoexcitatory neurons and the baroreflex in rats.
      ).
      To our knowledge, the present study is the first to describe the amplitude of positive inotropic responses generated by PAG disinhibition. Taking a translational perspective, literature reports that unilateral brain activation following stress exposure may be related to increases in the risk of sudden death and other cardiovascular effects, such as arrythmias (
      • Ziegelstein R.C.
      Acute emotional stress and cardiac arrhythmias.
      ) (
      • Davis A.M.
      • Natelson B.H.
      Brain-heart interactions: the neurocardiology of arrhythmia and sudden cardiac death.
      ) (
      • Xavier C.H.
      • Mendonça M.M.
      • Marins F.R.
      • da Silva E.S.
      • Ianzer D.
      • Colugnati D.B.
      • Pedrino G.R.
      • Fontes M.A.P.
      Stating asymmetry in neural pathways: methodological trends in autonomic neuroscience.
      ). Some of these studies shows that asymmetric midbrain activity during stress is linked to cardiac ectopies (
      • Critchley H.D.
      • Taggart P.
      • Sutton P.M.
      • Holdright D.R.
      • Batchvarov V.
      • Hnatkova K.
      • Malik M.
      • Dolan R.J.
      Mental stress and sudden cardiac death: asymmetric midbrain activity as a linking mechanism.
      ) (
      • Harris J.A.
      • Guglielmotti V.
      • Bentivoglio M.
      Diencephalic asymmetries.
      ) (
      • Toga A.W.
      • Thompson P.M.
      Mapping brain asymmetry.
      ), but the mechanisms underlying this abnormal heart function remain to be fully clarified. Considering that the PAG is a midbrain region involved in the physiological responses typically triggered from acute emotional stress (
      • Dampney R.
      Emotion and the cardiovascular system: postulated role of inputs from the medial prefrontal cortex to the dorsolateral periaqueductal gray.
      ) (
      • Xavier C.H.
      • Mendonça M.M.
      • Marins F.R.
      • da Silva E.S.
      • Ianzer D.
      • Colugnati D.B.
      • Pedrino G.R.
      • Fontes M.A.P.
      Stating asymmetry in neural pathways: methodological trends in autonomic neuroscience.
      ), we suggest that increases in cardiac output during defense reactions may rely on PAG-RPa pathway, which may also be involved in the aforementioned stress-evoked non-homeostatic cardiac function. In conclusion, PAG activation increases cardiac chronotropy and inotropy, and these responses seem to depend on a direct pathway from PAG reaching ventromedial medullary RPa neurons.

      Support

      Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) granted Xavier C.H. (Universal 406393/2018-4; PQ 308156/2018-8), Pedrino GR (PQ 312130/2019-8) and Fontes MAP (PQ 304388/2017-3); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

      Acknowledgements

      We thank and honor Dr. Arthur D. Loewy https://medicine.wustl.edu/news/obituary-arthur-d-loewy-professor-anatomy-neuroscience-74/ (in memoriam) https://www.legacy.com/obituaries/name/arthur-loewy-obituary?pid=187432032 for gently sending us offprints of his pioneer articles.

      Submission declaration

      The authors declare that the present work has not been published previously (except in the form of an abstract or as a part of an academic thesis), that it is not under consideration for publication elsewhere, that its publication has approved by all authors and, tacitly or explicitly, by the responsible authorities of the facilities in which the work was performed, and that, if accepted, the present work will not be published elsewhere in print or electronically in the same form in English or in any other language without the written consent of the copyright-holder.

      Declaration of competing interest

      No conflicts of interest, financial or otherwise, are declared by the authors.

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