6-cyano-7-nitroquinoxaline-2-3-dione and Hypercapnia

Both orexin neurons in the lateral hypothalamus and spinally-projecting pre-sympathetic neurons (PSNs) in the paraventricular nucleus of the hypothalamus (PVN) play an important role in the regulation of cardiovascular function under normal conditions and during cardiovascular challenges such as hypoxia and/or hypercapnia. We have previously established, using selective optogenetic excitation of orexin neurons and pathways, there is a heterogeneous neurotransmission from orexin neurons to PSNs in the PVN. This study was undertaken to test whether this pathway is altered by acute exposure to hypoxia alone and/or combined hypoxia and hypercapnia (H/H). To test this hypothesis, we selectively expressed channelrhodopsin-2 (ChR2) in orexin neurons in the lateral hypothalamus and photoactivated ChR2-expressing fibers to evoke postsynaptic currents in spinally-projecting PSNs in an in vitro slice preparation in rats. In accordance with previously published data, two subpopulations of spinally-projecting PSNs were established, including those with glutamatergic or GABAergic inputs from orexin neurons. Hypoxia alone did not alter the peak amplitude of either glutamatergic or GABAergic neurotransmission, however, H/H significantly inhibited both glutamatergic and GABAergic neurotransmission from orexin neurons to SPNs. In conclusion, H/H may modulate cardiovascular function by affecting heterogeneous pathways from orexin neurons to spinally-projecting PSNs in the PVN.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; Green Fluorescent Proteins; Hypercapnia; Hypoxia; In Vitro Techniques; Male; Neurons; Optogenetics; Orexins; Paraventricular Hypothalamic Nucleus; Pyridazines; Rats; Rats, Transgenic; Rhodopsin; Synaptic Transmission; Time Factors

Cardioinhibitory cardiac vagal neurons (CVNs) do not receive inspiratory-related excitatory inputs under normal conditions. However, excitatory purinergic and serotonergic pathways are recruited during inspiratory activity after episodes of hypoxia and hypercapnia (H/H). Prenatal nicotine (PNN) exposure is known to dramatically change cardiorespiratory responses and decrease the ability to resuscitate from H/H. This study tested whether PNN exposure alters excitatory neurotransmission to CVNs in the nucleus ambiguus during and after H/H. Spontaneous and inspiratory evoked excitatory postsynaptic currents were recorded in CVNs from rats that were exposed to nicotine (6 mg x kg(-1) x d(-1)) throughout the prenatal period. In contrast to unexposed animals, in PNN animals H/H recruited excitatory neurotransmission to CVNs during inspiratory-related activity that was blocked by the alpha3beta4 nicotinic acetylcholine receptor (nAChR) blocker alpha-conotoxin AuIB (alpha-CTX AuIB, 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM) and d(-)-2-amino-5-phosphonopentanoic acid (AP5, 50 microM), selective AMPA/kainate and N-methyl-d-aspartate receptor blockers, respectively. Following H/H, there was a significant increase in inspiratory-related excitatory postsynaptic currents that were unaltered by alpha-CTX AuIB or ondansetron, a 5-HT3 receptor blocker, but were subsequently inhibited by pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (100 microM), a purinergic receptor blocker and CNQX and AP5. The results from this study demonstrate that with PNN exposure, an excitatory neurotransmission to CVNs is recruited during H/H that is glutamatergic and dependent on activation of alpha3beta4-containing nAChRs. Furthermore, exposure to PNN abolishes a serotonergic long-lasting inspiratory-related excitation of CVNs that is replaced by recruitment of a glutamatergic pathway to CVNs post H/H.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Conotoxins; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agents; Female; Heart; Hypercapnia; Hypoxia; In Vitro Techniques; Male; Neurons; Nicotine; Nicotinic Antagonists; Ondansetron; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Serotonin; Serotonin Antagonists; Synaptic Transmission; Vagus Nerve; Valine

Inhibitory GABAergic and glycinergic neurotransmission to cardioinhibitory cardiac vagal neurons (CVNs) increase during inspiratory activity and likely mediate respiratory sinus arrhythmia, while the frequency of excitatory postsynaptic currents (EPSCs) in CVNs are unaltered during the different phases of respiration. However, following hypoxia and hypercapnia (H/H), the parasympathetic activity to the heart increases and thus far, identification of the pathways and neurotransmitters that are responsible for exciting CVNs post H/H are unclear. This study identifies different excitatory pathways to CVNs recruited post H/H. Spontaneous and inspiratory-related EPSCs were recorded in CVNs before, during, and after 10 min of H/H in an in vitro slice preparation that retains rhythmic respiratory activity. Before and during H/H, EPSCs in CVNs were completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5), selective AMPA/kainate and N-methyl-d-apartate (NMDA) receptor blockers, respectively. However, after H/H, there was a significant increase in EPSCs during each inspiratory burst. While some of the inspiratory-related EPSCs were blocked by the broad purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and the specific P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate monolithium trisodium salt (TNP-ATP) a P2X receptor blocker, most of the recruited excitatory neurotransmission to CVNs is serotonergic because odansetron, a selective 5-HT3 antagonist, abolished the majority of the spontaneous and inspiratory-related EPSCs evoked during recovery from H/H. The results from this study suggest that following episodes of H/H, two nonglutamatergic excitatory pathways, purinergic and serotonergic, activating P2X and 5-HT3 receptors, respectively, are recruited to excite CVNs in the post H/H recovery period.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Animals, Newborn; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hypercapnia; Hypoxia; In Vitro Techniques; Neurons; Nucleus Accumbens; Ondansetron; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Respiration; Serotonin; Serotonin Antagonists; Vagus Nerve; Valine

The respiratory role of excitatory amino acid (EAA) receptors within the Bötzinger complex (BötC) and the pre-Bötzinger complex (pre-BötC) was investigated in alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nL) of EAA receptor antagonists. Blockade of both N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors by 50 mM kynurenic acid (KYN) within the BötC induced a pattern of breathing characterized by low-amplitude, high-frequency irregular oscillations superimposed on tonic phrenic activity and successively the disappearance of respiratory rhythmicity in the presence of intense tonic inspiratory discharges (tonic apnea). KYN microinjections into the pre-BötC caused similar respiratory responses that, however, never led to tonic apnea. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1, 10 and 20 mM) within the BötC induced increases in respiratory frequency and decreases in peak phrenic amplitude; the highest concentrations caused tonic apnea insensitive to chemical stimuli. Blockade of non-NMDA receptors by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 and 20 mM) within the BötC produced only less pronounced increases in respiratory frequency. Responses to D-AP5 in the pre-BötC were similar, although less pronounced than those elicited in the BötC and never characterized by tonic apnea. In the same region, CNQX provoked increases in respiratory frequency similar to those elicited in the BötC, associated with slight reductions in peak phrenic activity. The results show that EAA receptors within the investigated medullary subregions mediate a potent control on both the intensity and frequency of inspiratory activity, with a major role played by NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Blood Pressure; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Hypercapnia; Hypoxia; Kynurenic Acid; Male; Medulla Oblongata; Microinjections; Neurons; Phrenic Nerve; Rabbits; Respiration; Time Factors; Vagotomy