Effects of a Lower Level of Control O\u3csub\u3e2\u3c/sub\u3e, Hyperoxia and Hypercapnia on Solitary Complex Neurons in Medullary Tissue Slices

Studies of neuronal O2-/CO2-chemosensitivity in the brainstem often employ tissue slices maintained in medium equilibrated with 95%O2, which we now know produces hyperoxia, increased production of reactive species, and oxidative stress. Accordingly, the objectives of this study were to determine if solitary complex (SC) neurons in medullary slices, incubated in 40%O2, exhibit spontaneous, stable activity and are responsive to hyperoxia and hypercapnia. Medullary slices (300-400µm, P3-P21) were harvested in 95%O2 + 5%CO2 (2-4°C) and immediately transferred to 40%O2 + 5%CO2 (22-24°C). Whole-cell recordings (~37°C) of 69 neurons show a mean resting membrane potential of -42mV with spontaneous and evoked action potentials that overshoot 0mV. Whole cell recordings were made within 2-7.5hr post-slicing. To date, 25 neurons were tested using hyperoxia (95% O2 + 5% CO2), hypercapnia (40% O2 + 10%CO2) and hyperoxic hypercapnia (90%O2 + 10%CO2). The majority of neurons (13) were insensitive to hyperoxia/hypercapnia. Eight neurons were stimulated by hyperoxia and five by hypercapnia. Of these, four were stimulated by hyperoxic hypercapnia. Two neurons were inhibited by hypercapnia and one was inhibited by hyperoxia. We conclude that medullary slices can be maintained in 40%O2 for many hours for studies of CO2/O2-chemosensitivity at normobaric pressure. (ONR N000140710890, NIH R01 HL 56683-09)

Effects of a Lower Level of Control O\u3csub\u3e2\u3c/sub\u3e, Hyperoxia and Hypercapnia on Solitary Complex Neurons in Medullary Tissue Slices

Studies of neuronal O2-/CO2-chemosensitivity in the brainstem often employ tissue slices maintained in medium equilibrated with 95%O2, which we now know produces hyperoxia, increased production of reactive species, and oxidative stress. Accordingly, the objectives of this study were to determine if solitary complex (SC) neurons in medullary slices, incubated in 40%O2, exhibit spontaneous, stable activity and are responsive to hyperoxia and hypercapnia. Medullary slices (300-400µm, P3-P21) were harvested in 95%O2 + 5%CO2 (2-4°C) and immediately transferred to 40%O2 + 5%CO2 (22-24°C). Whole-cell recordings (~37°C) of 69 neurons show a mean resting membrane potential of -42mV with spontaneous and evoked action potentials that overshoot 0mV. Whole cell recordings were made within 2-7.5hr post-slicing. To date, 25 neurons were tested using hyperoxia (95% O2 + 5% CO2), hypercapnia (40% O2 + 10%CO2) and hyperoxic hypercapnia (90%O2 + 10%CO2). The majority of neurons (13) were insensitive to hyperoxia/hypercapnia. Eight neurons were stimulated by hyperoxia and five by hypercapnia. Of these, four were stimulated by hyperoxic hypercapnia. Two neurons were inhibited by hypercapnia and one was inhibited by hyperoxia. We conclude that medullary slices can be maintained in 40%O2 for many hours for studies of CO2/O2-chemosensitivity at normobaric pressure. (ONR N000140710890, NIH R01 HL 56683-09)

12 TIPS for Implementing Peer Instruction in Medical Education

Peer Instruction (PI) is a vibrant instructional strategy, used successfully for over two decades in undergraduate physics and mathematics courses. It has had limited use and few publications in medical education. This 12 TIPS provides a focused review on the evidence supporting its use in higher education and rationale for its wider adoption in medical education. The authors detail important steps for its implementation with large classes. Based on several years of experience with PI in a US allopathic medical school, they feel that PI attends to core principles from the science of learning and provides students and faculty with immediate feedback on learning. It is also adaptable to on-line synchronous administration

Normobaric Hyperoxia (95% O\u3csub\u3e2\u3c/sub\u3e) Stimulates CO\u3csub\u3e2\u3c/sub\u3e-Sensitive and CO\u3csub\u3e2\u3c/sub\u3e-Insensitive Neurons in the Caudal Solitary Complex of Rat Medullary Tissue Slices Maintained in 40% O\u3csub\u3e2\u3c/sub\u3e

We tested the hypothesis that decreasing the control level of O2 from 95% to 40% reduces tissue partial pressure of oxygen (pO2), decreases extracellular nitric oxide (NO) and decreases intracellular superoxide (O2−) while maintaining viability in caudal solitary complex (cSC) neurons in slices (∼300–400 μm; neonatal rat P2–22; 34–37 °C). We also tested the hypothesis that normobaric hyperoxia is a general stimulant of cSC neurons, including CO2-excited neurons. Whole-cell recordings of cSC neurons maintained in 40% O2 were comparable to recordings made in 95% O2 in duration and quality. In 40% O2, cSC neurons had a significantly lower spontaneous firing rate but similar membrane potentials and input resistances as cSC neurons maintained in 95% O2. Tissue pO2 was threefold lower in 40% O2 versus 95% O2. Likewise, extracellular NO and intracellular O2− were lower in 40% versus 95% O2. 67% of neurons maintained in 40% O2 control were stimulated by hyperoxia (95% O2) compared to 81% of neurons maintained in 95% O2 that were stimulated during hyperoxic reoxygenation following acute exposure to 0–40% O2. cSC slices maintained in 40% O2 exhibited CO2-chemosensitive neurons, including CO2-excited (31.5%) and a higher incidence of CO2-inhibited (31.5%) neurons than previously reported. Likewise, a higher incidence of CO2-inhibited and lower incidence of CO2-excited neurons were observed in 85–95% O2. 82% of O2-excited neurons were also CO2-chemosensitive; CO2-excited (86%) and CO2-inhibited neurons (84%) were equally stimulated by hyperoxia. Our findings demonstrate that chronic (hours) and acute (minutes) exposure to hyperoxia stimulates firing rate in the majority of cSC neurons, most of which are also CO2 chemosensitive. Our findings support the hypothesis that recurring exposures to acute hyperoxia and hyperoxic reoxygenation—a repeating surge in tissue pO2—activate redox and nitrosative signaling mechanisms in CO2-chemosensitive neurons that alter expression of CO2 chemosensitivity (e.g., increased expression of CO2-inhibition) compared to sustained hyperoxia (85–95% O2)

Further Evidence of Redox Modulation of Neurons in a CO\u3csub\u3e2\u3c/sub\u3e-Chemosensitive Area: Normobaric Hyperoxia (95%O\u3csub\u3e2\u3c/sub\u3e) Stimulates CO\u3csub\u3e2\u3c/sub\u3e-Chemosensitive and –Insensitive Neurons in the Solitary Complex (SC)

We previously reported that CO2-excited neurons in SC, in medullary slices maintained in 95%O2, are stimulated by chemical oxidants and hyperbaric O2 (JAP 95:910-921, 2003; AJP 286:C940–951, 2004). Here we test the hypothesis that SC neurons, maintained in 40%O2 rather than 95%O2 (to reduce oxidative stress), are viable and stimulated by normobaric hyperoxia (95%O2). Slices (P1–21) were harvested in chilled ACSF gassed with 95%O2 and immediately transferred to ACSF (22–24°C) equilibrated with 40%O2-5%CO2 in N2. Whole cell recordings (35–37°C) were established in 40%O2 (n=44) and tested using hyperoxia (4095%O2) and hypercapnia (510 or 15%CO2). Hyperoxia stimulated 8/44 neurons and hypercapnia stimulated 5/44 neurons; of these 4/8 were stimulated by both O2 and CO2. Hyperoxia usually increased firing rate and decreased input resistance whereas hypercapnia increased firing rate and input resistance. Most neurons tested were insensitive to hyperoxia and hypercapnia (31/44). Compared to previous slice studies that used 95%O2 control, there is a smaller proportion of CO2-excited SC neurons in slices maintained in 40%O2. These data indicate that medullary slices are viable in 40%O2 and that hyperoxic stress stimulates SC neurons, including CO2-excited neurons. These data also suggest that oxidative stimuli increases the incidence of CO2-chemosensitivity in SC neurons (ONR N000140110179, NIH R01HL56683)

Normobaric Hyperoxia (95% O\u3csub\u3e2\u3c/sub\u3e) Stimulates CO\u3csub\u3e2\u3c/sub\u3e-Sensitive and CO\u3csub\u3e2\u3c/sub\u3e-Insensitive Neurons in the Caudal Solitary Complex of Rat Medullary Tissue Slices Maintained in 40% O\u3csub\u3e2\u3c/sub\u3e

We tested the hypothesis that decreasing the control level of O2 from 95% to 40% reduces tissue partial pressure of oxygen (pO2), decreases extracellular nitric oxide (NO) and decreases intracellular superoxide (O2−) while maintaining viability in caudal solitary complex (cSC) neurons in slices (∼300–400 μm; neonatal rat P2–22; 34–37 °C). We also tested the hypothesis that normobaric hyperoxia is a general stimulant of cSC neurons, including CO2-excited neurons. Whole-cell recordings of cSC neurons maintained in 40% O2 were comparable to recordings made in 95% O2 in duration and quality. In 40% O2, cSC neurons had a significantly lower spontaneous firing rate but similar membrane potentials and input resistances as cSC neurons maintained in 95% O2. Tissue pO2 was threefold lower in 40% O2 versus 95% O2. Likewise, extracellular NO and intracellular O2− were lower in 40% versus 95% O2. 67% of neurons maintained in 40% O2 control were stimulated by hyperoxia (95% O2) compared to 81% of neurons maintained in 95% O2 that were stimulated during hyperoxic reoxygenation following acute exposure to 0–40% O2. cSC slices maintained in 40% O2 exhibited CO2-chemosensitive neurons, including CO2-excited (31.5%) and a higher incidence of CO2-inhibited (31.5%) neurons than previously reported. Likewise, a higher incidence of CO2-inhibited and lower incidence of CO2-excited neurons were observed in 85–95% O2. 82% of O2-excited neurons were also CO2-chemosensitive; CO2-excited (86%) and CO2-inhibited neurons (84%) were equally stimulated by hyperoxia. Our findings demonstrate that chronic (hours) and acute (minutes) exposure to hyperoxia stimulates firing rate in the majority of cSC neurons, most of which are also CO2 chemosensitive. Our findings support the hypothesis that recurring exposures to acute hyperoxia and hyperoxic reoxygenation—a repeating surge in tissue pO2—activate redox and nitrosative signaling mechanisms in CO2-chemosensitive neurons that alter expression of CO2 chemosensitivity (e.g., increased expression of CO2-inhibition) compared to sustained hyperoxia (85–95% O2)

Further Evidence of Redox Modulation of Neurons in a CO\u3csub\u3e2\u3c/sub\u3e-Chemosensitive Area: Normobaric Hyperoxia (95%O\u3csub\u3e2\u3c/sub\u3e) Stimulates CO\u3csub\u3e2\u3c/sub\u3e-Chemosensitive and –Insensitive Neurons in the Solitary Complex (SC)

We previously reported that CO2-excited neurons in SC, in medullary slices maintained in 95%O2, are stimulated by chemical oxidants and hyperbaric O2 (JAP 95:910-921, 2003; AJP 286:C940–951, 2004). Here we test the hypothesis that SC neurons, maintained in 40%O2 rather than 95%O2 (to reduce oxidative stress), are viable and stimulated by normobaric hyperoxia (95%O2). Slices (P1–21) were harvested in chilled ACSF gassed with 95%O2 and immediately transferred to ACSF (22–24°C) equilibrated with 40%O2-5%CO2 in N2. Whole cell recordings (35–37°C) were established in 40%O2 (n=44) and tested using hyperoxia (4095%O2) and hypercapnia (510 or 15%CO2). Hyperoxia stimulated 8/44 neurons and hypercapnia stimulated 5/44 neurons; of these 4/8 were stimulated by both O2 and CO2. Hyperoxia usually increased firing rate and decreased input resistance whereas hypercapnia increased firing rate and input resistance. Most neurons tested were insensitive to hyperoxia and hypercapnia (31/44). Compared to previous slice studies that used 95%O2 control, there is a smaller proportion of CO2-excited SC neurons in slices maintained in 40%O2. These data indicate that medullary slices are viable in 40%O2 and that hyperoxic stress stimulates SC neurons, including CO2-excited neurons. These data also suggest that oxidative stimuli increases the incidence of CO2-chemosensitivity in SC neurons (ONR N000140110179, NIH R01HL56683)

Superoxide Production Increases in Nucleus Tractus Solitarius (NTS) Neurons in Rat Brain Slices during Acute Normobaric Hyperoxia and Hypoxia

We previously reported that hyperbaric hyperoxia stimulates firing rate of putative CO2-chemoreceptors in the solitary complex of the dorsocaudal medulla oblongata in rat brain slices (JAP 95: 910-921, 2003). We next reported that the typical control level of 95%O2 is a greater source of redox stress than ≤ 40%O2 leading to increased cell death in brain slices (J. Neurophysiol. 98:1030-1041, 2007). In the present study we used 20-40%O2 as the control to test the hypothesis that normobaric hyperoxia and hypoxia increase the rate of superoxide production (·O2-) in NTS neurons. Brain slices (400μm, 36-37oC) were maintained using 1- or 2-sided superfusion. Brainstem neurons maintained in 20-40%O2 (5%CO2, balance N2) exhibited i) whole-cell/intracellular activity for many hours, ii) CO2 chemosensitivity (10-15%CO2) and iii) were stimulated by hyperoxia (60-95%O2). ·O2- production was measured (3 min intervals) using the fluorogenic probe, dihydroethidium (2.5μM), continuously loaded via the superfusate. The rate of ·O2- production (slope of fluorescence intensity units/min, FIU/min) increased during acute hyperoxia (20 to 95%O2, 15-20min). Likewise, FIU/min increased during hypoxia (40/20% to 0%O2, 10-20min). ·O2- production during hypoxia was dependent on a lower threshold tissue pO2 that is estimated to be well below 20 Torr based on measurements of tissue slice pO2. ·O2- production during hypoxia was repeatedly induced using 95%N2-5%CO2 during either 1) 1-sided slice superfusion or 2) in combination with an O2-scavenger (1mM Na2SO3) during 2-sided slice superfusion. Myxothiazol (10μM; an inhibitor of Complex III) decreased ·O2- production during hypoxia but had little effect during hyperoxia. This suggests that mitochondrial Complex III is the primary source of ·O2- during hypoxia but not hyperoxia in NTS neurons. Preliminary experiments in CA1 hippocampus and Inferior olive indicate that these neurons do not increase their rate of ·O2- production during hypoxia/Na2SO3. We posit that the similar pattern of ·O2- production in NTS neurons activated by hypoxia and hyperoxia renders these cardio-respiratory neurons vulnerable to redox stimulation and/or stress during sleep disordered breathing (episodic hypoxia, reoxygenation and rebound hyperoxia) and during exposure to normobaric and hyperbaric hyperoxi