Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson's disease. If this is the case, there may be a protective role for antioxidant therapies

Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson's disease. If this is the case, there may be a protective role for antioxidant therapies

Phasic contractions of the mouse vagina and cervix at different phases of the estrus cycle and during late pregnancy

Background/Aims: The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca²⁺ release from sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) -dependent intracellular Ca²⁺ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking. Methodology/Principal Findings: Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K⁺ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca²⁺ stores, but were inhibited in low Ca²⁺ solution or in the presence of nifedipine, an inhibitor of L-type Ca²⁺channels. ICs were found in small numbers in the mouse cervix but not in the vagina. Conclusions/Significance: Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca²⁺ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges

Developmental changes in pacemaker currents in mouse locus coeruleus neurons

The present study compares the electrophysiological properties and the primary pacemaker currents that flow during the interspike interval in locus coeruleus (LC) neurons from infant (P7–12 days) and young adult (8–12 weeks) mice. The magnitude of the primary pacemaker currents, which consist of an excitatory TTX-sensitive Na⁺ current and an inhibitory voltage-dependent K⁺ current, increased in parallel during development. We found no evidence for the involvement of hyperpolarization-activated (IH) or Ca2⁺ currents in pacemaking in infant or adult LC neurons. The incidence of TTX-resistant spikes, observed during current clamp recordings, was greater in adult neurons. Neurons from adult animals also showed an increase in voltage fluctuations, during the interspike interval, as revealed in the presence of the K⁺ channel blocker, 4-AP (1 mM). In summary, our results suggest that mouse LC neurons undergo changes in basic electrophysiological properties during development that influence pacemaking and hence spontaneous firing in LC neurons

Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson’s disease. If this is the case, there may be a protective role for antioxidant therapies

Contractile responses recorded in response to the potassium channel blocker tetraethylammonium chloride (TEA) or an agonist (oxytocin) on cervical and vaginal smooth muscle with uterine data presented for comparative purposes.

<p>Application of TEA (10 mM) enhanced spontaneous contractions in uterine (<b>A</b>) and cervical (<b>B</b>) tissues and caused vaginal (<b>C</b>) and cervical tissues in diestrus and proestrus (data not shown) to become spontaneously active. Application of oxytocin (1 nM) to uterine (<b>D</b>), cervical (<b>E</b>) and vaginal (<b>F</b>) tissues caused a large contraction that gradually decreased revealing spontaneous contractions at high frequency for all three tissues.</p

Spontaneous contractions occur in the presence of CPA, an inhibitor of SR Ca²⁺ stores.

<p>Shown are the effects of inhibiting the SERCA using 10 µM CPA on contractions in uterine (<b>A, A1</b>), cervical (<b>B, B1</b>) and vaginal (<b>C, C1</b>) smooth muscle strips from non-prenant and pregnant mice. Recordings from vaginal tissues were made in the presence of TEA (10 mM) to reveal contractions. CPA (10 µM) applied for at least 30 minutes modulated but did not abolish the contractions in any of the three tissue types.</p

Anatomy of the mouse female reproductive tract.

<p>The diagram shows the regions at which the corresponding sections, labeled with smooth muscle α-actin conjugated to Cy3, were obtained (1 – vagina, 2 – cervix with surrounding vaginal tissue, <b>3</b> – uterine body, <b>4</b> – uterine horn). The uterine body and horn present a denser and wider muscle layer than the cervix and vagina.</p

Ketamine anesthesia helps preserve neuronal viability

The dissociative anesthetic ketamine that acts as an N-methyl-D-aspartate (NMDA) antagonist has been reported to improve neurological damage after experimental ischemic challenges. Here we show that deep anesthesia with ketamine before euthanasia by decapitation improves the quality of neonatal mouse neuronal brain slice preparations. Specifically we found that neurons of the locus coeruleus (LC) and hypoglossal motor neurons had significantly higher input resistances, and LC neurons that generally are difficult to voltage control, could be more reliably voltage clamped compared to control neurons