Activation state of the hyperpolarization-activated current modulates temperature-sensitivity of firing in locus coeruleus neurons from bullfrogs

Locus coeruleus neurons of anuran amphibians contribute to breathing control and have spontaneous firing frequencies that, paradoxically, increase with cooling. We previously showed that cooling inhibits a depolarizing membrane current, the hyperpolarization-activated current ( Ih) in locus coeruleus neurons from bullfrogs, Lithobates catesbeianus (Santin JM, Watters KC, Putnam RW, Hartzler LK. Am J Physiol Regul Integr Comp Physiol 305: R1451–R1464, 2013). This suggests an unlikely role for Ih in generating cold activation, but led us to hypothesize that inhibition of Ih by cooling functions as a physiological brake to limit the cold-activated response. Using whole cell electrophysiology in brain slices, we employed 2 mM Cs+ (an Ih antagonist) to isolate the role of Ih in spontaneous firing and cold activation in neurons recorded with either control or Ih agonist (cyclic AMP)-containing artificial intracellular fluid. Ih did not contribute to the membrane potential ( Vm) and spontaneous firing at 20°C. Although voltage-clamp analysis confirmed that cooling inhibits Ih, its lack of involvement in setting baseline firing and Vm precluded its ability to regulate cold activation as hypothesized. In contrast, neurons dialyzed with cAMP exhibited greater baseline firing frequencies at 20°C due to Ih activation. Our hypothesis was supported when the starting level of Ih was enhanced by elevating cAMP because cold activation was converted to more ordinary cold inhibition. These findings indicate that situations leading to enhancement of Ih facilitate firing at 20°C, yet the hyperpolarization associated with inhibiting a depolarizing cation current by cooling blunts the net Vm response to cooling to oppose normal cold-depolarizing factors. This suggests that the influence of Ih activation state on neuronal firing varies in the poikilothermic neuronal environment.

Postnatal emergence of serotonin-induced plateau potentials in commissural interneurons of the mouse spinal cord

Most studies of the mouse hindlimb locomotor network have used neonatal (P0–5) mice. In this study, we examine the postnatal development of intrinsic properties and serotonergic modulation of intersegmental commissural interneurons (CINs) from the neonatal period (P0–3) to the time the animals bear weight (P8–10) and begin to show adult walking (P14–16). CINs show an increase in excitability with age, associated with a decrease in action potential halfwidth and appearance of a fast component to the afterhyperpolarization at P14–16. Serotonin (5-HT) depolarizes and increases the excitability of most CINs at all ages. The major developmental difference is that serotonin can induce plateau potential capability in P14–16 CINs, but not at younger ages. These plateau potentials are abolished by nifedipine, suggesting that they are mediated by an L-type calcium current, ICa(L). Voltage-clamp analysis demonstrates that 5-HT increases a nifedipine-sensitive voltage-activated calcium current, ICa(V), in P14–16 CINs but does not increase ICa(V) in P8–10 CINs. These results, together with earlier work on 5-HT effects on neonatal CINs, suggest that 5-HT increases the excitability of CINs at all ages studied, but by opposite effects on calcium currents, decreasing N- and P/Q-type calcium currents and, indirectly, calcium-activated potassium current, at P0–3 but increasing ICa(L) at P14–16.

Development and Serotonergic Modulation of NMDA Bursting in Rat Trigeminal Motoneurons

The development of N-methyl-d-aspartate (NMDA)-induced burst discharge in rat trigeminal motoneurons (TMNs) between postnatal days P1 and P10 was examined using whole cell patch-clamp recording methods in brain slices. Bath application of NMDA (50 μM) induced a Mg2+-dependent rhythmical bursting activity starting around P8. Prior to the onset of bursting, the membrane potential depolarized and the input resistance increased. Hyperpolarization of the membrane potential with extrinsic current demonstrated a narrow window of membrane potential where maintained rhythmical burst discharge was evident. In P1–P4 neurons, NMDA application produced membrane depolarization and a minimal change in input resistance, but no burst activity at any membrane potential. Voltage-clamp analysis indicated that the bursting activity was related to the presence or absence of a voltage-dependent Mg2+ block and induction of a negative slope conductance (NSC) region in the I NMDA- V relationship. Regardless of age, reduction of extracellular Mg2+ from 1 mM to 30 μM enhanced I NMDA at voltages negative to −60 mV. However, in 1 mM Mg2+, P1–P4 neurons were devoid of a prominent NSC region compared with P8–P10 neurons, suggesting that the efficacy of depolarization in unblocking the NMDA receptors increased with age. NMDA bursting was not dependent on calcium influx through voltage-gated calcium channels (VGCC) but did require a minimal concentration of Ca2+ in the bath. Intracellular bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid application suppressed burst discharge completely, suggesting that intracellular Ca2+ directly, or via second-messenger systems, regulates NMDA receptor activity and bursting. Interestingly, NMDA bursting could be induced in P1–P4 neurons by simultaneous bath application of serotonin (5-HT, 10 μM), which by itself did not produce bursting, suggesting an “enabling” role for 5-HT. Voltage-clamp analysis demonstrated that the NMDA/5-HT bursting resulted from induction of an NSC in the I-Vrelationship of total membrane current. 5-HT by itself produced no such effect. The mechanisms for this effect were due to an enhancement of the NSC region of the I NMDA- V relationship and reduction of a presumed leak current by 5-HT. These data indicate that NMDA bursting in trigeminal motoneurons is developmentally regulated and subject to neuromessenger modulation. Control of the Mg2+ sensitivity of the NMDA receptor and voltage-dependent block by neuromessengers could be an effective means to control the efficacy of glutamatergic synaptic drive to motoneurons during rhythmical oral-motor activity at early postnatal ages.