BK potassium currents contribute differently to action potential waveform and firing rate as rat hippocampal neurons mature in the first postnatal week.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator of neuronal action potential firing and follows two distinct trends in early postnatal development: an increase in total expression and a shift from the faster activating STREX isoform to the slower ZERO isoform. We analyzed the functional consequences of developmental trends in BK channel expression in hippocampal neurons isolated from neonatal rats aged 1 to 7 days. Following overnight cultures, action potentials and currents were recorded using whole cell patch-clamp electrophysiology. These neurons undergo a steady increase in excitability during this time, and the effect of blockade of BK channel activity with 100 nM iberiotoxin changes as the neurons mature. BK currents contribute significantly more to total potassium current and single action potentials in neurons of 1-day old rats (with BK blockade extending action potential duration by 0.46 ± 0.12 ms) than in those of 7-day old rats (with BK blockade extending action potential duration by 0.17 ± 0.05 ms). BK currents contribute consistently to maintain firing rates in neurons of 1-day old rats throughout extended action potential firing; BK blockade evenly depresses firing frequency across action potential trains. In neurons from 7-day old rats, BK blockade initially increases firing frequency and then progressively decreases frequency as firing continues, ultimately depressing neuronal firing rates to a greater extent than in the neurons from 1-day-old animals. These results are consistent with a transition from low expression of a fast-activating BK isoform (STREX) to high expression of a slower activating isoform (ZERO).NEW & NOTEWORTHY This work describes the early developmental trends of large-conductance calcium-activated potassium (BK) channel activity. Early developmental trends in expression of BK channels, both total expression and relative isoform expression, have been previously reported, but little work describes the effect of these changes in expression patterns on excitability. Here, we show that early changes in BK channel expression patterns lead to changes in the role of BK channels in determining the action potential waveform and neuronal excitability.

GABA(B) receptors couple to Gαq to mediate increases in voltage-dependent calcium current during development.

Metabotropic GABA(B) receptors are known to modulate the activity of voltage-dependent calcium channels. Previously, we have shown that GABA(B) receptors couple to a non-Gi/o G-protein to enhance calcium influx through L-type calcium channels by activating protein kinase C in neonatal rat hippocampal neurons. In this study, the components of this signaling pathway were investigated further. Gαq was knocked down using morpholino oligonucleotides prior to examining GABA(B) -mediated enhancement of calcium influx. When Gαq G-proteins were eliminated using morpholino-mediated knockdown, the enhancing effects of the GABA(B) receptor agonist baclofen (10 µM) on calcium current or entry were eliminated. These data suggest that GABA(B) receptors couple to Gαq to regulate calcium influx. Confocal imaging analysis illustrating colocalization of GABA(B) receptors with Gαq supports this hypothesis. Furthermore, baclofen treatment caused translocation of PKCα (protein kinase C α) but not PKCß or PKCε, suggesting that it is the α isoform of PKC that mediates calcium current enhancement. Inhibition of calcium/calmodulin-dependent kinase II did not affect the baclofen-mediated enhancement of calcium levels. In summary, activation of GABA(B) receptors during development leads to increased calcium in a subset of neurons through Gαq signaling and PKCα activation without the involvement of calcium/calmodulin-dependent kinase II. Activation of GABA(B) receptors in the neonatal rat hippocampus enhances voltage-dependent calcium currents independently of Gi/o . In this study, knockdown of Gαq with morpholino oligonucleotides abolished enhancement of calcium influx and protein kinase Cα was activated by GABA(B) receptors. Therefore, we hypothesize that GABA(B) receptors couple to Gq to activate PKCα leading to enhancement of L-type calcium current.

Nonspecific, reversible inhibition of voltage-gated calcium channels by CaMKII inhibitor CK59.

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium-calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC50 for CK59 was approximately 50 µM, which is only fivefold larger than the reported IC50 values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC50 for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.

H+- and Na+- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga Chlamydomonas.

Although microtubules are known for dynamic instability, the dynamicity is considered to be tightly controlled to support a variety of cellular processes. Yet diverse evidence suggests that this is not applicable to Chlamydomonas, a biflagellate fresh water green alga, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by rises of intracellular H+ and Na+. These results from this model organism with characteristics of animal and plant cells provide novel explanations regarding how pH may drive cellular processes; how plants may respond to, and perhaps sense stresses; and how organisms with a similar sensitive cytoskeleton may be susceptible to environmental changes.

Amyotrophic lateral sclerosis patient IgG alters voltage dependence of Ca2+ channels in dissociated rat motoneurons.

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) have detected IgG within motoneurons of patients and these IgGs have been shown to alter voltage dependent calcium channel activity in various cell types. The current study investigates whether IgG from categorized SALS patients alter voltage dependent calcium currents in rat motoneurons in culture. Patients were categorized based on onset and progression pattern. IgG yields were 38% higher in SALS patients compared to control subjects. Incubation with 1 mg/ml IgG from SALS patients did not cause visible toxicity, alter input resistance, capacitance or the maximal calcium conductance in rat motoneurons when compared to motoneurons incubated with control IgG. However, the activation curve of calcium current was shifted to the left in motoneurons treated with SALS IgG compared to control IgG.

Writing Assignments with a Metacognitive Component Enhance Learning in a Large Introductory Biology Course.

Writing assignments, including note taking and written recall, should enhance retention of knowledge, whereas analytical writing tasks with metacognitive aspects should enhance higher-order thinking. In this study, we assessed how certain writing-intensive "interventions," such as written exam corrections and peer-reviewed writing assignments using Calibrated Peer Review and including a metacognitive component, improve student learning. We designed and tested the possible benefits of these approaches using control and experimental variables across and between our three-section introductory biology course. Based on assessment, students who corrected exam questions showed significant improvement on postexam assessment compared with their nonparticipating peers. Differences were also observed between students participating in written and discussion-based exercises. Students with low ACT scores benefited equally from written and discussion-based exam corrections, whereas students with midrange to high ACT scores benefited more from written than discussion-based exam corrections. Students scored higher on topics learned via peer-reviewed writing assignments relative to learning in an active classroom discussion or traditional lecture. However, students with low ACT scores (17-23) did not show the same benefit from peer-reviewed written essays as the other students. These changes offer significant student learning benefits with minimal additional effort by the instructors.

Levels of Ca(V)1.2 L-Type Ca(2+) Channels Peak in the First Two Weeks in Rat Hippocampus Whereas Ca(V)1.3 Channels Steadily Increase through Development.

Influx of calcium through voltage-dependent channels regulates processes throughout the nervous system. Specifically, influx through L-type channels plays a variety of roles in early neuronal development and is commonly modulated by G-protein-coupled receptors such as GABA(B) receptors. Of the four isoforms of L-type channels, only Ca(V)1.2 and Ca(V)1.3 are predominately expressed in the nervous system. Both isoforms are inhibited by the same pharmacological agents, so it has been difficult to determine the role of specific isoforms in physiological processes. In the present study, Western blot analysis and confocal microscopy were utilized to study developmental expression levels and patterns of Ca(V)1.2 and Ca(V)1.3 in the CA1 region of rat hippocampus. Steady-state expression of Ca(V)1.2 predominated during the early neonatal period decreasing by day 12. Steady-state expression of Ca(V)1.3 was low at birth and gradually rose to adult levels by postnatal day 15. In immunohistochemical studies, antibodies against Ca(V)1.2 and Ca(V)1.3 demonstrated the highest intensity of labeling in the proximal dendrites at all ages studied (P1-72). Immunohistochemical studies on one-week-old hippocampi demonstrated significantly more colocalization of GABA(B) receptors with Ca(V)1.2 than with Ca(V)1.3, suggesting that modulation of L-type calcium current in early development is mediated through Ca(V)1.2 channels.

Influx of calcium through L-type calcium channels in early postnatal regulation of chloride transporters in the rat hippocampus.

During the early postnatal period, GABA(B) receptor activation facilitates L-type calcium current in rat hippocampus. One developmental process that L-type current may regulate is the change in expression of the K(+)Cl(-) co-transporter (KCC2) and N(+)K(+)2Cl(-) co-transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L-type current, GABA(B) receptors, and expression of chloride transporters during development. The facilitation of L-type current by GABA(B) receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L-type channel antagonist and a GABA(B) receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L-type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L-type channels in the early postnatal period in hippocampal neurons.

Gamma-aminobutyric acid type B receptors facilitate L-type and attenuate N-type Ca(2+) currents in isolated hippocampal neurons.

Activation of presynaptic gamma-aminobutyric acid type B (GABA(B)) receptors inhibits neurotransmitter release at many synapses (both excitatory and inhibitory), and activation of postsynaptic GABA(B) receptors leads to a general inhibition of the postsynaptic cell in mature neurons. Although the action of GABA(B) receptors at the soma of excitatory hippocampal pyramidal cells has been resolved to be regulation of a potassium or calcium conductance, it is not clear that all neurons in the hippocampus demonstrate similar effects of GABA(B) receptor activation. In the current study, GABA(B) receptor-mediated effects on calcium currents in acute cultures composed of heterogeneous cells from the superior region of neonatal hippocampi were studied. In 54.5% of cells, the GABA(B) receptor agonist baclofen (10 microM) attenuated the whole-cell calcium current by 21.0% +/- 1.1%. In 29.9% of cells, baclofen facilitated the calcium current by 43.5% +/- 8.1%. The component of current attenuated by baclofen was blocked by the N-type calcium channel antagonist omega-conotoxin GVIA (3 microM). The component of current facilitated by baclofen was blocked by the L-type channel antagonist nimodipine (20 microM). For cells that showed calcium current facilitation, baclofen shifted the half-maximal activation by approximately -14 mV. The data indicate that activation of GABA(B) receptors in neurons of the superior hippocampus attenuates current through N-type channels and facilitates current through L-type channels. The two opposing effects of GABA(B) receptor activation may reflect the heterogeneity of the cultured cells or may be a developmentally regulated phenomenon.

Loss of T-type calcium current in sensory neurons of rats with neuropathic pain.

BACKGROUND: Pathophysiology in the primary sensory neuron may contribute to chronic neuropathic pain. Ca channels play a central role in neuronal processes, and sensory neurons are rich in low-voltage-activated calcium channels (LVACCs). However, the physiologic function of these channels is unknown. Their possible role in rebound burst firing makes them a candidate for increased excitability after neuropathic injury. METHODS: This study uses pharmacological methods to isolate LVACC in cells from the dorsal root ganglia of neuropathic and sham-operated rats, including the blockade of high-voltage-activated Ca channels with fluoride and selective toxins. LVACCs were examined with conventional whole cell patch clamp electrophysiology techniques. RESULTS: After chronic constriction injury of the peripheral axon, LVACC was significantly reduced compared to sham rats as shown by a 60% reduction in peak current density and an 80% reduction in total calcium influx. A depolarizing shift in the voltage dependence of activation and an increase in the rate of deactivation and inactivation appear to cause this reduction of LVACC. Either Ni2+ or mibefradil, blockers of LVACC, applied in the bath to normal dorsal root ganglion cells during current clamp significantly and reversibly increased excitability. CONCLUSIONS: These results suggest that loss of LVACC may contribute to decreased spike frequency adaptation and increased excitability after injury to sensory neurons. Through decreased Ca2+ influx, the cell becomes less stable and more likely to initiate or transmit bursts of action potentials. Consequently, modulation of Ca2+ currents at the dorsal root ganglion may be a potential method of therapeutic intervention.