A late phase of LTD in cultured cerebellar Purkinje cells requires persistent dynamin-mediated endocytosis

Long-term synaptic depression (LTD) of cerebellar parallel fiber-Purkinje cell synapses is a form of use-dependent synaptic plasticity that may be studied in cell culture. One form of LTD is induced postsynaptically through an mGlu1/Ca influx/protein kinase Cα (PKCα) cascade, and its initial expression requires phosphorylation of ser-880 in the COOH-terminal PDZ-ligand region of GluA2 and consequent binding of PICK1. This triggers postsynaptic clathrin/dynamin-mediated endocytosis of GluA2-containing surface AMPA receptors. Cerebellar LTD also has a late phase beginning 45–60 min after induction that is blocked by transcription or translation inhibitors. Here, I have sought to determine the expression mechanism of this late phase of LTD by applying various drugs and peptides after the late phase has been established. Neither bath application of mGluR1 antagonists (JNJ-16259685, LY-456236) nor the PKC inhibitor GF-109203X starting 60–70 min after LTD induction attenuated the late phase. Similarly, achieving the whole cell configuration with a second pipette loaded with the peptide PKC inhibitor PKC(19–36) starting 60 min postinduction also failed to alter the late phase. Late internal perfusion with peptides designed to disrupt PICK1-GLUA2 interaction or PICK1 dimerization failed to impact late phase LTD expression. However, late internal perfusion with two different blockers of dynamin, the drug dynasore and a dynamin inhibitory peptide (QVPSRPNRAP), produced rapid and complete reversal of cerebellar LTD expression. These findings suggest that the protein synthesis-dependent late phase of LTD requires persistent dynamin-mediated endocytosis, but not persistent PICK1-GluA2 binding nor persistent activation of the upstream mGluR1/PKCα signaling cascade.

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ

Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.

Altered Excitability of Intestinal Neurons in Primary Culture Caused by Acute Oxidative Stress

Neurons were isolated from the intestine of guinea pigs and grown in primary culture for ≤15 days. Using conventional whole cell recording techniques, we demonstrated that the majority of neurons express a prolonged poststimulus afterhyperpolarization (slow AHP). These neurons also had large-amplitude (∼100 mV), broad-duration (∼2 ms) action potentials and generated a hyperpolarization activated inward current ( Ih). Application of H2O2 (0.22–8.8 mM) hyperpolarized these neurons but not those lacking slow AHPs. The H2O2-induced hyperpolarization was followed by irreversible depolarization at higher concentrations (more than ∼1 mM) of H2O2 while it was maintained after washout of submillimolar H2O2. The ionic mechanisms underlying the hyperpolarization included the suppression of Ih and the activation of an inwardly rectifying outward current, which was blocked by glybenclamide (25–50 μM) and TEA (30 mM). In addition, H2O2 suppressed the slow AHP and its underlying current. Internal perfusion of catalase and glutathione opposed the H2O2-mediated decrease in IsAHP. Our results indicate that acute oxidative stress has neuron- and conductance-specific actions in intestinal neurons that may underlie pathophysiological conditions.

Modulation of the sarcolemmal L-type current by alteration in SR Ca2+ release

Modulation of the L-type current by sarcoplasmic reticulum (SR) Ca2+ release has been examined in patch-clamped mouse myotubes. Inhibition of SR Ca2+ release by inclusion of ryanodine in the internal solution shifted the half-activating voltage ( V 0.5) of the L-type current from 1.1 ± 2.1 to −7.7 ± 1.7 mV. Ruthenium red in the internal solution shifted V 0.5 from 5.4 ± 1.9 to −3.2 ± 4.1 mV. Chelation of myoplasmic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid perfusion shifted V 0.5 from 4.4 ± 1.7 to −3.5 ± 3.3 mV and increased the peak current. Extracellular caffeine (1 mM), which should enhance SR Ca2+ release, significantly decreased the peak Ca2+ current. In low (0.1 mM) internal EGTA, myotube contraction was abolished by internal perfusion with ryanodine or ruthenium red, whereas addition of caffeine to the extracellular solution lowered the contractile threshold, indicating that these modulators of SR Ca2+ release had the expected effects on contraction. Therefore, SR Ca2+ release appears to modulate the sarcolemmal L-type current, suggesting a retrograde communication from the SR to the sarcolemmal L-type channels in excitation-contraction coupling.

Perfusion-induced changes in cardiac contractility depend on capillary perfusion

The perfusion-induced increase in cardiac contractility (Gregg phenomenon) is especially found in heart preparations that lack adequate coronary autoregulation and thus protection of changes in capillary pressure. We determined in the isolated perfused papillary muscle of the rat whether cardiac muscle contractility is related to capillary perfusion. Oxygen availability of this muscle is independent of internal perfusion, and perfusion may be varied or even stopped without loss of function. Muscles contracted isometrically at 27°C ( n = 7). During the control state stepwise increases in perfusion pressure resulted in all muscles in a significant increase in active tension. Muscle diameter always increased with increased perfusion pressure, but muscle segment length was unaffected. Capillary perfusion was then obstructed by plastic microspheres (15 μm). Flow, at a perfusion pressure of 66.6 ± 26.2 cmH2O, reduced from 17.6 ± 5.4 μl/min in the control state to 3.2 ± 1.3 μl/min after microspheres. Active tension developed by the muscle in the unperfused condition before microspheres and after microspheres did not differ significantly (−12.8 ± 29.4% change). After microspheres similar perfusion pressure steps as in control never resulted in an increase in active tension. Even at the two highest perfusion pressures (89.1 ± 28.4 and 106.5 ± 31.7 cmH2O) that were applied a significant decrease in active tension was found. We conclude that the Gregg phenomenon is related to capillary perfusion.

Mechanisms of Potentiation by Calcium-Calmodulin Kinase II of Postsynaptic Sensitivity in Rat Hippocampal CA1 Neurons

Shirke, Aneil M. and Roberto Malinow. Mechanisms of potentiation by calcium-calmodulin kinase II of postsynaptic sensitivity in rat hippocampal CA1 neurons. J. Neurophysiol. 78: 2682–2692, 1997. Preactivated recombinant α-calcium–calmodulin dependent multifunctional protein kinase II (CaMKII*) was perfused internally into CA1 hippocampal slice neurons to test the effect on synaptic transmission and responses to exogenous application of glutamate analogues. After measurement of baseline transmission, internal perfusion of CaMKII* increased synaptic strength in rat hippocampal neurons and diminished the fraction of synaptic failures. After measurement of baseline responses to applied transmitter, CaMKII* perfusion potentiated responses to kainate but not responses to N-methyl-d-aspartate. Internal perfusion of CaMKII*potentiated the maximal effect of kainate. Potentiation byCaMKII* did not change the time course of responses to kainate, whereas increasing response size by pharmacologically manipulating desensitization or deactivation rate constants significantly altered the time course of responses. Nonstationary fluctuation analysis of responses to kainate showed a decrease in the coefficient of variation after potentiation by CaMKII*. These data support the hypothesis that CaMKII increases postsynaptic responsiveness by increasing the available number of active α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate channels and suggests that a similar process may occur during the expression of long-term potentiation.

Ins(1,3,4,5)P4 is effective in mobilizing Ca2+ in mouse exocrine pancreatic acinar cells if phospholipase A2 is inhibited

In enzymically isolated mouse pancreatic acinar cells, under conditions of whole-cell patch-clamp current recording, the effect of phospholipase C-coupled agonists can be mimicked by internal perfusion of the intracellular second messenger Ins(1,4,5)P3 (10 µM) or its analogue Ins(2,4,5)P3 (10 µM). The inositol trisphosphates mimic receptor activation by releasing Ca2+ from intracellular stores and by promoting Ca2+ influx across the surface membrane. This Ca2+-mobilizing role of inositol polyphosphates seems to be confined to the inositol trisphosphates because internal perfusion of Ins(1,3,4,5)P4 (10 µM) is not associated with any Ca2+-dependent current activation. In this study we investigate the effects of 4-bromophenacyl bromide (4BPB), a putative inhibitor of phospholipase A2 and arachadonic acid production, on inositol polyphosphate-induced Ca2+ signalling. At 10 µM, 4BPB has no effect on unstimulated Ca2+-dependent membrane currents. However, if 4BPB is applied to cells internally perfused with 10 µM Ins(1,4,5)P3 or Ins(2,4,5)P3 then the current responses are rapidly potentiated. In cells internally perfused with 10 µM Ins(1,3,4,5)P4, which has itself no effect on membrane currents, application of 4BPB resulted in the activation of Ca2+-dependent currents, seen either as repetitive spikes of current or as sustained current activations. The application of arachidonic acid blocks the current responses evoked by the inositol trisphosphates and by Ins(1,3,4,5)P4/4BPB. These results suggest that in enzymically isolated pancreatic acinar cells phospholipase A2 activity is exerting an inhibitory effect on inositol polyphosphate-mediated Ca2+ mobilization. 4BPB removes this inhibition and potentiates the responses to internally perfused inositol trisphosphates and, importantly, makes 10 µM Ins(1,3,4,5)P4 as effective as 10 µM Ins(1,4,5)P3 in mobilizing intracellular Ca2+ and in promoting Ca2+ influx.