Veronica Dunlap

Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: The role of postsynaptic acetylcholine receptors†

Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)‐mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time‐ and dose‐related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite‐associated receptor aggregates in nerve‐muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity‐dependent synapse reduction and does so, at least in part, by altering AChR stability. J. Neurosci. Res. 61:616–625, 2000. Published 2000 Wiley‐Liss, Inc.

Upregulation of GABAA Current by Astrocytes in Cultured Embryonic Rat Hippocampal Neurons

Embryonic rat hippocampal neurons were cultured on poly-d-lysine (PDL) or a monolayer of postnatal cortical astrocytes to reveal putative changes in neuronal physiology that involve astrocyte-derived signals during the first 4 d of culture. GABA-induced Cl− current (IGABA) was quantified using outside-out and whole-cell patch-clamp recordings beginning at 30 min, when cells had become adherent. The amplitude and density (current normalized to membrane capacitance) of IGABA in neurons grown on astrocytes became statistically greater than that recorded in neurons grown on PDL after 2 hr in culture (HIC). Although the current density remained unchanged in neurons on astrocytes, that in neurons on PDL decreased and became statistically lower beginning after 2 HIC. The differences in amplitude and density of IGABA in the two groups of neurons were maintained during the 4 d experiment. The upregulation effect of astrocytes on neuronal IGABA required intimate contact between the neuronal cell body and underlying astrocytes. Suppression of spontaneous Cac2+ elevations in astrocytes by bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid that was loaded intracellularly decreased their modulatory effects on IGABA. IGABA in all cells was blocked completely by bicuculline and exhibited virtually identical affinity constants, Hill coefficients, and potentiation by diazepam in the two groups. Outside-out patch recordings revealed identical unitary properties of IGABA in the two groups. More channels per unit of membrane area could explain the astrocyte enhancement of IGABA. The results reveal that cortical astrocytes potentiate IGABA in hippocampal neurons in a contact-dependent manner via a mechanism involving astrocyte Cac2+elevation.

The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activation in vitro†

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.

Opposing actions of protein kinase A and C mediate Hebbian synaptic plasticity

A compartmental nerve–muscle tissue culture system expresses Hebbian activity-dependent synapse modulation. Protein kinase C (PKC) mediates a heterosynaptic loss of efficacy, and we now show that protein kinase A (PKA) is involved in homosynaptic stabilization. Both work through postsynaptic changes in the acetylcholine receptor (AChR) as measured electrophysiologically and by imaging techniques.

Phosphorylation of the nicotinic acetylcholine receptor in myotube-cholinergic neuron cocultures

Acetylcholine receptor (AChR) stability in the postsynaptic membrane is affected by serine kinases. AChR are phosphorylated by protein kinase C (PKC) and PKA, and we have shown that activation of PKA and PKC have opposite effects on AChR stability and that this may play some role in the selective, activity‐dependent synapse loss that occurs during development of the neuromuscular junction. Myotube cultures with and without added spinal motor neurons were probed with immunoaffinity‐purified antibodies prepared against phosphorylated peptides with amino acid sequences from different AChR subunits. Different treatments activating PKC (phorbol 12‐myristate 13‐acetate; PMA) or PKA (dibutyryl cyclic adenosine monophosphate; cAMP) or blocking electrical activity (tetrodotoxin; TTX) of the cocultures were chosen because of their known effects, direct or indirect, on receptor stability. We asked whether the phospho‐specific antibody staining in conjunction with α‐bungarotoxin (BTX) identification of AChR aggregates could provide a direct demonstration of changes in receptor phosphorylation produced by the treatments. We found that PMA treatment did increase phosphorylation of the delta subunit and cAMP increased phosphorylation of the epsilon subunit relative to total BTX labeling in muscle‐nerve cocultures, but not in muscle‐only cultures. Blockade of electrical activity with TTX increased the incidence of aggregates that showed no phospho‐epsilon staining. Myotube cultures grown in the absence of neurons did not show the responses of myotubes in cocultures. The results show that manipulations that alter receptor stability also produce changes in receptor phosphorylation. We suggest that phosphorylation may be a mechanism mediating the changes in receptor stability.

Embryonic Rat Hippocampal Neurons and GABAA Receptor Subunit-Transfected Non-neuronal Cells Release GABA Tonically

Abstract. We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl−-filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl− channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with α1 and γ2 GABAA receptor subunits exhibited little baseline current variance when recorded with Cl−-filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons.

Pharmacological properties of fetal rat hippocampal GABAA receptors

Cells were cultured from embryonic (E) day 17 and 19 rat hippocampal tissues for one or more days in serum-supplemented growth medium. Intracellular recordings in the whole-cell configuration were made at room temperature. GABAA receptor agonists were applied by pressure pulses from closely positioned pipettes. Most of the cells recorded under these conditions responded to one or another ligand. When the equilibrium potential for Cl- was set near 0 mV, current responses to GABA and other GABAA receptor agonists reversed polarity near 0 mV, suggesting a dominant role for Cl- ions in the response. Many cells also responded to the general anesthetics (-)pentobarbital and alfaxalone (3 alpha-hydroxy-5 alpha-pregnane-11,20-dione). Micromolar concentrations of these drugs, like the transmitter GABA, elicited membrane current responses that reversed near 0 mV. Sequential exposure of individual neurons to both anesthetics revealed sensitivity to one but not the other agent as well as some responding to both. These results indicate: (1) the GABAA receptor function emerges pre-natally in rat hippocampal neurons; (2) barbiturates can have several sites for binding to in GABAA receptor complexes.