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 alpha-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.

N-methyl-D-aspartate subunit expression during mouse development altered by in utero alcohol exposure.

OBJECTIVE: Alcohol-related neurodevelopmental disorders are contributors to long-term learning disabilities. By using a model for fetal alcohol syndrome, we have shown that prenatal alcohol exposure results in adult learning deficits of unknown mechanisms. In the developing hippocampus, the N-methyl-D-aspartate (NMDA) receptor subunit NR2B triggers long-term potentiation, fundamental to learning and memory; this is supplemented by the less plastic NR2A subunit in the adult. To understand the mechanism of learning deficits in FAS, we evaluated NR2B and NR2A expression in embryonic and adult mice. STUDY DESIGN: Pregnant C57Bl6/J mice were treated on gestational day 8 with alcohol or control (saline solution). Embryos were harvested at 6 hours, 24 hours, and 10 days, and brains from adult offspring were collected at 3 months (after evaluation for learning deficit). Calibrator-normalized relative real-time polymerase chain reaction was performed for NR2B and NR2A with glyceraldehyde-3-phosphate dehydrogenase standardization. Statistical analysis included analysis of variance. RESULTS: At 6 hours, NR2B expression in the alcohol-exposed embryos was higher than in controls (P < .01). NR2A was not expressed in either group. By 24 hours there was no difference in NR2B (P = .3). However, at 10 days NR2B was lower in alcohol-exposed animals (P = .02). In the adult brains there was a relative decrease in NR2B (P = .03) and an increase in NR2A (P < .01). CONCLUSION: Prenatal alcohol exposure during development induces NR2B expression deregulation in the embryos that persists until adulthood, when a relative increase in the less modifiable subunit NR2A occurs. This alteration in NMDA receptor subunits may underlie the learning abnormalities in fetal alcohol syndrome.

Inflammatory-mediated model of cerebral palsy with developmental sequelae.

OBJECTIVE: Cerebral palsy (CP) is characterized by motor deficits. There is increasing evidence that CP may result from inflammatory and infection-mediated white matter damage. Our objective was to develop an inflammatory model for CP based on chronic lipopolysaccharide (LPS) exposure with a recognizable phenotype in offspring. STUDY DESIGN: On gestational days 15, 17, and 19 (approximately 28-36 wks human gestation; rat length of gestation is 21 days), pregnant rats were intracervically injected with 0.15 mg/kg LPS (in 0.1 mL saline) or 0.1 mL saline for controls. Neonatal tests for sensory-motor milestones were performed on postnatal days 1 to 21 (LPS n = 25; control n = 26). Adult males were tested at 8 weeks on open field and rotarod for motor activity. Immunohistochemistry studies were performed to assess olygodendrocyte (OL) damage. Statistical analysis included Mann-Whitney U and analysis of variance (ANOVA) with P < .05 considered significant. RESULTS: Immunohistochemistry revealed a decrease in the immature OL marker PLP on day 21 (P = .03) in LPS-exposed offspring, and an increase of the mature OL marker CNP on day 21 and at 8 weeks (P < .01, P < .001). LPS-exposed offspring were delayed achieving 3 motor milestones: negative geotaxis (P < .05), cliff aversion (P < .01), and surface righting (P = .05). They were also delayed in eye opening (P < .01). There was no difference between the 2 groups for the other tests. There was a trend towards decreased mean speed in LPS-exposed adults in open field testing (P = .08), but no differences observed in rotarod testing. CONCLUSION: Using an animal model for CP that mimics a chronic intrauterine inflammation that results in decreased levels of PLP, a marker for early oligodendrocytes consistent with white matter damage, we have demonstrated a phenotype relevant to the human CP manifestations in the neonatal period. Nevertheless, adult animals were able to compensate to the damage. Further refinement is needed to improve the understanding of pathogenesis, as well as allow for testing preventative therapies.

Pre- and postsynaptic mechanisms in Hebbian activity-dependent synapse modification.

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down-regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity-dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity-dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output.