No abstract

Protein kinase Mzeta (PKMzeta), a constitutively active, atypical PKC isoform, enhances synaptic strength during the maintenance of long-term potentiation (LTP). Here we examine the mechanism by which PKMzeta increases synaptic transmission. Postsynaptic perfusion of PKMzeta during whole-cell recordings of CA1 pyramidal cells strongly potentiated the amplitude of AMPA receptor (AMPAR)-mediated miniature EPSCs (mEPSCs). Nonstationary fluctuation analysis of events recorded before and after PKMzeta enhancement showed that the kinase doubled the number of functional postsynaptic AMPAR channels. After sustained potentiation, application of a PKMzeta inhibitor reversed the increase in functional channel number to basal levels, suggesting that persistent increase of PKMzeta is required to maintain the postsynaptic localization of a mobile subpopulation of receptors. The kinase did not affect other sites of LTP expression, including presynaptic transmitter release, silent synapse conversion, or AMPAR unit conductance. Thus PKMzeta functions specifically to establish and maintain long-term increases in active postsynaptic AMPAR number.

A hallmark of severe traumatic brain injury (TBI) is the development of post-traumatic epilepsy (PTE). However, the mechanisms underlying PTE remain poorly understood. In this study, we used a controlled cortical impact (CCI) model in rats to examine post-traumatic changes in neocortical excitability. Neocortical slices were prepared from rats at 7-9 days (week 1) and 14-16 days (week 2) after CCI injury. By week 2, we observed a substantial gray matter lesion with a cavity that extended to the hippocampal structure. Fluoro-Jade B staining of slices revealed active neuronal degeneration during weeks 1 and 2. Intracellular and extracellular recordings obtained from layer V revealed evoked and spontaneous epileptiform discharges in neocortices of CCI-injured rats. At week 1, intracellular recordings from pyramidal cells revealed evoked epileptiform firing that was synchronized with population events recorded extracellularly, suggestive of increased excitability. This activity was characterized by bursts of action potentials that were followed by recurrent, repetitive after-discharges. At week 2, both spontaneous and evoked epileptiform firing were recorded in slices from injured rats. The evoked discharges resembled those observed at week 1, but with longer burst durations. Spontaneous activity included prolonged, ictal-like discharges lasting up to 8-10 sec, and briefer interictal-like burst events (<1 sec). These results indicate that during the first 2 weeks following severe CCI injury, there is a progressive development of neocortical hyperexcitability that ultimately leads to spontaneous epileptiform firing, suggesting a rapid epileptogenic process.

1. The recruitment of evoked fast inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) was examined using whole cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex. Synaptic currents were evoked with graded electrical stimulation to assess the relative activation of IPSCs and EPSCs. Fast GABAA ergic IPSCs were selectively recorded by holding cells at potentials equal to EPSC reversal (approximately 0 mV). EPSCs were likewise isolated by holding cells at IPSC reversal potential (about -75 mV). 2. As stimulus intensities were increased, the magnitude of the postsynaptic currents also increased. Over the range of stimuli applied (2-10 V), EPSCs did not exhibit an upper limit. However, fast gamma-aminobutyric acid-A (GABAA-mediated IPSCs reached a maximum at intensities approximately 2 times threshold. 3. The limit on fast inhibition was unresponsive to alterations in N-methyl-D-aspartate (NMDA)-mediated excitation. Exposure to nominally magnesium-free solutions or to the NMDA antagonist 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid did not affect the fast IPSC maximum. Shifts in the input-output curves for submaximal activation of IPSCs were seen, which were attributed to polysynaptic excitation. 4.Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (non-NMDA) receptors with 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) completely abolished synaptically driven, fast GABAA-mediated inhibition. These findings suggested that neocortical inhibitory cells could be driven exclusively through non-NMDA transmission. 5. By comparison, in hippocampal CA1 pyramidal neurons maximal fast inhibition was sensitive to both NMDA and non-NMDA receptor blockade. 6. The results in neocortex were corroborated by direct intracellular recordings from layer V-VI interneurons. Non-NMDA receptor blockade with CNQX prevented synaptic activation of action potentials in these cells, even during cotreatment with magnesium-free solution. 7. Together, these results suggest that recruitment of GABA(A) ergic IPSCs in neocortex is ultimately driven via glutamatergic afferents arriving at non-NMDA receptors on interneurons. Properties limiting fast inhibition would favor the propagation of enhanced excitatory activity through the neuronal network.