Douglas S.F. Ling

Protein kinase Mζ is necessary and sufficient for LTP maintenance

Long-term potentiation (LTP), a persistent synaptic enhancement thought to be a substrate for memory, can be divided into two phases: induction, triggering potentiation, and maintenance, sustaining it over time. Many postsynaptic events are implicated in induction, including N-methyl-D-aspartate receptor (NMDAR) activation, calcium increases and stimulation of several protein kinases; in contrast, the mechanism maintaining LTP is not yet characterized. Here we show the constitutively active form of an atypical protein kinase C (PKC) isozyme, protein kinase M zeta (PKMζ), is necessary and sufficient for LTP maintenance.

Spontaneous Epileptiform Activity in Rat Neocortex after Controlled Cortical Impact Injury

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.

Activity-dependent depression of monosynaptic fast IPSCs in hippocampus: contributions from reductions in chloride driving force and conductance

Whole-cell recordings techniques were used to record pharmacologically isolated fast inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons from rat hippocampal slices. Repetitive extracellular stimulation up to 10 Hz progressively reduced steady-state fast IPSC amplitude. At low stimulation frequencies (up to 1 Hz), this attenuation was characterized by a positive shift of IPSC reversal potential with no change in IPSC conductance. Above 1 Hz stimulation, fast IPSC depression was associated with changes in both reversal potential and IPSC conductance. Use-dependent depression at low frequencies was prevented when cells were chloride-loaded using cesium chloride based intracellular solutions. These findings suggest that activity-dependent depression of fast IPSCs at low stimulus frequencies results entirely from a reduction in chloride driving force, stemming from intracellular chloride accumulation. Activity-dependent changes in fast IPSC conductance occur only at stimulation rates above 1 Hz.

Carbenoxolone Modifies Spontaneous Inhibitory and Excitatory Synaptic Transmission in Rat Somatosensory Cortex

Gap junction (GJ) coupling between neocortical GABAergic interneurons plays a critical role in the synchronization of activity in cortical networks in physiological and pathophysiological states, e.g., seizures. Past studies have shown that GJ blockers exert anticonvulsant actions in both in vivo and in vitro models of epilepsy. However, the precise mechanisms underlying these antiepileptic effects have not been fully elucidated. This is due, in part, to a lack of information of the influence of GJ blockade on network activity in the absence of convulsant agents or enhanced neuronal excitation. One key question is whether GJ blockers act on excitatory or inhibitory systems, or both. To address this issue, we examined the effects of the GJ blocker carbenoxolone (CarbX, 150 microM) on spontaneous inhibitory postsynaptic currents (sIPSCs) and excitatory postsynaptic currents (sEPSCs) in acute slices of rat somatosensory cortex. Results showed that CarbX decreased the amplitude and frequency of sIPSCs by 30.2% and 25.7%, respectively. CarbX increased the mean frequency of sEPSCs by 24.1%, but had no effect on sEPSC amplitude. During blockade of GABAA-mediated events with picrotoxin (20 microM), CarbX induced only a small increase in sEPSC frequency that was not statistically different from control, indicating CarbX enhancement of sEPECs was secondary to the depression of synaptic inhibition. These findings suggest that in neocortex, blockade of GJs leads to an increase in spontaneous excitation by uncoupling GABAergic interneurons, and that electronic communication between inhibitory cells plays a significant role in regulating tonic synaptic excitation.

Synchronous firing of inhibitory interneurons results in saturation of fast GABAA IPSC magnitude but not saturation of fast inhibitory efficacy in rat neocortical pyramidal cells

The kinetic properties of evoked fast inhibitory postsynaptic currents were examined to elucidate factors underlying the limit on the magnitude of fast inhibition in neocortex. Using whole‐cell voltage‐clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex, fast γ‐aminobutyric acid‐A (GABAA)ergic inhibitory postsynaptic currents were selectively recorded by holding cells at potentials equal to excitatory postsynaptic current reversal (∼0 mV). As stimulus intensity was increased, the magnitude and duration of the fast inhibitory postsynaptic current increased. Over the range of stimuli applied (2–10 V), fast GABAA‐mediated inhibitory postsynaptic currents reached a maximum peak conductance of 25.9 ± 4.2 nS (range 10.5–41.2 nS) at intensities approximately 2‐times threshold. As stimulus intensities were increased beyond this point of maximal conductance, the time constant of current decay increased as function of stimulus strength, while rise time remained unaffected. Exposure to nominally magnesium‐free solutions did not affect maximal peak conductances of fast inhibitory postsynaptic currents, but did cause an increase in the time constants of current decay by 66.3 ± 23.6%, resulting in an 85.6 ± 24.6% increase in the total charge flux carried by single inhibitory postsynaptic currents. This effect may be due to prolonged activation of postsynaptic GABAA receptors by excess GABA released in response to increased excitation. Exposure to the GABA uptake blocker, nipecotic acid, similarly prolonged current decay without affecting the maximal peak conductance.

Controlled Cortical Impact-induced Neurodegeneration Decreases after Administration of the Novel Calpain-inhibitor Gabadur

One aspect of secondary injury in traumatic brain injury is the marked increase in intracellular calcium and resultant over-activation of the calcium-dependent neutral cysteine protease calpain. Gabadur is a novel protease inhibitor with calpain-inhibition properties formulated from the classic protease inhibitor leupeptin linked to a pregabalin carrier. This construction allows the entire compound to cross the blood-brain barrier after peripheral administration to better target the site of injury. In this study, a single intraperitoneal dose of Gabadur was administered immediately following controlled cortical impact injury in rats. Neocortical slices were examined at 48 h post-injury via Fluoro-Jade B staining, revealing an improvement in cortical neurodegeneration in Gabadur treated rats. Levels of detrimental active calpain-2 measured via western blot were also decreased in rats receiving Gabadur. This data supports the benefit of targeted protease inhibition in the treatment of traumatic brain injury.