Peter L. Carlen

Prevalence and significance of neurocognitive dysfunction in hepatitis C in the absence of correlated risk factors.

Neurocognitive morbidity has been reported in individuals with chronic hepatitis C virus (HCV) infection, but the magnitude of such dysfunction in the absence of disease‐correlated factors known to affect the central nervous system (e.g., substance abuse, cirrhosis, depression, interferon treatment) and the impact of any such change on functioning is unclear. We investigated a cohort of individuals with HCV, all of whom were carefully screened to exclude relevant comorbidities, to elucidate virus‐related changes in the brain using neuropsychological tests and magnetic resonance spectroscopy (MRS). A cohort of 37 patients with chronic HCV infection was culled from 300 consecutive patients presenting to a tertiary care liver clinic. A comparison group of healthy controls (n = 46) was also assessed. Of 10 neurocognitive measures evaluated, the HCV group showed marginally poorer learning efficiency compared with controls; only 13% of patients demonstrated a clinical level of impairment on this test (defined as 1.5 SD below the normative standard). Although patients reported greater levels of fatigue and symptoms of depression, these factors did not correlate with the degree of learning inefficiency. With respect to MRS, the HCV group demonstrated increased choline and reduced N‐acetyl aspartate relative to controls in the central white matter. Indicators of liver disease severity did not correlate with either memory or MRS abnormalities. In conclusion, while our findings support an association between hepatitis C and indicators of central nervous system involvement in a cohort of patients carefully screened to eliminate other factors influencing neurocognitive integrity, the clinical significance of these effects is limited. (HEPATOLOGY 2005;41:801–808.)

Coupling potentials in CA1 neurons during calcium-free-induced field burst activity

Small amplitude depolarizations (fast prepotentials, spikelets) recorded in mammalian neurons are thought to represent either dendritic action potentials or presynaptic action potentials attenuated by gap junctions. We have used whole-cell recordings in an in vitro calcium- free model of epilepsy to record spikelets from CA1 neurons of the rat hippocampus. It was found that spikelet appearance was closely correlated with the occurrence of dye coupling between pyramidal neurons, indicating that both phenomena share a common substrate. Spikelets were characterized according to waveform (amplitude and shape) and temporal occurrence. Spikelet amplitudes were found to be invariant with neuronal membrane potential, and their pattern of occurrence was indistinguishable from patterns of action potential firing in these cells. Voltage and current recordings revealed a spikelet waveform that was usually biphasic, comprised of a rapid depolarization followed by a slower hyperpolarization. Numerical differentiation of spike bursts resulted in waveforms similar to recorded spikelet sequences, while numerical integration of spikelets yielded waveforms that were indistinguishable from action potentials. Modification of spikelet waveforms by the potassium channel blocker tetraethylammonium chloride suggests that spikelets may arise from both resistive and capacitive transmission of presynaptic action potentials. Intracellular alkalinization and acidification brought about by perfusion with NH4Cl caused changes in spikelet frequency, consistent with reported alterations of field burst activity in this model of epilepsy. These results suggest that spikelets result from gap junctional communication, and may be important determinants of neuronal activity during seizure-like activity.

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Chronic (18u2003h) exposure of cultured hippocampal slices to the type‐A GABA receptor blocker, bicuculline methiodide (BMI) 10u2003μm increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c‐fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100u2003μm). Extracellular recordings in CA1 stratum pyramidale, performed in BMI‐free solution, demonstrated that BMI‐exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post‐synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100u2003μm), but not its inactive analog, oleanolic acid (100u2003μm), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI‐induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Increased high-frequency oscillations precede in vitro low-Mg2+ seizures

Summary:u2002 Purpose: High‐frequency oscillations (HFOs) in the range of ≥80 Hz have been recorded in neocortical and hippocampal brain structures in vitro and in vivo and have been associated with physiologic and epileptiform neuronal population activity. Frequencies in the fast‐ripple range (>200 Hz) are believed to be exclusive to epileptiform activity and have been recorded in vitro, in vivo, and in epilepsy patients. Although the presence of HFOs is well characterized, their temporal evolution in the context of transition to seizure activity is not well understood.

Bidirectional multisite seizure propagation in the intact isolated hippocampus: The multifocality of the seizure “focus”

Localizing the seizure focus is difficult and frequently, multiple sites are found. This reflects our poor understanding of the fundamental mechanisms of seizure generation and propagation. We used multisite electrophysiological recordings in two seizure models and voltage-sensitive dye imaging, to spatiotemporally characterize the initiation and propagation of seizures in an intact epileptogenic brain region, the isolated hippocampus. In low-magnesium perfusate, seizures always originated in the temporal region, and propagated along the septotemporal axis to the septal region. After the seizure spread across the hippocampus, the bursts within a seizure became bidirectional, with different propagation patterns at different frequencies. When the intact hippocampus was separated along the septotemporal axis, independent bidirectional activity was observed in the two halves, and region-specific cuts to the tissue reveal that the CA3 region is critical for seizure generation and propagation. In a second seizure model, using focal tetanic stimulation of the septal and temporal CA3 region, seizures always originated at the stimulated site with bidirectionality later developing at different frequencies, as noted in the low magnesium model, behavior compatible with coupled neuronal network oscillators. These data provide novel insights into the dynamic multifocality of seizure onset and propagation, revealing that the current concept of a single seizure focus is complex.

Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus.

Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.

High frequency stimulation or elevated K+ depresses neuronal activity in the rat entopeduncular nucleus

High frequency stimulation (HFS) is applied to many brain regions to treat a variety of neurological disorders/diseases, yet the mechanism(s) underlying its effects remains unclear. While some studies showed that HFS inhibits the stimulated nucleus, others report excitation. In this in vitro study, we stimulated the rat globus pallidus interna (entopeduncular nucleus, EP), a commonly stimulated area for Parkinsons disease, to investigate the effect of HFS-induced elevation of extracellular potassium (K(+)(e)) on rat EP neuronal activity. Whole-cell patch-clamp recordings and [K(+)](e) measurements were obtained in rat EP brain slices before, during and after HFS. After HFS (150 Hz, 10 s), [K(+)](e) increased from 2.5-9.6+/-1.4 mM, the resting membrane potential of EP neurons depolarized by 11.1+/-2.5 mV, spiking activity was significantly depressed, and input resistance decreased by 25+/-6%. The GABA(A) receptor blocker, gabazine, did not prevent these effects. The bath perfusion of 6 or 10 mM K(+), with or without synaptic blockers, mimicked the HFS-mediated effects: inhibition of spike activity, a 20+/-9% decrease in input resistance and a 17.4+/-3.0 mV depolarization. This depolarization exceeded predicted values of elevated [K(+)](e) on the resting membrane potential. A depolarization block did not fully account for the K(+)-induced inhibition of EP neuronal activity. Taken together, our results show that HFS-induced elevation of [K(+)](e) decreased EP neuronal activity by the activation of an ion conductance resulting in membrane depolarization, independent of synaptic involvement. These findings could explain the inhibitory effects of HFS on neurons of the stimulated nucleus.

Hypoglycemic seizures during transient hypoglycemia exacerbate hippocampal dysfunction.

Severe hypoglycemia constitutes a medical emergency, involving seizures, coma and death. We hypothesized that seizures, during limited substrate availability, aggravate hypoglycemia-induced brain damage. Using immature isolated, intact hippocampi and frontal neocortical blocks subjected to low glucose perfusion, we characterized hypoglycemic (neuroglycopenic) seizures in vitro during transient hypoglycemia and their effects on synaptic transmission and glycogen content. Hippocampal hypoglycemic seizures were always followed by an irreversible reduction (>60% loss) in synaptic transmission and were occasionally accompanied by spreading depression-like events. Hypoglycemic seizures occurred more frequently with decreasing hypoglycemic extracellular glucose concentrations. In contrast, no hypoglycemic seizures were generated in the neocortex during transient hypoglycemia, and the reduction of synaptic transmission was reversible (<60% loss). Hypoglycemic seizures in the hippocampus were abolished by NMDA and non-NMDA antagonists. The anticonvulsant, midazolam, but neither phenytoin nor valproate, also abolished hypoglycemic seizures. Non-glycolytic, oxidative substrates attenuated, but did not abolish, hypoglycemic seizure activity and were unable to support synaptic transmission, even in the presence of the adenosine (A1) antagonist, DPCPX. Complete prevention of hypoglycemic seizures always led to the maintenance of synaptic transmission. A quantitative glycogen assay demonstrated that hypoglycemic seizures, in vitro, during hypoglycemia deplete hippocampal glycogen. These data suggest that suppressing seizures during hypoglycemia may decrease subsequent neuronal damage and dysfunction.

Curious and contradictory roles of glial connexins and pannexins in epilepsy

Glia play an under-recognized role in epilepsy. This review examines the involvement of glial connexins (Cxs) and pannexins (Panxs), proteins which form gap junctions and membrane hemichannels (connexins) and hemichannels (pannexins), in epilepsy. These proteins, particularly glial Cx43, have been shown to be upregulated in epileptic brain tissue. In a cobalt model of in vitro seizures, seizures increased Panxs1 and 2 and Cx43 expression, and remarkably reorganized the interrelationships between their mRNA levels (transcriptome) which then became statistically significant. Gap junctions are highly implicated in synchronous seizure activity. Blocking gap junctional communication (GJC) is often anticonvulsant, and assumed to be due to blocking gap junctionally-medicated electrotonic coupling between neurons. However, in organotypic hippocampal slice cultures, connexin43 specific peptides, which attenuate GJC possibly by blocking connexon docking, diminished spontaneous seizures. Glia have many functions including extracellular potassium redistribution, in part via gap junctions, which if blocked, can be seizuregenic. Glial gap junctions are critical for the delivery of nutrients to neurons, which if interrupted, can depress seizure activity. Other functions of glia possibly related to epileptogenesis are mentioned including anatomic reorganization in chronic seizure models greatly increasing the overlapping domains of glial processes, changes in neurotransmitter re-uptake, and possible glial generation of currents and fields during seizure activity. Finally there is recent evidence for Cx43 hemichannels and Panx1 channels in glial membranes which could play a role in brain damage and seizure activity. Although glial Cxs and Panxs are increasingly recognized as contributing to fundamental mechanisms of epilepsy, the data are often contradictory and controversial, requiring much more research. This article is part of a Special Issue entitled Electrical Synapses.

Acute Postischemic Seizures Are Associated with Increased Mortality and Brain Damage in Adult Mice

Postischemic seizures are associated with worsened outcome following stroke, but the underlying pathophysiology is poorly understood. Here we examined acute seizures in adult mice following hypoxia-ischemia (HI) via combined behavioral, electrophysiological, and histological assessments. C57BL/6 mice aged 4-9 months received a permanent occlusion of the right common carotid artery and then underwent a systemic hypoxic episode. Generalized motor seizures were observed within 72 h following HI. These seizures occurred nearly exclusively in animals with extensive brain injury in the hemisphere ipsilateral to the carotid occlusion, but their generation was not associated with electroencephalographic discharges in bilateral hippocampal and neocortical recordings. Animals exhibiting these seizures had a high rate of mortality, and post-HI treatments with diazepam and phenytoin greatly suppressed these behavioral seizures and improved post-HI animal survival. Based on these data, we conclude that these seizures are a consequence of HI brain injury, contribute to worsened outcome following HI, and that they originate from deep subcortical structures.