F. Edward Dudek

Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats.

Kainic acid (KA) injections destroy hippocampal pyramidal cells, induce recurrent collateral sprouting of the hilar mossy fibers (MFs), and lead to chronic seizures. In the present study, rats were injected systemically with KA (14 mg/kg) to determine whether the subsequent occurrence of seizures was correlated with MF sprouting, as indicated by Timms staining of proximal dendrites. At 4 weeks, 56% of the KA-treated rats had MF sprouting in the temporal (ventral) hippocampus, and 52% had shown seizures between 10 and 28 days. Both seizures and sprouting were seen in 32% of the treated animals. While Timms staining of MFs in the inner molecular layer was not directly correlated with seizure scores, animals which exhibited chronic seizures had significantly more sprouting than animals which did not have seizures.

Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy.

Human temporal lobe epilepsy is associated with complex partial seizures that can produce secondarily generalized seizures and motor convulsions. In some patients with temporal lobe epilepsy, the seizures and convulsions occur following a latent period after an initial injury and may progressively increase in frequency for much of the patients life. Available animal models of temporal lobe epilepsy are produced by acute treatments that often have high mortality rates and/or are associated with a low proportion of animals developing spontaneous chronic motor seizures. In this study, rats were given multiple low-dose intraperitoneal (i.p.) injections of kainate in order to minimize the mortality rate usually associated with single high-dose injections. We tested the hypothesis that these kainate-treated rats consistently develop a chronic epileptic state (i.e. long-term occurrence of spontaneous, generalized seizures and motor convulsions) following a latent period after the initial treatment. Kainate (5 mg/kg per h, i.p.) was administered to rats every hour for several hours so that class III-V seizures were elicited for > or = 3 h, while control rats were treated similarly with saline. This treatment protocol had a relatively low mortality rate (15%). After acute treatment, rats were observed for the occurrence of motor seizures for 6-8 h/week. Nearly all of the kainate-treated rats (97%) had two or more spontaneous motor seizures months after treatment. With this observation protocol, the average latency for the first spontaneous motor seizure was 77+/-38 (+/-S.D.) days after treatment. Although variability was observed between rats, seizure frequency initially increased with time after treatment, and nearly all of the kainate-treated rats (91%) had spontaneous motor seizures until the time of euthanasia (i.e. 5-22 months after treatment). Therefore, multiple low-dose injections of kainate, which cause recurrent motor seizures for > or = 3 h, lead to the development of a chronic epileptic state that is characterized by (i) a latent period before the onset of chronic motor seizures, and (ii) a high but variable seizure frequency that initially increases with time after the first chronic seizure. This modification of the kainate-treatment protocol is efficient and relatively simple, and the properties of the chronic epileptic state appear similar to severe human temporal lobe epilepsy. Furthermore, the observation that seizure frequency initially increased as a function of time after kainate treatment supports the hypothesis that temporal lobe epilepsy can be a progressive syndrome.

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.

Bag Cell Control of Egg Laying in Freely Behaving Aplysia

Neuroendocrine (bag cell) control of egg laying was studied in freely behaving Aplysia. Surgical lesions showed that bag cells are not necessary for egg laying, although they play a crucial role in its control, and that the pleurovisceral connectives are the afferent pathway to the bag cells. Recording in vivo showed that synchronous bag cell spikes progressively invade the network, leading to prolonged repetitive firing that initiates natural egg laying.

Grid-mapped freeze-fracture analysis of gap junctions in gray and white matter of adult rat central nervous system, with evidence for a "panglial syncytium" that is not coupled to neurons.

In white matter regions of the brain and spinal cord of adult mammals, gap junctions previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes and ependymacytes. The resulting functional syncytium was proposed to modulate the ion fluxes that occur during electrical activity of the associated axons. Gap junctions also have been reported linking neurons with glia, and functional neuronal-glial coupling has been postulated. To investigate the glial syncytium and the neuron-to-glial coupling hypotheses, we used grid-mapped freeze fracture, conventional thin-section electron microscopy, and light microscope immunocytochemistry to examine and characterize neurons and glia in gray and white matter of adult rat brain and spinal cord. We have obtained quantitative evidence for the abundance and widespread distribution of gap junctions interlinking the three primary types of macroglia throughout both gray and white matter of the mammalian central nervous system (CNS), thereby extending the concept to that of a functional panglial syncytium. In contrast to previous reports, we show that of more than 400 gap junctions in which both participating cells were identified, none were between neurons and glia. Thus, neuronal coupling and glial coupling involved separate and distinct pathways. Finally, putative water channels (i.e., square arrays) were confirmed to be abundant and in close association with gap junctions in astrocytes and ependymacytes. Because the astrocyte intermediaries extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, from the ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons to astrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abundance of water channels and intercellular ion channels, is optimally positioned and equipped to modulate water and ion fluxes across broad regions of the CNS.

‘NON-SYNAPTIC’ MECHANISMS IN SEIZURES AND EPILEPTOGENESIS

The role of ‘non‐synaptic’ mechanisms (i.e. those mechanisms that are independent of active chemical synpases) in the synchronization of neuronal activity during seizures and their possible contribution to chronic epileptogenesis are summarized. These ‘non‐synaptic’ mechanisms include electrotonic coupling through gap junctions, electrical field effects (i.e. ephaptic transmission), and ionic interactions (e.g. increases in the extracellular concentration of K+). Several lines of evidence indicate that granule cells and pyramidal cells of the hippocampus, and probably other cortical neurons, can generate synchronized electrical activity after active chemical synaptic transmission has been blocked. This synchronized activity is sensitive to alterations in the size of the extracellular space, thus suggesting that electrical field effects and ionic mechanisms contribute to this synchronized activity. Recent studies also indicate that ‘non‐synaptic’ synchronization is quite prominent early in development. Electrophysiological data from hippocampal and neocortical slices have led to a re‐interpretation of the fast prepotentials (i.e. partial spikes) recorded in cortical pyramidal cells, suggesting that they may not be due to dendritic spike generation. Improvement in freeze‐fracture ultrastructural techniques have led to a re‐assessment of previous data on gap junctions in the nervous system and opened new approaches to the quantitative analysis and characterization of gap junctions on glia and neurons. Finally, new methods of dye/tracer coupling have the potential to provide a more rigorous basis for evaluating gap junctions and electrotonic communication between neurons in the mammalian central nervous system. Therefore, recent data continue to suggest that gap junctions and electrotonic coupling play an important role in neural integration, although additional studies using new techniques will be needed to address some of the controversial issues that have arisen over the last several decades.

Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation

1 Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats. 2 All neurones that responded to vagal stimulation displayed EPSCs of relatively constant latency. Approximately 50 % of these also demonstrated variable‐latency IPSCs, and ∼31 % also displayed variable‐latency EPSCs to vagal stimulation. All neurones also had spontaneous EPSCs and IPSCs. 3 Evoked and spontaneous EPSCs reversed near 0 mV and were blocked by the glutamate AMPA/kainate receptor antagonists 6,7‐nitroquinoxaline‐2,3‐dione (DNQX) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) at rest. Evoked EPSCs had rapid rise times (< 1 s) and decayed monoexponentially (τ= 2.04 ± 0.03 ms) at potentials near rest. 4 At holding potentials positive to ∼−50 mV, a slow EPSC could be evoked in the presence of DNQX or CNQX. This current peaked at holding potentials near −25 mV and was blocked by the NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovaleric acid (AP5). It was therefore probably due to activation of NMDA receptors by vagal afferent fibres. 5 Fast IPSCs reversed near −70 mV and were blocked by the GABAA receptor antagonist bicuculline. In addition, bicuculline enhanced excitatory responses to vagal stimulation and increased spontaneous EPSC frequency. Antagonists to AMPA/kainate receptors reversibly blocked stimulus‐associated IPSCs and also decreased the frequency of spontaneous IPSCs. 6 These findings suggest that glutamate mediates synaptic transmission from the vagus nerve to neurones in the immature NTS by acting at non‐NMDA and NMDA receptors. NTS neurones may also receive glutamatergic and GABAergic synaptic input from local neurones that can be activated by vagal input and/or regulated by amino acid inputs from other brainstem neurones.1. Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.

Efficient unsupervised algorithms for the detection of seizures in continuous EEG recordings from rats after brain injury

Long-term EEG monitoring in chronically epileptic animals produces very large EEG data files which require efficient algorithms to differentiate interictal spikes and seizures from normal brain activity, noise, and, artifact. We compared four methods for seizure detection based on (1) EEG power as computed using amplitude squared (the power method), (2) the sum of the distances between consecutive data points (the coastline method), (3) automated spike frequency and duration detection (the spike frequency method), and (4) data range autocorrelation combined with spike frequency (the autocorrelation method). These methods were used to analyze a randomly selected test set of 13 days of continuous EEG data in which 75 seizures were imbedded. The EEG recordings were from eight different rats representing two different models of chronic epilepsy (five kainate-treated and three hypoxic-ischemic). The EEG power method had a positive predictive value (PPV, or true positives divided by the sum of true positives and false positives) of 18% and a sensitivity (true positives divided by the sum of true positives and false negatives) of 95%, the coastline method had a PPV of 78% and sensitivity of 99.59, the spike frequency method had a PPV of 78% and a sensitivity of 95%, and the autocorrelation method yielded a PPV of 96% and a sensitivity of 100%. It is possible to detect seizures automatically in a prolonged EEG recording using computationally efficient unsupervised algorithms. Both the quality of the EEG and the analysis method employed affect PPV and sensitivity.

Spontaneous motor seizures of rats with kainate-induced epilepsy: effect of time of day and activity state

Kainate treatment in rats can result in a chronic behavioral state that is similar to human temporal lobe epilepsy. We tested the hypothesis that, like some humans with epilepsy, rat with kainate-induced epilepsy have more spontaneous motor seizures during inactivity (i.e. little to no volitional movement, including apparent sleep) than during activity (i.e. apparent volitional movement, as in walking, grooming, eating, etc.). Rats were given intraperitoneal (i.p.) injections of kainate (5 mg/kg) every hour so that class III/IV/V seizures were elicited for > or = 3 h. Seizure behavior was video-monitored (24 h for 5-6 days, n = 32 rats at 3 months and n = 23 rats at 4 months after treatment) to examine the occurrence of seizures as a function of light versus dark (12-12-h light-dark cycle) and inactivity versus activity. Significantly more spontaneous motor seizures occurred during inactive versus active states (82% vs. 18%, P = 0.0001). Although more seizures occurred during the light period than the dark, the difference was not significant (62% vs. 38%, P > 0. 1). These data suggest that the frequency of spontaneous motor seizures in the rat with kainate-induced epilepsy depends primarily on activity state rather than time of day (i.e. time during the light-dark cycle). The effect of inactivity on the occurrence of seizures in the rat with kainate-induced epilepsy appears similar to some forms of human epilepsy.

Coupling in rat hippocampal slices: dye transfer between CA1 pyramidal cells.

Intracellular injections of Lucifer Yellow-CH (LY) into CA1 pyramidal cells were made in rat hippocampal slices to study dye transfer between neurons as evidence that these cells are electrotonically coupled. Extensive control procedures were performed which substantially reduced inadvertent staining. Over half of the neurons were dye-coupled after injections in stratus pyramidale. Dye coupling occurred even when spike amplitudes were greater than or equal to 70 mV throughout the impalement and was still present after chemical synapses were blocked with a low Ca2+ solution containing Mn2+. Somata of dye-coupled cells were usually located within 35 micrometers (post-fixation) of the injected cell and showed no preferred orientation. Fast prepotentials and dye coupling occurred independently. Neurons in superior cervical ganglia, which were sliced and injected using similar procedures, showed no dye coupling. Intradendritic injections of LY in stratum radiatum also yielded dye coupling between CA 1 pyramidal cells, although the dye coupling was less frequent. Within stratum radiatum, neither extracellular ejections nor intracellular injections of interneurons were associated with multiple staining. Thus, injection of LY into the soma or dendrite of a single CA1 pyramidal cell often resulted in multiple staining, and in many ensembles the somata were well spaced. Control experiments suggested that such dye transfer is not by an extracellular route. This implied that some CA1 cells are electrotonically coupled. Further electrophysiological and morphological studies are required to resolve the discrepancies among various techniques used to evaluate the amount of coupling in the hippocampus.