The piriform cortex is a temporal lobe structure with a very high seizure susceptibility. To investigate the spatiotemporal characteristics of epileptiform activity, slices of piriform cortex were examined by imaging electrical activity with a voltage-sensitive fluorescent dye. Discharge activity was studied for different sites of stimulation and different planes of slicing along the anterior-posterior axis. Epileptiform behavior was elicited either by disinhibition with a gamma-aminobutyric acid-A receptor antagonist or by induction with a transient period of spontaneous bursting in low-chloride medium. Control activity recorded with fluorescent dye had the same pharmacological and temporal characteristics as control activity reported previously with microelectrodes. Simultaneous optical and extracellular microelectrode recordings of epileptiform discharges showed the same duration, latency, and all-or-none character as described previously with microelectrodes. Under all conditions examined, threshold electrical stimulation applied throughout the piriform cortex evoked all-or-none epileptiform discharges originating in a site that included the endopiriform nucleus, a previously identified site of discharge onset. In induced slices, but not disinhibited slices, the site of onset also included layer VI of the adjoining agranular insular cortex and perirhinal cortex, in slices from anterior and posterior piriform cortex, respectively. These locations had not been identified previously as sites of discharge onset. Thus like the endopiriform nucleus, the deep agranular insular cortex and perirhinal cortex have a very low seizure threshold. Additional subtle differences were noted between the induced and disinhibited models of epileptogenesis. Velocity was determined for discharges after onset, as they propagated outward to the overlying piriform cortex. Propagation in other directions was examined as well. In most cases, velocities were below that for action potential conduction, suggesting that recurrent excitation and/or ephaptic interactions play a role in discharge propagation. Future investigations of the cellular and organizational properties of regions identified in this study should help clarify the neurobiological basis of high seizure susceptibility.
When near-threshold electrical stimulation is used to evoke epileptiform discharges in brain slices, a latent period of up to 150 msec elapses before the discharge begins. During this period most neurons are silent, and abnormal electrical activity is difficult to detect with microelectrodes. A fundamental question about epileptiform activity concerns how synchronous discharges arise abruptly in a relatively quiescent slice. This issue was addressed here by using voltage imaging techniques to study epileptiform discharges in rat piriform cortex slices. These experiments revealed two distinct forms of electrical activity during the latent period. (1) A steeply increasing depolarization, referred to here as onset activity, has been described previously and occurs at the site of discharge onset. (2) A sustained depolarization that precedes onset activity, referred to here as plateau activity, has not been described previously.Plateau and onset activity occurred in different subregions of the endopiriform nucleus (a region of high seizure susceptibility). When cobalt or kynurenic acid was applied focally to inhibit electrical activity at the site of plateau activity, discharges were blocked. However, application of these agents to other nearby sites (except the site of onset) failed to block discharges. Plateau activity represents a novel form of electrical activity that precedes and is necessary for epileptiform discharges. Discharges thus are generated in a sequential process by two spatially distinct neuronal circuits. The first circuit amplifies and sustains activity initiated by the stimulus, and the second generates the actual discharge in response to an excitatory drive from the first.
Neural networks capable of generating coordinated rhythmic activity form at early stages of development in the spinal cord. In this study, voltage-imaging techniques were used to examine the spatiotemporal pattern of rhythmic activity in transverse slices of lumbar spinal cord from embryonic and neonatal rats. Real-time images were recorded in slices stained with the voltage-sensitive fluorescent dye RH414 using a 464-element photodiode array. Fluorescence signals showed spontaneous voltage oscillations with a frequency of 3 Hz. Simultaneous recordings of fluorescence and extracellular field potential demonstrated that the two signals oscillated with the same frequency and had a distinct phase relationship, indicating that the fluorescence changes represented changes in transmembrane potentials. The oscillations were reversibly blocked by cobalt (1 mM), indicating a dependence on Ca(2+) influx through voltage-gated Ca(2+) channels. Extracellular field potentials revealed oscillations with the same frequency in both stained and unstained slices. Oscillations were apparent throughout a slice, although their amplitudes varied in different regions. The largest amplitude oscillations were produced in the lateral regions. To examine the spatial organization of rhythm-generating networks, slices were cut into halves and quarters. Each fragment continued to oscillate with the same frequency as intact slices but with smaller amplitudes. This finding suggested that rhythm-generating networks were widely distributed and did not depend on long-range projections. In slices from neonatal rats, the oscillations exhibited a complex spatiotemporal pattern, with depolarizations alternating between mirror locations in the right and left sides of the cord. Furthermore, within each side depolarizations alternated between the lateral and medial regions. This medial-lateral pattern was preserved in hemisected slices, indicating that pathways intrinsic to each side coordinated this activity. A different pattern of oscillation was observed in slices from embryos with synchronous 3-Hz oscillations occurring in limited regions. Our study demonstrated that rhythm generators were distributed throughout transverse sections of the lumbar spinal cord and exhibited a complex spatiotemporal pattern of coordinated rhythmic activity.
Brain slices serve as useful models for the investigation of epilepsy. However, the preparation of brain slices disrupts circuitry and severs axons, thus complicating efforts to relate epileptiform activity in vitro to seizure activity in vivo. This issue is relevant to studies in transverse slices of the piriform cortex (PC), the preparation of which disrupts extensive rostrocaudal fiber systems. In these slices, epileptiform discharges propagate slowly and in a wavelike manner, whereas such discharges in vivo propagate more rapidly and jump abruptly between layers. The objective of the present study was to identify fiber systems responsible for these differences. PC slices were prepared by cutting along three different nearly orthogonal planes (transverse, parasagittal, and longitudinal), and epileptiform discharges were imaged with a voltage-sensitive fluorescent dye. Interictal-like epileptiform activity was enabled by either a kindling-like induction process or disinhibition with bicuculline. The pattern of discharge onset was very similar in slices cut in different planes. As described previously in transverse PC slices, discharges were initiated in the endopiriform nucleus (En) and adjoining regions in a two-stage process, starting with low-amplitude "plateau activity" at one site and leading to an accelerating depolarization and discharge onset at another nearby site. The similar pattern of onset in slices of various orientations indicates that the local circuitry and neuronal properties in and around the En, rather than long-range fibers, assume dominant roles in the initiation of epileptiform activity. Subtle variations in the onset site indicate that interneurons can fine tune the site of discharge onset. In contrast to the mode of onset, discharge propagation showed striking variations. In longitudinal slices, where rostrocaudal association fibers are best preserved, discharge propagation resembled in vivo seizure activity in the following respects: propagation was as rapid as in vivo and about two to three times faster than in other slices; discharges jumped abruptly between the En and PC; and discharges had large amplitudes in superficial layers of the PC. Cuts in longitudinal slices that partially separated the PC from the En eliminated these unique features. These results help clarify why epileptiform activity differs between in vitro and in vivo experiments and suggest that rostrocaudal pyramidal cell association fibers play a major role in the propagation of discharges in the intact brain. The longitudinal PC slice, which best preserves these fibers, is ideally suited for the study their role.
The deep piriform region has an unusually high seizure susceptibility. Voltage imaging previously located the sites of epileptiform discharge onset in slices of rat piriform cortex and revealed the spatiotemporal pattern of development of two types of electrical activity during the latent period prior to discharge onset. A ramplike depolarization (onset activity) appears at the site of discharge onset. Onset activity is preceded by a sustained low-amplitude depolarization (plateau activity) at another site, which shows little if any overlap with the site of onset. Because synaptic blockade at either of these two sites blocks discharges, it was proposed that both forms of latent period activity are necessary for the generation of epileptiform discharges and that the onset and plateau sites work together in the amplification of electrical activity. The capacity for amplification was examined here by studying subthreshold responses in slices of piriform cortex using two different in vitro models of epilepsy. Under some conditions electrically evoked responses showed a nonlinear dependence on stimulus current, suggesting amplification by strong polysynaptic excitatory responses. The sites of plateau and onset activity were mapped for different in vitro models of epilepsy and different sites of stimulation. These experiments showed that the site of plateau activity expanded into deep layers of neighboring neocortex in parallel with expansions of the onset site into neocortex. These results provide further evidence that interactions between the sites of onset and plateau activity play an important role in the initiation of epileptiform discharges. The site of plateau activity showed little variation with different stimulation sites in the piriform cortex, but when stimulation was applied in the endopiriform nucleus (in the sites of onset of plateau activity), plateau activity had a lower amplitude and became distributed over a much wider area. These results indicate that in the initiation of epileptiform discharges, the location of the circuit that generates plateau activity is not rigidly defined but can exhibit flexibility.
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