RAPID COMMUNICATION Initiation of Spontaneous Epileptiform Activity in the Neocortical Slice

Tsau, Yang, Li Guan, and Jian-Young Wu. Initiation of spontaWith the use of different in vitro models the paroxysmal neous epileptiform activity in the neocortical slice. J. Neurophysevents appear different in local field potential recordings. In iol. 80: 978–982, 1998. Cortical local circuitry is important in bicuculine and high [K] models the paroxysmal events apepileptogenesis. Voltage-sensitive dyes and fast imaging were used pear as single spikes (PDS), and in the low [Mg] model a to visualize the initiation of spontaneous paroxysmal events in paroxysmal event is composed of a large initial spike (correadult rat neocortical slices. Although spontaneous paroxysmal lated to PDS) and a series of afterdischarges (ictal-like activevents could start from anywhere in the preparation, optical imity) . Because in all three in vitro models paroxysmal events aging revealed that all spontaneous events started at a few confined start with an initial population spike, we refer to the origin initiation foci and propagated to the whole preparation. Multielecof this spike as the initiation site of the paroxysmal event. trode recording over hundreds of spontaneous events revealed that often two or three initiation foci coexisted in each preparation (n Å 10). These foci took turns being dominant; the dominant M E T H O D S focus initiated the majority of the spontaneous paroxysmal events during that period. The dominant focus and dynamic rearrangement Sprague-Dawley rats (postnatal day P21–28) were deeply anesof foci suggest that the initiation of spontaneous epileptiform thetized with CO2 and decapitated. Neocortical slices (400 mm, events involves a local multineuronal process, perhaps with potenticoronal section, Bregma 4–6 mm) were prepared by a Vibratome ated synapses. (Campden Instruments) and incubated for 2 h in 95% O2-5% CO2 equilibrated artificial cerebral spinal fluid (ACSF) containing (in mM) 132 NaCl, 3 KCl, 2 MgCl2 , 2 CaCl2 , 26 NaHCO3, and 10 I N T R O D U C T I O N glucose, at pH 7.4. The slices were stained with 0.02 mg/ml of voltage-sensitive dye JPW1131 (RH 479) before the experiment. Intrinsic cortical mechanisms were suggested to be imDuring recording sessions the stained preparation was perfused in a submerged chamber with one of three modified ACSF solutions: portant in epileptogenesis (Connors 1984; McNamara 1994; 1) 0 [Mg] —ACSF but containing no added MgCl2 , 2) bicucuTraub et al. 1994). A small piece of isolated neocortex when line—ACSF but containing 1 mM MgCl2 and 20–40 mM bicucuperfused with convulsants (e.g., bicuculline) or high [K] or line (Research Biochemicals International) , and 3) high [K] — low [Mg] media is enough to autonomously generate allACSF but containing 5 mM KCl and 1 mM MgCl2. Dye-related or-none paroxysmal events ( the paroxysmal depolarization absorption signals (705 nm) were imaged by a 124-element shift, PDS) involving the activation of the whole preparation (12 1 12) photodiode array at a rate of 1,000 frames per second (Flint and Connors 1996; Silva et al. 1991; Wong and Prince (Wu and Cohen 1993). The raw image data has a low resolution 1990). Intrinsic bursting neurons (Chagna-Amitai and Con(12 1 12 pixels) . We used the contour display in NeuroPlex nors 1989; Connors 1984) in layers 4 and 5 were proposed (OptImaging, LLC, Fairfield, CT) to convert the raw data to pseuas potential pacemaker cells (Silva et al. 1991). However, docolor images. A 12.8 objective lens resulted in a field of view of 4.5-mm diam. A section of the gray matter containing all cortical it is unknown whether a spontaneous PDS is initiated from layers was covered by approximately five detectors (Fig. 1A) . The an individual cell’s activity or by an interaction among a dye signals were filtered at 400 Hz before digitizing. The fractional group of neurons. If an individual or small cluster of paceabsorption change (DI /I) for a typical initial spike was Ç0.1–1% maker cells can start a PDS then the initiation sites might of the resting light intensity (Fig. 1B) . Field potential recordings be small and randomly distributed in a preparation. Conwere made in layer II of the cortex with the use of glass microelecversely, if the initiation of PDS involves distributed interactrodes filled with 1 M NaCl and a tip resistance of 2–5 MV. tions among a large group of neurons, then the initiation of PDS would occur in a large and diffused area with no apparR E S U L T S ent individual site. In this report we attempt to distinguish these possibilities by imaging the spontaneous initiation of Confined initiation foci PDS events. Voltage-sensitive dye imaging (Davila et al. 1974; Ross Spontaneous paroxysmal activity emerged 20–40 min after the preparation was perfused with a modified ACSF. et al. 1977) provides adequate spatial and temporal resolution for these measurements. This method was used to study The PDS was a spontaneously occurring all-or-none population spike in the field potential recordings and in optical the spreading of paroxysmal events in neocortical slices (Albowitz and Kuhnt 1995; Sutor et al. 1994) and epileptiform recordings (Fig. 1B , PDS). The optical signal had a similar waveform and time course as the recordings from electrodes activity evoked by sensory stimuli in cortex in vivo (London et al. 1989). Here we use this imaging method to address in adjacent areas (e.g., Fig. 1B , top 2 traces) . To identify the initiation site of the paroxysmal events, the PDS spike the spontaneous initiation of epileptiform events in in vitro cortical tissue. Dual-electrode measurements were also used from each detector was normalized to its maximum amplitude and pseudocolor images were made from the contour to determine the spatial distribution of the initiation sites.