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Exp Brain Res (1999) 126:160–174 © Springer-Verlag 1999 R E S E A R C H A RT I C L E Raju Metherate · Scott J. Cruikshank Thalamocortical inputs trigger a propagating envelope of gamma-band activity in auditory cortex in vitro Received: 2 September 1998 / Accepted: 12 January 1999 Abstract To investigate how auditory cortex responds to thalamic inputs, we have used electrophysiological and anatomical techniques to characterize a brain slice containing functionally linked thalamocortical and intracortical pathways. In extracellular recordings, stimulation of thalamic afferents elicited a short-latency field potential and current sink in layer IV of the cortex, followed by 100–500 ms of polysynaptic activity containing rapid (gamma-band, 20–80 Hz) fluctuations. Paired intracellular and extracellular recordings showed that a short-latency excitatory postsynaptic potential (EPSP) corresponded to the fast extracellular potential, and that a slow intracellular depolarization with superimposed rapid fluctuations corresponded to the polysynaptic extracellular activity. Pharmacological manipulations demonstrated that glutamate receptors contributed to monoand polysynaptic activity, and that the gamma-band fluctuations contained intermixed rapid depolarizations and Cl–-mediated inhibition. The spread of evoked activity through auditory cortex was determined by extracellular mapping away from the excitatory focus (the site of the largest amplitude fast response). The short-latency potential traversed auditory cortex at 1.25 m/s and decreased over 1–2 mm, likely reflecting sequential activation of cells contacted by thalamocortical arbors. In contrast, polysynaptic activity did not decrease but propagated as a spatially restricted wave at a 57-fold slower velocity (0.022 m/s). Thus, stimulation of the auditory thalamocortical pathway in vitro elicited a fast glutamatergic potential in layer IV, followed by polysynaptic activity, including gamma-band fluctuations, that propagated through the cortex. Propagating activity may form transient neural assemblies that contribute to auditory information processing. R. Metherate (✉) · S.J. Cruikshank Department of Psychobiology, University of California, Irvine, 2205 Biological Sciences II, Irvine, CA 92697-4550, USA e-mail: [email protected] Tel.: +1-949-824-6141, Fax: +1-949-824-2447 Key words Thalamocortical · Synaptic · Auditory cortex · Gamma band · Glutamate · GABA Introduction A fundamental issue in cortical physiology concerns how information relayed by thalamic inputs is processed within cortical circuits. In the auditory system, acoustic stimuli generate short-latency (10–20 ms) cortical responses that reflect the physical characteristics of the stimulus, e.g., its frequency and intensity (Phillips 1993). In addition, longer-latency potentials (e.g., 100–300 ms) in auditory cortex can result from stimuli whose significance to the organism has been manipulated (e.g., eventrelated potentials and gamma-band oscillations; Rugg and Coles 1995; Pantev et al. 1991; Tallon-Baudry et al. 1997). Finally, recent theories have focused on cortical gamma-band oscillations as important mechanisms of sensory processing (Gray et al. 1989; Gray and Singer 1989). The cellular mechanisms underlying these different types of activity remain poorly understood (Volkov and Galazjuk 1991; Jefferys et al. 1996). Cortical brain slice preparations have greatly facilitated the study of cellular and synaptic mechanisms; however, they generally maintain only limited, shortdistance synaptic connectivity. Specialized preparations featuring long-distance pathways have been developed and successfully exploited to study thalamocortical synapses in the somatosensory system (Agmon and Connors 1991; Gil and Amitai 1996) and long-range intracortical pathways in visual and motor cortex (Hirsch and Gilbert 1991; Hess et al. 1994). However, in vitro preparations have rarely been used to study activation of intracortical pathways by thalamocortical inputs, presumably because such connections are disrupted during slice preparation. The experiments described here utilize a novel slice containing functionally linked thalamocortical and intracortical pathways in the auditory system, a preparation that will facilitate understanding mechanisms of thalamocortical information processing. A por- 161 tion of this work has appeared in abstract form (Metherate 1997). The brain was blocked in two steps while submerged in cold, oxygenated ACSF. First, with the whole brain placed ventral side down, a coronal or slightly angled (0–10°) cut was made to the anterior end with a handheld razor blade. The brain was then placed with the cut surface down on filter paper with a diagram indicating Materials and methods Slices from 10- to 25-day-old Sprague-Dawley rats or 14- to 57day-old FVB mice (Charles River) were maintained in vitro using conventional methods, and following the “Principles of laboratory animal care” (NIH publication 86–23) and University of California, Irvine, animal use regulations. Following decapitation under barbiturate or halothane anesthesia, brains were removed into cold artificial cerebrospinal fluid (ACSF, in mM): NaCl 125, KCl 2.5, KH2PO4 1.25, NaHCO3 25, MgSO4 1.2, CaCI2 2 and dextrose 10, bubbled with 95% O2/5% CO2, and then blocked to produce the optimal slice orientation for maintaining the auditory thalamocortical pathway. This orientation was near-horizontal, with the anterior end of the slice plane raised 0–10° above horizontal, and the lateral end raised ~15° above horizontal. This orientation was selected based partly on unpublished experiments by Dr. R.T. Robertson, University of California, Irvine, in which dioctadecyltetramethyllindocarbocyanine perchlorate (DiI) was placed in the medial geniculate nucleus (MG) or auditory cortex of rats and the resulting labeled pathway reconstructed in three dimensions using confocal microscopy. The results are consistent with published descriptions of the thalamocortical (Ryugo and Killackey 1974; Winer 1992) and corticothalamic (Rouiller and Welker 1991) pathways. In initial experiments, slices were cut with lateral angles ranging from 0 to 45° and the above range selected as optimal. Fig. 1A–C The auditory thalamocortical pathway in vitro. A Schematic horizontal section through a rat brain, showing the location of auditory cortex (AC, Te1–3) and the thalamocortical pathway. Inset is a coronal section showing the slice plane (dashed line) at a 15° angle from horizontal through the MG and AC (Te1, Te3) (Te1–3 temporal cortex, areas 1–3, Par parietal cortex, PRh perirhinal cortex, Ent entorhinal cortex, Hipp hippocampus, MG medial geniculate, LG lateral geniculate, VB ventrobasal complex, str superior thalamic radiation, ic internal capsule, CPu caudate putamen). Illustrations in A are modified from Paxinos and Watson (1986). B AChE staining in a juvenile (13-day-old) rat indicates that the thalamocortical slice includes primary auditory cortex. Staining results from AChE expression in lemniscal thalamocortical afferents from MGv and occurred primarily in layers III and IV. Typically, the anterior boundary (arrow) involved denser staining and was easier to delineate than the posterior boundary. Ci Biocytin-labeled thalamocortical pathway in the mouse brain. A biocytin injection (white asterisk) in str labeled fibers projecting to and/or from the auditory cortex and thalamus. Prior electrical stimulation at the injection site elicited a response in cortical layer IV (black asterisk; see Figs. 2, 3). Arrow points to retrogradely filled neurons in the MG that are shown at higher magnification in ii, iii Close-up of the cortical region near the asterisk in i shows labeled axons. Labeled axons occurred throughout auditory cortex, but are not visible at low magnification 162 Fig. 2A–D Stimulation of the MG or auditory thalamocortical pathway elicited a short-latency negative field potential in auditory cortex. All data derived from the same 600-µm-thick mouse slice. A Photograph of the living slice resting on a nylon mesh in the recording chamber. Visible structures include the hippocampus and boundaries between MG and LG nuclei. B Nissl-stained section from the same slice showing a close-up of the area outlined in A; note in particular the MG and str. C A grid of over 100 stimulation sites spaced ~250 µm apart was used to estimate the distribution of projections to auditory cortex (recording site indicated by asterisk). Large circles indicate effective sites where 100 µA ms stimulation elicited fast negative potentials of amplitude >75 µV. Small white circles indicate ineffective stimulus sites. Black circles indicate sites where stimulation elicited positive potentials in auditory cortex. D Sample responses from numbered sites in C demonstrate that stimulation of MG or str, but not the adjacent LG or VB, elicited robust, short-latency negative potentials in auditory cortex a 15° angle from horizontal (Fig. 1A, inset). A second cut was made at this angle, cutting from the back of the brain to the front. The brain was then glued, newly cut surface down, to the stage of a Vibroslice (Campden, WPI), with the lateral surface of the brain (containing the right auditory cortex) facing the blade. Wholebrain slices were cut starting from the ventral surface of the brain. As auditory cortex, localized relative to surface blood vessel patterns and the rhinal fissure, was approached during slicing, a thick bundle of white matter running from the thalamus to cortex became clearly visible. Generally, three to four slices (400 µm) of the right hemisphere were taken from this area and placed either in the recording chamber or in a holding chamber at room temperature. Recordings took place in an interface chamber (Haas model, Med Systems) maintained at 34°C with a continuous flow of warmed, humidified gas (95% O2, 5% CO2) passing over the slices. Recordings followed a waiting period of 2 h for the first slice and 1 h for subsequent slices. In later experiments it was found that increasing the slice thickness to 600 µm resulted in a fully connected pathway be- tween the MG and auditory cortex (Fig. 2). However, earlier experiments used 400-µm slices which contained only a partial thalamocortical pathway (see “Results”). To confirm the location of the thalamocortical pathway in the thinner slices, extracellular deposits of biocytin were made at points between the MG and auditory cortex and the resulting trajectories of projections mapped (Fig. 1C). Biocytin (5% in ACSF) was delivered iontophoretically (100- to 200-nA, 750-ms positive pulse every 1.5 s for 20– 30 mm). Slices remained in the recording chamber for 2–10 h after the injection and were then fixed. Intracellular and extracellular microelectrodes were pulled on a horizontal puller (P-97, Sutter Instruments). Intracellular recordings used either patch pipettes (4–6 MΩ, pulled in four stages) or sharp microelectrodes (60–100 MΩ). Extracellular microelectrodes contained ACSF or 0.15 M NaCl, sharp microelectrodes contained 3 M K acetate or 3 M KCl, and patch pipettes contained (in mM): 125 KMeSO3, 0.05 CaCl2, 2 MgATP, 0.5 NaGTP, 0.16 ethyleneglycoltetraacetic acid (EGTA), and 10 hydroxyethylpiperazine ethanesulfonic acid (HEPES) (adjusted to pH=7.2–7.3 with KOH). To examine the intracellular bases of responses, recordings were obtained in an interface-recording chamber using the “blind” whole-cell patch technique (Blanton et al. 1989; Metherate and Ashe 1994) or conventional sharp microelectrode methods. Recordings using patch electrodes were not corrected for junction potentials except when resting potentials were combined with sharp electrode recordings for statistics; 10 mV was then added to whole-cell measures (measurements of junction potentials with similar pipette solutions and KCl reference electrodes yielded values of 9–12 mV). Neural signals were recorded with extracellular (CyberAmp AI-401, Axon Instruments) and intracellular (Axoclamp 2B, Axon Instruments) amplifiers (bandpass 1–1000 Hz and DC 2000 Hz), monitored on a digital oscilloscope (Tektronix), digitized at 5 kHz and stored on computer (PowerMac, Apple Computer). Electrical stimuli (0.1–0.2 ms, 5–150 µA) were delivered via concentric bipolar electrodes (Ultrasmall, 25 µm inner core, 200 µm overall diameter; F. Haer) and a constant current unit (Axon Instruments or WPI). Experiments were software-controlled (AxoData) and analyzed off-line (AxoGraph, Axon Instruments). 163 Fig. 3A–C Subcortical stimulation elicited short-latency field potential and current sink in middle cortical layers. All data are from the same 400-µm-thick mouse slice. A Stimulation at five different sites (labeled S1–5), near where the str joined the internal capsule, elicited responses (traces 1–5) at the recording electrode (*) in cortical layer IV. Only stimulation at sites 2 and 3 produced prominent responses. Stimulus sites were ~250 µm apart. Stimulus artifacts are truncated; all traces are averages of five responses. B Stimulation of site S3 at different intensities (10–100 µA) produced graded increases in response amplitude with little change in response latency. C Laminar profile of response to stimulation at site S3. Field potentials (left column) were recorded sequentially by moving the electrode in 125-µm steps from the pia to the white matter. CSD profile (right column; upward deflection indicates current sink) obtained from field potentials revealed a prominent current sink in layer IV [note that more typical CSD results included layer I current sources and more superficial (layer III and upper layer IV) current sinks] To determine the laminar source of short-latency potentials elicited by subcortical stimulation, current source density (CSD) analysis was performed on responses recorded from 11–13 sites within a single cortical column in 125-µm steps from the pia to the white matter. The response at each recording site was the average of five trials. The CSD profile was obtained using standard one-dimensional techniques (Mitzdorf and Singer 1978; Agmon and Connors 1991). In brief, for a given recording site, the CSD was calculated by subtracting 2 times the voltage at that site from the sum of the voltages of the nearest neighbors on either side of the site, and then dividing by a distance constant (see Fig. 3C). Slices with biocytin injections were placed in 4% paraformaldehyde overnight, then in 0.1 M phosphate buffer (PB) for 1–7 days. Whole slices were washed in PB, incubated in ABC complex (Vector Labs) for 2–18 h, then rinsed in PB and in Tris buffer. Slices were preincubated in 0.02% diaminobenzidine (DAB, in Tris with 0.25% nickel ammonium sulfate) for 20 min, then reacted in 0.006% H2O2+DAB/nickel for 4–5 min. Sections were washed in Tris and in PB, then mounted on slides, dried, dehydrated, cleared and coverslipped. For acetylycholinesterase (AChE) histochemistry, rat brains were placed in 4% paraformaldehyde for at least 4 days. Following fixation, brains were blocked as described above and 100-µm sections were cut using a Vibratome (Polysciences). AChE histochemistry followed a modified (Koelle and Friedenwald 1949) method. Slices were rinsed in 0.1 M sodium acetate buffer, and incubated in medium containing the substrate acetylthiocholine iodide (1.0×10–4 M) and the nonspecific cholinesterase inhibitor, tetraisopropylpyrophosphoramide (1.14×10–4 M). Following 3 days of incubation, slices were rinsed in phosphate buffer and developed in 1% ammonium sulfide for approximately 30 s. Slices were then rinsed, mounted on slides, dried overnight, then dehydrated and coverslipped. Recorded neurons and extracellular recordings were assigned to cortical layers based on distances from the pia relative to the full cortical width, according to published values for rat (Roger and Arnault 1989) and mouse (Willard and Ryugo 1983) auditory cortex. Distances in the recording chamber were measured using a microscope reticle (25 µm resolution). Extracellular responses in figures are single traces to show rapid fluctuations, or averages of three to five responses. Stimulus artifacts are blanked. Extracellular traces in figures have been further filtered digitally at 500 Hz. Mean values are ±SEM. Drugs were dissolved in ACSF from frozen stocks and bath applied. CNQX stock was prepared with dimethyl sulfoxide (DMSO, final concentration 0.2%). Results The schematic in Fig. 1A depicts the slice orientation and structures relevant to this study. According to published reports (Ryugo and Killackey 1974; Winer 1992), the auditory thalamocortical pathway begins in the MG and courses anteriorly through the thalamus in the superior thalamic radiation (str) before it turns laterally to join the internal capsule. Fibers then fan out and traverse the caudate nucleus before reaching auditory cortex, where they turn posteriorly and course in deep layers before branching off to terminate primarily in layer IV. Auditory-responsive cortex includes primary cortex (area 41 of Krieg 1947 or Tel of Zilles and Wree 1985) as well as nonprimary auditory cortex (areas 20 and 36; Te2 and Te3; reviewed in Scheel 1988). Altogether, the auditory areas occupy ~15 mm2 of the lateral surface of the rat brain (Barth and Di 1990; Brett et al. 1994). In juvenile rats (<20 days old), primary sensory cortex and thalamus 164 can be visualized by staining for AChE (Robertson et al. 1991; Broide et al. 1996). In the auditory system, AChE is expressed by neurons in the ventral division of the MG (MGv) and delineates the extent of their terminal arbors in the middle layers of primary auditory cortex (Robertson et al. 1991). In 15 juvenile rats, slices cut as described to maintain the thalamocortical pathway displayed distinct AChE-positive bands in auditory cortex (Fig. 1B) as well as dense staining in the MG (not shown). For each animal, the maximum rostrocaudal extent of AChE staining in auditory cortex was determined, and this measure averaged 1.8±0.21 mm across animals (range 0.8–2.8 mm, animal age 10–17 days). The dorsoventral extent of staining, determined by counting the number of consecutive 100-µm slices with AChE-positive bands, ranged from 0.8 mm to over 1.8 mm. These data indicate that the slices used here likely included primary auditory cortex. However, the slices certainly included nonprimary auditory cortex as well. These areas abut primary cortex on ventral and caudal sides (and may completely surround primary cortex; Scheel 1988), and receive projections from the dorsal (MGd) and medial (MGm) divisions of the auditory thalamus (MGm also projects to primary cortex; Ryugo and Killackey 1974; Willard and Ryugo 1983; Scheel 1988; Winer 1992; Brett et al. 1994). While the physiological distinctions of the thalamocortical projections to primary versus nonprimary auditory cortex are of interest, we could not distinguish among them in the present experiments. The slice described here likely contained both primary and nonprimary auditory cortex. Because the thalamocortical pathway turns at several points, 400-µm-thick slices rarely contained a fully intact thalamocortical system, and stimulation of the MG per se evoked cortical responses in only a few cases. However, stimulation of the str reliably elicited cortical responses in about one slice per mouse. Since the str is a known part of the auditory thalamocortical pathway, it seemed likely that responses evoked in layer IV of auditory cortex resulted from activation of thalamocortical fibers. To confirm this, small deposits of biocytin were placed in the str at effective stimulation sites (Fig. 1C). These deposits predominantly labeled fibers projecting to and/or from auditory cortex (Fig. 1C, n=13/13 slices) as well as fibers projecting to and/or from the MG (Fig. 1C; n=11/13 slices). Labeled structures included cell bodies in the MG (Fig. 1Cii) and beaded axons in auditory cortex (Fig. 1Ciii). No other pathway was labeled consistently. Thus, the effective stimulation sites lay within the auditory thalamocortical pathway and, by extension, the responses elicited in auditory cortex resulted from stimulation of thalamocortical projections. In a subset of later experiments slice thickness was increased to 600 µm in juvenile mice (15–18 days old) in an attempt to maintain the entire thalamocortical pathway (Fig. 2A,B). In these experiments, usually one slice per animal contained a fully connected thalamocortical pathway, such that stimulation of the MG evoked clear, short-latency cortical responses (Fig. 2C,D; n=9 slices). For the experiment depicted in Fig. 2, stimuli were delivered to over 100 sites spaced ~250 µm apart (Fig. 2C; recording electrode in layer IV of auditory cortex at asterisk). Robust, short-latency negative potentials, as depicted in Fig. 2D and described below, resulted from stimulation of sites in the MG and along the thalamocortical pathway as it traversed the str, ic, and caudate, as well as in the lower cortical layers (large circles in Fig. 2C). Stimulation outside the auditory pathway, e.g., the visual (LG) or somatosensory (VB) thalamus, did not elicit responses in auditory cortex (Fig. 2D and small circles in Fig. 2C). Thus, responses to stimulation anywhere along the subcortical portions of the auditory thalamocortical pathway, including the MG, str, and caudate, were qualitatively similar (descriptions of responses are below). A comparison of the data from these slices with those from 400-µm-thick slices demonstrated that the distribution of effective stimulation sites overlapped completely (cf. Fig. 3). We conclude that either preparation enabled stimulation of the auditory thalamocortical pathway. In the present experiments, mouse and rat brains each provided specific advantages because of their respective sizes. As described above, the smaller mouse brain enabled capture of larger portions of the thalamocortical pathway, such that stimulation within the MG (600-µm slice) or str (400-µm slice) elicited cortical responses in about one slice per animal. By comparison, it was rarely possible to maintain functional connections from the str to auditory cortex in slices from rat brain. However, since the thalamocortical pathway widens after joining the internal capsule (Ryugo and Killackey 1974), responses could be elicited reliably from the internal capsule and caudate in two to three slices per rat or mouse. Rat slices were more useful for their larger cortical area, which facilitated mapping the intracortical spread of excitation. Despite these differences in preparation, similar cortical response configurations were recorded in mouse and rat slices, in slices that were 400 µm and 600 µm thick, and in response to stimulation of the MG, str, internal capsule, or caudate. Except where specified, stimulation sites within the thalamocortical pathway are referred to as thalamocortical or subcortical. Subcortical stimulation elicits fast and slow potentials and gamma-band fluctuations Electrophysiological results derive from 82 slices from rat brain and 58 slices from mouse brain. Stimulation of the MG in 600-µm-thick slices elicited a fast negative field potential in layer IV of the cortex (Fig. 2D; mean latency to peak 7.7±0.4 ms, amplitude 182±41 µV, 100 µA intensity; n=9 mouse slices). In 400-µm-thick slices, stimulation near the junction of the str and ic (mean distance 1.2±0.06 mm below the border of cortex and white matter) elicited a similar fast negative potential in layer IV (Fig. 3A; latency to peak 7.0±0 7 ms amplitude 225±39 µV, n=13 mouse slices). Movement of the stimulating electrode across str/ic in the rostrocaudal direction 165 Fig. 4A, B A slow potential with rapid, gamma-band fluctuations followed the fast potential in response to subcortical stimulation. A Averaged layer IV response is shown on top, three consecutive individual responses are below. Averaged power spectrum of the rapid fluctuations for the three individual responses shows peaks at 20–70 Hz (bottom spectra derived from ~200-ms portions of each trace), whereas the prestimulus baseline has no gamma-band activity (lower trace in spectrum). B Laminar profile of fast and slow potentials obtained by moving the electrode in 125-µm steps from the pia to the white matter. Both fast and slow potentials were positive in layer I and negative in layer IV. Slow potential and rapid fluctuations were most prominent in middle and upper layers. Note that the double-peaked slow potential in lower layer III is an atypical response. After completing laminar profile, response configurations were verified in layer III (bottom trace) and V (not shown). Data in A and B from mouse slices demonstrated a narrow (<500-µm-wide) region for effective stimulation (Fig. 3A, cf. Fig. 2C). With increasing stimulus intensity, the amplitude of the fast potential increased but its latency did not change appreciably (Fig. 3B), consistent with monosynaptic activation (Berry and Pentreath 1976). Movement of the recording electrode in small (125-µm) steps perpendicular to the pia demonstrated that the fast potential was of largest amplitude in the middle layers (Fig. 3C), became smaller in superficial and deep layers, and often reversed polarity in layers I–II (cf. Fig. 4B). Current source density analysis revealed a prominent current sink in layers III–IV, the region of dense thalamocortical terminations (Ryugo and Killackey 1974; Winer 1992), with current sources in upper layer V and supragranular layers (Fig. 3C, n=5). Similar CSD profiles resulted from stimulation of different loci within the thalamocortical pathway, i.e., str, internal capsule, or caudate. Thus, the fast potential reflects monosynaptic thalamocortical activation of layer IV neurons. Following the fast potential was a slow, negative extracellular potential of ~100–500 ms duration (Fig. 4A). Unlike the highly consistent monosynaptic response, whose amplitude increased gradually with increases in stimulus intensity, the slow potential often appeared as an all-or-none event near its threshold stimulus intensity, and had a latency, amplitude, and duration that varied somewhat from trial to trial (cf. average and individual trials in Fig. 4A). Low-stimulus intensities elicited the fast potential alone, whereas higher intensities elicited both fast and slow potentials. Further increasing the stimulus intensity decreased the latency to the slow potential, suggesting a polysynaptic origin. The slow potential occurred most prominently in middle and upper layers, and reversed polarity in superficial layers (Fig. 4B). The slow potential fatigued easily with repetitive stimulation, relative to the fast potential, but in general responded reliably with interstimulus intervals ≥15 s. In a representative sample of 23 mouse slices in 10 consecutive experiments, subcortical stimulation elicited both fast and slow potentials in 19, and a fast potential only in 2 slices. In an additional two slices, stimulation appeared to elicit only the slow potential without a preceding fast potential. However, in one of these cases, a simultaneous intracellular recording (see below) revealed a weak but clear intracellular fast potential. Thus, the fast potential usually precedes the slow potential and may contribute to its generation. Rapid, rhythmic fluctuations occurred at the peaks of slow potentials (Fig. 4A,B). Spectral analyses of the rapid fluctuations revealed peaks at 20–80 Hz (gammaband) with lesser components seen up to >100 Hz. The laminar profile of rapid fluctuations matched that of the slow potential (Fig. 4B), and rapid fluctuations never occurred in the absence of a slow potential. Rapid fluctuations also exhibited trial-to-trial variability in their time of occurrence (e.g., rapid fluctuations in Fig. 4A are prominent in individual trials but less apparent in the averaged response), and were not phase-locked to the stimulus (or else they would be apparent in the averaged response). As implied above and supported by additional evidence below, the slow potential and rapid fluctuations appear to reflect polysynaptic activity within small networks of neurons. As might be expected given the relative lability of the slow potential, it was more sensitive to conditions in the recording chamber than was the monosynaptic fast potential. At room temperature (22°C) the fast potential occurred reliably, although with a slower time course, whereas the slow potential occurred weakly or not at all. Warming the chamber increased the probability of occurrence and the amplitude of the slow potential, with the robust potential being observed above 30°C (standard recording temperature 34°C). Further, brief (e.g., 1- to 2-min) interruptions to the supply of warmed, humidified gas (95% O2, 5% CO2) flowing over the slice reversibly eliminated the slow potential while producing no or only partial reduction of the fast potential (longer duration interruptions to the gas supply eliminated both potentials). Finally, the slow potential disappeared when slices were submerged under ACSF flowing at the rate that was nor- 166 Fig. 5A, B Intracellular response to subcortical stimulation: correspondence with extracellular potentials. Aia Simultaneous extracellular and intracellular responses to subcortical stimulation recorded near the layer III/IV border (same slice as Fig. 4B). Fast and slow depolarizations corresponded to fast and slow extracellular responses. Intracellular rapid fluctuations could elicit action potentials. b Activity during gamma-band fluctuations is shown at higher resolution. c Cross-correlation of intracellular and extracellular records during the 230-ms trace in b reveals common oscillatory activity. d The cross-spectrum (power spectrum of the crosscorrelation) indicates power peaks for the common oscillation at 30 Hz and 50 Hz, as well as at lower frequencies (≥10 Hz) corresponding to the overall slow potential. ii For the same cell, intracortical stimulation on-beam (directly below the recording electrode) in layer VI elicited a fast EPSP followed by an IPSP, but no slow potential or rapid fluctuations. Cell was depolarized to –55 mV to reveal IPSP (but slow potentials or rapid fluctuations were not observed at any membrane potential). Whole-cell recording. B Fast and slow depolarizations increased in amplitude with membrane hyperpolarization and decreased with membrane depolarization from rest (–64 mV). Similar extracellular responses (top traces) indicate that changes are not due to response variability. Note late, slow AHP that reversed polarity negative to rest. Sharp microelectrode recording with K acetate. Data in A and B from mouse mal for interface recording (~1 ml/min), but recovered upon increasing the flow rate (e.g., to 5–10 ml/min). These data suggest that the slow potential is particularly sensitive to the temperature and degree of oxygenation of the slice. It was important to establish that the slow potential reflects normal physiological mechanisms, since activity resembling the slow potential can occur in tissue that is damaged, pharmacologically treated to reduce inhibition, or obtained from young animals with immature cortical inhibitory circuits (Chagnac-Amitai and Connors 1989a; Luhmann and Prince 1990; Gil and Amitai 1995; Metherate and Ashe 1995a). Several observations preclude these possibilities. First, only carefully handled slices reliably displayed the slow potential, and difficulties in slicing or handling resulted in no slow potential even though the fast potential remained. Second, in slices where subcortical stimulation elicited clear slow potentials, intracortical stimulation “on-beam” in layer VI elicited strong inhibition and no slow potential (e.g., Figs. 5A, 6A), similar to responses seen in numerous previous studies (Avoli 1986; Connors et al. 1988), including our own studies using coronal slices from auditory cortex (Cox et al. 1992; Metherate and Ashe 1994). Third, slow potentials in slices from rats up to 25 days old, and from mice up to 57 days old, were similar to those from younger animals. Finally, pharmacological blockade of GABAA receptors produced potentials that were qualitatively different from our typical slow potentials: stimulus-evoked bursts initially and, after longer periods of blockade, spontaneous and evoked paroxysmal discharge (Fig. 6C), as reported previously (Connors 1984; Chagnac-Amitai and Connors 1989a). Together, 167 Fig. 6A–C Effects of Cl–-loading and GABAA receptor/Cl– channel blockade on synaptic potentials. A Synaptic response in two neurons to intracortical on-beam stimulation with K acetate (left) or KCl (right) in the electrode. B For the latter cell in A (right, with KCl), subcortical stimulation elicited fast and slow depolarizations with robust excitation. Steady membrane hyperpolarization (to –90 mV) greatly increased the amplitude of the slow potential, which could still elicit spikes. Note that extracellular rapid fluctuations corresponded closely to intracellular fluctuations and spikes. C Bath application of picrotoxin first shortened the duration of the rapid fluctuations and increased the amplitude of the slow potential to produce an intracellular burst and extracellular negative-positive spike (early effect recorded after superfusion with 10 µM picrotoxin for 8–23 min), and later produced evoked paroxysmal discharge (full effect recorded at 25 min). Data in A–C from mouse slices these data demonstrate that slow potentials did not result from impaired or immature inhibition. However, it does appear that thalamocortical stimulation activates cortical inhibition much less strongly than does typical intracortical stimulation (see below), resulting in slow potentials that can resemble responses obtained with stimulus intensities insufficient to activate cortical inhibition (Jones and Baughman 1988; Sutor and Hablitz 1989; Metherate and Ashe 1995a), or with threshold doses of GABA receptor blockade (Chagnac-Amitai and Connors 1989a). Intracellular recordings Simultaneous extracellular and intracellular recordings were made to investigate the nature of evoked responses. An extracellular recording electrode was placed in layers III–IV, and intracellular recordings were made in layer IV within 50–200 µm of the extracellular electrode. Similar observations resulted from using patch pipettes containing KMeSO3-based solutions as with sharp microelectrodes containing K acetate. Neurons had resting membrane potentials >50 mV (mean 64±2.9 mV) and overshooting action potentials (63±3.0 mV from spike threshold; n=16). Subcortical stimulation elicited fast and slow intracellular depolarizations that closely corresponded to fast and slow extracellular negativities (Fig. 5A,B). In neurons where subcortical stimulation elicited a slow depolarization, moving the stimulating electrode to an intracortical, layer VI “on-beam” site (stimulation in the same cortical column as the recording electrode) resulted in a fast EPSP followed by robust inhibitory postsynaptic potentials (IPSPs) and no slow potential (Figs. 5Aii, 6A). Thus, intracortical inhibition was intact in these slices, but was bypassed, or engaged differently, by subcortical stimulation. Rapid intracellular fluctuations occurred during slow depolarizations at the same time as rapid extracellular fluctuations (Fig. 5A,B). Depolarizing peaks within the intracellular fluctuations often corresponded to negative phases of the extracellular record, and could elicit spike discharge (see expanded traces in Fig. 5Aib). For the cell in Fig. 5A, cross-correlating the expanded traces in Fig. 5Aib reveals a clear, though weak, correlation (Fig. 5Aic), and the cross-spectrum indicates common oscillations peaking at 30 and 50 Hz, as well as lower frequencies (≤10 Hz) corresponding to the overall slow potential (Fig. 5Aid). In other cells, intracellular and extracellular rapid fluctuations showed weaker correspondence in terms of individual peaks, but the overall envelope of the slow potentials invariably occurred at the same time. Membrane depolarization via steady current through the recording electrode reduced the magnitude of the slow potential, and membrane hyperpolarization increased it (Fig. 5B, n=3 of four neurons; in one neuron, the slow potential increased with membrane depolariza- 168 tion and decreased with hyperpolarization). Monitoring of extracellular potentials (Fig. 5B, top traces) confirmed that apparent changes in intracellular potentials with steady current did not result simply from response variability (data that did not meet this requirement were excluded). Unexpectedly, membrane depolarization to near spike threshold did not necessarily increase the number of spikes elicited during rapid fluctuations. While some rapid depolarizations continued to elicit spikes, rapid hyperpolarizing potentials remained that appeared to prevent increased excitation (in Fig. 5B, response at the most depolarized level still did not elicit spikes and exhibited rapid deflections in the hyperpolarizing direction). Finally, a slow afterhyperpolarization (AHP) that reversed polarity negative to rest often followed the rapid fluctuations (Fig. 5B, 6B), suggesting the presence of intrinsic and/or synaptically activated hyperpolarization (e.g., via Ca2+-activated and/or GABAB-mediated K+ currents). The observations that steady depolarization: (1) reduced the amplitude of the slow potential, but (2) did not reduce rapid hyperpolarizing fluctuations, and (3) did not necessarily increase the number of evoked spikes, suggested that rapid fluctuations contained fast inhibitory potentials. To test for this possibility, intracellular recordings were obtained with sharp electrodes containing KCl instead of K acetate. The diffusion of Cl– into the cell would be expected to shift the Cl– equilibrium potential above spike threshold, making Cl–-mediated potentials depolarizing and excitatory (Staley 1992). To confirm the effectiveness of this manipulation, neurons were tested by intracortical stimulation in layer VI. Such stimulation normally elicits fast (Cl–-dependent) and slow (K+-dependent) IPSPs (Fig. 6A, trace on left; cf. Connors et al. 1988; Metherate and Ashe 1994). Neurons impaled with KCl-filled electrodes for at least several minutes displayed long-duration depolarizations and no hyperpolarizing fast IPSPs in response to layer VI stimulation (Fig. 6A, trace on right), indicating that the Cl–-dependent fast IPSP was depolarizing. In these cells, subcortical stimulation elicited a slow potential that was significantly more excitatory than with control recording solutions (Fig. 6B). In control neurons depolarized with steady current to near spike threshold, the slow potential elicited 1.3±0.62 spikes (n=6 neurons; spike discharge for each neuron averaged from three to five responses). In contrast, in Cl–loaded neurons the slow potential elicited 7.7±1.10 spikes (n=5; t-test, P<0.001). Membrane potentials in the two groups were not different (control 54±3.5 mV; KCl, 54±2.1 mV; P>0.05). Also, upon steady hyperpolarization of Cl–-loaded neurons, stimulation produced very large amplitude depolarizations that could still elicit spiking (Fig. 6B). Finally, each negative peak in extracellular rapid fluctuations corresponded closely with either spike discharge or robust depolarizations in neurons recorded with KCl, in contrast to the weaker correspondence observed with control recording solutions. As an additional test for the involvement of fast inhibition in the rapid fluctuations, the GABAA receptor/Cl– channel blocker picrotoxin (10 µM; n=2 slices) was administered to the bath. If GABAergic IPSPs acted to retard depolarization and inhibit cell discharge during rapid fluctuations, then picrotoxin would be expected to reverse such actions. After a few minutes of perfusion with picrotoxin, stimulation no longer elicited rapid fluctuations riding on a long-duration slow potential, but elicited in their place a single, rapid depolarization leading to a burst of spikes, and a corresponding extracellular sharp, negative-positive deflection (Fig. 6C, early effect). These effects suggest that fast GABAergic IPSPs reduce excitation and can serve to extend the overall duration of rapid fluctuations. The full effect of picrotoxin occurred after a longer period of time and consisted of spontaneous and evoked paroxysmal discharge (Fig. 6C, full effect). Together with the above findings, these data indicate that fast, Cl–-mediated GABAergic IPSPs contribute to rapid fluctuations and normally act to inhibit cell discharge. Sensitivity of fast and slow potentials to glutamate receptor antagonists Selective antagonists were used to assess the involvement of glutamate receptors in the subcortically evoked fast and slow potentials. Bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino5-phosphonovaleric acid (APV, 25–50 µM) slightly reduced the fast potential, but completely and reversibly blocked the slow potential in intracellular (Fig. 7A; n=4) and extracellular (n=8) recordings. The α-amino3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate (AMPA/KA) glutamate receptor antagonist 6-cyano-7nitroquinoxaline-2, 3-dione (CNQX, 10–20 µM) initially blocked the slow potential (Fig. 7A, trace 4), and subsequently reduced the fast potential (Fig. 7A, trace 5, Fig. 7B; intracellular, n=3, extracellular, n=9). In the presence of CNQX, the residual fast response displayed nonconventional voltage dependence, becoming larger upon membrane depolarization and smaller upon membrane hyperpolarization, and was reduced by APV (Fig. 7Bi). The onset latency of the APV-sensitive component was similar to that of the control fast potential, and did not change with changes in stimulus intensity (Fig. 7Bii). Thus, the putative monosynaptic fast potential contained components mediated by both AMPA/KA and NMDA glutamate receptors. The time course of drug effects provided additional insight into the nature of fast and slow potentials. CNQX reduced the fast potential gradually over a 3- to 10-min period, and APV subsequently reduced the residual fast component gradually over a similar time period, consistent with previous findings using similar drug application methods and flow rates (cf. Fig. 3 in Metherate and Ashe 1995b). In contrast, each receptor antagonist reduced the slow potential abruptly and completely, often from one trial to the next (e.g., Fig. 7A, 30-s intertrial interval). While this may indicate that the slow potential 169 Fig. 7A, B Effects of glutamate receptor antagonists on fast and slow intracellular potentials. A Peak amplitude of the fast potential and the area of the slow potential were normalized to the average value for the last five responses before first drug application (fast potential=3.7 mV, slow potential=3100 mVms). The time course of effects is plotted in the graph on top at 30-s intervals, with the responses from the numbered time points shown below (spikes are truncated). APV (50 µM) slightly reduced the fast potential and, at the same time, abruptly and completely blocked the slow potential (traces 1, 2). Both potentials recovered after a few minutes wash (trace 3). CNQX (20 µM) reduced the fast potential gradually over ~5 min, but also blocked the slow potential abruptly (traces 4, 5). The amplitude of the residual fast potential in CNQX was increased by increasing the stimulus intensity from 70 µA to 150 µA (not shown); this residual response was blocked by APV (not shown). Partial recovery from both drugs was seen after wash (trace 6, stimulus intensity 150 µA). Sharp electrode recording with KCl, resting potential –52 mV. Bi In another cell, CNQX (20 µM) blocked the slow potential and reduced the fast potential elicited by subcortical stimulation (traces are averages of three to five responses, resting potential –75 mV). The magnitude of the residual fast potential in CNQX increased with depolarization, decreased with hyperpolarization, and was blocked by APV (50 µM). ii Superposition of the control fast potential with the APV-sensitive residual fast response elicited by stimuli of different intensities revealed similar onset latencies that did not change with stimulus intensity. Whole-cell recording. Data in A and B from mouse contains glutamatergic components, an additional interpretation is that glutamatergic potentials trigger the slow potential. Reduction of the triggering potential below a threshold value by either antagonist would result in the abrupt, complete disappearance of the slow potential. These data also provide further evidence that the slow potential and rapid fluctuations reflect polysynaptic activity. Intracortical spread of excitation evoked by thalamocortical inputs Two approaches were used to determine how fast and slow potentials spread though auditory cortex. Data presented here were obtained in slices from rat cortex, but mouse slices yielded qualitatively similar observations. First, for each slice, extracellular mapping across layer IV during subcortical stimulation identified the excitatory “focus,” i.e., the location of the largest amplitude fast potential. The spread of activity across cortex was then mapped by recording in layer IV at intervals of 0.2–1.0 mm away from the focus. Second, these results were reinforced by simultaneous recordings using two electrodes placed either in different layers within the same cortical column (e.g., Fig. 8A) or at different horizontal separations within layer IV (e.g., Fig. 8D), as well as 170 Fig. 8A–D Spread of fast and slow potentials across auditory cortex. A Extracellular recordings were made at the response focus in layer IV (single electrode, trace 1), then at 0.75 mm posterior to the focus, simultaneously in layers I and IV (two electrodes, traces in 2), and finally at 2.5 mm posterior to the focus, simultaneously in layers I and IV (traces in 3; arrows indicate time of stimulus). Schematic shows approximate positions of recording (1–3) and stimulation (S) sites. B Summary of 78 recording sites in 16 rat slices. Amplitudes of fast and slow potentials are normalized to the largest-amplitude fast and slow potential, respectively, for each slice (by definition the largest fast potential occurred at the focus). C Data from nine slices showing the onset latency of the slow potential at the focus and 1–2.5 mm off-focus (the most distant site tested for each slice). These data were used to estimate the slow potential’s propagation velocity (see “Results”). Pairs connected with dotted lines indicate data from simultaneous dual recordings (as in D), whereas solid lines indicate sequential recordings with a single electrode (as in A). D Dual extracellular recording in layer IV at the focus and 2.1 mm posterior to the focus. CNQX first completely blocked the off-focus slow potential (7 min), then the slow potential at the focus, and finally reduced the fast potential at the focus (27 min). Data in A–D from rat slices with pharmacological manipulations. These complementary approaches produced a consistent, though unexpected, picture of the auditory cortex response to subcortical stimulation. Recordings at the excitatory focus demonstrated fast and slow potentials, as described above. Moving the recording electrode to more posterior locations revealed a gradual decrement of the fast potential over ~2 mm. However, over this same distance, the slow potential did not decrease, and sometimes increased, in amplitude (Fig. 8A,B). In some cases, recordings at posterior distances >2 mm revealed a robust slow potential with an onset latency >100 ms, but essentially no fast potential (Fig. 8A,D). Anterior to the focus, both fast and slow potentials decreased within 1–2 mm (Fig. 8B). Dual extracellular recordings (n=23 paired recordings) in layers I and IV of the same cortical column demonstrated that the slow potential and gamma-band fluctuations occurred simultaneously throughout the middle and upper layers (Fig. 8A). Even at off-focus locations where no fast potential was visible, the slow potential and rapid fluctuations occurred simultaneously in middle and upper layers (Fig. 8A, records at –2.5 mm). Note that a functional consequence of the slow potential terminating itself (e.g., via an AHP) is that the excitation moving across the cortex remained spatially restricted, rather than forming an ever-expanding area of depolarization. The limited spread of excitation in the anterior direction may reflect anisotropic local circuitry rather than an absolute boundary (e.g., the edge of auditory cortex). Shifting the stimulating electrode anteriorly within the thalamocortical pathway could, within limits, shift the cortical focus anteriorly to a previously unresponsive area. Similarly, the restricted anterior spread of excitation was similar for foci in different locations. Finally, stimulating halfway between two recording electrodes (1–2 mm separation, all three electrodes in layer IV) produced a 2.3±0.3-fold larger short-latency response at the posterior electrode than at the anterior electrode (n=3). Thus, intracortical excitation within the slice preferentially projected in a posterior direction, although the extent to which slice orientation contributes to this phenomenon remains to be determined. 171 The fast and slow potentials differed greatly in their velocity of propagation within the cortex. The onset latency of the fast potential increased 1.2 ms over a cortical distance of 1.5 mm, a small but significant change (2.98±0.42 ms at the focus, 4.18±0.64 ms at –1.5±0.18 mm; one-tailed paired t-test, P<0.001, n=10). This indicates a velocity of 1.25 m/s. In contrast, when an onset latency could be determined for the slow potential both on- and off-focus, it increased 114±23.4 ms over a similar distance of 1.8±0.19 mm (Fig. 8C, n=9 slices; measurements obtained with simultaneous dual recordings (e.g., Fig. 8D, dotted lines in Fig. 8C) and sequential single-electrode recordings (e.g., Fig. 8A, solid lines in Fig. 8C). This indicates a velocity of 0.022 m/s, approximately 57-fold slower than the fast potential. Thus, the fast potential traversed auditory cortex relatively quickly, probably reflecting sequential activation of cells contacted by thalamocortical arbors, whereas the slow potential propagated relatively slowly, reflecting intracortical, polysynaptic transmission. Consistent with effects seen with single-electrode recordings, both APV and CNQX blocked the slow potential recorded off-focus as well as at the focus. Figure 8D shows that CNQX first blocked the slow potential off-focus (records at 7 min), then the slow potential at the focus, and finally reduced the fast potential at the focus (records at 27 min). The faster reduction of the off-focus slow potential further suggests that it propagated from the focus, i.e., the off-focus potential is further downstream in a polysynaptic chain than the on-focus potential. These data further support the conclusion that thalamocortical inputs initiate polysynaptic activity that propagates across auditory cortex. Discussion The results indicate that stimulation of auditory thalamocortical afferents in vitro initiates the following sequence of events: (1) glutamate released from thalamocortical afferents activates neurons in layer IV via AMPA/KA and NMDA receptors. (2) Monosynaptic potentials trigger polysynaptic activity that produces rapid, gammaband fluctuations. (3) Rapid fluctuations include excitatory and Cl–-mediated inhibitory events that may reflect coordinated, high-frequency discharge of cortical interneurons. (4) Polysynaptic activity, including gammaband fluctuations, propagates across cortex as a spatially restricted wave of activity. Nature of the fast potential elicited by thalamocortical stimulation Electrophysiological mapping experiments combined with tract tracing with biocytin demonstrated that the brain slice described here contains the auditory thalamocortical pathway, as described previously in anatomical studies (Ryugo and Killackey 1974; Willard and Ryugo 1983; Winer 1992). Stimulation of the MG or thalamocortical pathway elicited a short-latency response and associated current sink in cortical layer IV, consistent with the location of the major thalamocortical termination zone. While it is likely that the slice also contains corticothalamic fibers (Rouiller and Welker 1991), stimulation did not produce a current sink in layers V or VI, the location of corticothalamic cell bodies, which might reflect antidromic activation of these cells. Further, few antidromic responses occurred in intracellular recordings. While antidromic activity cannot be precluded, it does not appear to play a major role in the short-latency response, suggesting that corticothalamic fibers may not be strongly activated by the stimulus intensities employed here (cf. Agmon and Connors 1991). The fast potential has characteristics (latency, laminar profile, and responses to changes in stimulus intensity) that are expected for a monosynaptic thalamocortical potential. Its sensitivity to glutamate antagonists suggests that both AMPA/KA and NMDA receptors mediate thalamocortical synaptic transmission, as in the somatosensory thalamocortical slice (Agmon and O’Dowd 1992; Gil and Amitai 1996). Electrophysiological mapping of the fast potential indicates that it spreads over 3–5 mm in rat cortex, consistent with the activation of three to ten nonoverlapping arbors of thalamocortical afferents (Jensen and Killackey 1987; Winer 1992). Thus, the fast potential likely reflects monosynaptic activation of neurons by thalamocortical afferent fibers. Nature of the slow potential Several findings indicate that the slow potential reflects polysynaptic activity that propagates intracortically. The slow potential’s onset latency decreased with increases in stimulus intensity, as expected for activation of a polysynaptic chain of neurons (Berry and Pentreath 1976). Also, administration of receptor antagonists always reduced the slow potential before the fast potential, suggesting that the slow potential occurred downstream within a polysynaptic chain. The slow potential’s speed of propagation was significantly slower than that of the fast potential, which may reflect dependence of the former on unmyelinated intracortical pathways and multiple synaptic relays. Finally, receptor antagonists blocked the off-focus slow potential before the on-focus slow potential, suggesting that the off-focus response was “more polysynaptic” than that at the focus. Together, these data indicate that slow potential depends on intracortical, polysynaptic pathways. An alternative explanation is that propagating activity results from antidromic activation of corticothalamic cells that release transmitter from axon collaterals. This hypothesis is difficult to rule out, although, as summarized above, there was little evidence for antidromic activation in these experiments. Further, stimulation of longdistance intracortical (horizontal) pathways can also elicit propagating slow potentials (Hsieh et al. 1998), indi- 172 cating that antidromic activation of corticothalamic neurons is not required to elicit the slow potential. A final answer to this question must await in vivo experiments using sensory stimulation. However, recent experiments using electrophysiological and optical recording methods in vivo indicate that acoustic stimuli can generate longlatency gamma band activity (Franowicz and Barth 1995) and propagating activity (Horikawa et al. 1996) and can activate very large areas of auditory cortex (Phillips et al. 1994; Bakin et al. 1996). In previous in vitro studies, as in the present one, intracortical (on-beam) stimulation generally elicited strong IPSPs and no slow potential (Connors et al. 1988; Cox et al. 1992; Metherate and Ashe 1994). However, slow potentials bearing some resemblance to those described here can be elicited with intracortical stimulation of insufficient intensity to activate cortical inhibition (Jones and Baughman 1988; Sutor and Hablitz 1989; Metherate and Ashe 1995a), with paired-pulse stimulation that fatigues inhibition (Metherate and Ashe 1994; Metherate and Ashe 1995a), in young animals with immature inhibition (Luhmann and Prince 1990; Metherate and Ashe 1995a), or upon delivery of threshold doses of GABA receptor antagonist (<1 µM bicuculline; Chagnac-Amitai and Connors 1989a, 1989b; Gil and Amitai 1995). While the exact relationship between slow potentials in the present and previous studies is unclear, these findings suggest that thalamocortical inputs may engage intracortical inhibition in a different way, and less strongly, than does direct activation of intracortical fibers running vertically within a cortical column. Thus, activation of an afferent pathway that engages cortical inhibition weakly may produce a similar response to activating intracortical pathways without inhibition, or following reduction of inhibition. That thalamocortical and intracortical pathways activate cortical inhibition to different degrees is clearly demonstrated in the present study by comparing responses to subcortical versus intracortical stimulation in the same neuron (e.g., Figs. 5, 6). Responses in the present study may more closely reflect physiological activation of sensory cortex in vivo (Berman et al. 1991). Gamma-band fluctuations Rapid, gamma-band fluctuations occurred only during the slow potential and never by themselves, implying a shared underlying mechanism. Their voltage dependence and sensitivity to intracellular Cl– manipulations or GABA channel blockade indicate the likely involvement of Cl–-mediated, GABAergic IPSPs, intermixed with rapid excitatory depolarizations, most probably glutamatergic EPSPs. The relative proportions of GABAergic and NMDA receptor-mediated glutamatergic potentials could determine whether the slow potential’s overall amplitude either increased or decreased with membrane depolarization, and both results were observed in the present study. Intrinsic membrane currents, such as the depo- larization-activated persistent Na+ current (Whittington et al. 1995; Mittman and Alzheimer 1998), may also contribute to driving network activity (see also Llinas 1988; Llinas et al. 1991). The slow potential and gamma activity do not require a recurrent thalamocortical loop (Ribary et al. 1991), since the thinner slices (400 µm) used in this study do not normally contain a functional link to the MG itself. Fast oscillations in the intact animal could very well utilize corticocortical and thalamocortical loops (Ribary et al. 1991; Steriade et al. 1996a; Steriade et al. 1996b), but the activity described in the present study does not require them. It is possible that cortical gamma activity initiates recurrent activity in thalamocortical loops. Interestingly, physiological and computational findings suggest that sustained excitation of a network of interconnected inhibitory interneurons can result in gamma-band synaptic fluctuations (Whittington et al. 1995). Similar network properties may produce high-frequency activity in auditory cortex (Barth and MacDonald 1996; Demiralp et al. 1996), including sensory (click)-evoked, long-latency gamma band activity (Franowicz and Barth 1995). Several observations support the idea that a group of neurons acting as a functional unit, an “ensemble,” must be activated to produce the slow potential and gammaband fluctuations. The slow potential appeared in an allor-none manner during stimulation near threshold intensities, and disappeared in a similar manner at stimulation rates greater than once every 10–15 s. Similarly, the administration of glutamate antagonists reduced the slow potential abruptly and completely, as if the excitation required to activate the ensemble fell below a threshold value. This interpretation implies only that the triggering excitation is glutamatergic, and does not contradict evidence that the slow potential contains GABAergic IPSPs. Of course, ensemble activity may also contain glutamatergic EPSPs, and recent findings indicate that stimulation of metabotropic glutamate receptors produces gamma-band oscillations in hippocampal networks (Whittington et al. 1995; Jefferys et al. 1996) (note, however, that fast oscillations in the hippocampus respond differently to glutamate antagonists than do the fluctuations described in the present study; thus, the cellular mechanisms underlying fast oscillations may differ in different brain regions as well as in different brain states). Since thalamocortical inputs are glutamatergic, the simplest scenario is that they trigger ensemble activity that contains intermixed glutamatergic EPSPs and GABAergic IPSPs. Relevance to cortical function The glutamatergic, thalamocortical EPSP undoubtedly underlies the short-latency response to acoustic stimuli (Volkov and Galazjuk 1991; Phillips 1993). Future studies must determine how fast EPSPs contribute to the response properties of cortical neurons. The function of the slow potential is considerably less obvious. In terms 173 of its latency, time course, and sensitivity to repetition rate and glutamate antagonists, it more closely resembles event-related potentials that contribute to the detection of novel or behaviorally significant stimuli (Ehlers et al. 1992; Rugg and Coles 1995; Javitt et al. 1996). Recent studies of sensory processing have emphasized the importance of sensory-evoked gamma-band interactions among groups of cortical neurons (Gray et al. 1989; Gray and Singer 1989; Tallon-Baudry et al. 1997). Notably, acoustic stimulation in the human elicits a focus of gamma-band activity that moves across auditory cortex in an anterior to posterior direction over a distance of 1 cm or more (Pantev et al. 1991). While the function of propagating gamma activity is unknown, it could create transient neural ensembles that encode relationships among concurrent and previous stimuli (Lisman and Idiart 1995). The present study represents an initial step towards understanding the cellular bases of such activity. Acknowledgements Thanks are due to Drs. B. Aramakis, R. Robertson, I. Soltesz, and Ms. C. Hsieh for discussions and comments on the manuscript. We would also like to thank Ms. N. Patel for the histology and B. Aramakis for the AChE histochemical data. 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