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The Journal of Neuroscience, November 15, 2000, 20(22):8462-8473
Origins and Distribution of Cholinergically Induced
Rhythms in Hippocampal Slices
Ken Shimono1, Fernando Brucher2, Richard Granger2, Gary Lynch3, and Makoto Taketani1 1 Technology Development Center, Panasonic, Cypress, California 90630, and Departments of 2 Information and Computer Science and 3 Psychiatry and Human Behavior, University of California, Irvine, California 92697
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Regional variations and substrates of high-frequency rhythmic activity induced by cholinergic stimulation were studied in hippocampal slices with 64-electrode recording arrays. (1) Carbachol triggered
waves (17.6 ± 5.7 Hz) in pyramidal regions of 75% of the slices. (2) The waves had phase shifts across the cell body layers and were substantially larger in the apical dendrites than in cell body layers or basal dendrites. (3) Continuous, two-dimensional current source density analyses indicated apical sinks associated with basal sources, lasting ~10 msec, followed by apical sources and basal sinks, lasting ~20 msec, in a repeating pattern with a period in the range of 15-25 Hz. (4) Carbachol-induced
) oscillations in deep layers of the entorhinal cortex. (5) Cholinergically elicited
Key words: current source density;
INTRODUCTION
Rhythmic activity in the hippocampus of small mammals is described as falling into three frequency bands: 4-10 Hz (
), 10-30 Hz (
, 1999
Ascending cholinergic projections promote endogenous oscillations including those in the
How cholinergically driven high-frequency rhythms affect cortical operations depends on whether they are regionally specialized and how they are produced. Carbachol-elicited oscillations are reported to originate in restricted loci in the entorhinal cortex (Dickson and Alonso, 1997
MATERIALS AND METHODS
Preparation of multielectrode array. Procedures for the preparation of the recently introduced Multi-Electrode Dish (Panasonic; MED probe) are described by Oka et al. (1999)
For sufficient adhesion of the slice to the probe, the surface of the MED probe was treated with 0.1% polyethylenimine (Sigma, St. Louis, MO; P-3143) in 25 mM borate buffer, pH 8.4, for 8 hr at room temperature. The probe surface was rinsed three times with sterile distilled water. The probe (chamber) was then filled with DMEM and F-12 mixed medium, containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD; 16141-079) and 10% horse serum (Life Technologies; 16050-122), for at least 1 hr at 37°C. DMEM and F-12 mixed medium is a 1:1 mixture of DMEM and Ham's F-12 (Life Technologies; D/F-12 medium, 12400-024), supplemented with N2 supplement (Life Technologies; 17502-014) and hydrocortisone (20 nM; Sigma; H0888).
Preparation of hippocampal slices. A 17- to 24-d-old Sprague Dawley rat was killed by decapitation after anesthesia with halothane (2-bromo-2-chloro-1,1,1-trifluoroethane; Sigma; B4388), and the whole brain was removed carefully. The brain was immediately soaked for ~2 min in ice-cold, oxygenated preparation buffer of the following composition (in mM): 124 NaCl, 26 NaHCO3, 10 glucose, 3 KCl, 1.25 NaH2PO4, 2 CaCl2, and 2 MgSO4. Appropriate portions of the brain were trimmed and placed on the ice-cold stage of a vibrating tissue slicer (Leica, Nussloch, Germany; VT-1000S). The stage was immediately filled with both oxygenated and frozen preparation buffers. The thickness of each tissue slice was 350 µm. Each slice was gently taken off the blade by a painting brush, trimmed, and immediately soaked in the oxygenated preparation buffer for 1 hr at room temperature. Then a slice was placed on the center of the MED probe. The slice was positioned to cover the 8 × 8 array. After positioning the slice, the MED probe was immediately placed in a box filled with 95% O2 and 5% CO2 and allowed to recover at 32°C for 1 hr.
Electrophysiological recording. During electrophysiological recording, the slices on the MED probe were placed in a small CO2 incubator (Asahi Lifescience; model 4020) at 32°C. After recovery of the slice on the MED probe, the medium was replaced with DMEM and F-12 mixed medium without serum. The slices were on the interface, and a moisturized 95% O2 and 5% CO2 gas mixture was blown from above. In this condition, the responses were recorded for >2 hr.
Drugs were purchased from Research Biochemicals (Natick, MA; diazepam, bicuculline, CNQX, and 2-hydroxysaclofen) or Sigma (all other compounds). All drugs were bath applied at known concentrations and were prepared daily from frozen aliquots.
Spontaneous and evoked field potentials at all 64 sites were recorded simultaneously with the multichannel recording system (Panasonic; MED64 system) at a 20 kHz sampling rate. In the case of the evoked response, one of the planar microelectrodes out of the 64 available was used for the stimulating cathode. Bipolar constant-current pulses (10-50 µA; 0.1 msec) were produced by the data acquisition software via the isolator. The stimulating microelectrode was selected by the 64-switch box.
Current source density analysis. The well studied methods of current source density (CSD) analysis use the Laplacian transform (
2) on measured field potentials (
) to attempt to identify the locations and relative magnitudes of current sources and sinks (Im) (for review, see Howland et al., 1955
where
is the conductivity in each of the three orthogonal dimensions. The method is rarely used in its full three-dimensional form for electrophysiological measures (Nicholson, 1973
4 1, and 0 1 0) to produce a discrete approximation of the second spatial derivative. The medium was considered ohmic with a homogeneous conductance. The full correlation matrix was computed for all channels (64) in the time window considered (0-3 sec). Current-versus-time plots for single points in the slice were obtained by computing the 8 × 8 current source density for each time step and calculating the value at the desired location via bilinear interpolation. Well recognized limitations on the resolution affordable by current source density analysis arise from the relationship between the interelectrode distance (sampling resolution) and the radii of current sources or sinks occurring in the slice. The phenomenon of "aliasing," well known in the realm of computer graphics interfaces, refers to the occurrence of spurious data "ghosts" (aliases) when the distance between sampling points is larger than the size of the smallest phenomena to be represented. In computer screen graphics this occurs when the screen resolution is insufficient for the image being displayed (Fig. 1 left, middle). The appearance of spurious images on the screen is typically treated by "antialiasing" that incorporates low-pass filtering of the image with introduction of partially shaded pixels at the image's edges (Fig. 1, right). The present analysis uses low-pass filtering without full antialiasing; if the data contain phenomena with spatial frequencies too high to be resolved by the interelectrode sampling distances, the result is low-pass filtered to remove spurious aliases while also removing high-frequency data (Fig. 2).
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Figure 1. Aliases and antialiasing. Left, A high-resolution image with smooth edges is shown. Middle, The same image sampled with relatively low-resolution interpixel distance appears to have new features with high spatial frequency (the jagged stair steps). These "aliases" are introduced when the distance between adjacent sensors (pixels in this case) is insufficient to render the high-resolution image. Right, The same image sensed with the low-resolution pixels of the middle panel and antialiased is shown. Antialiasing includes a low-pass filter that removes the spurious alias features from the image and therefore may also (as in the present instance) eliminate some high-spatial frequency detail from the original image. The corresponding potential introduction of artifacts into array-sampled physiological data can be removed by the same antialiasing method (see Fig. 2 legend below) and will correspondingly eliminate some fine (high-spatial frequency) detail from the data.
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Figure 2. Effects of antialiasing. Top, An instance of intersensor spacing sufficient for the resolution of the sampled data is shown. Aliases appear as very high-frequency features; low-pass filtering at one-half the intersensor spatial frequency (double the intersensor distance) eliminates all aliases without reducing resolution of the sampled data. Bottom, Intersensor spacing insufficient for the resolution of the sampled data is shown. Spatial frequencies of aliases overlap the highest frequencies present in the data (black region). Left, Low-pass filtering at one-half the intersensor spatial frequency will fail to eliminate all the aliases and yet will eliminate some of the finest detail of the data. Right, Low-pass filtering at a lower spatial frequency successfully eliminates all spurious aliases and further reduces the resolution of the sampled data, eliminating much of the high-frequency fine detail that may be present in the measured data. Only lower frequency (larger) events remain.
RESULTS
Continuous, two-dimensional current source density analysis
Two-dimensional current source density analyses were conducted in the context of stimulation of the Schaffer-commissural afferents to field CA1 in a hippocampal slice preparation. Figure 3 illustrates a typical experiment. To prevent the GABAA- and GABAB-mediated inhibitory components, this experiment was performed with 50 µM picrotoxin and 100 µM 2-hydroxysaclofen. Figure 3A shows the placement of the slice on the electrode array (interelectrode spacing, 150 µm). Figure 3B shows a typical postsynaptic current elicited by a single stimulation pulse to the electrode indicated in white and measured from the electrode in gray. The figure illustrates the time course and magnitude of the postsynaptic current at the indicated electrode, which is in the proximal portion of the apical dendrites of CA1. The direction of the current sink and source is as indicated; after the initial fiber volley (lasting ~1 msec) a current sink increases over a period of ~5 msec and decreases over the subsequent 5 msec before returning to baseline at ~13 msec. It becomes a current source and lasts for approximately another 15-20 msec before returning to baseline at ~35-40 msec. The relative magnitudes and time courses of the sink and source are typical for such evoked responses.
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Figure 3. Measurement of evoked current. A, Placement of an 8 × 8 MED electrode array, with interelectrode spacing of 150 µm, centered in the apical dendritic field of CA1 in a hippocampal slice. The electrodes cover the basal dendrites of CA1, the apical dendrites of CA1, and the upper blade of the dentate gyrus granule cell field. B, EPSC elicited by a single pulse to the electrode marked in white in A and measured at the electrode in gray in A. To block GABA-mediated inhibitory components, 50 µM picrotoxin and 100 µM 2-hydroxysaclofen were applied. The time scale is typical for such evoked responses. Vertical dashed lines mark the time points for which measures will be taken across all 64 electrodes (see Fig. 4).
Two-dimensional current source density analysis was performed to obtain comparable measures of current sources and sinks of the same response. Figure 4 illustrates the continuous two-dimensional current source density analysis of the evoked response shown in Figure 3B. Each panel in Figure 4 illustrates the computed instantaneous sources and sinks in the slice, at selected times indicated by vertical dashed lines in Figure 3B. The array is positioned on the slice as shown in Figure 3A; the cell body layer of hippocampal field CA1 defines a horizontal curve approximately one-quarter of the way from the top of each panel in Figure 4. The dashed lines indicate the cell body layer of CA1 (top) and the boundary between stratum radiatum and stratum lacunosum/moleculare (bottom). Sinks are blue and sources are yellow, against a neutral background of black. It can be seen that after the initial fiber volley (lasting ~1 msec) a current sink in the apical dendrites of CA1 rises over a period of ~5 msec (see Figure 4, 6 msec panel) and falls over the subsequent 5 msec before disappearing at approximately the 11 msec panel. After an interim of ~2 msec during which currents are indistinguishable from neutral background, a source appears in the apical dendrites (Figure 4, 15 msec panel) and lasts for approximately another 15-20 msec before disappearing at ~35 msec. The time courses and magnitudes of the waveform from a single electrode (Fig. 3B) can be seen to correspond closely to the computed current source density sink-source series in Figure 4.
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Figure 4. Continuous two-dimensional current source density analysis of evoked response. Shown are the computed instantaneous two-dimensional current source density plots across all electrodes at each of the time points indicated in Figure 3B by vertical dashed lines. At the bottom right of each panel are the times in milliseconds after the application of a pulse to the white electrode of Figure 3A. As shown in the horizontal bar at the bottom right, sinks are blue and sources are yellow against a current-neutral background of black. The limits of the stratum radiatum in CA1 are indicated by horizontal dashed lines. The stimulating electrode cannot be recorded from, resulting in a singularity that is visible in the sink field in approximately the top middle of each frame. After an initial response because of the fiber volley, a current sink spreads rapidly through the dendritic zone of field CA1 (with the singularity appearing at the site of stimulation). The sink intensifies and expands over ~5 msec and then fades, disappearing at ~10 msec. After a brief pause, a current source appears in these dendrites, with its center slightly more distal than that of the current sink. The apical source intensifies, expands, dissipates, and disappears by ~35 msec, making the "period" of the evoked response ~35 msec. Both the apical sink and ensuing apical source are accompanied by a field of reversed polarity appearing in the basal dendrites of CA1 (top of each panel) that grows and dissipates with approximately the same time course as the apical events.
Revealed by the two-dimensional current source density method are spatial aspects of the current sources and sinks that are difficult to discern by other means. The EPSC sink spreads across the apical field of CA1, in the region of Schaffer-commissural fibers presumably stimulated by the initiating current pulse (Fig. 4, 3-9 msec panels); the ensuing current source occupies approximately the same zone (Fig. 4, 15-27 msec panels). Both the sink and subsequent source occurring in the apical dendrites are accompanied by currents of reversed polarity in the basal dendritic field of CA1 (Fig. 4, near the top of each panel). Thus the predominant evoked response can be characterized as a current sink-source dipole that occurs from 3 to 9 msec and reverses to form a current source-sink dipole from 15 to 27 msec. Other, smaller currents are present in the slice but are not discussed here.
As described, the antialiasing performed in these analyses to prevent the introduction of artifacts attributable to aliasing will cause the loss of data with sufficiently high spatial frequencies. The filtering passes only those phenomena with spatial frequencies at most one-half that of the sampling frequency; in the present instance the interelectrode distance was 150 µm, preventing the measurement of current sources or sinks smaller than ~300 µm across (150 µm radius). This could be a redoubtable shortcoming in a structure the size of hippocampal field CA1, and it is expected that fine spatial detail will be lost in the method. It is instructive, therefore, to examine the spatial resolution of the CSDs measured by this method. Figure 5 depicts the boundaries of the apical dendrites of field CA1 superimposed on the region of measurement. Figure 5, left, shows the slice and the position of the array (from Fig. 3); Figure 5, right, is the instantaneous CSD in the region of the electrode array (from Fig. 4), at 6 msec after stimulation at the indicated electrode. The dashed lines (Fig. 5, right) indicate the cell body layer of CA1 (top) and the boundary between stratum radiatum and stratum lacunosum/moleculare (bottom). The primary measured current sink (blue) occupies only the region where apical dendrites occur, and the reciprocal current source (yellow; top) occurs only in the region of basal dendrites. (An absence of current appears in the computed image at the location corresponding to the stimulating electrode; no recordings were taken from that electrode, and the resulting CSD processing leaves a gap.) It can be seen that the borders of the computed sink do not coincide precisely with the location of the anatomical limits of the dendritic field, inaccuracies that may result from the resolution limits of the method.
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Figure 5. Alignment of computed physiological phenomena with known anatomical structure. Left, Placement of the electrode array on the hippocampal slice (close-up of Fig. 3A). The top of the array coincides with the basal dendrites of CA1, the top-middle portion of the array overlays the field of apical dendrites in CA1, and the bottom third of the array overlays the upper blade of the dentate gyrus. Right, The physiological response computed by continuous two-dimensional current source density analysis 6 msec after the stimulation of a single electrode (indicated in blue). The image is a close-up of the 6 msec frame from Figure 4. The limits of the stratum radiatum in CA1 are indicated by dashed lines. It can be seen that the extent of the evoked current sink closely corresponds to the limits of the apical dendrites. An apparent hole in the current sink occurs at the site of the stimulating electrode, where no recording is performed and no current source density is computed. There is little current in the cell body layer itself, and current source appears in the basal dendrites (top of panel). There is little current at the apical tips of the CA1 dendrites (middle of panel) and some less intense current sources occurring in the stratum lacunosum/moleculare.
Distribution and current sources for carbachol-induced
Figure 6A contains a micrograph of a hippocampal slice and underlying 64-electrode array with 450 µm between recording positions (the "broad array"). Baseline activity was of low voltage and generally devoid of synchronous activity, and there were no reliable differences between subfields (Fig. 6C). Infusion of carbachol was followed by a gradually developing rhythmic activity in the pyramidal cell fields that was close to or within the
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Figure 6. Distribution of carbachol-induced 11 V2; C, D, 0.1 mV, 500 msec; E, 0.1 mV, 200 msec.
Fast Fourier transforms were used for quantitative analysis of the frequency and distribution of the carbachol-induced rhythms. For the example illustrated in Figure 6B, most of the power in the spectrum was found between 10 and 30 Hz with a definite peak at 20 Hz. The group mean for the dominant CA1 frequency for all slices was 18.8 ± 5.7 Hz (mean ± SD) and 19.2 ± 4.8 Hz for the
Arrays with 150 µm spacing (the "dense array"; Fig. 7A) provided finer spatial resolution of the rhythmic activity. Recording centered on the CA3-CA1 border revealed a surprisingly steep gradient of absolute potentials in the distal-to-proximal dimension with the largest potentials occupying a discrete region corresponding to the stratum moleculare of fields CA3a and b (Fig. 7B). It is noteworthy that potentials reversed across the cell layer boundary (see, e.g., Fig. 7B, top left quadrant). Lesser although still substantial voltage bands are also present in CA1. The frequency spectra for all channels are shown in Figure 7C. All the observed oscillatory activity was absent in the presence of blockers of either glutamate or GABA.
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Figure 7. Carbachol-induced
Continuous two-dimensional current source density analyses were conducted to define further the sources of these cholinergically induced oscillations. Figure 8 illustrates the instantaneous sources (yellow) and sinks (blue; against a neutral background of black), at 4 msec intervals from an arbitrarily chosen starting time of 0, in the same slice and during the same spontaneous activity shown in Figure 7. The outlines of the granule and pyramidal cell fields and the extents of their apical dendrites have been superimposed to illustrate the locations of events occurring during the time period. A large sink in apical CA1 is accompanied by a source in the basal dendrites of CA1. The sink and its accompanying basal source intensify and expand over the course of ~12 msec, and both subside to approximate baseline levels by ~16 msec after initial appearance. After a brief period characterized by currents that are barely distinguishable from background, a source arises in apical CA1, accompanied by a sink in the basal dendrites of CA1. The source (and its accompanying dipole) lasts for ~20 msec before dissipating. It is noteworthy that the apical source appears to have its center more distally located than that of the more focal apical sink preceding it, to within the resolving power of the interelectrode spacing of 150 µm. The sink-source dipole recurred and gave rise to the frequency plot shown in Figure 7; the time course from peak to peak of this recurring apical sink-source dipole was ~40 msec, consistent with a frequency of ~25 Hz. The source-sink and sink-source dipoles and their sharply defined boundaries between sources and sinks along the pyramidal cell body layer were recurring features across many experiments, as was the latency from one apical sink through an apical source to the next apical sink, as seen in Figure 9, which shows a typical record of the repetitive oscillations induced by carbachol in a slice. Shown are the averaged sinks and sources occurring in the apical dendrites, as measured from the electrodes marked in blue, and in the basal dendrites (red electrodes). The apical versus basal oscillations retain a relationship of opposite polarity throughout the measured period, and the frequency of the observed oscillations remains relatively stable at 23.1 ± 0.8 Hz across multiple 1 sec samples. The sustained occurrence of alternating current source-sink dipoles across the cell body layers of CA3 and CA1, within the range of 10-30 Hz (
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Figure 8. Current source density analyses of carbachol-induced activity. In the same slice as in Figure 7, the outlines of the pyramidal and granule cell fields and their apical dendrites are shown. The array includes parts of the pyramidal cell fields of CA3 and CA1 and their apical dendrites and part of the upper blade of the dentate gyrus. Each frame shows the instantaneous computed current source density in the region of the electrode array. From an arbitrarily chosen starting point, a sink appears in the apical dendrites of the border between fields CA3 and CA1, with an associated source across the cell boundary layer in the basal dendrites. Within a few milliseconds, an additional focal sink has appeared in apical CA1 with a corresponding basal source. The fields merge and intensify and then dissipate after ~12 msec. After a brief interim during which activity is not distinguished from background, a source appears in the apical dendrites at ~20 msec, with a corresponding sink in the basal dendrites. These expand and intensify before dissipating by ~20 msec later (40 msec), after which an apical sink reappears to reinitiate the cycle. The cycle repeats indefinitely (as indicated in the next figure), with an approximate frequency of 25 Hz.
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Figure 9. Relationship of recurring carbachol-induced oscillations in apical and basal dendrites. Left, Hippocampal slice on a broad (450 µm interelectrode spacing) array. The electrodes used to measure basal dendritic responses of pyramidal cell fields are indicated in red; those used to measure apical dendritic responses are in blue. Right, Averaged responses from apical (blue) and basal (red) dendritic fields shown over 500 msec (0.5 sec) of elapsed time. Carbachol-induced (20 µM) oscillations were sustained across all sampled periods, as in this typical response. Average apical and basal responses are reversed in polarity, i.e., are 180° out-of-phase. The frequency of the waves is 23.1 ± 0.8 Hz (mean ± SE).
Shifts between sinks and sources over larger areas were assessed with continuous two-dimensional current source density analyses using an electrode array with interelectrode spacing of 450 µm, as opposed to all previous current source density examples that used arrays of 150 µm distances. The resulting aliases and antialiasing (see Fig. 2) necessarily entail further loss of resolution; for an interelectrode spacing of 450 µm and concomitant antialiasing, the smallest events that can reliably be imaged are those with a radius of 450 µm (diameter of 900 µm) or larger. Figure 10 shows a typical cycle. At arbitrarily chosen time 0 msec, a weak current source in the apical dendrites of field CA3 is surrounded by diffuse sources centered on the pyramidal cell layers. Six milliseconds later a discrete but intense sink develops in the proximal st. radiatum of CA3 that is accompanied by sources in the basal dendrites of CA3. The sink grows, and a fully developed sink-source relationship centered on CA3b is in evidence at the 9 msec time point (Fig. 10). The sink then extends into the st. radiatum of CA1 accompanied by the appearance of a pronounced cell body/basal dendrite source at 15 msec. Note that by this time point the sink-source relationship in CA3 has begun to collapse. By 24 msec a pronounced current source has developed in the distal dendrites of field CA3 that grows in intensity and begins to extend into the distal dendrites of field CA1 (see Fig. 10, 30 msec time point). This source is reasonably stable and occupies much of the distal apical dendrites for the following 10 msec (see Fig. 10, 39 msec time point) and returns to the starting pattern at 42-45 msec. In all then, the wave involves an intense apical dendritic sink lasting ~10 msec followed by an apical source lasting for approximately twice that period. These events begin in CA3 and are seen in CA1 within 3-5 msec. Data of a similar type from the entorhinal-hippocampal system suggest that propagation of activity involves interactions between reverberating circuits (Iijima et al., 1996
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Figure 10. Evolution of current source density over time (42 msec). Each frame shows the computed current source density at a particular millisecond (indicated at the top left of the frame) during the recording of spontaneous activity with a broad (450 µm spacing) array. Blue indicates the maximum magnitude of sinks; yellow indicates the maximum magnitude of sources. The positions of the pyramidal and granule cell fields are indicated by dashed lines. Beginning at arbitrarily chosen time 0, an intense focal sink in the apical dendrites of field CA3 begins at ~6 msec, accompanied by sources in the cell and basal dendrite layers of CA3. The sink continues through ~12 msec before beginning to expand toward field CA1. The sink in apical CA3 dissipates at ~18 msec, followed ~3 msec later by the apical sink in CA1. The basal sources dissipate at approximately the same time as their associated apical sinks. An apical source begins in CA3 at ~24 msec, accompanied within a few milliseconds by a mild basal source in CA3; the apical source then expands toward CA1, with an accompanying basal sink in CA1. These source-sink pairs then dissipate after ~15-20 msec. Such cycles recur irregularly in this slice. (This series of frames and other related series can be viewed at http://www.med64.com/publications.htm.)
These observations show that relatively brief apical sinks are interposed between the longer lasting apical sources. Examination of averaged currents demonstrated that distribution of sources and sinks across the pyramidal cell subdivisions of the hippocampus varied between slices. Between-slice variability was also present in serial current source density analysis with major differences in the degree to which events were centered in CA3 versus CA1. However, the brief apical sink-longer apical source sequence for the apical dendrites was prominent in all slices exhibiting oscillations in the
Carbachol induces a 40 Hz rhythm in retrohippocampal cortex
The question of whether the rhythms elicited by carbachol are regionally differentiated was addressed by close examination of those slices in which the electrode array was positioned underneath a significant portion of the retrohippocampal cortex field along with the pyramidal cell fields of the hippocampus itself. Figure 11A shows a case in which the broad array (450 µm spacing) had a row underneath the deep layer of the entorhinal cortex. Carbachol-induced fast waves from the indicated positions in field CA1 and entorhinal cortex are compared in Figure 11B. As can be seen, the entorhinal oscillations have a higher frequency than do those from the hippocampus. Fast Fourier transforms indicate that the dominant frequency in the cortex is approximately twice that in field CA1. Results for all recording sites are summarized in Figure 11C. Rhythmic activity with a peak near 20 Hz is found via the apical dendrites of the hippocampal pyramidal cell fields, whereas 40 Hz activity is predominant at the entorhinal sites. It can also be seen that the 40 Hz oscillations are centered in the deep layers of the medial entorhinal cortex. Regions lying between the hippocampus and medial entorhinal cortex have both peaks. Indeed, it appears that the relative balance of 40 versus 20 Hz increases in an orderly manner across the series of steps included in the retrohippocampal cortex. Forty Hertz activity was recorded in the deep layers of the entorhinal cortex in each of the 15 slices that had appropriately positioned electrodes; it thus appears to be a characteristic response of this region to cholinergic stimulation. This is not unexpected considering that carbachol was reported to induce 40 Hz oscillations in the medial entorhinal cortex of isolated guinea pig brain (van der Linden et al., 1999
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Figure 11. Two distinct carbachol-induced rhythms in the hippocampus and cortex. A, Micrograph of a corticohippocampal slice on a broad array. Two sites are indicated in the apical dendrites of field CA1 (dashed circle) and the deep layers of entorhinal cortex (solid circle). B, Sample activity in response to the infusion of carbachol (50 µM) from the selected electrodes in entorhinal cortex and field CA1 (left) and power spectra for recordings over 3 sec at these two sites (right). Power values are given as × 10
Transmitter systems involved in carbachol-induced
Carbachol-induced high-frequency rhythms were completely eliminated by 20 µM atropine and greatly reduced by the AMPA receptor antagonist CNQX (20 µM). In the presence of bicuculline (10 µM), muscarinic stimulation produced high-frequency spiking in the stratum pyramidale and in some instances epileptiform discharges, but rhythmic activity was absent. Under these conditions, carbachol initiated repetitive bursting behavior and occasional seizures. The interval between the epileptiform bursts sometimes approximated the period of the
Benzodiazepines markedly enhanced the amplitude of
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Figure 12. Effect of benzodiazepines on carbachol-induced
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Table 1. Summary of eight experiments in which carbachol-induced high-frequency rhythms were present in a hippocampal slice before infusion of diazepam Orthodromic activation of pyramidal cells triggers
How the GABAergic cells responsible for apical sources are activated is clearly of importance for understanding the origins of
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Figure 13. Two-dimensional current source density analysis of
DISCUSSION
Simultaneous recording from a large number of sites revealed that cholinergic stimulation triggers
It is well established that carbachol blocks IPSPs, most probably by suppressing release (Behrends and Bruggencate, 1993
The dendrites of the apically directed interneurons are well situated to receive collaterals from pyramidal neurons, and it is reasonable to assume that the two collections of cells form a negative feedback circuit. Cycling activity would require tonic excitation of the pyramidal neurons. Cholinergic stimulation blocks potassium currents (Nakajima et al., 1986
A key requirement of the above model is that spiking of pyramidal cells triggers a current source in the apical dendrites with temporal characteristics appropriate for the
The above-described distribution of current sources responsible for
Finally, it should be noted that although carbachol induced rhythmic activity in the
FOOTNOTES Received Feb. 16, 2000; revised Aug. 23, 2000; accepted Aug. 29, 2000.
This work was supported in part by Grants N00014-98-1-0825 and N00014-98-1-0609 from the Defense Advanced Research Projects Agency and the Office of Naval Research. We thank Cheryl Cotman for her assistance with the current source density color maps and Laura Colgin for helpful discussions.
Correspondence should be addressed to Dr. Gary Lynch, Psychiatry Department, University of California, Irvine, CA 92697. E-mail: glynch{at}uci.edu.
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