Neuroscience 141 (2006) 1665–1677

DIFFERENTIAL BEHAVIORAL STATE-DEPENDENCE IN THE BURST PROPERTIES OF CA3 AND CA1 NEURONS

J. TROPP SNEIDER,a,b J. J. CHROBAK,a M. C. QUIRK,c the processes information one needs to ex- J. A. OLERa,d AND E. J. MARKUSa* amine the manner in which hippocampal neurons respond aBehavioral Neuroscience Division, Department of Psychology, Uni- to hippocampal and extra-hippocampal inputs. In addition versity of Connecticut, 406 Babbidge Road, Box U-20, Storrs, CT to location-specific discharge (O’Keefe and Dostrovsky, 06269, USA 1971), CA1 and CA3 pyramidal “place cells” can fire single bCognitive Neuroimaging Laboratory, McLean Hospital, 115 Mill spikes or a fast series of spikes commonly referred to as a Street, Belmont, MA 02478, USA burst. Typically bursts contain two to six spikes at short c Cold Spring Harbor Laboratories, 1 Bungtown Road, Cold Spring intervals, with a progressive attenuation in the amplitude of Harbor, NY 11724, USA the spikes within the burst (Kandel and Spencer, 1961; d Waiman Laboratory for Brain Imaging and Behavior, University of Ranck, 1973; Quirk and Wilson, 1999; Quirk et al., 2001). Wisconsin-Madison, 1500 Highland Avenue, Madison, WI 53705, USA Both the burst and attenuation phenomena have been postulated to play an important role in information process- Abstract—Brief bursts of fast high-frequency action poten- ing (Buzsáki et al., 1996; Lisman, 1997; Quirk et al., 2001; tials are a signature characteristic of CA3 and CA1 pyramidal Harris et al., 2002). Hippocampal bursts are often as- neurons. Understanding the factors determining burst and sumed to have distinct physiological (Pike et al., 1999; single spiking is potentially significant for sensory represen- Buzsáki et al., 2002) and/or computational functions tation, synaptic plasticity and epileptogenesis. A variety of models suggest distinct functional roles for burst discharge, (Lisman, 1997; Kepecs et al., 2002). Thus, bursts may and for specific characteristics of the burst in . enhance plasticity at both the pre-synaptic inputs that ini- However, little in vivo data demonstrate how often and under tiate a burst (Magee and Johnston, 1997; Pike et al., 1999) what conditions CA3 and CA1 actually exhibit burst and sin- as well as cause supra-linear summation of excitatory gle spike discharges. postsynaptic potentials (EPSPs) at postsynaptic targets The present study examined burst discharge and single that would enhance postsynaptic potentiation (Cousens spiking of CA3 and CA1 neurons across distinct behavioral states (awake-immobility and maze-running) in rats. In both and Otto, 1998; Williams and Stuart, 1999; Thompson, CA3 and CA1 spike bursts accounted for less than 20% of all 2000). Further the distinct temporal characteristics of a spike events. CA3 neurons exhibited more spikes per burst, burst in contrast to a single spike may serve unique com- greater spike frequency, larger amplitude spikes and more putational (neural coding) significance. Burst duration, the spike amplitude attenuation than CA1 neurons. number of spikes per burst, or burst frequency could each A major finding of the present study is that the propensity serve specific coding functions (Harris et al., 2001; Kepecs of CA1 neurons to burst was affected by behavioral state, while the propensity of CA3 to burst was not. CA1 neurons et al., 2002). exhibited fewer bursts during maze running compared with The functional significance of CA3 and CA1 bursts awake-immobility. In contrast, there were no differences in however is not clear. Harris and colleagues (2001) ana- burst discharge of CA3 neurons. Neurons in both subregions lyzed the relationship between single spikes and bursts in exhibited smaller spike amplitude, fewer spikes per burst, CA1 during locomotor activity (theta) and slow wave sleep. longer inter-spike intervals and greater spike amplitude at- CA1 bursting conveyed no unique information about spa- tenuation within a burst during awake-immobility compared with maze running. These findings demonstrate that the CA1 tial position as compared with single spikes, and the pro- network is under greater behavioral state-dependent regula- pensity to burst was most dependent on prior periods of tion than CA3. The present findings should inform both the- neuronal silence during both behavioral states (Harris oretic and computational models of CA3 and CA1 function. et al., 2001). © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Both CA1 and CA3 pyramidal neurons exhibit burst discharges during locomotion associated with hip- Key words: hippocampus, place cell, theta, , plasticity, epilepsy. pocampal theta, as well as during immobility and slow- wave sleep when the hippocampus exhibits sharp wave bursts (Buzsáki, 1989). Data however are quite limited The plays an important role in spa- about differences in the burst characteristics across be- tial navigation and the formation of certain types of mem- havioral states. Are there unique characteristics of the ories (Scoville and Milner, 1957; O’Keefe and Nadel, 1978; burst associated with distinct behavioral states? Further, Sutherland and Rudy, 1989; Cohen and Eichenbaum, since CA3 and CA1 pyramidal neurons have distinct cel- 1993; Vargha-Khadem et al., 1997). To understand how lular properties and receive quite distinct inputs (Fig. 1), *Corresponding author. Tel: ϩ1-860-486-4588; fax: ϩ1-860-486-2760. does this lead to differences in their bursts characteristics E-mail address: [email protected] (E. J. Markus). as a function of behavior? 0306-4522/06$30.00ϩ0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.05.052

1665 1666 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

CA1

EC V-VI Output to Neocortex

EC III Input from EC II Neocortex

DG

CA3

Fig. 1. The organization of EC inputs to the main subfields of the dorsal HPC (DG, CA3 and CA1) and the interconnections among these subfields. The principal glutamatergic drive onto CA3 pyramids (large pyramids) arises from the direct layer II input, the mossy fiber input from the DG and a massive recurrent input from CA3. The principal glutamatergic drive onto CA1 neurons arises from the direct layer III input and the Schaffer collateral input from CA3. Each major afferent input is associated with different classes of GABAergic neurons (shown as smaller circles innervating somatic or dendritic compartments) that can be activated in a feed-forward manner and which regulate bursting in somatodendritic compartments. The excitatory inputs to the GABAergic interneurons are illustrated to the somatic compartment for illustration purposes only. The bursting capacity of CA3 and CA1 pyramids is regulated by intrinsic membrane currents that can be altered by the dynamic interplay of glutamatergic and GABAergic input. Additional glutamatergic input to the DG, CA3 and CA1 arises from the amygdala and the nucleus reuniens of the thalamus. These inputs are largely absent from the dorsal hippocampus pyramids recorded in the present study. CA3 and CA1 neurons also receive a number of subcortical neuromodulatory inputs from the medial septum, hypothalamic nuclei and brain stem nuclei.

The present study examined differences in bursting pre-trained to alternate on the “U” shaped runway (Fig. 2) for food properties of CA1 and CA3 neurons in awake-behaving reinforcement (Noyes pellets; Research Diets, Inc., New Bruns- rats. In an effort to inform theoretic discussion of hip- wick, NJ, USA) on an automated system (custom software A. Kuzin, University of Connecticut, Stoyes, CT, USA). The rats were pocampal bursting and its role in sensory representation, given a 30-minute session each day to learn to alternate in the and epileptogenesis, the present apparatus (from Feeder A to Feeder B). The animals were trained report details the similarities and differences in the burst until they reached a criterion of 80 alternations for at least 4 days discharge of CA3 and CA1 neurons during distinct behav- of training. ioral states (awake-immobility and maze performance). Surgery

EXPERIMENTAL PROCEDURES After reaching criterion levels of performance, the animals re- ceived surgical implantation of an electrode microdrive for single Subjects unit recordings. Animals were anesthetized with a 4 ml/kg dose Thirteen female Sprague–Dawley rats (approximately 6–8 months of age; Harlan, Indianapolis, IN, USA) were used in this experiment. This research was approved by the University of Connecticut Institutional Animal Care and Use Committee and conformed to all American Psychological Association ethical stan- dards for the treatment and care of animals. The experimental procedures included measures to reduce the number of animals used and their suffering. Rats were singly housed in transparent plastic tubs, in a room with a 12-h light/dark cycle. All animals were weighed daily and extensively handled before any behav- ioral training. The recordings were conducted across the estrous cycle of these rats. No effects of the cycle were found on the burst firing characteristics of the cells (Tropp et al., 2005).

Apparatus and training procedure

All animals were mildly food deprived to 90–95% of their ad libitum body weights (Tropp and Markus, 2001). The testing environment consisted of a small room (2.1 mϫ2.1 m), which was dimly lit by lights from an open doorway. A “U” shaped runway was located in Fig. 2. Behavioral apparatus and training procedure. Animals were the center of the room and was raised 96 cm off the floor. The trained to alternate (from Feeder A to Feeder B) on a “U” shaped runway consisted of three black Plexiglas arms (widthϭ10 cm). runway to receive a food reward. The animal made a total of 40 The total path length of the runway was 228 cm. Rats were alternations during a recording session usually within 15 min. J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677 1667

(i.m.) of ketamine cocktail, consisting of ketamine (12.4 mg/ml), holder and running on the maze. A burst was defined as consec- acepromazine (0.1 mg/ml), and xylazine (1.27 mg/ml). Each ani- utive spikes with an interspike interval of less than 10 ms (see mal was placed in a stereotaxic apparatus (ASI Instruments, Ranck, 1973; Quirk and Wilson, 1999; Quirk et al., 2001). This Warren, MI, USA) and implanted with a miniature microdrive criterion for bursts was used for all cells recorded on the holder (O’Keefe and Recce, 1993; Wilson and McNaughton, 1993; Oler and the maze and for both CA1 and CA3 cells. Burst properties and Markus, 2000) containing four independently movable tetrode examined included: the average number of spikes in a burst, recording probes. Two small holes were drilled into the skull mean burst duration (ms), “burstiness” (number of burst spikes bilaterally over the dorsal hippocampus (3.2 mm posterior to divided by total spikes), ratio of burst events to single spike bregma and 2.2 mm lateral from the midline). The microdrive events, interspike interval (ms), amplitude of the first spike in a device was mounted onto the skull by small anchor screws and burst (millivolts), and amplitude of a single spike event (millivolts). dental acrylic. Each tetrode was composed of four twisted poly- Comparison of CA1 and CA3 subregion firing characteristics was imide-insulated, 14 ␮m nichrome wire (H. P. Ried, Palm Coast, conducted using a two-way ANOVA with the focus on the main FL, USA). The distance between the wire tips was approximately effect of subregion. 10 ␮m, such that local neuronal activity was reflected as a slightly To examine behavioral effects, and interactions between sub- different signal on each wire. A comparison of the recording signal region and behavior, a within cell repeated measures analysis was from each electrode allowed for a differentiation of the firing of one used. This was done only for recordings in which it was verified cell from another. The tips of the tetrode were gold-plated with an that the same cell was recorded while the animal was at rest and impedance of approximately 300–500 k⍀. After surgery, all ani- running on the maze (based on amplitude profile across the four mals were given an injection (i.m.) of a 0.25 ml/kg dose of Bu- tetrode channels; Tropp et al., 2005). In addition, the data were prenex (0.03 mg/ml) (Reckitt and Colman, Richmond, VA, USA) to examined using a Pearson’s correlation (r value) and plotted in minimize pain. They were allowed several days to recover before order to identify possible patterns in the relationship between food deprivation and retraining resumed. variables (e.g. holder versus maze comparisons). Recordings Criteria for pyramidal cells. Only cells with a minimum of 100 spikes were included. While this limited the number of cells Animals wore a multi-channel head stage device (Multichannel analyzed (to cells with fields on the maze; more active cells on the Concepts, Gaithersburg, MD, USA) that contained two arrays of holder), it was needed to provide a sufficiently large sample size infrared light emitting diodes. An overhead video tracking system for statistical analysis. provided information about the rat’s location and head direction (Dragon Tracker SA-3; Boulder, CO, USA). During the recording Cells recorded on maze. To be analyzed, complex spike session, the animals were connected to the recording apparatus cells (putative pyramidal cells) recorded on the maze had to have by a multi-wire cable that was attached to a pulley system in the a mean firing rate below 2.5 Hz (Markus et al., 1994) and a valid ceiling to counterbalance the weight of the head stage. This place field (minimum of 15 adjacent 0.5 cm bins with firing rate 2 design allowed for the animal to freely move on the apparatus. SD above cell mean (Tropp et al., 2005). Cells had to have a Rack-mounted amplifiers (Assembly Hunter: Neuralynx, Tucson, minimum of 100 spikes to be included in the analysis. In addition, AZ, USA) were used to detect the signals from the head stage. maze data were collected only if the animal moved faster than The signal was amplified 5000 times or less and filtered between 2.0 cm/s. This allowed for the analysis of maze data only when the 300 Hz and 6 kHz, which was sent to a PC-based analog to digital rat was moving (i.e. in theta) and not in other states (eating, signal-capture board (Data Translation, Marlboro, MA, USA). Fir- grooming, resting or other sharp wave states). ing and positional data were time-stamped by a synchronization Cells recorded on holder. Complex spike cells recorded at clock board (ComputerBoards, Mansfield, MA, USA). A 1.0 ms rest on the holder could not provide place field information. To sample of data was acquired at a rate of 25 kHz. An analysis of the differentiate them from interneurons, they were classified by a multi-single unit recordings from each probe was conducted off- spike width Ն288 ␮s (mean spike widthϭ355.28Ϯ4.69) and a line using a spike parameter clustering method (McNaughton et minimum of 100 spikes. If the cell was also recorded on the maze al., 1989; Mizumori et al., 1989; Markus et al., 1994)onaSun then the firing rate and place field criteria were also included. Blade 100 computer workstation (Sun Microsystems Inc., Palo Alto, CA, USA). The clustering was based on the relative ampli- Spike amplitude attenuation. A general feature of extracel- tudes of the signals and the spike durations (Wilson and Mc- lularly recorded action potentials of hippocampal pyramidal cells is Naughton, 1993). Data collection and analysis were conducted an activity-dependent attenuation in amplitude (Quirk and Wilson, using software written by M. Wilson, L. Frank, and M. Quirk (MIT, 1999). Cambridge, MA, USA) and W. E. Skaggs (UC-Davis, CA, USA). This effect has been observed in pyramidal cells that display complex spike bursts (Ranck, 1973; Quirk and Wilson, 1999; Experimental procedures Quirk et al., 2001). To assess spike amplitude attenuation, we compared the amplitude of the first and the third spike for bursts After recovery from surgery, animals were re-trained on the alter- that had three spikes or more (Quirk et al., 2001). nation task described above. The electrodes were slowly lowered into the dorsal hippocampus. The average latency to complete Amplitude of Third Spike one alternation (i.e. from Feeder A to Feeder B) provided a Attenuation of the Burst ϭ behavioral index of the animals’ activity/motivational level. Amplitude of First Spike Data were collected in two stages: (1) Recordings on holder: approximately 10 minutes of re- Cells had to have a minimum of three burst episodes in order to be cordings of cells were obtained while the animal sat quietly on a included in the attenuation analysis. small platform (diameterϭ19 cm) outside the maze room. (2) Recordings on maze: recordings of cells as the animal Histology performed 40 alternations on the maze (20 in each direction). After completion of all recording sessions, animals were killed with Data analysis CO2 and perfused intracardially with a 4% paraformaldehyde so- lution. The electrodes were raised and the brains were removed Burst properties. CA1 and CA3 pyramidal cells that dis- and placed in paraformaldehyde for 48 h. Coronal sections played complex spike bursts were recorded in rats resting on the (50 ␮m) were taken using a cryostat or a vibratome. The slices 1668 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

Fig. 3. Histology. Representative example of recording electrode placement in layers CA1 and CA3 of the hippocampus. were mounted and Nissl stained and examined under a micro- Firing rate and burst characteristics of CA1 and CA3 scope (see Fig. 3). Determination of recording location was based cells independent of behavior on the histology (electrode tracks) and/or physiological markers (cell layers, sharp waves, and ripples) and/or the amount the To examine differences between CA3 and CA1 burst and electrode was advanced. It should be noted that DG recordings single spikes independent of behavioral state, a compari- (track ending in the upper blade) and very anterior tip of the son of firing characteristics for the two subregions was Ͻ hippocampus ( 2.60 mm posterior to bregma) were not included conducted. in the analysis. There was no difference in mean firing rate (Hz) between CA1 (meanϮS.E.M.ϭ1.18Ϯ0.07) and CA3 RESULTS ϭ Ϯ ϭ Ͼ (mean 1.10 0.10) cells (ANOVA F(1,113) 0.43, P 0.1). Table 1A depicts the number of cells per animal and sub- CA3 cells exhibited a greater average number of spikes in ϭ Ͻ ϭ Ϯ region recorded, broken down by holder and maze. Ani- a burst (F(1,241) 7.03, P 0.01; CA1 mean 2.33 0.02; mals took about 14 min to complete the 40 alternations on CA3 meanϭ2.41Ϯ0.03), greater amplitude of the first ϭ Ͻ ϭ the maze (Tropp et al., 2005). spike in a burst (F(1,241) 14.88, P 0.001; CA1 mean The characteristics and differences between CA3 and 0.21Ϯ0.01; CA3 meanϭ0.25Ϯ0.01), greater amplitude of ϭ Ͻ CA1 bursts and single spikes independent of state are single spike events (F(1,241) 10.87, P 0.01; CA1 presented first. This is followed by a description of the meanϭ0.196Ϯ0.01; CA3 meanϭ0.23Ϯ0.01) [see Fig. ϭ effects of behavioral state and whether and how state 4(a–c)] and shorter interspike intervals (F(1,241) 11.67, interacts with bursts within each subregion (CA3 compared PϽ0.01; CA1 meanϭ5.91Ϯ0.06; CA3 meanϭ5.56Ϯ0.08) with CA1). as compared with CA1 cells [see Fig. 5(a–c)]. There were no significant differences for mean burst ϭ Ͼ ϭ Table 1A. Total number of cells from each individual animal for com- duration (ms) (F(1,241) 0.01, P 0.1) between CA1 (mean plex spike cells recorded on the holder and the maze for CA1 and CA3 7.84Ϯ0.11) and CA3 (meanϭ7.82Ϯ0.17) cells. There ϭ were no significant differences for burstiness (F(1,241) Rat ID Holder cells Maze cells 1.83, PϾ0.1) between CA1 (meanϭ.29Ϯ0.01) and CA3 ϭ Ϯ CA1 CA3 CA1 CA3 (mean .31 0.02) cells. Lastly, there were no significant differences for the ratio of bursts to single spikes events ϭ Ͼ ϭ Ϯ 253 2 — — — (F(1,241) 0.68, P 0.1) between CA1 (mean .15 0.01) 254 8 — 11 — and CA3 (meanϭ.16Ϯ0.01) cells. 361 11 — — — Taken together, CA3 neurons exhibited more spikes 3624—2—per burst, greater spike frequency (shorter ISI’s), and 4604364larger amplitude spikes (both first spike of burst as well as 485 1 — — — single spike amplitude). In contrast, there were no differ- 548 — 19 2 19 ences between CA3 and CA1 neurons in their tendency to 683 8 11 8 9 6858161burst or burst duration. 6962—1— 6982—5—Effect of behavioral state 7017472 To examine the effects of behavioral state on burst 763 27 5 26 5 Total 84 43 74 40 properties, a within-cell analysis was conducted for cells recorded on both the holder and maze using a repeated- J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677 1669

2.5 CA1 A * CA3

2.0 AVERAGE # SPIKES IN BURST

0.0

0.28 0.28 CA1 CA1 0.27 B **CA3 0.27 C CA3 0.26 0.26

0.25 0.25

0.24 0.24

0.23 0.23 (millivolt) (millivolt) 0.22 0.22

0.21 0.21

0.20 0.20 AMPLITUDE OF SINGLE SPIKE 0.19 0.19

AMPLITUDE OF FIRST SPIKE IN BURST 0.0 0.0

Fig. 4. Burst characteristics of CA1 and CA3 cells independent of behavior. (A) Average number of spikes in a burst: CA3 cells exhibited a greater average number of spikes in a burst (PϽ0.01) compared with CA1 cells. (B) Amplitude of first spike in burst: CA3 cells exhibited a greater amplitude (millivolt) of the first spike in a burst (PϽ0.001) compared with CA1 cells. (C) Amplitude of single spike: CA3 cells exhibited a greater amplitude of single spike events (PϽ0.01) compared with CA1 cells. measures ANOVA (state as the repeated factor and sub- Fig. 6b. While the animals spent on average 10 min on the region as the between factor; see Table 1B). A number of holder and 14 min running the maze, the decrease in CA1 different burst-related measures were examined. cells’ tendency to burst on the maze was largely a conse- quence of a reduction in the number of burst events on the Tendency to burst. This was examined in two man- maze rather than a large increase in single spikes (see ners, first the cells burstiness was defined by the number Table 2). of burst spikes divided by total spikes (Quirk et al., 2001). In addition, there was a significant correlation between An alternate index was defined as the ratio of burst to burstiness on the holder and the maze for both CA1 cells single spike events. This index treats the burst as an event (rϭ.58, PϽ0.001) and CA3 cells (rϭ.61, PϽ0.001); and for and reflects the proportion of burst to single spike events. the ratio of burst events to single spike events on the There was increased burstiness when the animal holder and the maze for both CA1 cells (rϭ.52, PϽ0.001) was resting on the holder as compared with when run- and CA3 cells (rϭ.60, PϽ0.001), indicating that cells that ning the maze (F ϭ15.07, PϽ0.001). However this (1,82) tended to burst on the holder were more likely to burst on was due to a change in CA1 cell burstiness while CA3 ϭ Ͻ maze. cells were unaffected (interaction F(1,82) 8.13, P 0.01; paired t-tests: CA1 PϽ0.001; CA3 PϾ0.10), see Fig. 6a. Burst characteristics. As can be seen in Fig. 7a, Similarly while there was a greater number of burst events there were a greater average number of spikes in a burst ϭ Ͻ to single spike events in rats resting on the holder than on the holder than the maze (F(1,82) 14.45, P 0.001) ϭ Ͻ ϭ running on the maze (F(1,82) 11.71, P 0.01), there was and this did not interact with subregion (F(1,82) 0.53, ϭ Ͻ Ͼ an interaction (F(1,82) 8.76, P 0.01), in which only CA1 P 0.1). There was no correlation between the average cells were greatly affected by the change in behavioral number of spikes in a burst on the holder and the maze state (paired t-tests: CA1 PϽ0.001; CA3 PϾ0.10) see for CA1 cells (rϭ.08, PϾ0.1) and CA3 cells (rϭ.23, 1670 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

6.2 CA1 A * CA3

6.0

5.8

5.6

5.4 INTERSPIKE INTERVAL (ms)

0.0

18 18

16 B CA1 16 C CA3

14 14

12 12

10 10

8 8

6 6

4 4

2 2

0 0 3.10 3 3 4.30 4 5 5 5 6.30 6 7 7 7 8 8 9 9.50 9 3 3 3 4 4 5 5 5 6 6 7 7 7 8 8 9 9 9 .5 .90 .7 .10 .50 .90 .7 .1 .50 .9 .3 .70 .1 .9 .10 .5 .90 .30 .7 .1 .50 .90 .3 .7 .10 .5 .9 .30 .7 .10 .5 .9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ISI (ms) ISI (ms)

Fig. 5. Interspike interval of CA1 and CA3 cells independent of behavior. (A) Interspike interval: CA3 cells exhibited shorter interspike intervals (ms) (PϽ0.01) compared with CA1 cells. (B) A histogram of the interspike interval of CA1 cells. (C) A histogram of the interspike interval of CA3 cells.

PϾ0.1). An examination of a random subset of cells correlated in either subregion, CA1 cells (rϭ.12, PϾ0.1) revealed a unimodal skewed distribution, with bursts and CA3 cells (rϭ.29, PϾ0.1). mostly of two spikes. This is in contrast with intracellular Interspike interval (ms) was shorter on the holder recordings that can show far larger average burst (meanϭ5.47Ϯ0.84) than on the maze (meanϭ5.84Ϯ0.07) ϭ Ͻ lengths (Harris et al., 2001). This could be an epiphe- (F(1,82) 24.78, P 0.001) and this did not interact with ϭ Ͼ nomenon due to far larger signal to noise ratio with subregion (F(1,82) 1.22, P 0.1). There was a significant intracellular recordings (allowing for even the smaller correlation between the interspike interval on the holder spikes in a burst that would otherwise be masked by and the maze for CA1 cells (rϭ.56, PϽ0.001) and for CA3 ϭ Ͻ background noise to be included), conversely, it could cells (r .37, P 0.05), (see Fig. 7b). Thus, despite the be related to increased bursting in anesthetized animals. overall reduction in interspike interval during awake-immo- There was no difference in burst duration (ms) on the bility, differences between cells in their interspike intervals holder (meanϭ7.72Ϯ0.15) and maze (meanϭ7.58Ϯ0.14) are preserved across behavioral states. ϭ Ͼ (F(1,82) 0.55, P 0.1), and no interaction between subre- Spike amplitude. As can be seen in Fig. 8, the am- ϭ Ͼ gion and burst duration (ms) (F(1,82) 0.00, P 0.1) (CA1 plitude of spikes was greater on the holder than on the Holder: meanϭ7.75Ϯ0.14; CA1 Maze: meanϭ7.61Ϯ0.20; maze. This was true both for the first spike in a burst ϭ Ϯ ϭ Ϯ ϭ Ͻ CA3 Holder: mean 7.69 0.33; CA3 Maze: mean 7.54 (F(1,82) 11.91, P 0.01) and the amplitude of single spikes ϭ Ͻ 0.19). Burst duration on the holder and the maze was not (F(1,82) 10.16, P 0.01). There was no interaction with J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677 1671

Table 1B. Total number of cells from each individual animal for the Table 2. Analysis of the number of single spikes and burst events for same complex spike cell recorded on the holder and the maze for CA1 CA1 and CA3 cells recorded on the holder and the maze and CA3 (within-cell analysis) CA1 (nϭ52) CA3 (nϭ32) Rat ID CA1 CA3 Holder 253 — — No. single spikes 490.8Ϯ52.44 447.4Ϯ71.95 254 8 — No. burst spikes 277.7Ϯ43.20 174.8Ϯ23.66 361 — — No. burst events 113.4Ϯ17.09 70.7Ϯ10.01 362 1 — Maze 460 4 1 No. single spikes 568.8Ϯ41.27 488.4Ϯ46.30 485 — — No. burst spikes 179.8Ϯ16.60 228.4Ϯ32.93 548 — 15 No. burst events 78.9Ϯ7.09 95.3Ϯ13.37 683 5 8 Ϯ 685 5 1 Values are means S.E.M. 696 1 — 698 2 — burst on the holder and the maze for CA1 cells (rϭ.88, 701 5 2 PϽ0.001) and CA3 cells (rϭ.95, PϽ0.001). There was a 763 21 5 significant correlation between the amplitude of a single Total 52 32 spike on the holder and the maze for CA1 cells (rϭ.93, PϽ0.001) and CA3 cells (rϭ.98, PϽ0.001). Taken to- subregion (both PϾ0.1). In addition, there was a significant gether spikes were larger during awake-immobility than correlation between the amplitude of the first peak in a locomotion, and this was true for both CA1 and CA3.

BURSTINESS RATIO BURST EVENTS TO SS EVENTS

1.0 0.6 CA1 CA3 0.5 0.8 AB 0.4 0.6

0.3 HOLDER HOLDER 0.4 0.2

0.2 0.1

0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 MAZE MAZE

0.4 0.4 CA1 CA1 * CA3 * CA3

0.3 0.3

0.2 0.2 BURSTINESS

0.1 0.1 BURSTS : SINGLE SPIKES

0.0 0.0 Holder Maze Holder Maze

Fig. 6. Tendency to burst at rest and on the maze. (A) Burstiness: The scatter-plot of CA1 and CA3 cells represents the proportion of bursts (burstiness) as measured by dividing the number of burst spikes by the total number of spikes. There was increased burstiness when the animal was resting on the holder compared with running on the maze (PϽ0.001). The line graph plots burstiness for CA1 and CA3 cells on the holder and the maze. In addition, there was a significant interaction (PϽ0.01) in which CA1 cell burstiness was affected by behavioral state (holder vs. maze). (B) Ratio of burst events to single spike events: The scatter-plot of CA1 and CA3 cells represents the ratio of burst events to single spike events. There were a greater number of burst events to single spike events in rats resting on the holder than running on the maze (PϽ0.01). The line graph plots the ratio of burst to single spike events for CA1 and CA3 cells on the holder and the maze. In addition, there was a significant interaction (PϽ0.01) in which CA1 cells were affected by the change in behavioral state (holder vs. maze). 1672 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

AVERAGE # SPIKES IN BURST INTERSPIKE INTERVAL

3.6 8.0 CA1 CA1 3.4 CA3 CA3

7.0 3.2 A B 3.0 6.0 2.8

2.6 HOLDER HOLDER 5.0 2.4

2.2 4.0 2.0

1.8 3.0 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.0 4.0 5.0 6.0 7.0 8.0 MAZE MAZE 2.8 7.0 CA1 CA1 * CA3 * CA3 2.6 6.0

2.4 5.0 2.2

4.0 2.0 INTERSPIKE INTERVAL (ms) AVERAGE # SPIKES IN BURST

0.0 0.0 Holder Maze Holder Maze

Fig. 7. Burst characteristics on the holder and the maze. (A) Average number of spikes in burst: The scatter-plot of CA1 and CA3 cells represents the average number of spikes in a burst. There were a greater average number of spikes in a burst on the holder than the maze (PϽ0.001). The line graph plots the average number of spikes in a burst for CA1 and CA3 cells on the holder and the maze (greater on the holder), which did not interact with subregion (PϾ0.1) (B) Interspike interval: The scatter-plot of CA1 and CA3 cells represents the interspike interval (ms), which was shorter on the holder than on the maze (PϽ0.001). The line graph plots the interspike interval for CA1 and CA3 cells on the holder and the maze (shorter on the holder), which did not interact with subregion (PϾ0.1). Comparing spike amplitude of single and burst 1999). Dividing the amplitude of the third spike by the spikes amplitude of the first spike assessed the amount of atten- uation in a burst. There was more spike amplitude atten- A repeated measures ANOVA was conducted to compare uation for CA3 cells in comparison to CA1 cells (F ϭ the amplitude of the first peak in a burst to the amplitude of a (1,217) 15.40, PϽ0.001, see Fig. 10a). In order to examine the single spike event for CA1 and CA3 cells (Fig. 9 a, b). It was effects of behavioral state, further analyses were con- found that a given cell exhibited a larger amplitude spike at ducted on spike amplitude attenuation for cells recorded the initiation of a burst than when it fired a single spike both on the holder and the maze (Fig. 10b). A two-way (F ϭ206.83, PϽ0.001). The amplitude of the CA3 cell (1,237) ANOVA for subregion and behavioral state revealed that in spikes was larger than the amplitude of CA1 cells addition to the CA1–CA3 difference in attenuation, there (F ϭ13.00, PϽ0.001). There was also a significant in- (1,237) was also more spike amplitude attenuation on the holder teraction between spike amplitudes and subregions than on the maze (F ϭ7.60, PϽ0.01). However, there (F ϭ9.85, PϽ0.01) indicating that the difference be- (1,62) (1,237) was no significant interaction between subregion and be- tween the amplitude of a single spike and the first spike in a havioral state (F ϭ0.21, PϾ0.1). In addition there was burst was much greater for CA3 cells than CA1 cells. In (1,62) no significant correlation between the spike amplitude at- addition, there was a significant correlation between the am- tenuation on the holder and the maze for CA1 cells plitude of the first peak in a burst to the amplitude of a single (rϭϪ.08, PϾ0.1) and CA3 cells (rϭ.26, PϾ0.1). spike event for both CA1 cells (rϭ.98, PϽ0.001) and CA3 cells (rϭ.97, PϽ0.001). Taken together, CA3 cells had greater amplitude than CA1 cells and were more affected by DISCUSSION whether the cell fired in a burst or a single spike. Complex spike bursts, a signature characteristic of both CA3 and CA1 neurons, are presumed to play distinct phys- Spike amplitude attenuation iologic and computational roles in hippocampal function A general property of pyramidal cell bursts is an activity- (see Lisman, 1997; Pike et al., 1999; Hirase et al., 2001). dependent attenuation in amplitude (Quirk and Wilson, The present study demonstrates differences in the burst J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677 1673

AMPLITUDE OF FIRST SPIKE IN BURST AMPLITUDE OF SINGLE SPIKE 0.45 0.45 A B 0.40 0.40 CA1 CA1 0.35 CA3 0.35 CA3

0.30 0.30

0.25 0.25 HOLDER HOLDER 0.20 0.20

0.15 0.15

0.10 0.10

0.05 0.05 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 MAZE MAZE

0.28 0.28 CA1 CA1 * CA3 * CA3 0.26 0.26

0.24 0.24

0.22 0.22

0.20 0.20

0.18 0.18 AMPLITUDE (millivolt) AMPLITUDE (millivolt) AMPLITUDE 0.16 0.16

0.00 0.00 HOLDER MAZE HOLDER MAZE

Fig. 8. Spike amplitude on the holder and the maze. The scatter-plots of CA1 and CA3 cells represent the amplitude of spikes (millivolt), which was greater on the holder than on the maze. This was demonstrated both for (A) the first spike in a burst (PϽ0.01) and (B) the amplitude of single spikes (PϽ0.01). The line graphs plot the amplitude for the first spike in a burst and amplitude of single spike event for CA1 and CA3 cells on the holder and the maze (greater on the holder), which did not interact with subregion (both PϾ0.1). properties of CA3 and CA1 neurons in vivo and most spike was emitted. The larger amplitude of the initial importantly differential effects of behavioral state on CA1 spike may be related to a stronger depolarization of the and CA3 bursts. cell, leading to both a larger amplitude spike and a burst, In both CA3 and CA1 spike bursts accounted for less or possibly due to a resetting of sodium channel activa- than 20% of all spike events, with the majority of events tion prior to a burst (Harris et al., 2001). Extending on being single spikes. Spike amplitude was greater when Harris and colleagues’ (2001, 2002) CA1 findings, we the cell was initiating a burst than when only a single found in both CA3 and CA1 individual differences in

0.7 0.7 A B 0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

CA1 0.1 0.1 BURST AMPLITUDE (millivolt) CA3 BURST AMPLITUDE (millivolt) 0.0 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 SINGLE SPIKE AMPLITUDE (microvolt) SINGLE SPIKE AMPLITUDE (microvolt)

Fig. 9. Spike amplitude of single and burst spikes. (A.) The scatter-plots represent a comparison of the amplitude (millivolt) of the first peak in a burst to the amplitude of a single spike event for (A) CA1 cells (black circles) and (B) CA3 cells (open circles). A given cell exhibited a larger amplitude spike at the initiation of a burst than when it fired a single spike (PϽ0.001). 1674 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

SPIKE AMPLITUDE ATTENUATION

1.0 1.3 CA1 CA1 A * CA3 B CA3 1.2

1.1

0.9

HOLDER 1.0

0.9 THIRD SPIKE/FIRST SPIKE

0.0 0.8 0.8 0.9 1.0 1.1 1.2 1.3 MAZE

1.0 CA1 * CA3

0.9

0.8 THIRD SPIKE/FIRST SPIKE

0.0 Holder Maze

Fig. 10. Spike amplitude attenuation. The amount of attenuation in a burst was determined by dividing the amplitude of the third spike by the amplitude of the first spike. (A) Spike amplitude attenuation independent of behavioral state: There was more spike amplitude attenuation for CA3 cells compared with CA1 cells (PϽ0.001). (B) Spike amplitude attenuation dependent on behavioral state: The scatter-plot represents the spike amplitude attenuation for cells recorded both on the holder and the maze, in which there was more spike amplitude attenuation on the holder than on the maze (PϽ0.01). The line graph plots the spike amplitude attenuation for CA1 and CA3 cells on the holder and the maze demonstrating more attenuation on the holder than on the maze regardless of subregion (interaction, PϾ0.1). tendency to burst were preserved across behavioral maze running as compared with awake-immobility, while states. However during awake-immobility there were the burstiness of CA3 was unchanged. This finding ex- more spikes per burst, shorter spiking frequencies and tends results reported by Harris and colleagues (2002) of a greater spike amplitude attenuation than during maze decrease in the probability of CA1 bursting during theta as running. compared with non-theta. The present findings demon- CA3 and CA1 bursts differed independent of behav- strate that this is not the case for CA3 neurons. These ioral state. CA3 bursts included: 1) more spikes; 2) a findings suggest that the dynamic range of bursts and their shorter ISI between spikes; and 3) a greater amplitude of signaling capacity is greater for CA1 than for CA3. Further, the first spike in a burst and of single spikes than in CA1. these findings suggest that subcortical afferents (e.g. cho- There was also more spike amplitude attenuation within a linergic, noradrenergic, serotonergic) may differentially CA3 burst than within CA1 bursts. Spike attenuation has regulate resting potential and/or burst generating mecha- been related to a decrease in the ability of somatic spikes nisms in CA1 and CA3 networks. to back-propagate into the dendrites of hippocampal cells (Buzsáki et al., 1996; Magee and Johnston, 1997). It is not The importance of bursting clear whether our findings of larger spike attenuation in CA3 are simply due to the larger initial spike in CA3, the The significance of bursts has been a topic of interest to shorter ISI in CA3 bursts, or some other factors that could researchers examining a range of neural circuits (Metzner affect back-propagation (e.g. bursts occurring during a et al., 1998; Reinagel et al., 1999; Swadlow and Gusev, large calcium spike could show a larger first spike and 2001). Within hippocampal pyramidal neurons, we report more attenuation). that the majority of CA3 and CA1 spikes were single spikes A major finding of the present study is that the propen- (only 20% in bursts). Given the relatively small percentage sity of neurons to burst was affected by behavioral state, in of time bursting, what determines whether a CA3 or CA1 CA1 but not in CA3. Within-cell comparisons demonstrated neuron bursts? One might suspect that the burst has spe- that the burstiness of CA1 neurons was reduced during cific significance for the representation of “space” (Otto et J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677 1675 al., 1991; Harris et al., 2001, 2002). Harris and colleagues input, CA3 neurons also receive dense associational in- reported that bursting does not preferentially occur in the puts from both ipsilateral and (in the rat) contralateral CA3 center of the place field. Rather, they report that the prob- (commissural input) as well as a prominent input from ability of burst initiation and the number of spikes within a layer 2 (Steward and Scoville, 1976; burst are largely determined by the length of preceding Tamamaki and Nojyo, 1993). In contrast to CA3, CA1 silence (see also Swadlow and Gusev, 2001). Whether this neurons receive a highly limited projection from ipsilateral is related to the present finding of decreased CA1 bursting and contralateral CA1 neurons (van Groen and Wyss, on the maze is unclear. 1990). CA1 receives a massive input from ipsilateral and Importantly, only CA1 neurons had a greater propen- contralateral CA3 as well as significant projections from sity to burst during awake-immobility than during maze- layer 3 of the entorhinal cortex and the nucleus reunions of running. Bursts during awake-immobility can reflect off-line the thalamus (Kloosterman et al., 2003). Each of these replay of prior experience (Wilson and McNaughton, 1994; synaptic inputs to CA3 and CA1 innervates a circum- Kudrimoti et al., 1999; Nadasdy et al., 1999; Lee and scribed portion of the dendritic field and is associated with Wilson, 2002) and the complex-spike bursts associated specific dendritic-targeting interneurons (Klausberger et with high-frequency discharges (CA1 ripples at 200 Hz al., 2003). The importance of this anatomical organization during sharp waves) are thought to contribute to synaptic is underscored by the fact that subcortical inputs (e.g. plasticity within the CA3, CA1 and hippocampal formation cholinergic, noradrenergic, serotonergic) can differentially output networks (Buzsáki et al., 1996; Chrobak and influence the efficacy of distinct afferent inputs directly Buzsáki, 1998; Csicsvari et al., 2000). The reason why the (e.g. Hasselmo and Bower, 1993; Hasselmo and Schnell, non-maze running increase in burstiness is limited to CA1 1994; Hasselmo et al., 1995, 1996) or via select modula- cells is not clear. However, Csicsvari and colleagues tion of distinct interneurons (Nitz and McNaughton, 1999, (2000) noted a much greater increase in firing rate in CA1 2004). While it has been demonstrated that there were no than CA3 during ripples (they did not differentiate single differences in the basic firing characteristics of hippocam- and burst spikes), also suggesting a greater dynamic pal cells across the estrous cycle (Tropp et al., 2005, range in CA1 output than CA3. Further assessment of the estrogen may exert some of its effects on the hippocampus conditions determining burst discharges during spatial ex- via regulation of brain-derived neurotrophic factor (BDNF), ploration, awake-immobility and slow-wave sleep would which can affect hippocampal function, synaptic plasticity provide experimental evidence for the role of bursts in and possibly the bursting properties of the cells (Scharf- off-line replay of sensory experience and synaptic plastic- man and MacLusky, 2005). While the mechanisms are still ity. For example we would predict increased synchrony of unclear, the result is that CA3 cells are less affected by EEG between CA1 and CA3 during rest in comparison to behavioral state than CA1 neurons. running on the maze.

Possible sources for differences in the behavioral Functional significance of differences in behavioral effects on CA3/CA1 burstiness modulation of CA3/CA1 burstiness Neurons within several forebrain structures have a propen- Burst probability in CA1 is more prominent during the sity to burst. The different cellular mechanisms by which non-theta state (Harris et al., 2001), although this is not the these neurons can produce fast-frequency spikes have case in CA3 (present results). This was true regardless of historically been an area of intense investigation (e.g. Lli- whether the ratio of burst to total spikes or the ratio of burst nas, 1988; Connors and Gutnick, 1990; Azouz et al., 1996; to single spike events was examined (Fig. 6). This lack of Schwindt and Crill, 1999; Williams and Stuart, 1999; Staff behavioral modulation was unexpected and indicates a et al., 2000; Bernard and Johnston, 2003). Within CA3 certain degree of autonomy/independence in CA3 pro- neurons, the activation of voltage-activated calcium chan- cessing, contrasting with the state-dependent modulation nels concentrated within the somatic compartment is of CA1 processing. thought to underlie burst firing (Wong and Prince, 1978; There is behavioral evidence that CA3 is more impor- Traub et al., 1991; Huguenard, 1996). CA1 neurons may tant than CA1 for forming rapid associations in novel situ- be more compartmentalized with dendritic compartments ations/tasks (Lee and Kesner, 2002). Examining place exhibiting bursting independent of the soma (Wong and cells also indicates differences between CA1 and CA3 in Stewart, 1992). Subtle differences in the distribution of representation of environmental change (Guzowski et al., voltage-activated CA2ϩ and Naϩ currents and the dynamic 2004; Lee et al., 2004a,b; Leutgeb et al., 2004). Specifi- interplay of these currents in response to synaptic inputs cally, CA3 place fields may be less affected than CA1 during theta may underlie greater state-dependent control fields by subtle changes in spatial landmarks and context. among CA1 neurons or the seeming indifference of CA3 In the present study the animals were overtrained on the neurons. maze task, and the place fields were presumably stable. It On a circuit level, it is quite clear that CA3 and CA1 is possible that the bursting of CA3 cells is affected by neurons have different subsets of synaptic inputs that behavior only during the initial exploration of an environ- could generate bursts (Fig. 1). CA3 neurons are the exclu- ment, once the situation becomes familiar the behavioral sive recipient of dentate granule cell input via the mossy modulation of bursting ceases. Conversely, CA1 cells re- fibers (Claiborne et al., 1986). In addition to the mossy fiber main responsive even in an over-trained task. 1676 J. Tropp Sneider et al. / Neuroscience 141 (2006) 1665–1677

Taken together, the data suggest in a familiar environ- Hasselmo ME, Schnell E (1994) Laminar selectivity of the cholin- ment CA1 neurons exhibit a greater plasticity and dynamic ergic suppression of synaptic transmission in rat hippocampal range, including their propensity to burst than CA3. These region CA1: computational modeling and brain slice physiology. J Neurosci 14:3898–3914. properties may further inform computational models that Hasselmo ME, Schnell E, Barkai E (1995) Dynamics of learning and stress the ability of CA3 neurons to complete previously at excitatory recurrent synapses and cholinergic modulation stored patterns (via an auto-associative network and/or in rat hippocampal region CA3. J Neurosci 15:5249–5262. reverberations) while CA1 is more affected by external Hasselmo ME, Wyble BP, Wallenstein GV (1996) and re- input (McNaughton and Morris, 1987; Lisman and Otmak- trieval of episodic memories: role of cholinergic and GABAergic hova, 2001). modulation in the hippocampus. Hippocampus 6:693–708. Hirase H, Leinekugel X, Czurko A, Csicsvari J, Buzsáki G (2001) Firing rates of hippocampal neurons are preserved during subsequent Acknowledgments—This work was supported by a National Re- sleep episodes and modified by novel awake experience. Proc Natl search Service Award: Predoctoral Fellowship (NIMH) #5F31- Acad SciUSA98(16):9386–9390. MH063551-03 (J.T.), UConn FRS445142 and NSF grant #IBN- Huguenard JR (1996) Low-threshold calcium currents in central ner- 9809958 (E.J.M). We thank Dr. Tanila for his helpful comments; vous system neurons. Annu Rev Physiol 58:329–348. 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(Accepted 16 May 2006) (Available online 14 July 2006)