1. EFFECTS OF INTRASEPTAL CARBACHOL ON BURST PROPERTIES OF CA1 PYRAMIDAL NEURONS S. Sava 1,2 *, G.J. Peters 1,3, E.J. Markus 1 1 Psych. Dept., Univ of Conn., Storrs, CT; 2 McLean Hospital/Harvard Medical School, Belmont, MA; 3 Psych. Dept., Univ of Delaware, Newark, DE. REFERENCES Bursting of CA1 cells was not affected by the novel configuration. EFFECT OF AGE AND BEHAVIORAL STATE Buzsáki G (1989). Two-stage model of memory trace formation: a role for "noisy" brain states. Neurosci. 31:551-570. Buzsáki G (2002). Theta oscillations in the hippocampus. Neuron 33:325-340. Harris KD, Hirase H, Leinekugel X, Henze DA, & Buzsáki G (2001). Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells. Neuron32:141-149. Kepecs A, Wang XJ, Lisman J (2002). Bursting neurons signal input slope. J Neurosci. 22:9053-6902. Lisman JE (1997) Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci. 20:38-43. O'Keefe J, Dostrovsky J (1971). The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res 34:171-175. Quirk MC & Wilson MA (1999). Interaction between spike waveform classification and temporal sequence detection. J of Neurosci Methods 94(1), 41-52. Quirk MC, Blum KI, & Wilson MA (2001). Experience-dependent changes in extracellular spike amplitude may reflect regulation of dendritic action potential back-propagation in rat hippocampal pyramidal cells. J Neurosci 21(1), 240-248. Ranck Jr JB (1973). Studies of single neurons in the dorsal hippocampal formation and septum in unrestrained rats. I. Behavioral correlates and firing repertoires. Exp. Neurology 41, 461-531. Tropp Sneider J, Chrobak JJ, Quirk MC, Oler JA, & Markus EJ (2006). Differential Behavioral state-dependence in the burst properties of CA3 and CA1 neurons. Neurosci 141:1665-1677. Wong RK, Prince DA (1978). Participation of calcium spikes during intrinsic burst firing in hippocampal neurons. Brain Res. 159:385-390.. ABSTRACT Single-unit recordings in the hippocampus show that, like cortical neurons, hippocampal pyramidal cells can fire single spikes or trains of high-frequency spikes, commonly called bursts (Rank, 1973). Hippocampal bursts are often assumed to have distinct physiological (Pike et al., 1999; Buzsáki et al., 2002) and/or computational functions (Lisman, 1997; Kepecs et al., 2002). The functional significance of bursts however is not clear. Tropp-Sneider and colleagues (2006) showed that the degree of CA1 bursting decreased when the animal ran on the maze compared to being at rest. These data may not indicate a functional change since bursting increases during sharp-wave oscillations (Buzsáki, 1989; Harris et al, 2001; Wong and Prince, 1978). The cholinergic agonist carbachol was infused into the medial septum, and hippocampal CA1 cells were recorded in freely moving rats in a familiar or novel environment. Septal activation disrupted the retrieval of a previously stored hippocampal place cell representation of a familiar environment regardless of age. When the environment was changed, medial septal activation disrupted the encoding process in young, but facilitated the encoding of the new information in aged rats. In contrast, burst properties of CA1 pyramidal cells were not affected by intraseptal carbachol. While burst firing was reduced when the animal was running on the maze, this was not related to the degree of environmental novelty or to septal activation. Supported by: UConn FRS445142; NIH #R29-A613941-01A1 to E.J.M. INTRODUCTION Hippocampal pyramidal cells exhibit “place-specific” discharge in relation to the location of the animal (O'Keefe & Dostrovsky, 1971). CA1 pyramidal neurons can fire single spikes or a fast series of spikes commonly referred to as a burst. Typically bursts contain 2-6 spikes at short intervals, with a progressive attenuation in the amplitude of the spikes within the burst (Ranck, 1973; Quirk and Wilson, 1999; Quirk et al., 2001). Both the burst and attenuation phenomena have been postulated to play an important role in information processing (Lisman, 1997; Quirk et al., 2001). Previous studies showed that behavioral state can affect the bursting of CA1 neurons; specifically, CA1 neurons have a greater propensity to burst during awake immobility than during maze running (Harris al., 2001; Tropp Sneider et al., 2006). The present study examined differences in the bursting properties of CA1 neurons in awake- behaving young and old rats during distinct behavioral states (awake immobility and maze performance). We also investigated the effects of intraseptal carbachol and novelty on the burst properties of CA1 cells. Are there differences in the burst properties of CA1 neurons as a function of behavioral state, age, or novelty? HISTOLOGY Burst = spike train having an interspike interval less than 10 ms. Within-cell examination of the effects of behavioral state, age and novelty: -Average number of spikes in burst -Burst spike interstimulus interval (ISI) -“Burstiness” (proportion of spikes in burst out of total number of spikes) -Attenuation (calculated only for bursts of 3 or more spikes) On holder, CA1 neurons exhibited higher proportion of spikes in bursts; bursts had more spikes and shorter ISI. There were no differences between burst properties of neurons recorded from young and old rats. GENERAL METHODS Summary of recorded cells by rat & condition Subjects Seven young (6-10 mo old) and eight old (22-24 mo) male Fischer 344 rats were used in this study. All animals were food deprived to approximately 90% of their ad libitum weights. Training Procedure The rats were given a 30 minute session each day to learn to alternate back and forth on a maze. After they reached criteria (80 alternations for at least 2 days of training), rats underwent surgery for microdrive implantation. Surgery Animals were anesthetized with ketamine-xylazine cocktail and implanted with a cannula aimed at the medial septum (0.5 mm anterior, 4.5 mm ventral form bregma), and a recording device to allow for extracellular single unit recordings aimed at the dorsal hippocampus (3.2 mm posterior, 2.2 mm lateral, 1 mm ventral from bregma). Each microdrive contained eight movable tetrodes. Re-training and recordings started one week after surgery. Recording Procedure Animals wore a multi-channel headstage device that contained two arrays of infrared light emitting diodes. An overhead video tracking system recorded the rat's location and head direction. Analysis of the multi-single unit recordings from each probe was conducted off-line manually, using a spike parameter clustering method ( McNaughton et al., 1989; Mizumori et al., 1989 ). The clustering was based on the relative amplitudes of the signals and the spike durations ( see Wilson & McNaughton, 1993 ). During analysis a velocity filter was used to assure that the data was collected only if the animal moved faster than 2.4cm/sec (Tropp et al., 2004). Hippocampal EEG was also recorded. Infusion Procedure A 0.5 µl volume was infused at a rate of 0.125 µl/min. One minute was allowed for drug diffusion before the injector was removed. Total dose of carbachol infused was 0.125 µg. -0.26mm +0.2mm +0.48mm +1mm +1.2mm cc CA1 * * Percentage increase in theta power Young Old Carbachol increases hippocampal theta power during drinking After all single unit recordings were done, each rat received a series of 5 carbachol infusions. The hippocampal theta power was measured while the rat was drinking, before and after carbachol administration. CARBACHOL OLD Maze preMaze post Maze preMaze post CARBACHOL YOUNG EFFECT OF CARBACHOL ON BURST PROPERTIES Infusion 10 min 9 min Amplitude of Third Spike Amplitude of First Spike Spike amplitude attenuation = EFFECT OF NOVELTY ON BURST PROPERTIES Infusion 10 min 21 trials 12 min Infusion 10 min 9 min HOLDER Despite altering the place field characteristics, carbachol did not affect the burst properties of CA1 neurons. There was less attenuation on the second run on the familiar maze. INTERSPIKE INTERVAL YOUNGOLD Control Carbachol ISI (ms) Drug, p=0.096 BURSTINESS Proportion of spikes in burst YOUNGOLD Control Carbachol Drug, p=0.062 SPIKE AMPLITUDE ATTENUATION YOUNGOLD Control Carbachol Amplitude attenuation Session by age, p=0.079 # SPIKES IN BURST # spikes in burst YOUNGOLD Control Carbachol Session, p=0.058 INTERSPIKE INTERVAL YOUNGOLD Control Carbachol ISI (ms) Drug, p=0.018 BURSTINESS Proportion of spikes in burst YOUNGOLD Control Carbachol NS SPIKE AMPLITUDE ATTENUATION YOUNGOLD Control Carbachol Amplitude attenuation Session, p=0.016 # SPIKES IN BURST # spikes in burst YOUNGOLD Control Carbachol Age, p=0.087 T-tests: *, p = 0.002 YOUNGOLD Proportion of spikes in burst Behav. State, p<0.001 * * BURSTINESS YOUNGOLD ISI (ms) Behav. State, p<0.001 * * INTERSPIKE INTERVAL YOUNGOLD # spikes in burst # SPIKES IN BURST Behav. State, p<0.001 * * SPIKE AMPLITUDE ATTENUATION YOUNGOLD Amplitude attenuation Behav. State, p<0.001 T-tests: *, p < 0.006; #, p = 0.06 T-test: *, p = 0.024 CONCLUSIONS CA1 neurons burst more during awake immobility than during maze running. Bursting of CA1 neurons does not seem to be affected by age, novelty, or intraseptal carbachol infusion.