Effects of 5-HT3 Receptor Antagonism on Hippocampal Cellular Activity in the Freely Moving Rat
Jeffrey Reznic and
Ursula Staubli
Center for Neural Science, New York University, New York, New York 10003
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ABSTRACT |
Reznic, Jeffrey and Ursula Staubli. Effects of 5-HT3 receptor antagonism on hippocampal cellular activity in the freely moving rat. J. Neurophysiol. 77: 517-521, 1997. Recent physiological studies conducted in the hippocampi of freely moving rats have revealed that systemic injections of the selective serotonin-3 (5-HT3) receptor antagonist ondansetron facilitate induction of long-term potentiation (LTP), increase the frequency of the theta electroencephalogram rhythm, and enhance retention of memory in hippocampally dependent tasks. To gain insight into the cellular mechanisms underlying these observations, in the present study we examined the effects of intraperitoneal injections of ondansetron on the firing rate of CA1 interneurons and pyramidal cells in the dorsal hippocampi of freely moving rats. Mean firing rates of a substantial proportion (17 of 27) of isolated neurons were significantly different before and after ondansetron injection (500 and 1,000 µg/kg). Of the interneurons that exhibited an effect, all (11 of 11) significantly decreased their mean firing rate, with an average change of
22.4 ± 3.9% (mean ± SE) across cells. Eighty-three percent (5 of 6) of pyramidal cells showing a change in mean firing rate displayed a significant increase in activity, with an average change of 56.3 ± 25.6% across cells. Ondansetron (1.0 mg/kg ip) had no detectable effect on spontaneous behavioral activity as measured by line crossings and rearings in an open-field apparatus. The present results show that pharmacological blockade of 5-HT3 receptors causes a reduction in firing activity of a subset of CA1 hippocampal interneurons, with concomitant increases in the firing rate of pyramidal cells. These changes may be directly related to the ondansetron-induced enhancement of LTP induction and memory formation observed in previous studies.
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INTRODUCTION |
Serotonergic brain stem projections to hippocampus have long been implicated in the regulation of behavioral states including the encoding of memory (Vertes 1986
). The terminals arising from the serotonin (5-HT) system are known to target and activate via 5-HT3 receptors, a distinct subpopulation of calbindin-positive interneurons in stratum radiatum, stratum lacunosum-moleculare, and stratum oriens(Battenberg et al. 1994
; Freund et al. 1990
; Tecott et al. 1993
). The 5-HT3 receptor is unique in that it is the only known serotonergic receptor directly linked to an ion channel (Derkach et al. 1989
), and converging lines of evidence indicate that pharmacological blockade of 5-HT3 receptors has significant memory-enhancing effects in rodents, primates, and humans (e.g., Crook and Lakin 1991
; Domeney et al. 1991
; Staubli and Xu 1995
). Tests for possible interactions between the 5-HT3 system and synaptic plasticity revealed that antagonists of this receptor facilitate the induction of long-term potentiation (LTP) both in hippocampal slices (Santamaria and Caille 1994
) and area CA1 of freely moving animals (Staubli and Xu 1995
). This, combined with the observation that 5-HT3 receptors are most abundant in the hippocampi of both rats and humans (Barnes et al. 1990
; Bufton et al. 1993
), suggests that the memory-enhancing effects of 5-HT3 receptor blockers might be mediated via an action on the cellular machinery underlying hippocampal LTP formation. Central among the factors controlling LTP induction are GABAergic interneurons that regulate the degree of local depolarization and hyperpolarization and thereby determine the amount of potentiation produced by afferent stimulation (Alger and Nicoll 1982
; Wigstrom and Gustafsson 1983). Thus, if GABAergic cells are the primary mediators of 5-HT3-antagonism-induced effects on hippocampal LTP and learning, it should be possible to observe a change in discharge patterns of interneurons in response to drugs that block the receptor. This was the goal of the present study, in which we examined the effects of systemic injections of ondansetron (Zofran, Glaxo Pharmaceuticals), a potent and selective 5-HT3 antagonist, on firing activity in area CA1 of freely moving rats.
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METHODS |
Male Long-Evans rats, 3 mo of age, housed individually in a reversed light-dark cycle and maintained at 85% free-feeding weight, were used. Before surgery the animals were handled daily, accustomed to receiving intraperitoneal injections of saline, and allowed to explore the recording cage (75 × 75 × 75 cm). While rats were under deep anesthesia with either a mixture of ketamine and xylazine or pentobarbital sodium, a driveable bundle of 10 microelectrodes (25 mm, formvar-insulated nichrome wire), essentially as described by Kubie (1984)
, was implanted several hundred micrometers above the dorsal CA1 pyramidal cell layer. Ten days later, the animals were acclimated to the attachment of the recording cable, which was mounted via a dual pulley arrangement with counterbalance, and screening for cells began. Signals were amplified 10 times via operational amplifiers (1 for each electrode) embedded in the cable, sent through a commutator, and further amplified (1,000 times) and displayed on an oscilloscope. Recordings were band-pass filtered (0.3-10 kHz for units; 0.001-5 kHz for electroencephalogram) and either 1) digitized at 23.6 kHz (Instrutech VR-100B) to be stored on video tape for off-line analysis with the use of customized software (developed by S. Fox) or 2) digitized at 31 kHz and analyzed with commercially available software (R. C. Electronics). Spikes were marked by either a pulse from a window discriminator (Bak DIS-1, time-amplitude) or software (R. C. Electronics) with the use of amplitude or action potential waveform criteria.
Once a suitable cell was found, experimental sessions began, with the rats behaving freely throughout the recording period. First, baseline firing was recorded for 15 min before and again after saline administration. Ondansetron was then injected at either 500 or 1,000 µg/kg, and recording continued for
45-60 min. In some animals, additional 15-min recording periods were conducted at 120 and 180 min after drug administration. For each recorded unit, mean firing rate histograms and autocorrelograms were constructed. Many cells displayed characteristic firing patterns during theta and nontheta states (Fig. 1). Neurons were classified according to criteria established in the literature (Fox et al. 1986
; Markus et al. 1994
), and were considered to be pyramidal if they exhibited firing rates <10 Hz during walking and displayed a long-duration action potential (>0.35 ms. measured from maximum to minimum voltage), with some of the action potentials occurring in bursts of progressively decreasing amplitude and interspike interval durations of <6 ms. Cells were considered interneurons if their action potentials were relatively constant in amplitude with short durations (
0.35 ms) and firing rates >10 Hz during walking. Durations were calculated from the average of 5-10 waveforms (Fig. 2D). The spike data were grouped into 1-s bins for subsequent statistical analyses. This binwidth was set to ensure independence of samples, which was confirmed by demonstrating that a surrogate autocorrelogram, calculated from spike arrival times taken at random from an exponential distribution, closely approximated that of the real cell. After completion of testing, animals were deeply anesthetized and perfused transcardially with 10% buffered formaldehyde, and standard 50-mm-thick cresyl-violet-stained sections were prepared to confirm the locations of the electrode tracks.

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| FIG. 1.
Representative examples of pyramidal cell and interneuron activity recorded in an awake rat during theta and nontheta states. A: interneuron firing at a preferred phase of the theta rhythm during all or most cycles. Top: raster display of CA1 hippocampal interneuron. Bottom: electroencephalogram recorded from contralateral hippocampal fissure/superficial molecular layer of the dentate gyrus in a walking rat. A : same neuron as in A during nontheta state firing randomly. B: CA1 pyramidal cell firing with a phase preference but during fewer theta rhythm cycles. B : same cell as in B during non-theta state. Scale: 0.15 s, 0.15 mV.
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| FIG. 2.
Majority of recorded interneurons shows a reversible decrease, and pyramidal cells an increase, in average firing rate after drug injection. A and B: changes in mean firing rate for an interneuron and a pyramidal cell, respectively. Insets: corresponding action potential waveforms. Scale: 0.5 ms, 0.1 mV (A); 3.0 ms, 0.1 mV (B); negative upward). Asterisk: significance level of P < 0.001 for changes in mean firing rate (see text). C: summary of % changes in mean firing rate between control and maximum response intervals for interneurons and pyramidal cells, with significance values indicated. Each bar along the horizontal axes indicates an individual cell. D: graph displaying firing rate vs. spike width of putative pyramidal cells ( ) and interneurons ( ).
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To rule out drug-induced alterations in spontaneous behavior as a possible explanation for changes in cellular discharge, open-field activities (line crossings and rearings) were assessed in an additional group of well-handled Long-Evans rats following an intraperitoneal injection of either saline (n = 5) or ondansetron (1,000 µg/kg, n = 5). Ten minutes after injection, each animal was placed into an evenly illuminated white Plexiglas chamber (120 × 120 × 40 cm) with the floor divided into 36 equal squares. The rat was allowed to explore for 10 min, then removed for 5 min to minimize the effect of habituation, and was replaced into the apparatus for a second 10-min period. Observations were conducted blind to drug condition (see Fig. 3).

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| FIG. 3.
Ondansetron has no effect on spontaneous behavior. A: activity scores expressed as number of line crossings (mean ± SE) during 2 10-min periods for rats injected with saline and drug. B: mean number of rearings for saline ( ) and drug ( ) groups measured at same times as in A, scored in 2-min intervals.
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RESULTS |
A total of 18 interneurons and nine pyramidal cells was recorded from five animals. To ensure the absence of changes in mean firing rates between the 15-min baseline versus the 15-min saline interval, independent-means t-tests were conducted for each cell, and those with significant differences were excluded from further analysis. Postdrug injection time periods were also organized into 15-min segments.
CA1 interneurons
Of the 18 interneurons recorded, 11 (61%) showed an effect following drug application, as determined by t-tests comparing baseline against the 15-min interval containing the greatest response (Fig. 2C). Paired Student's t-tests (2-tailed) were then used to evaluate changes in activity between baseline and saline conditions across all 11 cells, and revealed no significant differences. Therefore the average of these two means was used as the control value for subsequent comparisons. Next, a repeated measures analysis of variance was performed across these cells, with a significant effect for time being found [F(4,20) = 4.57;P < 0.01]. Post hoc multiple comparisons (k = 4) were then conducted in a Dunnett's-type procedure, comparing control against treatment intervals with the inclusion of a Bonferroni-corrected significance level (P < 0.0125) for multiple t-tests (for an example, see Fig. 2A). Ondansetron produced a significant decrease in mean firing rate relative to the control condition for the first [t(10) = 5.84; P < 0.001] and second [t(10) = 4.32; P < 0.002] 15-min segments, whereas the third [t(9) = 2.77; P < 0.025] and fourth [t(5) = 2.57; P < 0.05] periods approached significance. The average change between control and maximum response intervals across these cells was
22.4 ± 3.89 (mean ± SE).
CA1 pyramidal cells
Of the nine pyramidal cells recorded, six (67%) showed an effect following drug application, as determined by t-tests comparing baseline against the 15-min interval containing the greatest response. Repeated-measures analysis of variance for these neurons did not achieve statistical significance (F < 1.0) because of the small number of cells. We then sought to characterize the changes in mean firing rate for each neuron individually, with the use of repeated two-sample independent-groups z tests with the inclusion of a Bonferroni-corrected significance level (P < 0.0125) for multiple z tests. The large sample size within intervals (n = 900 for 15-min periods) involved in estimating the variances justifies the use of this test because the sample variance is very close to the population value. Five pyramidal cells significantly increased their mean firing rate and one decreased its rate in response to drug (z = 2.58 or greater; P < 0.01; Fig. 2C). Again, these changes were obvious in the first 45 min and lasted for at least one 15-min time segment, with most cells (5 of 6) showing significant changes for two or more periods (for an example, see Fig. 2B). The mean percent change between control and maximum response intervals across these cells was found to be 56.25 ± 24.56% (mean ± SE).
The firing rate of the majority of cells (59%) returned to baseline, mostly within 1 h after injection as determined by independent-means t-tests. The remaining cells were lost before recovery could be assessed. No relationship was evident between dosage and magnitude or direction of change for either cell group. Attempts to lesion the sites of the recording electrodes were unsuccessful, because the headcaps became detached from the skull before current was passed through the wires. Despite this, gross histological analysis confirmed that the locations of all recordings were within the CA1 region. Student's t-tests were performed to analyze the spontaneous activity in the open-field chamber after saline or drug injection, and revealed no significant differences in line crossings and rearing counts between the two groups according to traditional criteria (Fig. 3).
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DISCUSSION |
The present results show that pharmacological blockade of 5-HT3 receptors causes a reduction in firing activity of a subset of hippocampal interneurons, consistent with our prediction that the previously observed facilitatory action of ondansetron on learning, LTP induction, and theta rhythm frequency is prompted by a partial inhibition of GABAergic cells (Staubli and Xu 1995
). The interneurons involved could correspond to inhibitory circuits mediated by
-aminobutyric acid-A GABAA or by GABAB receptors, considering that antagonism of either of these binding sites is well known to promote LTP induction, although via distinctly different mechanisms (Arai and Lynch 1992
). It has been suggested that 5-HT could influence feedforward GABAB-receptor-mediated inhibition via inhibitory interneurons in stratum oriens, stratum radiatum, and stratum lacunosum-moleculare, whose axonal fields terminate largely onto the apical dendrites of pyramidal cells (Freund et al. 1990
). Within these strata, the coexistence of 5-HT3 receptor mRNA and GABA immunoreactivity has been demonstrated (Battenberg et al. 1994
), and interneurons situated near the stratum radiatum/lacunosum-moleculare border have been shown to express functional 5-HT3 receptors that lead to cellular depolarization on activation (Kauer and McMahon 1995
). It has been demonstrated that GABA acting through GABAA receptors modulates the theta rhythm (Soltesz and Deschenes 1993
; Ylinen et al. 1995
) and 5-HT3 receptor agonists increase spontaneous GABAA-receptor-mediated events (Ropert and Guy 1991
). A direct role of GABAB receptors in theta rhythm generation has not yet been demonstrated, possibly because of the difficulty in detecting distal GABAB-mediated events in intrasomatic recordings (Misgeld et al. 1995
). However, it has been suggested that the enhancement in theta rhythm frequency observed after ondansetron injection in freely moving rats is mediated, at least in part, via suppression of the slow GABAB-gated hyperpolarizing component occurring between successive peaks of the theta rhythm (Staubli and Xu 1995
). Regardless of the receptor subtype(s) involved, a reduction in GABAergic excitability is expected to augment the temporal and spatial summation of afferent excitation in principal cell dendrites (Arai and Lynch 1992
). This effect, which may be reflected by the elevated firing rates of pyramidal cells observed here, could thus be closely related to the changes underlying the promotion of LTP in vivo and memory facilitation subsequent to 5-HT3 receptor antagonism.
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ACKNOWLEDGEMENTS |
We gratefully acknowledge the assistance of S. E. Fox, E. Brazhnik, M. Palij, G. Quirk, and L. Maloney.
This research was supported in part by a grant from the Whitehall Foundation (F93-17) to U. V. Staubli and in part by National Eye Institute National Research Service Award Institutional Training Grant #5T32EY07136 to Robert Shapley, Ph.D. (J. Reznic).
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FOOTNOTES |
Address for reprint requests: U. Staubli, New York University, Ctr. for Neural Science, 4 Washington Pl., New York, NY 10003.
Received 21 May 1996; accepted in final form 16 September 1996.
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