Exp Brain Res (1989) 77:502516

�9 Springer-Verlag 1989

Firing relations of medial septal neurons to the hippocampal theta rhythm in urethane anesthetized rats

M. Stewart and S.E. Fox Department of Physiology, Box 31, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA

Summary. On the basis of spontaneous firing pat- terns and relations to the hippocampal theta rhythm, three cell types were identified within the medial septal nucleus and vertical limb of the nu- cleus of the diagonal band of Broca (MSN-NDB). In addition to the well known rhythmically bursting cells that fired in bursts on each cycle of the hippo- campal theta rhythm, two other cell types are dis- tinguished. "Clock" cells fired at high rates with a very regular, periodic firing pattern that was un- related to the theta rhythm. "Irregular" cells fired at much lower rates, especially during theta rhythm, and had a pseudo-random firing pattern. The firing of "irregular" cells was often significantly phase- locked to the hippocampal theta rhythm. Crude estimates of the relative proportions of these cell types suggest that the rhythmically bursting cells comprise about 75% of the cells of the MSN-NDB. These three cell types bear a remarkable resem- blance, in firing patterns and relative proportions, to the three principal cell types of the medial septal nuclei described in the freely moving rat (Ranck 1976). Measurements o f the preferred phases of firing of 128 rhythmically bursting septal neurons (including 22 atropine-resistant and 11 atropine- sensitive cells) indicate that there is no single pre- ferred phase of firing for the population. Rather the distribution of phases over the theta cycle is statisti- cally flat. Variations in recording locations cannot account for this distribution since large differences in preferred phase were found for pairs of cells at the same location. Similarly, plotting only the group of cells identified as projection cells by antidromic activation from the fimbria/fornix, failed to reveal a peak in the distribution. In contrast to the rhythmi- cally bursting cells, the distribution of preferred firing phases for the "irregular" cells with a signifi-

Offprint requests to: M. Stewart (address see above)

cant phase-locking to the theta rhythm did have a clear peak. The peak occurred near the dentate theta rhythm positivity, consistent with the hypothesis that they are driven by feedback from CAI complex- spike cells.

Key words: Theta rhythm - Medial septum - Phase histogram

Introduction

Initially described by Jung and Kornmfiller (1938), the hippocampal theta rhythm or rhythmical slow activity (RSA) has become one of the best character- ized electroencephalographic rhythms. Motivation for much of the research aimed at understanding the theta rhythm comes from two features of this EEG pattern. First, its remarkably large amplitude, due partly to the relatively simple structure of the hippo- campal cortex, suggests synchronous activity in vast numbers of synapses. Secondly, the hippocampal theta rhythm has been recorded only during certain "active" behaviors (Pickenhain and Klingberg 1967; Vanderwolf 1969; Winson 1974). In fact, lesions which eliminate the theta rhythm, without direct damage to the hippocampus, impair the perfor- mance of animals in tasks that are presumed to require the hippocampus (Winson 1978; Mitchell et al. 1982).

A complete description of the mechanism of production of the theta rhythm requires an under- standing of the pacemaking mechanism as well as the actual mechanism o fits. generation by extracellu- lar currents. A number of methods have already been brought to bear on the problem of identifying the synaptic inputs to individual hippocampal sub- fields that generate the EEG. Since the laminar

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ana tomy of the"hippocampus is fairly well under- stood, one of the most direct ways to identify the contr ibut ion o f a part icular group of neurons to the hippocampal theta rhy thm is by correlating the firing of the cells with the E E G (see e.g. Fox et al. 1986). Most of the existing data, however, are for the firing o f hippocampal neurons themselves in relation to the theta rhythm; very little data exist for retro- hippocampal or subcortical inputs to the hippo- campus.

Since the discovery by Petsche, Stumpf, and Gogolfik (1962) of rhyhmically bursting cells in the medial septal nuclei of rabbits, the medial septum has been considered the "pacemaker" for the hippo- campal theta rhythm. The precise mechanism by which the medial septal nuclei accompliSh this "pacemaker" function remains unclear. We have recently reported that the popula t ion of rhythmi- cally bursting septal cells is divisible into two groups based upon the effect o f atropine on the cells' firing patterns (Stewart and Fox 1989). Atropine-sensitive septal cells lose their rhythmicity after doses of atropine sufficient to eliminate the hippocampal theta rhythm. Atropine-resistant cells continue to discharge rhythmically in the absence o f any theta rhythm. Our proposed model of the septo-hippo- campal connections suggests that the septal "pace- maker" mechanism involves the rhythmic excitation and inhibition of h ippocampal interneurons (theta cells) by atropine-sensitive and atropine-resistant rhythmic septal cells, respectively. These rhythmic inputs to the theta cells cause the theta cells to discharge rhythmically, resulting in the modula t ion of pyramidal and granule cell firing th roughout the hippocampus and ultimately, resulting in the E E G theta rhythm. The phases of firing of the rhythmic septal cells, however, were not discussed. A descrip- tion of the phases of firing of the rhythmic cells of the septum is essential for a complete understanding of the "pacemaker" role of the medial septal nuclei.

This repor t describes the firing properties of neurons in the rat medial septal nucleus and vertical limb of the nucleus of the diagonal bond of Broca (MSN-NDB) in relation to the hippocampal theta rhythm. It differs in several respects f rom previous reports (Gogol/tk et al. 1968; Macadar et al. 1970; Apostol and Creutzfeld 1974; Gaztelu and Bufio 1982; Alonso et al. 1987). First, the activities of identified atropine-sensitive and atropine-resistant rhythmic septal neurons were recorded in relation to the theta rhythm. Identification of the preferred phases of firing of the cells in each of these two groups is necessary, since systematic phase differ- ences between the two groups may exist in light o f our suggestion that atropine-sensitive cells are excit-

a tory and atropine-resistant cells are inhibitory. Second, the firing o f "non- rhy thmic" M S N - N D B neurons in relation to the theta rhy thm was deter- mined. Third, bo th CA1 and dentate phases of the theta rhy thm were recorded in order to permit an accurate summary of the phase data, and finally, urethane anesthesia, without curare or eserine, was used to eliminate significant confounding variables and permit addit ion of this data to the existing large pool of electrophysiological data for the urethane- anesthetized rat. A por t ion of this work has been published in abstract form (Stewart and Fox 1987).

Material and methods

Male Sprague-Dawley albino rats (310-450 g) were anesthetized with urethane (1.0 g/kg iv or 1.Sg/kgip) and prepared with a tracheal cannula for connection to a source of moisturized air. When animals received urethane intravenously, they were fitted with the tracheal tube and intra-jugular cannula under ether anesthesia. The ether was gradually replaced with the calculated dose of urethane. Small supplemental doses of urethane were given, as needed, during the experiments.

Animals were held in Kopf stereotaxic frame and maintained at 37~ with an isothermal heating pad.(Braintree Scientific, Braintree, MA) and/or a shielded heating lamp. In the 48 rats in this study, small holes were drilled through the skull and enlarged when necessary with a rongeur. The dura at the base of a hole was carefully removed and the exposed brain was covered with Vaseline.

All implants were made using stereotaxic coordinates (Paxinos and Watson 1982). Unit recording electrodes (1-2 Mf~ teflon-insulated stainless steel, Microprobe Inc., Clarksburg, MD or ~ 1 Mf~ glass-insulated tungsten, Amassian et al. 1962) were lowered through the septal nuclei (AP level 9.2 9.8 mm anterior to interaural line) at an angle of 20 ~ offthe vertical in the coronal plane. Monopolar recordings of the hippocampal EEG were taken from each of a pair of stainless steel wires (125 # diameter) that were varnish insulated and cut square. The tips were vertically separated by about 1 mm and referred to a screw over cerebellum. The EEG electrode pair was lowered vertically (AP 4.7, ML 3.0) until the theta rhythm recorded on the deeper electrode was phase reversed with respect to that on the super- ficial electrode, and was maximal in amplitude. The deeper electrode tip was then near the hippocampal fissure (referred to here as "dentate" EEG) and the superficial electrode was dorsal to the pyramidal cell layer of CA1 (referred to here as "CAI" EEG) (Green and Rawlins 1979).

During experiments, extracellularly recorded medial septal units were identified on the basis of spontaneous firing patterns and stereotaxic coordinates. When stimulating electrodes were placed in the fimbria/fornix, units were occasionally driven antidromically. Electrode locations for the last unit on each pass were marked at the ends of the experiments by passing current through the recording electrodes and identifying the lesions directly or with the Prussian blue reaction. Units not at the lesion site were located by measuring back from a lesion in Nissl- stained 40 # frozen sections. All units were confirmed to be within the medial septal nucleus - nucleus of the diagonal band of Broca (vertical limb).

Atropine sulfate (Sigma) was prepared as a 25 or 50 mg/ml solution in water for intravenous (25 100mg/kg) or intra- peritoneal (50 mg/kg) injection into 23 of the 48 rats. Post- atropine recordings of septal unit activity and concurrent EEG

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were started at least five minutes after the full dose of atropine had been given and lasted for 10 min to one hour.

Unit activity and EEG recordings were amplified, filtered (single units: bandpass = 500 Hz to 10 kHz; - 2 4 dB/octave high pass, - 6 dB/octave low pass. EEG: bandpass=0.1 Hz to 10 kHz; - 6 dB/octave rolloffs), and stored on tape for off-line analysis. Phase histograms were constructed using either a DEC PDP 11/45 or a Hewlett Packard 9836U computer system. A detailed description of the construction of phase histograms has been published (Fox et al. 1986). Briefly, theta scores (Stewart and Fox 1989), estimates of rhythmic quality based on autocorrelations of cosine-windowed 1.024 seconds epochs of EEG made every 64ms, were used to identify periods of rhythmic theta frequency activity. Peaks of identified theta cycles were detected, and acceptable theta cycles were those whose periods fell within pre-set time windows (typically 176 272 ms). Cycles were averaged by normalizing slow wave voltage data and spike data collected during each theta cycle, redistributing them into 32 histogram bins. Final scaling yielded the slow wave voltage at each 11.25 degree segment of the average theta cycle. Significant phase-locking of the un i t to the theta rhythm was determined by the Runs and Rayleigh tests (Batschelet 1981; Fox et al. 1986). In order to generate a stable reference point on an averaged cycle of the theta rhythm, the minimum value of the averaged cycle was subtracted off to force the minimum to zero and permit the determination of a "mean phase". If the theta rhythm were actually sinusoidal, then adding an offset equal to the amplitude of the sinewave yields an expression of the form: r = a + a cos ~b. Equations of this form produce a cardioid on a polar plot and the mean phase is identical to the phase of the positive peak. Since the averaged theta cycles are not always as symmetrical as sine waves, small variations in the phase angles of the mean phase and the positive peak are common. The absolute difference between these values for the dentate theta rhythm in 176 recording samples was 8.0_+ 5.7 degrees (mean + std. dev.). The mean phase for the dentate theta rhythm was used as the reference for summarizing the mean phases of the spike histo- grams throughout this paper. In the same group of 176 record- ings, it was found that the absolute difference between the mean phases for the dentate and the CA1 theta cycle averages was 181.3 __ 37.3 degrees.

Results

Cell types

A total of 160 cells from the medial septal nucleus - vertical limb of the nucleus of the diagonal band of Broca (MSN-NDB) were recorded from 48 rats. The number of cells with signal to noise ratios of greater than 3 : 1 that were recorded from a single rat ranged from one, in some experiments where atropine was injected, to a maximum of 12. In addition to the well known rhythmically bursting cells of the MSN-NDB, two other cell types were identified on the basis of their spontaneous firing patterns and relations to the hippocampal theta rhythm. These three cell types are illustrated in Fig. 1.

"Clock" cells fired single spikes at high rates with a regular, periodic pattern. This is apparent in the interspike interval distribution (IID) for the "clock"

cell in Fig. 1. Their average firing rate for the 15 recorded cells of this type was 38.2_+ 12.2 spikes per second during theta rhythm. Stable non-theta firing rates were obtained for four of the cells. Bouts of non-theta EEG were relatively rare in all of these animals. Firing rates during theta rhythm were slightly higher than the corresponding non-theta rates in three of the cells. The percent increases of theta rates over non-theta rates were 6%, 10%, and 35%. The percent decrease in the firing rate of the fourth cell was 13%. The firing of "clock" cells was generally unrelated to the phase of the theta rhythm (see below). Orthodromic driving from the fimbria/fornix could be demonstrated in 4 of 6 cells tested, with latencies ranging from 7-40 ms. None of the cells of this type appeared to be driven antidromically.

The second non-rhythmic cell type, referred to as "irregular" cells, fired single spikes with a pseudo- random pattern. The l iD (Fig. 1) for an exemplary "irregular" cell is nearly a Poisson distribution. The firing rates of these cells were considerably slower than either the rhythmically bursting cells or the "clock" cells. The average firing rate for the 17 recorded cells of this type was 8.1 +6.5 spikes per second during theta rhythm. In 6 cases, stable non- theta firing rate measurements were possible. Firing rates during theta rhythm for 3 of these cells were higher (with percent increases of 4%, 26%, and 32%), and theta rates for the other 3 were lower (with percent decreases of 12%, 36%, and 49%) than their corresponding non-theta rates. These cells could occasionally be seen to fire in bursts with long inter-burst intervals. This occurred only during bouts of non-theta EEG. Averages triggered on the theta rhythm revealed that 10 of 17 of these cells had a phase relation to the theta rhythm (see below). Orthodromic driving of 3 of 9 cells was demon- strated to fimbria/fornix stimulation with latencies ranging from 2.5 to 4.8 ms. In one cell, antidromic driving with collision was identified. The latency of this driving at an intensity of 1.2 times threshold was 6.8 msec (collision interval = 8.0 ms; refractory period = 2.9 ms).

One hundred twenty-eight rhythmically bursting MSN-NDB cells were recorded, 22 of these were confirmed to be atropine-resistant and 11 were con- firmed to be atropine-sensitive. The remaining 95 rhythmic cells were not tested with systemic atropine because generally, only one cell can be tested per animal, since the effects of large systemic doses of atropine last for hours under urethane anesthesia (Stewart and Fox 1989). The average firing rate during theta rhythm for all 128 rhythmi- cally bursting cells was 26.1 • 14.9 spikes per second

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Fig. 1. Features~fthreeprin~ipa~ce~t~peswithinth~media~septa~nuc~eusandnuc~eus~fthediag~na~band~fBroca(vertica~im~). Each column shows two seconds of spontaneous firing of a representative cell along with the hippocampal EEG from the "CA I" and "dentate" recording sites, an interspike interval distribution (IID) for that cell, and a phase histogram for that cell. The IIDs reflect activity of the cells during hippocampal theta rhythm. An atropine-resistant rhythmically bursting cell is illustrated in the left column. Its rhythmic bursting is seen in the top trace (top panel) along with " C A I " EEG (middle trace of top panel) and "dentate" EEG (bottom trace of top panel). Notice the bimodal distribution of intervals in the IID (middle) reflecting the intra-burst and inter-burst intervals, and the unimodal distribution of spikes on an average cycle of the theta rhythm in the phase histogram (bottom). Atropine- resistant and atropine-sensitive rhythmic cells do not differ in their appearances in any of the three displays. Calibrations for top panel: 0.5 mV for the ceil, 1.0 mV for the EEG; 0.5 s. IID (middle panel): number of spikes= 6072, firing rate=26.6/s; bin width =2.5 ms. Phase histogram (bottom panel): number of spikes = 1737; number of theta cycles = 271. In the middle column are similar panels for a "clock" cell. Note the unimodal, "normal" distribution of intervals in the IID and the statistically flat distribution of spikes on an average theta cycle. These cells fired at a constant high rate, unrelated to the phase of the theta rhythm. Calibrations for top panel: 0.7 mV for the cell, 0.8 mV for CA1 EEG and 1.0 mV for dentate EEG; 0.5 s, IID (middle panel): number of spikes= 1081, firing rate = 32.2/s; bin width = 2.5 ms. Phase histogram (bottom panel): number of spikes = 445; number of theta cycles = 57. An "irregular" cell is illustrated in the right column. Note the slow firing rate of the cell necessitated the longer time axis for the IID. The total time displayed is 5 s and not 250 ms as had been used for the previous two columns. Notice that in spite of the nearly Poission (pseudo- random) distribution of intervals, there is a clear phase relation of the unit to the theta rhythm. When the cell fires, it is most likely to fire near the positive peak of the dentate theta rhythm. Calibrations for top panel: 0.25 mV for the cell, 0.5 mV for CA1 EEG and 1.0 mV for dentate EEG; 0.5 s, IID (middle panel): number of spikes = 750, firing rate = 2.5/s; bin width = 50 ms. Phase histogram (bottom panel): number of spikes =98; number of theta cycles = 197

(mean -t- std. dev.). For the identified groups of atro- pine-resistant and atropine-sensitive cells, the rates during theta rhythm were 30.0 +_ 15.6 and 22.8 +_ 9.6 spikes per second, respectively. For 33 rhythmic cells (of 128), stable non-theta firing rates could be ob- tained. Seven of the 33 cells were identified as atropine-resistant and 4 were identified as atropine- sensitive. While no significant change in firing rate from non-theta (21.9-t-18.3 spikes/s) to theta

(26.3 4-13.8 spikes/s) could be demonstrated for the group of rhythmically bursting cells, it was typically the case that the firing rate of these cells increased somewhat from non-theta to theta. The percent increase in firing rate from non-theta to theta for the identified atropine-resistant cells (N=7) was 44% while that for the identified atropine-sensitive cells (N =4) was 74%. Fourteen out of the 50 rhythmic cells tested for driving from the fimbria/fornix could

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Fig. 2. Summary distributions of the preferred phases of firing for 128 rhythmically bursting septal ceils relative to the dentate theta rhythm mean phase (0~ The dis- tribution of phases for all cells is shown in the top left. Chi-square and Rayleigh tests showed this distribution to be indistinguish- able from flat (random). Of this group of 128 cells, separate dis- tributions were made for atropine-resistant cells (top, right), atropine-sensitive cells (bottom, right), and anti- dromically driven cells (bottom, left)

be driven antidromically with collision. In general, the latencies were short, averaging 0.7_+0.1 ms for atropine-resistant cells and 1.1 + 0.4 ms for atropine-sensitive cells. The overall range was from 0.5 ms to as long as 9.0 ms, and reflects, to some extent, variation in the placement of the stimulating electrodes. None of the cells appeared to be driven orthodromically from the fimbria/fornix.

Often the sampling of MSN-NDB cells was intentionally biased and therefore prevented calcu- lation of the proportions of the three cell types. In 13 rats, however, all of the 49 cells that could be isolated (> 3:1 signal to noise) were recorded and classified. The resulting proportions of each of the three cell types was as follows: 38 of the 49 (78%) were rhythmically bursting cells; 4 of the 49 (8%) were "clock" cells; and 7 of the 49 (14%) were "irregular" cells.

Firing relations to the phase of the hippocampal theta rhythm

Rhythmic cells. The rhythmically bursting cells of the MSN-NDB have been known to show a relation to the hippocampal theta rhythm since their dis- covery by Petsche et al. (1962). The phase histo- grams of all 128 rhythmically bursting cells showed statistically significant phase-locking to the hippo- campal theta rhythm using both Runs and Rayleigh tests. The preferred phases of the rhythmically burst- ing septal cells are summarized in Figs. 2 and 3. The distribution of the mean phases of the histograms relative to the mean phases of the dentate theta

90

�9 atropine-resistant �9 atropine-sensitive O not tested with atropine 270

Fig. 3. Polar scatter plot of preferred phases for rhythmic cells versus mean vector lengths for the individual units. Distance from the origin of this plot represents mean vector length, where the outside circle is 1.0. The mean phases for the units are plotted as deviations from the dentate theta rhythm mean phase (0~ Filled circles represent identified atropine-resistant cells (N = 22), filled triangles represent identified atropine-sensitive cells (N = 11), and open squares represent the remaining cells which were not tested with atropine (N = 95)

rhythm is plotted in Fig. 2 for all rhythmically bursting septal cells (top left) and separately for the atropine-resistant (top right) and atropine-sensitive cells (bottom right). Those cells which were driven antidromically from the fimbria/fornix comprise a fourth histogram (bottom left). The mean phase of the dentate theta rhythm (0 ~ deviated from the positive peak only if the averaged dentate theta wave was asymmetrical (see Methods). The distribution

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containing all rhythmically bursting cells was not statistically different from flat (random) when tested with the chi-square or Rayleigh tests. Reyleigh tests of the remaining three distributions failed to identify peaks in any of them. It does however, appear that there is a tendency for the distributions of atropine- resistant and atropine-sensitive rhythmic cells to be shifted somewhat with respect to one another. The means of the two distributions are separated by about 53 degrees (mean for atropine-resistant cell distribution=9.5~ mean for atropine-sensitive cell distribution = -43.2~ but there is insufficient data at this point to be certain of differences.

To account, to some degree, for the tightness of grouping of spikes on the averaged theta cycle, the

length of the mean vector for each cell was calcu- lated. If all of the action potentials from a cell fell within a single bin on the histogram, the mean vector length would be 1.0. If instead, the action potentials were distributed uniformly across all 32 bins, the mean vector length would be zero. The mean vector length therefore indicates the degree of modulation of the firing rate of a cell. In Fig. 3, the mean vector length is plotted against the preferred phase for each of the 128 rhythmically bursting cells. Atropine- resistant and atropine-sensitive cells are indicated with different symbols. The lack of significant clustering of preferred phases or mean vector lengths is apparent. Average mean vector lengths were 0.43 +_0.16 for the entire group of 128 rhythmically

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Fig. 4. Phase histograms for 12 rhythmical- ly bursting septal cells recorded in two pas- ses in the same rat demonstrate variability in the shapes of the histograms and shifts in preferred phase as a function of recording location. The preferred phases of firing for the cells in pass no 2 (right) are more simi- lar than those in pass no 1 (left). These trends were entirely unpredictable given the stereotaxic or histological location of the re- cording site. Electrode tracks were 20 ~ off the vertical in the coronal plane and aimed for a spot near the border between vertical and horizontal limbs of the nucleus of the diagonal band of Broca. This is indicated in the coronal sketch at the bot tom of the right column. Calibration bar at the bottom of the sketch = 1.0 mm. Distances of record- ing locations from the target (lesion lo- cation) are given in microns to the right of each histogram. At two depths, one on each pass, two cells were isolated. These pairs of cells are enclosed with brackets. An average cycle of the dentate theta rhythm for this rat is shown below the fifth cell in pass no 2 (right column). Zero degrees indicates the dentate positive peak in this figure. Passes were separated by 200 # in the AP direction

bursting cells, and 0.52_+0.15 and 0.42___0.18 for atropine-resistant and atropine-sensitive cells, re- spectively.

-Clustering of preferred phases could not be demonstrated when the rhythmic cells were sorted on the basis of the antero-posterior, medio-lateral, or dorso-ventral coordinates of their locations within the MSN-NDB. To some extent, this can be seen in Fig. 4 which shows individual histograms for 12 cells recorded along 2 electrode tracks in a single rat. The phase relations of the different cells followed no apparent pattern. At two depths, two cells were isolated simultaneously on the micro- electrode. The mean phases for the pair of cells on the first pass are shifted by 154 ~ while in the second pass the pair of cells are shifted by only 39 ~ A total of eleven such cell pairs were recorded and the average difference in phase between cells of a pair was 60.8_+42.7 degrees and ranged from 1 to 154 degrees. It is interesting to note that the smallest shift (1 ~ was between a pair of cells which were both atropine-sensitive. Although these two cells were in phase with one another, no short latency (< 2.0 ms) interactions could be detected by cross-correlation of the spike trains. Phase shifts in pairs that con- tained at least one atropine-resistant cell were larger: atropine-resistant/atropine-resistant= 17 ~ , 62 ~ , and 81~ atropine-resistant/atropine-sensitive=62 ~ and 65 ~ .

Other cells. Twelve of the 15 "clock" cells showed no significant relation to the theta rhythm. The remain- ing three did show a statistically significant modu- lation of their firing by the Runs and Rayleigh tests. The preferred firing phases for these three cells were - 8 9 ~ + 130 ~ and + 141 ~ with respect to the mean

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Fig. 5. Summary distribution of the preferred phases of firing for 10 "irregular" MSN-NDB cells phase-locked to the theta rhythm. Note the peak in the distribution near the dentate positivity (0~ This distribution is strikingly similar to the distribution of phases for CA1 complex-spike cells (Fox et al. 1986) and may reflect feedback onto the "irregular" cells by the complex-spike cells

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phase of the dentate theta rhythm. The average mean vector length for all "clock" cells was 0.06_+0.07 and was only 0. t6+0.14 for the three cells with statistically significant phase-locking. The mean vector lengths indicate that this theta modu- lation was never nearly as pronounced as the degree of modulation in the rhythmically bursting cells.

The "irregular" cells were much more likely to show a significant phase relation to the theta rhythm. Ten of 17 of these cells were significantly phase-locked. The distribution of preferred phases for the "irregular" cells is shown in Fig. 5. There is a clear peak in this distribution near 0 ~ (dentate theta positivity). The mean vector lengths reflect this phase-locking: average mean vector length for all "irregular" cells was 0.22 ___ 0.14, and for the group of significantly phase-locked cells was 0.31 ___ 0.15.

Discussion

Since the initial report of Petsche et al. (1962), the rhythmically bursting cells of the medial septal nuclei have been extensively studied in an effort to characterize their role in the production of the hippocampal theta rhythm. This paper describes the firing properties of two "non-rhythmic" cell types within the medial septal nucleus and the vertical limb of the nucleus of the diagonal band (MSN- NDB). In addition, the rhythmically bursting septal cells as well as both of the other cell types are described in relation to the hippocampal theta rhythm.

Cell types

Three distinct cell types within the MSN-NDB can be defined by their firing patterns as well as their firing relations to the hippocampal theta rhythm. In addition to the rhythmically bursting neurons for which the medial septal nuclei are known, "clock" cells and "irregular" cells can also be recorded. "Clock" cells fire at continuously high rates; they are never seen to fire in bursts; and their firing generally bears no phase relation to the ongoing hippocampal theta rhythm. "Irregular" cells fire at much lower rates; they can occasionally be seen to fire in isolated bursts, but only during non-theta EEG; and their firing frequently has a significant phase relation to the theta rhythm.

A number of other reports have described "non- rhythmic" cells in the medial septal nuclei (Petsche et al. 1962; Ranck 1976; Wilson et al. 1976; Assaf and Miller 1978; Lamour et al. 1984). In general, the

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non-rhythmic cells of these reports appear similar to what we have called "irregular" cells, although some of the "non-bursting" cells reported by Lamour et al. (1984) may have been "clock" cells. Ranck (1976) recorded cells in the MSN-NDB of freely moving rats and classified them on the basis of firing pattern as well as their behavioral correlates. Ranck's "constant-firing" cells and "tight-group" cells appear to be completely analogous to "clock" cells and "irregular" cells in the urethane anesthe- tized rat. In fact, the relative proportions of these cell types in the two preparations are fairly similar: "constant-firing" and "clock" cells comprised 11% (awake) and 8% (urethane); "tight-group" and "irregular" cells comprised 25% (awake) and 14% (urethane); and rhythmic cells comprised 50% (awake) and 78% (urethane). The "clock" and "ir- regular" labels used in this report are better de- scriptors than the labels, "constant-firing" and "tight-group". The "constant-firing" label is ambig- uous, at least for our cells, because none of the three categories of cells showed dramatic changes in firing rate between theta and non-theta EEG states. In our urethane anesthetized rats, long periods of non-theta EEG were uncommon. "Irregular" cells discharged in bursts or "tight-groups" only during non-theta EEG, but recordings of such bursts were not necessary for the classification of these cells.

Electrophysiologically, the projection of the MSN-NDB to the hippocampus appears to consist predominantly of rhythmically bursting neurons, the antidromic driving of "clock" cells and "irregu- lar" cells apparently being rare in comparison with the driving of rhythmically bursting neurons (Assaf and Miller 1978; this report). Both atropine- resistant and atropine-sensitive rhythmic cells ap- pear to project to the hippocampus (Stewart and Fox 1989). Only 1 out of 9 "irregular" cells could be antidromically driven from the fimbria/fornix. Three others could be orthodromically driven at short latency, suggesting monosynaptic activation. In fact, the phases of firing of these cells suggest that they may be firing in response to feedback from CA1 complex-spike cells (see below). In comparison, 4 of 6 "clock" cells were orthodromically driven, but none was antidromically driven. The orthodromic driving of these cells was long latency and was presumably mediated by a multisynaptic pathway. Lamour et al. (1984) were apparently able to back- fire a considerably greater proportion of non-rhyth- mic cells. Without recordings of hippocampal theta rhythm for confirmation of the EEG state, it is not clear whether or not the "non-rhythmic" cells that they drove would have been rhythmic during bouts of hippocampal theta rhythm.

Although statistically significant changes in firing rate from non-theta to theta EEG states could not be detected for the rhythmically bursting cells, one point warrants mentioning. Nearly two-fold increases in firing rate are "characteristic" of hippo- campal theta cells in freely moving rats (Ranck 1973) and similar increases have been observed in urethane anesthetized rats (personal observation). Our recently proposed model of the "pacemaker" mechanism (Stewart and Fox 1989) suggests that two separate groups of rhythmically bursting septal cells send projections to the hippocampus, one excit- atory and the other inhibitory, which end on inter- neurons (theta cells). It is interesting that the percent increase in firing rate from non-theta to theta for the atropine-sensitive (excitatory) septal cells was greater than that for the atropine-resistant (inhibi- tory) septal cells: 74% compared to 44%. If the excitatory and inhibitory roles of atropine-sensitive and atropine-resistant septal cells that were de- scribed in the model are true, this difference in rate increases can account for the increase in firing of hippocampal theta cells.

Firing relations to the phase of the hippocampal theta rhythm

Rhythmic cells. Distributions of the preferred phases of firing for all recorded rhythmically bursting cells, for the atropine-resistant bursting cells, for the atropine-sensitive bursting cells, and for the anti- dromically driven bursting cells showed no clear peaks. In fact, chi-square and Rayleigh tests of the distribution of the firing phases for all rhythmic cells, and Rayleigh tests of the other three distri- butions, showed that the distributions were indis- tinguishable from distributions that were flat or random. Sorting the firing phases of cells on the basis of their antero-posterior, medio-lateral, or dorso-ventral locations within the MSN-NDB also failed to reveal a distinct peak or peaks in the distributions of preferred firing phases. It did appear that there might be a difference between the distri- butions of atropine-resistant and atropine-sensitive cells. Such a difference could conceivably account for the lack of a peak in the distribution of preferred firing phases for the entire population of rhythmic septal neurons. However, insufficient phase data exist for identified atropine-resistant and atropine- sensitive cells to confirm a significant difference between these two groups.

A number of other reports of the relations of rhythmic septal units to the hippocampal theta rhythm have been presented (Gogolfik et al. 1968;

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Macadar et al. 1970; Apostol and Creutzfeldt 1974; Gaztelu and Bufio 1982; Alonso et al. 1987). This report differs from those in several respects. None had data on the phases of firing of atropine-resistant and atropine-sensitive septal cells. With the excep- tion of Alonso et al. (1987) all of the animal prepa- rations used were treated with eserine or curare, or both. While eserine is commonly used to drive the theta rhythm, we have seen that it can adversely affect the rhythmicity of the bursting septal cells (unpublished observations), and curare has clear effects on the depth profiles of the hippocampal theta rhythm (Winson 1976). In the other studies, it was often the case that the theta rhythm recordings were not controlled for shifts in its phase with depth within the hippocampus. This would make it impossible to describe the preferred firing phases for a population of septal ceils recorded in different animals.

Unfortunately, it seems that the data on the phases of firing of rhythmic septal cells will not by themselves provide an explanation of the role of the septum in the production of the theta rhythm. The lack of a clear peak in the distribution of firing phases might, in fact, be used to support a gating function for septo-hippocampal connections rather than a pacemaker role. Consistent with this notion is the finding of "theta rhythm" in hippocampal slices when carbachol is added to the bath (Konopacki et al. 1987). The increased release of acetylcholine within the hippocampus by the septal cells, by itself, may be sufficient to permit oscillation within some intrinsic hippocampal circuitry. On the other hand, we have found that a change in the firing pattern from rhythmic to non-rhythmic in the population of atropine-sensitive (presumed cholinergic) septal neurons is sufficient to eliminate the hippocampal theta rhythm in rats anesthetized with urethane (Stewart and Fox 1989). This suggests that the theta rhythm, at least in urethane anesthetized rats, is normally paced by the rhythmic activity in the cholinergic septal cells.

Given the hypothesis that the atropine-sensitive and atropine-resistant septal cells rhythmically excite and inhibit hippocampal interneurons, the variability in the firing phases for the population of hippocampal interneurons (theta cells) taken across all hippocampal subfields could have been used to predict variability in the firing phases for the popu- lation of septal cells. In CA1, the phases of firing of theta cells are tightly grouped (Fox et al. 1986). The septal cells projecting to CA1 may be scattered amongst septal cells projecting to CA3 or to dentate, and the preferred firing phases for those cells projec- ting solely to CA1, for example, might show a

peaked distribution. Variability in firing phases of the group of septal cells projecting to any particular hippocampal subfield could be reduced within the population of theta cells by integration of their septal inputs. Determination of the phases of firing of septal cells identified as projecting to CA1, for example, would address such sorting directly.

Other cells. The firing relations of the "non-rhyth- mic cells of the MSN-NDB have not been described before. One of the advantages of the phase histo- gram, in comparison to other correlation methods, for determining the relation of a unit to the theta rhythm is best seen when the unit's firing rate is less than one spike on a given cycle of the theta rhythm (i.e. less than 4 to 5 per second in urethane anesthe- tized rats). By constructing phase histograms for all recorded cells, it was found that a large fraction of the "irregular" cells were statistically "phase- locked" to the theta rhythm. In general, their phases appeared to be grouped near the dentate positivity. This is identical to the phase of firing of CA1 complex-spike cells (Fox et al. 1986). Given that a number of "irregular" cells were orthodromically driven from the fimbria/fornix at short latency, and that the afferents to septum from CA1 are dis- tributed medially within the septum (Raisman 1966; DeFrance et al. 1971; Swanson and Cowan 1977), it is possible that the phase-locked firing of the "irreg- ular" cells is due to their excitation by hippocampal complex-spike cells and is not directly related to the firing of the rhythmically bursting MSN-NDB cells. Manipulations that would eliminate the phase- locked firing of hippocampal neurons, such as cooling or cutting the fimbria/fornix or large doses of atropine might be used to confirm this suggestion.

It appears that the principal projection cells of the MSN-NDB are the rhythmically bursting cells. This group can be subdivided into atropine-resistant and atropine-sensitive cells (Stewart and Fox 1989). In spite of the phase-locked firing of a number of "irregular" cells in the MSN-NDB it is unlikely that these cells make a substantial contribution to the theta rhythm in the hippocampus. Rather, "irregu- lar" cells may reflect the feedback of hippocampal complex-spike cells onto the septum. It is however, important to consider the role of "clock" cells and "irregular" cells in contributing to the activity of the rhythmically bursting septal cells. Before a complete picture of the connections required for the theta rhythm can be drawn, we will need to understand the interconnections between the four (or more) types of cells in the MSN-NDB and their contributions to the "pacemaker" activity of the medial septum.

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Acknowledgements. Supported by National Institutes of Health grants NS 17095 to S.E.F. and NS 07117 (neurophysiology training grant).

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Received January 30, 1989 / Accepted April 4, 1989