 |
INTRODUCTION |
The medial septum and the diagonal band of Broca nuclei (MS-DBB) contain cholinergic (Amaral and Kurz 1985
; Wainer et al. 1985
) and GABAergic neurons (Kölher et al. 1984
; Onteniente et al. 1986
) that project to the hippocampal formation. A significant proportion of these septohippocampal neurons (SHN) displays a rhythmically bursting (RB) activity that appears to be essential for the generation of hippocampal theta (Lamour et al. 1984
; Petsche et al. 1962
). MS-DBB lesions abolish hippocampal theta (Buzsaki et al. 1983
; Petsche et al. 1962
; Rawling et al. 1979
). Frequencies of RB septal neurons and hippocampal theta are tightly coupled and shift in parallel according to behavioral and sleep-waking states (Gaztelu and Buno 1982
; Morales et al. 1971
; Ranck 1976; Sweeney et al. 1992
). Rhythmically bursting activity in the SH pathway has attracted interest because there is evidence that it is involved in learning and memory (see references in Dutar et al. 1995
; Oddie et al. 1996
). MS-DBB lesions that eliminate hippocampal theta induce spatial memory deficits in rats (Winson 1978
). Aged rats with behavioral deficits exhibit altered electrophysiological properties of RB septal activity (Lamour et al. 1989
).
On the basis of pharmacological studies, two types of theta activity have been described (Kramis et al. 1975
). Type 1 theta, associated with voluntary movement, is resistant to atropine or to cholinergic depletion. Type 2 theta, sometimes present during immobility, is abolished by atropine and occurs spontaneously during urethan anesthesia. Serotonin (Vanderwolf and Baker 1986
) and
-aminobutyric acid (GABA) (Stewart and Fox 1989a
) play a role in type 1 theta generation. The cholinergic mediation of type 2 theta is supported by numerous observations made in the septum as well as in the hippocampus (see references in Smythe et al. 1992
). Nevertheless, more recent findings show that the former distinction between type 1 and type 2 theta needs to be reassessed. In fact, both types of theta have atropine-sensitive and resistant components (Brazhnik and Vinogradova 1986
; Stewart and Fox 1989a
), and type 1 and type 2 theta are still present after selective lesions of the cholinergic septohippocampal neurons (Bassant et al. 1995
; Jenkins et al. 1993
; Lee et al. 1994
).
Recently, the idea that the hippocampal theta results from the coactivation of cholinergic and GABAergic MS-DBB inputs has emerged (Smythe et al. 1992
; Stewart and Fox 1990
). Two populations of RB neurons have been identified in MS-DBB, based on the differential effect of atropine (Stewart and Fox 1989b
). Cells that lose RB activity after atropine might be cholinergic, whereas cells that retain RB activity might be GABAergic. One way to identify the rhythmically bursting SHNs as cholinergic or GABAergic neuronsis to lesion selectively one population of MS-DBB neurons and to look for the consequences of the lesion on RB activity. In the present study, septal unit activity and hippocampal theta rhythm have been recorded in rats after selective destruction of the cholinergic SHNs by the immunotoxin 192 IgG-saporin (Book et al. 1992
, 1994
). Experiments have been performed in urethan-anesthetized and unanesthetized rats.
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METHODS |
Animals
Thirty-two male Sprague-Dawley rats (Charles River, France) weighing 280-320 g were used. The rats were housed two by cage on a 12 h/12 h light/dark cycle with food and water available ad libitum.
Immunotoxin injection
192 IgG-saporin (Chemicon International) was prepared as previously described (Wiley et al. 1991
), dissolved in phosphate-buffered saline, and stored frozen (
70°C) until used.
Animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and placed in a stereotaxic apparatus. Two holes were drilled in the skull for 192 IgG-saporin injection in the right and left ventricles (coordinates: 1.2 mm anterior to bregma, 1.1 mm lateral to midline, 4.8 mm below the cortical surface). Pressure injections were performed through a glass micropipette (tip diameter 40 µm) attached to a 10-µl Hamilton syringe. Seventeen rats were injected with 192 IgG-saporin (total amount 4 µg in 10 µl of 0.9% saline) at the rate of 1 µl/min. The micropipette was allowed to remain in place for 10 additional minutes and was then slowly withdrawn from the brain. Control animals (n = 15) received an equivalent amount of saline. All the injected rats survived without loss of weight.
Surgery
URETHAN-ANESTHETIZED RATS.
Ten to 16 days after injection, rats were anesthetized with urethan (1.5 g/kg) and placed into a stereotaxic frame. The skull was opened above the MS-DBB area [AP = 8 to 9 anterior to interaural line, L = 0, according to the atlas of Paxinos and Watson (1986)
]. Electrocorticographic activity (ECoG) was recorded from a stainless steel jeweler's screw, inserted into the skull in contact with the dura overlying the right parietal cortex (4.5 posterior and 6 lateral to bregma). Another screw placed on the occipital crest served as reference. A bipolar electrode (twisted Teflon-coated tungsten wire, uninsulated tips separated 0.2 mm in depth) was implanted in the dorsal hippocampus (3.5 posterior and 4 lateral to bregma, depth: 2.7 mm below the dura). Hippocampal theta was recorded monopolarly from each wire with respect to the reference electrode. A stimulating electrode was placed in the fimbria-fornix (Fb-Fx) (AP = 7.7, H = +6, L = 1, coordinates relative to interaural zero) to identify the SHNs by their antidromic response to the Fb-Fx electrical stimulation.
UNANESTHETIZED RATS.
Rats were anesthetized with pentobarbital (60 mg/kg) and placed into a stereotaxic frame. The first step of the surgery was similar to that described above. In addition, a stainless steel hook soldered to fine flexible insulated silver wires was placed in the neck muscles for recording electromyogram (EMG). All the electrodes were soldered to a connector. A liquid bonding resin (Superbond, Sun Medical, Kyoto, Japan) was applied to the cleaned, dried skull surface. The hardened resin was subsequently covered with a layer of acrylic cement. Two metal tubes, temporarily held in place by anchored bars attached to the stereotaxic frame, were placed transversally slightly above the skull. The tubes were buried in a mound of acrylic cement in continuity with the first cement layer. The opening over the MS-DBB was covered by gelfoam soaked in saline. The well formed by the cement ridges was covered with sealing paste.
Recording sessions
URETHAN-ANESTHETIZED RATS.
Conventional amplification methods were used to record single-unit activity in the MS-DBB from micropipettes filled with 1 M NaCl and 2% Pontamine blue (tip diameter <1 µm, resistance 10-20 M
). Micropipettes allowed recordings with high signal-to-noise ratio (>4) so that neuron singularity was easily assessed by a window discriminator. Antidromic responses to Fb-Fx stimulation (square pulses of 0.2 ms, 40-600 µA) were identified using the fixed latency test and the collision test between spontaneously occurring spikes and the antidromically evoked spike. Electroencephalogram (EEG) was continually monitored while unitary activity was recorded. Recording sites of interest were marked by a local release of dye.
UNANESTHETIZED RATS.
Over a course of 7 days, the rat was gradually habituated to the restraint system. The rat's head was painlessly secured to the stereotaxic frame by inserting the anchored support bars into the ends of the metal tubes. The rat's body was comfortably supported in a hammock. On the day of recording, the dura over the septum was removed; lidocaine hydrochloride (Xylocaine; 0.02%) was applied locally onto the dura to avoid any discomfort. Medial septal neurons were recorded as described in the anesthetized rat. The state of arousal was monitored by EEG during the recording sessions. Slow-wave sleep and paradoxical sleep periods occurred frequently, showing that the immobilization was not painful. Each experiment lasted for three consecutive days, usually with one morning and one afternoon recording sessions of ~3 h each. When returned to its cage, at the end of the recording session, the rat would engage in normal feeding and grooming activities. The head restraint system was well tolerated by the rats, which were able to feed, drink, and sleep normally during the intersessions. Postmortem examination of the digestive track did not reveal any ulcer. Care and use of the animals reported on this study were approved by the local animal care and use committee.
Histology
At the end of the experiment, rats were deeply anesthetized with pentobarbital and perfused through the ascending aorta with 300 ml saline followed by 600 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were dissected out, postfixed at 4°C for an additional 3-4 h in the same fixative, and cryoprotected in phosphate-buffered 30% sucrose for 24 h. The brains were sectionned in the coronal plane on a freezing microtome, from the rostral septum to the cerebellum. Sections (40-µm thick) were collected in phosphate buffer. Brain sections with Pontamine blue deposits were stained with safranin. The preceding and the following sections were stained either for acetylcholinesterase (AChE) according to the method of Mesulam (1982)
or with a 192-IgG monoclonal antibody for the detection of the low-affinity nerve growth factor receptors (NGFr; Oncogen Science). For NGFr immunohistochemistry, sections were incubated in normal horse serum (3% in 0.1 M phosphate-buffered saline, 0.3% Triton X-100) for 30 min followed by the primary antiserum (NGFr antiserum 192-IgG, 4 µg/ml) at 4°C overnight. After incubation in the primary antiserum, sections were washed and incubated 30 min at 20°C in biotinylated secondary mice antisera, washed twice in PBS, and then incubated for 1 h in avidin-biotin-peroxydase complex. After a final wash in PBS, the sections were developed in 1-naphtol ammonium carbonated solution. Sections were mounted on gelatin-coated slides and air dried. The final step was a Crystal violet enhancement as previously described (Lantéri-Minet et al. 1993
). Staining appeared as an intense blue-violet color.
Parvalbumin (PARV) is colocalized with GABA in the septohippocampal projection neurons (Freund 1989
; Kiss et al. 1990
). In some cases, sections were stained with a PARV monoclonal antibody (dilution of the anti-PARV antiserum 1/2500; sections incubated 48 h and developed in 3-,3,'5-,5'-diaminobenzidine) to identify the GABAergic neurons. To check for the efficiency of the cholinergic lesion, a staining for AChE-positive fibers in the hippocampal formation was performed according to the method of Karnovsky and Roots (1964)
.
Data analysis
Electrode penetrations were reconstructed on camera lucida drawings, with respect to the dye deposit. The single-unit activity of neurons located in MS-DBB complex was analyzed as described previously (Sweeney et al. 1992
). The analysis was based on the following 1) Mean spontaneous neuronal activity and interspike interval histogram. 2) Burst parameters; a burst was defined as 2-20 action potentials, with a maximum of 40 ms interval between consecutive action potentials. The minimum interburst interval was set at 80 ms. The burst parameters consisted of the number of spikes per burst, mean interspike interval (ISI) within each burst and interburst interval. 3) Autocorrelation functions, based on the autocorrelogram histogram of Perkel et al. (1967)
. If recurrent events occurred as rhythmic bursts of action potentials, then the autocorrelogram showed periodic sinusoidal-like density peaks: the more regular the RB activity, the higher were the density peaks. To provide an objective classification of RB and non-RB neurons, a "rhythmicity index" was calculated: the autocorrelogram was smoothed out, and maximum and minimum amplitude of the curve were measured, averaged, then divided by the value of the spontaneous activity (imp/s). A higher index denoted more regular RB activity. The cutoff point of the rhythmicity index was set at 0.95 (Jobert et al. 1989
). 4) Latency of SHN antidromic responses.
In the unanesthetized rat, three states of arousal were identified: quiet wakefulness, slow-wave sleep, and rapid eye movement sleep (paradoxical sleep). The neurons were divided into three groups, depending on the state of vigilance observed while they were recorded, and the characteristics of single-unit activity in each group were compared.
Differences in percentages of RB neurons between lesioned and control rats and among states of vigilance were analyzed using
2 tests. Overall spontaneous activity, burst parameters, frequency of the bursts, latency of antidromic activation of neurons recorded from control, and 192 IgG-saporin-lesioned rats were compared by one-way analysis of variance (ANOVA) followed by Scheffé's test. Differences were considered significant if P < 0.05.
 |
RESULTS |
Anesthetized rats
Extracellular recordings were obtained from 356 neurons in control rats (n = 11) and 506 neurons in lesioned rats(n = 13). All the neurons were located in the medial septal nucleus and the vertical limb of the diagonal band of Broca. The mean number of cells recorded per electrode penetration was slightly (but not significantly) smaller in lesioned (9.5) as compared with control rats (10.8). Neurons were divided into two groups: unidentified neurons and SHNs identified by their antidromic response to the electrical stimulation of the Fb-Fx.
Characteristics of the neuronal activity of unidentified neurons are given in the Table 1. Changes in RB activity were observed in lesioned rats; the percentage of RB neurons was significantly lower (5.3 vs. 19.3%, P < 0.0001). The remaining RB activity had a frequency (number of bursts per second) significantly higher as compared with controls (4.4 vs. 3.7 Hz, P < 0.0001). The mean spontaneous activity (msa) for the overall population was lower in lesioned as compared with control rats. This decrease resulted from both the reduced number of fast firing RB neurons and the lower m.s.a. of non-RB neurons.
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TABLE 1.
Electrophysiological characteristics of SHNs and unidentified MS-DBB neurons in control and
lesioned urethan-anesthetized rats
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Characteristics of the neuronal activity of the SHNs are given in Table 1. As observed in unidentified neurons, the percentage of RB SHNs was significantly lower (17 vs. 41.5%, P < 0.001) in lesioned rats. The few SHNs that still displayed RB activity had a burst frequency significantly higher as compared with controls (4 vs. 3.7 Hz, P < 0.001; Fig. 1). The latencies of the antidromic responses of SHNs with a RB activity were significantly shorter in lesioned as compared with control rat (P < 0.05). Indeed, all the RB SHNs in lesioned rats (13 cells) had a latency shorter than 1.4 ms as compared with 59% (19 of 32 cells) in control rats.

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| FIG. 1.
Extensive loss of the medial septum and the diagonal band of Broca nuclei (MS-DBB) cholinergic neurons after intracerebroventricular (i.c.v.) 192 IgG-saporin injection in urethan-anesthetized rats. Top: microphotographs of coronal sections through the MS-DBB in a saline-treated rat (A and B) and a 192 IgG saporin-treated rat (C and D). Sections are stained for acetylcholinesterase (AChE) histochemistry (A-C) and with safranin (B-D). Note the complete loss of AChE-positive neurons and fibers after 192 IgG-saporin injection (C). The Pontamine blue deposits ( ) indicate the place where rhythmically bursting (RB) neurons have been recorded. Note that in the saline-treated rat, a RB neuron has been recorded in a region with many AChE-positive neurons (A), whereas in the 192 IgG-saporin-treated rat, a RB neuron has been recorded in an area devoid of AChE-positive neurons (C). Scale bar: 250 µm. Bottom, left: autocorrelation histograms of the neurons recorded in saline-treated (E) and 192 IgG-saporin-treated rat (F). The RB activity of each neuron is shown by the periodic density peaks of the autocorrelation histograms. The frequency (Fr) of the RB activity is higher in the lesioned as compared with saline-treated rat (4.2 vs. 3.8 Hz). The spike trains shown at the bottom of each autocorrelation histogram is a schematic representation of a part (5 s) of the discharge sequence used for the autocorrelogram computation. Notice that the number of spikes per burst and the burst duration are lower in lesioned (F) as compared with control rat (E; in this case, 3.5 vs. 8.4 spikes/burst and 67 vs. 81 ms, respectively). Right: the hippocampal theta recorded simultaneously is altered both in terms of amplitude and frequency in lesioned (H) as compared with saline-treated rat (G). Autocorrelation histogram ordinate: number of spike occurrences as a function of time after the spike at time 0; binwidth: 5 ms. RDH and LDH, right and left dorsal hippocampus, respectively.
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A comparison of burst pattern of RB SHNs in lesioned and control rats revealed significant differences. In lesioned rats, the number of spikes per burst was lower (5.1 vs. 7.8 in controls, P < 0.01), the burst duration was shorter (79 vs. 114 ms, P < 0.01) and the interburst interval was longer (183 vs. 155 ms, P < 0.05). The mean ISI was not different for the two groups.
Spontaneous hippocampal theta is present in urethan-anesthetized rats but does not occur continuously. It is intermingled with low-frequency activities (2-3 Hz). About 50% of the neurons were recorded in the presence of hippocampal theta and the other half during low-frequency activity. As a rule, RB neurons were recorded during hippocampal theta bouts, but non-RB neurons could also discharge during these periods. In lesioned rats, hippocampal theta activity (EEG) was dramatically altered (Fig. 1). In 5 of 13 rats, theta was almost completely abolished. In the other rats, the amplitude of the theta waves was 30-50% lower as compared with the mean control value and the frequency decreased significantly (2.5 ± 0.1 Hz vs. 3.6 ± 0.2 Hz, mean ± SE, P < 0.001).
Unanesthetized rats
Extracellular recordings were obtained from 167 MS-DBB neurons in control rats (n = 4) and 153 neurons in lesioned rats (n = 4). In unanesthetized rats, it was not possible to determine whether every neuron recorded could be fired antidromically from Fb-Fx. Consequently, the number of SHNs recorded was too small, especially in lesioned rats, for meaningful statistical analysis. Results included both unidentified MS-DBB neurons and SHNs. The msa for the overall neuronal population was significantly lower in lesioned as compared with control rats (10.1 ± 1.1 vs. 14.6 ± 1.1 imp/s, P < 0.01; Table 2). This difference was mainly due to the loss of RB neurons possessing a high level of activity. The percentage of RB neurons, indeed, decreased greatly in lesioned rats (3.3 vs. 21%, P < 0.001). At variance with the results obtained in urethan-anesthetized rats, there was no difference in the frequency of RB activity recorded in unanesthetized lesioned and control rats. In the absence of anesthesia, the msa and the burst frequency of the RB neurons were significantly higher as compared with urethan conditions (P < 0.01 and P < 0.001) both in control and lesioned rats. This result is in agreement with previous observations in unanesthetized rats (Sweeney et al. 1992
).
States of arousal were monitored while recording MS-DBB single-unit activity. Our experimental procedure does not allow arousal associated with movements. Under these conditions, spontaneous bouts of hippocampal theta were rare. Periods of theta rhythm associated with hypervigilance were induced by repeated noise (whistle). Three states of arousal were identified: wakefulness (W; high-frequency low-voltage EEG activity with bouts of hippocampal theta, sustained EMG activity), slow-wave sleep (SWS; high-amplitude slow waves intermingled with spindles), and rapid eye movement sleep [REM sleep; hypersynchronous theta waves, loss of neck muscle tone (EMG), jerks of jaw muscles and vibrissae at the end of the episode]. During W as well as during REM sleep, the frequency of theta rhythm was not significantly altered in lesioned rats as compared with controls (5.3 ± 0.7 vs. 5.7 ± 0.8 Hz and 6.3 ± 0.3 vs. 7 ± 0.9 during W and REM sleep, respectively), in agreement with our previous results obtained in freely moving rats (Bassant et al. 1995
). The average amplitude of theta recorded during W decreased by 25% in lesioned rats. The REM sleep-associated theta activity was virtually unchanged (hypersynchronous waves with high amplitude and high frequency) in three of the four lesioned rats, but the frequency was sometimes reduced during a brief period of time then recovered to its previous value, this phenomenon being periodic (Fig. 2). The REM-sleep associated theta was severely altered in the fourth rat, both in terms of amplitude and frequency.

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| FIG. 2.
Alteration of the RB activity in the MS-DBB of unanesthetized rats after i.c.v. 192 IgG-saporin injection. A and B: during wakefulness (documented by the occurrence of the hippocampal theta (C and D), a sizeable number of MS-DBB neurons exhibit a RB activity in saline-treated rats (neuron in A), whereas no RB neurons are found in lesioned rats, as shown by the lack of periodic peaks in the autocorrelation histogram of the neuron in B. E and F: examples of unit activity recorded during rapid eye movement (REM) sleep, documented by the hypersynchronous theta and the lack of muscle tone (G and H). Although the percentage of RB neurons decreases dramatically during REM sleep in lesioned rats, a few are still found (F), with a burst frequency similar to that of controls. Note that the hippocampal theta is still observed in the lesioned rat. Its amplitude, however, is decreased during wakefulness (D) and exhibits a waxing and wanning pattern during REM sleep (H). EMG: electromyogram; RDH and LDH, right and left dorsal hippocampus, respectively.
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MS-DBB neurons were divided into three groups (W, SWS, REM sleep) to study the relationship between levels of arousal and patterns of unit activity (Table 3). During W, no RB activity was found in lesioned rats (Fig. 2). During REM sleep, the percentage of RB neurons decreased dramatically (P < 0.01). The frequency of the burst remained unchanged (Fig. 2).
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TABLE 3.
Electrophysiological characteristics of MS-DBB neurons depending on the state of arousal (unanesthetized rats)
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Histochemistry
The loss of cholinergic MS-DBB neurons induced by intraventricular 192 IgG-saporin was checked on sections stained for AChE using the Mesulam procedure. All the rats included in the present study had a virtually complete loss of AChE-positive neurons (Fig. 1). In contrast, the PARV-containing neurons were completely spared (Heckers et al. 1994
). Immunohistochemistry for NGFr was more difficult to interpret. It is probable that the electrode penetrations resulted in high background staining that precluded a quantitative analysis of NGFr-positive neurons. However, the loss of NGFr-positive neurons was obvious in lesioned rats. A nearly complete loss of AChE fiber staining was also observed bilaterally in the hippocampus and in the cortex, indicating that all the cholinergic basal forebrain neurons were damaged by the toxin (Fig. 3). Consistent with previous findings (Bassant et al. 1995
; Heckers et al. 1994
), cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei (which do not express p75 NGF immunoreactivity) were unaffected by 192 IgG-saporin.

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| FIG. 3.
Location of rhythmically bursting activity in the MS-DBB after i.c.v. 192 IgG-saporin injection. A: coronal section of the horizontal limb of the DBB stained for AChE histochemistry. The Pontamine blue deposit (gray spot in the center of inset) indicates the place where a RB neuron has been recorded. AChE-positive neurons are completely absent. B: matching section stained immunocytochemically with an antibody against parvalbumine (PARV). Note that the RB activity has been recorded in the vicinity of a PARV-positive neuron. Scale bar: 100 µm. C and D: extent of cholinergic denervation after i.c.v. 192 IgG-saporin injection (AChE histochemistry). The dense network of AChE-positive fibers in the cortex (Cor), the hippocampal field (CA1), and the dentate gyrus (DG) of a saline-treated rat (C) has almost completely disappared in an immunotoxin-treated rat (D). Scale bar: 500 µm.
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Electrode tracks were reconstructed on camera lucida drawings to locate the position of each recorded neuron with respect to the dye deposit. In many cases, the Pontamine blue was released where a RB neuron had been recorded. Examination of the MS-DBB sections from lesioned rats revealed 1) that RB activity was still found in areas devoid of cholinergic neurons (Fig. 1) and 2) that RB activity was sometimes recorded in the vicinity of PARV-positive neurons, in the absence of AChE-positive neurons (Fig. 3).
 |
DISCUSSION |
The main result of the present study is that the proportion of RB neurons in MS-DBB decreases markedly after destruction of the cholinergic septohippocampal neurons by 192 IgG-saporin. A small percentage of neurons, however, remains rhythmic. Mean rate of neuronal activity decreases in lesioned rats, this alteration being mainly due to the reduced number of RB neurons. In urethan-anesthetized lesioned rats, neurons that still display RB activity have a significantly higher frequency than in control rats. Those identified as projecting to the hippocampus have a higher axonal conduction velocity and exhibit different burst parameters. Urethan-associated theta is impaired both in terms of frequency and amplitude. In unanesthetized lesioned rats, RB activity in MS-DBB is no longer observed during W and markedly reduced during REM sleep. At variance with the anesthetized preparation, the frequency of the RB activity that remains after lesion is similar to that of control. During W and REM sleep, the frequency of hippocampal theta is not altered, whereas its amplitude decreases.
Are the RB septal neurons cholinergic in nature?
Whereas the functional relationship between MS-DBB unit discharges and hippocampal theta rhythm is well demonstrated, the neurochemical nature of the rythmically bursting septal neurons has not been conclusively established yet. The hypothesis that they could be cholinergic has been proposed a long time ago (Lamour et al. 1984
; references in Bland 1986
). It was supported by indirect evidence such as the blockade of type 2 theta by systemic injection of atropine and its alteration by cholinergic agonists and antagonists injected in the medial septum (Monmaur and Breton 1991
). Intraventricular injection of hemicholinium-3 (which blocks choline uptake) severely reduces type 2 theta, whereas a subsequent administration of choline chloride restores it (Jobert et al. 1989
; Robinson and Green 1980
).
The decrease of rhythmically bursting activity observed in the present study after selective lesion of cholinergic MS-DBB neurons strongly suggests that the majority of the RB septal neurons are cholinergic. Is this hypothesis consistent with other available data? In vitro studies have provided evidence of two populations of MS-DBB neurons with different electrophysiological characteristics, one of the subpopulations being identified as cholinergic by AChE-histochemistry (Griffith and Matthews 1986
) and choline acetyltransferase (ChAT)-immunohistochemistry (Markram and Segal 1990
). The cholinergic neurons in the septal slice display electrophysiological characteristics such as a slow afterhyperpolarization potential (AHP) and a slow firing rate that would limit their ability to fire in a rhythmic manner with a high intraburst frequency. In contrast, the present in vivo study shows that RB activity (and thus high-frequency spiking) is strongly correlated with the presence of cholinergic neurons in the MS-DBB. It is likely that the cholinergic septal neurons may display RB activity under synaptic influence, when they are part of an in vivo network, as suggested by Markram and Segal (1990)
.
Electrophysiological characteristics of guinea pig MS-BDB neurons were also studied in an in vitro slice preparation by Matthews and Lee (1991)
. Extracellular recordings under microiontophoresed glutamate have distinguished three types of neurons: slow rhythmic firing cells with a "hump" in the falling phase of the action potential, which are likely to be cholinergic cells; fast firing rhythmic cells with a narrow action potential and no hump, which are likely to be noncholinergic cells; and burst firing cells (less frequently encountered) with an action potential bi- or triphasic. In the present study as well as in previous exhaustive in vivo studies from our laboratory, we have never observed reliable differences in the characteristics of action potentials using extracellular recordings in rats. At best, data obtained in 192 IgG-saporin-treated rats show that two subpopulations of RB neurons can be identified and characterized by criteria such as burst patterns and axonal conduction velocity. This classification, however, is rather poorly defined because, in spite of significant differences appearing when cell populations are compared, some individual cells in lesioned rats have characteristics that are similar to those of controls.
Two distinct fiber systems have been identified in the septohippocampal pathway; a very thin and delicate, possibly unmyelinated fiber network (type II) and a second much thicker axonal system (type I) (Nyakas et al. 1987
). The main target of the fine fibers appears to be the somata of the hippocampal pyramidal cells, whereas the main target of the coarse fibers appears to be the interneurons. Type II fibers originate from areas in MS-DBB that are rich in AChE-positive neurons, and their innervation patterns show coincidence with the distribution of ChAT immunostaining around the pyramidal and granular layers. It is assumed that type II fibers are cholinergic in nature. Our results show that RB neurons with low conduction velocity (and thus thin unmyelinated axon) are no longer found in lesioned rats. This finding is consistent with the hypothesis that the large majority of RB septal neurons are cholinergic.
An argument to be considered to the contrary is that the clear decrease of RB activity in the MS-DBB after destroying cholinergic neurons is not observed because the RB neurons are predominantly cholinergic, but rather because cholinergic input is important for the production of rhythmic activity in noncholinergic septal cells. Study of the intraseptal network (i.e., connections between cholinergic and noncholinergic neurons) has shown that ChAT-positive axons, likely collaterals from the cholinergic MS-DBB neurons, innervate predominantly noncholinergic cells (Leranth and Frotscher 1989
). GABAergic MS-DBB neurons express a high density of muscarinic acetylcholine receptors (Van der Zee and Luiten 1994
). They can therefore respond to the cholinergic activation arising from neighboring cholinergic neurons. However, if cholinergic imput was crucial for driving RB activity in noncholinergic neurons, then RB activity would have completely disappeared in 192 IgG-saporin-treated rats. In contrast, the present study shows that a small number of neurons retain RB activity. It is possible that the cholinergic input influences the bursting activity of noncholinergic septal neurons, not by inducing the rhythmical bursting but by modulating its frequency. Our results show, indeed, that the frequency of the RB activity that persists in lesioned rats is higher as compared with control. One explanation of this finding could be that the cholinergic input, in intact MS-DBB, excites GABAergic interneurons, which, in turn, reduce the activity of noncholinergic RB neurons.
Neurochemical identity of the neurons retaining RB activity in lesioned rats
Steward and Fox (1989b) reported that two populations of RB neurons are present in the MS-DBB of urethan-anesthetized rats. One group continues to burst at the theta frequency after a systemic injection of atropine sufficient to eliminate the hippocampal theta rhythm. The other cells lose their rhythmic firing pattern when hippocampal theta is eliminated by atropine. The authors suggest that the atropine-resistant RB cells are the septal GABAergic cells and the atropine-sensitive RB cells are the cholinergic ones. Intracellular recordings from septal neurons in vivo, during hippocampal theta, also reveal two categories of bursting neurons (Brazhnik and Fox 1992
). Some of them have a short action potential, consistent with those of noncholinergic septal cells (Markram and Segal 1990
). The others have electrical properties (long action potential, long AHP) consistent with those reported for cholinergic cells. Their frequency is lower and their bursting activity is abolished by scopolamine. Recently, Serafin et al. (1996)
have shown that noncholinergic MS-DBB neurons could fire in rhythmic burst at theta frequency in guinea pig basal forebrain slices.
Our results show that a few RB neurons can still be recorded in MS-DBB after complete destruction of the cholinergic neurons. In the absence of intracellular labeling and double staining, we cannot prove conclusively that these neurons are GABAergic. Several findings suggest, however, that it could be the case. It has been shown that the axons of the GABAergic SHNs have thick myelin sheaths allowing fast conduction velocity (Freund 1989
). Our results show that RB SHNs recorded after lesion have a significantly faster conduction velocity as compared with SHNs in control rats. The frequency of RB activity recorded in urethan-anesthetized lesioned rats is significantly higher than in controls. High-frequency of RB activity may be a characteristic of the GABAergic neurons (Brazhnik and Fox 1992
). Finally, dye deposits, aimed at locating RB neurons, have been found in the vicinity of PARV-positive neurons (i.e., GABAergic neurons), in areas otherwise devoid of cholinergic neurons. These findings support our assumption that RB septal neurons in lesioned rats might be GABAergic.
Septal rhythmic activity in lesioned rats under various experimental conditions
Type 2 theta was originally described as a cholinergically mediated rhythm, occurring during behavioral immobility and urethan anesthesia (Kramis et al. 1975
). Our results show an alteration of the urethan-associated theta after lesion of the cholinergic septohippocampal system. This finding confirms the cholinergic mediation of urethan-associated theta. In contrast, theta recorded during wakefulness is preserved, at least in term of frequency, although not a single RB neuron was recorded simultaneously in the MS-DBB. This indicates that theta associated with quiet wakefulness is much less dependent on cholinergic rhythmic inputs from the medial septum than urethan-associated theta. Consequently, it seems inappropriate to use the same term (type 2 theta) for these two types of theta, which probably depend on populations of "pacemaker" neurons different in terms of discharge frequency and neurotransmitter content.
At variance with results obtained in anesthetized rats, the RB activity remaining after lesion has a frequency similar to that of controls. If the cholinergic septal neurons influence the neighboring GABAergic RB neurons, as suggested above, this influence is probably much less important than the extrinsic modulation of cell discharges by the ascending brain stem input and is no longer disclosed in unanesthetized rats.
The RB activity is dramatically reduced during REM sleep in the MS-DBB of lesioned rats, whereas the hippocampal theta is only mildly altered (see also Bassant et al. 1995
). Under control conditions, a large number of septal neurons exhibit a RB activity during REM sleep, in the range of hippocampal theta frequency (Morales et al. 1971
; Sweeney et al. 1992
). The chemical control of this RB activity has not been identified yet. Nonselective lesions of the MS-DBB abolish the REM-sleep-related theta (Monmaur 1982
). The present findings show that the RB activity of the cholinergic MS-DBB neurons is not essential for the generation of REM-sleep-associated theta, even if it contributes to the regularity of its pattern.
Role of septal rhythmically bursting activity in generation of hippocampal theta
Lesions of the septal cholinergic system do not abolish the hippocampal theta but reduce its occurrence (Bassant et al. 1995
; Jenkins et al. 1993
) and decrease its power (Lee et al. 1994
). The present study shows, in addition, that theta can occur in unanesthetized rats even when the number of RB septal neurons is dramatically reduced. Two not necessary exclusive possibilities could account for this unexpected result: 1) noncholinergic RB neurons (presumably GABAergic), although few in number, are sufficient to induce theta rhythmicity in the hippocampus; or 2) rhythmic inputs from regions outside of the MS-DBB are able to generate hippocampal theta. Smythe et al. (1992)
have shown that GABAergic septal inputs reduce the overall level of inhibition of pyramidal hippocampal cells by inhibiting GABAergic hippocampal interneurons. They argue that such a mechanism can play a role in the generation of hippocampal rhythmic activity, providing that the excitatory cholinergic input acts synergistically (Bland et al. 1995
; Smythe et al. 1992
). When the septal cholinergic projections are eliminated (present study), the balance between excitatory and inhibitory mechanisms, which determines whether or not theta occurs, is probably altered. In this condition, it seems unlikely that the RB GABAergic neurons generate hippocampal theta all by themselves.
Rhythmic neurons discharging at the theta frequency have been recorded in several regions including the entorhinal cortex (Mitchell and Ranck 1980
; Stewart et al. 1992
; Vanderwolf et al. 1985
), the raphe nuclei (Kocsis and Vertes 1992
, 1997
), the nucleus pontalis oralis (Nunez et al. 1991
), the supramammillary nucleus (SUM), and the mammillary body (MB) (Kirk and McNaughton 1991
; Kocsis and Vertes 1994
). Kocsis and Vertes suggest that the SUM/MB, which occupies a key position in the crossroads of pathways between brain stem and limbic forebrain, might be involved in triggering theta activity. However, the finding that lesions of SUM failed to produce changes in the theta rhythm of freely moving rats indicates that its role is questionable (Thinschmidt et al. 1995
). Finally, RB activity recorded in the entorhinal cortex, even during cholinergic blockade, might be of importance to elicit hippocampal theta (Jeffery et al. 1995
). The results of the present study and those of recent reports suggest that many nuclei and pathways are involved in the generation of theta. Lesion of only one element of this network does not necessarily alter its capacity to maintain hippocampal theta.
Conclusions
Our results suggest that RB septal neurons are comprised of a large majority of cholinergic neurons and of a limited number of GABAergic neurons. The decrease of RB activity due to selective immunolesion of the cholinergic neurons is not sufficient to abolish hippocampal theta in unanesthetized rats. The present findings, together with behavioral studies that show that similar lesions have no or mild effects on learning and memory (Baxter et al. 1995
; Berger-Sweeney et al. 1994
; Torres et al. 1994
; Wenk et al. 1994
) suggest that the role of the cholinergic septohippocampal pathway as main pacemaker for hippocampal theta as well as its implication in memory processes should be reconsidered.