Brain Research 876 (2000) 37–47www.elsevier.com/ locate /bres

Research report

Age-related changes in rhythmically bursting activity in the medialseptum of rats

1 *´ ´Emmanuelle Apartis , Frederique Poindessous-Jazat, Jacques Epelbaum, Marie H. Bassantter´ ` ´Unite de Dynamique des Systemes Neuroendocriniens, INSERM U 159, 2 rue d’Alesia, 75014 Paris, France

Accepted 30 May 2000

Abstract

The effects of aging on the firing of septohippocampal neurons were estimated in unanesthetized, restrained young, old and very oldrats (respectively 3, 23 and 30 months). Extracellular recordings were obtained during various states of arousal. The mean spontaneousactivity for the overall neuronal population was not modified by aging. In contrast, the percentage of rhythmically bursting neurons wassignificantly lower in aged rats. During wakefulness, decrease of bursting activity was observed in old and very old rats (P,0.01 andP,0.001) whereas during rapid eye movement sleep it appeared only in the oldest group (P,0.01). The frequency of the bursts decreasedin 30-month-old rats during wakefulness while it remained unchanged in both aged groups during rapid eye movement sleep. In old rats,at a time when the cholinergic septal neurons already deteriorated, a third of neurons recorded during rapid eye movement sleep exhibiteda pattern of activity composed of long duration bursts with higher intraburst frequency than in young or very old rats. Our study showsthat rhythmically bursting septal activity is impaired in aged rats and that the amplitude of the changes depends on advancing age and onstates of arousal. Our findings suggest that age-induced loss and atrophy of cholinergic septal neurons contribute to the disorganization ofthe rhythmic activity but that functional alterations, influenced by the states of arousal, may also be considered. 2000 ElsevierScience B.V. All rights reserved.

Theme: Development and regeneration

Topic: Aging process

Keywords: Aging; Bursting activity; Cholinergic neuron; Paradoxical sleep; Sleep-waking cycle

1. Introduction (Refs. [14,20], for review). This rhythmicity appears to beessential for the generation of hippocampal theta rhythm

Numerous behavioral studies have described the cogni- and for memory and attention processes [12,48,57,62].tive impairments that occur in aged animals (Ref. [46], for Indeed, non selective lesioning of MS-DBB impairs spatialreview) but the electrophysiological correlates of these and non-spatial working memory (Ref. [62], for review)deficits are scarce. The septohippocampal system, impli- and selective lesioning of the cholinergic septohippocam-cated in a variety of behavioral processes including pal neurons alters attentional processing (Ref. [8], forattention and memory, is particularly useful for examining review).such parameters. In rodents, a significant proportion of A loss of markers selective for cholinergic neurons hascholinergic and GABAergic neurons located in the medial consistently been reported in MS-DBB of aged ratsseptum and the nucleus of the diagonal band of Broca [2,5,21,22,25,31,58]. Fewer changes in GABAergic cells(MS-DBB) exhibit a rhythmically bursting (RB) activity have been documented but a loss of parvalbumin-positive

neurons, likely to belong to a population of GABAergicseptal neurons, has been reported [33]. We had shown

*Corresponding author. Tel.: 133-1-40-78-9255; fax: 133-1-45-80- previously that RB activity in the septohippocampal sys-7293.

tem was altered in aged rats anesthetized with urethaneE-mail address: bassant@broca.inserm.fr (M.H. Bassant).1 [35,36]. However, anesthesia modifies RB activityˆPresent address: Service de Physiologie, Hopital Saint-Antoine, 75012Paris, France. [12,19,59] and does not allow to establish correlations

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02571-3

38 E. Apartis et al. / Brain Research 876 (2000) 37 –47

between states of arousal and rhythmic firing of septal dried skull surface. The hardened resin was subsequentlyneurons. In the present work, we took advantage of a covered with a layer of acrylic cement. Two metal tubes,recent restrained unanesthetized rat model [4] to assess the temporarily held in place by anchored bars attached to theeffect of aging on RB activity of MS-DBB neurons during stereotaxic frame, were placed transversally slightly abovewakefulness and rapid eye movement sleep (REM sleep). the skull. The tubes were buried in a mound of acrylicTwo groups of aged rats, 23 and 30-month-old, were cement brushed on the top of the first cement layer. Thecompared to 3-month-old rats in order to determine if opening over the MS-DBB was covered by gel foamchanges in RB activity were correlated to the morphologi- soaked in saline. The well formed by the cement ridgescal alterations occuring with advancing age. was covered with sealing paste. The rat was removed from

the stereotaxic frame and allowed to recover from anes-thesia. Intramuscular oxacilline (150 mg/kg and 80 mg/kg

2. Material and methods respectively in young and aged rats) was administeredevery 2 days during the recovery period. The head restraint

2.1. Animals system was well tolerated by the rats, who were able tofeed, drink and sleep normally.

Male Sprague–Dawley rats were obtained from CharlesRiver (France) and housed in our animal facility (accre- 2.3. Rat behavior during the recording sessionsdited by the National Committee for Assessment andAccreditation of laboratory animal care facilities) from the Rats were gradually habituated to the restraint systemage of 2 months. The rats were housed two per cage at during a week. The rat’s head was painlessly secured to the228C under a 12 h light /dark cycle and had free access to stereotaxic frame by inserting the anchored support barswater and food. Approximately 60% of the rats survived to into the ends of the metal tubes and its body wasan age of 23 months and 20% to an age of 30 months. comfortably supported by a hammock. Over a course ofAged rats used in the experiment had no apparent tumor about 3 days, the rat stood motionless during wakingmass or pituitary adenoma. Five young (3 months, body episodes and fell asleep easily. ECoG and EMG wereweight 350610 g), seven aged (23 months, 738620 g) recorded on a polygraph throughout the training session,and five very aged (30 months, 595640 g) rats were used allowing a constant observation of the rat’s behavior.

States of arousal were differentiated as followed: wakeful-2.2. Surgery to implant head-restraining system ness (W, high-frequency low-voltage ECoG activity inter-

mingled with periods of hippocampal theta induced byRats were anesthetized with pentobarbital (60 mg/kg for whistles and gentle peripheral sensory stimulus, sustained

young, 55 mg/kg for 23-month and 25 mg/kg for 30- EMG activity), slow wave sleep (SWS, high-amplitudemonth-old rats), placed into a stereotaxic frame and slow waves intermingled with spindles) and rapid eyemaintained at 378C with an isothermal heating pad. The movement sleep (REM sleep, hypersynchronous thetaskull was opened above the MS-DBB area. The midline waves, loss of neck muscle tone, jerks of jaw muscles andsuture was used to guide the laterality (L50). The coordi- vibrissae at the end of the episode). Slow wave sleep andnates relative to the interaural zero were AP58–9 mm for paradoxical sleep periods were identified in all animals,the young rats [49] and AP510–11 mm for the 23 and showing that the immobilization was not painful and30-month-old rats to take into account the age-related attesting that the recording conditions were well tolerated.antero-posterior growth of the brain and the angle of the Each experiment lasted for four consecutive days,skull in the stereotaxic frame. After exposing the dura usually with one morning and one afternoon of recordingabove the septum under microscopic control, a ligature sessions, each lasting about 3 h. When returned to its cage,was made by tying off the central sinus and the vein was at the end of the recording session, the rat would engage incut. normal feeding and grooming activities. All efforts were

Electrocorticogram (ECoG) activity was recorded from made to minimize animal suffering and to reduce thestainless steel jeweler’s screws inserted into the skull. The number of animals used. Care and use of animals used intip of one screw was placed onto the surface of the this study were approved by the local Animal Care andsensorimotor cortex over the right and left dorsal hip- Use Committee.pocampus (4 posterior and 3 lateral to bregma for youngrats, and 5 posterior and 4 lateral to bregma for aged rats). 2.4. Data collection and histologyA screw placed on the occipital crest served as reference.A stainless steel hook soldered to fine flexible insulated On the day of recording, the dura over the septum wassilver wires was placed in the neck muscles in order to removed; xylocaine (0.02%) was applied locally onto therecord the electromyogram (EMG). All the electrodes were dura to avoid any discomfort. Conventional amplificationsoldered to a connector. A liquid bonding resin (Super- methods were used to record single unit activity in thebond, Sun medical Co., Japan) was applied to the cleaned, MS-DBB from micropipettes filled with 1 M NaCl and 2%

E. Apartis et al. / Brain Research 876 (2000) 37 –47 39

Pontamine Blue (tip diameter,1 mm, resistance 10–20 potentials within a burst was defined as 1 ms and theMV). Micropipettes allowed recordings with high signal- minimum inter-burst interval was set at 40 ms [4]; (3)to-noise ratio (.4) so that neuron singularity could be autocorrelation functions, based on the autocorrelationeasily assessed by a window discriminator. The state of histogram. If recurrent events occured as rhythmic burstsarousal was continuously monitored by ECoG and EMG of action potentials, then the autocorrelation histogramrecordings while unitary activity was recorded. Recording showed periodic sinusoidal-like density peaks. To providesites of interest were marked by a local release of dye. At an objective classification of RB and non-RB neurons, athe end of the experiment, rats were deeply anesthetized ‘rhythmicity index’ was calculated: the autocorrelationwith pentobarbital and perfused through the ascending histogram was smoothed out, maximum and minimumaorta with saline (300 ml for young and 500 ml for aged amplitude of the curve were measured and averaged, thenrats) followed by 4% paraformaldehyde in 0.1 M phos- divided by the value of the spontaneous activity (imp/s). Aphate buffer (pH 7.4) (600 ml for young and 800–900 ml higher index denoted more regular RB activity. The cut-offfor aged rats). The brains were removed, post-fixed at 48C point of the rhythmicity index was set at 0.95. Data onfor an additional 3–4 h in the same fixative, and cryop- single unit activity were divided into three groups, depend-rotected in phosphate-buffered 30% sucrose for 24 h. The ing on the state of vigilance observed while the neuronsbrains were sectioned in the coronal plane on a freezing were recorded.microtome, from the rostral septum to the mid-dienceph-alon. Sections (40 mm thick) were collected in phosphate 2.6. Statistical analysesbuffer. Brain sections with Pontamine Blue deposits werestained with safranine. The adjacent sections were pro- Differences in percentages of RB neurons among 3, 23cessed for AChE histochemistry for labeling of AChE- and 30-month-old rats and among states of vigilance werecontaining neurons [43]. analyzed using Chi-square tests of Pearson. Considering

the individual variation in neurobiological alterations2.5. Data analysis observed in aged animals and the fact that measurements

made on a large number of cells from one individual areElectrode penetrations were reconstructed on camera not independent, comparisons between young and old rats

lucida drawings. The position of each recorded neuron in were conducted on a per animal basis, using one-waythe septal area was determined with respect to the dye ANOVA followed by post hoc Fisher PLSD. Differencesdeposits. were considered significant if P,0.05.

Electrophysiological data were digitized and analyzedoff-line with a CED-1401-plus system (Cambridge Elec-tronic Design). Samples of the ECoG (10 s duration) and 3. Resultsof the simultaneously recorded single unit activity weredigitized (converter sampling rate 10 msec) and sampled at 3.1. Methodological and general considerations100 Hz and 10 kHz respectively. The power spectrum ofthe ECoG was computed with a fast Fourier transform over The main problem encountered with aged rats was theira range of 0–30 Hz with 0.1 Hz resolution. Seven distinct sensitivity to anesthesia. Indeed, three old and seven very10 s samples of ECoG were analyzed and then averaged. old rats died during the post-operative period, in spite ofNumerical values were obtained for five frequency bands; the reduced doses of anesthesia used during the fixation ofdelta (0.1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz) and beta the head-restraining system. Thus, the hippocampal activi-(12–30 Hz). The relative power spectrum (each of the four ty was not recorded from deep electrodes, in order tobands / the sum of the power in all frequency bands) was shorten the duration of the surgery. In addition, the use ofcalculated. Only those ECoG epochs which exhibited a deep electrodes would have hindered the drying of thedistinct peak within the 4–8 Hz frequency were retained as spongy bone of aged rats before the bonding resin wasW or REM sleep. The theta frequency and the relative applied, creating a risk that the head-restraining systempower in the delta–gamma bands were compared among might be dislodged from the skull during a brisk move-the different age groups according to the vigilance states. ment of the animal. ECoG was recorded from the sen-

The analysis of single unit activity of MS-DBB neurons sorimotor cortex and appeared an acceptable way towas based on: (1) mean spontaneous neuronal activity identify the hippocampal theta. Two types of theta were(averaged number of action potentials per second); (2) observed in young and aged rats: one type associated withburst parameters, namely the number of spikes per burst, alert immobility [10,32,57] (see Fig. 1) and one typethe mean inter-spike interval, the frequency of discharge associated with rapid eye movement sleep (REM sleep)within each burst, the mean burst length, and the mean (see Fig. 2),interburst interval. A burst was defined as 2–20 action Extracellular recordings were obtained from 221 MS-potentials with a maximum of 30 ms between consecutive DBB neurons in young rats, 333 neurons in aged rats andaction potentials. The minimum interval between action 237 neurons in very aged rats. All neurons were located in

40 E. Apartis et al. / Brain Research 876 (2000) 37 –47

Fig. 1. Examples of rhythmically bursting activity (RB) in MS-DBB recorded from 3, 23 and 30-month-old rats during wakefulness. The state of arousal isdocumented by the pseudo-periodic pattern of ECoG activity in the theta range of frequency and the sustained muscle tone (EMG). UA: unitary activity.The RB activity of each neuron is shown by the periodic peaks of the autocorrelation histogram (AH). Notice that the frequency of RB activity (FrB)decreases with advancing age. Simultaneously, the peak of the power spectrum in the theta range (Fru) shifts towards lower values. AH ordinate: numberof spikes occurences as a function of time after the spike at time 0; binwidth: 5 ms.

the medial septal nucleus and the vertical limb of the by others [41,56]. During wakefulness, the power spectrumdiagonal band of Broca. The mean number of cells of sensorimotor ECoG was not modified. During REMrecorded per electrode penetration was not significantly sleep, the relative power in the beta band decreaseddifferent between the three groups (7.1, 6.9 and 6.4 for the (F 57.2, P,0.05) while the theta band increased sig-1.10

3, 23 and 30-month-old rats respectively). The proportions nificantly in 23 as compared to 3-month-old rats (F 51.10

of neurons recorded during W was significantly higher in 7.9, P,0.05). An immobility-related spike wave pattern —23 and 30-month-old rats (55% and 59% respectively) as referred to as high voltage spindles (HVS) — has been

2compared to 3-month-old rats (35%, Chi 538, d.f.52, observed from ECoG of rats. The occurrence of HVSP,0.001), indicating that sleep was less abundant in aged increases with aging [15]. In the present experiment, fourrats. REM sleep episodes tended to be less numerous in old and two very old rats showed elevated levels of HVSaged rats and the duration of the longer episodes decreased whereas no spindling was detected in young rats. Old ratsin 30-month-old rats (110625 s) as compared to 3-month- with HVS were not included in the study (see Discussion).old rats (173638 s, F 59.5 P,0.05). However, the The loss of cholinergic neurons was qualitatively as-1–8

number of cells recorded during REM sleep, expressed as sessed on brain sections stained for AChE histochemistry.the percentage of the total number of cells recorded during A reduction in the number of AChE-positive neurons wasall the sleep episodes (SWS and REM sleep), was similar clearly visible at 23 months of age and was more severe atin the three groups. 30 months of age (Fig. 3).

The hippocampal theta rhythm was marginally affectedby age during wakefulness (5.660.1 and 5.760.1 Hz in 23 3.2. Unit MS-DBB activity during wakefulnessand 30-month-old rats as compared to 6.060.1 Hz in3-month-old rats), the difference being significant only in Neuronal activity and burst patterns of RB neurons23-month-old rats (F 55.7, P,0.05). During REM during W are illustrated on Fig. 1 and their characteristics1.10

sleep, the frequency was similar in the three groups of rats are given in Table 1. The percentage of RB neurons was2(7.160.1 and 6.960.3 Hz in 23 and 30-month-old rats as significantly lower in 23-month-old rats (26%, Chi 510.5,

2compared to 6.961.0 Hz in 3-month-old rats) as observed d.f.51, P,0.01) and in 30-month-old rats (23%, Chi 5

E. Apartis et al. / Brain Research 876 (2000) 37 –47 41

Fig. 2. Examples of rhythmically bursting activity (RB) in MS-DBB recorded from 3, 23 and 30-month-old rats during REM sleep. REM sleep isdocumented by the hypersynchronous ECoG activity and by the lack of muscle tone. As observed in a large number of cases, the frequency of the RBactivity of septal neurons and of the ECoG are higher in the 23-month-old rat than in its younger and older counterparts (see Fig. 1 for legend).

12.4, d.f.51, P,0.001) as compared to 3-month-old rats 0.01), the larger number of spikes per burst (F 532,1.9

(45%). The mean frequency of RB activity decreased P,0.001) and the shorter intraburst interspike intervalprogressively, the difference being significant only in 30- (F 569, P,0.001) (Table 1, Figs. 2 and 3). These1.9

month-old rats (5.5 Hz, F 57.6 P,0.05) (Table 1 and changes were particularly marked for 11 of the 36 RB1–8

Fig. 1). The mean spontaneous activity of RB and non-RB neurons recorded during REM sleep (31%) whichneurons was similar in the three groups. The burst patterns exhibited high burst frequency (ranging from 7.4 to(number of spikes per burst, interspike interval, duration of 8.3 Hz) superior to the maximum value found inthe burst) were not affected by age. 3-month-old rats (7.3 Hz) as well as burst parameters

(number of spikes per burst, interval interspike, mean3.3. Unit MS-DBB activity during REM sleep firing level) which did not overlap those of young rats

(Fig. 4).Neuronal activity and burst patterns of RB neurons • In 30-month-old rats, the percentage of RB neurons

2during REM sleep are illustrated on Figs. 2 and 3 and their decreased (46%, Chi 58.7, d.f.51, P,0.01). Thecharacteristics are given in Table 1. The mean spontaneous burst frequency was similar to that of young rats. Theactivity for the non-RB neurons was not affected by age. patterns of the bursts fell between those recorded for

In contrast, changes in RB activity were observed but the 3 and 23-month-old rats, with a significant age-were not similar in the two groups of old rats: related difference limited to the interspike interval

value (Table 1). The mean spontaneous activity of RBneurons remained close to the 23-months value but the

• In 23-month-old rats, the percentage of RB neurons difference did not reach significant level as compared(72%) was relatively unchanged when compared with to the 3-months values. Two out of 13 RB neuronsthe percentage for young rats (82%). The level of recorded in very old rats exhibited a frequency (7.6–activity increased as shown by the higher mean 8.0 Hz) that exceeded the higher value found in youngspontaneous activity of RB neurons (F 518.9, P, rats (Fig. 4).1.9

42 E. Apartis et al. / Brain Research 876 (2000) 37 –47

Fig. 3. Age-related loss of AChE-positive neurons. Microphotographs of frontal sections through the medial septum (MS) and the diagonal band of Broca(DBD). Sections are stained for acetylcholinesterase (AChE) histochemistry. AChE-positive neurons are colored in brown (white arrow). Notice that theloss of AChE-positive neurons and fibers, already visible at 23-months of age, becomes more pronounced at 30-months. The pontamine blue deposits(black arrow) indicate the place where the rhythmically bursting (RB) neurons have been recorded during REM sleep. Examples of the discharge of eachRB neuron and of periodic ECoG recorded simultaneously are shown below microphotographs. Notice that the neuron recorded in the 23-month-old ratdischarges with bursts composed of a remarkably large number of spikes. Fr: frequency of the neuron (hertz). Scale bar: 250 mm.

Table 1aElectrophysiological characteristics of MS-DBB neurons in young and aged rats during wakefulness and REM sleep

States of Age Percentage Mean frequency Number Interspike Mean burst Mean Meanarousal of RB neurons of RB neurons of spikes interval duration spontaneous spontaneous

(Hz) per burst in the burst (ms) activity of RB activity of non(ms) neurons (imp/s) RB neurons

Wakefulness 3 M 45% 6.060.1 6.560.4 13.060.7 5965 3363 1763N55 (45/100)

††23 M 26% 5.760.2 7.060.3 12.060.5 6263 3762 1564N57 (59/226)

††† *30 M 23% 5.561 6.660.9 12.061 6162 3564 1864N55 (40/170)

REM sleep 3 M 82% 6.760.6 6.360.2 12.060.1 5663 3465 2363(40/49)

23 M 72% 7.060.1 9.560.5*** 8.060.8*** 6165 5763** 2667(36/50)

†† and ♦30 M 46% 6.660.3 8.761 10.060.9* 6064 5163 2464(13/28)

a †† †††Values are means6S.E. *P,0.05 **P,0.01, ***P,0.001 as compared to 3-month-old rats (analysis of variance). P,0.01, P,0.001 as compared to♦3-month-old rats (chi square test). P,0.05 as compared to 23-month old rats (chi square test). RB: rhythmically bursting neurons, imp. / s: impulses per

second, N: number of rats, in brackets: number of cells.

E. Apartis et al. / Brain Research 876 (2000) 37 –47 43

Fig. 4. Scatter-plots of the frequencies of the rhythmically bursting (RB) neurons during wakefulness and REM sleep. The continuous horizontal linesindicate the mean value for the entire group. Notice that during wakefulness the large majority of the values found in 23 and 30-month-old rats are belowthe 3-month mean value whereas the opposite is observed during REM sleep in the 23-month group. Moreover, 11 neurons from the 23-month group and 2neurons from the 30-month group have frequencies that exceed the higher values found in 3-month-old rats.

3.4. Unit MS-DBB activity during slow wave sleep in very old rats (15.3%). These results confirmed, forsingle neurons, the age-related loss of ability to fire in a

No RB activity was recorded during SWS irrespective of rhythmically bursting manner as observed in the overallage. The mean spontaneous activity was significantlyhigher in 23-month-old rats as compared to those aged 3and 30-months (25.764 imp/s, F 54.9, P,0.05)1.10

whereas it was similar in young and very aged rats(14.063 and 16.264 imp/s).

3.5. Neurons recorded in different states of arousal

A sizeable number of single neurons were followedduring different states of arousal (50, 53 and 39 neurons in3, 23 and 30-month-old rats respectively). Neurons withnon-rhythmically bursting activity during SWS exhibitedRB activity in 65.3% of the cases in 3-month-old rats but

2only in 36.7% in 23-month-old rats (Chi 53.9, d.f.51,2P,0.05) and 32% in 30-month-old rats (Chi 54.1, d.f.51,

P,0.05) (Fig. 5). Change from SWS to REM sleep wasaccompanied by a shift from non RB to RB activity in96% of the cases in 3-month-old rats. This percentage wasnot significantly altered in 23-month-old rats (83%) but

2decreased to 53% in 30-month-old rats (Chi 58.3, d.f.51,P,0.01) (Fig. 5). As a rule, RB neurons observed during

Fig. 5. Age-related loss of bursting activity at the level of single neuronsW and REM sleep (6 in young, 11 in old and 5 in very oldrecorded during different states of arousal. In young rats, a large numberrats) had a higher frequency of burst in REM sleep asof neurons devoid of RB activity during slow wave sleep (SWS) adopt

compared to W. The percentage of increase was sig- this pattern of discharge during wakefulness (W) and even more duringnificantly higher in old as compared to young rats (18.9 vs. REM sleep. This functional plasticity is significantly reduced with7.4%, F56.8, P,0.05) but did not reach significant level advancing age.

44 E. Apartis et al. / Brain Research 876 (2000) 37 –47

population and the reinforcement of rhythmicity during correlation in our experimental conditions because HVSREM sleep at 23 months of age. episodes were associated with twitching of the facial

muscles which prevent durable recordings of MS-DBBneurons.

4. Discussion4.3. Overall level of activity

The present work demonstrates that alterations in RBseptal activity in old and very old rats depend on the states Mean spontaneous activity of septal neurons was notof arousal. During wakefulness, loss of RB activity is altered in unanesthetized aged rats. This result is consistentobserved as soon as 23 months of age whereas during with that of previous studies in the MS-DBB and theREM sleep this loss occurs only in 30-month-old rats. The cerebral cortex of anesthetized Sprague–Dawley ratsburst frequency decreases during wakefulness in 30- [30,34] and the hippocampus of freely-moving Fisher 344month-old rats but remains unchanged in both groups of rats [6]. Other studies, however, reported decreases inaged rats during REM sleep. Interestingly, at the age of 23 mean spontaneous activity in the frontal cortex of anes-months, when cholinergic deficit is already taking place, thetized Sprague–Dawley rats [1] and the pyramidal layersabout one third of RB neurons recorded during REM sleep of the hippocampus of anesthetized Fisher 344 rats [39].have a high level of activity composed of long bursts with This indicates that aging does not induce a generalized andshort interspike intervals. These new findings suggest that marked reduction in neuronal activity. Rather, changesage-induced changes in neuronal activity cannot be totally seem to be region and cell-specific [7]. For example,explained by cell death but might result from various discharge rates of hippocampal interneurons recordedfunctional impairments revealed by distinguishing between during different locomotion states in 24-month-old Fisherthe states of arousal. 344 rats were higher in stratum lacunosum, lower in

stratum oriens, and unchanged in stratum pyramidale [44].

4.1. States of arousal in aged rats4.4. RB activity in the medial septum

Results of previous investigations into age-relatedchanges in sleep–wake stages appear contradictory but this Two types of neurons with different electrophysiologicalmay depend on the rat strain studied. Indeed, the daily characteristics have been recorded in vitro in MS-DBB.amount of wakefulness and sleep was found stable in One type has been identified as cholinergic by histoch-21–27-month-old Long Evans rats and 30-month-old emistry while the other is likely to be GABAergicBrown Norway [38,63] whereas a tendency toward an [28,29,42]. In vivo intracellular recordings have alsoincrease of wakefulness and a moderate reduction of SWS revealed two categories of bursting neurons in MS-DBB,and REM sleep was found in 22-month-old Wistar rats and one category with characteristics compatible with a28–32-month-old Sprague–Dawley rats [52,60]. In the cholinergic nature and the other with a non-cholinergicpresent experimental conditions, aged Sprague–Dawley nature [13,14]. Moreover, results obtained after selectiverats were more frequently awake during recording ses- lesioning of the cholinergic septohippocampal neuronssions. SWS and REM sleep were nevertheless observed in have confirmed that the septal RB neurons are comprisedboth aged groups but the duration of the longer REM sleep of a majority of cholinergic neurons and of a limitedperiods was significantly reduced as compared with young number of GABAergic neurons [4]. There is evidence thatrats. both intrinsic and extrinsic mechanisms govern the

rhythmic activity in MS-DBB. On the one hand, a numberof septal neurons are probably endowed with intrinsic

4.2. Age-related ECoG alterations: high-voltage spindles properties of ‘pacemaker’ cells because they retain RBactivity in the deafferented septum [12]. On the other hand,

The occurrence of high-voltage spindles (HVS) in- sustained RB activity in MS-DBB requires synchronizingcreases with aging in Fisher 344 and Wistar rats [15,50]. inputs arising from other brain regions. For example,We have also observed elevated HVS levels in aged stimulations of the brainstem reticular formation induceSprague–Dawley. It has been established that HVS activity marked RB activity in MS-DBB and hippocampal thetais associated with memory impairments [50] and might (Refs. [11,61], for review)result, at least in part, from damage to the cholinergicneurons in the nucleus basalis [16]. While it would havebeen interesting to determine whether neuronal activity in 4.5. Age-related loss of RB activityMS-DBB was more severely impaired in aged rats withHVS activity, it was not possible to establish such a The decrease in RB activity observed with age might

E. Apartis et al. / Brain Research 876 (2000) 37 –47 45

result from damages within MS-DBB (cholinergic and oralis in generating periodic activity during REM sleepGABAergic neurons) and/or from the loss of external whereas other areas would send inputs to nucleus rostralsynchronizing inputs. pontis oralis during wakefulness. In such a case, one must

The number of cholinergic neurons in the MS-DBB was suppose that all reticular nuclei are not affected by age toreduced in 24 and 30-month-old rats [2,5,17,25]. These the same extent, since RB septal activity is more disruptedresults suggest that the decrease of RB activity in MS- during wakefulness than during REM sleep.DBB might be due to the loss and/or severe degeneration Busting activity in MS-DBB and hippocampal thetaof cholinergic neurons. However, the decrease in the frequency increase with movements such as walking andnumber of RB neurons found in the present study (40% rearing [24,45,51]. The loss of RB activity in old ratsduring waking at 23 months, 49% and 40% during waking might have been related to the reduced motor activity inand REM sleep at 30 months) are larger than the decrease animals of advanced age. This explanation, however, is notin the number of cholinergic cells observed in Sprague– relevant in the present case as the septal unit activity wasDawley rats of the same age (19 and 27% at 24 and 30 recorded in motionless rats.months) [22]. This shows that the loss of cholinergicneurons cannot be the only cause of the decline in RBactivity. Accordingly, Fischer and colleagues have stated 4.6. Enhancement of RB activity during REM sleepthat the magnitude of the morphological changes is notsufficient enough to explain the severe spatial learning The large majority of septal neurons recorded duringdeficits observed in aged Sprague–Dawley rats [22]. REM-sleep in 23-month-old rats show an unexpected highBesides decreasing in number, the cholinergic neurons also level of activity. MS-DBB neurons displayed features such

21endure morphological alterations such as shrinkage, en- as low-threshold Ca [3] and inhibitory and excitatorylargements, loss of immunoreactivity for galanin, reduction synaptic currents with fast kinetics [55] that probablyin muscarinic M2 receptor binding [2,17,18,21,22,26] all favoured RB activity. Voltage-gated calcium channels,

21signs of progressive degenerative process that might impair which control the influx of extracellular Ca into neurons,their functional properties. However, in both 23 and 30- have been studied in dissociated MS-DBB neurons frommonth-old rats, a number of neurons remain capable of 24 to 26-month-old rats. It has been found that low-voltage

21discharging rhythmically since they recover their RB activated Ca currents increased with age [47]. Theseactivity, apparently lost during wakefulness, when REM currents are important for repetitive firing and their in-sleep occurs. This finding suggests that disorganization of crease might explain that a number of neurons displaybursting activity in MS-DBB results from subtle changes enhanced bursting activity in 23-month-old rats and, to arather than frank neurodegeneration. lesser extent, in very old rats.

GABAergic neurons may also contribute to the loss of In Sprague–Dawley rats, the number of GABA-im-RB activity observed in aged rats. Indeed, intrinsic con- munoreactive cells in MS-DBB did not change at 26nections between cholinergic and GABAergic neurons months of age whereas the number of cholinergic neuronsexist in the MS-DBB [37]. This network allows for was already decreased [33]. One might have hypothesizedinterplay between excitatory and inhibitory inputs that that the bursting activity during REM sleep is due tocontribute to the pacing of RB activity [4,12,14]. The GABAergic neurons since they tend to fire at higher raterhythmicity of the GABAergic cells may decrease because than the cholinergic cells, at least in young rats [4,14].the cholinergic input which synchronizes their activity in However, the finding that REM sleep-associated RByounger rats is reduced along aging. Furthermore, an activity is considerably reduced after lesioning theage-related decrease in GABA-elicited inhibition has been cholinergic SM-DBB neurons supports the notion that thedescribed in vivo in the lateral septum of the mouse [23] majority of neurons displaying RB activity during REM-and in vitro in the hippocampus of the rat [9]. Such a sleep are cholinergic [4]. It is therefore unlikely that thereduction in GABAergic neurotransmission might occur in maintenance and/or reinforcement of RB activity duringMS-DBB and alter the rhythmicity by modifying the REM sleep in 23-month-old rats result only from preservedbalance between cholinergic and GABAergic inputs. GABAergic RB neurons. Another possibility is that com-

In addition to changes in MS-DBB, a loss of external pensatory events occur during aging. Enlargements ofsynchronizing inputs may also occur. The cholinoceptive / septal cholinergic neurons have been reported in variouscholinergic systems of the brainstem are involved in rat strains [5,25]. It is not clear whether the enlargement ofgenerating rhythmic activity in the septohippocampal septal neurons could influence the level of activity and thesystem [61]. The most effective sites are the nucleus rostral pattern of discharge as the synaptic density does notpontis oralis and the pedunculopontine tegmental nucleus. change with age in MS-DBB [54]. In any case, ‘restorativeVertes and Kocsis have suggested that the mechanisms and/or compensatory events’ might occur in response toinducing rhythmicity in the septohippocampal system aging as suggested by Armstrong and colleagues [5]. Itdiffer according to the state of arousal; pedunculopontine remains to be seen why the compensatory events appeartegmental nucleus would influence nucleus rostral pontis only during REM-sleep.

46 E. Apartis et al. / Brain Research 876 (2000) 37 –47

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