Temporal matching of sensory-motor behavior and limbic θ rhythm deteriorates in aging rats

Neurobiology of Aging, Vol. 5, pp. 7-17, 1984. ©AnkhoInternationalInc. Printedin the U.S.A.

0197-4580/84$3.00 + .00

Temporal Matching of Sensory-Motor Behavior and Limbic 0 Rhythm Deteriorates in Aging R a t s 1'2 W I L L I A M B. F O R B E S A N D F O T E O S M A C R I D E S

Worcester Foundation f o r Experimental Biology, 222 Maple A v e n u e , Shrewsbury, M A 01545 R e c e i v e d 5 D e c e m b e r 1983 FORBES, W. B. AND F. MACRIDES. Temporal matching of sensory-motor behavior and limbic 0 rhythm deteriorates in aging rats. NEUROBIOL AGING 5(1) 7-17, 1984.--The correlations between investigatory sniffing and rhythmic slow-wave activity (RSA) in the dorsal hippocampal formation were studied during free behavior in Fischer 344 rats aged 3, 18, 30, and 36 months. The amount and vigor of spontaneous exploratory behavior was reduced in older animals, and the frequency distributions of investigatory sniffing and hippocampal RSA both shifted with age toward the lower end of their normal ranges. In the youngest animals, (1) the dominant frequency of sniffing matched that of hippocampal RSA (frequency entrainment) more often than would be predicted by chance; (2) preferred phase differences between sniffing and hippocampal RSA were reliably observed in the 5-9 Hz range; and (3) these preferred phase differences varied linearly as a function of frequency, implying an underlying latency relationship. These correlations changed progressively with age as follows: (1) the incidence of frequency entrainments decreased; (2) the frequency range within which preferred phase differences were observed became lower and narrower; and (3) the incidence of preferred phase differences decreased. However, animals of all ages exhibited similar preferred phase differences for those frequencies at which significant preferences were expressed. These findings are discussed in relation to the hypothesis that alterations of forebrain 0 rhythms may accompany aberrations of the medial septum-diagonal band-nucleusbasalis complex and may be importantly involved in aging-related impairments of cognitive and learning abilities. Aging

Theta

Olfaction

Sniffing

Limbic system

RECENT neuroanatomical and biochemical findings indicate that the medial septal nucleus, the nuclei of the diagonal band, and the nucleus basalis form a complex of subcortical neurons in the basal forebrain which collectively project to most of the limbic, olfactory, and neocortical structures of the telencephalon and account for much of the cholinergic input to these structures [10, 19, 32, 36, 37, 41, 42, 51, 74]. This complex also appears to contain a population of catecholaminergic neurons [18] and a variety of peptidergic neurons [17, 23, 36, 57]. Several lines of evidence suggest that aging-related changes in this complex, particularly in its cholinergic component, may contribute to abnormalities of cognition, memory, orientation, and behavior which sometimes occur with normal aging, and that more severe neurochemical, neuroanatomical, and degenerative aberrations of this complex may account for the debilitating symptoms of Alzheimer's disease and may be generally involved in senile dementia [5, 16, 50, 70]. For example, loss of neurons in the nucleus basalis of Meynert and substantia innominata has been reported in healthy aged humans and in Alzheimer's patients [25, 45, 47, 50]. Furthermore, reduced activity of choline acetyitransferase has been reported in the brains of normal aged humans and patients with Alzheimer's disease (e.g., [3,53]). These findings are complemented by reports

Sensory-motor behavior

Hippocampus

that cholinomimetic drugs, including physostigmine and arecoline, are effective in ameliorating aging-related memory deficits in monkeys and humans (e.g., [59,61]). Similar findings have been reported in aged rats, including decreases in brain choline acetyltransferase activity [58,60] and improved mnemonic performance in response to cholinomimetic drug treatments [7]. These findings lend substantial support to the proposition that changes in basal forebrain cholinergic systems may be important etiological factors in aging-related behavioral deficits. Cholinergic and possibly other neurons located in the medial septal area and predominantly in the vertical limb of the diagonal band are thought to function as pacemakers for the hippocampal 0 rhythm [4, 24, 30, 48], whose appearance as 4--12 Hz rhythmic slow-wave activity (RSA) has been associated with attentiveness [8,27], or with phenomena involving a heightened or focused state of vigilance such as voluntary movement [66], spatial guidance [46], and learning [1, 9, 31]. We recently have found that neurons located throughout the horizontal limb of the diagonal band and in the rodent homolog of the nucleus basalis of Meynert, like those in the vertical limb and medial septal nucleus, exhibit rhythmic bursting activity in the 0 range [41], and thus collectively may drive 0 activity in a variety of olfactory, limbic,

~Portions of these findings were presented at the Society for Neuroscience 13th annual meeting, Boston, MA, November 6-11, 1983. ~This work was supported by National Institutes of Health Research Grants AG00779 and NS12344.

8

FORBES AND MACRIDES

and neocortical structures (cf., [22, 32, 38, 39]). Furthermore, lesions in this complex which impair learning also reduce or abolish 0 activity in the hippocampal formation and related cortical structures (e.g., [44]). These various findings suggest that alterations in forebrain 0 rhythms may accompany aberrations of the medial septum-diagonal bandnucleus basalis complex and may be importantly involved in aging-related impairments of cognitive and learning abilities. We recently found that during odor discrimination reversal learning, rats exhibit a preferred latency relationship between the onsets of their sniffing cycles and a pa,ticular phase of the pacemaker activity which drives the hippocampal 0 rhythm [38]. Our observations sugges~ that neither the sniffing rhythm nor the hippocampal 0 rhythm is driven directly by the other, but that the two become temporally correlated through an intermediate neural process. This process involves periodic adjustments of the sniffing rhythm to bring it into register with relatively more stable, ongoing trains of hippocampal 0 activity. This temporal matching is strongest shortly before attainment of criterion-level performance, when the animals are most likely to be evaluating the behavioral relevance of the stimuli and are in the process of modifying their responses to those stimuli. In our earlier studies using hamsters we found (1) that during free exploratory behavior rhythmic sniffing occurs with the same dominant frequency as hippocampal RSA more often than would be expected by chance, (2) that during such frequency entrainments sniffing exhibits a preferred latency relationship to this 0 rhythm similar to the latency relationship subsequently found in rats, and (3) that this temporal correlation is strongest during investigation of novel or socially-relevant odors [33,75]. These findings are consistent with the hypothesis that temporal matching of sensory-motor behavior and forebrain 0 activity may be important for higher-order processing of sensory information (cf., [ 13, 26, 27, 28, 33, 34, 35, 54, 55]). The purpose of the present study was to determine whether rats, like hamsters, exhibit significant correlations between their investigatory sniffing and the hippocampal 0 rhythm during free behavior, and to evaluate possible agingrelated changes in the frequency characteristics of these two rhythmic activities or in the temporal relationship between the two rhythms. METHOD

Twenty-three male Fischer 344 rats, aged 3 (N=8), 18 (N=5), 30 (N=5), or 36 (N=5) months, obtained from the National Institute on Aging contract colony (Charles River Breeding Laboratories, Wilmington, MA) were used in this study. Nasal airflow was monitored using a removable thermocouple which was held in a chronically indwelling cannula constructed of 25 gauge stainless steel tubing implanted in the left nasal passage. The thermocouple was enclosed in a stainless steel sheath (250 b~m outer diameter) which, when in place, extended approximately 0.75 mm beyond the tip of the cannula. The output of the thermocouple was inverted electronically so that inhalation (cooling of the probe) produced a positive voltage swing. The EEG was recorded from the left dorsal hipoocampus using a bipolar electrode implanted so as to span the stratum pyramidale of field CA l (deep tip non-inverting). Electrodes were constructed of either 125 or 250 >m diameter stainless steel wire and had a vertical tip separation of 0.75-1.0 mm. Similar recording electrodes were implanted in the right main olfactory bulb (MOB) so as to span the ventral mitral body layer. The

analyses of the EEG recorded in the MOB will be presented in a later report. All surgical procedures were conducted under barbiturate anesthesia. At least 10 days were allowed for recovery from surgery before recording began. Following completion of recording, electrode placements and the condition of the nasal epithelium were verified histologically. For recording, animals were placed in a Plexiglas chamber {64× 38 x 36 cm) with a corncob-chip floor enclosed within a Faraday cage. Though no specific olfactory stimuli were presented, the animals were exposed continuously to the odors of the appartus, their own odors, and the odors of the animals which preceded them in the chamber. Recordings were made for about 20 minutes per day for 4 days. Unity gain field effect transistor followers attached to the skull were used to eliminate cable-sway artifacts. The cable leads were connected through a commutator/counterbalance assembly. The inhalation and EEG signals were recorded simultaneously on polygraph paper and analog magnetic tape, and subsequently analysed using a general-purpose digital computer. The frequency response of the amplifier/ tape system was set at 0.3-35 Hz for inhalation and 0.31000 Hz for EEG. By inspection of the polygraph records, 1300-3000 seconds of data from each animal were selected for digitization. In selecting t h e s e data. the only criteria applied were that ( I ) the recordings be artifact free, (2) there be a high incidence of investigatory sniffing (4 Hz or faster), and (3) roughly comparable amounts of data be obtained across age groups. No attempt was made to select on the basis of other behavioral criteria nor to equate the relative incidence of rapid 17 Hz or faster) versus slower {4-6 Hz) sniffing across animals. Due to a marked aging-related decrease in the amount of exploratory behavior (see Results), these selection procedures sampled a smaller percentage of investigatory sniffing in the young animals than in the older animals. The data were digitized at a sampling rate of 512 Hz. The fast-Fourier transform ot' each l-second epoch was computed yielding coefficients with a resolution of I Hz and a Nyquist frequency of 256 Hz. The Fourier coefficients were used to compute the dominant frequency of sniffing and hippocampal EEG, as well as their respective phases. The phase of a component was defined as the phase, with respect to the beginning of the epoch, of the equivalent sine wave. All phase angles and phase angle differences were expressed as modulo 360 (that is, within the 0 ° to 360 ° range). For each l-second epoch of the initially-selected data, the dominant frequency (i.e., the frequency component with the greatest relative amplitude) within the 2-14 Hz range was determined for each signal. Epochs in which the dominant frequency of the thermocouple signal fell outside the 0 range (4-12 Hz) were excluded from further analysis. Thus, only those epochs verified to contain investigatory sniffing bouts were retained. These data subsequently were used to analyse the frequency characteristics, incidences of frequency entrainments, and temporal relationships of sniffing and hippocampal RSA as described in the Results section. Nonparametric statistics were used to test the significance of aging-related changes in the phenomena studied. RESUI,TS

Sni[fin~' and EEG Freq,e,cic,~ When animals were placed in the recording chamber they typically exhibited an initial period of activation, during which they moved about the chamber sniffing the bedding

S N I F F I N G A N D 0 IN A G I N G R A T S

9

TABLE 1 INCIDENCE DISTRIBUTIONS OF I-SECOND ANALYSIS EPOCHS HAVING DOMINANT SNIFFING AND HIPPOCAMPAL EEG FREQUENCIES IN THE 0 RANGE (4-12 Hz) Age (months) 3

18

30

36

Frequency

n

%

n

%

n

%

n

%

Sniffing

4 5 6 7 8 9 10 11 12

230 220 277 261 216 145 36 3 1

17 16 20 19 16 10 3 0 0

321 321 298 211 93 17 1 0 0

25 25 24 17 7 I 0 0 0

178 180 116 50 I0 1 0 0 0

33 34 22 9 2 0 0 0 0

345 326 243 114 31 6 1 0 0

32 31 23 11 3 1 0 0 0

Hippocampal EEG

4 5 6 7 8 9 10 11 12

59 98 232 544 397 42 9 5 3

4 7 17 39 29 3 1 0 0

71 109 252 569 219 23 9 4 4

6 9 20 45 17 2 1 0 0

37 51 108 257 68 7 2 1 1

7 10 20 48 13 1 0 0 0

32 83 340 545 59 4 1 0 0

3 8 32 51 6 0 0 0 0

For each dominant frequency, the mean incidences of epochs for animals within age groups are shown (n). These values are also expressed as percentages of the grand total for each age group. There was an aging-related decrease in the incidence of higher (8-12 Hz) dominant frequencies for both signals, and a relative increase in the incidence of lower dominant frequencies. This shift to lower frequencies was more pronounced for sniffing than for hippocampal EEG and coincided with an aging-related decrease in the vigor of exploratory activity.

and walls. Following this e x p l o r a t o r y period they might g r o o m or rest, though additional investigatory sniffing bouts might o c c u r periodically during the recording session. In all age groups, the amount o f exploratory activity was greatest on the first and second days of recording, declining somewhat as the animals b e c a m e a c c u s t o m e d to the recording chamber. There was a clear aging-related d e c r e a s e in the amount and vigor of spontaneous e x p l o r a t o r y b e h a v i o r that was particularly noticeable in the two oldest groups. This behavioral change was a c c o m p a n i e d by a d e c r e a s e d incidence of relatively high f r e q u e n c y sniffing and hippocampal RSA. Table 1 shows the distributions o f e p o c h s selected for analysis having dominant sniffing and hippocampal E E G frequencies in the 4-12 H z range for e a c h age group. In the youngest group, the sampling procedures yielded a roughly uniform incidence o f sniffing in the 4-8 H z range with a m o d e r a t e amount of 9 H z sniffing and s o m e yet higher freq u e n c y sniffing as well. There was a progessive aging-related decrease in the incidence of 7-12 H z sniffing with a concomitant relative increase in the incidence of 4 and 5 H z sniffing. The hippocampal E E G d o m i n a n t frequencies during these periods of investigatory sniffing exhibited much tighter distributions, and 7 H z was the most c o m m o n dominant freq u e n c y in all age groups. H e r e too, h o w e v e r , the older groups showed relatively less 8 H z or faster and m o r e 6 H z or slower hippocampal R S A in c o m p a r i s o n with the youngest group. The heterogeneity o f the age groups was statistically significant for both sniffing and hippocampal

R S A (p<0.001 and p < 0 . 0 5 , respectively; Kruskal-Wallis o n e - w a y A N O V A s based on individual m e a n frequencies). E v e n the oldest animals were capable of exhibiting long trains o f 8 H z hippocampal R S A w h e n induced to struggle by holding them just a b o v e the floor of the recording c h a m b e r , suggesting that the o b s e r v e d reduction in the incidence of higher f r e q u e n c y hippocampal R S A might be related to a lessening of the overall vigor o f m o v e m e n t s during free behavior (cf., [69]) rather than to an aging effect on the capacity o f the hippocampal RSA p a c e m a k e r m e c h a n i s m to attain high frequencies. Entrainment Phenomena In all but one animal, the d o m i n a n t f r e q u e n c y o f sniffing matched that o f hippocampal R S A (frequency entrainment) more often than would be predicted on the basis o f chance. F u r t h e r m o r e , there was a progessive trend toward f e w e r such frequency entrainments in the older animals. The incidence of f r e q u e n c y entrainments was evaluated statistically as follows. F o r each animal, a t w o - w a y incidence table was constructed having dominant sniffing f r e q u e n c y on one axis and dominant hippocampai E E G f r e q u e n c y on the other. The incidence in each cell that would be e x p e c t e d by chance o v e r the 4-12 H z range was c o m p u t e d as the product o f the row and c o l u m n totals for each f r e q u e n c y divided by the grand total (as one would c o m p u t e e x p e c t e d incidences in a Chi-square analysis). The o b s e r v e d and e x p e c t e d incidences

l0

FORBES A N D MACRIDES

1.75 .so 1.25

iil

1.00 ~. t...

o')

3

18

30 36

Age (months)

FIG. 1. Aging-related changes in the incidence of frequency entrainments between sniffing and hippocampal RSA. The two rhythms are said to be frequency entrained in a given 1-second analysis epoch if the dominant frequency of sniffing matches that of hippocampal RSA. Data are expressed as mean (_+S.E.M.) ratios between the observed number of frequency entrainments and the number expected by chance (i.e., based on the assumption that the frequencies of the two rhythms are statistically independent). The heterogeneity of the age groups is statistically significant 09<0.01; Kruskal-Wallis one-way ANOVA).

of frequency entrainments at each frequency were taken directly from the cells on the diagonal. Both the observed and expected incidences were then summed across frequencies for each animal. The ratio of the sums was used as a descriptor of the incidence of frequency entrainments for an animal. If the two rhythms were independent of each other, this ratio would have an expected value of 1.0. The mean (___S.E.M.) values of the ratio for the four age groups are shown in Fig. 1. The youngest animals exhibited 53% more frequency entrainments between sniffing and hippocampal RSA than would be predicted on the basis of chance, whereas the oldest animals exhibited only 10% more entrainments than chance would predict. The heterogeneity of the age groups was statistically significant (p<0.01; Kruskal-Wallis oneway ANOVA).

Preferred Temporal Relationship-Young Animals In accord with our previously reported observations [38], the phase differences between sniffing and the hippocampal RSA were not random across epochs but tended to assume frequency-dependent values more often than would be predicted on the basis of chance. The analyses described below were employed to quantify (1) the preferred phase differences between sniffing and hippocampal RSA as a function of domim,nt sniffing frequency in the 0 range (4-12 Hz), (2) the magnitude and significance of the preferences at each dominant frequency, and (3) the coefficients of the linear trend in phase differences across frequencies. For each 1-second epoch, the dominant frequency component of sniffing was identified, and the phase difference between sniffing and hippocampal RSA was determined by subtracting the phase angle of the sniffing component from the phase angle

of the hippocampal E E G component at that frequency using polar arithmetic. The phase differences were expressed as vectors of unit length and summed across epochs for each dominant frequency. The angles of the resultant vectors were used as descriptors of the preferred phase difference at each frequency and their magnitudes were used to compute the statistical significance of the uniformity of the phase differences (i.e., the degree of preference) using a modification of the Rayleigh test [76]. Animals in the youngest group exhibited significantly preferred phase differences between sniffing and the hippocampal RSA most consistently in the 5-9 Hz frequency range. Table 2 shows the values of the preferred phase differences for each animal over the entire 4-12 Hz range. Though the computational procedures yielded a preferred phase difference value at each frequency for each animal, the values are shown in Table 2 only if the distribution of phase differences was significantly non-random (p<0.05). Only 2 of the 8 3-month old animals exhibited significantly preferred phase differences between sniffing and the hippocampal RSA at 4 Hz; 3 of the 8 exhibited significantly preferred phase differences at 10 Hz. At least 5 of the 8 animals exhibited significantly preferred phase differences at each frequency in the 5-9 Hz range, and at 6 and 7 Hz all 8 animals exhibited significant preferences. The frequency-dependent linear trend in the preferred phase differences is also evident by inspection of Table 2. Because of the circular nature of these data, special computational procedures, as described in our previous reports [38,75], were called for to determine the regression coefficients for this linear trend. In Fig. 2 the preferred phase differences between the sniffing and hippocampal E E G signals are shown along with the derived regression lines over the 5-9 Hz range for each of the 3-month old animals. It may be seen that the variation in the preferred phase differences across frequencies was well fit by a linear function for all animals. The observation that the preferred phase differences vary linearly as a function of frequency implies an underlying preferred latency relationship (i.e., temporal offset) between the two signals. That latency relationship may be characterised briefly as follows. For each animal, there is a particular phase angle of the hippocampal E E G signal, ~ , which tends to occur r msec following the onsets (phase 0°) of individual sniff cycles, Our earlier observations indicate that this is brought about by modulation of the animals' sniffing behavior to achieve the preferred temporal relationship with ongoing trains of hippocampal RSA. The parameters ~" and ~ , are derived, respectively, from the slopes and y-intercepts of the regression equations graphed in Fig. 1 according to the following algebraic relationship: ~o(t) = ~ , + 0.36"r'f mod(360),

(1)

where cO0(/) is the preferred phase difference at frequencyf. Across animals, the latency coefficient, r, might differ slightly due to variation in the rostral-caudal placement of the thermocouple. The phase coefficient, ~,, varies as a function of hippocampal electrode location with respect to the phase reversal layers within the hippocampal formation [38]. This implies that, across animal, sniffonset is latency-related to an invariant phase of the pacemaker activity which drives hippocampal RSA. A more complete discussion of these points is given in [38]. The derived values of the parameters ~-and ~ for each of the 3-month old animals are given in Fig. 1. The mean

SNIFFING

AND 0 IN AGING

RATS

11

TABLE

2

PREFERRED PHASE DIFFERENCES IN DEGREES BETWEEN SNIFFING AND HIPPOCAMPAL EEG AS A FUNCTION OF DOMINANT SNIFFING FREQUENCY IN THE 4-12 Hz RANGE FOR EACH OF THE 3-MONTH OLD ANIMALS F r e q u e n c y (Hz) Animal 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8

4

5

6

7

8

126 220

122 160 93 166 -195 126

81 67 56 71 71 135 86 70

21 2 26 68 44 88 33 11

0 313

-

-

-----

-

-

-

-

9

.

297 258 309 232 315 -246

-

-331 32 350 -

-

10

.

.

11

12

.

-

-

-

-

-

-

-

-

-

-

-

-

-208 251 185 -

-

-----

-----

-

-

-

-

T h o u g h the computational p r o c e d u r e s yielded a preferred p h a s e difference at e a c h f r e q e u n c y for each animal, the values are s h o w n only if the preference was statistically significant (0<0.05; Rayleigh test). Significant f r e q u e n c y - d e p e n d e n t preferred phase differences were m o s t reliably found in the 5-9 H z range for this age group.

w

[ 2'°F

':!r

3-I 'r . 130.6

~

:::L #.

3-3

3-2 ~

6

l

I

l

I

i

6

7

6

9

=

141.7

8.1

358.1

5

r

r • 163.9

I

I

i

i

i

368

i

i

i

I

1

I

Frequency (Ifz) 3-6

~ •

i

i

i

i

0

'r • 166.7 0.1

30.2

I

i

I

i

~

,

1

~" =

i

i

i

i

I

i

3-8

3-7 ~

i

21.0

133.3 O8

~

3

r • 166.8

7.1

I

I

I

I

I

FIG. 2. Preferred p h a s e differences b e t w e e n sniffing and hippocampai R S A as a function of d o m i n a n t sniffing frequency for the 3-month old animals. Statistically significant p h a s e difference preferences (p <0.05; Rayleigh test) are indicated by filled circles; open circles, p > 0 . 0 5 . T h e variation in preferred p h a s e differences across frequencies is well fit by a linear function for all animals. T h e coefficients o f the linear regression lines were used to derive the p a r a m e t e r s ~" (msec) and qb (degrees) for each animal, representing, respectively, the preferred latency (i.e., temporal offset) b e t w e e n the two signals, and the p h a s e angle o f the R S A to w h i c h the o n s e t s (phase 0 °) o f sniffs are related by this preferred latency (see text).

12

FORBES AND MACRIDES

TABLE 3 PREFERRED PHASE DIFFERENCES IN DEGREES BETWEEN SNIFFING AND HIPPOCAMPAL EEG AS A FUNCTION OF DOMINANT SNIFFING FREQUENCY IN q'HE 4-12 Hz RANGE FOR EACH ANIMAL IN THE THREE OLDEST GROUPS Frequency IHz) 4

5

6

7

8

9

10

11

12

Expected ~o(J)

187

134

80

27

334

280

227

173

120

Animal 18-1 18-2 18-3 18-4 18-5

. 61 171 100 115

. 93 116 72 --

. 55 68 48 349

. 278 II ---

-----

i I

30-1 30-2 30-3 30-4 30-5

112 192 305 -54

52 126 201 156 133

-353 . . . . . . 300 --

--. . --

---

36- l 36-2 36-3 36-4 36-5

224 --259 --

170 94 1112 159 134

168 --139 --

--

------

.

. 108 151 75 115

6 78

------

270

i

--

I i i I I I I

E

m

m

m

m

m

m

m

m

As for Table 2, only statistically significant preferences are shown. Expected values of the frequency-dependent phase differences, q~.(/), were derived from the data of the 3-month old animals using equation [2] as described in the text. The correspondence with expected preferred phase differences was reasonably good for the 18-month old animals in the 5-7 Hz range. In the two oldest groups, there was a further narrowing and lowering of the frequency range within which there was good correspondence with the expected values.

value (_+S.D.) o f ~- for t h e s e animals w a s 148.3_+ 13.5 m s e c . The values o f ~ varied b e t w e e n 359. I ° (equivalent to 0.9 °) and 121.0 ° with an angular m e a n value (_+angular deviation; A . D . ) o f 40.8_+41.2 ° (see [76] pp. 313-316, for a d e s c r i p t i o n o f t h e s e angular statistics). In all but one o f t h e s e animals, the ventral (non-inverting) tip o f the h i p p o c a m p a l e l e c t r o d e was located in stratum radiatum or stratum moleculare o f dorsal field CA1. In o n e animal (3-6) the ventral tip was situated in the superficial part o f the s t r a t u m m o l e c u l a r e o f the d e n t a t e gyrus.

PreJ~rred Temporal Relationship-Effects of Aging Animals o l d e r than 3 m o n t h s also e x h i b i t e d f r e q u e n c y d e p e n d e n t p r e f e r r e d p h a s e d i f f e r e n c e s b e t w e e n the sniffing and h i p p o c a m p a l E E G signals. H o w e v e r , with increasing age, statistically significant p r e f e r e n c e s w e r e exhibited o v e r a p r o g r e s s i v e l y l o w e r and n a r r o w e r range o f f r e q u e n c i e s . Table 3 s h o w s the p r e f e r r e d p h a s e d i f f e r e n c e values as a function o f f r e q u e n c y for the t h r e e o l d e r age groups. A s for Table 2, only statistically significant p r e f e r e n c e s are s h o w n . W h e r e a s m o r e than half o f the 3 - m o n t h old animals e x h i b i t e d significant p r e f e r e n c e s t h r o u g h o u t the 5-9 H z range, the equivalent ranges w e r e 4--7 H z for the 18-month g r o u p and 4-6 H z for the 30-month group. The 3 6 - m o n t h group reliably e x h i b i t e d significantly p r e f e r r e d p h a s e d i f f e r e n c e s only at 5 Hz.

Since the f r e q u e n c y range o v e r w h i c h p r e f e r r e d p h a s e d i f f e r e n c e s w e r e o b s e r v e d was t r u n c a t e d in the older animals, it was not possible to c o m p u t e reliably the parame t e r s r and ~T for t h e s e animals using the s a m e analytic p r o c e d u r e s that had b e e n applied to the y o u n g e r a n i m a l ' s data. In o r d e r to evaluate w h e t h e r the t e m p o r a l relationship b e t w e e n sniffing and the h i p p o c a m p a l RSA signals w a s similar in old and y o u n g animals, the f r e q u e n c y - s p e c i f i c preferred p h a s e d i f f e r e n c e s o b s e r v e d in the o l d e r animals w e r e c o m p a r e d to the e x p e c t e d p h a s e d i f f e r e n c e s for young animals. The e x p e c t e d p r e f e r r e d p h a s e d i f f e r e n c e b e t w e e n sniffing and h i p p o c a m p a l RSA at e a c h f r e q u e n c y was det e r m i n e d by inserting the m e a n values o f 7 and ~ for 3-month old animals into e q u a t i o n (1), yielding the equation: • ,,(J) - 40.8-53.4:[ mod(360).

(2)

The values thus d e r i v e d are s h o w n at the top o f Table 3. F o r the 18-month old animals, all but 1 o f the 11 statistically significant o b s e r v e d values o f q~o(/) in the 5-7 H z range fell within the same q u a d r a n t (i.e., w i t h i n + 4 5 °) as the e x p e c t e d value. At 4 Hz, 3 o f the 4 o b s e r v e d values differed from the e x p e c t e d value by m o r e than 45 °. F o r the 30- and 36-month old groups, the c o r r e s p o n d e n c e b e t w e e n o b s e r v e d and exp e c t e d values o f ~ , ~ ) was r e s t r i c t e d to yet l o w e r and narr o w e r f r e q u e n c y ranges. A m o n g t h e s e animals, the only statistically significant values o f ~,,q) at 5 H z w h i c h fell out-

S N I F F I N G A N D 0 IN A G I N G RATS

13

side the same quadrant as the expected values were those o f animals 30-1 and 30-3. Histology revealed that the noninverting tip of the hippocampal electrode was just dorsal to stratum pyramidale of dorsal field CA1 for animal 30-1, and in the deep part of stratum moleculare of the dentate gyrus for animal 30-3. In that these placements were the most divergent among all animals studied and corresponded to the extrema of a zone o f RSA phase reversal [71], it is reasonable to conclude that the values o f ~o(f) for these two animals differed from the expected values because of variations in electrode placement. Thus, all l0 of the animals in the 30- and 36-month old groups showed significantly preferred phase differences between sniffing and hippocampal RSA at 5 Hz which were consistent with those observed in 3-month old animals. At frequencies other than 5 Hz, significantly preferred phase differences were not reliably observed in the oldest group and, where they were seen, corresponded poorly with the expected values. These findings indicated that the three oldest groups of animals exhibited a preferred latency relationship between sniffing and hippocampal RSA which was similar to that seen in the 3-month old animals, though within progressively lower and narrower frequency ranges. Subsequently, we sought to analyze aging-related changes in the relative incidence of the preferred temporal relationship over the ranges of predominant sniffing frequencies for each age group. This was done by first determining for each group the 3-Hz frequency band which encompassed the highest incidences of dominant sniffing frequenices: 5-7 Hz for the 3-month old group and 4-6 Hz for the three older groups (cf., Table l). Then, within the appropriate frequency range, we computed for each animal the magnitude of the preference for a particular phase angle of the hippocampal E E G signal at a latency of 148.3 msec after phase 0° of its sniff cycles, i.e., at the mean preferred latency of the 3-month old group. If the underlying nature of the preferred latency relationship between the two signals were indeed similar across ages, the preferred phase angle at that latency for the older animals would be near the mean value of qb~ for the youngest group and the magnitude of the preference would reflect the overall incidence of the preferred latency relationship. F o r each l-second analysis epoch having a dominant sniffing frequency in the appropriate range, the phase difference between the sniffing and hippocampal E E G signals, ~(o-~), was found by vector subtraction as described above. The phase angle of the hippocampal EEG signal 148.3 msec after phase 0 ° of the sniffing cycles, ~0, was computed according to the formula: qb0 = ~0-s, + 5 3 . 4 f mod(360),

(3)

where f is the dominant frequency of sniffing in the epoch and 53.4 is the slope of equation (2). The values of qb0 for each epoch were treated as vectors of unit length and summed to yield a resultant vector having the preferred phase angle, ~'T, and magnitude R. The statistic R/n, where n is the number of epochs contributing to the sum, indicates the magnitude of the preference (cf., [76]). The value of R/n varies between 0.0 (no preference) and 1.0, and is independent of n. These statistics could thus be used to determine for each animal the preferred phase angle of the hippocampai EEG signal occurring 148.3 msec following the onsets of sniff cycles and the magnitude of the preference for that phase angle. Figure 3 shows the mean values (-+ S.E.M.) of R/n for the four age groups. There was a trend toward lower incidence

.20 .15 t~

.10 .05 i

3

I

18

i

i

30 36

Age (months) FIG. 3. Aging-related changes in the strength of the preferred temporal relationship between sniffing and hippocampal RSA during spontaneous exploratory behavior. Analyses were conducted over the 3-Hz ranges which encompassed the major proportion of the dominant sniffing frequencies for each group: 5-7 Hz for the 3-month old group and 4-6 Hz for the three oldest groups. The statistic R/n is descriptive of the magnitude of the preference for a particular phase angle of the hippocampal EEG signal at a latency of 148.3 msec after the onsets of sniffcycles. The values plotted are the means (+_S.E.M.) for each age group. There is a decreasing trend with age, indicating an aging-related diminution in the relative incidence of the preferred temporal relationship.

of the preferred latency relationship with increasing age, and the heterogeneity of the groups was stati'fically significant (p<0,05; Kruskal-Wallis one-way A N O ~ A ) . Table 4 gives the values of ~'7 for each animal (only statistically significant values are shown). The mean value (-+A.D.) of qb'T for the 3-month old animals was 42.2-+24.0 ° and was essentially the same as their mean value of qb (40.8_+41.2°). Among the older animals, values of qb' based on statistically significant phase preferences were generally within the range of variability seen in the 3-month old animals except for the two older animals with electrode placements outside stratum radiatum-stratum moleculare of the dorsal hippocampus (animals 30-1 and 30-3). Thus, when it was exhibited, the temporal relationship between sniffing and hippocampal RSA appeared to be similar across age groups despite agingrelated changes in the freqeuncy characteristics of these two rhythms and in the strength of their temporal relationship during spontaneous exploratory behavior. DISCUSSION The results of this study demonstrate that freely behaving rats exhibit significant correlations between their investigatory sniffing and the RSA recorded in the dorsal hippocampus. In young rats, the relationship between these two rhythms is similar to that observed in hamsters and in rats under other experimental conditions. As we found in the hamster [75], the dominant frequencies of sniffing and hippocampal RSA match more often than would be predicted by chance based on their respective frequency distributions. Frequency entrainments between sniffing and hippocampal 0 activity have been reported previously in freely behaving rats but were not quantified nor evaluated statistically [26]. Freely behaving rats also exhibit preferred phase differences

14

FORBES AND MACRIDES TABLE 4 PREFERRED PHASE ANGLESOF HIPPOCAMPALRSA AT A LATENCYOF 148.3msec FOLLOWINGTHE ONSETS OF SNIFF CYCLES 3 months Animal 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8

18 months

30 months

n

qb'

Animal

n

qb',

Animal

n

852 1024 701 453 1055 951 1118 870

35.8 29.6 17.7 57.5 41.0 99.0 43.5 21.3

18-1 18-2 18-3 18-4 18-5

445 1317 969 1951 1170

-10.8 58.5 345.2 9.7

30-1 30-2 30-3 30-4 30-5

468 614 590 488 637

36 months qb'T 323.2 37.2 137.9 52.4 --

Animal

n

~'~

36-1 36-2 36-3 36-4 36-5

1109 747 581 1352 1073

89.8 26.8 -88.1 --

As for Fig. 3, analyses were conducted over the 3-Hz ranges which encompassed the major proportion of dominant sniffing frequencies for each group (n=number of epochs in these ranges). Only statistically significant (/,<0.05; Rayleigh test) preferred phase angles, ~'T, are shown. The mean value (_+A.D.) of qb' for the 3 month animals was 42.2+24.0 °.

between sniffing and hippocampal RSA which vary linearly as a function of dominant sniffing frequency. The existence of a linear relationship such as this implies that the two signals are related by a preferred temporal offset which is invariant across frequencies. The mean preferred latency under the present experimental conditions corresponds within 1 msec to that observed in Long-Evans rats during odor discrimination reversal learning [38]. The correlations between sniffing and hippocampal RSA change progressively with age as follows: (1) the incidence of frequency entrainments decreases; (2) the frequency range within which preferred phase differences are observed becomes lower and narrower; and (3) the incidence of preferred phase differences decreases. Though the frequency range and relative incidence of the preferred temporal relationship are thus reduced, our data indicate that animals of all ages exhibit a similar preferred temporal relationship between the two signals for those frequencies at which preferences are reliably expressed. Thus, the robustness of the preferred temporal relationship between sniffing and hippocampal RSA declines with age, but the underlying nature of the relationship appears to be similar in young and old animals. In addition to this aging-related decline in the temporal matching between sniffing and hippocampal RSA, both rhythms exhibit aging-related decreases in mean frequency in conjunction with a decline in the amount and vigor of exploratory activity. Though these various changes may have a common physiological basis, the aging-related weakening of the correlations between the two rhythms is not due simply to the changes in their frequency distributions nor in the amount of investigatory sniffing. For example, the weakness of the preferred temporal relationship at 6 Hz in the oldest group of animals was clearly not due to a lack of investigatory sniffing at that frequency in our data base, since the incidence of their 6 Hz sniffing was comparable to that of the youngest group's 5 and 8 Hz sniffing (cf., Table 1), for which the preferred temporal relationship was quite strong (cf., Table 2). Furthermore, our analysis of aging changes in the strength of the preferred temporal relationship was based on the data in each group's predominant sniffing frequency band and was thus insensitive to group differences in sniffing frequency. It also should be noted that our definition

of the 0 frequency range followed the convention of including the upper end of the range for hippocampal RSA in rats, thus extending into the c~ band (cf., [38]). However, the significant correlations between sniffing and hippocampal RSA occurred within the middle and lower portion of the defined 0 range, corresponding to the frequency range of hippocampal RSA which can be blocked by atropine [30,67] and which thus is most likely to be driven by cholinergic projections from the medial septal nucleus and the nucleus of vertical limb of the diagonal band (cf., [4,24]). The present findings complement previous neuroanatomical and biochemical evidence for aging-related changes in the medial septum-diagonal band-nucleus basalis complex [5, 16, 50, 70] and may provide a basis for developing a model system in which to study forebrain mechanisms which underlie aging-related abnormalities of cognition or learning. During discrimination reversal learning, the preferred latency relationship is stronger when animals are assessing the behavioral relevance of odorants than when they are most reliably discriminating the same odorants but are not modifying their behavioral responses to them [38]. Likewise, during free behavior, the preferred temporal relationship between sniffing and hippocampal RSA appears to be characteristic of periods when animals are evaluating the relevance of odors (cf., [33,75]). The aging-related weakening of the correlations between investigatory sniffing and the hippocampal 0 rhythm, together with the general decrease in the amount and vigor of exploratory behavior, likely reflect aging-related reductions in the predisposition or ability of these macrosmatic mammals to evaluate stimulus relevance or modify their behavior in a novel environment. Several lines of evidence suggest that these higher order processes may involve the temporal patterning of sensory inputs in relation to endogenous 0 activity in the basal forebrain. Investigatory sniffing in rodents is a stereotyped sensory-motor pattern which normally casts olfactory, somatosensory and visual inputs into discrete, coordinated sampling epochs [26, 27, 33, 34, 35, 68]. With each cycle of inhalation, the animals typically execute a cycle of vibrissae sweeping and of head bobbing (i.e., alternating movement and fixation of the head and neck) so that each sniff is accompanied by a new sample of somatosensory input from the

S N I F F I N G AND 0 IN AGING RATS

15

head region and by a saccadic shift of visual stimuli on the retina. Neurons in the basal portion of the nucleus of the vertical limb of the diagonal band, in the nucleus of the horizontal limb of the diagonal band, and in the rodent homolog of the nucleus basalis of Meynert (hereafter referred to collectively as the basal c o m p o n e n t of the medial septumdiagonal band-nucleus basalis complex) project to most of the olfactory and neocortical structures, including the olfactory bulb, somatosensory cortex, and visual cortex [10, 32, 37, 41,74]. Neurons in this basal component, including ones confirmed with antidromic stimulation to project into the olfactory bulb, exhibit endogenous rhythmic bursting activity throughout the 4--12 Hz range [40]. The 0 bursting activity in the basal component, like investigatory sniffing, is not invariably correlated with hippocampal RSA (e.g., can exhibit a slower or faster dominant frequency). Although 0 activity recorded with extra-hippocampal EEG electrodes often has been assumed to represent volume-conducted hippocampal RSA (e.g., [ 12,71 ]), intrinsic 0 activity has been demonstrated in paleo-, meso-, and neocortical structures (e.g., [22, 29, 39, 65]) and may be driven by neurons of the basal component. Furthermore, a highly coherent 0 rhythm in neocortex accompanies the orientation response [64] and the coherence of middle and low frequency 0 activity in different sensory and motor neocortices has been shown to increase during elaboration of a conditioned response (cf., [2]; [20,21]). Several regions of olfactory cortex and associational neocortex that receive projections from the basal component also receive direct inputs from the hippocampal formation ([52, 62, 63]; J. E. Marchand, T. A. Shoenfeld and F. Macrides, in preparation), and output neurons of the hippocampal forma-

tion have been shown to discharge in relation to complex sensory, situational and/or spatial contingencies but to synchronize their firing with hippocampal RSA when they do discharge [11, 72, 73]. These various observations suggest that changes in the strength of correlations among temporally patterned sensory-motor activity and the limbic, olfactory, and neocortical 0 rhythms may reflect, or underlie, changes in the higher-order processing of information about the environment (cf., [28, 34, 54]). It is interesting in this regard that aging-related changes in the temporal characteristics of investigatory eye movements have been reported in humans [14, 15, 56], and that neurons located throughout the entire extent of the basal component project to the striate cortex in monkeys [19]. In rodents, the distribution of neurons in the basal component that project to the olfactory bulb or olfactory cortex overlaps extensively with the distribution of neurons projecting to visual neocortex ([10,32]; J. E. Marchand, T. A. Schoenfeld and F. Macrides, in preparation). Analyses of the functional roles of forebrain 0 activity in olfactory processing thus are likely to be generalizable to other modalities and may also provide insights into the functions of the medial septum-diagonal band-nucleus basalis complex in microsmatic mammals. The stereotyped nature of investigatory sniffing in rodents, its significant but variable correlations with the hippocampal 0 rhythm, and the existence of a distinguishable 0 rhythm in the olfactory system make these macrosmatic mammals excellent models in which to study the possible interrelationships among aberrations of this complex, alterations of forebrain 0 rhythms, and aging-related impairments of cognitive and learning abilities.

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