The Development of Response to Continuous Auditory
Stimulation in Rats Treated Neonatally with 6-Hydroxydopamine YVONNE BRACKBILL Department of Psychology University of Florida Gainesville, Florida THOMAS C. DOUTHITT Adult Psychiatry Branch National Institute o f Mental Health Bethesda, Maryland
This study investigated biochemical substrates of the continuous stimulation effect, i.e., the developmental phenomenon whereby continuous stimulation depresses arousal level. Neonatal rats were injected intracisternally with 6-hydroxydopamine. Motility, heart rate, and brain amines were measured in theses animals, as well as in injected and noninjected control animals, at 14 and 28 days of age in an own-control design with and without continuous auditory stimulation. Brain norepinephrine was significantly and inversely related to the magnitude of the continuous stimulation effect and experimental-control differences increased with age. These results indicate that an intact catecholaminergic system is necessary for the mediation of increased arousal during stimulation.
Recent studies of human infants have shown that continuous stimulation produces a marked drop in state or arousal level as indexed by a decrease in heart rate, respiration rate, crying, and motor activity along with an increase in time spent asleep (Brackbill, 1970, 1971, 1973; Brackbill, Adams, Crowell, & Gray, 1966). This phenomenon begins immediately upon stimulation and is sustained over the course of stimulation without any apparent homeostatic adjustment other than that attributable to the hunger/feeding cycle. Several stimulus parameters are now known to influence the extent to which the continuous stimulation effect occurs. The effect is transmodal, that is, it occurs under continuous auditory stimulation, continuous visual stimulation, continuous proprioceptive-tactile stimulation, and so on (Brackbill, 1971). Further, the effect is cumulative, that is, the level of arousal changes as an inverse function of the number of
Received for publication 18 December 1974 Revised for publication 3 March 1975 Developmental Psychobiology, 9(1): 5-15 (1976) 0 1 9 7 6 by John Wiley & Sons, Inc.
5
6
BRACKBILL AND DOUTHITT
sensory modalities continuously stimulated (Brackbill, 1971) with continuous auditory and visual stimulation depressing arousal level t o a greater extent than either mode o f stimulation alone. Finally, the intensity of the effect is, t o some extent, a direct function of the intensity of stimulation, e.g., 80-dB white noise depresses arousal levcl to a greater extent than does 60.~d13white noise (Brackbill, in press). In addition to the stimulus parameters known t o influence the continuous stimulation effect, several organi.smic variables are also related to the probability of its occui-rence and intensity. Taken together, these suggest that the effect occurs to the extent that the stimulated organism is neurologically immature. Relations among these variables include the following: (1) The effect is age-dependent, occurring in babies and very young children, but apparently not in adult human beings (Scott, 1972) or in adult rats (Van Twyver, Levitt, & Dunn, 1966) except under very intense stimulation. (2) Among neonatal animals, the effect occurs in rat pups but not in guinea pigs (Brackbill, in press). (3) The effect does not appear to depend on central nervous system (CNS) integrity in that it has been found in an anencephalic infant (Brackbill, 1971). The question is, then, what neurophysiological mechanisnis d o mediate the effects 01' continuous stimulation on arousal level and why is arousal level so peculiarly sensitive to stimulus input prior to neurological maturation? The most promising directions for inquiry come from recent biochemical studies of immature animals showing that for organisms whose development is still inmature at time of birth, levels of those neurotransmitters now known t o be involved in various manifestations of arousal level-e.g., activity, exploration, sleep-are low during the neonatal period and only gradually approach adult levels (Coyle & Axelrod, 1971; Eiduson, 197 1 ; Loizou, 1972). Because central catecholaminergic mechanisms have been implicated in mediation of arousal level via midbrain pathways, we suspected that increasing levels of norepinephrine (NE) might underlie the developmental behavioral change in response to continuous stimulation. In the present study we proposed to determine the validity of this hypothesis by preventing the normal developmental increase of this catecholaminergic neurotransmitter and assessing the effects of this interference on arousal level under nonstimulated and stirnubated conditions. Specifically, the procedure was to inject into the CNS of 2 4 h r old rat pups 6-hydroxydopamine (6-OHDA), a substance causing degeneration of catecholamine nerve temiinals (Breese & Traylor, 1972) and t o measure subsequently motility and heart rate in these animals at 14 or 28 days in an own-control design with and without continuous auditory stimulation.
Method
Design We examined 3 independent variables: ( I ) chemical interference with normal atecholanunergic function; (2) stage of maturation at time of testing; and (3) presence or absence of continuous auditory stimulation.
6-OHDA AND AROUSAL DURING STIMULATION
7
N E Utilization. Experimental animals were injected intracisternally 24 hr after birth with 6-OHDA, thereby impairing normal development of central catecholaminergic nerve transmission. Stage of Maturation. Chronological testing ages of 14 and 28 days were chosen to represent early and late stages of neurological maturation. The design was cross-sectional, with 30 animals at each age; 10 experimental subjects; 10 injected control subjects; and 10 noninjected control subjects. Loss of subjects reduced the final N from 60 to 53. (See Table 1). Continuous Auditory Stimulation. Each subject served as his own control under 2 stimulus conditions: continuous auditory stimulation (white noise at 80 dB and no auditory stimulation beyond the ambient background level (59 dB). We recorded 3 principal dependent variables: motility (gross motor activity); heart rate (HR); and biochemical assay of NE and its precursor, dopamine (DA). Serotonin (5-HT) was also measured to determine if 6-OHDA administration might also have damaged the tryptaminergic system. Motility. Gross motor movement was measured by means of a stabiiimeter device constructed as follows: The clear plastic testing boxes were equipped with a grid floor consisting of 18 parallel copper wires spaced at 1.3-cm intervals and wired alternately so that the animal's movement across the grid floor produced changes in resistance as the circuit was opened or closed by the animal's contact with it. Voltage changes (in the range of 100-300 pV) thus produced were recorded polygraphically. Motility was scored every 2.5 sec in terms of maximum pen deflection over .5-cm units. Scores were summed over 10-sec intervals. Heart Rate. The HR was measured by means of an implanted electrode arrangement described below and recorded on a Grass Model 5 polygraph at a paper speed of 25 mmlsec. Biochemical Assays. Fluorometric assays of whole brain NE were carried out by means of the method developed by Laverty and Taylor (1968) and for 5-HT by the method developed by Bogdanski, Pletscher, Brodie, and Udenfriend (1956).
Subjects and Procedure Subjects were Osborne-Mendel rat ( R a m s noruegicus) pups. The pregnant females were received from the National Institutes of Health Animal Breeding Facility 5 days before term to allow accommodation to the laboratory before littering. They were housed individually in 30.8 x 46.2 x 15.4 cm plastic cages and provided with paper nesting material. Cages were kept under a 12-hr light: 12-hr dark cycle at a temperature
TABLE 1. Design, n, and Mean Weight at Testing. 28 Days
14 Days Group
E IC NIC
n 9 10
to
Weight (g)
n
Weight (g)
34.9 36.6 41.1
7 7 12
97.1 86.7 97.2
8
BKACKBILL AND DOUTHITT
of 23.5"C and an ambient noise level of 56?2dB. Food and water were provided ad lib. Litters were culled t o 9 pups within 24 hr of delivery to insure equivalence of tnaternal attention. Mothers were removed from their litters after 22 days. At approximately 24 hr of age, each litter of 9 was divided into 3 groups and treated as follows. Two pups were assigned to the Experimental (E) group and injected ititracisternally with .01 mg o f 0-OHDA MBr (Regis) in .1% L-ascorbic acid in .01 ml N saline (pH adjusted to 4.3). Two animals, assigned to the Injected Control (IC) g o u p , were injected with the vehicle alone. Two animals assigned t o the Noninjected Control (NIC) group were not treated. Each group was constituted o f approximately equal numbers of rnales and females. Results were not differentially affected by gender. The remaining 3 animals were resewed as spares or used for other purposes. All animals were marked for litter number, subject number, and treatment group by injecting different combinations of black and colored India ink under the pads o f 1 or more teet. Animals were handled on this and a11 other occasions with Tritlex vinyl examining gloves. Cannibalism or other gross indices of abnormal tnaternal behavior were not observed. Forty-eight hours before testing, the electrodes necessary to record HR were placed in the animal. The procedure followed was generally that recommended by Hofer (M. A. Hofer, personal communication; Hofer & Reiser, 1969). After the animal was anesthetized with ethyl ether, 2 male Amphenol pins (220-P02-100), each connected t o 30-gauge stainless steel multifilament surgical wire, were sewn under the skin. The upper electrode was placed I cm above the right foreleg; the lower electrode was placed on the left side, at the level o f the lower rib. Each electrode was stabilized with collodion 15 min before testing began. The testing chamber was a 47.4 x 56.4 x 66.7 cm Elconap incubator (Model B-2) equipped with a Sony cassette tape player. Within the incubator was a plastic testing box (17.9 x 28.2 x 12.8 cm) equipped with a grid floor stabilimeter. Temperature in the incubator was maintained at 33k1 "C, the temperature recommended for individual animals o f this age b y M.A. Hofer (personal communication). Five tninutes before the testing session began, the animal was placed in the testing box and electrodes connected to the recording cable. A 5-min pretest accomodation was sufficient (M. A. Hofer, personal communication) for HR t o return t o normal resting level following a brief phasic change. The 20-min testing session itself was divided into two 1 O-min periods: continuous auditory stimulation (white noise played at 80 dB SPL), and no extra auditory stimulation beyond the ambient noise level o f 59 52dB. The sequence of administering the sound (S)/no sound (NS) phases was counter-balanced over drug treatments, stimulus conditions, and age groups. The FIR and voltage changes from the stabilimeter floor were directly recorded on a Grass Model 5 polygraph using ari electroencephalogram (EEG) preamplifier for the stabilimeter output with a time constant of . i sec. After testing, animals were returned t o home cages. Twenty-four hours later, each was weighed and decapitated. The brain was immediately removed, frozen on dry ice, and stored at -20°C for subsequent biochemical assay. Whole brain monoarnine levels were determined b y homogenizing the brains in .7N petcliloric acid with .S% ethylenediamine tetra-acetate. The homogenates were transferred i n t o plastic tubes containing .5 ml of 5% ascorbic acid and centrifuged at
6-OHDA AND AROUSAL DURING STIMULATION
9
12,000 g for 20 min. The clear supernatant fluids were transferred to 45-ml centrifuge tubes containing 250 mg alumina. The pH was adjusted to 8.6 with 3M hydroxymethylamino methane buffer. The tubes were shaken for 5 min. After centrifugation, the supernatant 5-HT was extracted and fluorometrically assayed according to Bogdanski et al. (1956). The alumina containing NE was washed twice with distilled water following which the supernatant fluid was adjusted t o pH 7 and discarded. The catecholamines were eluted from the alumina with 3 ml of . l N HCl and analyzed by the method of Laverty and Taylor (1968). Behavioral and physiological results were analyzed statistically by ANOVA for repeated measures. Biochemical results were evaluated by means of Student's t-test. Pearson product moment coefficients were used to assess degree of relationship between biochemical and behavioral/physiological data.
Results
Psychophysiological Measures Results at 14 Days. A significant drop in motility occurred under continuous auditory stimulation (F=4.95, df = 1/23, p < .05) being most pronouned for the drug-treated group (Table 2, Fig. 1). A significant Sound x Sequence-of-Sound interaction ( F = 22.80, df= 1/23, p < .001) also occurred, the direction of which indicated an order effect, i.e., continuous stimulation had a greater pacifying effect if it preceded no sound. The steadily declining level of arousal throughout the 20-min session suggests, however, that an initial 5-min adaptation period was insufficient t o familiarize animals with the testing chambers and allow return of motility to base-line levels. it also may suggest that continuous stimulation may overcome short-term stress-induced excitement. The effect of continuous stimulation on HR appeared as a significant Sound x Sequence-of-Sound interaction ( F =8.26, df = 1/23, p < .Ol). An insufficient adaptation period to the testing chamber is the most probable cause of the sequence effect (Table 2, Fig. 2). A significant Drug Treatment effect (F = 4.27, df= 2/23, p < .05) was attributed to the higher HR levels for the E and IC groups than the NIC group. The closer correspondence between IC and E means than between IC and NIC means suggests that the physical insult of injecting .01 ml of an acidic solution into the neonatal brain has effects that outlast the neonatal period. Results at 28 Days. At this age, sound-related differences among groups in motility were pronounced (Fig. 1). Continuous auditory stimulation continued to have a quieting effect on the 6-OHDA treated animals. However, it had a significant arousing effect on the control animals ( F =3.60, df= 2/19, p < .05). Again, adaptation effects appeared as a significant Sound x Sequence-of-Sound interaction ( F = 9.92, df = 1/19, p < .Ol). The effects of continuous stimulation on HR interacted significantly with Drug Treatment (F = 5.34, df = 2/20, p < .05) and with Sequence of Sound ( F = 4.21, df = 1/20, p < .06). Thus, experimental animals continued t o respond in an infantile fashion to continuous stimulation whereas control animals now responded with higher HR's. However, this directional reversal in responsiveness only manifested itself after an adaptation period longer than the allotted S min. Figure 2 shows the maturation-linked reversal in responsiveness during the period maximally free of adaptation effects.
9
HR
Motility
HR
14 days
28 days
28 days 7
7
9
Motility
14 days
n
Measure
Age
4.21 1.12 473.64 c14.80 4.30 t 1.04 442.56 k 13.50 t
NS
E
2.54 1.13 479.76 k12.80 2.71 t .58 411.78 i19.50
*
S
7
6
10
10
n
480.18 t10.90 5.01 t 1.20 427.86 i 12.40
+ 1.08
3.98
NS
IC
Group
10
3.92 1.09 485.94 t 10.40 5.65 ? .44 443.82 i16.80
12
12
10
n S
+
TABLE 2. HR and Motility during Periods of NS and S Stiniulatiori (Mearzs 2 S E).
S
2.54 +.67 444.12 59.80 5.15 t.71 433.68 ? 8.20
NS 2.64 k.46 443.76 t 7.30 4.41 t.88 426.30 t9.40
NIC
6-OHDA AND AROUSAL DURING STIMULATlON
11
-2J E
IC NIC 14 Days
E
IC NIC
28 Days
Fig. 1. Continuous stimulation effects on motility. Negative difference scores indicate a decrease from NS to S conditions.
EXPIC NIC
480-
.-----. ... .... .
470-
460-
450-
*... 440-
k 4 430a n
'.._
".
e
z
f
420-
41 0-
400-
390-
No Sound Sound 14 Days
No Sound Sound 28 D a y s
Fig. 2. Continuous stimulation effect on HR. Scores are from 2nd half of session.
BRACKBILL AND DOUTHITT
12
*
TA RLI' 3. Whole Brain N E and 5-HT I,evels (Mean SE in ngfg). 1:
n
NF
5-HT
14 d e ys
9
28days
5
18.89a t12.55 26.80" t13.37
215.22 t 8.82 256.20 k21.15
~
IC
-
Age
NIC
11
NE
S-lfT
n
NI,
9
141.44 t20.32 163.33 t21.26
208.67 t13.05 235.33 t18.37
8
111.25 i12.04 191.61 230.69
5-H7
~~
3
:'Dtffers significantly from both control groups a t both a g e s 9
6
179.00 + 7.40 256.67 138.22
< .01 (t-test).
Biochernical Assays The effectiveness of 6-OHDA in destroying N E function is obvious in that these animals had drastically lower NE levels than either control group at either age (Table 3). The NE level for drug-treated animals was noticeably less than that for control animals with n o overlap between these distributions. The correlation between NE and triotility sound/no sound difference scores was -.34 ( t = 2.17, df = 35, p < .02S; 1 tailed test) and in the directjonliypothesized, i.e., the lower the level o f N E , the greater the effectiveness of continuous stimulation in reducing gross motor activity. Likewise, a significant correlation between NE and IIR difference scores indicates the magnitude of the continuous stimulation effect is also inversely related to N E level (Y = -.28, t = 1.71, df = 35, p = .OS; 1-tailed test). The levels of 5-HT recovered showed no effect of prior drug administration. All mean values were within normal, age-appropriate limits and were uncorrelated with motility (-.06) or HR (-.04).
Discussion This study hypothesized that a low level of endogenous N E is responsiblc for the developmental phenomenon whereby continuous stimulation depresses arousal Icvel. The hypothesis is supported by results indicating that the continuous stimulation effect is prolonged in rats whose normal utiliLation of this adrenergic neurotransmitter is blocked. Specifically, our results indicated both that the magnitude of the continuous stimulation effect is correlated with the N E level and that the experimental-control differences in the effect increase with age. Results for both psychophysiological measures indicated that continuous stimulation depressed arousal level. Nevertheless, this conclusion must be qualified by the occurrence of sequence effects, which in turn relate to the stress of introducing young animals into the novel environment o f testing boxes. Clearly, the 5-rnin adaptation period proposed by Hofer and Reiser (1969) was insufficient t o dissipate tllis stress and to allow psychophysiological measures to return t o resting levels. (Note that no sequence effects have been found in continuous stimulation studies with human infants.)
6-OHDA AND AROUSAL DURING STIMULATION
13
Whatever stressful effects may have resulted from the intracisternal injection procedure did not significantly affect experimental results gathered at later ages. (A tendency toward slightly higher HR in the IC group than NIC group at age 14 days is not reflected in the activity measure at that age or in either psychophysiological measuure at age 28 days.) The fact that the NIC and IC groups did not differ significantly on psychophysiological measures-or on brain amine levels-strengthens the conclusions that the results are attributable t o the experimental manipulation of central catecholaminergic NE synthesizing capacity. Both motility and HR were depressed by continuous stimulation. This directional agreement between somatic and autonomic measures confirms previous results with human infants showing that although dissociation of measures o f arousal may often occur with adult subjects, it does not characterize experimental outcomes at early age levels. Campbell, Lytle, and Fibiger (1969) and Mabry and Campbell (1973) have proposed that NE has an excitatory or facilitating effect on arousal-presumably at the brain stem level, in the reticular formation-whereas the cholinergic (ACh) system from the forebrain area and the 5-HT system in the midbrain area act t o attentuate or inhibit arousal. They further proposed that both the ACh and 5-HT inhibitory systems become functionally mature at a later development point than does the NE-mediated excitatory system. If this conceptualization is correct then our findings suggest continuous sensory stimulation might facilitate 5-HT activity, e.g., by accelerating its rate of synthesis, and/or might inhibit NE activity. This theoretical view and our empirical results with human infants showing increased quiet sleep accompanied by decreased active sleep under continuous stimulation also agree with Jouvet’s (1969) sleep model, in which he proposed that increased 5-HT activity is a principle correlate of slow wave sleep whereas ACh and NE activation are more closely allied to paradoxical or rapid eye movement (REM) sleep. The developmental aspects of response to continuous stimulation are especially interesting to consider. We find it unlikely that the continuous stimulation effect entirely disappears with maturity but, rather, believe that to demonstrate the effect requires extremes of stimulus intensity not generally encountered under real-life conditions-or most laboratory conditions, for that matter. Thus, Licklider (1961) reported many dental patients using audioanalgesia fall asleep during or after a dental procedure while listening to music or white noise through their earphones set as high as 116 dB. Oswald (1960) produced EEG signs of sleep in human adults by subjecting them to intense rhythmic auditory, tactile, and visual stimulation. SimiIarly, Pavlov found that the elicitation of conditioned responses could be inhibited by conditional stimuli that were too strong (transmarginal inhibition) and, further, that a frequent correlate of this procedure was sleep. Finally, as noted earlier, both Van Twyver et al. (1966) and Scott (1972) found decreased REM sleep and increased slow wave sleep in adult rats and adult human beings, respectively, but at white noise levels maintained at 92-93 dB for several consecutive hours. These considerations suggest that the developmental course of the continuous stimulation effect is not dichotomous-present in earliest stages and then gone forever, like the sucking reflex-but rather that the ease with which it is manifested depends on the intensity of the stimulus in relation to the degree t o which inhibitory mechanisms have developed in the organism being
14
BRACKBILL AND DOUTHITT
stimulated. For a neurologically immature organism, low levels of stimulation are effective because inhibitory ability functions in a rudimentary fashion if at all: for the neurologically mature organism extreme intensities of stimulation are required to overcome fully functional inhibitory processes. T h s line of reasoning is very similar to Pavlov’s early speculation, formulated on the basis of behavioral rather than biochemical observations, that the ontogeny of responsivemess precedes the ontogeny of inhibitory capacity.
Notes This investigation was supported in part by Research Scientist Award MH 5925 from the National Institute of Mental Health and in part by National Science Foundation research grant GB 35155. The authors wish t o thank Dr. Jorge Pcrez-Cruet of the Laboratory of Clinical Science, National Institute of hlcntal Health, for carrying out t h e biochemical assays, and Kelvin Gleeson for his research assistance. Request reprints from: Dr. Yvonne Brackbill, Department of Psychology, University of Florida, Gainesville, Florida 32611, U.S.A.
References Bogdanski, D. F., Pletscher, A., Brodie, B. B., and Udenfriend, S. (1956). Identification and assay of serotonin in the brain. J. Pharmacol. Exp. Ther., I 1 7 : 82-88. Brackbill, Y. (1970). Acoustic variation and arousal level in infants. Psychophysiology, 6 : 5 17-526. Brackbill, Y. (1971). Cumulative effects of continuous stimulation o n arousal level in infants. C’lild Dev.. 42: 17-26. Brackbill, Y. (1973). Continuous stimulation reduccs arousal level: Stability of the effect over time. Child Dev., 4 4 : 43-46. Brackbill, Y. (in press). Continuous stimulation and arousal level in infancy: Effects of stimulus intensity and stress. CliildDev. Brackbill, Y., Adams, G., Crowell, D. H., and Gray, M. L. (1966). Arousal level in neonates and prcschool children under continuous auditory stimulation. J. Exp. Child Psychol., 4 : 178-1 88. Breesc, G. R., and Traylor, T. D. (1 972). Developmental characteristics of brain catecholamines and tyrosine hydroxylase in the rat: Effects of 6-hydroxydopamine. Rr. J. Pharmacol.. 4 4 : 210-222. Campbell, B.A., Lytle, L.D.. and Fibiger, H.C., (1969). Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science, 1 6 6 : 635-63 7 . Coyle, J.T. and Axelrod, J . (1971). Development of the uptake and storage of L-[’HI norepinephrine in the rat brain. J. Neurochem. 18: 2061-2075. Eiduson, S. (1971). Biogenic amines in the developing brain. In D.C. Pease (Ed.), UCLA Forum in .Wedical Sciences, No. 14. Cellular Aspects of Neural Growth and Diffeventiation. Los Angeles: University of California Press. Pp. 391-41 8. IIofer, M. A., and Reiser, M. 1:. (1969). The development of cardiac rate regulation in pre-weanling rats. Psychosom. Mcd., 21 : 372-388. Jouvet, M. (1969). Biogenic amines and t h e states of sleep. Science, 163: 3 2 4 1 . h v e r t y , R., and Taylor, K.M. (1968). The fluorometric assay of catecholamine:; and related compounds. A nal. Biochem., ;?2: 26 9-2 19. Licklider, J. C. R. (1961). O n psychophysiological models. In W. A. Rosenblith (Ed.), Scnsory Communication. Cambridge: Massachusetts Institute of Technology Press. Pp. 49-72. Loizou, L.A. (1972). The postnatal ontogeny of monamine-containing neurons in the central nervous system of the albino rat. Brain Res., 4 0 : 395418.
6-OHDA AND AROUSAL DURING STIMULATION
15
Mabry, P.D., and Campbell, B. A. (1973). Serotonergic inhibition of catecholamine-induced behavioral arousal. Brain Rex, 49: 381-391. Oswald, I. (1960). Falling asleep open-eyed during intense rhythmic stimulation. Br. Med. J . , I : 1450-1455. Scott, T.D. (1972). The effects of continuous, high intensity, white noise on the human sleep cycle. Psychophysiobgy, 9 : 227-232. Van Twyver, H.B., Levitt, R.A., and Dunn, R . S . (1966). The effects of high intensity white noise on the sleep pattern of the rat. Psychonom. S c i , 6 : 355-356.
Stimulation in Rats Treated Neonatally with 6-Hydroxydopamine YVONNE BRACKBILL Department of Psychology University of Florida Gainesville, Florida THOMAS C. DOUTHITT Adult Psychiatry Branch National Institute o f Mental Health Bethesda, Maryland
This study investigated biochemical substrates of the continuous stimulation effect, i.e., the developmental phenomenon whereby continuous stimulation depresses arousal level. Neonatal rats were injected intracisternally with 6-hydroxydopamine. Motility, heart rate, and brain amines were measured in theses animals, as well as in injected and noninjected control animals, at 14 and 28 days of age in an own-control design with and without continuous auditory stimulation. Brain norepinephrine was significantly and inversely related to the magnitude of the continuous stimulation effect and experimental-control differences increased with age. These results indicate that an intact catecholaminergic system is necessary for the mediation of increased arousal during stimulation.
Recent studies of human infants have shown that continuous stimulation produces a marked drop in state or arousal level as indexed by a decrease in heart rate, respiration rate, crying, and motor activity along with an increase in time spent asleep (Brackbill, 1970, 1971, 1973; Brackbill, Adams, Crowell, & Gray, 1966). This phenomenon begins immediately upon stimulation and is sustained over the course of stimulation without any apparent homeostatic adjustment other than that attributable to the hunger/feeding cycle. Several stimulus parameters are now known to influence the extent to which the continuous stimulation effect occurs. The effect is transmodal, that is, it occurs under continuous auditory stimulation, continuous visual stimulation, continuous proprioceptive-tactile stimulation, and so on (Brackbill, 1971). Further, the effect is cumulative, that is, the level of arousal changes as an inverse function of the number of
Received for publication 18 December 1974 Revised for publication 3 March 1975 Developmental Psychobiology, 9(1): 5-15 (1976) 0 1 9 7 6 by John Wiley & Sons, Inc.
5
6
BRACKBILL AND DOUTHITT
sensory modalities continuously stimulated (Brackbill, 1971) with continuous auditory and visual stimulation depressing arousal level t o a greater extent than either mode o f stimulation alone. Finally, the intensity of the effect is, t o some extent, a direct function of the intensity of stimulation, e.g., 80-dB white noise depresses arousal levcl to a greater extent than does 60.~d13white noise (Brackbill, in press). In addition to the stimulus parameters known t o influence the continuous stimulation effect, several organi.smic variables are also related to the probability of its occui-rence and intensity. Taken together, these suggest that the effect occurs to the extent that the stimulated organism is neurologically immature. Relations among these variables include the following: (1) The effect is age-dependent, occurring in babies and very young children, but apparently not in adult human beings (Scott, 1972) or in adult rats (Van Twyver, Levitt, & Dunn, 1966) except under very intense stimulation. (2) Among neonatal animals, the effect occurs in rat pups but not in guinea pigs (Brackbill, in press). (3) The effect does not appear to depend on central nervous system (CNS) integrity in that it has been found in an anencephalic infant (Brackbill, 1971). The question is, then, what neurophysiological mechanisnis d o mediate the effects 01' continuous stimulation on arousal level and why is arousal level so peculiarly sensitive to stimulus input prior to neurological maturation? The most promising directions for inquiry come from recent biochemical studies of immature animals showing that for organisms whose development is still inmature at time of birth, levels of those neurotransmitters now known t o be involved in various manifestations of arousal level-e.g., activity, exploration, sleep-are low during the neonatal period and only gradually approach adult levels (Coyle & Axelrod, 1971; Eiduson, 197 1 ; Loizou, 1972). Because central catecholaminergic mechanisms have been implicated in mediation of arousal level via midbrain pathways, we suspected that increasing levels of norepinephrine (NE) might underlie the developmental behavioral change in response to continuous stimulation. In the present study we proposed to determine the validity of this hypothesis by preventing the normal developmental increase of this catecholaminergic neurotransmitter and assessing the effects of this interference on arousal level under nonstimulated and stirnubated conditions. Specifically, the procedure was to inject into the CNS of 2 4 h r old rat pups 6-hydroxydopamine (6-OHDA), a substance causing degeneration of catecholamine nerve temiinals (Breese & Traylor, 1972) and t o measure subsequently motility and heart rate in these animals at 14 or 28 days in an own-control design with and without continuous auditory stimulation.
Method
Design We examined 3 independent variables: ( I ) chemical interference with normal atecholanunergic function; (2) stage of maturation at time of testing; and (3) presence or absence of continuous auditory stimulation.
6-OHDA AND AROUSAL DURING STIMULATION
7
N E Utilization. Experimental animals were injected intracisternally 24 hr after birth with 6-OHDA, thereby impairing normal development of central catecholaminergic nerve transmission. Stage of Maturation. Chronological testing ages of 14 and 28 days were chosen to represent early and late stages of neurological maturation. The design was cross-sectional, with 30 animals at each age; 10 experimental subjects; 10 injected control subjects; and 10 noninjected control subjects. Loss of subjects reduced the final N from 60 to 53. (See Table 1). Continuous Auditory Stimulation. Each subject served as his own control under 2 stimulus conditions: continuous auditory stimulation (white noise at 80 dB and no auditory stimulation beyond the ambient background level (59 dB). We recorded 3 principal dependent variables: motility (gross motor activity); heart rate (HR); and biochemical assay of NE and its precursor, dopamine (DA). Serotonin (5-HT) was also measured to determine if 6-OHDA administration might also have damaged the tryptaminergic system. Motility. Gross motor movement was measured by means of a stabiiimeter device constructed as follows: The clear plastic testing boxes were equipped with a grid floor consisting of 18 parallel copper wires spaced at 1.3-cm intervals and wired alternately so that the animal's movement across the grid floor produced changes in resistance as the circuit was opened or closed by the animal's contact with it. Voltage changes (in the range of 100-300 pV) thus produced were recorded polygraphically. Motility was scored every 2.5 sec in terms of maximum pen deflection over .5-cm units. Scores were summed over 10-sec intervals. Heart Rate. The HR was measured by means of an implanted electrode arrangement described below and recorded on a Grass Model 5 polygraph at a paper speed of 25 mmlsec. Biochemical Assays. Fluorometric assays of whole brain NE were carried out by means of the method developed by Laverty and Taylor (1968) and for 5-HT by the method developed by Bogdanski, Pletscher, Brodie, and Udenfriend (1956).
Subjects and Procedure Subjects were Osborne-Mendel rat ( R a m s noruegicus) pups. The pregnant females were received from the National Institutes of Health Animal Breeding Facility 5 days before term to allow accommodation to the laboratory before littering. They were housed individually in 30.8 x 46.2 x 15.4 cm plastic cages and provided with paper nesting material. Cages were kept under a 12-hr light: 12-hr dark cycle at a temperature
TABLE 1. Design, n, and Mean Weight at Testing. 28 Days
14 Days Group
E IC NIC
n 9 10
to
Weight (g)
n
Weight (g)
34.9 36.6 41.1
7 7 12
97.1 86.7 97.2
8
BKACKBILL AND DOUTHITT
of 23.5"C and an ambient noise level of 56?2dB. Food and water were provided ad lib. Litters were culled t o 9 pups within 24 hr of delivery to insure equivalence of tnaternal attention. Mothers were removed from their litters after 22 days. At approximately 24 hr of age, each litter of 9 was divided into 3 groups and treated as follows. Two pups were assigned to the Experimental (E) group and injected ititracisternally with .01 mg o f 0-OHDA MBr (Regis) in .1% L-ascorbic acid in .01 ml N saline (pH adjusted to 4.3). Two animals, assigned to the Injected Control (IC) g o u p , were injected with the vehicle alone. Two animals assigned t o the Noninjected Control (NIC) group were not treated. Each group was constituted o f approximately equal numbers of rnales and females. Results were not differentially affected by gender. The remaining 3 animals were resewed as spares or used for other purposes. All animals were marked for litter number, subject number, and treatment group by injecting different combinations of black and colored India ink under the pads o f 1 or more teet. Animals were handled on this and a11 other occasions with Tritlex vinyl examining gloves. Cannibalism or other gross indices of abnormal tnaternal behavior were not observed. Forty-eight hours before testing, the electrodes necessary to record HR were placed in the animal. The procedure followed was generally that recommended by Hofer (M. A. Hofer, personal communication; Hofer & Reiser, 1969). After the animal was anesthetized with ethyl ether, 2 male Amphenol pins (220-P02-100), each connected t o 30-gauge stainless steel multifilament surgical wire, were sewn under the skin. The upper electrode was placed I cm above the right foreleg; the lower electrode was placed on the left side, at the level o f the lower rib. Each electrode was stabilized with collodion 15 min before testing began. The testing chamber was a 47.4 x 56.4 x 66.7 cm Elconap incubator (Model B-2) equipped with a Sony cassette tape player. Within the incubator was a plastic testing box (17.9 x 28.2 x 12.8 cm) equipped with a grid floor stabilimeter. Temperature in the incubator was maintained at 33k1 "C, the temperature recommended for individual animals o f this age b y M.A. Hofer (personal communication). Five tninutes before the testing session began, the animal was placed in the testing box and electrodes connected to the recording cable. A 5-min pretest accomodation was sufficient (M. A. Hofer, personal communication) for HR t o return t o normal resting level following a brief phasic change. The 20-min testing session itself was divided into two 1 O-min periods: continuous auditory stimulation (white noise played at 80 dB SPL), and no extra auditory stimulation beyond the ambient noise level o f 59 52dB. The sequence of administering the sound (S)/no sound (NS) phases was counter-balanced over drug treatments, stimulus conditions, and age groups. The FIR and voltage changes from the stabilimeter floor were directly recorded on a Grass Model 5 polygraph using ari electroencephalogram (EEG) preamplifier for the stabilimeter output with a time constant of . i sec. After testing, animals were returned t o home cages. Twenty-four hours later, each was weighed and decapitated. The brain was immediately removed, frozen on dry ice, and stored at -20°C for subsequent biochemical assay. Whole brain monoarnine levels were determined b y homogenizing the brains in .7N petcliloric acid with .S% ethylenediamine tetra-acetate. The homogenates were transferred i n t o plastic tubes containing .5 ml of 5% ascorbic acid and centrifuged at
6-OHDA AND AROUSAL DURING STIMULATION
9
12,000 g for 20 min. The clear supernatant fluids were transferred to 45-ml centrifuge tubes containing 250 mg alumina. The pH was adjusted to 8.6 with 3M hydroxymethylamino methane buffer. The tubes were shaken for 5 min. After centrifugation, the supernatant 5-HT was extracted and fluorometrically assayed according to Bogdanski et al. (1956). The alumina containing NE was washed twice with distilled water following which the supernatant fluid was adjusted t o pH 7 and discarded. The catecholamines were eluted from the alumina with 3 ml of . l N HCl and analyzed by the method of Laverty and Taylor (1968). Behavioral and physiological results were analyzed statistically by ANOVA for repeated measures. Biochemical results were evaluated by means of Student's t-test. Pearson product moment coefficients were used to assess degree of relationship between biochemical and behavioral/physiological data.
Results
Psychophysiological Measures Results at 14 Days. A significant drop in motility occurred under continuous auditory stimulation (F=4.95, df = 1/23, p < .05) being most pronouned for the drug-treated group (Table 2, Fig. 1). A significant Sound x Sequence-of-Sound interaction ( F = 22.80, df= 1/23, p < .001) also occurred, the direction of which indicated an order effect, i.e., continuous stimulation had a greater pacifying effect if it preceded no sound. The steadily declining level of arousal throughout the 20-min session suggests, however, that an initial 5-min adaptation period was insufficient t o familiarize animals with the testing chambers and allow return of motility to base-line levels. it also may suggest that continuous stimulation may overcome short-term stress-induced excitement. The effect of continuous stimulation on HR appeared as a significant Sound x Sequence-of-Sound interaction ( F =8.26, df = 1/23, p < .Ol). An insufficient adaptation period to the testing chamber is the most probable cause of the sequence effect (Table 2, Fig. 2). A significant Drug Treatment effect (F = 4.27, df= 2/23, p < .05) was attributed to the higher HR levels for the E and IC groups than the NIC group. The closer correspondence between IC and E means than between IC and NIC means suggests that the physical insult of injecting .01 ml of an acidic solution into the neonatal brain has effects that outlast the neonatal period. Results at 28 Days. At this age, sound-related differences among groups in motility were pronounced (Fig. 1). Continuous auditory stimulation continued to have a quieting effect on the 6-OHDA treated animals. However, it had a significant arousing effect on the control animals ( F =3.60, df= 2/19, p < .05). Again, adaptation effects appeared as a significant Sound x Sequence-of-Sound interaction ( F = 9.92, df = 1/19, p < .Ol). The effects of continuous stimulation on HR interacted significantly with Drug Treatment (F = 5.34, df = 2/20, p < .05) and with Sequence of Sound ( F = 4.21, df = 1/20, p < .06). Thus, experimental animals continued t o respond in an infantile fashion to continuous stimulation whereas control animals now responded with higher HR's. However, this directional reversal in responsiveness only manifested itself after an adaptation period longer than the allotted S min. Figure 2 shows the maturation-linked reversal in responsiveness during the period maximally free of adaptation effects.
9
HR
Motility
HR
14 days
28 days
28 days 7
7
9
Motility
14 days
n
Measure
Age
4.21 1.12 473.64 c14.80 4.30 t 1.04 442.56 k 13.50 t
NS
E
2.54 1.13 479.76 k12.80 2.71 t .58 411.78 i19.50
*
S
7
6
10
10
n
480.18 t10.90 5.01 t 1.20 427.86 i 12.40
+ 1.08
3.98
NS
IC
Group
10
3.92 1.09 485.94 t 10.40 5.65 ? .44 443.82 i16.80
12
12
10
n S
+
TABLE 2. HR and Motility during Periods of NS and S Stiniulatiori (Mearzs 2 S E).
S
2.54 +.67 444.12 59.80 5.15 t.71 433.68 ? 8.20
NS 2.64 k.46 443.76 t 7.30 4.41 t.88 426.30 t9.40
NIC
6-OHDA AND AROUSAL DURING STIMULATlON
11
-2J E
IC NIC 14 Days
E
IC NIC
28 Days
Fig. 1. Continuous stimulation effects on motility. Negative difference scores indicate a decrease from NS to S conditions.
EXPIC NIC
480-
.-----. ... .... .
470-
460-
450-
*... 440-
k 4 430a n
'.._
".
e
z
f
420-
41 0-
400-
390-
No Sound Sound 14 Days
No Sound Sound 28 D a y s
Fig. 2. Continuous stimulation effect on HR. Scores are from 2nd half of session.
BRACKBILL AND DOUTHITT
12
*
TA RLI' 3. Whole Brain N E and 5-HT I,evels (Mean SE in ngfg). 1:
n
NF
5-HT
14 d e ys
9
28days
5
18.89a t12.55 26.80" t13.37
215.22 t 8.82 256.20 k21.15
~
IC
-
Age
NIC
11
NE
S-lfT
n
NI,
9
141.44 t20.32 163.33 t21.26
208.67 t13.05 235.33 t18.37
8
111.25 i12.04 191.61 230.69
5-H7
~~
3
:'Dtffers significantly from both control groups a t both a g e s 9
6
179.00 + 7.40 256.67 138.22
< .01 (t-test).
Biochernical Assays The effectiveness of 6-OHDA in destroying N E function is obvious in that these animals had drastically lower NE levels than either control group at either age (Table 3). The NE level for drug-treated animals was noticeably less than that for control animals with n o overlap between these distributions. The correlation between NE and triotility sound/no sound difference scores was -.34 ( t = 2.17, df = 35, p < .02S; 1 tailed test) and in the directjonliypothesized, i.e., the lower the level o f N E , the greater the effectiveness of continuous stimulation in reducing gross motor activity. Likewise, a significant correlation between NE and IIR difference scores indicates the magnitude of the continuous stimulation effect is also inversely related to N E level (Y = -.28, t = 1.71, df = 35, p = .OS; 1-tailed test). The levels of 5-HT recovered showed no effect of prior drug administration. All mean values were within normal, age-appropriate limits and were uncorrelated with motility (-.06) or HR (-.04).
Discussion This study hypothesized that a low level of endogenous N E is responsiblc for the developmental phenomenon whereby continuous stimulation depresses arousal Icvel. The hypothesis is supported by results indicating that the continuous stimulation effect is prolonged in rats whose normal utiliLation of this adrenergic neurotransmitter is blocked. Specifically, our results indicated both that the magnitude of the continuous stimulation effect is correlated with the N E level and that the experimental-control differences in the effect increase with age. Results for both psychophysiological measures indicated that continuous stimulation depressed arousal level. Nevertheless, this conclusion must be qualified by the occurrence of sequence effects, which in turn relate to the stress of introducing young animals into the novel environment o f testing boxes. Clearly, the 5-rnin adaptation period proposed by Hofer and Reiser (1969) was insufficient t o dissipate tllis stress and to allow psychophysiological measures to return t o resting levels. (Note that no sequence effects have been found in continuous stimulation studies with human infants.)
6-OHDA AND AROUSAL DURING STIMULATION
13
Whatever stressful effects may have resulted from the intracisternal injection procedure did not significantly affect experimental results gathered at later ages. (A tendency toward slightly higher HR in the IC group than NIC group at age 14 days is not reflected in the activity measure at that age or in either psychophysiological measuure at age 28 days.) The fact that the NIC and IC groups did not differ significantly on psychophysiological measures-or on brain amine levels-strengthens the conclusions that the results are attributable t o the experimental manipulation of central catecholaminergic NE synthesizing capacity. Both motility and HR were depressed by continuous stimulation. This directional agreement between somatic and autonomic measures confirms previous results with human infants showing that although dissociation of measures o f arousal may often occur with adult subjects, it does not characterize experimental outcomes at early age levels. Campbell, Lytle, and Fibiger (1969) and Mabry and Campbell (1973) have proposed that NE has an excitatory or facilitating effect on arousal-presumably at the brain stem level, in the reticular formation-whereas the cholinergic (ACh) system from the forebrain area and the 5-HT system in the midbrain area act t o attentuate or inhibit arousal. They further proposed that both the ACh and 5-HT inhibitory systems become functionally mature at a later development point than does the NE-mediated excitatory system. If this conceptualization is correct then our findings suggest continuous sensory stimulation might facilitate 5-HT activity, e.g., by accelerating its rate of synthesis, and/or might inhibit NE activity. This theoretical view and our empirical results with human infants showing increased quiet sleep accompanied by decreased active sleep under continuous stimulation also agree with Jouvet’s (1969) sleep model, in which he proposed that increased 5-HT activity is a principle correlate of slow wave sleep whereas ACh and NE activation are more closely allied to paradoxical or rapid eye movement (REM) sleep. The developmental aspects of response to continuous stimulation are especially interesting to consider. We find it unlikely that the continuous stimulation effect entirely disappears with maturity but, rather, believe that to demonstrate the effect requires extremes of stimulus intensity not generally encountered under real-life conditions-or most laboratory conditions, for that matter. Thus, Licklider (1961) reported many dental patients using audioanalgesia fall asleep during or after a dental procedure while listening to music or white noise through their earphones set as high as 116 dB. Oswald (1960) produced EEG signs of sleep in human adults by subjecting them to intense rhythmic auditory, tactile, and visual stimulation. SimiIarly, Pavlov found that the elicitation of conditioned responses could be inhibited by conditional stimuli that were too strong (transmarginal inhibition) and, further, that a frequent correlate of this procedure was sleep. Finally, as noted earlier, both Van Twyver et al. (1966) and Scott (1972) found decreased REM sleep and increased slow wave sleep in adult rats and adult human beings, respectively, but at white noise levels maintained at 92-93 dB for several consecutive hours. These considerations suggest that the developmental course of the continuous stimulation effect is not dichotomous-present in earliest stages and then gone forever, like the sucking reflex-but rather that the ease with which it is manifested depends on the intensity of the stimulus in relation to the degree t o which inhibitory mechanisms have developed in the organism being
14
BRACKBILL AND DOUTHITT
stimulated. For a neurologically immature organism, low levels of stimulation are effective because inhibitory ability functions in a rudimentary fashion if at all: for the neurologically mature organism extreme intensities of stimulation are required to overcome fully functional inhibitory processes. T h s line of reasoning is very similar to Pavlov’s early speculation, formulated on the basis of behavioral rather than biochemical observations, that the ontogeny of responsivemess precedes the ontogeny of inhibitory capacity.
Notes This investigation was supported in part by Research Scientist Award MH 5925 from the National Institute of Mental Health and in part by National Science Foundation research grant GB 35155. The authors wish t o thank Dr. Jorge Pcrez-Cruet of the Laboratory of Clinical Science, National Institute of hlcntal Health, for carrying out t h e biochemical assays, and Kelvin Gleeson for his research assistance. Request reprints from: Dr. Yvonne Brackbill, Department of Psychology, University of Florida, Gainesville, Florida 32611, U.S.A.
References Bogdanski, D. F., Pletscher, A., Brodie, B. B., and Udenfriend, S. (1956). Identification and assay of serotonin in the brain. J. Pharmacol. Exp. Ther., I 1 7 : 82-88. Brackbill, Y. (1970). Acoustic variation and arousal level in infants. Psychophysiology, 6 : 5 17-526. Brackbill, Y. (1971). Cumulative effects of continuous stimulation o n arousal level in infants. C’lild Dev.. 42: 17-26. Brackbill, Y. (1973). Continuous stimulation reduccs arousal level: Stability of the effect over time. Child Dev., 4 4 : 43-46. Brackbill, Y. (in press). Continuous stimulation and arousal level in infancy: Effects of stimulus intensity and stress. CliildDev. Brackbill, Y., Adams, G., Crowell, D. H., and Gray, M. L. (1966). Arousal level in neonates and prcschool children under continuous auditory stimulation. J. Exp. Child Psychol., 4 : 178-1 88. Breesc, G. R., and Traylor, T. D. (1 972). Developmental characteristics of brain catecholamines and tyrosine hydroxylase in the rat: Effects of 6-hydroxydopamine. Rr. J. Pharmacol.. 4 4 : 210-222. Campbell, B.A., Lytle, L.D.. and Fibiger, H.C., (1969). Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science, 1 6 6 : 635-63 7 . Coyle, J.T. and Axelrod, J . (1971). Development of the uptake and storage of L-[’HI norepinephrine in the rat brain. J. Neurochem. 18: 2061-2075. Eiduson, S. (1971). Biogenic amines in the developing brain. In D.C. Pease (Ed.), UCLA Forum in .Wedical Sciences, No. 14. Cellular Aspects of Neural Growth and Diffeventiation. Los Angeles: University of California Press. Pp. 391-41 8. IIofer, M. A., and Reiser, M. 1:. (1969). The development of cardiac rate regulation in pre-weanling rats. Psychosom. Mcd., 21 : 372-388. Jouvet, M. (1969). Biogenic amines and t h e states of sleep. Science, 163: 3 2 4 1 . h v e r t y , R., and Taylor, K.M. (1968). The fluorometric assay of catecholamine:; and related compounds. A nal. Biochem., ;?2: 26 9-2 19. Licklider, J. C. R. (1961). O n psychophysiological models. In W. A. Rosenblith (Ed.), Scnsory Communication. Cambridge: Massachusetts Institute of Technology Press. Pp. 49-72. Loizou, L.A. (1972). The postnatal ontogeny of monamine-containing neurons in the central nervous system of the albino rat. Brain Res., 4 0 : 395418.
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Mabry, P.D., and Campbell, B. A. (1973). Serotonergic inhibition of catecholamine-induced behavioral arousal. Brain Rex, 49: 381-391. Oswald, I. (1960). Falling asleep open-eyed during intense rhythmic stimulation. Br. Med. J . , I : 1450-1455. Scott, T.D. (1972). The effects of continuous, high intensity, white noise on the human sleep cycle. Psychophysiobgy, 9 : 227-232. Van Twyver, H.B., Levitt, R.A., and Dunn, R . S . (1966). The effects of high intensity white noise on the sleep pattern of the rat. Psychonom. S c i , 6 : 355-356.
