Noradrenergic pathways and sleep-waking states in cats

EXPERIMENTAL NEUROLOGY 41, 233-245 (1973)

Noradrenergic Pathways and Sleep-Waking States in Cats

JAAK PANKSEPP, JOHN E. JALOWIEC, P. J. MORGANE, A. J. ZOLOVICR, ANL, WARREN C. STERN r

Laboratory of Newophysiologg, Worcester Foundation for Experimental Biology, Slzrewshtry, Massachusetts 01545

Received March 20,1973; Revision Received June 16,1973

Four female cats were surgically prepared with an array of recording electrodes (cerebral cortex, hippocampus, lateral geniculate nuclei, neck and eye muscles) for electrophysiological recording of sleep-waking patterns. Polygraphic recordings were obtained before and after administration of 6-hydroxydopamine into the region of the ventral noradrenergic pathway at the mesencephalic level. Records were scored in five categories : active awake, quiet awake, light slow wave sleep, deep slow wave sleep and REM (rapid eye movement) sleep. The acute effect of 6-hydroxydopamine was a shift toward electrocortical arousal; this probably reflects release of endogenous amines within ascending noradrenergic systems. The chronic effect was a small increase in deep slow wave sleep and REM sleep. Bio- chemical analyses of regional norepinephrine and serotonin levels indicated significant depletion of norepinephrine in all parts sampled (occipital cortex, temporal cortex, pyriform lobe, basal forebrain region, striatum, hypothalamus, cerebellum, and brain stem). Serotonin was depleted to a lesser extent in occipital cortex, temporal cortex, pyriform lobe, cerebellum, and brain stem. Since the greatest depletion of norepinephrine was found in the basal forebrain (to 24% of control levels), the ventral noradrenergic pathway was selectively influenced to some extent. The data thus appear to indicate that activation of the ventral pathway induces electrocortical arousal, while depletion of amines within this pathway may induce somnolence.

INTRODUCTION

Noradrenergic neural systems have been implicated in the control of

tonic cortical activation. Depletion of brain norepinephrine leads to electrocortical synchronization and somnolence ( 1 2, 14, 21) while the potentiation of adrenergic activity leads to cortical and behavioral arousal (6). More recently, Jouvet suggested that dopatnine is also involved in behavioral

1 Supported by grants MH 10625, MH 02211 and MH 23945, NIMH. Dr. Pank- sepp’s present address is Department of Psychology, Bowling Green State University, Bowling Green, Ohio 43403.

233

Copyright @ 1973 by Academic Press. Inc. All rights of reproduction in any form reserved.

234 PANKSEPP ET AL.

arousal (5). With respect to the control of sleep, Jouvet proposed that noradrenergic circuitry participates in the elaboration of rapid eye movement (REM) sleep, the evidence being that L-dopa can reinstate REM sleep in reserpinized cats and localized lesions of the locus coeruleus, a mainly noradrenergic brain stem nucleus, decreases REM sleep but has little effect on slow wave sleep (6, 7).

Histofluorescence mapping of monoaminergic pathways in the brain has demonstrated that the dorsolateral locus coeruleus projects rostrally primarily via a neurochemically homogeneous fascicle designated as the dorsal noradrenergic bundle (1, 10, 16, 19). This pathway runs in the dorsal tegmentum, ventrolateral to the medial longitudinal fasciculus and distributes diffusely, via the medial forebrain bundle, throughout the cerebral cortex and hippocampus. A parallel but neuroanatomically distinct ventral noradrenergic pathway arises from brain stem cell groups Al-AS and A7 as defined by histofluorescence mapping (3) and ascends along a more ventral trajectory through the mesencephalic tegmentum. At the di- encephalic level, the ventral pathway forms a ventral component of the medial forebrain bundle and distributes diffusely throughout the hypothalamus, preoptic region, and basal forebrain. Although there is evidence that the dorsal pathway may participate in the genesis of REM sleep (7)) the function of the ventral pathway, if any, in the organization of sleep- waking patterns has yet to be assessed. Presently this pathway has only been implicated in “reward” and self-stimulation processes (5).

In our experiments we analyzed the role of the ventral noradrenergic bundle in sleep-waking behavior of cats. To this end, we damaged the ventral pathway at mesencephalic levels, where the two noradrenergic bundles are anatomically discrete with localized, cannulae-guided administration of 6-hydroxydopamine ( l&20), and we monitored electroencephalo- graphic indices of sleep-waking activity for 5 wk thereafter.

METHODS

Four adult female cats were anesthetized with Nembutal and a-chloralose. Recording electrodes were implanted in the neocortex, hippocampus, lateral geniculate nuclei, dorsal neck muscles, and frontal air sinus (to record eye movements). In all animals, bilateral 21-gauge stainless steel cannulae were also aimed for the mesencephalic trajectory of the ventral noradrenergic bundle. The guide cannulae were lowered to 3.0 mm above the intended site of injection, and the injection needles were precut to project this distance past the tips of the guides. The coordinates according to the Reinoso-Suarez atlas (13) were : anterior-posterior +1 mm; lateral +3.5 mm; depth 4-7.5 mm. Two weeks were allowed for postoperative recovery before the start of testing.

NORADRENERGIC PATHWAYS 235

I 1 1 5 10” 15 ‘eU

SUCCESSIVE TEST DAYS

FIG. 1. Sleep-waking activity as percent of total recording time (7-8 hr) before and after administration of 6-hydroxydopamine. Wake-l refers to active awake and Wake-2 to quiet awake. Animals were tested every other day. Shading indicates increases in respecive states above mean control baseline levels. Levels of significance are noted for all points reliably different from control levels (correlated t test).

Recordings were carried out in an electrically shielded, dimly lit, sound attenuated cubicle. A flexible cable was attached to the electrode plug cemented to the skull and connected to a counterweighted 15lead slip-ring system. Recording was with Grass Node1 5 and 7 polygraphs at paper speeds of 2.5 to 3 mm/set. The records were visually scored in lo-12 set epochs into five categories. (1) Active Awake was characterized by gross body movement artifacts; (2) Quiet Awake was characterized by a desynchronized EEG in the absence of REM indicators (isoelectric neck EMG, PGO spikes, hippocampal theta) : (3) Light Slow Wave Sleep (SWS-1) was characterized by intermittent bursts of spindle and slow wave activity interspersed with lmrsts of cortical desynchronization ; (I)

Deep Slow Wave Sleep (SWS-2) was characterized by continuous electrocortical siow wave activity and complete bodily quiescence, (5) REM sleep

236 PANKSEPP ET AL.

TABLE 1

MEAN DURATION AND FREQUENCY (& SEM) OF REM PERIODS BEFORE AND AFTER ADMINISTRATION OF ~-HYDR~~YDOPAM~NE~

Experimental condition

REM periods/8 hr Duration (min)

Control 9.3 (IkO.7) 5.8 (4~0.3) Post 6-OHDA-1 10.7 (f0.6) 6.5 (f0.3) Post 6-OHDA-2 11.2 (zkO.7) 6.2 (f0.3)

a Each value is based on 32 recording sessions in four animals.

was characterized by a desynchronized cortex, total neck atonia, rapid eye movements, PGO spikes recorded from the lateral geniculate nucleus, and persistent hippocampal theta activity.

Recordings of sleep-waking activity were obtained for each cat during 7-8 hr recording sessions every other day. Food and water were withheld during the test sessions. Eight baseline recordings were obtained, one of which was extended to 24 hr. During one additional session, animals were tested immediately after administration of d-amphetamine sulfate (0.25 mg/kg ip). The amphetamine test was included to assess whether acute activation of the ventral pathway (i.e., liberation of norepinephrine immediately after 6-hydroxydopamine administration) is similar to amphetamine arousal, and to determine whether injury to the ventral pathway attenuates amphetamine arousal. After these control measures had been obtained, each animal was injected bilaterally with 6-hydroxydopamine hydrobromide (4.0 mg/ml) dissolved in deoxygenated 0.9% saline solution containing ascorbic acid (1 mg/ml) to reduce oxidation. Injections (8 pg/ cannula) were infused slowly across 4-min periods. Recording commenced immediately after treatment, and was continued for 8 hr. After nine recording sessions, animals were again administered 6-hydroxydopamine as described, and tested for 9 additional days. During this second recording period, one test session followed intraperitoneal administration of d-amphetamine sulfate (0.25 mg/kg ip) and one session lasted 24 hr.

Upon completion of behavioral testing, all animals were anesthetized with chloroform, and their brains were removed. The mesencephalon of each animal was fixed in 10% formalin for subsequent histological analysis. Histological identification of the cannulae placements and degree of tissue damage were estimated from serially prepared frozen sections (20 pm) stained by the Khiver-Barrera method. Eighteen other brain parts were dissected and frozen for determination of regional monoamines. Bilateral samples were obtained from nine brain regions: occipital cortex, temporal cortex, anterior pyriform lobe (includin, u the amygdaloid complex), poste-


238 PANKSEPP ET AL.

TABLE 3

MEAN (& SEM) LATENCY IN MINUTES TO FIRST SWS-2 AND REM PERIODS

Experimental condition S\VS-2 REM

Control (Sessions 1-8) Post 6-OHDA-1 (Sessions 10-17) Post 6-OHDA-2 (Sessions 19-26) Amphetaminea Amphetamine* 6-OHDA-1 (Acute)c 6-OHDA-2 (Acute)d

38 (&S) 72 (f8) 29 (f4) 50 (f4)* 21 (f3)* 41 (f3)*

108 (&lo) 221 (f45) 60 (f26)* 213 (f35)

260 (f30) 397 (*17) 90 (*35)* 300 (*79)

* P < 0.01, correlated I test. n 0.25 mg/kg ip given 16 days prior to 6-OHDA-1. * 0.25 mg/kg ip given 10 days after 6-OHDA-2. c Recorded on the day of the first 6-OHDA injection (Session 9). d Recorded on the day of the second 6-OHDA injection (Session 18).

rior pyriform lobe (including hippocampus), basal forebrain region, striatum, hypothalamus, cerebellum, and brain stem (pons and medulla). Norepinephrine and serotonin levels were determined spectrophotofluoro- metrically by a modification (17) of the method of Maickel, Cox, Saillant, and Miller (9).

RESULTS

Sleep-waking patterns, as percent of total recording time, for all tests except days when amphetamine was administered, are summarized in Fig. 1. Statistics refer to correlated t tests between mean individual scores for control days and the results of individual test days. During the control period, no significant variation in sleep-waking durations was observed. Administration of 6-hydroxydopamine increased electrocortical arousal. Quiet Waking and SWS-1 were increased, while SWS-2 and REM were markedly reduced, Thus, animals exhibited a tendency for increased waking which was not accompanied by motor activity. On subsequent days, the reverse pattern emerged. Animals exhibited an increase in SWS-2 and REM, and a concomitant reduction in waking and SWS-1. Throughout testing, no abnormalities in the sleep-waking indices were observed: neck atonia, hippocampal theta, and lateral geniculate spikes appeared as before the drug treatment. Behavior also seemed normal in all apparent respects.

After 9 days of recording (19 days after the first 6-hydroxydopamine injection), the 6-hydroxydopamine treatment was repeated, and a pattern of electrocortical arousal was again elicited, though to a somewhat lesser extent: SWS-1 was still increased and REM decreased, while the effects on Quiet Waking and SWS-2 were small (Fig. 1, day 18). The second in-

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240 PANKSEPP ET AL.

FIG. 2. Left cannulae tract for one cat as seen in a cross section through the mesencephalon at the level of superior colliculus and interpeduncular nucleus. The arrow indicates the tip of the injection needle and the outlining indicates our estimate of the extent of acellularity at the site of injection. Stain: cresylviolet and 111x01 blue. Magnification : X 10.

jection did not appear to modify chronic sleep-waking patterns beyond changes produced by the first injection.

Closer analysis of sleep records indicated that the increase in REM sleep after 6-hydroxydopamine administration was attributed to a slight increase

SORADREKERGIC PATHWAYS

F~ti. 3. Right cannulae tract for the same animal as in Fig. 2 as seen in a cross section through the mesencephalon at the level of superior colliculus and interpeduncular nucleus. The arrow indicates the tip of the injection needle and the outlining indicates our estimate of the extent of acellularity at the site of injection. Stain: cresyl violet and 1~x01 blue. Magnification : X 9.

in the duration and the number of REM periods (Table 1). Comparison of 24-hr records before and after 6hydroxydopamine indicated that total amount of REM sleep increased from 10.470 to 12.7% of total recording time. No significant change in total slow-waye sleep was observed for the 24-hr recording session (55.5-57.5~).

The effect of amphetamine before and after G-hydroxydopamine is summarized in Table 2. Comparison of these results with those in Fig. 1 indicates that amphetamine decreased both SWS-2 and REM while in-

242 PANKSEPP ET AL.

creasing Quiet Waking. Although it appears that the effect of amphetamine was somewhat less after 6-hydroxydopamine than before, the effect was by no means eliminated.

Latencies to the first period of SWS-2 and REM during testing are pre- sented in Table 3. Prior to 6-hydroxydopamine treatment, amphetamine increased the latency to onset of SWS-2 and REM by 70 and 149 minutes, respectively. After 6-hydroxydopamine, amphetamine increased these laten- ties by 39 and 172 min. Similarly, the acute effect of 6-hydroxydopmaine was to delay both the onset of SWS-2 and REM, (222 and 325 min, respectively), the effects being somewhat less after the second injection (61 and 250 min, respectively). The chronic effect of 6-hydroxydopamine treatment was to speed entry into SWS-2 and REM. The effect of amphetamine was attenuated by 6-hydroxydopamine, but only with respect to SWS-2.

Regional levels of norepinephrine and serotonin are summarized in Table 4. Control values represent baseline data from 16 normal cats for which regional biochemistry has been obtained in our laboratory. Since no bilateral differences in brain amine levels were observed, bilateral samples have been pooled. Marked depletions of both amines were observed, though the effects on norepinephrine were generally greater than on serotonin.

Histological analysis indicated that the tips of cannulae were located symmetrically just dorsolateral to the decussation of the brachium con- junctivum in the area of the ventral pathway. Cross sections of the mesencephalon of one animal are depicted in Figs. 2 and 3. It should be noted that the injection sites are several millimeters below the more prominent damage inflicted by the guide cannulae. The zone of acellularity has been outlined on each photomicrograph.

DISCUSSION

The decrease in both serotonin and norepinephrine levels indicates that a very small dose of 6-hydroxydopamine, though aimed specifically into the trajectory of the ventral noradrenergic pathway, can affect both the dorsal noradrenergic and medial serotonergic systems situated several millimeters from the site of injection. Whether these changes are the result of cannulae inflicted damage, direct chemical effects, or indirect compensatory effects cannot be determined from the present data. Widespread decreases of brain norepinephrine, dopamine, and serotonin have also been noted by Petitjean et al. (11) after intraventricular administration of 6-hydroxydopamine in cats. Despite the widspread biochemical effects of our treatment, and the consequent difficulty in formulating chemistry-function rela- tionships, there is evidence that the ventral noradrenergic pathway was damaged more than the dorsal pathway. First, cannulae tips were found to


be located in the described trajectory of the ventral system (19). Second, norepinephrine levels were reduced most markedly in basal forebrain samples-the only region among those sampled which is innervated rela- tively exclusively by the ventral pathway (5, 19). Hence, we feel it is probable that activity changes in the ventral noradrenergic system induced by 6-hydrodopamine played a significant role in producing the present findings.

Although the changes in sleep-waking behavior resulting from administration of 6-hydroxydopamine were small, they were consistent for all animals. The acute effect is similar to a small injection of amphetamine, a shift toward electrocortical arousal, which probably reflects reIease of endogenous norepinephrine (2,4) within the ventral noradrenergic system. Long-term changes in sleep-waking patterns were just the reverse of the acute effects. Deep Slow Wave Sleep and REM sleep were increased at the expense of Quiet Waking and Light Slow Wave Sleep. The animals also tended to fall asleep faster and to be somewhat less reactive to the arousal produced by amphetamine. Reduced sensitivity to amphetamine has also been observed by Stern et al. (15) in rats treated with 6-hydroxydopamine. The present data thus indicate that depletion of norepinephrine within the ventral pathway alters the balance of vigilance control toward increased somnolence, leading to the possibility that the ventral noradrenergic system normally participates in maintenance of behavioral arousal. Furthermore, the present findings suggest that the increase in REM sleep found after inhibition of norepinephrine synthesis with alpha-methyltyrosine in cats (8, 16) may be primarily due to effects on the ventral branch of ascending noradrenergic neural systems.

Still, we have to consider whether the above changes in sleep-waking after injection of 6-hydroxydopamine into the ventral noradrenergic system may have been due to damage of other neurochemical systems. It would appear unlikely that the observed effects were due either to attenuation of activity within serotonergic or dorsal noradrenergic systems. hlanipula- tions which selectively decrease brain serotonin generally decrease both Slow Wave Sleep and REM sleep (6)) and selective damage to the dorsal noradrenergic system decreases REM sleep (7). In fact, were it not for counteracting effects of damage to those systems, the tendency for increased somnolence in the present animals may have been greater than was actually observed. The present effects, however, may have been due to damage of nearby dopamine systems. Although destruction of the histochemically defined dopamine containing cell bodies of group A9 “does not induce a significant change in the spectrum of the EEG recording during the sleep- walking cycle” (6), the dopamine cells of group A8 have been implicated in the “tonic cortical activation which accompanies waking” (6). Accord-

244 PANKSEPP ET AL.

ingly, we cannot be absolutely certain that the effects reported herein are due to destruction confined to the ventral noradrenergic system. Destruction of dopamine systems may have beeli critical. Presently, it can only be con- cluded that administration of 6-llydrc)sydopamille into the known trajectory of the ventral noradrenergic pathway in the mesencephalic reticular forma- tion does produce a reliable shift of sleep-waking states toward increased somnolence as defined by electrophysiological criteria.

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17. ‘kIohrpsoN, J. H., C. A. SPEZIA, and hf. ANGuLO. 1970. Fluorometric detection of serotonin using o-phthaldialdehyde : an improvement. E.rperietttia 15 : 327-329.

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Noradrenergic pathways and sleep-waking states in cats