Brain Research, 539 (1991) 287-303 Elsevier

287

BRES 16263

Neurotoxic lesion of the mesencephalic reticular formation and/or the posterior hypothalamus does not alter waking in the cat Michel Denoyer, Marcelle Sallanon, Colette Buda, Kunio Kitahama and Michel Jouvet D~partement de M~decine Exp~rimentale, LN.S.E.R.M. U52, C.N.R.S. URA 1195, Universit~ Claude Bernard, Lyon (France) (Accepted 21 August 1990) Key words: Waking; Sleep; Mesencephalic reticular formation; Posterior hypothalamus; Cat; Ibotenic acid

In order to re-evaluate the role of two putative waking systems, we injected a neural cell body toxin, ibotenic acid (IA) (45 pg/pl), into the mesencephalic reticular formation (MRF) and/or the posterior hypothalamus (PH). On the one hand, when the cell body destruction was only restricted to the MRF, the IA microinjection was followed by a temporary high voltage and slow neocortical electroencephalogram (EEG) during the first 24 postoperative hours and by a subsequent long term increase in waking which lasted 8-12 h. After the first postoperative day, there were no motor disturbances, no aphagia nor adypsia, no alteration of cortical activation and no modification of thermoregulation or of the sleep-waking cycle. On the other hand, the IA microinjection into the PH induced a hypothermia during the first postoperative night and a dramatic transient hypersomnia immediately after the disappearance of the anesthesia (14-24 h after the IA injection). On the third day, all cats recovered control level of paradoxical sleep (PS), slow wave sleep (SWS) and cerebral temperature. They presented normal motor behavior but they were not able to eat by themselves during the first postoperative week. Finally, when the lesions of the MRF and the PH were realized in one single operation, the cats were first motionless in a comatose state for 2-3 days. This state was accompanied by a transitory hypotbermia and the suppression of a spontaneous or evoked cortical low voltage fast activity. However, from the 2nd postoperative week, both behavioral and EEG waking re-occurred. By contrast, the two successive operations (MRF followed by PH) did not induce a comatose state. We did not observe any deficit in motor behavior, and the sleep-waking cycle was quite normal as from the second postlesion day. In the MRF-PH-lesioned cats, the injection of a-methyl-p-tyrosine (150 mg/kg) induced a large decrease in waking and a moderate increase in PS. In the MRF-lesioned cats, IA produced a large area of cell body loss, centered in the MRF, that extended from levels A2 to A6 of stereotaxic planes and sometimes encroached upon the red nucleus and the substantia nigra. In the PH-lesioned cats, the histological analysis revealed a great loss of cell bodies in the PH extended from levels A8 to A12.5. The damage included the lateral and posterior hypothalamic areas and sometimes the tuberomamillary nucleus. In MRF- and PH-lesioned cats, the cell body loss extended from levels A2 to A12.5. These findings indicate that neither the MRF nor the PH play a necessary role for initiating or maintaining behavioral or EEG arousal, and lead us to postulate multiple systems for waking. INTRODUCTION It is known, from the classical studies of Moruzzi and M a g o u n in 1949, that electrical stimulation of the medial brainstem induces cortical and behavioral arousal reactions 3s. Later, Lindsley et al. showed that the coagulation of the mesencephalic reticular formation ( M R F ) and/or the posterior hypothalamus (PH) was followed, in the cat, by a long lasting comatose state associated with the d i s a p p e a r a n c e of spontaneous or induced cortical arousal 29. Taken together, these results led Moruzzi to the concept of a 'waking system' localized either in the ascending reticular formation and/or in the PH. It was hypothesized that: 'The ascending reticular system and a group of neurons lying in the posterior hypothalamus are e n d o w e d with a tonic activating influence. They are p r o b a b l y concerned with the maintenance of wakefulness '39.

H o w e v e r , the absence of waking after such lesions is not a direct p r o o f that either the M R F or P H are necessary and sufficient for wakefulness, since coagulation destroys not only p e r i k a r y a but also several axonal pathways which might play some crucial roles. Likewise, the electrical stimulation is not a m o r e direct proof, since the stimulation can excite both ascending or descending axonal pathways which pass through the MRF. Recently, kainic acid ( K A ) and ibotenic acid ( I A ) have been r e p o r t e d to destroy neuronal cell bodies without altering axonal pathways and terminals 25. The selective nature of the neurotoxin lesion m a k e s it a useful tool for the evaluation of the i m p o r t a n c e of particular neuronal populations in s l e e p - w a k i n g states. This technique has already been utilized to destroy P H o r M R F in o r d e r to study some acute or p e r m a n e n t effects of these lesions. Thus, the destruction o f the P H cell bodies by local injections of I A did not result in any chronic deficit of

Correspondence: M. Denoyer, D6partement de Mddecine ExpErimentale, I.N.S.E.R.M. U 52, C.N.R.S. URA 1195, Universit6 Claude Bernard, 8 avenue Rockefeller, 69373 Lyon, France. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

288 wakefulness s2, whereas microinjections of KA into the

MATERIALS AND METHODS

MRF, in the unanesthetized cat, induced a state of intense excitation and protracted arousal 24. Furthermore, destruction with K A of the cell bodies located in the dorsolateral pontine tegmentum and in the caudal portion of the mesencephalic reticular formation, did not produce any significant alteration of waking 64. Although these findings appeared to counter the concept of a 'waking system' located in areas such as the M R F or the PH, they were not believed to be a significant proof against this hypothesis 57. The purpose of the present study was to resolve this contradiction. In this aim, we first destroyed the perikarya of the entire mesencephalic reticular formation or of the posterior hypothalamus with ibotenic acid. Secondly, we damaged the neuronal cell bodies of both the M R F and the PH either in one single operation or in two successive operations ( M R F followed by PH). Changes in the quantities and pattern of E E G sleep-waking cycle as well as the behavioral disturbances induced by the different lesions were assessed over 3 - 4 weeks. Some

of these

preliminary note m.

results have been reported

in a

Twelve adult male and female cats (2.5-4 kg) were operated under deep pentobarbital anesthesia (25 mg/kg i.v.) The solution of IA (Sigma) (45 ~g/~l) dissolved in phosphate buffer (1 M, pH: 7.4) was injected, under control of cerebral temperature, bilaterally into the ventrolateral part of the posterior hypothalamus and/or into the mesencephalic reticular formation through a 5 ~ul Hamilton syringe (external diameter of the needle: 0.4 ram) under stereotaxic guidance. The total volume bilaterally injected varied from 0.9 to 2 ,ul and the coordinates were: A 1.5 to 5; L 2.5 to 3.5; H 0.5 to H -1.5, for the MRF, A 8 to 10; L 2 to 2.5; H - 4 for the PH, A 1.5 to 5; L 2.5 to 3.5; H -0.5 to -1.5 and A 7 to 11; L 2 to 2.5; H - 4 for the MRF and PH, according to the atlases of Berman and Jones~ and Berman4. The injections were made over a period of 2-6 min (0.05 /*l/min) according to the dose. Subsequently, the cats were implanted for polygraphic recordings of PGO activity (from the lateral geniculate nucleus), electroencephalogram (from frontal and occipital cortices), electromyogram (from the neck muscles), eye movements (from the orbits) and temperature recordings by means of intracerebral thermistor in the caudate nucleus and subcutaneous thermistor in the neck of the cat. Output of the thermistors and integrated output of the eleetromyogram activity were superimposed on a myotherm 8602. The data were recorded every 2 s on a Linseis recorder. Polygraphic control recordings (23 h/day) were performed over 3-4 weeks. The scoring of these recordings was carried out according to previously described criteria for waking, light slow wave sleep (SWS 1), deep slow wave sleep (SWS 2) and paradoxical sleep (PS). The results obtained were compared to the laboratory

Fig. 1. Waking behavior in cats BI13, 0114, TlI3 and HlI3 on the 15th postoperative day.

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Fig. 2. Polygraphic characteristics of wakefulness, slow wave sleep and paradoxical sleep before the lesion (A), the first day after IA microinjection in the MRF (B), 9 days after the MRF lesion (C), the first day after the second microinjection of IA in the posterior hypothalamus (D) and 8 days after the PH lesion. No PS epoch was observed in this cat (B120) during the first postoperative day of recording after IA microinjection in the MRE EOG, electrooculogram; EMG, electromyogram; ECG, electroencephalogram; LGN, lateral geniculate nucleus.

290 bank control values from 363 recorded days of unlesioned cats. Only two cats (B 119, B 120) were implanted for baseline recording for 4-5 days. Subsequently, bilateral destruction of the MRF was performed under anesthesia by microinjection of IA. Animals were recorded for 14 days and afterwards they were again operated under anesthesia for the lesion of the PH. Thereafter, the cats were recorded for 3 weeks. In 4 cats (O114, T113, B119, B120) a-methyl-p-tyrosine (a-MPT) was intraperitoneally (i.p.) injected (I50 mg/kg) 2 weeks after the MRF and PH destruction. At the end of the experiment, the animals were perfused under deep Nembutal anesthesia, through the ascending aorta with 1 liter of Ringer lactate solution, followed by 2500 ml of an ice cold fixative containing 4% paraformaldehyde, 0.2% glutaraldehyde, 0.2% picric acid in 0.1 M phosphate buffer (PB, pH 7.4). After perfusion, the brain was removed and sliced into frontal blocks 1-2 cm thick. The tissue blocks were postfixed for 1 day by immersion in a second fixative, consisting of 2% paraformaldehyde and 0.2% picric acid in 0.1 M PB at 4 °C. They were then soaked in 0.1 M PB containing 30% sucrose at 4 °C for 2 days and afterwards 25/~m frozen sections were cut on a cryostat. One out of 4 sections was immediately stained by Cresyl violet.

Histological analysis of the lesions was done on frontal serial sections stained with Cresyl violet and with immunohistochemistry of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) according to a method previously described22'-''

RESULTS

Behavior MRF lesion. The IA injection in the MRF induced a

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Fig. 3. Hypnograms showing the effects upon sleep-waking cycle induced by a bilateral injection of ibotenic acid in the MRF (A) or in the PH (B) under pentobarbital anesthesia during the first postoperative day. The IA microinjection in the MRF was followed by an increase in SWS during the first 16 h of the day, while the IA microinjection in the posterior hypothalamus induced a dramatic increase of PS during the same time. Abscissa, time in hours; ordinate, waking (W), light slow wave sleep (SWS 1), deep slow wave sleep (SWS 2) and paradoxical sleep (PS).

hypothermia (32-35 °C) during the first night except in two cats, J l l l and X l l l . After the 2nd day only cats Fl16 and B120 showed a moderate increase in brain temperature (40-40.5 °C) until the 7th day. In 5 cats ( J l l l , X l l l , F116, Bl19, B120) the MRF lesion was followed, during the second postoperative day by an intense agitation (circling, undirected walking) while cat Bl13 remained comatose for 3 days. It developed cortical epileptic seizures which were controlled by diazepam (1 mg/kg i.m.). After 3 days the animals were capable of orientation to visual, auditory and somatosensory stimulations and showed good motor coordination (Fig. 1). Only cats B120 and Fl16 were handicapped by a lack of motor coordination at the level of the hind legs, respectively, during 1 and 2 weeks. They remained in an outstretched position for 6-10 days when they were sleeping, while all the other animals adopted the normal sphinx and curled sleep position from the third day. Cats J l l l and X l l l were able to eat normally as from the second postoperative day. The others accepted food from the experimenter during the first week before being able to eat by themselves. PH lesion. At stable ambient temperature (25-26 °C), all the cats developed a transitory hyperthermia during or immediately after the end of the operative procedure. However, during the first night, cerebral temperature gradually decreased to 33-30 °C. Afterwards, cerebral temperature rapidly re.turned to normal levels (between 37 and 39 °C) 15-26 h after the end of the injection. When the cats recovered from anesthesia, they were hypotonic but able to sit and walk. All cats except K92 were hyperactive to sensory stimulations and presented aggressive behavior with stereotypical behavior of chewing during the first postoperative day which gradually disappeared. Four cats, Sl15, K92, C96, X106 showed a major aphagia for 7 days. They were force-fed with liver. Cats K88 and Gl15 presented a normal food intake as early as the 2nd postoperative day. One step MRF-PH lesion. In cats Tl13 and Ol14, the lesion induced a comatose state lasting 2-3 days. The animal lay in an outstretched position. This state was associated with cortical epileptic seizures which were

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Fig. 4. Effects of MRF cell body lesion (A), PH cell body lesion (B), one step MRF-PH perikarya lesion (C) and two step MRF-PH perikarya lesion (D) on cortical waking. Mean daily percentages of wakefulness (ordinates) during the postoperative days for each lesioned cat (abscissa). C: percentages of waking state (+ S.D.) obtained from the laboratory bank control values (n = 363).

controlled by diazepam injections (1 mg/kg i.m.). All the cats were hypothermic during the first 3 postoperative days and then recovered a subcontrol cerebral temperature (between 37.5 and 39.5 °C). Only cat Hl13 was not comatose. However, cortical and motor epileptic seizures occurred between the 6th and 8th day. In all cats, after the third day, spontaneous and/or evoked behavioral waking reactions gradually reappeared, but a lack of motor activity persisted up to the end of the first week. They were force-fed during this week. However, the 3 cats were subnormal at the end of the third week: they were able to stand, to walk, eat by themselves and presented normal sleep positions. Two step MRF-PH lesion. The effects of the MRF lesion have been described in the first group. The additive lesion of the PH induced a fall in cerebral temperature (32-36 °C) during the first postoperative night and day. Afterwards, temperature, food intake, waking and sleeping behavior were normal. We noticed a slight increase in aggressive behavior in B120.

Effect of the lesion upon sleep: qualitative aspect MRF lesion. Cortical epileptic seizures occurred only in cat Bl13 (3 days). The others showed an E C G composed of high voltage slow activity and spindles with continuous discharge of PGO waves during the first 15-24 postoperative hours. From the first day, spontaneous and evoked periods of cortical low voltage fast activity could be obtained (Fig. 2). Polygraphic patterns of PS, SWS and waking (W) were normal as from the 2nd postoperative day. PH lesion. Cortical epileptic seizures were never observed in this group. A continuous discharge of PGO waves occurred during the first 8-10 postoperative hours. The E C G was composed of slow waves and spindles. Short periods of cortical desynchronization could be obtained by intensive external stimulation but no spontaneous activation occurred. As from the 20th hour after the lesion, normal pattern of PS with desynchronized E E G occurred in Sl15 and X106, while PS with synchronized ECG was present for 30 h in other cats.

292

TABLE I

TABLE 11

Percent o f vigilance stages and mean daily cerebral temperature on 7 postoperative days (D) after ibotenic acid injections in the mesencephalic reticular formation ( M R F lesion)

Percent of" vigilance stages and mean daily cerebral temperature on 7 postoperative days (D) after the posterior hypothalamus lesion (PI1 lesion), the mesencephalic reticular formation and the posterior hypothalamus lesion in one step (MRF-PH lesion) and in two steps (MRF/PH lesion)

Daily percentages of paradoxical sleep (P), slow wave sleep (S), waking (W) and mean daily cerebral temperature (T) over 7 postoperative days (D1, D3, D6, D9, D12, D15, D18) in each cat which had received a bilateral microinjection of IA in the MRF. Percentages of SWS, PS (laboratory bank control values, n = 363) and T (n = 4) in control animals are indicated.

Daily percentages of paradoxical sleep (P), slow wavc sleep (S), waking (W) and mean daily cerebral temperature (T) over 7 postoperative days (DI, D3, D6, D9, DI2, D15, DI8) in each cat which had received a bilateral microinjection of IA in the PH (C96, K92, K88, Sl15, GII5, X106), in the MRF and the PH in one single operation (Hl13, TII3, 0114) and in the MRF and the PH in two successive operations (MRF followed by PH) (B119, B120). Percentages of SWS, PS (laboratory bank control values, n = 363) and T (n = 4) in control animals are indicated in Table I.

D1 M R F lesion Jill P(%) S (%) W (%) T (°C) Xlll P(%) S (%) W (%) T(°C) B113 P(%) S (%) W (%) T(°C) Fl16 P(%) S (%) W(%) T (°C) B119 P(%) S (%) W (%) T(°C) B120 P(%) S (%) W(%) T (°C) Control P(%) S (%) W(%) T (°C)

D3

D6

D9

D12

D15

3.5 40.5 56.0 -

12.6 68.2 19.2 39

9.6 44.7 45.7 38.8

10.1 38.9 51.0 38.5

10.1 51.2 38.7 38.8

12.5 51 36.5 38.8

0 67.5 32.5 -

2.9 47.6 49.5 39.1

11.3 51.2 37.5 38.7

15.8 48.7 35.5 38.9

16.5 46.1 37.4 39.0

10.3 43.1 46.6 -

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8.4 78.6 13.0 39.4

8.6 56 35.4 39.2

13.2 40.1 46.7 38.9

15.9 44 40.1 38.9

14.3 47.9 37.8 38.2

0 95 5 -

1 1 98 40.6

1.6 66.9 31.5 40.3

3 23.4 73.6 40.2

9.3 40.7 51.0 39.4

9.7 42.8 48.5 39

0 75.2 24.8 37.9

1 99 39.3

20.9 79.1 39.7

8.4 43 48.6 39.1

11.7 54.6 33.7 39.1

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40

1.7 13.3 85.0 39.9

4.2 22.4 73.4 39.5

9.8 46.1 44.1 39

11.3 47.4 41.3 39.1

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D18

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D3

D6

D9

DI2

D15

D18

33.5 60.5 6 -

8.1 63.2 28.7

10.7 53.1 36.2 -

35.2 64.7 .

7 11.5 12.8 81.3 65.9 52.9 11.7 22.6 34.3 . . .

13.7 53.2 33.1

20.4 66.0 13.6

15 38.2 46.8

22.1 28.1 49.8

19.4 25.4 55.1

11.8 69.7 18.5 -

10.9 70.5 19.6 -

PH lesion

16 45.9 38.1

C96 P(%) S (%) W (%) T (°C) K92 P(%) S (%) W(%) T (°C) K88 P(%) S(%) W(%) T (of)

12.4 39 48.6 39.1

12.8+2.8 51.6 + 16.6 35.6_+ 10 38.9 + 0.2

One step MRF-PH lesion. Two cats (Tl13, 0114) showed cortical epileptic seizures during the 3 or 4 days after the injection of IA. The ECG was characterized by theta activity (low voltage slow activity). This state was accompanied by the suppression of the spontaneous or evoked cortical low voltage fast activity. In cat Hl13, ECG was composed of slow waves and spindles over 3 days while no cortical activation occurred when there was no EMG activity accompanied by PGO waves (PS without cortical desynchronization). The polygraphic pattern of PS, SWS and W presented normal features from the 5th postoperative day. Two step MRF-PH lesion. After the 2nd lesion, the

8115 P(%) S (%) W (%) T (°C) Gl15 P(%) S (%) W (%) T(°C) XI06 P(%) S(%) W (%) T(°C)

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15.6 26.7 57.7 .

19.4 24.2 56.4 .

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37.4 54.3 8.3 34.5

1.7 83.4 14.9 39

12.5 75 12.5 39.2

11.1 74.4 14.5 39.2

11 65.1 23.9 38.9

22.2 64.1 13.7 35.1

8.9 70.3 20.8 38.0

8.6 68.8 23.6 39.4

12.4 62.8 24.8 38.6

15.6 64.0 20.4 38.6

25.7 70.8 3.5

ll.7 33.4 54.9 38.1

5.7 27 67.3 38.2

15.3 31.9 52.8 38.5

13.1 39.1 47.8 38.5

12.2 35 52.8 38.8

11.1 38.6 50.3 38.8

16.5 49 34.5 34.5

7.1 51.5 41.4

7.1 26.6 66.3 37.6

11.8 42.5 46.7 -

13.8 48.3 37.8 37.6

12.7 39.6 47.7 -

38

0 16.7 83.3 39.6

2.8 36.1 61.1 39

2.5 49 48.5 39.6

1.7 49.4 49.9 39.3

2.3 44.2 53.5 39.1

38.3

0 10 90 39.8

2 43.1 54.9 38.5

4.7 61.5 34.8

1.6 49.7 48.7 38.9

7.6 57.5 34.9 39.2

5.6 59 35.4 36.7

9.7 52.7 37.6 39.5

3.8 54.7 41.5 39,7

13.4 48.5 33.1

12.1 52.9 35.9 39.4

8.7 53.6 38.7 38.8

13.9 66.6 19.5

9.9 88.5 1.6 36.9

12 78.4 9.6 38.8

15.6 67.5 16.9 39.1

16.1 66 18.9 38.7

13.2 68.6 19.2 39

10.6 73.8 15.6 39.4

19 63.4 17.6 38.9

MRF-PH lesion HlI3 P(%) S (%) W(%) T (°C) TlI3 p(%) S (%) W (%) T(°C) O114 p(%) S(%) W(%) T (°C)

MRF/PH lesion BlI9 P(%) S (%) W (%) T (°C) BI20 P(%) S (%) W(%) T (°C)

293 TABLE III Main daily percentages of vigilance stages and cerebral temperature on the postoperative days D1, D3, D6, D9, D12, D15 and D18, in each group of the lesioned animals Mean daily percentages (_+ S.D.) of paradoxical sleep (P), slow-wave sleep (S), waking (W) and mean daily cerebral temperature (T) on 7 postlesion days in each group of animals (MRF-lesioned cats, PH-lesioned cats, MRF-PH-lesioned cats in one step, MRF/PH-lesioned cats in two steps). D1

D3

D6

D9

D12

D15

D18

MRF lesion P(%) S(%) W(%) T(°C)

0.9_+1.5" 69.6_+19.5 29.5_+18.3 39_+1

5.3+4.5* 34.9_+32.5 60.6_+35.5 39.5+0.6**

7.1_+3.6" 43.7_+16.9 50.4_+18.8 39.4_+0.6*

10.1_+4 40+8.1 49.9_+11.7 39.1_+0.5

12.5_+2.8 47.3_+4.5 40.4_+5.3 39.1+0.2

11.7_+1.8 46.2+3.4 42.3_+5.3 38.7+0.3

14.2_+1.8 42.4_+3.5 43.4_+5.4 39.1_+0

PH lesion P(%) S(%) W(%) T (°C)

29.1_+7.6"* 63.4-+5.1" 9+4** 34.8 _+0.3*

8.7_+4.1" 61.6+19.5 29.6_+16.1 38.4 _+0.4

10.8_+3.1 52.7+19.4 36.7_+19.7 38.9 _+0.5

14.2_+2.9 49.2_+18.7 36.6+16 38.8 _+0.3

15.1_+3.8 49.9_+14.4 35_+12 38.7 + 0.2

14.5_+3.5 43.4_+19 42.1_+16.7 38.8 + 0

11 +0.1 54.5_+16 34.9_+15.3 38.8 _+0

16.5_+0 49_+0 34.5_+0 36.9+1.8"*

2.4_+3.3** 26.1_+18.2 71.6_+21.5" 39.7-+0.1"

4+2.2** 35.3_+6.8 60.8_+4.7** 38.4_+0.6

6.3_+4* 51_+7.9 43.3_+6 39.6-+0

5.7_+5.7 49.1_+0.6 45.5_+5.4 38.6-+0.7

7.5_+4.2 47.1+7.9 45.4_+7.8 39.1_+0.1

10.9+1.1 65.6+12.9 23.6+14 39.1 _+0.4

9.7+5.9 61.6+6.4 29.2+12.3 39.4 _+0.3*

14.7+1.3 57.2+8.7 28.5_+9.6 38.7 _+0

12.6+0.6 60.8+7.9 27.6+8.4 39.2 _+0.2

9.7+1 63.7+10.1 27.1_+11.6 39.1 _+0.3

16.4+2.6 65+1.6 18.6_+1" 38.9 + 0.1

MRF-PH lesion in one step P(%) S(%) W(%) T(*C) MRF/PH lesion in two steps P(%) S(%) W(%) T (°C)

7.8+2.1" 73.7+14.7 18.5+16.9 36.8 _+0.1"

Significant differences with control values (cf. Table I) are computed with non-parametric Kolmogorov-Smirnov test (*P < 0.05; **P < 0.01). cats did not develop cortical epileptic seizure activity. The E C G was c o m p o s e d only of high voltage, slow wave activity and spindles with a continuous P G O wave discharge during the first 10 postoperative hours. Spontaneous cortical low voltage fast activity could occur on the first p o s t o p e r a t i v e day as shown in Fig. 2. During the first day, B l 1 9 and B120 p r e s e n t e d PS epochs with synchronized E C G . A s early as the 2nd postoperative day polygraphic characteristics of SWS, PS and W were normal.

TABLE IV Effect of a-MPT injection upon relative duration of sleep-waking activity in MRF- and PH-lesioned cats Mean percentages + S.D. of paradoxical sleep (PS), slow-wave sleep (SWS), waking (W) during the day before injection, the day of injection and the day following the injection of a-MPT (150 mg/kg i.p.) in the MRF- and the PH-lesioned cats.

PS SWS W *P< 0.1 (t-test).

Day before injection

Day of injection

Day after injection

7.8 + 4.4 52.9 + 14.8 39.2 + 18.3

14.2 + 4* 67.7 + 9.7 19.5 + 7.5*

8.1 + 3.8 62.8 + 15.7 27.4 + 13.6

Effect o f the lesion upon sleep: quantitative aspect M R F lesion. T h e I A injection in M R F was followed by an increment in SWS and a suppression of PS during the first day (Fig. 3A). A f t e r w a r d s , cats B120, B l 1 9 , F l 1 6 presented a significant increase in waking, respectively, during 6, 7 and 3 p o s t o p e r a t i v e days, while the percentages in waking state, in cats F l l l and X l l l , were not different from the control value as from the second postoperative day (Fig. 4A). T h e waking average returned to control value in all cats from the second week. A s shown in Table I, time spent in PS and in SWS was significantly diminished during the first w e e k in cats B120 and Bl19. F l 1 6 exhibited a longer decrease in PS (8 days) while SWS was less impaired. T h e percentages of PS and SWS were not different from the control level in the o t h e r cats. P H lesion. I A cell b o d y destruction of the P H resulted in a dramatic biphasic transient h y p e r s o m n i a (Fig. 3B). This h y p e r s o m n i a was also o b s e r v e d after the disappearance of the effects of p e n t o b a r b i t a l anesthesia. During this period, the frequency and the duration of PS epoch were significantly increased. The m e a n percentages of SWS, PS and waking were 44.7 + 6.3, 51.9 + 8.1 and 3.4 + 2.6, respectively. Subsequently, PS d e c r e a s e d while a m a r k e d increase in SWS occurred during 8 - 1 9 h.

294 After this early period of hypersomnia, the evolution of the quantitative aspect of sleep varied greatly from one cat to another (Fig. 4B and Table I1). During the 2 postoperative weeks, waking level was similar to the control level (G115, C96), slightly increased (X106, K88) or decreased (K92, Sl15). As shown in Table 11, there was a permanent increase

A

Jlll

X111

of PS in only one cat (K88) while SWS was increased in K92 and S115. One step M R F - P H lesion. After the comatose state period (2-4 days) Tl13 and 0114 showed an increase in waking accompanied by a decrease in SWS and an absence of PS during the first week (Fig. 4C). Afterwards, SWS was slightly increased while PS remained at

Bl13

F~

As

B

X~

Fig. 5. (See for legend next page.)

~

G~

Km

Kgg

°

CN

0

295 a low level. Cat H l 1 3 exhibited a decrease in waking up to the third p o s t o p e r a t i v e day, which was followed by normal a m o u n t of the vigilance state (Table II). Two step M R F - P H lesion. The results obtained after the M R F lesion were described in group 1 ( M R F lesion).

C

D

HllB

The P H lesion (after the M R F ) was followed by an increase in SWS for only 24-36 h while PS quantities were quite normal. Cat B l 1 9 exhibited, as from the 2nd postoperative day, percentages of W, SWS and PS similar to control

"1"113

B 120

BI'I~

Fig. 5. Schematic drawing of the bilateral cell body lesions, induced by IA microinjection in the MRF (A), in the PH (B), in the MRF and PH in one step (C) and in the MRF and PH in two steps (D). Levels correspond to those of stereotaxic atlases of Berman 4 and Berman and Jones 5.

296

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-

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•

~,~

°

, ~ .~

~d

r

~

,~ 4,4

297 value, while cat B120 presented a significant decrease in waking compensated by an increase of SWS (Fig. 4D and Table II). Finally, the mean percent of vigilance stages in each group of lesioned animals for 7 postoperative days are presented in Table III. Effect of a - M P T in the MRF- and PH-lesioned cats The fact that the amount of each stage of vigilance was inconsistent from one cat to another produced an increase in standard deviation. However, the injection of a-MPT was followed by a significant decrease in the waking state and an increase in PS. The results are presented in Table IV. No significant modification of the sleep-waking cycle was observed the day after a-MPT injection. Histological results M R F lesion. The MRF neurons were extremely sensitive to IA since the neuronal loss extended from A2 to A6 in all animals (Fig. 5). The neuronal cell loss was often accompanied by a great proliferation of glial cells in the center of the lesion (Fig. 6). The dorsoventral and mediolateral diameters of the lesion were approximately 3 mm. The red nucleus was destroyed in every cat except in X l l l and J l l l . In cats Bl13 and B120, there was a more rostral extension which encroached upon the caudal part of the hypothalamus (Fig. 5). The lesion was more extensive in cat Fl16 since it included almost all the substantia nigra and the ventral tegmental area. This observ~ition was confirmed by immunohistochemistry of AADC. As already reported, AADC-IR cells are widely distributed in the PH and MRF, and their fibers innervate all parts of the brain. As shown in Fig. 7, owing to the immunohistochemistry of AADC, the microinjection of IA destroyed only perikarya and did not alter the fibers of passage and terminals. In cats B120 and Bl19, dopaminergic neurons were only partially affected by the injection of IA. Thus, the area of cell loss involved the retrorubral nucleus and a portion of the substantia nigra compacta and the VTA for cat Bl19 while the lesion of cat B120 included a part of the lateral substantia nigra at the level of the red nucleus (A4-A5) and a small portion of the VTA. In the other cats ( J l l l , X l l l , Bl13), the majority of the neurons within the substantia nigra were spared.

P H lesion. The bilateral lesions extended from A9 to A12 in all cats. They included the lateral hypothalamic area, the posterior hypothalamic area, the ventro- and dorsomedial hypothalamic nucleus. In the case of large lesions (C96, K92, $115), a part of the dorsal hypothalamic area was damaged and the destruction encroached upon the ventromedial nucleus of the thalamus. In cats K92, $115 and X106 a part of the tuberomammillary nucleus was spared. In all cats the caudal magnocellular cells were either largely or completely (C96, Gl15) destroyed as demonstrated by 5-HT and AADC immunochemistry (Fig. 7). One step MRF-PH lesion. The combined lesion of the MRF-PH perikarya extended from A2 to A l l . 5 in 3 cats (Ol14, Hl13, Tl13). The MRF destruction was extensive and only few cell bodies persisted medially in cat Hl13. In cat Tl13, the substantia nigra and the VTA were severely damaged while in the others, the majority of dopaminergic neurons were still intact except at the level of A6 for cat Hl13. The destruction of the posterior hypothalamus included the tuberomamillary nucleus, lateral and posterior hypothalamic areas and the ventromedial nucleus. We also noticed a partial damage of the dorsal hypothalamic area. In cat Hl13, the lesion of these structures was total whereas the medial parts of the dorsal and ventral hypothalamus were spared in cat Tl13 as well as the ventromedial nucleus in cat 0114. In all cats, caudal magnocellular neurons of the PH were destroyed (Fig. 5). Two step MRF-PH lesion. The MRF destruction has been already described in the first group (MRF lesion). In summary, the MRF lesion extended from level A2 to A6 in two cats (B120, Bl19) including the red nucleus. The cell bodies of the substantia nigra and VTA were only partially damaged but the retrorubral nucleus was totally destroyed in cat Bl19. The anteroposterior levels of the posterior hypothalamus lesion ranged from A8 to A12 in cat B120 and from A7 to A l l in Bl19. The destruction of the PH neuronal cell bodies included the posterior and lateral hypothalamic areas except the infundibulum. The ventromedial nucleus was damaged in two cats while the dorsomedial nucleus was intact in cat Bl19. Moreover, the most caudal part of the tuberomamillary nucleus was not destroyed in cat B120 (Fig. 5).

Fig. 6. A,a, B,b: photomicrographs of Cresyl violet-stained frontal sections at the level of the MRF in a control animal (A) and in an experimental animal (B) which received bilateral injection of 0.6/zi of IA (45/zg//zl) in the MRF. Bars: 300/~m. Higher power photomicrograph of the lesion site (b) showing the disappearance of the MRF cell bodies when compared with the non-lesioned control animal (a). Bars: 75 /~m. C,c, D,d: photomicrographs of frontal Cresyi violet-stained sections at the level of the PH in a control animal (C) and in an experimental animal (D) which received a bilateral injection of 0.6/zl of IA (45/zg//~l) in the PH. Bars: 300/zm. Higher power photomicrograph of the lesion site showing the disappearance of the perifornical cell bodies of the PH (d) when compared with a non-lesioned control animal (c). Bars: 75 /~m. MRF, mesencephalic reticular formation; HLA, lateral hypothalamic area.

298

• i

i~iiii!ill¸ i!

•

4

Fig. 7. Photomicrographs of AADC-immunostained frontal sections showing a part of the ventrolateral PH (A,a), a part of the caudal PH at the level of the tuberomammillary nucleus (B,b) and a part of the substantia nigra at the level of the red nucleus (C,c), in the unlesioned control cat (A-C) and in the experimental animal which received a bilateral microinjection of IA in the PH or in the MRF (a-c). Note the disappearance of cell bodies without damage of passing fibers in a-c. Bars: 475 j~m. F, fornix; HLA, lateral hypothalamic area: Ira, medial lemniscus; SN, substantia nigra; MM. medial mammillary nucleus.

DISCUSSION

e f f e c t s o f M R F a n d P H l e s i o n o n w a k i n g b e f o r e interpreting our main results with regard to those obtained

W e shall first discuss t h e d i f f e r e n c e s b e t w e e n t h e

e i t h e r by c o a g u l a t i o n o r cell b o d y l e s i o n s .

299

Effect of MRF lesion When the lesion was restricted only to the MRF ( X l l l , J l l l ) , the IA microinjection produced a persistence of high voltage and slow neocortical EEG during the first 24 postoperative hours and a subsequent long term increase in waking which appeared immediately after recovery from the anesthesia. This state lasted 8-12 h. The animals displayed pupillary dilation, accelerated respiration and circling behavior. According to Kitsikis and Steriade 24, the KA microinjection in the MRF was followed, in unanesthetized cats, by a waking state with hallucinatory-type behavior. In our cases, because we injected the IA in anesthetized preparations, we have never observed such hallucinatory type behavior. The MRF lesion did not produce chronic behavioral and electrographic abnormalities. Thus, as from the first postoperative day, there were no motor disturbances, no aphagia or adypsia, no alteration of cortical activation and no modification in the sleep-waking cycle. When the lesion encroached upon other mesencephalic structures such as substantia nigra, red or retrorubral nucleus as demonstrated by Nissl staining and confirmed by A A D C and TH immunohistochemistry (Fl16, Bl19, B120), the same acute effects were observed, followed by two chronic specific symptoms: motor disturbances and increase of waking in the first week. Firstly, the marked decrease of TH and AADC immunoreactive neurons of the SN suggest that the dopaminergic nigral perikarya are very sensitive to IA. Secondly, our results are in accordance with the fact that the nigral neurons are involved in motor control. However, it is surprising that the almost complete destruction of the substantia nigra and the ventral tegmental area, in cat Fl16, was followed only by a paralysis of the hind legs during the first week and only by a lack of motor coordination during the 2nd week. Furthermore, our findings indicate that the partial destruction of the substantia nigra induces a significant increase in waking. By contrast, electrolytic coagulation of the ventral mesencephalic tegmentum (groups A9 and A10) produced a behavioral state of 'coma' (akinetic animals) but did not alter the percentage of E E G arousal 15. Such differences between the neurotoxic and coagulation lesions could be due to the damage of fibers of passage in the substantia nigra. Further studies are necessary to determine the origin of the pathways passing through this nucleus.

Effect of PH lesion The acute effects of the PH lesion differed from those of the MRF lesion. Indeed, the IA microinjection into the PH was followed by a hypothermia, a dramatic increase of PS during the first postoperative day and by a lack of PS cortical activation for 1-2 days.

A dramatic hypothermia was the first marked symptom which took place during the first postoperative night. This phenomenon could be related to the inhibition of the neuronal cell bodies of the PH since the injection of muscimol, a GABA agonist, in the same structure is also followed by a fall in body temperature 28. Moreover, when the cerebral temperature was recorded during the IA microinjection, we observed a concomitant hyperthermia accompanied by shivering. This could be due to the intense excitation of cell bodies of a 'shivering area' by IA. From these observations, our findings are consistent with numerous studies suggesting that PH is involved in cold defense mechanisms. Indeed, electrical stimulation of the dorsomedial regions of the PH produces shivering in the cat 59, while its destruction by coagulation abolishes or markedly impairs shivering 6°. Thus, hypothermia induced by IA injection in the PH could be explained by the lack of the possibility to mediate convergent efferent responses like shivering. However, another explanation seems possible since inactivation of neuronal cell bodies of the lateral PH can induce an impairment of the cutaneous vasoconstriction leading to an increase of heat loss at specific heat exchangers. This phenomenon associated with the effect of anesthesia would then lead to hypothermia. Thereafter, the animal with PH lesion quickly recovers from hypothermia due to the existence of numerous other thermosensitive areas which may trigger heat production (see ref. 7 for reviews). Since there are many reciprocal interactions between sleep and thermoregulation, it is necessary to discuss the point whether the increase of PS amount was only due to the hypothermia. Recently, Jouvet et al. have demonstrated that hypothermia, below 30 °C, induces an almost permanent paradoxical sleep state in pontine cats deprived of thermoregulatory hypothalamic system 2°. They speculated that the hypersomnia was due to the differential thermosensitivity of both executive and permissive structures of PS. On the other hand, numerous studies have suggested that in the normal animal, PS is impaired by the cold defense mechanisms like vasoconstriction and shivering (see ref. 46 for review). Thus, it is possible that hypothermia might increase PS after a transitory inhibition of the cold defense mechanism by IA microinjection in the PH. However, this hypothesis may be rejected since in some cases (K92) the hypothermia could occur and disappear before the appearance of the hypersomnia. It is more likely that in our cases, the IA microinjection in the PH either resulted in an inactivation of some neural population which might exert a tonic inhibitory influence on PS executive structures, or induced the release of some hormonal factor which might facilitate PS. On the other hand, our findings show that the first PS

300 epochs exhibited E C G synchronization since there was no spontaneous desynchronization during the first 24 to 30 postoperative hours. Afterwards, the return of cortical desynchronization appeared only during PS while waking was still synchronized. This suggests a greater participation of the reticulo-hypothalamo-cortical system in the maintenance of desynchronization during waking than during PS. Furthermore, during the first postoperative day, all cats except K92 displayed aggressive behavior. Although the effects of electrical stimulation upon behavior are well documented, little is known about the effects of lesioning. However, Wheately demonstrated that destruction of the ventromedial hypothalamic nucleus could induce permanent savage behavior in the cat 65. In conclusion, the acute effects of IA administration in the PH are strikingly different from those obtained in the MRF. Concerning the chronic effect of IA, whatever the location of the lesion, all the cats presented normal motor behavior. It is important to note that all the animals were interested in their surroundings except cat Sl15. However, they were not able to eat by themselves during the first week. Our findings confirm previous reports regarding aphagia after the lateral hypothalamus destruction with neurotoxin 12'33. Aggressive behavior persisted only in cat X106. It is well known that the PH is the location of HA-containing neurons in the cat 26'42. Systemic or local injections of various histaminergic and anti-histaminergic drugs have led to the hypothesis that histamine is responsible for the maintenance of waking 27'37. However, even after total destruction of the caudal magnocellular neurons in cat C96, there was no alteration of E E G waking, SWS or PS. Only two cats (K92, Sl15) exhibited a significant decrease of waking, respectively, during 1 and 3 weeks. Although the amount of destroyed tissue was roughly equal in all the animals at the level of the middle and anterior parts of the PH, the caudal part of the PH at A8 level (according to the atlas of Berman and Jones 5) was spared in cats K92 and Sl15. From our present findings, there seems to be no clear correlation between destroyed histaminergic neurons and impairment of waking state. Since the injection of muscimol, a potent G A B A agonist, in the caudal part of the PH produced a profound and prolonged insomnia with behavioral hyperactivity28, it may be speculated that the neuronal destruction of this area would compensate the otherwise slight decrease of waking induced by the lesion of the tuberal level of the lateral hypothalamic area (A9 to A12 level). M R F and PH combined lesion MRF-PH-lesioned cats were first motionless in a coma-

tose state for 2-3 days. This state was accompanied by a transitory hypothermia (34 °C) and by the suppression of a spontaneous or evoked cortical low fast voltage activity. Only Hl13 did not develop cortical epileptic seizures in the acute period. It is interesting to note that cat HI 13 showed a normal amount of waking as soon as the spontaneous cortical arousal was possible (third postoperative day) and there was also no alteration in SWS or PS. Cats Ol14 and Tl13 presented a decrease in PS while waking and SWS remained at the control level from the 2nd week. During the first week, all the cats were handicapped by a lack of motor coordination and displayed little spontaneous activity. However, at the end of the experiments, the animals were able to stand, walk, groom and eat by themselves. In contrast to neuronal lesions produced in one step, the two successive operations (Bl19, B120) were not followed by any deficit in motor behavior. In these cats, the waking behavioral manifestations were similar to those which followed the PH lesion. These results are in accordance with those of Adametz I who demonstrated that electrolytic tegrnental lesions of the rostral midbrain, made in one single operation, led to coma, while the same lesion with an interval of 3 weeks between the two coagulations, was followed by an early return of motor functions. After the two step MRF-PH lesion, there was no chronic alteration of spontaneous or evoked cortical activation. However, our findings indicate that cat B120 exhibited a slight decrease in waking. This effect was similar to that observed in two other cases (K92, Sl15) after PH destruction. It could be related to the cell body integrity of the caudal part of the PH in these 3 cats. General discussion Our results differ considerably from those obtained by a coagulation of the same structures: the neuronal cell somata destruction of the MRF and/or the PH was not followed by any alteration in behavioral and E E G waking as observed by coagulation studies. Electrolytic lesions of the mesencephalic tegmentum with or without lesion of the substantia nigra produced a 'behavioral' coma and a large deficit in waking over 20-25 days ~5. In addition, since the early works of Ranson 47 and Nauta 4°, numerous studies have shown that the coagulation of the PH produced somnolence and a motor behavior deficit in the rat 34'54, cat 61 and monkey 11. Finally, Lindsley et al. have reported that lesions in the ponto-midbrain tegmentum, midbrain tegmentum and hypothalamus induced a postoperative lethargy in which the animal displayed little or no spontaneous activity over a 20-day survival period e9. The marked different effects on cortical and behavioral arousal following IA or coagulation lesions could be

301 explained in two ways. On the one hand, when coagulation is obtained by anodal currents, a high mortality rate after noradrenergic A1 lesion was induced, while a relatively low postoperative mortality and morbidity rate was observed with bilateral cathodal lesions 3. It was suggested that this was related to toxic effects of ferric ion deposition. However, this hypothesis cannot account for the differences between neurotoxin and coagulation since Adametz had observed a long lasting comatose state after cathodal coagulation of the midbrain tegmentum 1. On the other hand, it is more likely that the considerable decrease in waking state induced by coagulation is due to the destruction of the ascending or descending axonal pathways which would appear to be involved both in cortical activation and behavioral responsiveness of waking. There is evidence that coagulation of the rostrai mesencephalic tegmentum is very effective in inducing lethargy and the disappearance of spontaneous activity. This may be explained by the convergence of both thalamic and extrathalamic noradrenergic, cholinergic, serotonergic ascending pathways and the dopaminergic projections to the forebrain at this level 6'15"17'32"44'51'55. Similarly, the somnolence observed after coagulation of PH is probably due to the damage of the fibers in the medial forebrain bundle which collects efferents from diverse caudal structures 4~. However, we have noticed that IA injections in the MRF and PH do not produce the same immediate effects, suggesting a different role of these two structures in the sleep-waking cycle. It is probable that this difference is due to the existence of close connections between the ventrolateral posterior hypothalamus and the mediobasal preoptic area known for its control of PS onset 53'66. Our findings do not favor the hypothesis of the existence of a necessary waking system at the level of the MRF and/or PH. Thus, a question arises concerning the location of neuronal candidates underlying the tonic ascending activation. Several cell bodies in the brainstem and diencephalon have been implicated in the control of cortical activation. On the one hand, the noradrenergic neurons of the locus coeruleus give rise to widespread projections to the cortex and the thalamus 3°'32. They may represent an ascending modulatory neurotransmitter system that is important in wakefulness ~9. However, noradrenergic neurons do not seem to be involved in the induction and maintenance of neocortical desynchronization since their destruction does not result in a marked deficit in EEG waking ~3"~6'5°. It is of interest to note that the administration of a-MPT, an inhibitor of tyrosine hydroxylase, in the MRF-PH-lesioned cats induced an increase in PS amount and a decrease of wakefulness. This result indirectly indicates that a buibar catecholamine system

(A1/A2, C1/C2) might participate in the maintenance of cortical activation via the posterior hypothalamus since neither pontine NA- nor diencephalic DA-containing neurons seem to be necessary. On the other hand, a considerable body of evidence has accumulated supporting the hypothesis that acetylcholine is involved in neocortical activation 9'21'31'36'62. The effects of ACh on cortical activation are thought to be mediated by cholinergic neurons which give rise to ascending projections to the forebrain 18'55 or to the thalamus 14"43'58. Since the mesopontine tegmentum neurons provide the major source of cholinergic innervation for thalamic nuclei 35, the reticulo-thalamo-cortical pathway may play a key role in cortical activation. Thus, a great deal of information is available concerning the abolition of the thalamocortical neuronal rhythmic oscillation, and the increase in neuronal responsiveness to synaptic inputs to the thalamus, during the shift to waking (see ref. 57 for review). However, the dorsolateral pontomesencephalic neuron destruction with KA was not followed by any alteration of neocortical activation 64. Large lesions of the thalamus including the reticular and the intralaminar nuclei with KA had no effect on the cortical desynchronization of waking 8. Taken together, these results suggest that the cholinergic reticulo-thalamic system does not play a crucial role in the EEG desynchronization during the waking state as has been traditionally supposed. Recent data obtained in the rat indicate that the basal forebrain, via widespread thalamus and cortical projections 3s,45 is the most likely candidate to mediate the effects of ACh upon neocortical activation, but no details are provided concerning the time spent daily in the waking state after lesioning of the basal nucleus with IA s. From these data, it is likely that each putative structure for neocortical activation represents only one component of a wide ascending system. Thus, when a 'waking system' is destroyed, another system could compensate the loss. Recent electrophysiological studies have reported some similarities between histaminergic, noradrenergic, cholinergic and serotonergic populations: first, the presence of neurons which exhibited slow and tonic discharge patterns and increased firing rates during waking state 2'48A9"63 and, secondly the release of these neurotransmitters induced a marked reduction in spike frequency adaptation of cortical pyramidal cells while ACh and NA caused a slow depolarization of thalamocortical relay cells 36. Likewise, single unit recording studies suggest that unknown MRF neurons display a neuronal discharge specific to waking 56 and MRF stimulation exerts a depolarizing effect on the thalamocorticai neurons. In conclusion, behavioral and EEG waking appear to be the result of a complex interplay of structures located

302 in the e n t i r e b r a i n s t e m and in the d i e n c e p h a l o n . T h e o r g a n i z a t i o n of t h e s e systems and their h i e r a r c h y r e m a i n u n k n o w n . It is p r o b a b l e that the stimulation o f o n e of these w a k i n g systems causes a r e p e r c u s s i o n t h r o u g h the others

while

its suppression

is rapidly

followed

by

Acknowledgements. This work was supported by I.N.S.E.R.M. (U 52), C.N.R.S. (UA 1195) and DRET (Grant 89-203). The authors would like to express their appreciation to Dr. Weber and Dr. Nagatsu for the gifts of AADC antiserum and TH antiserum, respectively. We also thank V. Cucchiaro for secretarial work and Dr. J. Carew for the revision of the manuscript.

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