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Neuronal activity of identified posterior hypothalamic neurons projecting to the brainstem peribrachial area of the cat
Neuroscienee Letters, 107 (1989) 145-150
145
Elsevier Scientific Publishers Ireland Ltd. NSL 06516
Neuronal activity of identified posterior hypothalamic neurons projecting to the brainstem peribrachial area of the cat D. Par6, Y. Smith, A. Parent and M. Steriade Laboratoires de Neurophysiologie et de Neurobiologie, Facultb de Mbdecine, Universit~ Laval, Quebec, Que. (Canada)
(Received 5 June 1989; Revisedversion received 6 August 1989; Accepted 9 August 1989) Key words." Peribrachial area; Hypothalamus; Sleep-wakingcycle
Following horseradish peroxidase injections in the brainstem peribrachial (PB) area, massiveretrograde labeling was found in the posterior hypothalamic region. Single-unit recordings of posterior hypothalamic neurons with antidromicallyidentified projections to the PB area revealed that these neurons have higher firing rates in waking than in slow-wavesleep and dissimilar discharge patterns as compared with intralaminar thalamic neurons. The results are discussed in the context of reciprocal hypothalamc~brainstem circuits.
Recent morphological [8, 14] and electrophysiological [4, 9] studies indicate that a cholinergic contingent arising in the brainstem peribrachial (PB) area and laterodorsal tegmental (LDT) nucleus innervates thalamic nuclei and enhances the synaptic responsiveness of thalamic neurons to cortical and subcortical inputs. Moreover, the discharge rate of thalamically projecting PB neurons is significantly higher during behavioral states associated with an increased level of thalamic excitability, that is during waking (W) and paradoxical sleep (PS) [1]. Because several results support the idea that the hypothalamus contains one or more neuronal groups involved in the regulation o f states of vigilance [2, 6, 11, 15], it was of interest to verify if the PB area receives hypothalamic projections. We used two methods: the retrograde transport of horseradish peroxidase conjugated to the lectin wheatgerm agglutinin ( W G A - H R P ) , and the search for hypothalamic neurons antidromically invaded from the PB area. In addition, the discharge rate and pattern o f hypothalamic-PB neurons were studied during W and EG-synchronized sleep (S) and compared to those of intralaminar thalamic neurons. W G A - H R P injections (Sigma; 10% in phosphate buffer 0.01 M, p H 7.4) were perCorrespondence: M. Steriade, Laboratoire de Neurophysiologie, D6partement de Physiologie, Facult6 de M6decine, Universit6 Laval, Quebec., Canada G I K 7P4.
0304-3940/89/$ 03.50 © 1989 ElsevierScientific Publishers Ireland Ltd.
146
formed under ketamine (Ketaset. 40 mg/kg, i.m.) anesthesia using a 0.5 i~1 Hamilton microsyringe. In 5 cats, unilateral 0.01 Ill W G A HRP injections were directed at the PB area. After a 48 h survival time, the animals were perfused under barbiturate anesthesia (Somnotol, 40 mg/kg, i.p.) with 500 ml of a 0.9% saline solution followed by I liter of a fixative (1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) and finally by 1 liter of phosphate buffer containing 10% sucrose at 4"C. The brains were sectioned on a freezing microtome at 40 izm in the frontal plane• The selected sections were processed according to the tetramethylbenzidine (TMB) procedure [7] and counterstained by means of Neutral red. In 3 out of 5 cases, we succeeded in obtaining an injection site restricted to the PB area. A representative example of the retrograde labeling observed in the hypothalamus following such an injection is illustrated in Fig. I. Most of the retrogradely
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Fig. 1. Hypothalamic projections to the peribrachial area of the cat. A E: camera lucida drawings showing the distribution of retrogradely labeled neurons at various levels of the hypothalamus following a WGAHRP injection in the peribrachial area. The injection site is depicted in F. Horizontal bar indicates mm. AQ, aqueduct; BP. brachium pontis; CA, caudate nucleus; EN, entopeduncular nucleus; F, fornix; FF, field of Forel; HDA, dorsal hypothalamic area; HL, lateral habenular nucleus; HLA, lateral hypothalamic area; MM, mammillary bodies; MT, mammillothalamic tract; NST, nucleus of the stria terminalis" OT, optic tract; PP, cerebral peduncle; PUL, pulvinar thalamic nucleus; RE, reticular thalamic nucleus; RFB, retroflex bundle; SUB, subthalamic nucleus; VM, ventromedial hypothalamic nucleus; V3, third ventricle; ZI, zona incerta.
147
labeled hypothalamic cells were located in the posterior part of the hypothalamus. At this level, numerous labeled elements were observed in the ventromedial and ventrolateral hypothalamic area, the tuberomammillary nucleus, the perifornical nucleus and the periventricular region. At more rostral levels of the hypothalamus, only the medial part of the paraventricular nucleus was intensely labeled. Because most of the retrogradely labeled hypothalamic neurons were located in the posterior region of the lateral hypothalamic area, our microelectrode explorations were aimed to this part of the hypothalamus. Single neurons were recorded with tungsten microelectrodes (1-5 Mr2 atl kHz) in two chronically implanted, headrestrained cats. Physiological variables used to identify wake and sleep states (EEG, EOG and EMG) were also recorded. To backfire hypothalamic neurons, an array of four stimulating electrodes was placed in the PB area. The criteria used for antidromic identification were: a fixed response latency, collision with spontaneously occurring action potentials, and the ability to follow high-frequency (250 Hz) stimulation (Fig. 2).
1 ms
Cells Io-
i
i
i
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Fig. 2. Antidromic identification of neurons located in (i) the lateral part of the posterior hypothalamus (Hypoth. lat.), (ii) the intralaminar thalamic (IL th.) (centromedian-parafascicular (CM-Pf, top) or centrolateral-paracentral (CL-Pc, bottom)) nuclei, and (iii) the ventromedial thalamic (VM th) nucleus. Stimulating electrodes were located in the pontomesencephalic tegmentum ipsilateral to the recorded neurons. Examples of antidromic responses are depicted in A (two a-b CM-PF neurons) and B (a posterior hypothalamic neuron). Stimuli artefacts are indicated by arrows. Open arrows in B point to the break between the initial segment and somatodendritic components of the antidromic responses. Oblique arrow in B2 shows collision of antidromic response with a preceding spontaneous discharge. Lower left corner, histogram of antidromic response latencies of neurons located in various diencephalic sites.
148 Eight posterior hypothalamic and 16 intralaminar thalamic neurons were antidromicaily invaded from the PB area (Fig. 2). In contrast with intralaminar thalamic cells, which discharge tonically during EEG-desynchronized states and with stereotyped high-frequency spike bursts during S [3], posterior hypothalamic neurons generated isolated action potentials in both EEG-desynchronized and EEG-synchronized states. This difference in the discharge pattern of hypothalamic and intralaminat cells is apparent in the grouped interspike interval histograms illustrated in Fig. 3B. Whereas in posterior hypothalamic neurons the proportion of interspike intervals shorter than 5 msec (i.e. reflecting very high intraburst frequencies) remained below 1% in all states (Fig. 3Bi), in intralaminar neurons it reached 48% in S, while remaining below 3% in W (Fig. 3B2). According to the Wilcoxon matched-pairs signed-ranks test, the firing rate of posterior hypothalamic neurons was significantly higher in W than in S ( P < 0.05). In our sample, the median discharge rate was 25.65 H z i n W and 13.23 H z i n S. Accumulating evidence links the hypothalamus to behavioral state control. On the
w
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Fig. 3. Discharge pattern of lateral hypothalamic neurons with antidromicaUy-identifiedprojections to the mesopontine tegmentum. A: oscilloscopictraces depicting the discharge of a lateral hypothalamic neuron in the waking state (W) and during EEG-synchronized sleep (S). B: grouped interspike interval histograms of a sample of 8 hypothalamic neurons (1) and 16 intralaminar neurons (2) during W and S states• For details see text.
149
basis of postmortem case studies, Von Economo [2] reported that caudal hypothalamic lesions were associated with somnolence while damage to the rostral hypothalamus gave rise to insomnia. Since then, investigations using single-unit recordings, lesion and stimulation methods as well as pharmacological manipulations have strengthened these views [6, 11, 12]. The projection from the posterior hypothalamus to the rostral midbrain core [10] and, in this study, to the PB area, is reciprocated by a PB-hypothalamic pathway [5, 16]. Lesions of either the PB area and adjacent central tegmental field [13] or the posterolateral hypothalamic area [12] provoke a state of hypersomnia that lasts for only 3-4 days. These data emphasize that neither the hypothalamus nor the upper brainstem reticular core can be conceived as waking 'centers'. Rather, distributed networks consisting of interconnected cell aggregates must be envisaged as necessary for the induction and maintenance of the waking state. This work was supported by the Medical Research Council of Canada (MT-3689 and MT-5781). D.P. and Y.S. were MRC fellows. We thank D. Drolet, P. Gigu6re, C. Harvey and L. Bertrand for their assistance.
1 Datta, S., Par& D., Oakson, G. and Steriade, M., Thalamic-projecting neurons in brainstem cholinergic nuclei increase their firing rates one minute in advance of EEG desynchronization associated with REM sleep, Soc. Neurosci. Abstr., 15 (1989)452. 2 Economo, C., Von, Die Encephalitis Lethargica, Deuticke, Vienna, 1918. 3 Glenn, L.L. and Steriade, M., Discharge rate and excitability of cortically projecting neurons in the intralaminar thalamic nuclei during waking and sleep states, J. Neurosci., 2 (1982) 138~1404. 4 Hu, B., Steriade, M. and Desch6nes, M., The effects of brainstem peribrachial stimulation on neurons of the lateral geniculate nucleus, Neuroscience, 31 (1989) 13-24. 5 Jones, B.E. and Yang, T.-Z., The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat, J. Comp. Neurol., 242 (1985) 56-92. 6 Lin, J.S., Sakai, K., Vanni-Mercier, G. and Jouvet, M., A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats, Brain Res., 479 (1989) 225-240. 7 Mesulam, M.M., Axonal transport enzyme histochemistry and light microscopic analysis. In Tracing neural connections with horseradish peroxidase, Wiley, New York, 1982, pp. 1-151. 8 Par6, D., Smith, Y., Parent, A. and Steriade, M., Projections of upper brainstem reticular cholinergic and non-cholinergic neurons of cat to intralaminar and reticular thalamic nuclei, Neuroscience, 25 (1988) 69-86. 9 Par& D., Steriade, M., Desch6nes, M. and Bouhassira, D., Prolonged enhancement of anterior thalamic synaptic responsiveness by stimulation of a brainstem cholinergic group, J. Neurosci., in press. 10 Parent, A. and Steriade, M., Afferent projections from the periaqueductal gray, medial hypothalamus and medial thalamus to the midbrain reticular core, Brain Res. Bull., 7 (1981) 411-418. 11 Sallanon, M., Denoyer, M., Kitahama, K., Aubert, C., Gay, N. and Jouvet, M., Long-lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus in the cat, Neuroscience, in press. 12 Sallanon, M., Sakai, K., Buda, C., Puymartin, M. and Jouvet, M., Augmentation du sommeil paradoxal induite par l'injeetion d'acide ibot6nique dans l'hypothalamus ventrolat6ral post6rieur, chez le chat, C.R. Acad. Sci. Paris, S6rie III, 303 (1986) 175-179. 13 Steriade, M., Cellular mechanisms of wakefulness and slow wave sleep. In A. Mayes (Ed.), Sleep
150 Mechanisms and Functions in H u m a n s and Animals, Van Nostrand Reinhold, Wokingham, U.K., pp. [61 2[6. 14 Steriade, M., Pard, D., Parent, A. and Smith, Y., Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey, Neuroscience, 25 (I988) 47 67. 15 Vanni-Mercier, G., Sakai, K., Salvert, D. and Jouvet, M., Waking-state specific neurons in the posterior hypothalamus of the cat. In W.P. Koella, E. Rtither and H. Schulz (Eds.), Sleep "84, Fisher, New York, 1985, pp. 238 240. 16 Woolf, N.J. and Butcher, L.L., Cholinergic systems ill the rat brain: IIl, Projections from the pontomesencephalic tegmentum to the thalamus, rectum, basal ganglia, and the basal forebrain, Brain. Res. Bu11.,16(1986) 603 637.
145
Elsevier Scientific Publishers Ireland Ltd. NSL 06516
Neuronal activity of identified posterior hypothalamic neurons projecting to the brainstem peribrachial area of the cat D. Par6, Y. Smith, A. Parent and M. Steriade Laboratoires de Neurophysiologie et de Neurobiologie, Facultb de Mbdecine, Universit~ Laval, Quebec, Que. (Canada)
(Received 5 June 1989; Revisedversion received 6 August 1989; Accepted 9 August 1989) Key words." Peribrachial area; Hypothalamus; Sleep-wakingcycle
Following horseradish peroxidase injections in the brainstem peribrachial (PB) area, massiveretrograde labeling was found in the posterior hypothalamic region. Single-unit recordings of posterior hypothalamic neurons with antidromicallyidentified projections to the PB area revealed that these neurons have higher firing rates in waking than in slow-wavesleep and dissimilar discharge patterns as compared with intralaminar thalamic neurons. The results are discussed in the context of reciprocal hypothalamc~brainstem circuits.
Recent morphological [8, 14] and electrophysiological [4, 9] studies indicate that a cholinergic contingent arising in the brainstem peribrachial (PB) area and laterodorsal tegmental (LDT) nucleus innervates thalamic nuclei and enhances the synaptic responsiveness of thalamic neurons to cortical and subcortical inputs. Moreover, the discharge rate of thalamically projecting PB neurons is significantly higher during behavioral states associated with an increased level of thalamic excitability, that is during waking (W) and paradoxical sleep (PS) [1]. Because several results support the idea that the hypothalamus contains one or more neuronal groups involved in the regulation o f states of vigilance [2, 6, 11, 15], it was of interest to verify if the PB area receives hypothalamic projections. We used two methods: the retrograde transport of horseradish peroxidase conjugated to the lectin wheatgerm agglutinin ( W G A - H R P ) , and the search for hypothalamic neurons antidromically invaded from the PB area. In addition, the discharge rate and pattern o f hypothalamic-PB neurons were studied during W and EG-synchronized sleep (S) and compared to those of intralaminar thalamic neurons. W G A - H R P injections (Sigma; 10% in phosphate buffer 0.01 M, p H 7.4) were perCorrespondence: M. Steriade, Laboratoire de Neurophysiologie, D6partement de Physiologie, Facult6 de M6decine, Universit6 Laval, Quebec., Canada G I K 7P4.
0304-3940/89/$ 03.50 © 1989 ElsevierScientific Publishers Ireland Ltd.
146
formed under ketamine (Ketaset. 40 mg/kg, i.m.) anesthesia using a 0.5 i~1 Hamilton microsyringe. In 5 cats, unilateral 0.01 Ill W G A HRP injections were directed at the PB area. After a 48 h survival time, the animals were perfused under barbiturate anesthesia (Somnotol, 40 mg/kg, i.p.) with 500 ml of a 0.9% saline solution followed by I liter of a fixative (1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) and finally by 1 liter of phosphate buffer containing 10% sucrose at 4"C. The brains were sectioned on a freezing microtome at 40 izm in the frontal plane• The selected sections were processed according to the tetramethylbenzidine (TMB) procedure [7] and counterstained by means of Neutral red. In 3 out of 5 cases, we succeeded in obtaining an injection site restricted to the PB area. A representative example of the retrograde labeling observed in the hypothalamus following such an injection is illustrated in Fig. I. Most of the retrogradely
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Fig. 1. Hypothalamic projections to the peribrachial area of the cat. A E: camera lucida drawings showing the distribution of retrogradely labeled neurons at various levels of the hypothalamus following a WGAHRP injection in the peribrachial area. The injection site is depicted in F. Horizontal bar indicates mm. AQ, aqueduct; BP. brachium pontis; CA, caudate nucleus; EN, entopeduncular nucleus; F, fornix; FF, field of Forel; HDA, dorsal hypothalamic area; HL, lateral habenular nucleus; HLA, lateral hypothalamic area; MM, mammillary bodies; MT, mammillothalamic tract; NST, nucleus of the stria terminalis" OT, optic tract; PP, cerebral peduncle; PUL, pulvinar thalamic nucleus; RE, reticular thalamic nucleus; RFB, retroflex bundle; SUB, subthalamic nucleus; VM, ventromedial hypothalamic nucleus; V3, third ventricle; ZI, zona incerta.
147
labeled hypothalamic cells were located in the posterior part of the hypothalamus. At this level, numerous labeled elements were observed in the ventromedial and ventrolateral hypothalamic area, the tuberomammillary nucleus, the perifornical nucleus and the periventricular region. At more rostral levels of the hypothalamus, only the medial part of the paraventricular nucleus was intensely labeled. Because most of the retrogradely labeled hypothalamic neurons were located in the posterior region of the lateral hypothalamic area, our microelectrode explorations were aimed to this part of the hypothalamus. Single neurons were recorded with tungsten microelectrodes (1-5 Mr2 atl kHz) in two chronically implanted, headrestrained cats. Physiological variables used to identify wake and sleep states (EEG, EOG and EMG) were also recorded. To backfire hypothalamic neurons, an array of four stimulating electrodes was placed in the PB area. The criteria used for antidromic identification were: a fixed response latency, collision with spontaneously occurring action potentials, and the ability to follow high-frequency (250 Hz) stimulation (Fig. 2).
1 ms
Cells Io-
i
i
i
0 $
I0
1.5
i
20
~
i
i
3.0
4.0
oth.
IL th. VM th.
u
,
t00 ms
lat.
2ms
Fig. 2. Antidromic identification of neurons located in (i) the lateral part of the posterior hypothalamus (Hypoth. lat.), (ii) the intralaminar thalamic (IL th.) (centromedian-parafascicular (CM-Pf, top) or centrolateral-paracentral (CL-Pc, bottom)) nuclei, and (iii) the ventromedial thalamic (VM th) nucleus. Stimulating electrodes were located in the pontomesencephalic tegmentum ipsilateral to the recorded neurons. Examples of antidromic responses are depicted in A (two a-b CM-PF neurons) and B (a posterior hypothalamic neuron). Stimuli artefacts are indicated by arrows. Open arrows in B point to the break between the initial segment and somatodendritic components of the antidromic responses. Oblique arrow in B2 shows collision of antidromic response with a preceding spontaneous discharge. Lower left corner, histogram of antidromic response latencies of neurons located in various diencephalic sites.
148 Eight posterior hypothalamic and 16 intralaminar thalamic neurons were antidromicaily invaded from the PB area (Fig. 2). In contrast with intralaminar thalamic cells, which discharge tonically during EEG-desynchronized states and with stereotyped high-frequency spike bursts during S [3], posterior hypothalamic neurons generated isolated action potentials in both EEG-desynchronized and EEG-synchronized states. This difference in the discharge pattern of hypothalamic and intralaminat cells is apparent in the grouped interspike interval histograms illustrated in Fig. 3B. Whereas in posterior hypothalamic neurons the proportion of interspike intervals shorter than 5 msec (i.e. reflecting very high intraburst frequencies) remained below 1% in all states (Fig. 3Bi), in intralaminar neurons it reached 48% in S, while remaining below 3% in W (Fig. 3B2). According to the Wilcoxon matched-pairs signed-ranks test, the firing rate of posterior hypothalamic neurons was significantly higher in W than in S ( P < 0.05). In our sample, the median discharge rate was 25.65 H z i n W and 13.23 H z i n S. Accumulating evidence links the hypothalamus to behavioral state control. On the
w
A
B 2%
w
80
'° 1
70" 60'
70
50.
W S
30
40' 30
2o
20
10 •
10
60
i ........
- 100-500<
ms
<5
t i - I00-500<
ms
Fig. 3. Discharge pattern of lateral hypothalamic neurons with antidromicaUy-identifiedprojections to the mesopontine tegmentum. A: oscilloscopictraces depicting the discharge of a lateral hypothalamic neuron in the waking state (W) and during EEG-synchronized sleep (S). B: grouped interspike interval histograms of a sample of 8 hypothalamic neurons (1) and 16 intralaminar neurons (2) during W and S states• For details see text.
149
basis of postmortem case studies, Von Economo [2] reported that caudal hypothalamic lesions were associated with somnolence while damage to the rostral hypothalamus gave rise to insomnia. Since then, investigations using single-unit recordings, lesion and stimulation methods as well as pharmacological manipulations have strengthened these views [6, 11, 12]. The projection from the posterior hypothalamus to the rostral midbrain core [10] and, in this study, to the PB area, is reciprocated by a PB-hypothalamic pathway [5, 16]. Lesions of either the PB area and adjacent central tegmental field [13] or the posterolateral hypothalamic area [12] provoke a state of hypersomnia that lasts for only 3-4 days. These data emphasize that neither the hypothalamus nor the upper brainstem reticular core can be conceived as waking 'centers'. Rather, distributed networks consisting of interconnected cell aggregates must be envisaged as necessary for the induction and maintenance of the waking state. This work was supported by the Medical Research Council of Canada (MT-3689 and MT-5781). D.P. and Y.S. were MRC fellows. We thank D. Drolet, P. Gigu6re, C. Harvey and L. Bertrand for their assistance.
1 Datta, S., Par& D., Oakson, G. and Steriade, M., Thalamic-projecting neurons in brainstem cholinergic nuclei increase their firing rates one minute in advance of EEG desynchronization associated with REM sleep, Soc. Neurosci. Abstr., 15 (1989)452. 2 Economo, C., Von, Die Encephalitis Lethargica, Deuticke, Vienna, 1918. 3 Glenn, L.L. and Steriade, M., Discharge rate and excitability of cortically projecting neurons in the intralaminar thalamic nuclei during waking and sleep states, J. Neurosci., 2 (1982) 138~1404. 4 Hu, B., Steriade, M. and Desch6nes, M., The effects of brainstem peribrachial stimulation on neurons of the lateral geniculate nucleus, Neuroscience, 31 (1989) 13-24. 5 Jones, B.E. and Yang, T.-Z., The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat, J. Comp. Neurol., 242 (1985) 56-92. 6 Lin, J.S., Sakai, K., Vanni-Mercier, G. and Jouvet, M., A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats, Brain Res., 479 (1989) 225-240. 7 Mesulam, M.M., Axonal transport enzyme histochemistry and light microscopic analysis. In Tracing neural connections with horseradish peroxidase, Wiley, New York, 1982, pp. 1-151. 8 Par6, D., Smith, Y., Parent, A. and Steriade, M., Projections of upper brainstem reticular cholinergic and non-cholinergic neurons of cat to intralaminar and reticular thalamic nuclei, Neuroscience, 25 (1988) 69-86. 9 Par& D., Steriade, M., Desch6nes, M. and Bouhassira, D., Prolonged enhancement of anterior thalamic synaptic responsiveness by stimulation of a brainstem cholinergic group, J. Neurosci., in press. 10 Parent, A. and Steriade, M., Afferent projections from the periaqueductal gray, medial hypothalamus and medial thalamus to the midbrain reticular core, Brain Res. Bull., 7 (1981) 411-418. 11 Sallanon, M., Denoyer, M., Kitahama, K., Aubert, C., Gay, N. and Jouvet, M., Long-lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus in the cat, Neuroscience, in press. 12 Sallanon, M., Sakai, K., Buda, C., Puymartin, M. and Jouvet, M., Augmentation du sommeil paradoxal induite par l'injeetion d'acide ibot6nique dans l'hypothalamus ventrolat6ral post6rieur, chez le chat, C.R. Acad. Sci. Paris, S6rie III, 303 (1986) 175-179. 13 Steriade, M., Cellular mechanisms of wakefulness and slow wave sleep. In A. Mayes (Ed.), Sleep
150 Mechanisms and Functions in H u m a n s and Animals, Van Nostrand Reinhold, Wokingham, U.K., pp. [61 2[6. 14 Steriade, M., Pard, D., Parent, A. and Smith, Y., Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey, Neuroscience, 25 (I988) 47 67. 15 Vanni-Mercier, G., Sakai, K., Salvert, D. and Jouvet, M., Waking-state specific neurons in the posterior hypothalamus of the cat. In W.P. Koella, E. Rtither and H. Schulz (Eds.), Sleep "84, Fisher, New York, 1985, pp. 238 240. 16 Woolf, N.J. and Butcher, L.L., Cholinergic systems ill the rat brain: IIl, Projections from the pontomesencephalic tegmentum to the thalamus, rectum, basal ganglia, and the basal forebrain, Brain. Res. Bu11.,16(1986) 603 637.