A neuroanatomical gradient in the pontine tegmentum for the cholinoceptive induction of desynchronized sleep signs

Brain Research, 414 (1987) 245-261 Elsevier

245

BRE 12656

A neuroanatomical gradient in the pontine tegmentum for the cholinoceptive induction of desynchronized sleep signs Helen A. Baghdoyan, Margarita L. Rodrigo-Angulo*, Robert W. McCarley** and J. Allan Hobson Laboratory of Neurophysiology, Department of Psychiatry, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 11 November 1986)

Key words: Desynchronized sleep; pontine tegmentum; Acetylcholine; Carbachol; Cat; Microinjection

Microinjection of cholinergic agonists into the pontine tegmentum was used to evoke a state which was polygraphically and behaviorally similar to desynchronized (D) sleep. This study was designed to test the hypothesis that the production of this pharmacologically induced D sleep-like state (D-ACh) was dependent upon the anatomical locus of drug administration within the pontine tegmentum. Four dependent variables of D sleep were measured: D latency, D percentage, D duration and D frequency. Multiple regression analysis and analysis of variance were performed to evaluate the relationship between the three-dimensional coordinates of the injection site (posterior, vertical and lateral) and these 4 dependent measures. The intrapontine site of drug administration accounted for a statistically significant amount of the variance in D latency, D percentage and D duration. There was no significant relationship between the anatomical site of saline injection and the dependent measures of D sleep. A significant increase in D frequency following microinjection of cholinergic agonists was found to be independent of injection site. Pontine injection sites which yielded the shortest D latencies were found to be the same sites from which the highest D percentages were evoked. Rostrodorsal pontine tegmental injection sites were most effective in producing the highest percentages of D-ACh with the shortest latencies to onset. Injections made more caudally and ventrally within the pontine tegmentum produced lower percentages of D-ACh with longer lateneies to onset. These data suggest the existence of an anatomical gradient within the pontine tegmentum for the cholinoceptive evocation of a D sleep-like state, and further support the concept that D sleep is generated, in part, by pontine cholinergic mechanisms.

INTRODUCTION

tems involved in regulating many physiological and behavioral functions t4,35,42.

Cholinergic hypotheses of sleep induction were first tested by Hernandez-Peon et al. 17 who reported that both synchronized (S) sleep and desynchronized (D) sleep could be produced by direct application of acetylcholine (ACh) crystals to brain regions in or near the limbic forebrain-limbic midbrain circuit described by Nauta 36. Techniques for administering pharmacological agonists directly into the central nervous system have since evolved, and the method of central microinjection has been used productively to examine the roles of various neurotransmitter sys-

Microinjection of cholinergic agonists directly into the pontine tegmentum has been shown to produce a state which is polygraphically and behaviorally similar to physiological D sleep (for review see refs. 3, 18, 20, 30). Recent attention has focused on the precise anatomical sites of drug administration within the brainstem at which cholinergic agonists elicit D sleep signs and D sleep-like behavior 2'5'6'13,28,54. As a result of these studies it is now known that: (1) microinjection of cholinergic agonists produces a D sleep-like state when applied to the pontine reticular formation

* Present address: Departament de Morfologia, Facultad de Medicina, Universidad Autonoma de Madrid, 28029 Madrid, Spain. ** Present address: Psychiatry (166A), Veterans Administration Medical Center, 940 Belmont Street, Brocton, MA 02401, U.S.A. Correspondence: H. A. Baghdoyan, Laboratory of Neurophysiology, Department of Psychiatry, Harvard Medical School, 74 Fenwood Road, Boston, MA 02115, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

246 but not when administered to the midbrain or medullary reticular formationS; (2) there are sites within the pontine tegmentum from which individual electrographic components of D sleep (e.g. ponto-geniculo-occipital (PGO) waves, rapid eye movements, or muscular atonia) can be evoked independently from the other components of D sleep6,28,s4,56; and (3) even within the pontine tegmentum there are sites from which D sleep can be suppressed by microinjection of cholinergic agonists 5'6'33. The purpose of the present study was to perform a systematic and quantitative analysis of the relationship between the anatomical locus of drug administration within the pontine tegmentum and the potency of D sleep sign enhancement, as measured by 4 dependent variables: (1) the latency to onset of the D sleep-like state, (2) frequency of occurrence and (3) duration of the D sleep-like episodes, and (4) the percentage of time spent in the D sleep-like state following drug injection. The results show that the latency to onset, the duration, and the percentage of the cholinoceptively induced D sleep-like state varied significantly and systematically according to the site of microinjection. MATERIALS AND METHODS

Surgical preparation of animals Twelve adult (3.5-5 kg) male cats were surgically prepared for polygraphic recording of behavioral state and microinjection of drug solutions by previously described methods 4,5. Animals were anesthetized with pentobarbital (35 mg/kg i.p.) and implanted with electrodes for recording the electroencephalogram (EEG), electrooculogram (EOG), electromyogram (EMG) and PGO waves. Stainlesssteel guide tubes (24 gauge) were stereotaxically aimed at various sites throughout the pontine tegmentum (theta = 60 degrees) and cemented in place with dental acrylic.

Microinjection of pharmacological agents During the microinjection procedure, the cats were always awake. The animals were placed in head restraint and a stainless-steel cannula (31 gauge) was inserted into the fixed guide tube. The injection cannula protruded 3-5 mm below the end of the guide tube. Injections were made using a 1.0 pl Hamilton

syringe mounted in a manual microdrive and attached to the injection cannula with polyethylene tubing (PE-20). All injections were unilateral, given in a volume of 250-500 nl, and delivered over a period of 30-35 s. Following the injection, the cannula was removed and the flow in the delivery system was checked to ensure that the solution had been administered. The cats were then released from head restraint and placed in a recording chamber (2' x 2' × 3'). The animals received either carbachol chloride (4/~g) or a mixture of acetylcholine chloride (5/~g) and neostigmine bromide (20/~g) (ACh/N) dissolved in sterile saline. Five to 7 days were allowed between microinjections. A total of 41 drug injections were made into 20 different pontine sites. Twenty-one control injections of saline were made into 11 pontine sites.

Polygraphic recording of behavioral states Cats were given 7-10 days to recover from surgery and an additional 2-3 days to adapt to the recording chamber. Polygraphic recordings were begun immediately after the injection and lasted for 4 h. All recordings were conducted in freely moving animals between 11.00 and 17.00 h under constant illumination. The behavioral states of wakefulness (W), S sleep and D sleep were scored according to standard polygraphic criteria 52. The drug-induced D sleep-like state was defined by the simultaneous occurrence of somatic muscular atonia, rapid eye movements, PGO waves and E E G desynchrony, as well as the behavioral appearance of sleep. This state was termed D-ACh to distinguish it from physiological D sleep (Fig. 1). The polygraphic indices of behavioral state were scored in 25-s epochs, yielding a total of 576 bins which were scored following every injection (trial).

Data analysis Multiple regression analysis and analysis of variance as were used to examine the relationship between the anatomical site of drug administration and the effect of the drugs or saline on D sleep signs. The independent variables were the three-dimensional coordinates (posterior, vertical, lateral) of the injection sites. The electrographic and behavioral measures provided 4 dependent variables: D latency, D

247

I

,4. EMG EOG LGB(left) LGB(right) EEG

L.. .......

~. ......... ,,~ . . . . ~ .

_.~................ ~

...........

B EMG ::: . . . .

.,,,d_ .,L . . . . . . .

LGB(righl)

I Fig. 1. Polygraphic record of the cholinoceptively induced D sleep-like state as compared with physiologically occurring D sleep. Two 80-s segments of polygraphic variables recorded from the same cat under control (A) and drug injection (B) conditions. A: shows the normal transition into D sleep from S sleep. Note the progressive diminution of muscle tone (EMG), the increasing frequency of PGO waves (LGB left and right), and the loss of spindles in the cortex (EEG) as the animal enters D sleep. B: shows the abrupt entry into DACh, which occurs directly from wakefulness with no intervening S sleep. Note the rapid loss of muscle tone (EMG) which is followed by PGO waves (LGB left and right) occurring first singly and then clustered, and the continually desynchronized cortex (EEG). This episode of D-ACh occurred during the third hour post-injection.

percentage, D frequency and D duration. D latency was defined as the time in minutes from the onset of injection to the onset of the first D sleep or D - A C h episode. D percentage was defined as the percent of recording time spent in D sleep or D - A C h , and was calculated for the first hour post-injection as well as for the entire post-injection recording period (4 h). D frequency was the n u m b e r of D sleep-like episodes per trial, and D duration was the duration in min of each D sleep-like episode.

Luxol fast blue. Every histological section containing a lesion from the injection site was visually projected and drawn, and the injection sites were reconstructed in three dimensions according to the sagittal plates of Berman 8. A photomicrograph of a typical injection site is shown in Fig. 2.

Histological localization of injection sites

Table I summarizes the range and mean values for D latency, D duration, D frequency and D percentage during the first hour following microinjection of drugs or saline (control). Examination of these data without regard to the anatomical site of drug administration within the pontine tegmentum (Table I) shows that the mean D latency after drug administra-

Following microinjection experiments, the animals were deeply anesthetized with pentobarbital (40 mg/kg i.p.) and perfused transcardially with heparinized saline followed by 10% formalin. Brains were e m b e d d e d in celloidin and cut in 25/~m sagittal sections which were stained with Cresyl violet and

RESULTS

Centrally administered cholinergic agonists alter the temporal organization of D sleep

248 TABLE I Range and mean values for D latency, D percentage, D duration and D frequency ?

"3 ;" 3 ~.~.A

Condition

.. D latency (min) D percentage (hour 1)

Range

Mean +S.D.

drug (n=41) 0.8 to 122.8 41.8 + 32.8 control(n=21) 26.6to 93.0 50.5 + 20.0 drug control

0.0to 91.7 27.8 + 29.6* 0.0to 20.8 9.2 + 7.0

D duration

drug control

0.4 to 180.1 8.4 + 11.0 0.4 to 12.4 5.2+ 2.3

D frequency

drug control

2.0to 50.0 14.1 + 10.6" 1.0to 9.0 5.6+2.1

*P < 0.005, t-test.

Fig. 2. Photomicrograph of an injection site. A projection drawing (top) from a sagittal section of the cat brainstem. The boxed area appears in the photomicrograph below. Rostrai is to the right and caudal is to the left. The arrow indicates the location of the injection site. LC, locus coeruleus; 7G, genu of the facial nerve; FTG, gigantocellular tegmental field; 6N, abducens nerve; TRC, tegmental reticular nucleus, central division; TB, trapezoid body; P, pyramidal tract.

tion was not significantly different from control (t = 1.05, df = 60, P < 0.25). Contrastingly, drug administration did p r o d u c e a significant overall increase in D percentage during the first hour (Table I) as corn-

p a r e d with control levels (t = 2.82, df = 60, P < 0.005). A s will be shown below, the variability in D latency and D percentage following drug injection can be explained by taking into account the a n a t o m ical site of drug administration within the pontine tegmentum. A s shown in Table I the average D duration was not significantly changed following drug injection (t = 1.36, df = 60, P < 0.2). Microinjection of cholinergic agonists did induce a significant increase in D frequency (Table I; t = 3.61, df = 60, P < 0.001). Following drug injection, 63% of the trials had 10 or m o r e episodes of D - A C h , whereas following saline injection there were no trials having 10 or m o r e D sleep episodes. The occurrence and duration of all D - A C h and D sleep episodes are shown for each trial in Fig. 3. T h e large amount of inter-trial variability in the latency to onset of the first D - A C h episode, the percentage of D - A C h , the duration of D - A C h episodes and the frequency of occurrence of D - A C h , which was r e p o r t e d in Table I, is also evident in Fig. 3A. Following saline injections (Fig. 3B, Table I) there was much less variability in these parameters. D latency depends upon injection site To examine the hypothesis that the site of drug administration within the pontine t e g m e n t u m could account for the large a m o u n t of variability in D latency (Table I), the D latency values were divided into 3 groups (low, m e d i u m and long latency) and the anatomical distribution of injection sites within these

249

A EXPERIMENTAL TRIAL S I 2 3 4 5 6 7 8 9 IO

•

I II • l l i l l • I I III l l l l l l il

II

Ill

it

In

12 l U l l 15 14 J5 i6 17 II 18 llll 19 I I

II

21

I •

I

•

I I

I I I I I

lllll •

I l •

I

I

l- m i l l

.

IIIIII

I

.

.

.

III

III

II I

i I

Illl

I

0

i

ll

I

! •

--

II I I I I Ill I I I III lllll lllll I I I I 1 •

•

IIII

I

I

II

IIIII I n

I

I

B CONTROL TRIALS

III II • I

I III

ll •

I •

I

I

4

I

0

I

• ~

I

I

L 2 s 4 5

I

I • • I • __IJJn__ - Ill I I Illl I ill, l l l l l I I l l • I • • J IN l n i l • I I Ii llll-I II n I l I n l I n I • 3t • 52 I illlll Ill. i l l 33 I I I I I III 54 I II Ill Ill II i 55 I II I I I 1 ilaJ 56 • lll I • 57 II 1 I • l l l l 58 I I In I 59 I I I I 40 41_ I I I I ll~ I _ L i ll I

5

2

I

22 25 24 2 5 26 27 28 29 5O

I

I

I

l

I

2

3

•

l

•

I I

n

•

_

I

I

m

15

_

il

i

1

lima • •

•

•

• l

_

II

16

•

t7

m

_

n

|

nn I i

II l _ __

n

I I

All i U ~ _

_ H _ II I I II _ _ _ 1 I

I

!

•

_ l

18 ~N 19 ~ ~ • ~0 _ _ _ _ 2t ~

n In I g l

n

• •

• ill

• •

n _

I

•

n

l

9 _ _ i ~ _ _ t2 ,3 14 ~

II

lm

8

•

I

_

r _ _

I

I I

I II

6

4

• •

_

! 1 l

I

I

I

I

i

0

I

2

3

4

TIME POST INJECTION (hrs) Fig. 3. Effect of cholinergic agonists on the temporal distribution of D sleep signs. Each black bar represents the occurrence and duration of one D - A C h or D sleep episode. All 62 microinjection trials (ordinate) are shown from the time of injection onset (Time 0 Post Injection) to the end of the fourth hour post-injection (abcissa). A: D - A C h for the 41 drug injection trials. Note that in some trials the frequency of occurrence of D - A C h episodes was increased (Trials 1,7,8,12), whereas in others the duration was increased (Trials 4,11,13,32,39). Following some drug injections, the occurrence of D sleep signs actually was suppressed (Trials 3,6,17). B: D sleep following 21 saline injection trials.

T A B L E II

Mean stereotaxic coordinates of injection sites yielding short, medium and long D latencies, and high, medium and low D percentages

D latency ( • i n )

Group

Condition

n

Mean + S.D.

Stereotaxic coordinates (mean + S.D.) Vertical

Posterior

Lateral

Short

drug

13

10.3 + 4.6

-7.1 + 1.4

2.5 + 0.8

1.1 + 0.2

0

-

-

(0-20) control

-8.5 + 1.5

3.6 + 1.4

1.1

36.8 _+ 6.8

- 8 . 2 _ 1.6

2.9 + 1.0

1.0 + 0.3

83.0 + 20.8

-8.5 + 1.2

4.6 + 1.6

1.2

9

57.2 + 43.9

- 8 . 4 + 1.6

3.8 + 1.4

1.2+0.4

High drug (51-100%) control

12

69.2 + 11.2

-6.6 + 1.1

2.4 + 0.9

1.0+0.3

0

-

-

-

Medium (16-50%)

10

26.4 +_ 6.7

- 8 . 6 -+ 1.5

3.0 +_ 0.9

1.2 -+ 0.3

4

20.1 + 1.4

- 8 . 0 + 1.4

2.1 + 1.6

1.0 _+ 0.2

Medium

drug

15

33.5 + 10.8

control

12

drug

13

control

-

+ 0.3

(21-50) Long (>51) D percentage (hour 1)

drug control

Low (0-15%) * P < 0.01, t-test.

+

0.4

drug

19

2.3 + 4.0*

- 8 . 6 + 1.1

4.6 + 1.6

1.2 _+ 0.4

control

17

6.6 + 5.0

-8.4 + 1.6

3.5 + 1.3

1.1 _+0.4

250

~

DLATENCY

c>

i o~-f~--~

) ~+~

19

DPERCENTAGE

08

~I

~9

Fig. 4. Anatomical localization of pontine injection sites and relationship of site of D latency and D percentage. Injection sites are shown as circles on a series of sagittal schematic drawings of the brainstem. The brainstem schematics were taken from Bermans and range from 0.3 to 1.9 mm lateral. This region includes the gigantocellular tegmental field, trapezoid body, genu of the facial nerve, abducens nucleus and nerve, magnocellular tegmental field and nucleus praepositus hypoglossi. Cross marks (+) on each drawing indicate stereotaxic zero in the anterior-posterior plane and stereotaxic -I-10.0 in the vertical plane. Each circle indicates that an injection was made at that site, and multiple injections into the same area are marked by circles which are slightly offset from one another. A: injection sites are coded into 3 groups based upon the latency to onset of the first D-ACh episode obtained following microinjection into that site. Solid circles indicate sites from which latencies of 0-20 rain were obtained (n = 13), striped circles denote sites which yielded latencies in the 21-50 rain range (n = 15) and stippled circles represent sites from which the latency to onset of D-ACh was greater than 51 min (n = 13). B: injection sites are coded into 3 groups based upon the percentage of time spent in D-ACh during the first hour post-injection. Solid circles indicate sites from which 51-100% D-ACh was obtained (n = 12), striped circles denote sites which yielded values in the 16-50% range (n = 10) and stippled circles represent sites from which 0-15% D-ACh was produced (n = 19).

groups was examined. These data are shown in Table II (upper half) and Fig. 4A. In Fig. 4 A injection sites are c o d e d according to the value for D latency o b t a i n e d following each microinjection trial. Injection sites that yielded the shortest latencies (solid circles) t e n d e d to be g r o u p e d rostrally and dorsally, whereas the sites from which the longest latencies were o b t a i n e d (stippled circles) were m o r e caudal and ventral. The u p p e r half of Table II shows that injection sites from which the shortest D latencies ( 0 - 2 0 min) were o b t a i n e d were located m o r e dorsally (-7.1 vs - 8 . 5 ) and m o r e rostrally (2.5 vs 4.6) than injection sites which p r o d u c e d the longest D latencies (>51 min). Table II (upper half) also shows that in no case did microinjection of saline (control) p r o d u c e a D latency of less than 20 min. Thus, although the overall mean D latency following drug injection was not significantly decreased (Table I), there was a dramatic reduction in D latency following microinjection of cholinergic agonists into certain brainstem sites. To evaluate statistically the relationship between injection site and D latency, multiple regression analysis was p e r f o r m e d using the three-dimensional coordinates of the injection sites (posterior, vertical and lateral dimensions of the brainstem) as i n d e p e n d e n t variables and D latency as the d e p e n d e n t measure. This test revealed that the intrapontine site of drug administration accounted for 40% of the variance in D latency (r = 0.633) and that this effect was statistically significant ( F = 8.249; df = 3,37; P < 0.0002). Saline (control) injections were m a d e into the same anatomical range of sites as drug injections (Table III). A similar multiple regression analysis of the con-

TABLE III Range and mean values for the stereotaxic coordinates o f the injection sites Condition

Range

Mean + S.D.

Vertical (mm)

drug (n = 41) -5.5 to-10.0 -8.0 + 1.5 control (n = 21) -5.5 to-10.0 -8.3 + 1.6

Posterior(mm)

drug control

1.8 to 1.8to

7.2 6.5

3.6+ 1.6 3.3 + 1.2

Lateral (mm)

drug control

0.3 to 0.3 to

1.9 1.9

1.1 + 0.3 1.1 +0.4

251 trol data was used to examine the possible effect of saline injection site on D latency. N o statistically significant relationship between D latency and the anatomical site of saline administration was found (r -0.545; F = 2.400; df = 3,17; P = 0.1036).

B

A ~2C lOC tic

To examine the contribution of the individual anatomical coordinates (posterior, vertical and lateral) toward the significant effect of injection site on D latency, the partial regression coefficient for each of these independent variables was tested for significance. The contributions of the posterior coordinate and the vertical coordinate were statistically significant (t = 3.882, P < 0.0004 and t -- 1.855, P < 0.0717, respectively), but the contribution of the lateral coordinate was not statistically significant (t = 0.395, P < 0.6948). Fig. 5A and Table II show the relationship between D latency and the posterior coordinate of the injection site, and demonstrate that shorter D latencies were obtained by drug injections into more rostral sites. Of the 13 drug trials which produced D latencies in the 0 - 2 0 rain range, 11 (85%) were located between 1.8 and 3.0 posterior (Table II). The Pearson p r o d u c t - m o m e n t correlation revealed that the posterior coordinate accounted for 31% of the variance in D latency (r = 0.5606), and that this effect was statistically significant (dr -- 1,39; P < 0.001). Fig. 5B and Table II show the relationship between D latency and the vertical coordinate of the injection site. Eight of the 13 (62%) short latency trials (0-20 min) were obtained following drug injections located between - 5 . 5 and - 7 . 0 vertical (Table II). Eleven percent of the variance in D latency was accounted for by the vertical coordinate (r = 0.3324), which was statistically significant (df = 1,39; P < 0.05). Thus, the shortest D latencies were obtained following drug injections into sites which were located rostrally and dorsally within the pontine tegmentum.

D percentage is dependent upon injection site As with D latency, D percentage showed a large amount of variability following drug injection (Table I, Fig. 3A). Would it also be possible to explain this variability on the basis of the site of drug administra-

!

eC

6(

Injections into rostral and dorsal pontine sites most effectively reduce D latency

From) -~

14C

14C

4(

C~ 2( c

L~

'I: : ""

,_g°4

1

.%,

KX • C 9(

•

II

•

6oi

•

10

tram)

e~ ml

8

1 ~ ";

I • $

•

•

5oi 4oi 3oi

3O

ZOl 101

I0

1

2--

3--~-

5

Posterior (mm)

6

?=

0

~,.~ _~ -:~:_~.~ Vert/col(mm )

Fig. 5. Relationship between D latency and D percentage and the posterior and vertical coordinates of the injection sites. Brainstem schematics show the stereotaxic coordinates for the anatomical dimension relevant to each graph. The box inside the brainstem schematic indicates the anatomical region into which all 41 injections were made. The least squares method was used to calculate the regression line for each of these functions. Correlations are given in the text. A: D latency was dependent upon the posterior coordinate of the injection site. The shortest D lateneies were obtained following injections into rostral sites. The slope of the regression line was statistically significant by analysis of variance (F = 19.22; (if = 1,39; P < 0.001). B: D latency also varied according to the vertical coordinate of the injection site, with injections into dorsal sites yielding the shortest D latencies. The slope of the line was statistically significant (F = 5.21; df = 1,39; P < 0.05). C: D percentage was significantly dependent upon the posterior coordinate of the injection site (F = 20.41; df = 1,39; P < 0.001). Highest D percentages were produced by injections into rostral sites• D: D percentage also varied significantly according to the vertical coordinate of the injection site, with injections into dorsal sites giving the highest D percentages (F = 24.27; df = 1,39; P < 0.001). Taken together, these data show that the shortest D latencies and the highest D percentages were produced following drug injections into rostrodorsal pontine sites.

tion within the pontine tegmentum? To begin to answer this question, D percentage values were divided into 3 groups (low, medium and high percentages; Table II, lower half) and the anatomical distribution of these groups was examined (Fig. 4B).

252 Fig. 4B illustrates the 3 groups of injection sites which were based upon the percentage of recording time during the first hour post-injection spent in DACh. Sites from which microinjection produced the highest D percentages (solid circles) tended to be found more rostrally and dorsally, whereas sites which yielded the lowest D percentages (stippled circles) appeared to be located more caudally and ventrally. Table II (lower half) shows that microinjection of cholinergic agonists both increased and decreased D percentage as compared with control levels. The increase in D percentage following drug injection is reflected by the fact that in no case was a control injection able to produce D percentages in the high (51-100%) range. Medium range values for D percentage (16-50%) were not significantly changed from control following drug injection (t = 1.81, df = 12, P < 0.10). Comparison of the drug injection and control values in the low (0-15%) range revealed that the cholinergic agonists actually lowered D percentage below control levels (t = 2.86, df = 34, * P < 0.01). This decrease in D percentage was not apparent when the overall D percentage was examined (Table I). Whether microinjection of a cholinergic agonist into the pontine tegmentum induced an increase or decrease in the mean D percentage depended upon the site of drug administration (Table II, lower half). Injection sites which yielded the highest D percentages (51-100%), when compared with the sites that produced the lowest D percentages (0-15%), were more dorsal (-6.6 vs -8.6) and more rostral (2.4 vs 4.6). Multiple regression analysis was used to examine the relationship between D percentage and injection site. The three-dimensional coordinates of the injection sites (posterior, vertical and lateral) were the independent variables and D percentage during the first hour post-injection was the dependent variable. This analysis revealed that 66% of the variance in D percentage was accounted for by the anatomical site of drug microinjection. Thus, D percentage during hour 1 was significantly dependent on the site of drug injection (r = 0.810; F = 23.464; df = 3,37; P < 0.0001). Following control (saline) injections D percentage was not significantly related to injection locus (r = 0.496; F = 1.845; df = 3,17; P = 0.1773).

Injections into rostral and dorsal pontine sites evoke the highest D percentages Analysis of the contribution of each individual anatomical coordinate (posterior, vertical and lateral) toward the significant effect of injection site on D percentage revealed that both the posterior and vertical dimensions were the important variables (t = 5.054, P < 0.0001 and t = 5.293, P < 0.0001, respectively). The effect of the lateral coordinate, however, was not significant (t = 1.047, P < 0.3017). Fig. 5C and Table II describe the relationship between D percentage during the first hour post-injection and the posterior coordinate of the injection site, and show that injections into more rostral sites produced higher D percentages than injections into more caudal sites. Nine of the 12 drug injections (75%) that produced D percentages in the high (51-100%) range were located between 1.8 and 3.0 posterior (Table II). The Pearson product-moment correlation showed that the posterior coordinate accounted for 34% of the variance in D percentage (r = -0.5831), and that this effect was statistically significant (df = 39, P < 0.001). Fig. 5D and Table II show that dorsal injection sites yielded higher D percentages than ventral sites. Ten of the 12 drug injections (83%) that evoked D percentages in the 51-100% range were located between -5.5 and -7.0 vertical (Table II). The vertical coordinate accounted for 38% of the variance in D percentage (r = -0.6194; df = 1,39; P < 0.001). Thus, the highest D percentages were obtained following drug injections into the rostral and dorsal pontine tegmentum. D duration but not D frequency depends upon pontine injection site As shown in Table I there was no change from control in the mean D duration following microinjection of cholinergic agonists. However, some drug injection trials were characterized by a marked increase in D duration (Fig. 3A, Trials 13, 29, 32, 39) while other drug injection trials produced many short duration D sleep-like episodes (Fig. 3A, Trials 3, 7, 12). Could these observed differences in D duration be explained by examining the relationship between D duration and site of injection? Multiple regression analysis was performed to address this question using the three-dimensional coor-

253

D LATENCY ,4

B

-~00~ C

D -5O

9.0

-'~00

"

\~9 \

-~00

_

.,o.,

"

_ \(o \

_

Fig. 6. Three-dimensional representation of pontine injection sites in relationship to D latency. The distribution of the injection sites is displayed on a three-dimensional graph that represents the brainstem. The axes labelled Vertical, Posterior, and Lateral show the 3 stereotax!c planes of the brain in mm. In B - D , the point labelled "0" on the posterior and lateral axes represents stereotaxic zero. The posterior axis runs from rostral (0.0) to caudal (8.0) along the midline of the brainstem, the lateral axis runs from medial (0.0) to lateral (2.0), and the vertical axis runs from dorsal (--5.0) to ventral (--10.0). Thus, these graphs depict the left half of the brainstem with the point "0" being the most rostral, medial and ventral. A: a stereotaxically accurate perspective drawing of the cat brainstem. The portion of brainstem shown here includes the area in which microinjections were made and provides an orientation for the graphs in B - D . Note that the scale is quite different between A and B - D . In A, the point labelled "0" on the lateral axis marks the midline. The point labelled "0" on the posterior axis marks anterior-posterior zero and vertical --10.0. Each calibration tick on the axes indicates 1 mm. B: solid squares represent pontine sites from which microinjection evoked D-ACh with latencies of 0-20 min (n = 13). C: triangles indicate injection sites which produced D latencies in the 21-50 min range (n = 15). D: circles code for injection sites which yielded D latencies of greater than 51 min (n = 13).

254 dinates of the i n j e c t i o n sites as the i n d e p e n d e n t variables a n d D d u r a t i o n as the d e p e n d e n t variable. This test r e v e a l e d that the i n t r a p o n t i n e site of drug a d m i n istration a c c o u n t e d for 2 3 % of the v a r i a n c e in D du-

significant ( F = 3.745; df = 3,37; P = 0.0191). Following saline a d m i n i s t r a t i o n , there was n o significant relationship b e t w e e n D d u r a t i o n a n d the location of the i n j e c t i o n site (r = 0.329; F = 0.690; df = 3,17; P

r a t i o n (r = 0.483) a n d that this effect was statistically

= 0.5707).

D PERCENTAGE A.

B

_50 8°i5 _~004,." ~0

G

~

\~u,~

I

B

- ~ 80 ¸

-~oo*q.

"~J

o

Fig. 7. Three-dimensional representation of pontine injection sites in relationship to D percentage in hour 1. Injection sites are shown on the same graphs used in Fig. 6. A: a stereotaxically accurate perspective drawing of the cat brainstem including the area into which injections were made. B: solid squares represent pontine sites from which microinjection evoked 51-100% D-ACh during the first hour post-injection (n = 12). C: triangles indicate injection sites which produced 16-50% D-ACh (n = 10). D: circles code for injection sites which yielded 0-15% D-ACh (n = 19).

255 The significant increase in D frequency following drug injections (Table I) was found to be independent of injection site. Multiple regression analysis showed that only 2% of the variance in D frequency could be accounted for by the site of drug administration (r = 0.145); this effect was not statistically significant (F = 0.266; df = 3,37; P = 0.8498). Thus, in contrast to D latency, D percentage and D duration, the pharmacologically induced increase in D frequency was not dependent upon the site of drug administration within the pontine tegmentum. Salineinjected controls also showed no significant relationship between D frequency and injection site (r = 0.895; F = 0.895; df = 3,17; P = 0.4640).

Three-dimensional anatomical distribution of the most effective injection sites The anatomical distribution of the injection sites and their relationship to D latency and D percentage in hour 1 are shown in Figs. 6 and 7. Injection sites which produced a D sleep-like state within 0-20 min (Fig. 6B) tended to be grouped together in a narrow band in the rostral pontine tegmentum. Injections into sites surrounding the region illustrated by Fig. 6B produced D-ACh with longer latencies, ranging from 21 to 50 min (Fig. 6C), and as injections were made even farther away from the effective region of Fig. 6B, the latencies to onset of D-ACh were longer than >51 min (Fig. 6D). Similarly, injection sites from which the highest amounts of D-ACh (51-100%) were produced tended to be grouped together in the rostrodorsal aspect of the pontine tegmentum (Fig. 7B). Injection sites which yielded 16-50% D-ACh (Fig. 7C) also tended to be rostral but more ventral than the sites producing higher percentages. Finally, the sites from which microinjection of cholinergic agonists evoked the lowest amounts of D-ACh (0-15%) were located in the caudal and ventral pontine tegmentum (Fig. 7D; see also Table II, bottom). Thus, there was a region within the pontine tegmentum where microinjection of cholinergic agonists evoked D-ACh characterized by a short latency (Fig. 6B) and a high percentage (Fig. 7B). When injections were made farther away from that optimal region, the latency of D-ACh increased (Fig. 6C, D) and the percentage of time spent in D-ACh decreased (Fig. 7C, D).

The relationship between D percentage and D latency Microinjection of low doses of neostigmine into the pontine tegmentum has previously been shown to reduce D latency without increasing D percentage 4. At high dosages, microinjection of carbachol into some pontine sites increased D percentage without reducing D latency5. Given the complexity of these findings, the present study sought to examine systematically the relationship between D percentage and D latency. Fig. 8A shows that following drug injections the highest D percentages during the 4 h post-injection were significantly correlated with the shortest D latencies (r = -0.6812; df = 1,26; P < 0.001). For example, 12 of the 13 drug injection trials (92%) which induced short D latencies (0-20 min) exhibited the highest D percentages (51-100%), while only two of the 15 drug trials (13%) which produced D latencies in the medium (21-50 min) range had high D percentages. Thus, pontine sites where microinjection of a cholinergic agonist evoked D-ACh with the shortest latencies were the same sites where these drugs produced the highest percentage of D-ACh. Following saline injection (Fig. 8B) there was no significant correlation between D latency and D percentage (r = 0.0753; df = 1,14; P < 0.10).

~k B

::t A

~k

7'o ~* /,°

7016015OF

% fy:13 2 -'" L °

,o

oo

ao

,oo

oo~x

°"

,~o 40 D Latency /mm)

Fig. 8. Relationship between D latency and D percentage. D percentage during the 4-h recording period is shown on the ordinate, and D latency is shown on the abscissa. The least squares method was used to calculate the regression line. Correlations are given in the text. A: following drug administration, the relationship between D percentage and D latency was statistically significant by analysis of variance (F = 25.91; df = 1,39; P < 0.001). B: following saline injection there was no statistically significant relationship between D percentage and D latency ( F = 0.02; df = 1,19; P < 0.5).

256

Equivalency of the effects of carbachol and A Ch/N Carbachol and ACh/N were both tested in the same sites and the responses were found to be equivalent. Following ACh/N the mean D latency + S.D. was 19.8 + 19.6 min and the mean D percentage was 58.7 + 41.1% (n = 3). Following carbachol microinjection into the same sites, D latency was 19.4 + 17.4 min and D percentage was 55.2 + 32.5% (n = 4). DISCUSSION

An anatomical gradient for cholinoceptive evocation of a D sleep-like state In contrast to previous papers from this laboratory describing the localization of the cholinoceptively induced D sleep-like state to the pontine tegmentum 1'5'19A9'56, the present report focuses upon DACh evoked from a population of injection sites within the pontine tegmentum. These data documented the quantitative strength of the response to cholinomimetics (D-ACh) as measured by the speed of induction (D latency) and the amount of time spent in the D sleep-like state (D percentage). The present data show that the shortest D latencies and highest D percentages were obtained following microinjection of cholinergic agonists into the rostrodorsal pontine tegmentum. When injections were made more caudally in the pontine tegmentum, D latencies were longer and D percentages were lower. Similarly, more ventral injection sites yielded longer latencies and lower percentages of D-ACh. Three-dimensional plots of these injection sites suggest a response gradient radiating away from an optimal region in the anterodorsal pontine tegmentum from which the cholinoceptive D sleep-like state was most effectively evoked. At points increasingly distant from this optimal region, the intensity of the response (i.e. D latency, D percentage) declined progressively and then the response was reversed (i.e. D sleep was suppressed). These data indicate that while the pontine tegmentum contains an extensive field of cholinoceptive neurons whose pharmacological activation at any point within the field produces the D sleep-like state, quantitatively the response varied as a function of distance from the optimal region of this field. This response gradient could, in part, be a function of diffusion. For example, when injections were

made outside of the optimal region for eliciting DACh, diffusion of the drug into the optimal region would result in longer D latencies. The farther away an injection were made from the optimal site, the longer the latency would be. The response gradient could also be a function of the specific types of neurons that were activated by the drugs. For example, immediately surrounding the effective region for D-ACh there are pontine sites where the microinjection of carbachol or ACh/N produced D percentages that were below control levels, or sites where D sleep generation was actively suppressed. It can be speculated that this suppression of D sleep resulted from not only injecting too far from the optimal site, but also from activating neuronal systems which suppress the physiological signs of D sleep. Microinjection of carbachol into those sites which suppressed D sleep produced abnormal waking states which often included individual D sleep traits. For example, microinjection of carbachol into the caudodorsal pontine tegmentum induced continuous rapid eye movements but a total suppression of the state of D sleep 7. This injection site was located in the nucleus praepositus hypoglossi, which is known to project to the oculomotor complex 15 and which is contained in the region where microinjection evoked D-ACh with a long latency and a low percentage. The cholinergic system has been previously implicated in the generation of abnormal waking states26,53. From an injection site in the peribrachial (rostrolateral) pontine brainstem, microinjection of carbachol suppressed D sleep and evoked a syndrome typified by continuous, state-independent PGO wave clusters 6,56. This region of the rostrolateral pontine brainstem contains cells which fire in bursts with fixed latencies preceding ipsilateral PGO waves, project to the dorsal lateral geniculate bodies, and are considered to be the output elements in the PGO wave generation system 37,43,45. The injection sites where state-independent rapid eye movements 7 and PGO waves 56 could be cholinoceptively evoked were located 3-4 mm away from the optimal region for cholinoceptive evocation of the D sleep-like state. However, in other injection sites located within 1-2 mm of the optimal region described in the present report cholinergic agonists

257 have been shown to suppress D sleep while evoking motor atonia during behavioral wakefulness 12'33'54. These sites are rostral to the optimal region, just ventral to the caudal pole of the locus coeruleus and surrounding the ventral tegmental nucleus of Gudden. Based on electrophysiological and neuroanatomical studies, neurons in this region have been hypothesized to be involved in the generation of motor atonia during D sleep 44'46. Taken together, the three-dimensional localization of the effective sites for short D latencies and high D percentages shown in the present report, and the previously reported occurrence of several electrographic syndromes dependent upon site of drug administration, strongly support the concept of a cholinoceptive gradient for D sleep evocation within the pontine tegmentum. The present data reveal that the optimal region for cholinoceptive evocation of a D sleep-like state is located in the rostrodorsal pontine tegmentum. This optimal region is surrounded by an effective region located slightly more caudally and ventrally, which in turn is bordered by sites from which the state of D sleep is actively suppressed. Multiple regression analyses consistently showed that along the mediolateral plane the injection site did not exert a statistically significant effect on DACh. It is likely that this negative finding results from the fact that only a narrow range of sites along the mediolateral dimension were sampled in this study. Table III shows that the range of injection sites spanned approximately 5 mm in both the rostrocaudal and dorsoventral planes, but injection sites spanned only about 2 mm laterally from the midline. All injections were confined to the medial pontine tegmentum, and the lateral pontine tegmentum remains to be systematically examined using the microinjection technique. Therefore, the lack of a statistically significant effect of injection site on D sleep signs along the mediolateral plane can not be interpreted to mean that there is no cholinoceptive gradient for D sleep sign evocation in this dimension, nor does this negative finding imply that the lateral pontine tegmentum is not involved in the physiological generation of D sleep. Recent evidence suggests, in fact, that the dorsolateral pontine tegmentum may play an important role in the D sleep generation. Sakai44 has demonstrated the existence of cells which fire selectively

during D sleep in the dorsolateral pontine tegmentum, from approximately posterior (P) 1 to P4, lateral (L) 2 to L3, and vertical (V) -1.5 to -3, and has also documented the presence of cholinergic cell bodies in this region. Friedman and Jones 11 showed that D sleep was disrupted by thermolytic lesions localized to the lateral caudal pontine tegmentum, approximately from P3 to P6, L2 to LA, and V -2 to -7. Most recently, kainic acid lesions of cell bodies in the dorsolateral pontine tegmentum have severely disrupted D sleep 23.

Methodological issues The role of diffusion in the spacial resolution of the microinjection technique. By correlating the anatomical coordinates of the injection site with 4 dependent measures of D-ACh, this study has shown that the ability of centrally administered cholinomimetics to induce D-ACh depends, in part, upon the site of drug administration within the pontine brainstem. One of the methodological assumptions underlying such an approach is that injected pharmacological agents act primarily within the restricted brain sites into which they are administered. Thus, an important methodological question is: how far do these drugs diffuse when injected into the brainstem? Myers34, using dyes of different molecular weights, showed that in the thalamus and hypothalamus of the rat, a volume of 0.5/~1 had an average spread of 1.0 mm over 10-25 min. Routtenberg 42 used carbachol to investigate hypothalamic control of drinking behavior and found that volumes in the 0.25-0.50/~1 range produced the most consistent results with the least amount of tissue damage. Gnadt and Pegram 13 estimated the spread of carbachol in the rat pontine brainstem by measuring the diameter of a spot on an autoradiogram after microinjecting 0.1/al of a solution containing a mixture of tritiated choline and carbachol. In rats sacrificed 1 h post-injection it appeared that the diffusion distance was 1-1.5 mm. We have shown that in the cat different behavioral and electrographic syndromes can be evoked by carbachol microinjection (4/zg in 0.50/xl) into sites which are within 2-3 mm of each other 6,2°. In the present study, the mean anatomical sites producing short and medium range D latencies were separated by 1.4 mm for the vertical coordinate and 1.1 mm for the posterior coordinate (Table II). Like-

258 wise, the average anatomical sites from which high and medium range D percentages were evoked were separated by 2.0 mm in the vertical plane and 0.6 mm in the posterior plane (Table II). Thus, taken together, these data suggest that the resolving power of the chemical microinjection technique is within 2 mm when volumes no greater than 0.50/~1 are used.

The comparability of carbachol and acetylcholine plus neostigmine. One purpose of this study was to test the hypothesis that the anatomical site of drug administration within the pontine tegmentum accounted for a statistically significant amount of the variability in the response to the cholinergic drugs. Animals received injections of either carbachol or a mixture of acetyicholine and neostigmine. In order to determine that the drug effects on the dependent measures of D sleep were a function of injection site and not due to administering different pharmacological agents, it was important to show that carbachol and ACh/N, at the dosages used in this study, produced equivalent effects when injected into the same site in the same animal. While the n values are small, these data suggest that there were no differences between the effects of carbachol and ACh/N on the dependent measures of D sleep when injected into the same site in the same animal.

The cholinergic hypothesis olD sleep generation The hypothesis that cholinergic mechanisms play a role in the generation of D sleep has been considered by many investigators. Jouvet 24,25 suggested, on the basis of lesion, transection and pharmacological studies, that cholinergic and aminergic mechanisms were involved in both wakefulness and D sleep generation. Hernandez-Peon et al. 17 reported that ACh could evoke both S sleep and D sleep when applied directly to limbic forebrain and midbrain regions. Karczmar et al. 27, working in the reserpinized cat, concluded that when the activity in aminergic systems was low, stimulation of cholinergic mechanisms produced D sleep. Hobson et al. 21 and McCarley and Hobson 31 proposed a synaptic model, based on unit recording studies, which posited that the generator neurons for D sleep were cholinergic, and that oscillations in the sleep cycle resulted from an interaction between aminergic and cholinergic brainstem systems. Pompeiano et al. 39-41, in a series of electrophysiological and pharmacological studies using the

decerebrate cat, provided substantial evidence that rapid eye movements and postural atonia are generated by cholinergic mechanisms. In the present report, an optimal region for D sleep sign evocation by microinjection of cholinergic agonists has been localized to the rostrodorsal pontine tegmentum. Anatomical and electrophysiological data suggest that this effective pontine zone for pharmacological evocation of a D sleep-like state is both cholinoceptive and cholinergic. Using a light microscopic autoradiographic technique, Wamsley et al. 57 have demonstrated radiolabeled quinuclidinyl benzilate (QNB) binding in the rostral pontine tegmentum of the rat, indicating the presence of muscarinic, cholinergic receptors. Iontophoretic studies in the ketamine- and chloralose-anesthetized cat have shown that neurons in this region responded to acetylcholine by either excitation, inhibition, or a biphasic inhibitory-excitatory response 16. Also consistent with the cholinergic hypothesis was the finding of Kimura et al. 29 which demonstrated the presence of the synthetic enzyme for ACh, choline acetyltransferase (CHAT), in cells of the rostral pontine tegmentum. In the cat, discharge of these pontine tegmental neurons increased before and during physiologically occurring D sleep 18'2t. In further support of the cholinergic hypothesis of D sleep generation, brainstem regions implicated in the physiological production of all the major electrographic D sleep signs have been shown to contain cholinergic cells. For example, the magnocellular neurons of the medullary reticular formation, proposed to play a role in generating the E E G desynchrony of D sleep 5~, have been shown to stain positively for ChAT 29,44. Neurons in a region ventral to the locus coeruleus (LC), called the peri-LC alpha, have been implicated in generating the muscular atonia of D sleep 44,46, and have been found to be ChAT immunopositive 22,44. The peribrachial region of the dorsolateral, rostral pontine tegmentum has been shown to be involved in generating PGO waves 37'43, and to contain a high concentration of cholinergic neurons 22'32'44'5°. Finally, the brainstem ocular motor nuclei which generate the eye movements of waking and REM sleep also contain ChAT-positive neuronslO,29,32. Despite the pharmacological and neuroanatomical evidence supporting the hypothesis that D sleep is

259 generated, in part, by cholinergic/cholinoceptive pontine mechanisms, 3 major questions concerning the cholinergic hypothesis remain unanswered: First, how are the various putative n e u r o ~ i g e ' ~ r - : ator subpopulations for the different comp0ilefifs o L D sleep activated in a temporally coordinated w a y t o produce the constellation of events comprising" the state of D sleep? Second, what are the electrophysiological discharge profiles of immunohistochemically identified pontine cholinergic neurons across the sleep cycle? Cells which increase their firing rates prior to and during D sleep (D-on cells) have been localized to pontine regions containing cholinergic cell bodies ~, but immunohistochemicaUy identified ChAT-containing neurons have not been simultaneously demonstrated to be D-on cells. Third, what are the precise receptor mechanisms involved in cholinoceptive D sleep generation? Although some neurons in the effective zone for pharmacological evocation of a D sleep-like state have been demonstrated to be cholinoceptive, the concentration of QNB binding sites was low 57. Furthermore, atropine did not block the responses of pontine reticulospinal neurons to iontophoretically applied ACh,

REFERENCES 1 Amatruda, T., Black, D., McKenna, T., McCarley, R.W. and Hobson, J.A., Sleep cycle control and cholinergic mechanisms: differential effects of carbachol injections at pontine brainstem sites, Brain Research, 98 (1975) 501-515. 2 Baghdoyan, H.A., McCarley, R.W. and Hobson, J.A., Is there an optimal site for desynchronized sleep generation by cholinergic microstimulation of the pons?, Sleep Res., 13 (1984) 40. 3 Baghdoyan, H.A., McCarley, R.W. and Hobson, J.A., Cholinergic manipulation of brainstem reticular systems: effects on desynchronized sleep generation. In A. Wauquier, J.M. Gaillard, J.M. Monti and M. Radulovacki (Eds.), Sleep: Neurotransmitters and Neuromodulators, Raven, New York, 1985, pp. 15-27. 4 Baghdoyan, H.A., Monaco, A.P., Rodrigo-Angulo, M.L., Assens, F., McCarley, R.W. and Hobson, J.A., Microinjection of neostigmine into the pontine reticular formation of cats enhances desynchronized sleep signs, J. Pharmacol. Exp. Ther., 231 (1984) 173-180. 5 Baghdoyan, H.A., Rodrigo-Angulo, M.L., McCarley, R.W. and Hobson, J.A., Site-specific enhancement and suppression of desynchronized sleep signs following cholinergic stimulation of three brainstem regions, Brain Research, 306 (1984) 39-52.

and in some cases ACh inhibited glutamate-induced firing of these neurons 16. In addition, both increases and decreases in the firing rates of pontine tegmental neurons occurred during the carbachol-induced D : si~ep-like state48. ThtiS,~although the phenomenon of cholinoceptive D sleep sign enhancement produced by pontine microinjection of cholinomimetics is now extensively documented 1,4,5,9,12,13,19,28,33,47,49,54-56, the foregoing questions demonstrate that the cellular and molecular mechanisms by which D sleep signs are cholinoceptively evoked must continue to be investigated. ACKNOWLEDGEMENTS Supported by National Research Service Award MH 14275 (H.A.B.) and Grants M H 13923 (J.A.H.), MH 39683 and the Veterans Administration (R.W.M.). The authors wish to thank Dr. Alexander Karczmar for his critical reading of the manuscript, and Tony Monaco for help with data collection. We dedicate this paper to our histologist, Andrew Galdins, who provided the celloidin-imbedded sections. Mr. Galdins died while the manuscript was in progress.

6 Baghdoyan, H.A., Rodrigo-Angulo, M.L., McCarley, R.W. and Hobson, J.A., Cholinergic induction of desynchronized sleep by carbachol microinjection shows intrapontine site differentiation, Sleep Res., 11 (1982) 51. 7 Baghdoyan, H.A., Rodrigo-Angulo, M.L., Mitchell, K., Shea, V. and Hobson, J.A., Novel behavioral states produced by carbachol microinjection, Sleep Res., 11 (1982) 52. 8 Berman, A.L., The Brain Stem o/the Cat, The University of Wisconsin Press, Madison, 1968, 175pp. 9 Cordeau, J.P., Moreau, A., Beaulnes, A. and Laurin, C., EEG and behavioral changes following microinjections of acetylcholine and adrenaline in the brainstem of cats, Arch. ltal. Biol., 101 (1963) 30-47. 10 CueUo, A.C. and Sofroniew, M.V., The anatomy of CNS cholinergic neurons, Trends Neurosci., 7 (1984) 74-78. 11 Friedman, L. and Jones, B.E., Computer graphics analysis of sleep-wakefulness state changes after pontine lesions, Brain Res. Bull., 13 (1984) 53-68. 12 George, R., Haslett, W.L. and Jenden, D.J., A cholinergic mechanism in the brainstem reticular formation: induction of paradoxical sleep, Int. J. Neuropharmacol., 3 (1964) 541-552. 13 Gnadt, J.W. and Pegram, G.V., Cholinergic brainstem mechanisms of REM sleep in the rat, Brain Research, 384 (1986) 29-41. 14 Goodchild, A.K., Dampney, R.A.L. and Bundler, R., A

260 method for evoking physiological responses by stimulation of cell bodies, but not axons of passage, within localized regions of the central nervous system, J, Neurosci. Meth., 6 (1982) 351-363. 15 Graybiel, A.M., Organization of oculomotor pathways in the cat and rhesus monkey. In R. Baker and A. Berthoz (Eds.), Control of Gaze by Brain Stem Neurons, Developments in Neuroscience, Vol. 1, Elsevier, Amsterdam, 1977, pp. 79-88. 16 Greene, R.W. and Carpenter, D.O., Actions of neurotransmitters on pontine medial reticular formation neurons of the cat, J. Neurophysiol., 54 (1985) 520-531. 17 Hernandez-Pe0 n, R., Chavez-Ibarra, G., Morgane, P.J. and Timo-Iaria, C., Limbic cholinergic pathways involved in sleep and emotional behavior, Exp. Neurol., 8 (1963) 93-111. 18 Hobson, J.A. and Steriade, M., The neuronal basis of behavioral state control. In J.M. Briikhard, V.B. Mountcastle, and F.E. Bloom (Eds.), Handbook of Physiology, American Physiological Society, Vol. 4, 1986, pp. 701-823. 19 Hobson, J.A., Goldberg, M., Vivaldi, E. and Riew, D., Enhancement of desynchronized sleep signs after pontine microinjection of the muscarinic agonist bethanechol, Brain Research, 275 (1983) 127-136. 20 Hobson, J.A., Lydic, R. and Baghdoyan, H.A., Evolving concepts of sleep cycle generation: from brain centers to neuronal populations, Behav. Brain Sci., 9 (1986) 371-448. 21 Hobson, J.A., McCarley, R.W. and Wyzinski, P.W., Sleep cycle oscillation: reciprocal discharge by two brainstem neuronal groups, Science, 189 (1975) 55-58. 22 Jones, B.E., Paradoxical sleep and chemically identified neurons of the reticular formation. In A. Waquier, J.M. Galliard, J.M. Monti and M. Radulovacki (Eds.), Sleep: Neurotransmitters and Neuromodulators, Raven, New York, 1985, pp. 43-56. 23 Jones, B.E., The need for a new model of sleep generation, Behav. Brain Sci., 9 (1986) 409-411. 24 Jouvet, M., Recherches sur les structures nerveuses et les mecanisms responsables des differentes phases du sommeil physiologique, Arch. Ital. Biol., 100 (1962) 125-206. 25 Jouvet, M., Cholinergic mechanisms and sleep. In P.G. Waser (Ed.), Cholinergic Mechanisms, Raven, New York, 1975, pp. 455-476. 26 Karczmar, A.G., Brain acetylcholine and animal electrophysiology. In K.L. Davis and P.A. Berger (Eds.), Brain Acetylcholine and Neuropsychiatric Disease, Plenum, New York, 1979, pp. 265-310. 27 Karczrnar, A.G., Longo, V.G. and DeCarolis, A.S., A pharmacological model of paradoxical sleep: the role of cholinergic and monoamine systems, Physiol. Behav., 5 (1970) 175-182. 28 Katayama, Y., DeWitt, D.S., Becker, D.P. and Hayes, R.L., Behavioral evidence for a cholinoceptive pontine inhibitory area: descending control of spinal motor output and sensory input, Brain Research, 296 (1984) 241-262. 29 Kimura, H., McGeer, P.L., Peng, J.H. and McGeer, E.G., The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat, J. Comp. Neurol., 200 (1981) 151-201. 30 Marczynski, T.J., Topical application of drugs to subcortical brain structures and selected aspects of electrical stimulation, Ergebn. Physiol. Biol. Chem. Exp. Pharmakol., 59 (1967) 83-159. 31 McCarley, R.W. and Hobson, J.A., Neuronal excitability

over the sleep cycle: a structural and mathematical model, Science, 189 (1975) 58-60. 32 Mesulam, M.-M., Mufson, E.J., Levey, A.I. and Wainer, B.H., Atlas of cholinergic neurons in the forebrain and upper brainstem of the macaque based on monodonal choline acetyltransferase immunohistochemistry and acetylchohnesterase histochemistry, Neuroscience, 12 (1984) 669-686. 33 Mitler, M.M. and Dement, W.C., Cataleptic-like behavior in cats after micro-injections of carbachol in pontine reticular formation, Brain Research, 68 (1974) 335-343. 34 Myers, R.D., Injection of solutions into cerebral tissue: relation between volume and diffusion, Physiol. Behav., 1 (1966) 171-174. 35 Myers, R.D., Handbook of Drug and Chemical Stimulation of the Brain, Van Nostrand Reinhold Company, New York, 1974, 759 pp. 36 Nauta, W.J.H., Hippocampal projections and related neural pathways to the mid-brain in the cat, Brain, 81 (1958) 319-340. 37 Nelson, J.P., McCarley, R.W. and Hobson, J.A., REM sleep burst neurons, PGO waves, and eye movement information, J. Neurophysiol., 50 (1983) 784-797. 38 Nie, N.H., Hull, C.H., Jenkins, J.G., Steinbrenner, J. and Bent, D.H., Statistical Package for the Social Sciences (SPSS), McGraw Hill, New York, 1975, 327 pp. 39 Pompeiano, O., Cholinergic activation of reticular and vestibular mechanisms controlling posture and eye movements. In J.A. Hobson and M.A.B. Brazier (Eds.), The Reticular Formation Revisited, Raven, New York, 1980, pp. 473-512. 40 Pompeiano, O. and Hoshino, K., Tonic inhibition of dorsal pontine neurons during the postural atonia produced by an anticholinesterase in the decerebrate cat, Arch. Ital. Biol., 114 (1976) 310-340. 41 Pompeiano, O. and Valentinttzzi, M., A mathematical model for the mechanism of rapid eye movements induced by an anticholinesterase in the decerebrate cat, Arch. Ital. Biol., 114 (1976) 103-154. 42 Routtenberg, A., Intracranial chemical injection and behavior: a critical review;, Behav. Biol., 7 (1972) 601-641. 43 Saito, H., Sakai, K. and Jouvet, M., Discharge patterns of the nucleus parabrachialis lateralis neurons of the cat during sleep and waking, Brain Research, 134 (1977) 59-72. 44 Sakai, K., Neurons responsible for paradoxical sleep. In A. Waquier, J.M. Gaillard, J.M. Monti and M. Radulovacki (Eds.), Sleep: Neurotransmitters and Neuromodulators, Raven, New York, 1985, pp. 29-42. 45 Sakai, K. and Jouvet, M., Brain stem PGO-on cells projecting directly to the cat dorsal lateral geniculate nucleus, Brain Research, 194 (1980) 500-505. 46 Sakai, K., Sastre, J.-P., Kanamori, N. and Jouvet, M., State-specific neurons in the ponto-medullary reticular formation with special reference to the postural atonia during paradoxical sleep in the cat. In O. Pompeiano and C. Ajmone Marsan (Eds.), Brain Mechanisms and Perceptual Awareness, Raven, New York, 1981, pp. 405-429. 47 Shiromani, P. and McGinty, D.J., Pontine sites for cholinergic PGO waves and atonia: localization and blockade with scopolamine, Soc. Neurosci. Abstr., 9 (1983) 1203. 48 Shiromani, P. and McGinty, D.J., Pontine neuronal responses to local cholinergic infusion: relation to REM sleep, Brain Research, 386 (1986) 20-31. 49 Silberman, E.K., Vivaldi, E., Garfield, J., McCarley, R.W. and Hobson, J.A., Carbachol triggering of desyn-

261

50

51

52

53

chronized sleep phenomena: enhancement Via small volume infusions, Brain Research, 191 (1980) 215-224. Sofroniew, M.V., Priestley, J.V., Consolazione, A., Eckenstein, F. and Cuello, A.C., Cholinergie projections from the midbrain and ports to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistoehemistry, Brain Research, 329 (1985) 213-223. Stefiade, M., Sakai, K. and Jouvet, M., Bulbo-thalamie neurons related to thalamocortical activation processes during paradoxical sleep, Exp. Brain Res., 54 (1984) 463-475. Ursin, R. and Sterman, M.B., A Manual for Standardized Scoring of Sleep and Waking States in the Adult Cat, Brain Information Service/Brain Research Institute, Univ. Calif., Los Angeles, 1981, 103 pp. Vanderwolf, C.H., Neoeortical and hippocampal activation in relation to behavior: effects of atropine, eserine, phenothiazines and amphetamine, J. Comp. Physiol. Psy-

chol., 88 (1975) 300-323. 54 van Dongen, P.A.M., Locus ceruleus region: effects on behavior of eholinergic, noradrenergie and opiate drugs injected intracerebrally into freely moving eats, Exp. Neurol., 67 (1980) 52-78. 55 Velasco, M., Velasco, F., Cepeda, C. and Romo, R., Effect of a push-pull perfusion of carbaehol on the pontine reticular formation multiple unit activity. In P. Passouant and I. Oswald (Eds), Pharmacology of the States of Alertness, Pergamon, New York, 1979, pp. 231-233. 56 Vivaldi, E., MeCarley, R.W. and Hobson, J.A., Evocation of desynchronized sleep signs by chemical microstimulation of the pontine brainstem. In J.A. Hobson and M.A.B. Brazier (Eds.), The Reticular Formation Revisited, Raven, New York, 1980, pp. 513-529. 57 Wamsley, J.K., Lewis, M.S., Young III, W.S. and Kuhar, M.J., Autoradiographic localization of museafinie eholinergie receptors in rat brainstem, J. Neurosci., 1 (1981) 176-191.