THE JOURNAL OF COMPARATIVE NEUROLOGY 371~345-361 (1996)

Cholinergic and Noncholinergic Tegmental Pedunculopontine Projection Neurons in Rats Revealed by Intracellular Labeling

K. TAKAKUSAKI, T. SHIROYAMA, T. YAMAMOTO, AND S. T. KITAI Department of Anatomy and Neurobiology, The University of Tennessee,

College of Medicine, Memphis, Tennessee 38163

ABSTRACT Morphological features of rat pedunculopontine projection neurons were investigated in in

vitro preparation by using intracellular labeling with biocytin combined with choline acetyltrans- ferase (ChAT) immunohistochemistry. These neurons were classified into two types (Type I and II), based on their electrical membrane properties: Type I had low-threshold Ca2+ spikes, and Type I1 had A-current. All Type I neurons (n = 17) were ChAT immunonegative (ChAT-). Type I1 neurons were either ChAT immunopositive (CUT+; n = 49) or ChAT- (n = 20). In terms of topography in the tegmental pedunculopontine nucleus (PPN), Type I neurons were dispersed throughout the extent of the nucleus, whereas Type I1 neurons tended to be located more in the rostral and middle sections. Both Type I and I1 neurons consisted of small (long axis < 20 pm), medium (20-35 pm), and large ( > 35 pm) cells. The small cells were round or oval; medium cells were round, triangular, or fusiform; and the large cells were primarily fusiform in shape. In terms of the soma size, there was a difference in Type I (15-38 pm) and Type I1 (11-50 pm) neurons, but no significant difference was found between Type I1 ChAT+ and ChAT- cells. Both types of neurons had three to six primary dendrites, but the dendritic field was more prominent in Type I1 neurons. Most of the axons originated from one of the primary dendrites, which gave off axon collaterals, some of which projected out of the nucleus. The intrinsic collaterals were thin and branched partly within the dendritic field of the parent cell. The extrinsic collaterals were thicker and could be grouped into three categories: 1) collaterals arborizing in the substantia nigra; 2 ) collaterals ascending mainly toward the thalamus, pretectal, and tectal area; and 3) collaterals descending toward the mesencephalic and/or pontine reticular formation. It was noted that the collaterals of both C U T + and C U T - neurons were traced into the substantia nigra. There was no significant difference in antidromic latencies between Type I (m = 1.47 msec) and Type I1 (m = 1.36 msec) neurons following electrical stimulation of the substantia nigra.

Indexing terms: brainstem, biocytin, ChAT immunohistochemistry, brain slice preparation

o 1996 wiley-Liss, Inc.

The tegmental pedunculopontine nucleus (PPN) consists of large and medium-sized, loosely arranged cells that surround the superior cerebellar peduncle at the pontomes- encephalic junction. The PPN has been subdivided on the basis of cellular densities into the pars compacta (PPNc) and the pars dissipata (PPNd; Olszewski and Baxter, 1954). Cytochemical studies have demonstrated the presence of numerous cholinergic and noncholinergic neurons in the PPN as well as in the laterodorsal tegmental nuclei (LDT) in the rat (Armstrong et al., 1983; Mesulam et al., 1983; Butcher and Woolf, 1984; Spann and Grofova, 19921, cat (Kimura et al., 1981; Jones and Beaudet, 1987), monkey (Mesulam et al., 1984; Smith and Parent, 1984), and human (Mesulam et al., 1989).

Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) are considered to be major targets of PPN cholinergic neurons. Neurons were retrogradely labeled in the PPN of rats after injections of various tracers in the substantia nigra (SN; Jackson and Crossman, 1983; Woolf and Butcher, 1986; Beninato and Spencer, 1987; Clarke et

Accepted January 25,1996. K. Takakusaki is currently a t the Department of Physiology, Asahikawa

Medical College, Asahikawa 078, Japan. T. Yamamoto is currently at the Second Department of Internal Medicine,

Tohoku University School of Medicine, Sendai 980, Japan. Address reprint requests to S.T. Kitai, Department of Anatomy and

Neurobiology, The University of Tennessee, Memphis, 855 Monroe Avenue, Room 515, Memphis TN 381.63.

O 1996 WILEY-LISS. INC.

346 K. TAKAKUSAKI ET AL.

were then decapitated; the brain was removed and rinsed with cold, oxygenated Krebs solution. The brain was blocked with a razor and was sliced parasagittally (500 pm) with a tissue slicer (Vibratome series 1000; TPI). The PPN was sliced medially from the most lateral part of the initial ascending limb of the superior cerebellar peduncle to the SN. Slices were preincubated in Krebs solution for about 1 hour at room temperature before recording. The recording chamber was constructed to allow Krebs solution (36°C) to flow continuously below the slice at a rate of about 1.0 ml/minute and to allow a warm, moist gas mixture (95% 02, 5% GO2) to flow over the top surface of the slice. Composi- tion of Krebs solution was as follows (concentration in mM): NaCll24.0, KCl3.0, CaClz 2.4, NaHC03 26.0, MgS04 1.3, KH2P04 1.2, glucose 10.0.

Recording and stimulation Glass micropipettes filled with 1.0% biocytin in 0.5 M KCl

and 0.05 M Tris buffer, pH 7.0-7.4, were used for recording (Hirokawa and Armstrong, 1988). The DC resistance of the recording electrodes was between 80 and 120 MO. A high-input impedance DC amplifier with an active bridge circuit (model IR183;, Neurodata) was used to simulta- neously measure the membrane potential and inject cur- rent pulses intracellularly through the recording electrode. Biocytin was injected by using hyperpolarizing current pulses (amplitude 0.1-0.4 nA, duration 500-600 msec/ second, 10-20 minutes). The output of the amplifier was fed into a digital oscilloscope and was stored on videotape. Data were analyzed by using Axodata software.

For electrical stimulation to activate PPN neurons anti- dromically, a glass-coated carbon fiber electrode with a tip diameter of 6 pm and an impedance of 500 KO (Takakusaki et al., 1994) was placed in the midpart of the SNc. The tip of the stimulating electrode penetrated to a depth of 200-300 pm. The following stimulus parameters were used: inten- sity of 5-40 pA, duration of 0.2 msec, and frequency of 1 Hz. The criteria used to define antidromic responses were 1) a fixed latency (Fig. 6A,B), 2) the ability to follow high-frequency stimulation up to 250 Hz (Fig. 6B), and 3) collision with spontaneous action potentials (Fig. 6A).

Histology and cell reconstruction After recording and biocytin injection, the slices were

transferred to a fixative solution of 4% paraformaldehyde and 15% saturated picric acid in phosphate-buffered saline (PBS; 0.1 M), pH 7.4, and stored overnight at 4°C. After several rinses in Tris-buffered saline (TBS; 0.1 M), pH 7.5, the slices were stored overnight in 30% sucrose in PBS and were then cut into 30-pm-thick sections with a cryostat. Sections were rinsed several times in TBS (0.1 M), pH 7.5, and were then transferred to TBS containing 2% H202 (30 minutes). After another thorough rinsing, sections were incubated for 2 hours with Avidin-Texas red ( M O O ) in TBS containing 0.5% Triton X-100 (TBS-T) in order to label the biocytin-filled neurons. Sections were then examined by using an epifluorescence microscope to identify the biocytin- filled neurons conjugated with Avidin-Texas red. The sec- tions containing the biocytin-filled neurons were then processed for ChAT immunohistochemistry by using the peroxidase-antiperoxidase (PAP) method (Sternberger, 1979). After a 1 hour preincubation in 10% normal goat serum (NGS) in TBS-T, the sections were incubated for 48 hours at 4°C in monoclonal anti-ChAT antibody (150; Boehringer Mannheim) with 20% NGS and 2% bovine serum albumin in TBS-T. They were then incubated in goat

al., 1987; Scarnati et al., 1987a; Gould et al., 1989; Spann and Grofova, 1989). Lavoie and Parent (1994b) demon- strated the topographical distribution of PPN neurons in the monkey, showing that most pedunculonigral fibers originate in the PPNd, and only a few originate in the PPNc. These findings in the primate agree with those obtained in the rat (Spann and Grofova, 1989, 1991,1992) and suggest that the PPNd is related more directly to the basal ganglia than to the PPNc. Results from anterograde tract-tracing studies also support the existence of the pedunculonigral cholinergic projection, showing that pedun- culonigral fibers arborize mainly in the SNc but not in the substantia nigra pars reticulata (SNr; Saper and Loewy, 1982; Jackson and Crossman, 1983; Moon Edley and Graybiel, 1983; Gould et al., 1989; Lavoie and Parent, 199413). Furthermore, ultrastructural studies have shown that choline acetyltransferase (ChAT) immunopositive (ChAT+) fibers form asymmetrical synaptic contacts with both cell bodies and proximal dendrites of SNc neurons (Martinez-Murillo et al., 1989; Bolam et al., 1991). The postsynaptic targets of these contacts are dopaminergic cells, as revealed by tyrosine hydroxylase (TH) immunohis- tochemistry (Bolam et al., 1991).

Electrophysiological studies suggest the presence of cho- linergic and noncholinergic pedunculonigral projections in rats. Electrical stimulation of the PPN is reported to produce monosynaptic excitatory responses in SNc neurons in in vivo preparations (Scarnati et al., 1984, 1987a) and also in in vitro slice preparations (Futami et al., 1995). It has been suggested that this excitatory effect is partly mediated by glutamate (Scarnati et al., 1986). Futami et al. (1995) demonstrated in rat slice preparations that micro- stimulation of the PPN evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in DA neurons in the SNc. The EPSPs were partially suppressed by applications of antiglutamatergic agents, and the glutamatergic resistant EPSPs were further suppressed by applications of anticho- linergic agents. These results indicate the presence of excitatory cholinergic and glutamatergic projections from PPN neurons to SNc-DA neurons.

The present study was designed to demonstrate the morphological features of pedunculonigral projections from both cholinergic and noncholinergic neurons in the PPN. These neurons were also defined as Type I and I1 based on their electrical membrane properties. For this purpose, we employed intracellular recording and the biocytin labeling technique combined with ChAT immunohistochemistry in in vitro rat brain slice preparations. Before intracellular injection of biocytin, we characterized the electrophysiologi- cal phenotypes (Kang and Kitai, 1990; Takakusaki and Kitai, 1995) of PPN neurons and examined whether they were antidromically activated by microstimulation of the SNc. By using these electrophysiological and immunohisto- chemical procedures, we were able to demonstrate the trajectories of single pedunculonigral axons originating from both C U T + and ChAT immunonegative (ChAT-) PPN neurons in addition to the morphological features of these neurons. Furthermore, we have shown the relation- ship between the topography of PPN neurons and their axonal projection sites.

MATERIALS AND METHODS In vitro slice preparation

Male Sprague-Dawley rats (70-150 g) were anesthetized by using methoxyflurane (Metofane; Pitman-Moore) and

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 347

anti-rat immunoglobin (I&; 1:40) with TBS-T for 60 minutes, after which they were incubated with rat PAP (1:200) in TBS-T for 1 hour. After these two steps were repeated, the sections were reacted with diaminobenzidine (DAB; 0.05%) in TBS-T with H20z (0.002%). After each incubation step, sections were rinsed carefully several times. The reacted sections were mounted on gelatin-coated glass slides and coverslipped with 1,4-diazabicyclo[2.2.2] octane in Tris-buffered glycerol and were examined with an epifluorescence microscope to determine whether the biocy- tin-filled (Texas red-labeled) neurons were also ChAT+ (DAB labeled). To make a serial reconstruction of the recorded neurons, they were processed for nickel-DAB reaction. After the removal of the coverslips, the sections containing the biocytin-labeled neuron were rinsed in TBS five times (10 minutes each) and were incubated in the avidin-biotinylated horseradish peroxidase complex (ABC) solution ( 1 : l O O ; ABC kit, Vector) for 1 hour. The sections were then rinsed in TBS three more times (10 minutes each) and were incubated in TBS (50 ml, 0.1 M), pH 7.5, containing 10 mg DAB for 10 minutes. The sections were further incubated in this solution with an addition of nickel-ammonium sulfate (1.5 ml, 250 mM) and 0.3% HzOz (335 pl), after which they were rinsed in TBS several times, dehydrated, and coverslipped with Permount. The biocytin- labeled PPN neurons were drawn and reconstructed by using the camera lucida technique with x 100 objectives. The axonal fields of labeled neurons were also mapped at low magnification to locate their positions by using corre- sponding plates in the atlas of Paxinos and Watson (1986). Subsequently, their location was further verified with neutral red staining.

RESULTS Identification of PPN neurons

Recordings were obtained from 107 neurons (17 Type I and 90 Type 11) that had action potential amplitudes greater than 80 mV. All 107 neurons were labeled by intracellular injection of biocytin and confirmed to be within the PPNd: none was in the PPNc. Therefore, we refer to the PPNd as the PPN in this report. PPN neurons are divided into two types based on their electrophysiologi- cal membrane properties (Kang and Kitai, 1990). Type I neurons are characterized by the presence of low-threshold Ca2+ spikes (LTS; Fig. lA), and Type I1 neurons are characterized by the presence of either transient outward current (A-current; Fig. 1Ba) or A-current followed by LTS (Fig. 1Bb). Only 16 of 90 Type I1 neurons had LTS in addition to A-current.

Of 107 neurons that were intracellularly injected with biocytin, 83 neurons were further processed for ChAT immunohistochemistry. Forty-nine neurons were success- fully doubled labeled with ChAT; therefore, they were considered to be cholinergic. All ChAT+ neurons were Type 11. None of the Type I neurons were labeled for ChAT (n =

14). Examples of ChAT+ and C U T - neurons are shown in Figure 2. The biocytin-labeled neuron in Figure 2A has a fusiform shape and was identified electrophysiologically as a Type I1 neuron. This neuron was also labeled by ChAT (Fig. 2B, arrowheads) and was located in the rostral part of the PPN, surrounded by many ChAT+ neurons. Two biocytin-labeled neurons are observed in one section, as shown in Figure 2C. The neuron in the center was electro- physiologically defined as Type I and ChAT-. The other neuron (upper left) was Type I1 and C U T + (Fig. 2D).

Morphological and electrophysiological features of Type I and Type I1 neurons are summarized in Table 1.

Location and morphology of labeled PPN neurons

The locations of biocytin-labeled cells (n = 107) were plotted on either a medial or a lateral parasagittal plane of brainstem (Fig. 3Aa,Ab,Ba,Bb): both C U T + (Type 11) and ChAT- (Type I1 and Type I) neurons were located mainly within the PPN. Type I neurons were dispersed relatively equally throughout the PPN (Fig. 3Ca). On the other hand, the distribution of Type I1 neurons was skewed, with most located in the rostral and the middle areas and with their number decreasing gradually from rostral to caudal (Fig. 3Cb); 57% (24/42), 54% (29/37), and 45% (5/11) of neurons were C U T + in the rostral, middle, and caudal areas of the PPN, respectively.

Figure 4 shows camera lucida drawings of representative PPN neurons. Figure 4B,C shows Type I neurons (ChAT-), and all of the others are Type I1 neurons (ChAT+: Fig. 4A,E-I; ChAT-: Fig. 4D,J). The cells in Figure 4A,B have small somata (long axis < 20 pm) and exhibit three or four primary dendrites that extend in rostral and caudal direc- tions from the soma. Each primary dendrite is 2-3 pm in diameter and divides into two or three secondary dendrites. These small cells are usually round or oval in shape. The cells in Figure 4C-G have medium-sized somata (20 pm 5 long axis < 35 pm). They have three to five primary dendrites and are round, triangular (pyramidal), fusiform, or polygonal in shape. The cells in Figure 4 H J are large cells with a long axis of more than 35 pm and with four to six long and thick primary dendrites. Each primary den- drite is 3-5 pm in diameter and divides into two or three secondary dendrites, which taper gradually. Some of these dendrites extend more than 500 pm from the soma (Fig. 41). Irregularly spaced swellings, which resemble varicosi- ties, are present on secondary dendrites. Large cells are mostly fusiform in shape.

Type I and Type I1 neurons differ in their dendritic arborization: The dendritic arborization and the dendritic field are more prominent in Type I1 neurons, and the primary dendrites of Type I1 neurons are thicker than those of Type I neurons. In general, axons of both Type I and Type I1 neurons originate from a primary dendrite (Figs. 4A,C,FJ, 7,s).

The soma size (long and short axis) and the numbers of primary dendrites of all biocytin-labeled neurons are sum- marized in Table 1. In Type I neurons, the long and short axes range from 15 to 38 pm (mean 27.0 pm) and from 8 to 25 pm (mean 15.0 pm), respectively. In Type I1 neurons, the long and short axes range from 11 to 50 pm (mean 30.9 pm) and from 5 to 28 pm (mean 15.8 pm), respectively. There is a difference between Type I neurons and ChAT+ Type I1 neurons in the long somatic axis (P < 0.5, unpaired t test). However, no significant diffa-ences in size were observed between Type I neurons and ChAT- Type I1 neurons or between ChAT+ and ChAT- Type I1 neurons. Figure 5 shows that both Type I and Type I1 small and medium-sized neurons (long axis < 35 pm) are located throughout the PPN, but large neurons (long axis 2 35 pm) are located mostly in the rostral and middle PPN. In Type I1 neurons, 56% of small and medium-sized neurons and 58% of large neurons are ChAT+ (Fig. 5B). Figure 4

348 K. TAKAKUSAKI E T AL.

A Type I neuron 0 nA

B Type I1 neurons

a "A" type 0 nA

J-- \

100 ms -50 mV

...-------

b "A+LTS" type 0 nA

-50 mV

Fig. 1. Electrophysiological identification of the tegmental peduncu- lopontine nucleus (PPN) neurons. A: Response pattern of a Type I neuron to an injection of hyperpolarizing current pulses demonstrating low-threshold Ca2+ spikes (LTS), which is indicated by an arrow.

B: Response pattern of a Type I1 neuron to an injection of hyperpolariz- ing current pulse (a) with A-current indicated by the downward arrow. Response of a Type I1 neuron (b) with A-current (downward arrow) and LTS (upward arrow).

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 349

Fig. 2. Immunohistochernical identification of PPN neurons. A A biocytin-filled Type I1 neuron visualized hy avidin-conjugated Texas red. B: The same neuron was immunopositive to choline acetyltransfer- ase (CUT'; arrowheads). C: Two biocytin-filled neurons visualized by

Texas red. D: In the same cells as in C, the upper neuron was ChAT+ (arrowheads), whereas the other neuron (lower) was imrnunonegative to ChAT (CUT-). Scale bars = 50 pm in B (also applies to A) and D (also applies to C).

shows that PPN neurons average two to six primary dendrites, those with larger somata having more primary dendrites than those with small somata. The numbers of primary dendrites of Type I and Type I1 neurons are significantly different (P < 0.01, unpaired t test).

Antidromic responses of PPN neurons to stimulation of the SNc

Antidromic action potentials were evoked following stimu- lation of the SNc in 39 (42.9%; 5 Type I and 34 Type 11) of 91 neurons tested. In the 34 Type I1 neurons, 20 neurons were C U T + . Examples of antidromic responses are shown in Figure 6A,B. Figure 6A shows antidromically activated spikes following SNc stimulation. The latency of 1.3 msec was measured from the onset of the stimulus pulse to the

onset of the antidromic spikes. The antidromic response was blocked when a spontaneous action potential preceded the SNc stimulation by 3-5 msec (Fig. 6A). Figure 6B shows another neuron with an antidromically activated spike with a fixed latency of 2.9 msec. When a second stimulus was delivered within an interval of 4 msec, the second anti- dromic spike sometimes failed to be generated. When the second spikes were activated, a prominent inflection could be observed on the rising phase of the spike (indicated by double arrows).

Figure 6C shows the rostrocaudal distributions of PPN neurons with antidromic activation (n = 39; Fig. 6Ca) and without antidromic activation (n = 52; Fig. 6Cb). The antidromically activated neurons were located mainly in the rostra1 two-thirds of the PPN (Fig. 6Ca). However, neurons without antidromic activation were distributed

350 K. TAKAKUSAKI ET AL.

TABLE 1. Morphological Characteristics and Axonal Conduction Velocities of Tegmenta Pedunculopontine Nucleus Neurons'

Tvue I1 neurons

Measure Type I neurons All CUT+ ChAT-

Cell numbers C U T + (n) ChAT- (n) C U T Unidentified (n) Size

Long axis (pm) Short axis (pm)

Primary dendrite (n) SNc antichromic cells (n) SNc latency (ms) Conduction velocity (mls)

17 0 (0%)

14 (826) 3 (18%)

27.0 2 6.8 (15-38) 15.0 i 4.3 (8-25) 2.65 t 0.79 (2-5)

5 1.47 f 1.07 (0.63.2) 0.77 f 0.16 (0.5-0.94)

90 49 (55%) 49 20 (22%) 20 21 (23%)

32 1 f 9.4 (14-50) 15.8 f 4.2 (5-25) 3.41 ? 0.96 (2-7)

34 20 8 1.25 i 0.62 (0.4-2.6) 0.77 t 0.16 (0.41-1.04)

31 2 f 11.6 (1545)

3.15 i 0.93 (2-5)

1.36 i 0 66 (0.G3.4) 0.78 t 0.25 (0.43-1.22)

30.9 f 9.4 (11-50) 15.8 t 4.3 (5-28) 3.36 f 0.92 (2-7)

1.36 f 0.66 (0.4-3.4) 0.77 i 0 21 (0.41-1.29)

14.9 2 4.5 ( a m

'CUT, choline acetyltransferase; SNc, substantia nigra pars compacta

h Location of biocvtin-labeled neurons

I Unidentified ,

I \ \ I ' Rostral ' Middle ' Caudal ' b Location of biocytin-labeled neurons

,

Unidentified

I I I I Rostral Middle Caudal

Fig. 3. Location of biocytin-labeled neurons in the PPN. A. Sche- matic drawing (a) of a parasagittal section of medial plane. Enlarge- ment (b) of a boxed area in Aa demonstrating the location of biocytin- labeled neurons. B: Schematic drawing (a) of a parasagittal section of lateral plane. Enlargement (b) of boxed area in Ba demonstrating the location of biocytin-labeled neurons. In both medial and lateral sec- tions, Type I neurons are denoted by open (ChAT-) and solid (unidenti- fied ChAT immunoreactivity) squares. Type I1 neurons are denoted hy hatched (ChAT+), open (ChAT-), and solid (unidentified ChAT immu-

over the entire extent of the PPN (Fig. 6Cb). The latency of antidromic responses ranged from 0.4 to 3.5 msec (mean 1.38 0.71 msec; Table 1, Fig. 6D). About 80% of neurons (27137) were activated within 2.0 msec. There was no difference in the antidromic latencies of Type I (mean * S.D., 1.47 * 1.07 msec; n = 5) and Type I1 neurons (1.36 * 0.66 msec; n = 34). The latency of antidromic responses was plotted against the rostrocaudal location in the PPN (Fig. 6E), and it is apparent that the latency is longer in caudally located neurons. By using a straight-line estima-

c Rostro-caudal distribution

a

b z - a - 18 -

16

2 14

v1 1 2 -

5 10-

v - 0

8 -

6 -

4-

2 -

O J

Type I neurons (N=l7)

dImib3- I Rostral I Middle [ I

Type I1 neurons (N=90)

ChAT positive neurons (n=49)

noreactivity) circles. The PPN is divided into three areas, i.e., rostral, middle, and caudal. C: Rostrocaudal distribution of numbers of Type I (a) and Type I1 (b) neurons. Distribution of ChAT+ neurons is indicated by the hatched bars in b. Cb, cerebellum; IC, inferior colliculus; ML, medial lemniscus; NRPc, pontine reticular nucleus caudal: NRPo, pontine reticular nucleus oval; PPN, tegmental pedunculopontine nucleus; RN, red nucleus; SC, superior colliculus; SNr, substantia nigra pars reticulata; Sp, superior cerebellar peduncle; 7N, facial nucleus.

tion of the distance between the SNc stimulation sites and the location of PPN neurons, conduction velocities for the pedunculonigral axons were calculated to range between 0.41 and 1.29 mls (0.77 2 0.20; n = 39). Pedunculonigral axons were found in 30 (77%; Table 2) of 39 neurons antidromically activated by SNc stimulation. Three neu- rons were found not to have pedunculonigral axons, but ascending axons were traced into the tectal, the pretectal, and/or the thalamic areas. We could not identify the axons of the remaining six neurons.

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 351

A & + --

100 &m

\

Dorsal

4

Fig. 4. A 4 Camera lucida drawings of PPN neurons. B,C show Type I ChAT- neurons, A , D J show Type I1 neurons, A,E-I show C U T + neurons, and D,J show ChAT- neurons. Double arrows in A,C,FJ point to the axons.

Axonal destinations and terminals of PPN neurons

Axons of biocytin-labeled neurons were found in 65 of 107 PPN neurons (7 Type I and 58 Type 11). Pedunculoni- gral axons were found in 43 (4 Type I and 39 Type 11) neurons. Representative examples of axonal patterns of PPN projection neurons are illustrated in Figures 7 and 8. Figure 7A is a camera lucida drawing of a neuron in the rostra1 part of the PPN with pedunculonigral axon branches (Fig. 7B). Electrophysiologically, this neuron was identified as Type I1 and was antidromically activated by SNc stimula- tion with a latency of 0.65 msec. This is the same C U T +

neuron that is shown in Figure 2A. It had a fusiform shape with a long axis of 40 pm and a short axis of 20 pm and with four primary dendrites, and the dendritic field extended to about 500 ym in a rostrocaudal direction. The stem axon of this neuron originated from one of the primary dendrites. It gave off three axon collaterals, which projected rostrally to the SNc, and several small branches, which arborized in the retrorubral field (RRF). Two major axons reached the SNc, where labeled fibers arborized profusely in the caudal part. The stem axon was traced dorsorostrally into the thalamus.

Figure 8 is a camera lucida drawing of two biocytin- labeled PPN neurons within one slice. These neurons were

352 K. TAKAKUSAKI ET AL.

Fig. 5. Rostrocaudal distribution of Type I (A) and Type I1 (B) neurons in relation to their size. In both Type I and Type I1 neurons, small to medium cells (long axis less than 35 pm) are distributed in rostral, middle, and caudal areas. Large cells (long axis greater than 35 pm) are distributed in rostral and middle areas of the PPN. Hatched bars in B indicate ChAT+ cells.

located in the middle-caudal part of the PPN (Fig. 8B). The dorsally located neuron was C U T + (Fig. 2C), and the ventrally located neuron was ChAT- (Fig. 2D). The ChAT+ neuron had a large soma (35 x 25 pm) with five primary dendrites and two main axonal branches (ascending and descending) originating from one dendrite. The ascending axonal branch coursed through the area ventral to the inferior colliculus and was traced into the pretectal area. The pedunculonigral axon branched from the ascending axon and gave off several collaterals before entering into the SNc from the dorsal surface. The descending axon projected toward the rostral part of the pontine reticular formation (PRF).

The C U T - neuron had a polygonal shape with a large soma (38 x 20 pm), five primary dendrites, and ascending and descending axons. The axon arose from one of the primary dendrites and bifurcated into rostrally and cau- dally projecting branches. The rostrally projecting axon branch bifurcated within the PPN, and both branches projected to the SNc. One of the branches bifurcated further in the PPN and ran rostrally toward the SNc. Just outside of the PPN, one of the axons gave off a thick ascending axon collateral, which was traced into the thala- mus. The pedunculonigral axons arborized profusely within the SNc, but some arborized in the dorsal part of the SNr.

Like the C U T + neuron, this neuron had a descending axon that projected to the rostral part of the PRF. Both Type I (noncholinergic) and Type I1 (cholinergic) neurons were found to have pedunculonigral projections. Some of them also had ascending and/or descending axons, but peduncu- lonigral axons were usually thinner than the ascending and descending axons. The axons that entered the SNc from its dorsal surface tended to arborize rostrally and ventrally. However, axons that entered the SNc from its caudal end did not usually send branches ventrally (Figs. 7, 8). In addition, arborization within the SNc was more profuse in the medial part.

The axons of 65 PPN neurons were classified into three groups: 1) pedunculonigral axons, 2) axons ascending mainly toward the thalamus, pretectal-parafascicular nuclear ar- eas and tectum, and 3) axons descending toward the mesencephalic and/or PRF. PPN neurons, therefore, were divided into seven groups according to their axonal projec- tion patterns, as shown in Table 2. Thirty-eight neurons had single axonal projections, 22 had bifurcating axonal projections, and five (one Type I and four Type 11) had trifurcating axonal projections. It is interesting to note that most neurons possessed axon collaterals within the PPN and retrorubral area (Figs. 7-9). Figure 9A shows the arborization of fibers originating from C U T + neurons observed in the rostral part of the PPN. Thick fibers with varicosities (Fig. 9A, arrowheads) can be seen closely apposed to C U T + cell bodies (Fig. 9B, arrows). The varicosities have a diameter of about 1-3 km.

Forty percent (431107) of PPN neurons had pedunculonigral axons. These neurons were both C U T + (47%; 23/49; all Type 11) and ChAT- (50%; 17/34; four Type I and 13 Type 11) in the sample. It is interesting to note that PPN neurons with short peduncu- lonigral axons (n = 20) were located mainly in the rostral half of the PPN. However, neurons with ascending and/or descending axons in addition to pedunculonigral axons were located throughout the entire rostral caudal extent of the PPN. There was a connection between the location of cells within PPN and the percentage of cells with peduncu- lonigral axons. Fifty percent (25150) of pedunculonigral projection neurons were found in the rostral, 39% (17144) in the middle, and 8% (1113) in the caudal areas ofthe PPN. Figure 9C shows the biocytin-labeled fibers with varicosi- ties originating from C U T + neurons, which bifurcate and project rostrally in the caudal part of the SNc. Figure 9D demonstrates a rostroventrally running fiber from a ChAT neuron in the rostral part of the SNc. Because this fiber was very thin and was faintly labeled, varicosities or terminal boutons could not be distinctly demonstrated. A bifurcating pedunculonigral fiber originating from a ChAT- neuron is shown in Figure 9E. This fiber entered into the SNc from its caudal part, bifurcated in the SNc, and displayed varicosities.

In total, 33 neurons had ascending axons projecting toward the thalamus, pretectal-parafascicular nuclear areas, and tec- tum. Twenty-four neurons not only had ascending axons but also had axons descending toward the SNc. Only nine neurons were found to have single ascending axons. Neu- rons located in the rostral PPN had a tendency to send their axons rostrally, and those located in the caudal PPN projected axons dorsally or caudally, although they were

Pedunculonigral projection.

Ascending and descending axonal projections.

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS

- C 10-

3 5 - u VJ i

353

Typen(N=34)

A 6

D

a 5 - N=39,Mean+S.D. 1 . 3 8 ~ 0 7 1 rns

Type I1 ChAT positive

Type I1 ChATnegative & unidentified

0 0.5 1.0 1.5 2 0 2 5 3.0 3 5 4 0 4.5 Latency of antidromic response (ms)

3 4 E 3 2 e 4 rns

4 SNc stim. (35 p A ) = 1 2

6 1 B :Om"

4 4 SNc stim. (25 pA x 2) E T-I T-I1 ChATpositive (N=20) N=39.

C 0 0 ChAThegative ( N = l I ) r=0.61, p<O.OOOl = Unidentified(N=d)

3.5 7 - a Cells with antidromic response (N=39)

- 151 = TypeI(N=5) A a

v 3.0 -

2 2 . 5 - $2 2 2.0 - 3

b Cells without antidromic response ( ~ ~ 5 2 )

m 2 1 0 v

2 5 3

3 0 I I I I

Rostral Middle Caudal I I I I

Rostral Middle Caudal

Fig. 6. Antidromic responses of PPN neurons evoked by substantial nigra pars compacta (SNc) stimulation. A: Antidromic spikes (five sweeps) superimposed with a fixed latency of 1.3 msec. When spontane- ous spikes preceded, SNc stimulation failed to generate the antidromic response (collision). B: Antidromic responses induced by double-shock stimulations (interval, 4 msec) with a latency of 2.9 msec (traces superimposed five times). The second stimuli sometimes failed to generate antidromic responses. Note the prominent initial segment- somadendritic (IS-SD) break (indicated by double arrows) of antidromic response. C: Rostrocaudal distribution of PPN cells with antidromic

responses (a) and without antidromic responses (b). D. Latency histogram of antidromic response. In C and D, Type I and Type I1 neurons are shown by solid and hatched bars, respectively. E: Relation- ship between the rostrocaudal location and antidromic latency of antidromically activated PPN neurons. Type I neurons are denoted by open (ChAT-) and solid (unidentified ChAT immunoreactivity) squares. Type I1 neurons are denoted by hatched (CUT+) , open (ChAT-), and solid (unidentified ChAT immunoreactivity) circles. Upward arrows in A and B indicate stimulation onset.

TABLE 2. Axonal Destinations of Tegmenta Pedunculopontine Nucleus Neurons'

PPN neuron and projected toward the pretectal area. Within the PPN, the main axon gave off a descending collateral (Fig. lOA,C), which ramified (not shown) and terminated within the PPN. Figure 10D shows an axon from another C U T + Type I1 neuron that ascended dorso- caudally towards the caudal part of the superior colliculus. Figure 10E demonstrates a ChAT- PPN Type I1 neuron that projected toward the superior colliculus (rostral por- tion).

Descending axons were found in 20 (three Type I and 17 Type 11) neurons (Table 2). Only eight (two Type I and six Type 11) neurons had single descending axons. Twelve neurons had ascending axons and/or axons toward the SNc in addition to descending axons as shown in Figure 7. Figure 10F shows a C U T + neuron that projected ventrally to the rostral part of the PRF and ramified there. The higher magnification in Figure 10G shows that one of the collaterals in the PRF had varicosity that apposed a cell body. Figure 10H shows an axon forming varicosities in the rostral part of the PRF. This axon originated from a ChAT- Type I neuron in the PPN.

SNc antidromic

(%I 14 (82.4) 12 (92.3)

1 (33.3)

3 (75)

3 (33.3)

0 (01

0 (0) 33 (60.0) 30 (81.1)

SNe stimulated

Type I Neurons (all Ch-)

SNc 2 Ascendingand SNc 1 SN and descending 0 Ascending, SNc,

and descending 1 Ascending (Th, SC,

etc.1 1 Descending (MRF,

PRF) 2 Ascending and

descending 0 Total 7 Total SNc 4

Type I1 (Ch+:Ch-)

18 (11:7)

Type1 + Type I1

20 17 14 (92) 3 (1:l)

15 3

13 3

4 (2:O)

8 (5:2)

6 (3:Ol

4 (2:l) 57 (33.13) 39 (23:lO)

3 55 37

'Ch-, choline acetyltransferase (ChAT) negative; Chi , ChAT positive; SNC, substantia nigra pars compacta; Th, thalamus; SC, superior colliculus; MRF, mesencephalic reticular formation; PRP, pontine reticular formation.

exceptions. Figure 10 demonstrates the axonal projection patterns obtained from five PPN neurons. Figure 10A,B shows an ascending axon that originated from a C U T +

354

A K. TAKAKUSAKI ET AL.

Dorsal

Fig. 7. Camera lucida drawing of a C U T + PPN neuron. A. A reconstructed PPN neuron from a series of eight parasagittal sections. B: Schematic drawing of a parasagittal section indicating the location of the neuron in PPN. ML, medial lemniscus; PPN, tegmental pedunculopontine nucleus; PRF, positive reticular formation; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.

DISCUSSION Cholinergic and noncholinergic PPN neurons

Intracellular recordings were obtained from 107 neurons (17 Type I and 90 Type 11) that had spike heights of more than 80 mV. One hundred and seven neurons were intracel- lularly labeled with biocytin and located within the PPN. Some of these neurons were double labeled with ChAT immunohistochemistry. The general distribution pattern of ChAT+ neurons in the PPN identified in this study agrees with previous reports of the data obtained from various species, including the rat (Armstrong et al., 1983; Mesulam et al., 1983; Sofroniew et al., 1985; Beninato and Spencer, 1986; Woolf and Butcher, 1986; Rye et al., 1987; Goldsmith and van der Kooy, 1988; Gould et al., 1989; Spann and Grofova, 1992), cat (Mitani et al., 1988; Steriade et al., 1988; Hall et al., 1989), monkey (Mesulam et al., 1984; Smith and Parent, 1984; Satoh and Fiberger, 1985; Steriade et al., 1988; Lavoie and Parent, 1994c), and human (Mizukawa et al., 1986; Mesulam et al., 1989). Most of the cholinergic neurons we identified were located around the ascending limb of the superior cerebellar peduncle at the mesencephalic junction and also rostroventrally toward the SN, but very few were located in the RRF. It has been reported that a few cholinergic neurons were found in the RRF and even in the SN (Gould and Butcher, 1986; Martinez-Murillo et al., 1989; Spann and Grofova, 1992). These C U T + neurons were intermingled with ChAT- (noncholinergic) neurons in the PPN. These findings do not

support the idea that the PPN consists only of cholinergic neurons, as had been suggested by some investigators (Rye et al., 1987; Lee et al., 1988).

It was found that all C U T + neurons were Type I1 neurons, as defined electrophysiologically (Kang and Kitai, 1990; Takakusaki and Kitai, 1995). The morphological features of ChAT neurons are consistent with previous observations in the rat and other species (Sugimoto et al., 1984; Isaacson and Tanaka, 1986; Rye et al., 1987; Mesu- lam et al., 1989; Kang and Kitai, 1990; Spann and Grofova, 1992). In this study, about 30% (20/69) of Type I1 neurons were ChAT-. ChAT+ and ChAT- Type I1 neurons do not differ in their morphological characteristics (Table 1) or in their electrophysiological membrane characteristics (Taka- kusaki and Kitai, 1995). However, ChAT+ Type I1 neurons were larger than those of Type I, which, without exception, were ChAT-. These results suggest that there are two distinct subpopulations of noncholinergic neurons (i.e., noncholinergic Type I1 neurons vs. noncholinergic Type I neurons), which agrees with a previous description of two morphologically distinct subpopulations of noncholinergic PPN neurons, one with small cell bodies and the other with large cell bodies (Spann and Grofova, 1992).

It has been demonstrated in rats that numerous glutama- tergic (Clements and Grant, 1990; Clements et al., 19911, GABAergic (Ottersen and Storm-Mathisen, 1984; Kosaka et al., 1988), and peptidergic (e.g., substance P; Vincent et al., 1983; Standaert et al., 1986) neurons are found in the

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 355

S N r

Fig. 8. Camera lucida drawing of two PPN neurons. A Reconstruc- tion of C U T + (upper) and CUT- (lower) neurons made from a series of 12 parasagittal sections. B Schematic drawing of a parasagittal section indicating the location of two neurons in PPN. IC, inferior

mesopontine tegmentum, including the PPN and the LDT. Lavoie and Parent (1994a) found that about 40% of monkey PPN cells express combined cholinergic and glutamatergic immunoreactivity with the other neurons expressing either glutamate or ChAT immunoreactivity alone. They also demonstrated that the majority of glutamate-positive neu- rons are of a medium size, although some are large. These morphological studies, combined with our findings, would indicate that Type I neurons are likely to be glutamatergic.

Type I and Type I1 neurons possess different numbers of primary dendrites, and the dendritic arborizations and dendritic fields of Type I1 neurons are also more prominent, as illustrated in Figure 4. The difference in the number of primary dendrites may be due to the difference in cell size between Type I and Type I1 neurons, because the number of primary dendrites might be expected to increase in propor- tion to cell size. It is also possible that the prominent dendritic arborization of Type I1 neurons relates to the existence of high-threshold Ca2+ conductances. It has been suggested that the conductance for generating LTS predomi- nates in the cell body, whereas the conductance for generat- ing high-threshold Ca2+ spikes (HTS) is mainly in the dendrites (Llinas, 1988). We recently found that there are two types of high-threshold Ca2+ conductances in Type I1 neurons but not in Type I neurons. One is a dihydropyridin-

colliculus; ML, medial lemniscus; PPN, tegmental pedunculopontine nucleus; PRF, pontine reticular formation; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.

sensitive Ca-dependent oscillatory conductance and the other is an o-conotoxin sensitive HTS (Takakusaki and Kitai, 1995).

Antidromic responses of PPN neurons We observed 39 PPN neurons that were antidromically

activated by SNc stimulation with latencies from 0.4 to 3.4 msec and with conduction velocities in the range of 0.41- 1.29 m/s. This range of conduction velocities is quite similar to the conduction velocity of pedunculonigral neu- rons (0.45 and 1.1 m/s) reported in in vivo preparations by Scarnati et al. (1987b) and by Granata and Kitai (1991). The slow conduction velocity of pedunculonigral axons is probably because they are unmyelinated and have a rela- tively small diameter of about 0.25 pm (Beninato and Spencer, 1988).

In 30 of the antidromically activated neurons, we were able to identify pedunculonigral axons. In three of the nine neurons that were activated antidromically but for which we could not identify nigropetal axons, we were able to trace the axons into the tectum and/or the thalamus. The responses of these three neurons were judged to be anti- dromic, because the activation was of a fixed latency and followed high-frequency stimulation (250-200 Hz). How-

Figure 9

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 357

ever, these three neurons were located at the rostral end of the PPN; therefore, they might have been inadvertentlyac- tivated directly by nigral stimulation. The dendrites of these neurons extended into the dorsal part of the SN, as has previously been reported by Martinez-Murillo et al. (1989). On the other hand, our morphological analysis of intracellularly labeled PPN neurons demonstrated nigrop- eta1 axons in seven PPN neurons that failed to respond to antidromic stimulation. Three of these seven neurons were located adjacent to the SN, and four were located in the caudal PPN. It is possible that direct (dendritic) activation of PPN neurons might have blocked antidromic invasion of the rostrally located neurons. It is perhaps probable that the microstimulation (less than 40 PA) employed in this study failed to activate the axons of all of the PPN neurons recorded, because some of these axons were very thin and unmyelinated; therefore, they might be expected to have a higher threshold for activation.

Topographical arrangements of PPN neurons Of 107 PPN neurons studied, 40% had pedunculonigral

axons. Fifty-nine percent of pedunculonigral neurons were ChAT-, and 41% were CUT+. These results are consistent with those of retrograde labeling studies of pedunculonigral neurons in the rat, where as much as 40% of the PPN cholinergic neurons could be retrogradely labeled (Woolf and Butcher, 1986; Gould et al., 1989). Even though our sampling method was biased by the inherent limitation in the intracellular recording technique, our results indicate a topographical arrangement of pedunculonigral neurons in the PPN. We have made a conscious effort to record evenly throughout the rostrocaudal extent of the PPN. It was found that most pedunculonigral neurons were located in the rostral and middle one-thirds of the PPN; those neu- rons having only pedunculonigral axons (n = 20; Fig. 8B) were located in the rostral half. Antidromic responses were observed from 51%, 40%, and 20% of neurons recorded in the rostral, middle, and caudal one-thirds of the PPN, respectively. Pedunculonigral axons were found in 58%, 30%, and 8% of neurons located in each of these respective areas. These findings fit well with the previous study on the cat, indicating that cholinergic neurons were located mainly in the rostral two-thirds of the PPN, and they discharge tonically with virtually no burst firing (Steriade et al., 1990a). Lavoie and Parent (1994~) also observed a similar distribution of C U T + and ChAT- retrogradely labeled pedunculonigral neurons in the squirrel monkey. That is, cholinergic neurons were found to make up about 35%, 25%, and 15% of neurons in the rostral, middle, and caudal one-thirds of the PPN, respectively, whereas the number of ChAT- retrogradely labeled neurons increased toward the caudal extent of the PPN. These findings, however, do not

Fig. 9. Photomicrographs of PPN axons. A Axonal arborization in PPN originating from a C U T + neuron. Varicosities are indicated by arrowheads. B: Enlargement of a boxed area in A demonstrating a fiber with varicosities closely opposed to a C U T + cell body (indicated by arrowheads). C: Axons in the caudal part of SNc that originated from a ChAT+ PPN neuron. D: An axon (indicated by arrows) in the rostral part of SNc that originated from a ChAT+ PPN neuron. E: A bifurcating axon in the middle part of SNc that originated from a ChAT- Type I PPN neuron. F A higher magnification showing varicosity-like structures (indicated by arrowheads) of an axon in the boxed area in E. a, Anterior; d, dorsal. Scale bars = 50 pm in A,C,E, 20 pm in B,D,F.

agree with those of Rye et al. (19871, which indicated that projection to the endopeduncular -nucleus and the SN originated almost entirely from noncholinergic neurons in the midbrain extrapyramidal area (MEA).

A topographical arrangement of PPN neurons with as- cending and/or descending axons has also been observed previously. It has been reported that there are massive ascending pedunculothalamic arborizations, which are most profuse in the interlaminar and midline nuclei (Hallanger and Wainer, 1988; Pare et al., 1988; Steriade et al., 1988; Lavoie and Parent, 1994b). Projections to lateral geniculate (Leger et al., 19751, superior colliculus (Woolf and Butcher, 1986), pretectal, and parafascicular nuclear areas (Gray- biel, 1977) have also been reported. Descending axons originating from the PPN have also been demonstrated to project to the pontine and medullary reticular formation (Mitani et al., 1988; Rye et al., 1987; Semba et al., 1990; Semba and Fibiger, 1992). In this study, ascending axons were found in 33 PPN (19 ChAT+) neurons, with axons directed toward the superior colliculus, pretectal area, and parafascicular nuclear area. Descending axons toward the PRF were also found in 17 (eight ChAT') neurons. Neu- rons located in the rostral PPN tended to have ascending axons, and those in the caudal PPN tended to have descend- ing axons.

Pedunculonigral projection It has been demonstrated previously that the PPN is

interconnected with the basal ganglia structures, such as the globus pallidus, the entopeduncular nucleus, the subtha- lamic nuclei, and the SNr as well as the SNc (Nauta and Mehler, 1966; Jackson and Crossman, 1983; Woolf and Butcher, 1986; Semba and Fibiger, 1992; Bolam and Bevan, 1994; Lavoie and Parent, 1994b). The present results demonstrate that pedunculonigral fibers arising from both C U T + and ChAT- neurons arborize profusely within the SNc. It was also found that each pedunculonigral projection cell has at least two or more distinct nigropetal axons (see, e.g., Figs. 7,8), and a great number of axons from different pedunculonigral cells converge in the same area. These results indicate a divergence of information from a single PPN neuron to the SNc and a convergence of information from many PPN neurons to a single SN neuron. Further- more, axons entering the SNc from its dorsal or dorsocau- dal part tended to arborize rostrally or ventrally, as has been observed in the monkey (Lavoie and Parent 1994b).

There are numerous data to support the notion that acetylcholine acts as a neurotransmitter in the pedunculoni- gral projection. Terminal boutons displaying ChAT immu- noreactivity and forming asymmetric contacts with SNc neurons have been observed in both ferrets and rats (Gould and Butcher, 1986; Scarnati et al., 1988; Tokuno et al., 1988; Martinez-Murillo et al., 1989; Bolam et al., 1991). Futami et al. (1995) demonstrated in in vitro rat slice preparations that PPN stimulation evoked monosynaptic EPSPs in the dopaminergic neurons in the SNc. EPSPs were partially suppressed by applications of antiglutamater- gic agents (kynurenic acid and/or CNQX), and the glutama- tergic-resistant EPSPs were further suppressed by applica- tions of anticholinergic agents (atropine, mecamylamine, and pirenzepine). These results suggest that the PPN exerts an excitatory influence on dopaminergic neurons in the SNc that is mediated by both glutamatergic and cholin- ergic transmitters.

TEGMENTAL PEDUNCULOPONTINE PROJECTION NEURONS 359

Our intracellular single-cell labeling technique combined with immunocytochemical analysis clearly demonstrated that both cholinergic and noncholinergic PPN neurons have ascending axons, descending axons, nigropetal axons, or any combination of these. The existence of a dual projection (ascending thalamopetal and descending pontore- ticulopetal axons) was described by Semba et al. (19901, and there have also been reports of dual projections from a single PPN cholinergic neuron to the basal ganglia and the thalamus (Woolf and Butcher, 1986; Scarnati et al., 1987a). However, the present study is the first to demonstrate a triadic projection from a single PPN neuron with ascending (thalamus, tectal area), descending (brainstem), and nigrop- eta1 axons.

Functional implications of the PPN projection system

The PPN cholinergic neurons have been shown to project to the thalamic areas, mainly to the “nonspecific” thalamic nuclei (Woolf and Butcher, 1986; Hallanger and Wainer, 1988; Steriade et al., 1988; Semba and Fibiger, 1992; Lavoie and Parent, 1994b) and to the pontine and medullary reticular formations (Jackson and Crossman, 1983; Rye et al., 1987; Mitani et al., 1988; Woolf and Butcher, 1989; Semba, 1993). It has been suggested that PPN cholinergic neurons are involved in the modulation of thalamocortical neurons to induce electroencephalography (EEG) desynchro- nization (El Mansari et al., 1989; Steriade et al., 1991) and the pontogeniculooccipital (PGO) wave (McCarley et al., 1978; Sakai and Jouvet, 1980; Steriade et al., 1990b) through their ascending projections. On the other hand, the descending projection of the PPN to the brainstem is thought to be involved in the initiation of postural atonia, similar to that observed during rapid eye movement (REM) sleep in both conscious (Amatruda et al., 1975; Hobson et al., 1983; Yamamoto et al., 1990) and decerebrate (Taka- kusaki et al., 1993; 1994) cats and in the generation of rhythmic activities, such as locomotion, respiration, and mastication as well as the sleepiwaking cycle (Garcia-Rill and Skinner, 1988).

Lavoie and Parent (199413) have recently hypothesized a “dual function” for the ascending projections from the PPN. Massive projections to the nonspecific thalamic nuclei allow the PPN to exert prominent modulatory effects on general brain functions, such as arousal, consciousness, and the sleep/waking cycle. On the other hand, the projec- tions of the PPN to basal ganglia structures appear to play a role in more specific subcortical functions, such as the

Fig. 10. Photomicrographs of PPN axons. A: An ascending axon (indicated by arrowheads) in the pretectal area that originated from a ChAT+ neuron. A descending axon collateral (indicated by black and white arrowheads) is given off from the stem axon in the PPN. B A larger magnification of the ascending axon in a n enclosed area. C: A higher magnification of the descending axon collateral (indicated by an arrow) in the enclosed area in A. D: A caudally projecting ascending axon (indicated by arrowheads) originating from a ChAT+ Type I1 neuron. E: Dorsally projecting ascending axon (indicated by arrow) originating from a ChAT- Type I1 neuron. F: A ChAT+ neuron with a n axon descending to the rostral part of the pontine reticular formation. G A higher magnification of the enclosed area in F shows terminal bouton-like structures (indicated by arrowheads) closely opposed to the soma of a pontine reticular cell. H: A fiber with varicosities (indicated by arrowheads) originating from a ChAT- Type I neuron in the rostral part of the pontine reticular formation. Scale bars = 100 pm in A,D, 50 pm in B,C,F, 200 pm in E, 20 pm in G,H.

control of motor behavior. Jackson and Crossman (1983) hypothesized that the PPN may represent a direct link between the basal ganglia and lower (brainstem and spinal cord) motor systems. In our electrophysiological study of the rhythmic firing of PPN neurons, we found that Type I1 neurons (both cholinergic and noncholinergic) have intrin- sic Na- and Ca-dependent oscillatory conductances com- pared to Type I neurons (noncholinergic), which lack Na- or Ca-dependent conductances (Takakusaki and Kitai, 1996). These findings suggest that Type I1 neurons possess intrin- sic membrane conductances that could lead to a more stable firing pattern than Type I neurons and, thus, to more rhythmic and sustained firing. This would then lead to a sustained control of tonic activity, such as waking and REM sleep.

Semba et al. (1990) postulated that the dual projections of single PPN cholinergic neurons are significant in the initiation of REM sleep via the descending projection system and also in the blockade of rhythmic thalamocorti- cal activity and the modulation of sensory transmission via the ascending projection system. We now have clear evi- dence that both cholinergic and noncholinergic PPN neu- rons can simultaneously influence basal ganglia, thalamic, tectal, and brainstem activities. Taken together with our studies on the ionic mechanisms for the generation of spontaneous firing in PPN neurons (Kang and Kitai, 1990; Takakusaki and Kitai, 1995), we postulate that the PPN may operate as an integrative interface between higher and lower brain centers in order to execute sustained and appropriate motor actions by regulating the level of state- dependent behavior.

ACKNOWLEDGMENTS We thank Dr. Y. Kuga for his help in the immunohisto-

chemical identification of intracellularly labeled neurons and Drs. C. Richards and C. Wilson for their critical review of this paper. Special thanks to Ms. B. Doggett for her assistance in the preparation of this paper. This study was supported by USPHS grant NS20702 and by the Human Frontier Science Program.

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