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Serotonergic dorsal raphe nucleus projections to the cholinergic and noncholinergic neurons of the pedunculopontine tegmental region a light and electron microscopic anterograde tracing and immunohistochemical study

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THE JOURNAL OF COMPARATIVE NEUROLOGY 382:302–322 (1997)
Serotonergic Dorsal Raphe Nucleus
Projections to the Cholinergic and
Noncholinergic Neurons of the
Pedunculopontine Tegmental Region:
A Light and Electron Microscopic
Anterograde Tracing and
Immunohistochemical Study
TERESA L. STEININGER,1 BRUCE H. WAINER,4,5 RANDY D. BLAKELY,2
AND DAVID B. RYE3,4,6*
1Committee on Neurobiology, The University of Chicago, Chicago, Illinois 60637
2Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232
3Neuroscience Program, Emory University, Atlanta, Georgia 30322
4Department of Neurology, Emory University, Atlanta, Georgia 30322
5Department of Pathology, Emory University, Atlanta, Georgia 30322
ABSTRACT
The serotonergic dorsal raphe nucleus is considered an important modulator of statedependent neural activity via projections to cholinergic neurons of the pedunculopontine
tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections
from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry
at the light and electron microscopic levels, to determine whether PPT neurons receive
serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled
and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light
microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars
compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3)
approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites;
and (4) at least 2–4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal
raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate
cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital
waves that precede and accompany REM sleep is discussed. J. Comp. Neurol. 382:302–322,
1997. r 1997 Wiley-Liss, Inc.
Indexing terms: serotonin transporter; brainstem; biotinylated dextran; double
immunohistochemistry; REM sleep
The pedunculopontine tegmental nucleus (PPT), situated in the dorsolateral mesopontine tegmentum, together
with the caudally adjacent laterodorsal tegmental nucleus
(LDT), contain a prominent group of cholinergic neurons
which project widely throughout the brainstem and forebrain (De Lima and Singer, 1987; Hallanger et al., 1987;
Hallanger and Wainer, 1988; Rye et al., 1988; Woolf and
r 1997 WILEY-LISS, INC.
Grant sponsor: National Institutes of Health; Grant number: NS-1766114; Grant number: DA-07390.
*Correspondence to: Dr. David B. Rye, Department of Neurology, Emory
University, Suite 6000 WMRB, Atlanta, GA 30322. Email: dbr@neuro.emory.edu
Received 23 July 1996; Revised 16 January 1997; Accepted 22 January
1997
DORSAL RAPHE AFFERENTS TO THE PPT
Butcher, 1989; Woolf et al., 1990). Innervation of thalamicspecific relay, nonspecific and reticular nuclei by these
cholinergic cells (De Lima and Singer, 1987; Hallanger et
al., 1987; Hallanger and Wainer, 1988), are viewed as
important substrates in the induction and maintenance of
prolonged states of electroencephalograph (EEG) desynchronization, such as those accompanying wakefulness
and rapid eye movement (REM) sleep (McCormick, 1989;
Steriade et al., 1990a, 1993; Steriade, 1992). Converging
lines of evidence also implicate the PPT in the transfer of
phasic ponto-geniculo-occipital (PGO) waves in the thalamus that appear prior to and during REM sleep (McCarley
et al., 1978; Hu et al., 1989; Steriade and McCarley, 1990;
Steriade et al., 1990b; Steriade, 1992). Elucidation of the
neural and pharmacological substrates influencing cholinergic PPT neurons has therefore garnered particular attention given their potential importance in governing states of
EEG desynchronization and the evocation of PGO spikes.
In addition to representing a sine qua non for the occurrence of REM sleep, PGO waves have been proposed as the
neural correlate underlying dream-like imagery (Steriade
and McCarley, 1990; Steriade et al., 1990b; Steriade,
1992), hallucinations (Kitsikis and Steriade, 1981; Steriade and McCarley, 1990) and orienting responses (Bowker
and Morrison, 1976). Hyperpolarization of tonically active
PPT/LDT neurons, and thereby inhibition of cortical
‘‘arousal,’’ is felt to be mediated by serotonin, gammaaminobutyric acid (GABA), acetylcholine (Muhlethaler et
al., 1990; Steriade and McCarley, 1990; Jones, 1991;
Steriade, 1992; Leonard and Llinas, 1994), adenosine
(Rainnie et al., 1994), and norepinephrine (Williams and
Reiner, 1993), while hyperpolarization of a separate population of neurons displaying low-threshold calcium spikes
contributes to a burst firing mode and PGO waves. Histamine (Muhlethaler et al., 1990) and glutamate (Sanchez et
al., 1991) are proposed excitatory transmitters in the
PPT/LDT. Very little is known, however, concerning the
origin of these transmitters and the specific cell types of
the PPT region with which they are in synaptic contact
Abbreviations
AQ, Aq
cll
Cnf
cp
CTF
DR
DTg
IP
LC
LDT
ll
MEA
mes5
ml
mlf
PAG
PB
PL
PPT
PTF
RRF
scp
SN
xscp
3n
4n
4N
4V
cerebral aqueduct
commissure of the lateral lemniscus
cuneiform nulceus
cerebral peduncle
central tegmental field
dorsal raphe
dorsal tegmental nucleus
interpeduncular nucleus
locus coeruleus
laterodorsal tegmental nucleus
lateral lemniscus
midbrain extrapyramidal area
mesencephalic tract of the trigeminal nerve
medial lemniscus
medial longitudinal fasciculus
periaqueductal gray matter
parabrachial nucleus
paralemniscal nucleus
pedunculopontine tegmental nucleus
pontine tegmental field
retrorubral field
superior cerebellar peduncle
substantia nigra
decussation of the superior cerebellar peduncle
oculomotor nerve
trochlear nerve
trochlear nucleus
fourth ventricle
303
(e.g., cholinergic versus noncholinergic/tonically active versus bursters).
The spectrum of putative afferents to the mesopontine
tegmentum region, containing PPT cholinergic neurons, is
diverse. Comingling of cholinergic cells and smaller noncholinergic cells, and their close apposition to several other
functionally distinct nuclei and fiber tracts has precluded
a precise determination of which afferents actually terminate in the region, and of these, which terminate on
cholinergic elements. Putative serotonergic afferents from
the dorsal raphe nucleus (DR) to cholinergic PPT/LDT
neurons have been proposed as one of the important
neural substrates governing both the normal and pathological expression of REM sleep and PGO waves (Steriade and
McCarley, 1990). The synaptic organization of dorsal raphe
and/or serotonergic pathways to the PPT/LDT is nevertheless
poorly described. Anatomical tracer injections into the mesopontine tegmentum, containing the PPT, retrogradely label
DR neurons (Semba and Fibiger, 1992; Steininger et al.,
1992), but fail to discriminate as to which specific cells
receive DR afferents. Injections of tritiated amino acids
(Bobillier et al., 1975; Pierce et al., 1976; Moore et al.,
1978; Conrad et al., 1991) and Phaseolus vulgaris leucoagglutinin (PHA-L; Vertes, 1991; Vertes and Kocsis, 1994)
into the DR delineate efferents which putatively innervate
the PPT. Whether these efferents are actually serotonergic
is unclear because only 40–60% of the total population of
DR neurons employ serotonin as a neurotransmitter
(Moore, 1981; Descarries et al., 1982). Moreover, descriptions of mesopontine tegmental projections from the DR
generally lack detail because the cholinergic identity of
PPT neurons was not examined. Synapses between serotonergic terminals and cholinergic elements have been described in the rat PPT and LDT, but the majority contact
noncholinergic dendrites (Honda and Semba, 1994). Because these findings were not quantified, it is unclear as to:
(1) the relative distribution of serotonergic efferents between various cholinergic and noncholinergic elements; (2)
what extent serotonergic terminals contribute to the total
synaptic innervation of cholinergic PPT neurons; and (3)
whether these serotonergic efferents arise from the DR.
In the present study, the anterograde transport of
biotinylated dextran from the DR was therefore combined
with immunocytochemistry for choline acetyltransferase
(ChAT), the synthetic enzyme for acetylcholine, to provide
a detailed description of projections to the cholinergic PPT
and LDT nuclei at the light microscopic level. At the
electron microscopic level, the synaptic connectivity of anterogradely labeled terminals in the pars compacta of the PPT at
the electron microscpic level was established and quantified in
detail. To investigate the putative serotonergic nature of
these projections, immunohistochemistry for the serotonin
transporter protein (SERT; Qian et al., 1995) was utilized
as a marker for serotonergic axons and terminals, in
combination with ChAT immunohistochemistry, and investigated in a like manner at both light and electron microscopic levels in a separate set of experiments.
MATERIALS AND METHODS
Injection of anterograde tracer
Male Sprague-Dawley rats (250–350 g, Harlan) were
anesthetized with chloral hydrate (400 mg/kg, i.p.) and
placed in a stereotaxic apparatus. Stereotaxic injections of
10% biotinylated dextran amine (10,000 MW, lysine fix-
304
T.L. STEININGER ET AL.
able; Molecular Probes, Eugene, OR) were made into the
dorsal raphe nucleus using a micropipette at a lateral
approach of 20°. The tracer was ejected iontophoretically
with a positive current of 5 µA, at a 7 seconds on/7 seconds
off cycle, for 30 minutes. After a survival period of 7–10
days, the animals were perfused and the brains sectioned
as described below. All animal care and procedures employed as part of these studies were approved by the
institutional animal care and use committee (IACUC).
Perfusion/fixation
Animals were deeply anesthetized with chloral hydrate
and given an injection of sodium heparin (600 U i.p.) 15
minutes prior to the procedure. The animals were perfused
transcaridally with fixative consisting of 300 ml of 3%
paraformaldehyde and 0.1–0.2% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4 (PB), over a period of 15 minutes.
The brains were postfixed in situ for 1 hour before removal
from the skull. Brains were then dissected into blocks
containing the injection site and the PPT, and sectioned at
40–50 µm on a vibrating microtome in PB. Tissue sections
were collected into staining dishes and rinsed several
times in 0.05 M Tris-buffered saline pH 7.6 (TBS).
containing 0.025% sodium nitroprusside and 0.005% hydrogen peroxide. The reaction was allowed to proceed for
15–30 minutes and was stopped by several rinses in 0.01
M PB pH 6.6. Following the biotinylated dextran (BD)/
ChAT visualization procedures, sections were processed
for light and electron microscopy as described below.
Control sections, in which the primary antiserum was
omitted, were processed in the same manner. The utility of
these chromogen combinations at the light and electron
microscopic level has been previously demonstrated by
ourselves (Bolam et al., 1986; Levey et al., 1986; Rye et al.,
1987) and others (Zaborszky and Heimer, 1989; Bolam et
al., 1991; Lapper and Bolam, 1992; Corvaja et al., 1993;
Zaborszky et al., 1993).
SERT and serotonin (5-HT)
immunohistochemistry
The injected and transported biotinylated dextran was
visualized by incubation of the sections in avidin-biotinperoxidase complex (ABC, Vectastain Elite kit, Vector
Labs, Burlingame, CA) in TBS for 24–48 hours and rinsing
in TBS (4 3 15 minutes). Two visualization procedures
were followed: (1) visualization with 3,38 diaminobenzidine tetrahydrochloride (DAB, Sigma Chemicals, St. Louis,
MO) or (2) visualization with nickel-enhanced DAB (NiDAB; Hsu and Soban, 1982; Hancock, 1986). For DAB
visualization, sections were rinsed in 0.05 M Tris buffer pH
7.6, and incubated in 0.05% DAB and 0.01% hydrogen
peroxide in 0.05 M Tris buffer for 5–10 minutes. The
reaction was halted by extensive rinses in TBS. For
nickel-enhanced DAB visualization, sections were rinsed
in buffer containing imidazole (0.01 M) and sodium acetate
(0.12 M; IAB; pH 7.2–7.4), and incubated in a solution of
0.05% DAB, 2.5% nickel ammonium sulfate and 0.005%
hydrogen peroxide in IAB for 5–10 minutes, then extensively rinsed in IAB, followed by rinses in TBS.
Rats were perfused as above with 3% paraformaldehyde
and 0.05–0.1% glutaraldehyde in PB. Immunohistochemistry was performed as follows. Sections were incubated in
4% normal goat serum in TBS (diluent) for 2 hours to block
nonspecific staining, then incubated in primary antiserum
(affinity-purified, rabbit polyclonal anti-SERT antibody
CT-2; 0.5–1.0 µg/ml) that was directed at the carboxy
terminus of the SERT protein (Qian et al., 1995) or
rabbit-anti-5-HT (Incstar, Stillwater, MN) for 48 hours.
Following four 15-minute rinses in TBS, sections were
then incubated overnight in biotinylated goat-anti-rabbit
(Vectastain Elite kit, Vector Labs), and then rinsed in TBS
and incubated in avidin-biotin-peroxidase complex (Vectastain Elite kit, Vector Labs) for 4 hours. Following rinses
in TBS and 0.05 M Tris, the bound peroxidase was
visualized with either NiDAB or DAB as described above.
Control sections, in which the primary antiserum was
omitted, were processed in the same manner. ChAT immunohistochemistry was then performed with the following
modifications to the above procedure: (1) the diluent
consisted of 4% normal goat serum in TBS; and (2) the
secondary antibody was goat-anti-rat. ChAT immunoreactivity was visualized with either DAB (following visualization of SERT with NiDAB) or BDHC (following visualization of SERT with DAB) as the chromogen. Following the
double immunohistochemical procedures, sections were processed for light and electron microscopy as described below.
ChAT immunohistochemistry
Preparation for light microscopy
Anterograde tracing was combined with immunohistochemistry for ChAT. Following the above procedure, sections were incubated in 4% normal rabbit serum in TBS
(diluent) for 2 hours to block nonspecific staining. Sections
were then incubated for 24–48 hours at 4°C in primary
antibody (monoclonal rat-anti-ChAT [AB8] 1:250; Levey et
al., 1983) in diluent. Following the incubation and four
15-minute rinses in TBS, sections were incubated overnight in secondary antibody (rabbit-anti-rat, Incstar) 1:200
in diluent; rinsed in TBS (4 3 15 minutes) and incubated
for 4 hours in rat peroxidase-anti-peroxidase (PAP, Sternberger, Baltimore, MD) 1:200 in diluent. Following four 15
minute rinses in TBS, the bound peroxidase was visualized with either DAB (after tracer visualization with
NiDAB) or benzidine dihydrochloride (BDHC; after tracer
visualization with DAB; Levey et al., 1986). The DAB
reaction was performed as above. For the BDHC visualization, sections were rinsed in 0.01 M PB pH 6.6 and
incubated in 0.01% BDHC (Sigma), in 0.01 M PB pH 6.6,
Following immunohistochemical procedures, the sections were mounted onto gelatin-coated slides, air-dried,
dehydrated in ethanol, cleared in xylene and coverslipped
with DPX mountant (BDH Laboratory Supplies, Poole,
England). Tissue sections were examined in the light
microscope and drawings were made with the aid of a
camera lucida drawing tube. Nuclear boundaries were
determined with the aid of the rat brain atlas of Paxinos
and Watson (1986), with mesopontine tegmental nuclear
divisions following the convention adopted by Rye et al.
(1987). The subnuclear organization of the dorsal raphe
follows the terminology of Steinbusch et al. (1981) and
Steinbusch and Nieuwenhuys (1983).
Visualization of tracer
Preparation for electron microscopy
Following the staining procedures, sections were postfixed in 2% glutaraldehyde in PB for 2–12 hours, and
osmicated in 1% osmium tetroxide in either 0.1 M cacodylate buffer, pH 7.5 (for NiDAB/DAB staining) or PB pH 6.6
DORSAL RAPHE AFFERENTS TO THE PPT
(for DAB/BDHC staining). Sections were then rinsed,
dehydrated in ascending concentrations of ethanol and
then propylene oxide, infiltrated with resin (Durcupan
ACM, Fluka, Ronkonkoma, NY), mounted onto microscope
slides, and coverslipped with a plastic slide, which was
removed following curing of the resin at 60°C for 48 hours.
Blocks containing the region of the dorsal PPT pars
compacta (PPT-pc; Fig. 2C) were cut out and glued to blank
cylinders of resin for sectioning. Ultrathin sections (70–80
nm) were cut on an ultramicrotome (Sorvall MT2B),
collected onto Formvar-coated single slot grids, stained
with uranyl acetate and lead citrate, and examined in an
electron microscope (Zeiss EM10 or Jeol JEM 100C).
Electron microscopic analysis
The region of the dorsal PPT-pc, illustrated in Figure 2C,
was analyzed at the electron microscopic level. Given the
variability in penetration of immunoreagents, only the
most superficial 5–10 µm of each tissue block was examined. Three or more blocks of the PPT-pc were examined
from each animal in order to determine: (1) the proportion
of ChAT-immunoreactive (IR) versus non-ChAT-IR spines,
dendrites, terminals or somata contacted by a labeled
afferent; (2) the density of afferent labeling in the neuropil;
and (3) the density of afferent labeling contacting ChAT-IR
elements. Two analyses were performed separately for
both anterogradely labeled and SERT-immunoreactive
(SERT-IR) terminals. In the first analysis, a sampling
region from the dorsal PPT-pc was examined in the
electron microscope and all labeled terminals encountered
in the ultrathin section were photographed at approximately 310,000 magnification. The numbers of BDlabeled or SERT-IR synaptic profiles in contact with
ChAT-IR and non-ChAT-IR spines, dendrites, terminals
and somata were counted. Dendrites postsynaptic to labeled elements were further classified as small (,1 µm
diameter), medium (.1 µm but ,2 µm) or large (.2 µm).
These absolute numbers were then converted into percentages to express the proportion of labeled terminals in
contact with ChAT-IR elements (number of synaptic profiles with ChAT-IR elements/total number of synaptic
profiles; see Table 1).
In the second analysis, photomicrograph montages were
made of randomly selected regions of sampled ultrathin
sections, at approximately 37,000 magnification (an average of 3,000 µm2 was analyzed for each tissue block), to
determine the density of afferent labeling in the neuropil
and those contacting ChAT-IR elements. If more than 33%
of the sampling region was taken up by blood vessels or
myelinated axon bundles (e.g., commissure of the lateral
lemniscus or superior cerebellar peduncle), new coordinates were determined. Electron micrographs were then
made of BD-labeled or SERT-IR terminals, and analysis
derived from 8–10 overlapping photographic prints. The
following were counted in each montage: (1) labeled axon
terminals; (2) labeled axon terminals forming synapses
with ChAT-IR spines, dendrites or somata; (3) labeled axon
terminals forming synapses with non-ChAT-IR spines,
dendrites or somata; (4) unlabeled axon terminals; (5)
unlabeled axon terminals forming synapses with ChAT-IR
spines, dendrites or somata; (6) -unlabeled axon terminals
forming synapses with non-ChAT-IR spines, dendrites or
somata; and (7) labeled and unlabeled dendrites. Quantitation was also performed both for labeled synaptic profiles
(S) and for total labeled terminals (synaptic profiles plus
305
TABLE 1. Synaptic Targets of Anterogradely Labeled and Serotonin
Transporter-Immunoreactive Terminals in the PPT-pc1,2
Anterogradely labeled
terminals
Unlabeled dendrites
small (,1 µm)
medium (.1 µm; ,2 µm)
large (.2 µm)
total
ChAT-IR3 Dendrites
small
medium
large
total
Unlabeled terminals
Total
SERT-IR4 terminals
n
% of total
n
% of total
125
50
4
179
59.5
23.8
1.9
85.2
35
8
1
44
72.9
16.7
2.1
91.7
17
5
3
25
6
210
8.1
2.4
1.4
11.9
2.9
3
0
0
3
1
48
6.2
0
0
6.2
2.1
1Data
are shown for those terminals having distinct synaptic specializations.
pedunculopantine tegmental nucleus pars compacta.
choline acetyltransferase immunoreactivity.
4SERT-IR, Serotonin transporter immunoreactivity.
2PPT-pc,
3ChAT-IR,
terminal appositions, S 1 N; see Table 2). The absolute
numbers were then converted into percentages to express
the following characteristics of each labeled afferent: (1)
the density of terminals in the neuropil (labeled terminals/
labeled 1 unlabeled terminals) and (2) the density of
terminals contacting ChAT-IR elements (labeled terminals
contacting ChAT-IR elements/total terminals contacting
ChAT-IR elements; see Table 2). The density of afferent
labeling on ChAT-IR elements was also expressed as a
terminal-covering ratio for ChAT-IR dendrites, which was
calculated in a manner similar to that of a somatic
terminal-covering ratio described in the accompanying
paper (Steininger et al., 1997).
Measurements of terminals and postsynaptic elements
were made from photographic prints. The length of dendritic membrane and the length of terminals (both synaptic profiles and terminal appositions) contacting the dendrite were measured by using an image analysis program
(NIH Image). The terminal-covering ratio was determined
by dividing the total length of labeled terminal by the total
length of dendritic membrane.
RESULTS
Anterograde tracing
from the dorsal raphe nucleus
Biotinylated dextran injection sites in the dorsal
raphe. Iontophoretic injections of the anterograde tracer
BD were made into the DR. Four distinct subdivisions
within the DR are recognized on the basis of cytoarchitecture and cell packing density (Steinbusch et al., 1981;
Steinbusch and Nieuwenhuys, 1983; Fig. 1A): a dorsomedian division, the ventromedial division, paired lateral
divisions and a pars caudalis. Injections of biotinylated
dextran amine were directed at the dorsomedian subdivision, because our previous retrograde tracing studies
indicated that the majority of dorsal raphe and/or serotonergic inputs to the PPT originate from cells in this division
(Steininger et al., 1992). The locations of small, iontophoretic applications of BD were confirmed on both cyto- and
chemoarchitecture grounds, as well as by determining that
resultant patterns of labeling in brain regions outside the
mesopontine tegmentum recapitulated those previously
described for DR efferents (Vertes, 1991; Vertes and Kocsis, 1994). In all cases, moderate to dense anterogradely
labeled fibers were observed in the bed nucleus of the stria
306
T.L. STEININGER ET AL.
TABLE 2. Frequency of Anterogradely Labeled and SERT-Immunoreactive Terminals Synapsing or Contacting CHAT-Immunoreactive
Dendrites in the PPT-pc1
Terminals synapsing
with CHAT-IR dendrites
Case
Anterogradely labeled
terminals
B14
B23
B29
total
SERT-immunoreactive
terminals
TR7
TR20
TR21
total
Terminals apposed to
CHAT-IR dendrites (S 1 N)
Synaptic terminals
in the neuropil
ChAT-IR
dendrites
(n)
Unlabeled
terminals
(n)
Labeled
terminals
(n)
Frequency
of labeling
(%)
Unlabeled
terminals
(n)
Labeled
terminals
(n)
Frequency
of labeling
(%)
Unlabeled
terminals
(n)
Labeled
terminals
(n)
Frequency
of labeling
(%)
114
93
64
271
93
47
46
186
4
2
1
7
4.1
4.1
2.1
3.6
152
87
76
315
6
4
2
12
3.8
4.4
2.6
3.7
1,651
1,420
1,751
4,822
62
53
31
146
3.6
3.6
1.7
2.9
49
38
71
158
46
24
45
115
1
1
2
4
2.1
4.0
4.3
3.4
75
48
72
195
1
3
3
7
1.3
5.9
4.0
3.5
643
1,110
1,158
2,911
22
24
19
65
3.3
2.1
1.6
2.2
1Data shown are for terminals that synapse with ChAT-immunoreactive dendrites (left set of columns) and for all terminals, both nonsynapsing and synapsing (N 1 S), apposed to
ChAT-immunoreactive dendrites (center columns). Data in the rightmost column are for terminals making synaptic contact with both ChAT-immunoreactive and nonimmunoreactive elements in the neuropil (i.e., inclusive of all synaptic terminals in the neuropil).
terminalis, amygdala, ventral striatum, lateral hypothalamic area, intralaminar and posterior thalamic nuclei,
the posterior hypothalamus, ventral tegmental area, locus
coeruleus, the parabrachial nucleus (both lateral and
medial subnuclei), and pontine reticular formation (particularly medial regions). Moderate anterogradely labeled
fibers were observed in the striatum, piriform cortex,
nucleus accumbens, medial septum/diagonal band, substantia innominata, dorsomedial and ventromedial hypothalamic nuclei, lateral geniculate, zona incerta, entopeduncular nucleus, substantia nigra and medullary
gigantocellular field (particularly the ventromedial zone
immediately dorsal to the inferior olive). A few anterogradely labeled fibers were observed in the olfactory bulb,
frontal cortex, suprachiasmatic nucleus, the thalamic reticular nucleus and other thalamic nuclei, hippocampus
and the lateral habenula. Taken together, these observations, made us feel entirely confident in attributing the
present findings to specific uptake and transport of BD by
DR neurons.
In order to determine whether the anterogradely labeled
terminals make direct synaptic contact with the cholinergic PPT neurons, anterograde tracing was combined with
ChAT immunohistochemistry. Two double-staining visualization techniques were followed. In one procedure, the
anterograde tracer was visualized with DAB, which yielded
a smooth, brown reaction product, and ChAT immunoreactivity was visualized with BDHC, which yielded a granular
blue reaction product. Alternatively, the anterograde tracer
was visualized with NiDAB, which yielded a dense, amorphous, black reaction product, and ChAT immunoreactivity was visualized with DAB (brown homogeneous reaction
product). With both methodologies, the anterogradely labeled fibers and varicosities were easily distinguishable
from the ChAT-immunoreactive cells and dendrites on the
basis of the color and texture of the reaction products. The
possible retrograde labeling of neurons, which has been
observed with the biotinylated dextran tracer, was minimized in these studies by using iontophoretic injections
methods and micropipetters with small tip diameters. The
location of retrogradely labeled neurons in each case is
described for each case below.
Visualization of the anterograde tracer yielded a Golgilike dark brown (DAB) or black (NiDAB) staining of cell
bodies and processes of the neurons in the DR that had
taken up and transported the BD (Fig. 2A). Anterogradely
labeled fibers and varicosities were dense in regions
immediately surrounding the injection site. Labeled varicose fibers were also observed in the ependymal layer
surrounding the cerebral aqueduct. The cases having
injection sites largely restricted to the DR were utilized for
analysis and these are depicted in Figure 1. An example of
a BD injection site (Case B26) is illustrated in the photomicrograph in Figure 2A.
Case B14. The injection site in case B14 (Fig. 1B) was
the largest of the analyzed cases. The injection was
centered at a midcaudal level of the DR and involved
nearly the entire DR. The injection extended from the level
of the trochlear nucleus, rostrally, to the level of the
transition from the cerebral aqueduct to the fourth ventricle (at the rostral level of the LDT), caudally. A few
neurons that had taken up the tracer were located in the
ventrolateral periaqueductal gray (PAG), lateral to the
lateral subdivision of the DR. Anterograde labeling was
most extensive in this case as was the amount of retrograde labeling. A distinct cluster of retrogradely labeled
neurons were observed bilaterally, rostral to the injection
site in the ventrolateral central gray, immediately dorsal
to the oculomotor nucleus. Scattered retrogradely labeled
neurons (about 5–10 neurons per 40-µm section) were also
observed in the pontine reticular formation, central tegmental field and dorsal periaqueductal gray. Retrograde labeling in other parts of the brain was minimal (,20 neurons
total), and limited primarily to the lateral hypothalamic
and preoptic areas.
Case B23. The injection site in case B23 (Fig. 1B) was
the second largest and well localized to the DR, and
encompassed nearly the entire nucleus, including the
ventral aspect which was not included in the injection site
of case B14. This injection was centered at a mesencephalic level immediately caudal to the trochlear nucleus,
extending from the level of the rostral trochlear nucleus to
the level of the fourth ventricle in the rostral pons. The
handful of retrogradely labeled neurons observed in this
case were situated in the ventrolateral PAG, immediately
dorsal to the oculomotor nucleus, as in case B14.
Case B26. The injection site in case B26 (Figs. 1C, 2A)
was smaller than those in cases B14 or B23, and was
centered in the lateral portion of the dorsomedial subdivision of the DR (Steinbusch et al., 1981; Steinbusch and
DORSAL RAPHE AFFERENTS TO THE PPT
307
Fig. 1. Biotinylated dextran injection sites in the dorsal raphe
nucleus (DR). Camera lucida drawings of coronal sections at four
rostrocaudal levels of the DR illustrate the location and extent of the
tracer injections. The boundaries of the DR nucleus are illustrated in
column A. The injection sites of case B14 (dotted area) and case B23
(striped area) are illustrated in column B. In column C, smaller
injection sites of case B26 (dotted area) and B29 (striped area) are
illustrated. Scale bar 5 1 mm.
Nieuwenhuys, 1983), at a mesencephalic level caudal to
the trochlear nucleus. The injection site extended from the
level of the caudal region of the oculomotor nucleus to the
level of the rostral LDT (caudal midbrain). Few (,10)
retrogradely labeled neurons were observed in the mesopontine tegmentum. The ultrastructural preservation was
not optimal for electron microscopic analysis; therefore,
this case was utilized to illustrate anterograde labeling at
the light microscopic level only.
Case B29. The injection site in case B29 (Fig. 1C) was
the smallest, and centered in the dorsomedial and ventromedial subdivisions of the DR (Steinbusch et al., 1981;
Steinbusch and Nieuwenhuys, 1983). The anteroposterior
extent of the injection was limited to sections, including
the bulk of the trochlear nucleus through a mid-PPT level.
Retrograde labeling outside the injection site was limited
to a few neurons in the PAG adjacent to the DR.
Anterograde labeling in the mesopontine tegmentum. Coronal sections of the mesopontine tegmentum
processed for the visualization of BD and ChAT were
examined in the light microscope. A moderate-to-dense
plexus of anterogradely labeled varicose axonal processes
were observed in the region of the cholinergic PPT neurons. The majority of projection axons were thin, varicose
and infrequently studded with boutons. Thick tortuous
axons that exhibited clusters of larger boutons were occasionally observed. A representative case (B26) was used to
illustrate the pattern of anterograde labeling in the mesopontine tegmentum following DR injections. Camera lucida drawings were made to illustrate the relationship of
anterogradely labeled varicosities to ChAT-immunoreactive (ChAT-IR) neurons at four rostrocaudal levels of the
mesopontine tegmentum (Fig. 3A–D).
Rostral (Fig. 3A). The major target of DR fibers at this
level was the midbrain central gray, particularly the
rostral DR and ventrolateral periaqueductal gray. Few
anterogradely labeled fibers were observed in the lateral
and dorsal PAG at this level. Moderate-to-dense anterograde labeling was also observed in tegmental regions rich
in dopaminergic neurons, such as the caudal aspects of the
ventral tegmental area and substantia nigra pars compacta, as well as the retrorubral field (RRF). Anterogradely
308
T.L. STEININGER ET AL.
Fig. 2. Combined light microscopic visualization of anterograde
labeling and choline acetyltransferase (ChAT) immunostaining (Case
B26). A: Coronal section of the DR, depicting the appearance of a
typical biotinylated dextran injection site. The neurons that have
taken up and transported the tracer are filled with 3,38 diaminobenzidine (DAB) reaction product. B: A coronal section through the dorsal
part of the pedunculopontine tegmental nucleus (PPT) pars compacta.
The fibers of the commissure of the lateral lemniscus (arrows) are
prominent landmarks of the pars compacta. The ChAT-immunoreactivity (IR) neurons are visualized with benzidine dihydrochloride (BDHC;
granular reaction product). ChAT immunoreactivity is also visible in
the trochlear nerve in the upper left corner of the photomicrograph. C:
Higher magnification photomicrograph montage of the PPT region
depicted in B. Numerous anterogradely labeled fibers and boutons are
seen in the vicinity of the ChAT-IR neurons. Arrows mark the same
fascicles of the commissure of the lateral lemniscus as in B, and the
asterisks mark the location of the identical blood vessel in B and C for
orientation purposes. Scale bars 5 500 µm in A; 250 µm in B; 100 µm
in C.
labeled fibers were a bit less dense dorsal to the RRF in the
central tegmental field (CTF). Few axons were observed in
the vicinity of the most anterior PPT neurons located
ventrolaterally in the tegmentum between the RRF dor-
sally, and substantia nigra (SN), ventrally. Anterograde
labeling in the contralateral tegmentum was less dense,
but described a similar pattern. The amount of contralateral anterograde labeling appeared to vary according to
DORSAL RAPHE AFFERENTS TO THE PPT
309
Fig. 3. Anterograde labeling in the mesopontine tegmentum in
case B26. A–D: Camera lucida drawings of coronal sections at four
rostrocaudal levels in (BD)/ChAT double-stained sections, illustrating
the pattern of DR innervation in this brain region. The shape and
location of cholinergic neurons and anterogradely labeled varicosities
are illustrated. At rostral levels of the PPT (A,B), very little of
theanterograde labeling is directed to the region of the cholinergic
neurons. The situation is much different at caudal levels (C,D), where
much of the labeling coincides with cholinergic PPT neurons (and the
adjacent MEA and LDT), and less anterograde labeling is observed
elsewhere in the section, e.g., cuneiform nucleus (Cnf) and pontine
tegmental field (PTF). Scale bar 5 500 µm.
the extent of involvement of the contralateral DR at the
injection site.
Mid-PPT (Fig. 3B). The injection site was centered at
this level, immediately caudal to the trochlear nucleus.
Dense patches of anterogradely labeled varicosities were
observed in a region that extended ventrolaterally from
the central gray to the central tegmental field immediately
dorsal to the PPT, and also in the tegmentum between the
midline and the PPT neurons. While cholinergic neurons
were more numerous than in the rostral level, they
remained rather loosely arranged and had not yet reached
the density observed in the PPT-pc. A few anterogradely
labeled fibers and boutons were seen adjacent to cholinergic neurons.
Midcaudal PPT (Fig. 3C). At this level of the mesencephalon, the majority of anterogradely labeled varicose
fibers were concentrated in the region of the PPT, as
illustrated in the photomicrographs in Figure 2B and 2C.
At this level, cholinergic neurons were concentrated in the
PPT-pc, situated within the commissure of the lateral
310
lemniscus (cll), lateral to the ascending limb of the superior cerebellar peduncle (scp). Many labeled varicosities
were observed in putative contact with ChAT-IR cell bodies
and dendrites. Labeled fibers were quite dense in the
dorsal part of the pars compacta and the pars dissipatus
ventromedial to the superior cerebellar peduncle, as compared to ventral aspects of the PPT-pc. Moderate amounts
of anterogradely labeled fibers were observed in the midbrain extrapyramidal area (MEA), just medial to the
cholinergic neurons in the PPT-pc. Moderately dense anterograde labeling was observed in the tegmentum just
lateral to the median raphe and in the DR caudal to the
injection site. Few anterogradely labeled fibers were seen
ventral to the pontine tegmental field (PTF).
Caudal PPT (Fig. 3D). Anterogradely labeled fibers
were concentrated within and dorsal to cholinergic somata
in the LDT. Labeled fibers were less dense in the lateral
parabrachial subnuclei (PB) immediately caudal to the
PPT-pc. A few anterogradely labeled varicose axons were
observed in the caudal DR. A moderately dense plexus of
labeled axons were present in the neuropil containing the
remaining cholinergic PPT neurons, located ventromedial
to the superior cerebellar peduncle and ventral to the LDT
(i.e.; the ‘‘subcoeruleal region’’). The labeling observed in
the medial tegmentum more rostrally was not observed at
this level. Moderate anterogradely labeling was also observed in the pontine tegmental field ventral to the PPT.
No significant anterograde labeling was observed in the
pontine nuclei.
Anterograde labeling: Electron microscopy
Ultrastructural appearance. Anterograde tracing visualized with DAB (smooth brown reaction product) was
paired with ChAT immunoreactivity visualized with BDHC
(blue granular reaction product). Alternatively, anterograde tracing visualized with NiDAB (dense black reaction
product) was paired with ChAT immunoreactivity visualized with DAB. At the electron microscopic level, the DAB
reaction product was amorphous and associated with
organelle membranes and inner plasmalemma, whereas
the BDHC reaction product appeared as large crystalline
granules dispersed in the cytoplasm, such that the combination of DAB with BDHC resulted in reliably distinct
reaction products. In sections developed using the combination of NiDAB and DAB, the NiDAB and DAB reaction
products were similarly amorphous. The NiDAB reaction
product in the anterogradely labeled terminals, however,
was more abundant and electron-dense than the DAB
reaction product in the ChAT-immunoreactive elements.
The distinction between NiDAB and DAB reaction products has been previously described by other investigators
(Lapper and Bolam, 1992; Zaborszky et al., 1993).
The NiDAB reaction product was observed in a few
neurons in the PPT region that were retrogradely labeled
from the injection (in case B14 only). The appearance of
the NiDAB reaction product in these retrogradely labeled
elements was reliably distinct from the DAB reaction
product seen in ChAT-IR elements, i.e., the reaction product was more dense and completely filled the somatodendritic compartment, as opposed to the patchy, flocculent
electron-dense reaction product observed in ChAT-IR somata and dendrites (Fig. 5J, K). A few DAB- or NiDABlabeled myelinated axons were also observed (Fig. 5I),
although the distinction between ChAT-IR and anterogradely labeled myelinated axons was less clear.
T.L. STEININGER ET AL.
Quantitative analysis. Two analyses were performed:
(1) the postsynaptic targets of anterogradely labeled terminals were determined and quantitated; and (2) the proportion of anterogradely labeled versus unlabeled terminals
synapsing with ChAT-IR neurons was determined and
compared to the pattern observed for noncholinergic elements in the surrounding neuropil.
For the first analysis, photomicrographs were made of
each anterogradely labeled terminal encountered in these
blocks. A total of 353 anterogradely labeled terminals were
photographed. Only those exhibiting clear synaptic specializations were selected for further analysis, for a total of
210 anterogradely labeled terminals. Anterogradely labeled terminals were typically bulb- or club-shaped, and
ranged in size from 0.23 to 3.74-µm-long (parallel to
synaptic membrane; mean 5 0.94 µm) by 0.15 to 1.58-µmwide (perpendicular to synaptic membrane; mean 5 0.61
µm). Of the 210 anterogradely labeled terminals analyzed,
179 (85.2%) made synaptic contact with unlabeled dendrites, 25 (11.9%) made synaptic contact with ChAT-IR
dendrites, six (2.8%) made synaptic contact with unlabeled
terminals (Table 1), and no synapses were observed between anterogradely labeled terminals and neuronal somata.
The unlabeled dendrites contacted by anterogradely
labeled terminals (Fig. 4) ranged from 0.15 to 2.72 µm in
diameter. In a few instances, two anterogradely labeled
terminals contacted the same dendritic target (Fig. 4F).
Although synaptic vesicle profiles in the anterogradely
labeled terminals were partially obscured by the DAB or
NiDAB reaction product, the shape of these vesicles appeared to be round in most of the labeled terminals. No
clear distinctions in vesicle type could be made; however, it
appeared that some terminals contained smaller, more
densely packed vesicles (Fig. 4G). Of the 179 anterogradely labeled terminals contacting unlabeled dendrites,
125 (69.8%) synapsed with small (,1 µm diameter) dendrites (Fig. 4B–D,E,G–I), 50 (23.8%) synapsed with medium dendrites (between 1 and 2 µm; Fig. 4A,F) and four
(2.8%) synapsed with large dendrites (.2 µm).
Synaptic specializations were both symmetric (Fig. 4A–C)
and asymmetric (Fig. 4D–I). Asymmetric synapses were
preferentially located on small dendrites, while symmetric
synapses were more prevalent on medium and large
dendrites. Of the synapses with small dendrites, 68 were
asymmetric and 57 were symmetric. Of the synapses with
medium dendrites, 19 were asymmetric, 30 were symmetric and one was unclear. Of the synapses with large
dendrites, one was asymmetric and three were symmetric.
Approximately 16% of the asymmetric synapses were
associated with subjunctional dense bodies.
Of the 210 anterogradely labeled terminals analyzed, 25
(11.9%) were found to make synaptic contact with ChAT-IR
elements, and in all cases, these were dendrites of ChAT-IR
neurons. Of these 25 synapses, 17 (68%) were with small
ChAT-IR dendrites (Fig. 5B–F), five (20.0%) were with
medium ChAT-IR dendrites (Fig. 5A) and three (12%) were
with large ChAT-IR dendrites. Synapses between anterogradely labeled terminals and ChAT-IR dendrites were
both asymmetric (Fig. 5C,D) and symmetric (Fig. 5A,B,E,F).
Of the synapses made with small ChAT-IR dendrites, 12
were asymmetric and five were symmetric. Of the synapses with medium ChAT-IR dendrites, one was asym-
DORSAL RAPHE AFFERENTS TO THE PPT
Fig. 4. Electron photomicrographs depicting synaptic contacts,
between anterogradely labeled axon terminals (t) and unlabeled
dendrites (d), that illustrate the morphology of anterogradely labeled
terminals and the caliber of unlabeled dendrites that are contacted.
Synaptic specializations are symmetric (A–C) and asymmetric (D–I).
311
Note that the reaction product densely fills these terminals. In F, two
anterogradely labeled terminals appose a single unlabeled dendrite;
however, synaptic specializations are not apparent for the apposition
on the right. Scale bar 5 0.5 µm.
312
metric, three were symmetric and one was unclear. Of the
synapses with large ChAT-IR dendrites, one was asymmetric and two were symmetric. Of the 210 anterogradely
labeled terminals analyzed, six (2.9%) made synapses with
unlabeled terminals (axoaxonic synapses). The synapses
were either symmetric (n 5 5) or asymmetric (n 5 1). The
postsynaptic terminals contained either pleomorphic
vesicles (Fig. 5G) or small round clear vesicles (Fig. 5H).
None of the postsynaptic terminals were seen to be in
synaptic contact with other elements in the plane of
section.
In the second analysis, 271 ChAT-IR dendritic profiles
were examined from the same tissue sampled in the first
analysis (Table 2). A total of 327 terminals were apposed to
these ChAT-IR dendrites, and of those terminals, only 193
exhibited clear synaptic specializations. Of these 193
synaptic profiles, seven were anterogradely labeled, yielding a frequency of 3.6% of anterogradely labeled input to
the ChAT-IR dendrites (which varied among cases from
2.1% to 4.1%). The proportion was found to be similar
(3.7%) when labeled terminal appositions (i.e., those exhibiting no apparent synaptic specialization) were included.
In the neuropil surrounding these ChAT-IR dendrites,
4,968 synaptic profiles were identified. Of these, 146 were
anterogradely labeled, giving a frequency of labeling of
2.9% (range of 1.7–3.6%) in the neuropil.
Serotonin transporter immunoreactivity
It was of interest to determine whether inputs from the
dorsal raphe to the PPT might be serotonergic, since
serotonin has been implicated in behavioral state control,
and since only a portion (40–60%) of neurons in the
cytoarchitectonically defined DR are serotonergic (Moore,
1981; Descarries et al., 1982; Léger and Wiklund, 1982).
We therefore employed SERT immunohistochemistry as a
marker for the serotonergic terminals. As illustrated in
Figure 6C,D, a complete absence of immunostaining was
observed in the PPT, following preabsorption of the antiSERT antibody with SERT fusion protein (used as antigen
at 100 µg/ml). Based on the high quality of immunostaining achieved with this antibody, it was our expectation
that SERT immunohistochemistry would yield superior
ultrastructure and staining when compared to immunohistochemistry using commercially avaiable antibodies to
serotonin.
Light microscopy. Serotonin transporter immunoreactivity (SERT-IR) visualized with DAB (smooth, brown)
was paired with ChAT immunoreactivity visualized with
BDHC (granular, blue), and SERT immunoreactivity visualized with NiDAB (dense, black) was paired with ChAT
immunoreactivity visualized with DAB (smooth, brown).
SERT-immunoreactive neurons were observed in the brainstem raphe nuclei and SERT-IR varicose axons were
observed in many brain regions. The appearance and
distribution of SERT-IR neurons in the dorsal raphe
nucleus (Fig. 6A) were identical to that of 5-HT-immunoreactive neurons (not illustrated); i.e., they are present in
four clusters (dorsomedian, ventromedian, lateral and
caudal), are medium to large in size, and fusiform or ovoid
in shape.
SERT-IR fibers were dense in the region of the PPT,
particularly in the dorsal region of the pars compacta (Fig.
6B). At least two classes of SERT-IR fibers were observed.
The majority of axons were characterized by a uniformly
thin caliber and were lightly studded with small boutons. A
T.L. STEININGER ET AL.
second type of labeled axon displayed a varying, but
typically greater caliber along its course and was interrupted less regularly by fairly large varicosities. Putative
contacts were observed between SERT-IR varicosities and
ChAT-IR elements. The pattern of SERT immunoreactivity in the PPT region was different than the pattern of
anterograde labeling from the dorsal raphe, in that more
SERT-IR fibers were seen in the ventrolateral tegmentum
encompassing the rostral third of the PPT.
Electron microscopy. SERT-IR elements were identified by the presence of amorphous electron-dense DAB
reaction product, which was present throughout the cytoplasm, and accumulated at the mitochondrial, vesicular
and internal surface of neuronal membranes, and ChAT-IR
elements were identified by the presence of BDHC crystals
(Fig. 7A). SERT-immunoreactive fibers were less dense in
sections prepared for electron microscopy, likely secondary
to the limited penetration of the antibody with the omission of Triton-X from the diluent. The majority of the
SERT-IR elements in the dorsal PPT-pc consisted of thin
unmyelinated axons and preterminal axons. No SERT-IR
dendrites or somata were observed in the PPT-pc. SERT-IR
terminals (Fig. 7A–G) varied in size from 0.38 to 4.05 µm
in length (parallel to synaptic membrane; mean 5 1.0 6
0.89 µm) and 0.15 to 0.80 µm in width (perpendicular to
synaptic membrane; mean 5 0.45 6 0.15 µm).
The analysis performed in SERT/ChAT double-stained
tissue was the same as that performed for the anterograde
tracing experiments described above: (1) the postsynaptic
targets of SERT-IR terminals were determined and quantitated (see Table 1), and (2) the proportion of SERT-IR
versus unlabeled terminals synapsing with ChAT-IR neurons was determined and compared to the proportion of
SERT-IR terminals synapsing in the surrounding neuropil
(see Table 2). In the first analysis, greater than 170
SERT-IR vesicle-containing axons were photographed and
48 exhibiting synaptic specializations were selected for
analysis. Of these 48 SERT-IR synaptic profiles, 44 (91%)
were found to synapse with unlabeled dendrites (Fig.
7B–F), three (6%) were found to synapse with ChAT-IR
elements (Fig. 7A), and one (2%) synapsed with an unlabeled terminal (Fig. 7G). Of the non-ChAT-IR dendrites
contacted by SERT-IR synaptic profiles, 35 (80%) were
small (,1 µm diameter), with 16 describing symmetric
and 16 asymmetric specializations with the morphological
subtype of three profiles undeterminable. Medium-sized
(.1 µm but ,2 µm diameter) non-ChAT-IR dendrites
contacted by SERT-IR synaptic profiles totaled eight (18%)
of total dendrites contacted), with specializations evenly
distributed between symmetric and asymmetric types.
One (2% of total dendrites contacted) non-ChAT-IR dendrite contacted by a SERT-IR synaptic profile was large
(.2 µm diameter). All three ChAT-IR dendrites contacted
by SERT-IR synaptic profiles were small with one symmetric and two asymmetric specializations. Numerous
ChAT-IR myelinated axons were observed (Fig. 7H), but
SERT-IR myelinated axons were only rarely observed.
SERT-IR terminals were frequently seen in apposition to
unlabeled terminals in the neuropil. No contacts were observed between SERT-IR terminals and neuronal somata.
In a separate analysis, 158 ChAT-IR dendrites were
examined in SERT/ChAT double-stained material (Table
2). A total of 202 terminals were apposed to these ChAT-IR
dendrites, and of those terminals, only 119 exhibited clear
synaptic specializations. Of these 119 synaptic profiles,
DORSAL RAPHE AFFERENTS TO THE PPT
Fig. 5. A–F: Electron photomicrographs illustrating synaptic contacts between anterogradely labeled axon terminals (t) and ChAT-IR
dendrites (d). G–H: Anterogradely labeled terminals in synaptic
contact with both an unlabeled dendrite (d) and an unlabeled terminal
313
(t). I: Labeled myelinated axons (a). J–K: Retrogradely labeled dendrites (d). Note the contrast between the appearance of the reaction
product in these non-ChAT-IR dendrites and in the ChAT-IR dendrites
illustrated in A–F. Scale bar 5 0.5 µm.
314
T.L. STEININGER ET AL.
Fig. 6. A and B: Serotonin transporter (SERT)/ChAT doubleimmunostaining at the light microscopic level. A: A coronal section of
the DR showing the location and appearance of SERT-IR neurons.
(Subdivisions of the dorsal raphe are recognized as follows: dm,
dorsomedian; vm, ventromedian; lat, lateral.) B: SERT-IR fibers and
ChAT-IR neurons in the dorsal part of the PPT pars compacta.
Numerous SERT-IR fibers are in putative contact with ChAT-IR
somata. C: Coronal tissue section stained solely for SERT-IR demon-
strates dense SERT-immunoreactive fibers in the PPT-pars compacta
(marked by the fibers of the commissure of the lateral lemniscus,
arrows). D: In an adjacent section, the same region shows no immunoreactivity following preabsorption of the primary antiserum (rabbitanti-SERT) with excess SERT fusion protein. Arrows in D indicate all
fascicles in a similar location to that seen in C. Scale bars 5 500 µm for
A; 100 µm for B–D.
four were SERT-IR, yielding a frequency of 3.4% of synapses on ChAT-IR dendrites that were SERT-IR. This
frequency was found to vary among cases from 2.1% to
4.3%. The frequency was similar when terminal appositions (i.e., those not having clear synaptic specializations)
were included in the analysis. In the neuropil surrounding
these ChAT-IR dendrites, 2,976 synaptic terminals were
identified. Of those terminals, 65 were SERT-IR, giving a
frequency of 2.2% (ranged from 1.6% to 3.3%) of all
synapses on neuropil elements that were SERT-IR.
lish the presence of SERT containing terminals in the
PPT-pc; and (4) demonstrate an ultrastructural similarity
in the PPT-pc between terminals anterogradely labeled
from the dorsal raphe and those containing SERT. Approximately 12% of anterogradely labeled dorsal raphe terminals in the PPT-pc synapsed with ChAT-IR dendrites and
85% synapsed with unlabeled dendrites, while 3% synapsed with unlabeled terminals. Axosomatic synapses
were not observed. Anterogradely labeled terminals constituted 2–4% of the total synapses on cholinergic dendrites,
as well as in the surrounding neuropil, indicating that
raphe inputs to the cholinergic PPT neurons were no more
frequent than raphe input to noncholinergic elements in
the PPT region. Parallel light and electron microscopic
visualizations of SERT immunoreactivity as a specific
marker for serotonergic terminals confirmed the presumptive serotonergic nature of labeled DR efferents. Dual
labeling of either the anterograde tracer or SERT-IR with
DISCUSSION
The results of the present study: (1) confirm the existence of a projection from the dorsal raphe to the PPT-pc;
(2) provide the first quantitative assessment of an identified afferent to neurochemically defined elements (i.e.,
cholinergic versus noncholinergic) in the PPT-pc; (3) estab-
DORSAL RAPHE AFFERENTS TO THE PPT
Fig. 7. SERT/ChAT double-immunostaining at the ultrastructural
level. A: An apposition between a small SERT-IR terminal (t) and a
ChAT-IR dendrite (d), which is identified as such by the presence of
large (BDHC) crystals (arrows). B–F: SERT-IR terminals (t) in apposition to unlabeled dendrites (d). In B (the synapse on the left) and F,
synaptic specializations are clearly asymmetric, while other examples
315
are symmetric. G: A SERT-IR terminal in apposition to an unlabeled
terminal. H: A ChAT-IR myelinated axon (a), which contains a large
BDHC crystal. I: A ChAT-IR dendrite containing crystalline BDHC
receives an asymmetric synaptic contact from an unlabeled terminal
containing round clear vesicles. Scale bars 5 1 µm for A; 0.5 µm for
B–I.
316
T.L. STEININGER ET AL.
ChAT reveals novel information concerning the synaptic
organization of the PPT and extends previous findings,
which noted only that the majority of 5-HT-IR terminals in
the rat PPT are in synaptic contact with non-ChAT-IR
dendrites (Honda and Semba, 1994).
Technical considerations
Biotinylated dextran as an anterograde tracer. Biotinylated dextran (BD; also, biotinylated dextran-amine,
BDA) has recently been introduced as a neuronal tracer
(Veeman et al., 1992; Rajakumar et al., 1993; Wouterlood
and Jorritsma-Byham, 1993; Naito and Kita, 1994). Anterograde tracing with BD compares favorably with the widely
used anterograde tracer, PHA-L in light microscopic (Veenman et al., 1992) and electron microscopic studies (Wouterlood and Jorritsma-Byham, 1993). The advantages of BD
over PHA-L are: (1) enhanced anterograde labeling; (2)
ease of detection (e.g., a single incubation in avidin-biotinperoxidase complex [ABC] versus a 2–3-step immunohistochemical procedure); (3) improved ultrastructure reflecting shorter incubation times; and (4) better tissue
penetration of ABC compared to immunohistochemical
reagents for PHA-L visualization (Wouterlood and Jorritsma-Byham, 1993). The single disadvantage of BD is
the greater tendency for this tracer to be transported
retrogradely, as compared to PHA-L. In theory, axon
collaterals of retrogradely labeled neurons might also be
labeled, thereby yielding false-positive results. Iontophoretic application of BD, which we employed here, as
opposed to pressure injection, minimizes this potential
problem (Veenman et al., 1992). Iontophoretic injection of
BD produced excellent anterograde labeling and a minor
amount of retrograde labeling, which increased proportionately with increasing injection micropipette tip diameters,
as has been suggested by others (Veenman et al., 1992).
The extent of retrograde labeling probably had little effect
on the results because the pattern of DR efferents visualized with BD: (1) essentially mirrored that which we have
observed using PHA-L, where retrograde labeling is not
observed (Steininger and Wainer, 1991); and (2) varied
little across the cases analyzed, independent of the degree
of retrograde labeling. That the anterogradely labeled
terminals had a similar morphology and pattern of postsynaptic targets in all three cases analyzed, and that this
pattern was recapitulated by SERT-IR terminals, further
substantiates the results.
SERT immunoreactivity as a specific marker
for serotonin (5-hydroxytryptamine; 5-HT)
SERT belongs to a family of Na1/Cl2 dependent amine
transporters, that also includes transporters for noradrenaline and dopamine. SERT exists on the plasma membrane
of serotonergic presynaptic terminals and plays a prominent role in terminating the action of synaptically released
5-HT (Amara and Kuhar, 1993; Rudnick and Clark, 1993;
Barker and Blakely, 1994). The recent cloning of the SERT
gene (Blakely et al., 1991) led to the development of
antibodies directed at an epitope highly specific to the
SERT peptide and fusion protein (Qian et al., 1995). These
antibodies demonstrated SERT-IR cells and axons throughout the rat, monkey and human brain in a pattern entirely
consistent with the known localization of 5-HT uptake,
SERT antagonist binding and 5-HT immunoreactivity
(Qian et al., 1995). We therefore felt entirely confident
employing these antibodies as specific markers for seroton-
ergic terminals in the present study. In all experimental
material generated as a portion of this analysis, we always
confirmed that the pattern of SERT-IR cells and axons
throughout the brain exhibited a strict correspondence
with 5-HT visualized in adjacent sections. SERT expression has previously been attributed to both neuronal and
glial elements (Anderson et al., 1992). SERT mRNA, for
example, has been localized to neurons of the midbrain
raphe (Blakely et al., 1991; Fujita et al., 1993; Lesch et al.,
1993), as well as the frontal cortex which is devoid of
serotonergic cell bodies, but rich in serotonergic innervation (Lesch et al., 1993). The latter finding suggests either
transport of mRNA from dorsal raphe neurons for local
translation, or a postsynaptic and possibly glial localization, given the well-known uptake of 5-HT by glia. In the
present analysis, however, SERT-IR elements in the dorsal
PPT-pc consisted only of thin unmyleinated axons and
preterminal axons with no SERT-IR dendrites, neuronal
somata or glia observed. The absence of SERT-IR in glia in
the rat PPT-pc might reflect a unique feature of the rat
PPT-pc or a true false-negative secondary to methodological considerations. Glia in the PPT-pc, for example, might
express a unique SERT, generated by alternative splicing,
which lacks the epitope recognized by our antibodies.
Serotonin uptake in cultured glia may alternatively reflect
a feature unique to cultured, reactive astrocytes and less
so of glia in vivo.
Single and dual labeling immuno-electron microscopy. The first methodological consideration relates to
the potential sampling bias against distal dendrites of
cholinergic neurons, which is a well-recognized limitation
of immuno-electron microscopy. Although the majority of
ChAT-IR dendrites visualized here were small (,1 µm,
mean average diameter, see Table 1), the proportion and
density of a labeled afferent (e.g., anterogradely labeled or
SERT-IR) contacting ChAT-IR dendrites should be considered minimal estimates. Underestimation of distal dendrites may be a particular concern in dual immunolabeling, with BDHC as the second chromogen secondary to the
limited penetration of the large, granular BDHC precipitate within small caliber dendrites, its relative solubility,
and inconsistency in the BDHC visualization reaction, as
has been previously noted (Lapper and Bolam, 1992). In
the present studies, however, ChAT-IR dendrites visualized with BDHC were as abundant as those visualized
with DAB (e.g., BDHC labeled dendrites were 7.7% of
dendrites in the neuropil, compared to 6–14% seen in DAB
material (see Steininger et al., 1997).
A second methodological consideration relates to falsepositives that might occur secondary to difficulties in
discriminating between two chromogens employed in dual
immunolabeling procedures. Sequential immunoperoxidase staining with DAB and BDHC avoids this problem,
since the two electron-dense markers contrast sharply, and
are therefore reliably distinct at both the light and electron
microscopic levels (Bolam et al., 1986, 1991; Levey et al.,
1986; Zaborszky and Heimer, 1989). Combinations employing NiDAB and DAB as the two chromogens have also been
extensively utilized in light and electron microscopic dual
antigen localization studies (Zaborszky and Heimer, 1989;
Lapper and Bolam, 1992; Zaborszky et al., 1993). At the
electron microscopic level, the NiDAB and DAB reaction
products were both amorphous, but were judged as distinct on the basis of the greater abundance and electrondense nature of the NiDAB reaction product, as has been
DORSAL RAPHE AFFERENTS TO THE PPT
noted by other investigators (Lapper and Bolam, 1992;
Zaborszky et al., 1993). Misidentification of NiDAB (i.e.,
anterogradely labeled) as DAB (i.e., ChAT-IR) in terminals, and vice versa, would not be expected to significantly
alter the results of our analysis, because in material
processed only for ChAT-IR: (1) the frequency of ChAT-IR
terminals in the PPT-pc (0.4–0.7%; Steininger et al., 1997)
is much less than that of DR terminals (2–4%); and (2)
innervation of ChAT-IR dendrites or somata by ChAT-IR
terminals was not observed (Steininger et al., 1997; see,
however, Honda and Semba, 1995). The labeled synaptic
profiles contacting ChAT-IR dendrites analyzed in the
present studies are therefore likely to represent anterograde labeling or SERT-IR, and not ChAT-IR. Several
additional sources for false-positives in dual immunolabeling techniques exist and have been previously discussed in
detail (Bolam et al., 1986; Levey et al., 1986). Tissue
processed to control for interactions between the individual immunolabeling procedures indicated that potential false-positive staining was not encountered in the
present studies. The reliability of the results is further
supported by the observation that the two methods of
visualizing DR efferents to the PPT-pc (e.g., anterograde
labeling and SERT immunolabeling) produced very much
the same results independent of the dual chromogen
combination (e.g., NiDAB/DAB or DAB/BDHC) employed.
Anterograde labeling of the dorsal
raphe-PPT-pc projection
Light microscopy. Injections of BD in the dorsal
raphe anterogradely labeled terminals were concentrated
in caudal and dorsal aspects of the PPT (i.e., the PPT-pc)
and the LDT. Anterogradely labeled terminals were less
evenly distributed throughout the tegmentum encompassing the full extent of the PPT, when compared to 5-HT-IR
or SERT-IR axons and varicosities. This observation reflects either suboptimal labeling of DR efferents with the
anterograde tracer or the presence of additional innervation to the PPT from other serotonergic nuclei such as the
median raphe, supralemniscal nucleus (i.e., the B9 cell
group) or raphe magnus (e.g., see Semba and Fibiger, 1992;
Honda and Semba, 1994). Previous investigations have
noted that anatomical tracer injections involving, but not
restricted to, the PPT retrogradely label DR neurons
(Semba and Fibiger, 1992; Steininger et al., 1992), while
anterogradely labeled DR axons invest the dorsolateral
mesopontine tegmentum containing the PPT (and other
cell populations; Bobillier et al., 1975; Pierce et al., 1976;
Moore et al., 1978; Conrad et al., 1991; Vertes, 1991; Vertes
and Kocsis, 1994). These descriptions of the DR, as a
source of PPT afferents, lack specificity because the methodologies, applied by themselves, cannot differentiate between the several distinct ‘‘nuclei’’ within the PPT region
where an additional partial comingling of cholinergic and
noncholinergic exists (Rye et al., 1987). Retrograde tracer
injections, for example, cannot be restricted to a single cell
population in the PPT region, so that this methodology
identifies only putative afferents to cholinergic PPT neurons. Moreover, as has been previously demonstrated that
lack of an appreciation for the detailed cyto- and chemoarchitecture of the dorsolateral mesopontine tegmentum can
lead to misidentification of cholinergic PPT neurons as the
primary recipients of various labeled afferents (see, e.g.,
Rye et al., 1987, 1996). The combination of anterograde
labeling and immunocytochemistry, for ChAT employed here,
317
avoids these methodological concerns in revealing that cholinergic PPT neurons were invested by a moderately dense
plexus of anterogradely labeled DR fibers, in accord with our
previous experience with PHA-L (Steininger and Wainer,
1991). Numerous labeled varicosities were observed in putative contact with ChAT-IR somata and proximal dendrites
(Fig. 2B,C).
Electron microscopy. The postsynaptic targets of raphe-tegmental projections were determined by ultrastructural analysis of anterograde tracing combined with ChAT
immunohistochemistry. Quantitative analysis demonstrated that 85% of terminals synapsed with unlabeled
dendrites, 12% with ChAT-IR dendrites and 3% with
unlabeled terminals. These results might suggest that
anterogradely labeled synapses with ChAT-IR elements
were relatively infrequent. Because ChAT-IR dendrites
comprise only 6–14% of dendrites in the neuropil (Steininger et al., 1997), only 12% of terminals synapsed with
ChAT-IR dendrites indicates a relatively even distribution
of the projection between cholinergic and noncholinergic
dendrites. The proportion of anterogradely labeled terminals synapsing with ChAT-IR dendrites (2–4%), for example, was determined to be similar to that of anterogradely labeled terminals synapsing in the neuropil.
Analysis of ChAT-IR dendrites demonstrated that anterogradely labeled terminals accounted for 2–4% of their total
synaptic contacts. This proportion should be considered a
minimum for dorsal raphe inputs to the PPT, due to
experimental limitations such as the inability to involve
the entire DR in the injection site, penetration of visualization reagents, and difficulties in the preservation of membrane and synaptic structures.
Because this is the first quantitative study of afferent
innervation of the cholinergic PPT neurons, there is no
basis for comparison of the proportion of inputs from the
dorsal raphe with other sources of synaptic input. The
proportion of input to PPT neurons from the DR (2–4%),
which was likely to be an underestimate of the true level of
innervation, however, represents an appreciable input.
This raphe-PPT input, for example, approximates that
described for thalamocortical synapses on pyramidal cell
dendrites (6–20%; White and Hersch, 1981), 1.5–6.8% for
corticocortical neurons (White and Hersch, 1982), and only
0.3–0.9% for corticostriatal synapses on medium spiny
cells (Hersch and White, 1982).
SERT-immunoreactive terminals
in the PPT-pc
In the present light microscopic experiments, a rather
dense plexus of SERT-IR axonal processes was observed in
the region of cholinergic PPT neurons. Labeled axons and
varicosities were more evenly distributed along the full
rostral-to-caudal extent of ChAT-IR PPT somata when
compared to the distribution of anterogradely labeled DR
efferents, as has been discussed above. This observation
reflects either less than maximal labeling of DR efferents
with the anterograde tracer or the presence of additional
serotonergic innervation to the PPT from other nuclei,
such as the median raphe, supralemniscal nucleus (i.e.,
the B9 cell group) or raphe magnus (e.g., see Semba and
Fibiger, 1992; Honda and Semba, 1994). At the ultrastructural level, SERT immunoreactivity was localized primarily to presynaptic axonal elements and much less so to
axonal processes. No dendritic or somatic structures were
found to be SERT-IR. SERT-IR terminals were indistin-
318
guishable from anterogradely labeled terminals on the
basis of their morphology, size, vesicle morphology and
presence of mitochondria. Furthermore, the type of synaptic specialization and the distribution of postsynaptic
targets were similar for both classes of terminals. Both
SERT-IR and anterogradely labeled DR terminals, for
example, primarily contacted small caliber, noncholinergic
and cholinergic dendrites in equal proportions, and were
equally as likely to describe symmetrical as well as asymmetrical membrane specializations. The two classes of
labeled terminals were also found to constitute 2–4% of the
synaptic input to cholinergic dendrites. These observations are consistent with the hypothesis that the majority,
if not all, DR efferents directed at the PPT-pc are serotonergic. Of the SERT-IR terminals analyzed, however, fewer
were found to synapse with ChAT-IR dendrites when
compared to anterogradely labeled DR efferents (i.e., 6%
vs. 12%). The most parsimonius explanation for this
discrepancy is that SERT-IR may have been suboptimally
labeled secondary to methodological considerations. Immuno-electron microscopy, for example, is well recognized
to be plagued by perfusion/fixation conditions that may
adversely affect antigenicity and/or penetration of immunoreagents, especially when compared to the less stringent
conditions required for visualization of BD. Alternatively,
decreased labeling for SERT-IR, when compared to DRlabeled efferents, may reflect the presence of other neurochemical inputs (i.e., nonserotonergic projections) from the
DR to ChAT-IR dendrites in anterogradely labeled material. Although it is known that 40–60% of subprimate DR
neurons do not employ 5-HT as a neurotransmitter (Moore,
1981; Descarries et al., 1982; Léger and Wiklund, 1982),
quantitative data on the neurotransmitter content of the
nonserotonergic DR neurons and/or potential coexpression
of neurotransmitters are either conflicting or sparse. As
many as 10% of neurons in the DR appear to be GABAergic
(Stamp and Semba, 1995), with dopaminergic neurons
described as sparse and diffusely distributed throughout
the DR (personal observations; Ochi and Shimizu, 1978;
Hökfelt et al., 1984; Geffard et al., 1987). Substance P
colocalizes with 5-HT in a substantial population of DR
neurons; however, the degree to which substance P localizes by itself to a unique population of DR neurons is
unknown (Chan-Palay et al., 1978; Hökfelt et al., 1984;
Baker et al., 1991). The projection patterns of these
neurons and the remainder of non-5-HT DR somata are
unknown, and whether they might project to cholinergic
PPT neurons will need to be addressed in future studies.
The ultrastructural characteristics of SERT-IR and anterogradely labeled DR terminals in the PPT-pc were
similar to those described previously for 5-HT terminals in
the rat LDT (Honda and Semba, 1994). The morphology
and micro-environment of SERT-IR and/or DR-labeled
terminals analyzed here also demonstrated features in
common with 5-HT terminals described in several other
brain regions. In several nuclei of the basal ganglia (Pasik
et al., 1984; Corvaja et al., 1993), for example, a high
proportion (up to 40%) of 5-HT terminals demonstrated
subjunctional dense bodies, compared to the 16% of asymmetrical terminals containing subjunctional dense bodies
recognized here. Although no attempts were made to
quantify and distinguish features of the micro-environment of SERT-IR and/or DR-labeled terminals from nonlabeled terminals, it was our impression that they were more
frequently apposed to unlabeled axon terminals and fewer
T.L. STEININGER ET AL.
displayed classic synaptic junctions when compared to
unlabeled terminals. These features have previously been
noted in serial thin sections of 5-HT terminals in the rat
striatum (Soghomonian et al., 1989) and hippocampus
(Oleskevich et al., 1991). The findings that 5-HT innervation is largely nonjunctional and possibly presynaptic,
support arguments that not only in these brain regions,
but also in the PPT, 5-HT may act by volume transmission
at pre- as well as postsynaptic sites (Fuxe and Agnati,
1991). Immuno-electron microscopy for simultaneous visualization of pre- and postsynaptic markers of serotonergic
innervation will be a necessary anatomical approach to
further validate hypotheses regarding volume transmission. Finally, in noting that ,50% of SERT-IR and/or
DR-labeled synaptic contacts make asymmetrical membrane specializations in the PPT-pc, our results vary from
the rat LDT where membrane specializations described by
5-HT-IR were mostly (,70%) asymmetrical (Honda and
Semba, 1994). Asymmetrical synaptic specializations also
appear to be characteristic of 5-HT-containing terminals in
other brain regions, such as the striatum (Pasik et al.,
1984; Corvaja et al., 1993), globus pallidus (Pasik et al.,
1984), substantia nigra (Corvaja et al., 1993), and hippocampus (Oleskevich et al., 1991). Our tendency to find a
lesser frequency of asymmetric membrane specializations
reflects either a unique feature of the rat PPT-pc, age or
strain differences in the rats investigated, or possibly an
inability to accurately distinguish synaptic features secondary to inadequate tissue preservation of ‘‘masking’’ by
peroxidase-benzidine precipitates.
Physiological and behavior significance
of 5-HT in the PPT-pc
The effect of serotonin on guinea pig PPT neurons
(Muhlethaler et al., 1990; Leonard and Llinas, 1994) and
rat LDT neurons (Luebke et al., 1992) has been studied
with intracellular electrophysiological recording in the in
vitro slice preparation. These studies demonstrate that
serotonin potently hyperpolarizes up to 90% of physiologically (Muhlethaler et al., 1990) or histologically identified
(Leonard and Llinas, 1994) cholinergic PPT neurons via
activation of an outward K1 conductance. This effect
appears to be mediated by postsynaptic 5-HT1 receptors,
because hyperpolarization was unaffected by tetrodotoxin,
blocked by the 5-HT antagonistic properties of spiperone
(Leonard and Llinas, 1994), and mimicked by the agonists
(1)-8-OH-DPAT (Leonard and Llinas, 1994) or 5-carboxamidotryptamine (Luebke et al., 1992). These findings are
difficult to reconcile with autoradiographic ligand binding
studies which have failed to localize 5-HT1 receptors in the
PPT or LDT (Pazos and Palacios, 1985). Moreover, while
intense 5-HT2 receptor immunoreactivity has been localized to cholinergic PPT and LDT neurons, but not noncholinergic neurons, in the rat (Morilak et al., 1993; Morilak
and Ciaranello, 1993), this receptor subtype appears to
mediate depolarizing responses (Bobker and Williams,
1991). The serotonin receptor subtype-mediating hyperpolarization of cholinergic PPT neurons, therefore, remains
unclear and is certain to attract renewed attention as more
specific and sensitive techniques for investigation of multiple 5-HT receptor subtypes become available.
The intrinsic electrophysiological properties of cholinergic LDT/PPT neurons recognized by different laboratories
are in disagreement, so it remains unclear how the excitability and discharge patterns of cholinergic cells are
DORSAL RAPHE AFFERENTS TO THE PPT
affected by the presence or absence of serotonin. Many
cholinergic LDT cells from neonatal rat, for example,
display low-threshold bursting secondary to activation of a
voltage-sensitive calcium current in vitro (Luebke et al.,
1992). It has therefore been hypothesized that during
waking, when serotonergic dorsal raphe neuronal activity
is maximal (McGinty and Harper, 1976; Trulson et al.,
1981), bursting cholinergic neurons are relatively quiescent unless subject to sufficient depolarizing influences
(Steriade et al., 1990b; Steriade, 1992). Conversely, during
REM sleep when serotonergic dorsal raphe neurons cease
firing (McGinty and Harper, 1976; Trulson et al., 1981),
inhibition of cholinergic bursting neurons is removed. On
the basis of these data, generation of a burst firing mode
that favors PGO wave production during REM sleep
requires deinactivation of the low-threshold calcium current by transient hyperpolarizing synaptic events. Both
local cholinergic (Luebke et al., 1992; Leonard and Llinas,
1994) and GABAergic basal ganglia afferents (Datta et al.,
1991; Rye et al., 1996; Rye and Bliwise, 1997) have been
proposed as sources for these hyperpolarizing influences.
Further evidence in support of a serotonergic dorsal raphe
modulation of the discharge pattern of bursting cholinergic LDT/PPT neurons, and thereby PGO wave production,
derives from several observations: (1) DR neurons cease
firing immediately prior to the onset of each PGO spike
(McGinty and Harper, 1976); (2) PGO waves are ‘‘released’’
in wake, and non-REM (NREM) sleep follows chemical
depletion of serotonin (Dement et al., 1972; Jouvet, 1972),
reversible cooling or lesions of the raphe (Jouvet, 1972), or
parasaggital cuts that isolate the raphe from the PPT
(Simon et al., 1973); (3) PGO burst neurons are antidromically activated from the lateral geniculate nucleus (McCarley et al., 1978; Sakai and Jouvet, 1980; Nelson et al., 1983;
Sakai, 1985), whose exclusive source of tegmental afferents derives from cholinergic neurons in the PPT, and to a
lesser extent, LDT (Sakai, 1980; Hallanger et al., 1987;
Bickford et al., 1995; Wilson et al., 1995; J.R. Wilson and
D.B. Rye, personal observations); and (4) PGO waves can
be completely suppressed by injections of cholinergic (nicotinic) antagonists into the lateral geniculate nucleus (RuchMonachon et al., 1976; Hu et al., 1988), and lesions (Sakai,
1980; Webster and Jones, 1988) or reversible cooling
(Laurent and Alayaguerrero, 1975) of the PPT region. The
modest serotonergic dorsal raphe innervation to cholinergic PPT neurons demonstrated in the present study provides the first anatomical evidence in support of the
hypothesis that 5-HT derived from the DR modulates
phasic REM sleep phenomenology via modulation of the
discharge of bursting cholinergic neurons. Inconsistent
with this hypothesis are independent in vitro analyses of
PPT neurons in the adult rat (Kang and Kitai, 1990) and
guinea pig (Leonard and Llinas, 1994), and in vivo recordings in the adult cat (Steriade et al., 1990b), that suggest
that the vast majority of low-threshold burst neurons are
noncholinergic while nonbursting neurons are cholinergic.
The validity of this hypothesis is also questioned by
observations that the release of PGO waves requires
lesions of several serotonergic raphe nuclei rather than
lesions involving only the dorsal raphe (Simon et al., 1973).
Our findings of a more robust and uniform innervation of
the PPT region, particularly its rostal portion, by SERT-IR
when compared to labeled DR efferents suggest the existence of an additional serotonergic innervation to the PPT.
In fact, other investigators have noted potential serotoner-
319
gic afferents to the PPT from the median raphe, supralemniscal nucleus (i.e., the B9 cell group) and/or raphe magnus (e.g., see Semba and Fibiger, 1992; Honda and Semba,
1994). It remains to be determined if these putative
afferents to the PPT are indeed serotonergic, and what
their role is, if any, in modulating REM sleep.
Models of serotonergic dorsal raphe/PPT interactions
and REM sleep control that focus solely on PGO burst/
cholinergic neurons largely ignore the status of the substantial populations of nonbursting and/or noncholinergic (Rye
et al., 1987) neurons in the LDT/PPT region. Based on in
vivo studies, for example, PGO burst neurons represent
only 9% of all neurons in the LDT/PPT region, with
non-PGO-related neurons and PGO-related, but nonbursting, neurons constituting 76% and 15% of the total recorded cell population, respectively (Steriade et al.,
1990a,b). Tonically active, non-PGO-related neurons with
efferent projections outside of the lateral geniculate nucleus
may modulate tonic aspects of REM sleep such as muscle
atonia and/or EEG desynchronization (Steriade and McCarley, 1990). The majority of these cells, particularly those
which are cholinergic, are also hyperpolarized by 5-HT
(Luebke et al., 1992; Leonard and Llinas, 1994). The
effects of 5-HT on noncholinergic neurons in the LDT/PPT
region are less well described, possibly because their small
size precludes ready intracellular sampling and voltage
clamping. In the neonatal rat LDT, 6/7 bursting noncholinergic neurons were hyperpolarized by 5-HT, whereas only
2/8 nonbursting, noncholinergic neurons were similarly
hyperpolarized (Luebke et al., 1992). In the adult guinea
pig PPT, the hyperpolarizing effects of 5-HT on noncholinergic neurons are less ubiquitous, and when observed are
generally weak (Leonard and Llinas, 1994; C. Leonard,
personal communication). One might have predicted a
more widespread response, given the present observations
that dorsal raphe/serotonergic afferents to noncholinergic
elements in the PPT region are as prevalent as those on
cholinergic dendrites. These noncholinergic neurons are
neurochemically and connectionally heterogeneous, and it
has also been speculated that at least some are GABAergic
interneurons (Scarnati et al., 1988; Jones, 1991). Putative
dorsal raphe/serotonergic projections to noncholinergic
interneurons may indirectly affect the functioning of cholinergic PPT neurons. Alternatively, dorsal raphe/serotonergic afferents of the PPT region may contact noncholinergic, presumptively glutamatergic, neurons which project
to the basal ganglia (Rye et al., 1987; Lee et al., 1988;
Lavoie and Parent, 1994a,b), midline thalamic nuclei
(Hallanger et al., 1987), and pontine (Mitani et al., 1988;
Yasui et al., 1990; Lai et al., 1993) and medullary (Rye et
al., 1988; Shiromani et al., 1990; Yasui et al., 1990)
reticular fields. It will be critical to determine if neurons
classified as to their cholinergic/noncholinergic status, as
well as their bursting status, display unique patterns of
efferents, and if they do, whether they are differentially
affected by serotonin. Such information will come when
antidromic identification of a neuron’s projection pattern is
combined with a determination of its neurochemical identity in the in vitro slice preparation.
In summary, serotonergic/dorsal raphe projections to the
PPT region are particularly dense in the dorsal, compact
portion of the nucleus where they primarily contact small
caliber, noncholinergic and cholinergic dendrites in equal
proportions. This afferent source of the PPT region is likely
to play a key role in neuromodulation of behavioral state
320
T.L. STEININGER ET AL.
given the DR’s well-described state-related alterations in
activity; i.e., serotonergic/dorsal raphe ‘‘tone’’ is a potent
determinant of the ultimate discharge pattern of a majority of cholinergic and noncholinergic neurons in the PPT
region. Specific REM sleep-related physiological activity is
then dependent not only on the intrinsic electrophysiological properties of individual cholinergic and noncholinergic
neurons, but also on their unique afferent connectivity
(i.e., excitatory or inhibitory synaptic inputs). Putative
sources of these afferent connections are numerous and
originate from widespread brain regions (Steininger et al.,
1992). A more comprehensive picture of the determinants
of PPT activity will emerge when detailed quantitative
analyses of the synaptology of identified afferents, as
presented here, is applied to some of these putative
afferent sources. Determination of the uniqueness of the
efferent connections of neurons contacted by these afferents may ultimately identify specific neuronal populations
and neural circuits that subserve individual components of
REM sleep (e.g., see Discussion in Rye et al., 1996).
Elucidation of the detailed synaptic circuitry of the PPT
region is likely to impact directly upon our understanding
of diseases characterized by abnormalities of REM sleep
such as depression (McCarley, 1982; Steriade and McCarley, 1992; Gillin et al., 1993), schizophrenia (Karson et al.,
1991; Yeomans, 1995), narcolepsy (Aldrich, 1991), and
REM sleep behavior disorder (Rye and Bliwise, 1997).
ACKNOWLEDGMENTS
The authors express their thanks to Drs. Steven Hersch
and Allan Levey for their critical insights into the experimental design and analysis as well as reading of the
manuscript. The secretarial assistance of Ms. Katy Hair
and the technical assistance of S. Edmunds and H. Rees
are also gratefully acknowledged. This work was supported by the National Institutes of Health, NS-17661-14
(T.L.S.,B.H.W.), DA-07390 (R.D.B.), and a Cotzias Fellowship Award from the American Parkinson’s Disease Association (D.B.R.).
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dorsal, light, electro, regions, raphe, immunohistochemical, stud, projections, microscopy, tracing, pedunculopontine, noncholinergic, neurons, tegmental, serotonergic, nucleus, cholinergic, anterograde
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