Colocalization of γ-aminobutyric acid and acetylcholine in neurons in the laterodorsal and...

www.elsevier.com/locate/brainresBrain Research 992 (2003) 205–219

Research report

Colocalization of g-aminobutyric acid and acetylcholine in neurons in the

laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and

electron microscopic study

Hong-Ge Jiaa, Jack Yamuya,*, Sharon Sampognab, Francisco R. Moralesa, Michael H. Chasea,b

aDepartment of Physiology, UCLA School of Medicine, Los Angeles, CA 90095, USAbBrain Research Institute, UCLA, Los Angeles, CA 90095, USA

Accepted 21 August 2003

Abstract

Cholinergic and g-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in

the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in

this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light

microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the

laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons

in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was

observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed

symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and

GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA

immunoreactive, whereas only 6–8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA

immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive

terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles,

approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT

and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/

PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing

possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of

behavioral states.

D 2003 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: biological rhythms and sleep

Keywords: Brainstem; GABA; Acetylcholine; Immunohistochemistry; Active sleep; Colocalization

1. Introduction of the activity of cholinergic neurons in these regions

The laterodorsal tegmental nucleus (LDT) and the

pedunculopontine tegmental nucleus (PPT) in the brain-

stem play a critical role in active (REM) sleep and

wakefulness [19]. In these nuclei, there are large numbers

of cholinergic and GABAergic neurons [13,20,29,56,57].

Active sleep is thought to be generated, in part, as a result

0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.08.062

* Corresponding author. Tel.: +1-310-825-3348; fax: +1-310-206-3499.

E-mail address: [email protected] (J. Yamuy).

because they discharge at higher rates immediately prior

to and during active sleep than during quiet sleep [23,45]

and the destruction of these neurons results in the abolition

of active sleep [21,55].

Several lines of evidence indicate that GABAergic neu-

rons in the LDT/PPT are important in the control of sleep

and wakefulness. Neurons in these nuclei that were presum-

ably GABAergic, originally designated PGO-off cells, in-

creased their firing rate during active sleep [46]. In addition,

studies that have used Fos immunohistochemistry as a

H.-G. Jia et al. / Brain Research 992 (2003) 205–219206

functional marker indicate that LDT/PPT GABAergic neu-

rons discharge during active sleep [29,49]. Furthermore, we

have recently reported that the microinjection of g-amino-

butyric acid (GABA)A agonists into the PPT enhances active

sleep [50]. Therefore, it is reasonable to expect that local

interactions between GABAergic and cholinergic neurons

play a role in the mechanisms that control the states of

active sleep and wakefulness.

In the present study, using a combination of immunocy-

tochemical techniques, we describe the essential morpho-

logical features of cholinergic and GABAergic neurons and

their processes in the LDT/PPT. In addition, we focus on a

surprising observation that was made during the course of

these experiments, namely, that choline acetyltransferase

(ChAT) and GABA are colocalized in the somas and

synaptic terminals of LDT/PPT neurons. This finding indi-

cates that a subpopulation of cholinergic neurons are also

GABAergic, which provides new data that must be inte-

grated into current hypotheses dealing with the mechanisms

of action of mesopontine neurons in the control of active

sleep and wakefulness.

Fig. 1. Immunofluorescence photomicrographs of GABA+ and ChAT+ labeled neu

ChAT-IR (A–B; rhodamine B sulfonyl chloride, red), GABA-IR (C–D; dichloro

double-labeled neurons (E–F; yellow cells indicated by arrowheads). A, C, and

2. Materials and methods

2.1. Animals

Seven adult male cats, weighing 3.5–5.0 kg, were

utilized in the present study. Three cats were used for light

microscopy; four cats were employed for electron micros-

copy (EM). All experimental procedures were conducted in

accordance with the Guide for the Care and Use of Labo-

ratory Animals (NIH Publications No. 80-23) and approved

by the Chancellor’s Animal Research Committee of the

University of California Los Angeles Office for the Protec-

tion of Research Subjects.

2.2. Perfusion and tissue preparation

Under deep anesthesia (Nembutal, 50 mg/kg, i.v.), cats

were perfused transcardially with 1 l of heparinized saline,

followed by 3 l of a fixative solution containing 4%

paraformaldehyde (for light microscopy) or a solution con-

taining 2% paraformaldehyde and 2% glutaraldehyde (for

rons in the LDT and PPT of the cat. (A–F) Immunofluorescence staining for

triazinylamino fluorescein-green, using tyramide signal amplification) and

E in the LDT; B, D, and F in the PPT. Scale bars: A–F = 100 Am.

Fig. 2. Confocal photomicrographs of a neuron in the LDT: (A) GABA immunoreactivity; (B) ChAT immunoreactivity. The soma-dendritic profile of this

neuron, shown at several depths, exhibits immunoreactivity for both GABA and ChAT. In addition, there is a dense collection of GABA+ terminals

surrounding this neuron. Scale bar: 25 Am.

H.-G. Jia et al. / Brain Research 992 (2003) 205–219 207

EM) in 0.1 M phosphate buffer (PB, pH 7.4). The brainstem

was removed and postfixed overnight in the same fixative

used for perfusion. Tissue blocks for light microscopy were

Fig. 3. (A and B) ChAT immunostaining using single BDHC immunoperoxidase.

magnification, two ChAT immunoreactive neurons (indicated by numerals 1 and 2

GABAwhich yields a diffuse brown staining, and BDHC for ChAT which yields a

(arrow) and several small GABA-IR single labeled neurons (arrowheads) in the L

segment of a dendrite (between two arrowheads) contains smooth brown DAB st

neuron in the PPT. Scale bars: A, D = 100 Am; B, C, E and F = 50 Am.

placed in 0.1 M PB containing 25% sucrose for cryopro-

tection. The brainstem was cut in 15-Am-thick sections on a

Reichert-Jung cryostat. The blocks of tissue for EM were

The reaction product appears as dark-blue granules; B depicts, at a higher

in A); (C–F) double-labeling using two different chromogens, i.e., DAB for

dark-blue granular reaction product. (C–D) A large double-labeled neuron

DT (C) and PPT (D); (E) a double-labeled neuron in the LDT. Note that a

aining but no BDHC granular staining; (F) an example of a double-labeled

Fig. 5. Electron micrographs of pre-embedding immunocytochemistry for

GABA highlighting GABA-IR terminals and dendrites. (A) Note the

different packing densities of vesicles in GABA-IR terminals (T1 and T2)

in the LDT. The dendrite (d) has a lower immunoreactive density than that

present in the two terminals. (B) A GABA-IR terminal (T) forms synaptic

contact with a GABA-IR dendrite in the PPT. Scale bar = 1 Am in A, 0.5 Amin B.


cut into 50-Am-thick sections on a vibratome. The sections

were treated with 1% sodium borohydride in 0.1 M PB for

30 min and rinsed in PB.

2.3. Immunocytochemical procedures

2.3.1. Double immunostaining for light microscopy

Double immunofluorescence was performed using tyra-

mide signal amplification (Renaissance TSA-Direct, NEN,

Boston, MA) and lissamine rhodamine B sulfonyl chloride

(LRSC). After treatment with 6% normal donkey serum,

sections were incubated overnight in the first primary anti-

body at a dilution of 1:1000 for rabbit anti-GABA (Chem-

icon International, Temecula, CA) or 1:8000 for guinea pig

anti-GABA (Protos Biotech, NY). Either biotinylated don-

key anti-rabbit (for rabbit anti-GABA) or anti-guinea pig (for

guinea pig-anti-GABA) IgG was applied for 1 h at room

temperature. After rinsing, the tissue was treated with 0.5%

of the blocking reagent from the fluorescein-tyramide kit for

30 min; it was then incubated in streptavidin-horseradish

peroxidase for 30 min, washed and then incubated in green

fluorescein-tyramide diluted 1:100 in amplification diluent

for 6 min. Sections were then washed and the second primary

goat ChAT antibody diluted 1:1000 (Chemicon Internation-

al) was applied overnight. The second primary antibody was

revealed by an hour of incubation with LRSC-conjugated

donkey anti-goat IgG (1:200; Jackson, West Grove, PA). The

sections were washed and mounted on glass slides in

Vectashield (Vector Laboratory, Burlingame, CA). Sections

were viewed and photomicrographs were taken by using a

Nikon fluorescent microscope equipped with a spot camera.

Selected sections were also viewed through a Leica TCS

confocal microscope in order to confirm the location of

GABA and ChAT immunoreactivity within individual cells

in the LDT/PPT. Double immunofluorescent images were

displayed as dual-color merged images in TIFF format using

Adobe Photoshop software.

Fig. 4. Electron micrograph of pre-embedding immunocytochemistry for

GABA of a GABA-IR cell body in the LDT. Note that the GABA-IR cell is

small sized, has a round shape, and a bulky nucleus with a deep indentation.

Scale bar = 1 Am.

In double immunofluorescence labeling experiments,

when TSA was replaced by FITC-conjugated donkey anti-

guinea pig or anti-rabbit IgG, GABA-IR cell bodies were

rarely observed in the LDT/PPT; in this case, the content of

GABA in cell bodies in this region was probably too low to

be detected by standard, indirect, immunofluorescence

labeling.

To confirm the results of the immunofluorescence experi-

ments, another double labeling procedure was performed

using two different chromogens. Briefly, after being treated

with 3% hydrogen peroxide (to inactivate endogenous

peroxidases) and 2% normal donkey serum, the sections

were incubated overnight in guinea pig anti-GABA anti-

body (1:4000 in 0.05 M PBS containing 0.2% Triton X-

100). The following day, the sections were incubated for 2

Table 1

Number of neuronal elements of pre-embedding immunoreactivity for

GABA in the LDT and PPT

Terminals Dendrites Fibers Cell bodies Total

LDT 61.7% (79) 29.7% (38) 4.7% (6) 3.9% (5) 100% (128)

PPT 64.8% (70) 22.2% (24) 11.1% (12) 1.9% (2) 100% (108)


h at room temperature in biotinylated donkey anti-guinea

pig IgG (1:300). Finally, sections were incubated in ABC

complex for another 2 h. Bound peroxidase was revealed

by placing the sections in a solution containing 0.05%

diaminobenzidine (DAB) and 0.003% H2O2 in 0.05 M

Tris–HCl buffer (TB; pH 7.6) for 15–30 min. The brain-

stem sections were then processed for ChAT immunocyto-

chemistry following the procedure described above, with

the exception that bound peroxidase was revealed by

benzidine dihydrochloride (BDHC), which produces a

granular reaction product [26]. In order to avoid the

possibility that the combination of the DAB and BDHC

methods could confound the results from double-labeling

experiments (see Discussion), the following procedures

were carried out. In the BDHC method, pH was restricted

to the range of 6.6–6.8 to maintain the blue color of the

BDHC reaction product. Additionally, in each experiment,

a number of brainstem sections were processed for ChAT

Fig. 6. Electron micrographs of pre-embedding immunocytochemistry for cholin

immunogold staining for GABA (immunogold particles) illustrating typical GAB

LDT, one (T1) of which is also ChAT-IR. (B) A non-labeled (non-GABA, non-ChA

in the LDT. (C) A GABA-IR terminal in the PPT forms an asymmetric synaptic

asymmetric synapse with a ChAT-IR dendrite (d) in the LDT. Scale bars = 0.5 Am

immunocytochemistry using the BDHC method while

omitting GABA immunostaining using the DAB immuno-

peroxidase method. The sections were thoroughly washed

between each step with phosphate-buffered saline (PBS)

and, thereafter, were mounted, dehydrated, cleared and

coverslipped. Photomicrographs were taken using an Olym-

pus BX60 microscope.

2.3.2. Pre-embedding immunocytochemistry for electron

microscopy

GABA and ChAT were separately revealed by pre-

embedding immunocytochemistry in two sets of histological

sections that included the LDT/PPT. The immunocytochem-

ical procedures for EM were the same as those utilized for

GABA light microscopic immunostaining, except that Tri-

ton X-100 was not used.

Following the completion of the immunocytochemical

reactions, the sections were washed with 0.1 M PB for 30

e acetyltransferase (ChAT, DAB reaction) combined with post-embedding

A and ChAT immunoreactive profiles. (A) Two GABA-IR terminals in the

T) terminal (T) forms an asymmetric synapse with a GABA-IR dendrite (d)

contact with a non-labeled dendrite (d). (D) A ChAT-IR terminal forms an

in A, B and C, 1 Am in D.

Fig. 7. Histograms illustrating quantitative GABA data from electron

micrographs of randomly selected areas of the LDT and PPT. (A)

Frequency of various densities of gold particles in 134 terminals in the

electron micrographs. The lower density group (left) contains gold particles

from 0 to 30 grains/Am2. Note that most terminals of this group exhibit less

than 10 grains/Am2. The higher density group (right) contains more than 40

grains/Am2. Therefore, 40 grains/Am2 was used as the labeling threshold for

GABA. (B) Comparison of gold particle density in GABA positive (z 40

grains/Am2) terminals (left column, n= 90) and their post-synaptic or

contacted dendrites (right column, n= 91). MeansF standard deviations of

gold particle density are presented for the two group profiles. Student’s

t-test: p< 0.0001. (C) Frequency of various densities of gold particles in

dendrites in the electron micrographs. Most dendrites contained less than 20

grains/Am2, although a few contained >40 grains/Am2 (positive criterion for

terminals).


min and then postfixed for 1 h in osmium tetroxide (1% in

PB). Subsequently, the sections were dehydrated through a

graded series of ethanol alcohol and propylene oxide,

embedded in Epon-812 on glass slides, and cured at 60

jC for 24 h. Uranyl acetate (2%) was added to the 70%

ethanol alcohol for 40 min to improve contrast. After

curing, the sections were examined under a light micro-

scope, and the area that included the LDT/PPT was

removed and glued to plastic cylinders for ultrathin (500

A) sectioning. The ultrathin sections that were immunos-

tained for GABA were mounted on Formvar-coated 50

mesh copper grids. After being counterstained with uranyl

acetate and lead citrate for 5 min each, the sections were

examined directly in a JEOL 200CX electron microscope.

The ultrathin sections immunostained for ChAT prior to

embedding were mounted on 200-mesh uncoated nickel

grids and stained by post-embedding immunogold to reveal

the endogenous GABA.

2.3.3. Post-embedding GABA immunocytochemistry

The post-embedding immunocytochemical procedures

for GABA immunocytochemistry have been previously

described in detail [17,18,34]. Briefly, grids were washed

with Tris-buffered saline containing 0.1% Triton X-100

(TBST), pH 7.6, incubated overnight at room temperature

in either rabbit (1:600) or guinea pig (1:8000) anti-GABA

serum, rinsed in TBST (pH 7.6) and then transferred to

TBST (pH 8.2). Grids exposed to rabbit anti-GABA serum

were incubated for 1 h in goat anti-rabbit IgG conjugated

with 15-nm gold particles (Amersham, NJ, 1:25 in TBST,

pH 8.2). Grids exposed to guinea pig anti-GABA serum

were incubated for 1 h in donkey anti-guinea pig IgG

conjugated with 18-nm gold particles (Accurate Chemical

and Scientific, NY, 1:25 in TBST, pH 8.2). Following

rinsing, ultrathin sections were counterstained with uranyl

acetate and lead citrate for 5 min each and then examined

using the electron microscope.

2.3.4. Control experiments

In every experiment, including both the pre- and post-

embedding stages of immunocytochemistry, control sections

were used in which phosphate-buffered saline or non-

immune normal serum (from the same animal species as

that of the secondary antibody) were substituted for the

primary antibody. All of these control brainstem sections

were devoid of immunoreactivity.

2.4. Analysis procedures

To conclude that any particular profile was GABA

positive in post-embedding immuno-electron microscopy,

the following procedure was employed: electron micro-

graphs were first taken from randomly selected areas of

ultrathin sections of the LDT/PPT. Only one ultrathin section

was selected from each block to avoid repeatedly counting

each profile. Micrographs were then scanned by using a


ScanJet-II scanner and Photoshop 6.0 software on a Macin-

tosh G3 computer. Gold particles within each profile, ex-

cluding those that were associated with mitochondria in

order to avoid possible false positive data [34], were counted

and profile areas were measured by using the NIH Image

analysis software. The number of grains per square microm-

eter was then employed to assess whether profiles were

GABA positive or negative.

Fig. 8. Electron micrographs of pre-embedding for ChAT combined with post-em

and ChAT in the LDT. (A) Two GABA/ChAT double-labeled terminals (T1 and T2

symmetric synapse. (B) A GABA/ChAT double-labeled terminal (T). (C) The s

decreases because the section was deeper in the pre-embedded tissue than the sec

Arrowheads in B and C indicate the same synapse in serial sections. Scale bars =

3. Results

3.1. Light microscopy

In studies using fluorescent double-labeling, GABA-

immunoreactivity appeared green (fluorescein-tyramide);

ChAT-IR was revealed in red (LRSC; Fig. 1A–F).

GABA-IR was observed both in somas and neuronal pro-

bedding immunogold staining for GABA showing colocalization of GABA

) which contain large dense-cored vesicles (arrows). Arrowheads point to a

ame terminal (TV) from an adjacent ultrathin section, in which ChAT-IR

tion in B; note that post-embedded immunogold staining remains positive.

1 Am in A, 0.5 Am in B and C.


cesses. Most GABA-IR neurons were small (10–20 Am),

although some medium to large-sized cells (20–40 Am)

were also observed. Both GABA antibodies that were used

yielded the same results.

ChAT-IR was present mainly in cell bodies and proximal

dendrites. Throughout the LDT/PPT, ChAT and GABA

immunoreactivities were observed in the same cell. GABA

was colocalized with ChAT in cells that were considered to

be typically cholinergic neurons on the basis of their size

and shape (20–30 Am in diameter, fusiform or multipolar in

shape [20]). The proportion of GABA/ChAT double-labeled

neurons among all ChAT-IR neurons could not be deter-

mined precisely in this immunofluorescent study because

staining of the fibers and terminals often obscured soma

staining (Fig. 1C–F). However, based on an examination of

more than 20 sections from three cats, it appeared that

Fig. 9. Electron micrographs from two adjacent serial ultrathin sections showing G

single-labeled GABA-IR terminals. Arrowheads point to synapses. Scale bars = 0

approximately 50% of the ChAT-IR neurons were also

GABA-IR.

Because the intense GABA-IR staining of fibers often

interfered with the observation of soma staining when

traditional immunofluorescent microscopy was used, dou-

ble-labeled sections were also examined with a confocal

microscope. This technique provides for the visualization of

immunostaining at different depths of the tissue section,

which is critical in order to determine whether the antigen is

located within the cytoplasm of a particular cell. Confocal

images for GABA and ChAT immunoreactivities in the

LDT/PPT were identical to those observed using the immu-

nofluorescent microscope (Fig. 2A and B). Note, that by

focusing at different tissue depths, the colocalization of

GABA and ChAT immunoreactivities can clearly be seen

within the soma of the same neuron (Fig. 2). In addition,

ABA/ChAT colocalization (T1 and TV) in the PPT. T2 and T2Vare adjacent.5 Am in A and B.

Table 2

Postsynaptic targets of ChAT-IR and ChAT/GABA-IR terminals in LDT

and PPT

LDT PPT

ChAT+ ChAT/

GABA+

Total ChAT+ ChAT/

GABA+

Total

Non-Dena 146

(64.6%)

58

(25.7%)

204

(90.3%)

76

(67.9%)

22

(19.6%)

98

(87.5%)

ChAT-Den 16

(7.1%)

6

(2.6%)

22

(9.7%)

12

(10.7%)

2

(1.8%)

14

(12.5%)

Total 162

(71.7%)

64

(28.3%)

226

(100%)

88

(78.6%)

24

(21.4%)

112

(100%)

a Den, dendrites; Non-Den, Non-immunoreactive dendrites.


GABA-IR terminals were also observed both within the

neuropil and surrounding ChAT-IR cells.

In single immunostained sections processed with the

BDHC method, ChAT-IR was observed as blue or dark-blue

granules with virtually no smooth background staining (Fig.

3A and B). In sections that were immunostained using two

color chromogens to reveal different antigens (GABA with

DAB and ChAT with BDHC), double-labeled somas were

also present in which BDHC dark blue granules of ChAT

immunoreactivity were visualized against the DAB diffuse

brown product of GABA immunoreactivity (Fig. 3C–F).

3.2. Electron microscopy

3.2.1. Pre-embedding immunocytochemistry for GABA

Labeling with GABA antibody produced weak to mod-

erate patterns of reaction in somas and dendrites and strong

patterns of reaction in many axon terminals as well as in un-

myelinated and thin myelinated fibers (Figs. 4 and 5). Most

of the GABA-IR neurons were small to medium-sized (10–

25 Am in diameter), rounded or fusiform in shape, with a

bulky, deeply indented nucleus (Fig. 4). Axo-somatic syn-

apses on GABA-IR cell bodies were uncommon. Most of the

terminals that contacted GABA-IR dendrites were non-

GABA-IR. GABA-IR terminals comprised approximate

30–40% of all terminals in the neuropil of the LDT/PPT;

Fig. 10. Electron micrographs showing GABA/ChAT double-labeled

terminals in contact with ChAT-IR dendrites. (A) A GABA/ChAT-IR

terminal (T) forms a synapse with a ChAT-IR dendrite (d) in the LDT. (B) A

ChAT-IR terminal (T1) and a GABA/ChAT-IR terminal (T2), respectively,

contact a ChAT-IR dendrite in the PPT. Scale bars = 1 Am in A and B.

they contained pleomorphic and round vesicles and formed

symmetric synapses with non-GABA-labeled dendrites and

somas. Synapses between GABA-IR terminals and GABA-

IR dendrites were only occasionally observed (Fig. 5B).

A total of 128 GABA-IR profiles in the LDT and 108

GABA-IR profiles in the PPT were identified in six non-

serial sections (one section per block, two blocks from each

of three animals). In both nuclei, more than 60% of all

profiles were determined to be terminals, while approxi-

mately 22–30% were dendrites. Table 1 summarizes the

percentage distribution of each type of profile for all

GABA-IR elements.

3.3. Pre-embedding immunocytochemistry for ChAT com-

bined with post-embedding immunocytochemistry for GABA

In ultrathin sections, ChAT-IR neurons, dendrites, fibers

and terminals were identified by the presence of diffuse

electron-dense DAB reaction product; GABA-IR elements

were characterized by the presence of immunogold particles

(Fig. 6). These two patterns of immunoreactivity were easily

distinguished.

ChAT-IR perikarya contained abundant organelles, in-

cluding rough endoplasmic reticulum, Golgi apparatus,

mitochondria, microtubules and neurofilaments. ChAT-IR

dendrites contained mitochondria and microtubules. Rough

endoplasmic reticulum was also present in proximal den-

drites. Labeled dendrites were the most frequently encoun-

tered ChAT-IR profile; their diameters ranged from 0.3 to

3.0 Am. ChAT-IR axon terminals were also observed in the

LDT/PPT; they contained round and pleomorphic clear

vesicles and some large dense-cored vesicles and were

0.2–1.4 Am in diameter. ChAT-IR axon terminals formed

Table 3

Postsynaptic targets of GABAergic terminals in the LDT and PPT

Bodies Dendrites Spines Total

ChAT� ChAT+ ChAT� ChAT+ ChAT� ChAT+

LDT 4

(4.1%)

3

(3.1%)

68

(69.4%)

21

(21.4%)

2

(2%)

0 98

(100%)

PPT 3

(2.9%)

2

(1.9%)

78

(75.7%)

19

(18.5%)

1

(1%)

0 103

(100%)


symmetric and asymmetric synaptic contacts principally

with unlabeled somas or dendrites. A few ChAT-IR termi-

nals formed synapses with ChAT-IR somas or dendrites.

Tissue from animals that were perfused with the fixative

containing 2% glutaraldehyde was particularly well suited

for GABA immunostaining. With this procedure, immuno-

gold particles were found to overlay axons and terminals;

very few particles were observed in conjunction with somas,

dendrites, or glial processes. Both GABA antibodies pro-

duced the same pattern of immunostaining.

In order to determine whether a particular axon termi-

nal was GABA positive, randomly selected electron

Fig. 11. Electron micrographs showing GABA-IR terminals forming synapses (

terminal (T) and a soma in the LDT; (B) a synapse between a terminal and a dendri

dendrite in the PPT. Note that T1 is GABA-IR and T2 is a non-GABA-IR. Scale

micrographs from six sections were used to measure the

density of gold particles in different profiles (one section

per block, two blocks from each of three animals). Since

GABA antibodies tend to adhere to mitochondria, this

pattern of background staining could be misinterpreted as

specific staining. Therefore, in order to avoid the inclu-

sion of false-positive data, gold particles which were

present on mitochondria were not included in these

measurements [34].

As shown in Fig. 7A, based on the densities of gold

particles in 98 terminals, two different populations of termi-

nals were observed. In the terminals that exhibited a low

arrowheads) with ChAT-IR soma and dendrite. (A) A synapse between a

te also in the LDT; (C) two terminals (T1 and T2) in contact with a ChAT-IR

bars = 0.5 Am in A and B, 0.5 Am in C.

Table 4

The proportion of ChAT-IR somas and large dendrites that received GABA-

IR and non-GABA-IR terminals

ChAT-IR LDT PPT

Somas L-Dendrites Somas L-Dendrites

GABA+ 6 (15%) 4 (11.8%) 3 (9.4%) 4 (9.7%)

GABA+/� 12 (30%) 16 (47.1%) 10 (31.2%) 17 (41.5%)

GABA� 10 (25%) 6 (17.6%) 9 (28.1%) 13 (31.7%)

Non-Syn 12 (30%) 8 (23.5%) 10 (31.3%) 7 (17.1%)

Total 40 (100%) 34 (100%) 32 (100%) 41 (100%)


density of gold particles (left, 0–30 particles/Am2), most

contained less than 10 grains/Am2. In the population of

terminals that exhibited a high density of gold particles, all

contained more than 40 grains/Am2. Therefore, the threshold

for GABA-IR labeling was set at 40 grains/Am2, i.e.,

terminals were recognized as being immunoreactive to

GABA if they displayed a number of particles equal to or

greater than this threshold. The density of immunogold

particles in GABA terminals was much higher than the

density of particles in the dendrites (Fig. 7B). As shown in

Fig. 7C, most of the dendrites contained less than 10 grains/

Am2. Therefore, it was not possible to differentiate between

positive and negative GABA-immunoreactive dendrites,

except for a few dendrites that contained a high density of

immunogold particles. Some non-labeled terminals made

asymmetrical synapses with these dendrites (Fig. 6B). Sim-

ilarly, because of the low density of gold particles in the

somas, it was difficult to confirm the presence of GABA-IR

somas in the post-embedding immunostained material.

Immunogold-stained terminals for GABA exhibited fea-

tures that were similar to those of the pre-embedded GABA-

immunostained terminals described above. Most of these

terminals were small (0.74F 0.29 Am2) and contained small

clear vesicles which were pleomorphic and round (Fig. 6A).

The packing density of these vesicles varied considerably,

from sparse to very dense. Large dense-core vesicles were

also observed in some immunoreactive profiles, including

those terminals in which GABA and ChATwere found to be

colocalized (Fig. 8A, see below). GABA-IR terminals usu-

ally formed symmetric synapses and, occasionally, asym-

metric synapses with unlabeled somas or dendrites (Fig. 6C).

3.4. Colocalization of ChAT and GABA in axon terminals in

the LDT and PPT

A number of terminals displaying ChAT-IR also exhibited

a high density of GABA-immunogold particles (Figs. 6A,

8 and 9). These double-labeled terminals were consistently

observed in serial sections, as illustrated in Figs. 8B,C and 9,

which is a clear indication that these results were not

artifactual. The double-labeled terminals displayed the same

general morphological characteristics of the single ChATand

GABA-labeled terminals described above.

Two hundred and twenty six (226) and one hundred and

twelve (112) ChAT-IR terminals were examined in the LDT

and PPT, respectively; they were selected from six ultra-thin

sections in order to analyze the percentage of double-labeled

terminals and their postsynaptic profiles (one section per

block, two blocks from each of three animals). Of the 226

ChAT-IR terminals in the LDT, 64 (28.3%) were also GABA-

IR. ChAT/GABA-IR terminals were also present in the PPT

(Fig. 9). Of the 112 ChAT-IR terminals in the PPT, 24 (21.4%)

were GABA-IR. These double-labeled terminals usually

formed symmetric synapses with non-labeled dendrites (Figs.

8 and 9). A few double-labeled ChAT/GABA-IR terminals

were observed that made contact with ChAT-IR dendrites

(Fig. 10). The percentages for each type of the various

postsynaptic target profiles of ChAT-IR and ChAT/GABA-

IR terminals in the LDT/PPT are presented in Table 2.

The number of GABA-IR terminals was much higher

than the number of ChAT-IR terminals. Only 8.6% (8/98)

GABA-IR terminals in the LDT and 5.8% (6/103) in the

PPT were also ChAT-IR.

3.5. Postynaptic targets of GABAergic terminals in the LDT

and PPT

A total of 98 GABA-IR presynaptic terminals were

examined in the LDT and 103 in the PPT. These terminals

established symmetrical synaptic contacts with somas (ap-

proximately 7% in the LDT, 5% in the PPT), dendrites

(approximately 91% in the LDT, 94% in the PPT) and

spines (2% in the LDT, 1% in the PPT) of neurons. The

majority of neuronal profiles innervated by these GABA-IR

terminals were non-ChAT-IR (75.5% in the LDT, 79.6% in

the PPT; see Table 3).

3.6. ChAT-IR profiles received GABAergic and non-

GABAergic innervation

GABA-IR and non-GABA-IR terminals formed synap-

ses with ChAT-IR dendrites and somas (Fig. 11) in both

the LDT/PPT. Randomly selected ChAT-IR somas, large-

sized dendrites and medium-to small-sized dendrites were

counted to determine the proportion of the different pro-

files receiving GABA-IR and non-GABA-IR inputs in the

LDT/PPT.

For the present determinations, we used the following

criteria, (a) a soma was defined as a ChAT-IR profile

containing a nucleus, (b) a large-sized dendrite was defined

as a ChAT-IR profile containing rough endoplasmic reticu-

lum, but no nucleus, and (c) small-to-medium-sized den-

drites were defined as ChAT-IR profiles larger than 0.2 Amin diameter and devoid of rough endoplasmic reticulum or

synaptic vesicles.

In ChAT-IR profiles in the LDT and PPT, more than 40%

of the somas and 50% of the large-sized dendrites received

GABA-IR synaptic terminals. Of these GABA-IR terminals

that synapsed with ChAT-IR profiles, the vast majority (over

95%) were non-ChAT-IR. Only 3–5% of these GABA-IR

terminals were GABA/ChAT double-labeled (Fig. 10).

Table 5

Number of small- and medium-sized dendrites that received GABA-IR and

non-GABA-IR terminals in the LDT/PPT

LDT PPT

GABA+ 29 (32.2%) 44 (36.1%)

GABA+/� 23 (25.6%) 28 (22.9%)

GABA� 19 (21.1%) 13 (10.7%)

Non-Syn 19 (21.1%) 37 (30.3%)

Total 90 (100%) 122 (100%)


Somas and large-sized dendrites also received inputs from

non-labeled terminals. In addition, there were somas and

large-sized dendrites which did not form synaptic contacts

with any terminals (Table 4).

Of 90 small-to-medium-sized ChAT-IR dendrites in the

LDT, 32.2% received only GABAergic synaptic terminals,

25.6% received both GABAergic and non-GABAergic syn-

aptic terminals, 21.1% received only non-GABAergic ter-

minals and in 21.1%, there was no synaptic input. The

synaptic relationships in the PPT were similar to those in

the LDT (see Table 5).

4. Discussion

In the present study, GABA and ChAT immunoreactivity

was examined in the LDT/PPT of the adult cat. At the light

microscopic level, these substances were found to be

colocalized in numerous neurons of these nuclei. At the

electron microscopic level, it was shown, for the first time,

that GABA and ChATwere colocalized in axon terminals in

the LDT/PPT and that GABAergic terminals establish

synaptic contacts with cholinergic somas and dendrites in

the LDT/PPT.

4.1. Methodological considerations

The tyramide signal amplification (TSA) technique

[8,18,27] was used to detect GABA-immunoreactive neu-

rons in the LDT/PPT. Although the intense staining of

GABAergic fibers interfered, to some degree, with obser-

vation of GABA immunoreactivity in somas, the TSA

amplification and double immunofluorescence techniques

revealed that many cholinergic neurons also displayed

GABA immunoreactivity. These results were corroborated

using different chromogens, i.e., DAB and BDHC double-

labeling procedures. Most importantly, the colocalization of

GABA and ChAT immunostaining in LDT/PPT cells was

confirmed by confocal microscopy.

Double antigen localization using DAB and BDHC has

been extensively employed [9,10,14,26,40,51,53]. One ca-

veat of the BDHC immunoperoxidase technique is that the

color of the BDHC product can be converted to a smooth

brown if the pH of the tissue is higher than 7, which would

make it impossible to distinguish DAB from BDHC. To

avoid this problem, the pH of the tissue was maintained in

the range 6.6–6.8. Control, single BDHC reactions, which

were performed in conjunction with each DAB/BDHC

double-labeling experiment, exhibited blue or dark blue

granules that could be easily distinguished from DAB

smooth immunostaining.

The combination of pre-embedding immunocytochemical

labeling of ChAT with post-embedding immunogold label-

ing of GABA [4,5,7,17,18] allowed us to detect ChAT and

GABA immunoreactivities within the same axon terminals

in ultrathin sections. The immunostaining of ChAT and

GABA was not considered to be artifactual because ChAT-

IR terminals, which were devoid of gold particles, were also

present in the same section where double-labeling was

observed. In addition, anti-GABA and anti-ChAT antibodies

were from different species of animals, thus making cross-

reaction unlikely. Finally, the two GABA antibodies that

were used yielded identical results.

The number of immunogold particles in cell bodies and

dendrites, as reported previously [17,41,52], was very low

and, in most cases, similar to the number in the background;

consequently, we were unable to examine the ultrastructural

relationships between ChAT-IR terminals and GABA-IR

somas and dendrites.

4.2. GABA-IR and ChAT-IR ultrastructural features in the

LDT and PPT

GABAergic neurons in the LDT/PPT have been exten-

sively examined using both light microscopy [1,13,32,35,

36,47,54,56] and electron microscopy [36,54,56]. In the

present study, GABA-IR neurons, terminals and fibers were

observed in the LDT/PPT in both light and electron micros-

copy studies. GABA-IR terminals comprised about 40% of

all terminals in these regions. Most of these terminals formed

symmetric synapses, i.e., putatively inhibitory, with non-

GABA-IR profiles. These data highlight the importance of

GABA-mediated synaptic inhibition in the LDT/PPT.

The observations made in the present study regarding the

characteristics of cholinergic neurons in the LDT/PPT are, in

general, in agreement with the previous reports [16,44].

These neurons exhibit abundant cytoplasmic organelles

and a large, clear nucleus with a few infoldings. Both

ChAT-IR somas and dendrites receive many synapses.

ChAT-IR axon terminals were present in the LDT/PPT which

formed both asymmetric and symmetric synapses, usually

with non-ChAT-IR dendrites.

4.3. Colocalization of GABA and ChAT

The present study is the first in which GABA and ChAT

colocalization has been demonstrated in cell bodies and

terminals in the LDT/PPT. Colocalization of these substan-

ces was evident when using the tyramide signal amplifica-

tion technique and also when GABA was detected with

DAB and ChATwith BDHC. In contrast, Ford et al. [13] did


not find neurons double-immunostained for glutamic acid

decarboxylase (GAD) and ChAT in the LDT/PPT of the rat.

They immunostained for GAD using BDHC and for ChAT

using DAB. Possible explanations for this discrepancy are

that different antibodies were used in the two studies (anti-

GABA vs. anti-GAD) and/or that the method employed by

Ford et al. was less sensitive. Colocalization of GABA and

ChAT has been described in cells in other regions of the

nervous system [3,4,6,7,12,25,38,48].

In electron microscopy studies, colocalization of GABA

and ChAT was found in axon terminals of the LDT/PPT. To

the best of our knowledge, this pattern of colocalization of

GABA and ChAT has been reported only in the rat inferior

olive [7] and in the cat striate cortex [4]. Because we

observed colocalization in cell bodies in the LDT/PPT, we

suggest that double-labeled terminals originate in local

somas, although there are GABAergic projections to the

LDT/PPT [25,39].

Double-labeled terminals formed symmetric and, occa-

sionally, asymmetric synapses with non-immunostained

dendrites. These dendrites are likely to contain glutamate,

enkephalin or monoamines, all of which have been reported

to be present in neurons in the LDT/PPT [2,29,54,55,56].

4.4. GABAergic synaptic input to cholinergic neurons

GABAergic terminals formed symmetric synaptic con-

tacts with approximately 40–50% of the cholinergic cell

bodies and dendrites. Over 95% of the GABAergic terminals

that contacted ChAT-IR profiles did not display ChAT-IR.

Because only 20–24% of the total number of GABAergic

terminals innervated cholinergic profiles, most of the

GABAergic terminals innervated non-cholinergic neurons.

Based on the high concentration of GABAergic neurons in

the LDT/PPT, most of the GABAergic axon terminals

probably arise from local neurons. However, it is also

possible that a portion of these GABAergic terminals orig-

inates from other regions, for example, the internal pallidum

and substantia nigra reticulata [11,37,42].

4.5. Functional implications

In the nervous system of the adult animal, GABA

functions as an inhibitory neurotransmitter [33]. Acetylcho-

line (ACh) can act either as an excitatory or as an inhibitory

neuromodulator, depending on the nature of the postsynaptic

receptor [22,24]. Therefore, the result of the concomitant

release of GABA and ACh in the synaptic cleft of any

particular synapse will depend on the nature of the postsyn-

aptic receptors [30]. If the postsynaptic neuron expresses

muscarinic inhibitory receptors and GABA receptors, the

result would be synergistic inhibition. If the receptors for

ACh mediate excitatory actions, a mixed effect would occur

with results that are difficult to predict. It is also possible that

different patterns of presynaptic discharge may act to release

only one or another of these neurotransmitters.

From a behavioral perspective, cholinergic neurons that

also contain GABA may be involved in the initiation of

active sleep and/or in other functions of the LDT/PPT, for

example, locomotion or cognition [15,43]. In light of the

present results, we hypothesize that in the initiation and/or

maintenance of active sleep, there is a GABAergic compo-

nent that acts synergistically with the well-established

cholinergic drive that generates active sleep.

Recently, we have determined that the injection of

muscimol, a GABA agonist, into the PPT induces active

sleep [50]; in addition, GABAergic neurons in the LDT/PPT

express c-fos during active sleep [29,49]. It has also been

reported that excitation during a period of hyperpolarized

membrane potential promotes a bursting pattern of dis-

charge in a portion of cholinergic and non-cholinergic

neurons within the LDT/PPT [28]. This bursting behavior

is due to the de-inactivation of a low-threshold calcium

inward current that occurs during hyperpolarization [28]. It

has been proposed that these bursting neurons are implicat-

ed in the generation of ponto-geniculo-occipital waves that

occur during active sleep [31].

It is therefore possible that, in spite of their hyperpolariz-

ing postsynaptic effect, GABA and ACh act in concert to

increase the responsiveness and alter the pattern of discharge

of cholinergic and non-cholinergic neurons of the LDT/PPT

that are involved in the initiation of active sleep. The finding

that terminals in the LDT/PPT contain both ACh and GABA

raises several questions regarding the pattern of postsynaptic

modulation of the activity of their target neurons as well as

the role of these colocalized substances in the control of the

states of active sleep and wakefulness.

Acknowledgements

This work was supported by the following grants from

the U.S. Public Health Service: MH43362, NS 09999,

NS23426, HL60296, AGO4307, and MH59284.

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Colocalization of γ-aminobutyric acid and acetylcholine in neurons in the laterodorsal and...

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