www.elsevier.com/locate/brainres
Brain Research 1066
Research Report
Localization of GABA (g-aminobutyric acid) markers
in the turtle’s basal optic nucleus
John Martina,b, Michael Arielc,*
aCenter for Anatomical Science and Education, Saint Louis University School of Medicine, St. Louis, MO 63104, USAbDepartment of Surgery, Saint Louis University School of Medicine, St. Louis, MO 63104, USA
cDepartment of Pharmacological and Physiological Science, Saint Louis University, 1402 S. Grand Blvd., St. Louis, MO 63104, USA
Accepted 15 October 2005
Available online 9 November 2005
Abstract
Recent physiological data have demonstrated that retinal slip, the sensory code of global visual pattern motion, results from complex
interactions of excitatory and inhibitory visual inputs to neurons in the turtle’s accessory optic system (the basal optic nucleus, BON)
[M. Ariel, N. Kogo, Direction tuning of inhibitory inputs to the turtle accessory optic system, Journal of Neurophysiology 86 (2001)
2919–2930. [6], N. Kogo, T.X. Fan, M. Ariel, Synaptic pharmacology in the turtle accessory optic system, Experimental Brain Research 147
(2002) 464–472. [23]]. In the present study, the inhibitory neurotransmitter g-aminobutyric acid (GABA), its synthetic enzyme, glutamic acid
decarboxylase (GAD-67) and its receptor subtypes GABAA and GABAB receptors were localized within the BON. GABA antibodies revealed
cell bodies and processes, whereas antibodies against GAD revealed a moderate density of immunoreactive puncta throughout the BON. GAD
in situ hybridization labeled BON cell bodies, indicating a possible source of inhibition intrinsic to the nucleus. Ultrastructural analysis
revealed terminals positive for GAD that exhibit symmetric synaptic specializations, mainly at neuronal processes having small diameters.
Neurons exhibiting immunoreactivity for GABAA receptors were diffusely labeled throughout the BON, with neuronal processes exhibiting
more labeling than cell bodies. In contrast, GABAB-receptor-immunoreactive neurons exhibited strong labeling at the cell body and proximal
neuronal processes. Both these receptor subtypes are functional, as evidenced by changes of visual responses of BON neurons during
application to the brainstem of selective receptor agonists and antagonists. Therefore, GABAmay be synthesized by BON neurons, released by
terminals within its neuropil and stimulate both receptor subtypes, supporting its role in mediating visually evoked inhibition contributing to
modulation of the retinal slip signals in the turtle accessory optic system.
D 2005 Elsevier B.V. All rights reserved.
Theme: Sensory systems
Topic: Subcortical visual pathways
Keywords: Accessory optic system; GAD; Ultrastructure; Synapse; Retinal slip; Brainstem; Direction-sensitive
1. Introduction
The turtle’s accessory optic system (the basal optic
nucleus, BON) is a collection of neurons in the ventrolateral
mesencephalon. It has been shown that neurons in the BON
receive excitatory inputs from direction-sensitive retinal
ganglion cells distributed across the entire contralateral retina
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.10.040
* Corresponding author. Fax: +1 314 977 5127.
E-mail address: [email protected] (M. Ariel).
[23,43]. The convergence of these inputs onto BON neurons
forms an average, full-field motion signal, called retinal slip,
which is relayed to vestibular and oculomotor pathways for
the control of eye and head movements that compensate for
movements of the visual field. Neurons in the BON also
receive non-retinal inputs [24]. Retinal application of
lidocaine increased the frequency of spontaneous BON
inhibitory events, suggesting that a tonic retinal output
normally blocks inhibitory pathways to the BON from
within the brainstem. One possible source of this inhibition
(2005) 109 – 119
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119110
is the pretectum, whose neurons receive retinal inputs [39]
and are direction-sensitive to full-field motion [16].
A direction-sensitive inhibitory synaptic input to the BON
cells also exists [6]. The preferred directions of these
inhibitory responses are often similar to the preferred
direction of the excitatory inputs from the retina. Brainstem
application of bicuculline, an antagonist to an g-aminobutyric
acid receptor (GABAA), blocks spontaneous inhibitory
synaptic events in the BON as well as responses to pretectal
microstimulation recorded there [24]. Recently, it was shown
that the substantial synaptic current evoked in BON cells by
GABA can shunt current sufficiently to attenuate coincident
excitatory events recorded in the same cell [4]. Removal of
this inhibition by application of bicuculline increased the
preferred direction response, as would be anticipated if
direction-sensitive excitation is released from inhibition.
These physiological data on GABA effects in the turtle
accessory optic system are accompanied by little neuroana-
tomical information, although immunohistochemical studies
in various other species have demonstrated GABAergic
neuronal elements in the accessory optic system and
pretectum [12,14,18,35,38,42]. In addition, in situ hybridiza-
tion has been utilized to demonstrate the expression of GAD-
67 mRNA in neurons of the medial terminal nucleus, the
mammalian counterpart to the BON [34]. The present study
utilized in situ hybridization and immunohistochemical
techniques to demonstrate a GABAergic presence within the
turtle BON. Furthermore, GABAA and GABAB receptor
agonists and antagonists were applied while recording
direction-sensitive extracellular spike activity in BON cells
in the in vitro brainstem preparation in order to demonstrate
that both types of GABA receptors exert physiological effects.
2. Material and methods
2.1. Surgery
Thirty-seven turtles (Trachemys scripta elegans) were
used. The animals were housed at room temperature on a
16/8 h light–dark cycle in a large aquarium with compo-
nents for swimming and basking. The experimental proto-
cols reported here were reviewed and approved by the Saint
Louis University Animal Care Committee in accordance
with the National Institutes of Health Guide for the Care
and Handling of Laboratory Animals and monitored by the
Department of Comparative Medicine of the Saint Louis
University School of Medicine.
To prepare for electrophysiology, turtles were anesthe-
tized first by hypothermia and then with 12.5 mg of
thiopenthal (i.m.). The brains and eyes were dissected free
from the cranial cavities, and the telencephalon was
removed (for details, see [22,33]). The eyes were hemi-
sected and drained of vitreous, and the preparation was
placed ventral side up in a superfusion chamber containing
(in mM) 96.5 NaCl, 2.6 KCl, 2.0 MgCl2, 31.5 NaHCO3, 20
d-glucose and 4.0 CaCl2 adjusted to pH 7.4 at room
temperature and bubbled with 95% O2 5% CO2.
To generate fixed tissues for anatomical studies, turtles
were anesthetized first by hypothermia and then with 1.0 ml
of 0.5% tricaine after which they were perfused trans-
cardially first with a rinse of 50 ml superfusate followed by
200 ml of 0.1 M phosphate buffer (PB) containing 4%
paraformaldehyde. The pH of all solutions was adjusted to
7.4 unless noted otherwise. The solution used to fix brains
destined for electron microscopic analysis also contained
0.1% glutaraldehyde. Perfused brains were removed from
the cranial cavity, immersed in the same fixative for 24 h and
then placed overnight in 0.1 M PB containing 25% sucrose.
Sections were cut in the transverse plane on a freezing
microtome at 50 Am and thawed in 0.1 M PB. Sections
destined for ultrastructural analysis were again incubated in
25% sucrose–PB and freeze-thawed in liquid nitrogen to
enhance penetration of the antibody.
2.2. Electrophysiology
Single unit extracellular recordings were made using
Epoxylite-coated, etched tungsten recording electrodes (0.5–
5 MV at 135 Hz) placed just beneath the ventral brainstem
surface about 0.5 mm anterolateral to the exit of the
oculomotor nerve [33]. Spike activity was amplified 1000�and filtered between 0.3 and 3 kHz, before being passed
through a window discriminator and audio monitor. The eye
contralateral to the recording was stimulated with a full-field
checkerboard pattern drifting in one of 12 directions for three
5-s stimulus presentations [1]. Only well-isolated units with
direction-sensitive responses were studied (that is, preferred
direction spike response was 2 times greater than null
direction response, see [33]). While the retinal eyecups
remained in the control perfusate, drug solutions (baclofen or
saclofen) were applied to the brain compartment while
monitoring the visual responses to different directions or to
different speeds along the preferred/null axis.
2.3. Immunohistochemistry
Primary antibodies were obtained from Chemicon
International (Temecula, CA) and diluted in 0.1 M PB
containing 0.2% Triton-X and used at the following
concentrations: mouse anti-GABA monoclonal antibody
(1:4000), rabbit anti-glutamate decarboxylase (GAD) poly-
clonal antibody (1:3000), mouse anti-GABAA receptor, h-chain monoclonal antibody (1:8000) and guinea pig anti-
GABAB receptor polyclonal antibody (1:6000).
Free-floating sections were rinsed and then immersed in
primary antibody. The following day, sections were rinsed
in 0.1 M PB containing 0.2% Triton-X prior to immersion in
biotinylated anti-mouse, -rabbit or -guinea pig immunoglo-
bin G (1:2000) for 1 h. The sections were rinsed again and
processed with ABC reagents as per manufacturer’s
instruction (Vectastain Elite ABC Kit) for 1 h. The sections
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119 111
were then rinsed in 0.1 M PB prior to immersion in 0.1 M
PB containing 0.05% diaminobenzidine and 0.003% hydro-
gen peroxide. Sections were then rinsed again, mounted on
gelled slides, air dried and coverslipped.
Controls for the specificity of the immunoreactions
included omission of the primary antibody. Under this
condition, staining was abolished for all antibodies used in
this study. In most cases, adjacent sections were stained for
Nissl substance with cresyl violet to facilitate the identifi-
cation of the BON, which corresponds with the terminal
field of retinal fibers labeled by an intravitreal injection of
HRP CTB-WGA [27]. Immunoreactive cell bodies were
scored relative to the numbers of cell bodies present in
adjacent Nissl-stained sections as less than 33%, 33–66%,
and more than 66%, respectively. Between the cell bodies,
the neuropil was evaluated based on the density of labeled
processes. We considered BON labeling to be high for the
highest number of labeled processes observed for any
antiserum and low for the lowest number. Any intermediate
labeling in the neuropil was scored as moderate.
2.4. Probe synthesis and in situ hybridizations
Sense (control) and antisense riboprobes were tran-
scribed from previously characterized 3.2-kb GAD-67 or
2.3-kb GAD-67 DNA templates in plasmids kindly supplied
by A. Tobin [15,40]. The cDNA was incorporated into
ampicillin-resistant Escherichia coli cells, from which
templates were isolated and linearized with SalI (3.2-kb
cDNA) and BamHI (2.3-kb cDNA). The riboprobes were
synthesized and labeled with digoxigenin–uridine triphos-
phate (UTP) by in vitro transcription using T3 (3.2-kb
cDNA) and SP6 (2.3-kb cDNA) RNA polymerases.
Hybridizations were carried out on free-floating sections
rinsed in sterile 0.1 M PB, treated with acetic anhydride
(AA) for 15 min, rinsed again in 0.1 M PB and then
placed in a prehybridization buffer consisting of Tris–HCl
(20 nM), EDTA (1 mM), NaCl (300 mM), formamide
(50%), dextran sulfate (10%) and Denhardt’s solution (1�)
for at least 30 min. After an overnight incubation at 55 -Cin a mixture of 1–5 Al of stock probe preparation, nucleic
acids (salmon sperm DNA [10 Ag/ml], yeast total RNA
[40], yeast tRNA [25 Ag/ml]), sodium dodecyl sulfate
(10%) and sodium thiosulfate (10%) in hybridization
buffer, the sections were allowed to cool to room
temperature, rinsed several times in 4� SSC, 0.1� SSC,
at various temperatures and rinsed again in 0.1 M Tris–
HCl and 0.1 M Tris–HCl containing 3% normal goat
serum and 0.3% Triton X.
Hybridized neurons were visualized by exposing the
sections to anti-digoxigenin antibodies conjugated to
alkaline phosphatase (Boehringer) overnight at a dilution
of 1:1000 at room temperature, after which the sections
were rinsed in Tris–HCl buffer (2 � 5 min) and exposed
for 5 h in the dark to a mixture containing nitroblue
tetrazolium (used at 45 Al/10 ml), 5-bromo-4-chloro-3-
indoyl phosphate toluidinium (BCIP; used at 35 Al/10 ml)
(Vector, product #SK-5400) and levamisole (Vector,
product #SP-5000 used at 80 Al/10 ml). The sections
were then rinsed in Tris–HCl buffer and PB, mounted
and air dried. Mounted sections were then dehydrated
briefly in ascending alcohols, cleared in xylene and
coverslipped.
2.5. Preparation of GAD-immunolabeled sections for
ultrastructural analysis
Sections from 8 turtles were labeled with antiserum to
GAD and prepared for electron microscopic analysis. These
sections were flattened onto non-gelatinized microscope
slides. They were then immersed in 0.1 M PB containing
1% osmium tetroxide for 30 min at room temperature,
washed in water, dehydrated through a series of ascending
concentrations of ethanol and then placed in propylene oxide.
The sections were then transferred to Spurr’s resin overnight,
mounted on plastic sheets, flattened with glass coverslips and
cured for 72 h at 60 -C. Under light microscopic guidance,
selected areas were excised from the tissue, mounted on
preformed resin blocks using cyanoacrylate glue and
sectioned on an ultramicrotome. Ultrathin sections were
collected on single slot, Formvar-coated, copper grids stained
with 2% uranyl acetate in 50% ethanol and lead citrate and
examined in a Zeiss 106 electron microscope.
2.6. Analysis of immunolabeled processes
Ultrastructural data were obtained with the aid of a
drawing tube attached to the electron microscope, a
digitizing tablet and computer software (Sigma Scan,
version 3.10; Jandel Corp.). Immunonegative and immuno-
positive synaptic elements were traced, and the area and
lengths of major and minor axes of presynaptic and
postsynaptic elements were measured and saved. Measure-
ments were restricted to only those terminals that contained
postsynaptic densities or presynaptic vesicles separated by a
synaptic cleft from a postsynaptic membrane. Because the
orientations of the EM profiles are unknown, the minor axes
of the processes were used to estimate diameter. Selected
images were photographed and reproduced by Adobe
Photoshop software.
3. Results
3.1. GABA and GAD localization
Specific immunohistochemical staining with antibodies
against GABA and the synthetic enzyme GAD was
observed in the turtle BON, but regional variations in the
distributions of the immunoreactivity were not observed
within the nucleus. The relative densities of label in the
BON are summarized in Table 1.
Table 1
Relative densities of immunostained cell bodies and neuropil
Antigen Cell bodies Neuropil
GABA + (19%) ++
GAD � +++
GAD mRNA +++ (91%) �GABAA receptor + (33%) ++
GABAB receptor +++ (94%) �The neuropil density was scaled compared to the density of GAD-
immunoreactive terminals, which was the most intense labeling in the
BON. The cell body density was rated in relation to the total number of
Nissl-stained cell bodies in adjacent sections. Relative densities: � absent;
+ low; ++ moderate; +++ high.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119112
3.1.1. GABA
GABA-immunopositive fascicles, cell bodies and pro-
cesses are illustrated in Fig. 1B. GABA-immunoreactive (-ir)
fascicles were intensely labeled in the dorsal region of the
BON. GABA-ir fascicles also were observed lateral to the
BON along the brainstem surface between the nucleus and
the optic tract. Additionally, GABA-ir fascicles were
observed in sections rostral to the BON along the ventral
surface of the brainstem in a position corresponding to that of
the basal optic tract. GABA-ir cell bodies comprised of 19%
of neurons counted in adjacent Nissl-stained sections. Single
GABA-ir processes of various calibers were also observed,
some extending from GABAergic cell bodies.
3.1.2. GAD
Punctate immunoreactivity to GAD was present within
the BON and surrounding ventrolateral tegmentum. The
density of these GAD-ir puncta was uniform within the
neuropil of the BON (Fig. 1C). Although BON cell bodies
were not directly stained, unlabeled somata were often
surrounded by GAD-ir puncta.
In order to identify cell bodies that produce GAD, in situ
hybridization using an antisense probe for GAD-67 mRNA
was performed (Fig. 1D). The alkaline phosphatase reaction
product filled most of their somata but was concentrated near
the unstained nucleus. Cell counts from six turtles revealed
that 91% of BON neurons were labeled with that technique.
Hybridized neurons were found throughout the nucleus and
along all axes (rostro-caudal, medial–lateral, dorso-ventral).
In contrast, hybridization using the sense probe for GAD-67
mRNA did not label neurons.
3.1.3. Ultrastructural analysis of GAD immunolabeling
Because GAD-ir puncta would produce a dense outline
around some unlabeled BON somata as viewed with the
light microscope, an ultrastructural study was performed
to observe whether the puncta here related to BON somata
by synaptic contacts. When viewed in the electron
microscope, the GAD-ir labeling appeared as a dark
reaction product (Fig. 2) concentrated in presynaptic
terminals associated with small diameter processes and
juxtaposed to both dendrites and somata of BON cells
(Fig. 3).
GAD-ir synaptic profiles were observed in small diameter
processes that frequently made presynaptic contacts with
membranes of BON neurons. Cell bodies not immunoreac-
tive to GAD were observed, consistent with results observed
in the light microscope. The selectivity of the antibody was
apparent due to the observance of anti-GAD-labeled
processes located in proximity to unlabeled processes. Of
275 labeled terminals examined with the electron micro-
scope, 68% were located along the membrane surfaces of
processes with a minor axis less than 1.0 Am in length, and
32% were located along membrane surfaces that had a minor
axis length equal to or greater than 1.0 Am, including those
along membrane surfaces of cell bodies (Fig. 4B). Likewise,
of 354 unlabeled terminals examined, 58% were located on
processes less than 1.0 Am, while 42% were observed along
membrane surfaces that had a minor axis length equal to or
greater than 1.0, also including those along membrane
surfaces of cell bodies (Fig. 4A). These results indicate that
anti-GAD-ir, as well as unlabeled terminals, are primarily
located along the distal ends of BON cells.
GAD-ir and unlabeled terminals were compared along
the membrane of 10 somatic profiles viewed in the electron
microscope. These profiles were from different fusiform cell
bodies of medium to large size (range of 15.4–158.6 Am2
with a mean area of 76.3 Am2). Each profile exhibited
between 1 and 5 synapses from GAD-ir terminals and 2–10
synapses from unlabeled terminals. Our analysis shows that
there were more unlabeled than GAD-ir terminals along
BON cell bodies (P < 0.005). On average, one GAD-ir
terminal was observed for every four unlabeled terminals
along a BON cell body.
3.2. GABA receptor subtypes
GABAA and GABAB receptor immunoreactivity was
observed on BON cell bodies and processes. However,
consistent differences in the localization of the two different
receptor subtypes were seen.
3.2.1. GABAA receptor localization
Structures immunoreactive to GABAA receptors were
observed throughout the BON. GABAA-ir processes within
the BON were numerous and were arranged in a fascicle
extending along the ventrolateral surface between the optic
tract and the BON (Fig. 1E). The number of labeled cell
bodies comprised of 33% of neurons counted in adjacent
Nissl-stained sections. Fusiform or round cell bodies of a
broad variety of sizes were immunolabeled (range of major
axis length 7.9–37.9 Am; mean major axis length = 20.1
Am). This range for GABAA-ir cell bodies was similar to
that observed for Nissl-stained cells.
3.2.2. GABAB receptor localization
BON cells exhibited robust GABAB receptor immuno-
reactivity. The immunolabeled cell bodies were mainly
fusiform in shape and comprised of 94% of the neurons
Fig. 1. Photomicrographs of transverse sections demonstrating GABA markers within the basal optic nucleus (BON). Dashed lines indicate the approximate
boundaries of the BON, as determined by the Nissl-staining of adjacent sections. (A) Nissl-stained section of the BON. Inset shows a low micrograph section
through the entire mesencephalon at the level of the BON. The arrow shows the location of the BON just above the ventral surface. (B) Antibodies against
GABA labeled few cell bodies and processes in the BON. Asterisk shows GABA-immunoreactive fascicles. Inset shows a cell body (arrow) and neuronal
processes (arrowheads) immunoreactive to GABA. (C) Antibodies against GAD labeled small tiny structures referred to as puncta. Inset shows GAD-
immunoreactive puncta (arrowheads) surrounding a pale region, possibly a BON cell body. (D) In situ hybridization of antisense GAD mRNA labeled neurons
in the BON. Inset shows GAD mRNA staining in cell cytoplasm surrounding the cell nucleus. Analysis revealed that the majority of BON neurons contain
GAD mRNA. (E) Antibodies against GABAA receptors reveal a loose network of immunoreactive processes and few cell bodies in the BON. Asterisk shows
immunoreactivity in the basal optic tract. The inset shows a cell body (arrow) and process (arrowhead) immunopositive for GABAA receptors. (F) Antibodies
against GABAB receptors label a majority of BON cell bodies and few processes. Inset shows granule-like structures on three BON cell bodies. Scale bar = 250
Am. Inset scale bar = 30 Am.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119 113
counted in adjacent Nissl-stained sections. The GABAB
receptor immunoreactivity was found on cell bodies of a
broad variety of sizes (range of major axis length 7.9–52.8
Am; mean major axis length = 18.5 Am). In addition,
numerous (10–20) GABAB-receptor-immunoreactive
puncta abutted cell bodies and proximal processes through-
out the BON (Fig. 1F).
3.2.3. Effects of GABAB receptor drugs
Twenty nine well-isolated, direction-sensitive units in
the BON were studied by brainstem applications of the
GABAB drugs. During the GABAB agonist baclofen (50
AM, n = 8; 100 AM, n = 4; 200 AM, n = 7), spike
responses decreased by 88.5% (n = 19, two other cells
showed no change), indicating that the GABAB receptor is
present and functional on the BON cell membrane.
GABAB antagonists were applied to test whether these
receptors respond to GABA during natural visual stimu-
lation (Fig. 5). During application of the GABAB
antagonist saclofen (50–100 AM), spike responses in-
creased in most (n = 4) of the recorded neurons (Fig. 6),
although one cell showed a small decrease. The average
spike increase was 131%. The results with the antagonist
phacophen (100 AM; n = 2) were similar to that of
Fig. 2. Electron photomicrographs of synaptic terminals in the basal optic
nucleus. (A) Presynaptic terminal labeled using anti-GAD making a
symmetrical contact with a small diameter neuronal process. Note the dark
reaction product surrounding the synaptic vesicles and mitochondria. (B)
Unlabeled presynaptic terminal making an asymmetric synaptic contact.
Scale bar = 0.5 Am.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119114
saclofen, but the response increase was only 38%. This
evidence indicates that functional GABAB receptors
modulate visual processing in the BON (Figs. 5,6).
Fig. 3. Electron micrographs of GAD-immunoreactive terminals in selected
areas of the basal optic nucleus (BON). (A) Numerous GAD-ir terminals
(arrows) and unlabeled terminals (arrowheads) making synaptic connec-
tions on small diameter neuronal processes in the BON. (B) A medium size
BON cell body receiving a GAD-ir (arrow) and unlabeled (arrowheads)
synaptic terminals. Scale bars = 1.0 Am.
4. Discussion
The localization of markers for the inhibitory neuro-
transmitter GABA has been described and quantified in the
basal optic nucleus. It was shown that GABA, its synthetic
enzyme GAD and its receptor subtypes, GABAA and
GABAB, are present in the BON. These findings support
previous physiological experiments that have shown that
retinal slip, the sensory code of global visual pattern motion,
is processed by interactions between excitatory and inhib-
itory visual inputs in the turtle accessory optic system
[6,24]. Although these techniques did not reveal regional
differences within the BON, GABAergic synapses seen in
the electron microscope were more numerous on distal
dendrites than on BON somata and proximal dendrites. One
potential source for these GABAergic synapses may be
within the nucleus itself as most BON cells localize GAD
mRNA. Finally, GABAB receptors were modulated by
selective GABAB receptor drugs, suggesting that GABA’s
role in visual processing within the BON may be mediated
by more than one receptor subtype.
4.1. Is there an intrinsic or extrinsic source of inhibition to
the BON?
The present study revealed immunoreactivity to GABA
within cell bodies in the BON. This finding is consistent with
studies in other vertebrates that have identified GABA
markers in the accessory optic system (in mammal [18], in
pigeon [12], in frog [42]) and in identifying GABA’s role in
optokinetic nystagmus (in mammal [21]; in bird [10,41]; in
reptile [3]). Although only a few immunoreactive cell bodies
to GABA were revealed in the present study, their presence
argues that some cells in the BON are GABAergic. Many
more BON cells localized GAD mRNA than GABA-ir,
however, consistent with the possibility that GABA is present
in most BON neurons, but were not uniformly detected by our
protocol. This finding is consistent in the rat medial terminal
nucleus (MTN, the mammalian homolog to the BON) in that
the number of GAD mRNA neurons (up to 98%) [34] is
higher than the number of GABA-ir neurons (72%) [19].
Although mRNA for GAD-67 was localized to many BON
neurons, one cannot be sure how many of these neurons
synthesize active GAD-67 enzyme which then synthesizes
GABA for synaptic release. It is also not knownwhether such
GABAergic cell bodies serve as interneurons that release
GABA locally within the BON or project elsewhere to inhibit
other brainstem structures. Likewise, other structures send
axons to the BON, which form GAD puncta that release
GABA onto BON cells to inhibit their activity. Thus, the
source of the GAD puncta in the BON is not known.
Fig. 4. Histograms showing the number of unlabeled terminals (A) and GAD-ir (B) along the membrane of basal optic nucleus (BON) neurons. (A) The gray
bars (n = 109) represent the number of unlabeled terminals along cell bodies, while the white bars (n = 245) represent unlabeled terminals along neuronal
processes. A total of 354 unlabeled terminals were observed on BON neurons. (B) Gray bars (n = 30) represent the number of GAD-ir terminals along cell
bodies, while the white bars (n = 245) represent GAD-ir terminals along neuronal processes. A total of 275 GAD-ir terminals were observed on BON neurons.
In both panels A and B, the majority of terminals made synaptic contact with postsynaptic processes less than 1.0 Am in diameter, while nearly one-fifth of all
terminals made synaptic contact on processes with diameters between 1.0 and 5.0 Am.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119 115
Although an AOS–pretectal inhibitory connection has
not been identified in the turtle, the present finding of
GABAergic cells in the BON, along with physiological
evidence that these two nuclei have complementary
direction-tuning [16], suggests that the turtle BON may be
responsible for the GABA inhibition of the pretectum. It has
already been shown that stimulating the pretectum evoked
shunting GABA inhibition in the BON [4,24]. This
reciprocal relationship of AOS–pretectal interactions is
consistent with findings in other vertebrates. Physiological
interactions between pretectal and accessory optic system
neurons have been shown in birds [31] and rats [29], while
anatomical projections have been identified using anterog-
rade and retrograde tracers (in bird [11]; anuran [28];
urodele [30]; and mammal [17]). In anuran, Li [26]
combined immunostaining of GABA with retrograde
labeling of rhodamine beads showed 66% of GABAergic
nucleus of the basal optic root (nBOR, the homolog to the
BON) neurons project to the pretectum. Similarly, 72% of
neurons in the rat MTN have been shown to be GABAergic,
and these neurons project to the pretectum [19]. Moreover,
GABAergic projections have been demonstrated from the
pretectum to the AOS [37].
In the present study, numerous GAD puncta were shown
to be axon terminals in the electron microscope, as was also
observed in the rat [37]. Previous studies have characterized
AOS synaptic morphology on the basis of synaptic vesicle
shape into either terminals with round synaptic vesicles (R
terminals), presumably excitatory synapses, or flat synaptic
vesicles (F terminals), presumably inhibitory synapses (in
frog [20]; in rat [36,37]; in pigeon [32]). In this study, only
round vesicles were observed. However, GAD-ir and non-
labeled synaptic terminals were identified in the BON. We
noted no differences regarding the postsynaptic distributions
of these terminals, which is consistent with other studies (in
frog [20]; in rat [25,37]; in pigeon [32]).
It is not known how much of the BON output is
excitatory or inhibitory. GABAergic BON neurons might
inhibit the pretectum, the contralateral BON or just provide
local inhibition with its nucleus. There is evidence that BON
output is excitatory within the cerebellar cortex [5].
Although GAD mRNA is localized to many BON cells,
the low proportion of GAD mRNA negative cells may still
be sufficient to provide non-inhibitory signals to its more
caudal targets in the brainstem. This is supported in findings
in other species where only 18% of nBOR neurons in
Fig. 5. Effects of selective GABAB agonist on visual responses of a unit recorded in the basal optic nucleus. Baclofen (50 AM) was applied to the brainstem for
20 m. Recovery data were collected 1 h later. (Left) Each histogram shows spike responses to 2–3 sets of speed stimuli. Each set presented the same
checkerboard pattern imaged onto the retina contralateral to the neuronal recording. A stimulus set was a series of preferred and null motions of increasing
speed. (Right) Polar plots of the direction-tuning of the same cell. The preferred direction used for the speed stimuli was the direction that elicited the largest
response on the polar plots. Note that no spikes occurred during baclofen so the polar plot is just one filled circle at the origin.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119116
pigeon were projection neurons [28] and only 10% of rat
MTN neurons projected to non-pretectal targets [13].
4.2. The role of GABAB receptors in the BON
The presence of GABAA receptors on BON processes
and cell bodies is consistent with physiological studies in
which the application of the GABAA antagonist bicuculline
to the bathing medium disrupts direction-sensitive inhibitory
responses of BON neurons [6]. However, there were no
previous data suggesting a role of the GABAB receptors in
the turtle BON. Although the immunolabeling of GABAB
receptors is very intense near the soma and proximal
processes, it is not known whether GABAB receptors are
presynaptic or postsynaptic receptors. An ultrastructural
study may determine the precise location of GABAB
receptors in the turtle BON.
If GABAB receptors are presynaptic, they might exert
negative control on either GABA or glutamate release,
resulting in changes in excitation or inhibition. Punctate
Fig. 6. Effects of selective GABAB antagonist on visual responses of a unit recorded in the basal optic nucleus. Saclofen (100 AM) was applied to the brainstem
for 15 min. Recovery data were collected 40 min later. Data are displayed as in Fig. 5. In this example, the spike responses during saclofen increased 35%
above the control.
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119 117
structures exhibiting GABAB immunoreactivity, albeit near
BON cell bodies and proximal processes, may be axon
terminals of glutamatergic retinal or GABAergic pretectal
cells. Alternatively, GABAB receptors may be postsynaptic.
Unlike GABAA, the GABAB receptor is metabotropic and
would have a slower, longer lasting inhibitory or modulatory
effect. Our evidence that baclofen decreases and saclofen
increases BON spike activity supports a postsynaptic role for
the GABAB receptor. Intracellular recordings comparing the
effects of these GABAB drugs with bicuculline are necessary
to better understand the complex role that GABA plays in
visual processing in the turtle accessory optic system.
Unlike the finding of GABAA receptors in the BON,
localization of GABAB receptors was a surprise because
bicuculline appears to block all the inhibitory events
recorded in BON neurons [24]. Those neuronal recordings,
however, were made in the whole-cell configuration by
rupturing a membrane patch in the pipette and dialyzing the
BON cell’s cytoplasm. GABAB receptors, being metabo-
tropic, require specific intracellular machinery to allow the
J. Martin, M. Ariel / Brain Research 1066 (2005) 109–119118
receptor to mediate a physiological response. This recording
technique may have disabled GABAB responses or GABAB
receptors may have never been inserted into the postsynap-
tic BON membrane, as suggested by immunohistochemis-
try. However, the physiological experiments performed here
measured extracellular BON spike potentials and showed
that GABAB drugs did modulate the visual responses.
4.3. Two functional compartments of BON cells?
Ultrastructurally, GAD-ir terminals make symmetric
synaptic contacts with BON cells, while non-GABAergic
terminals, which are presumed to represent glutamatergic
retinal terminals, make asymmetric synaptic contacts. These
findings are consistent with ultrastructural studies of
inhibitory and excitatory synapses in other brain structures.
The present study also revealed that GABAergic and non-
GABAergic inputs are nearly similarly distributed along
distal dendrites of BON cells. Those different patterns of
distribution along the dendritic arbor may be significant to the
membrane interactions of the direct excitatory retinal input
and the modulatory GABAergic controls. These synaptic
inputs share a common preferred direction [6] so the synaptic
release of both systems can be simultaneous and thereby
evoke shunting inhibition of the BON neuronal membrane
[4]. The strength of this shuntingmay be related to the relative
distribution of GABAergic and non-GABAergic synapses, as
observed in the present study. In that case, shunting may be
stronger on the distal dendrites than near the soma.
In conclusion, GABA neurotransmission plays an im-
portant role in visual processing of retinal slip signals.
GABAA receptor blockade within the retina clearly blocks
direction-sensitive processing which affects optokinetic
nystagmus [2,7–9]. On the other hand, the role of GABA
in the accessory optic system is less clear but is not the
simple convergence of direction-sensitive retinal inputs that
sum into a retinal slip signal.
Acknowledgments
We thank Allen J. Tobin (UCLA) for kindly providing
the GAD cDNA, the R. Mark Buller laboratory (Saint Louis
University) who helped make the riboprobes and Daniel S.
Zahm (Saint Louis University) and Evelyn Williams for
their assistance with the immunohistochemistry and in situ
hybridization. In addition, we thank Mr. Tian Xing Fan for
helping with the electrophysiology experiments and to Dr.
Zahm for comments on the manuscript.
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