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: ARIELM@SLU.EDU (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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