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Aims and significance

This project addresses the physiological mechanisms underlying the ability of the retina to adjust its
sensitivity to different mean light intensities. When we move from a dark to a bright environment
(and vice versa), our visual system experiences an initial loss of sensitivity and acuity. In order to
regain functionality, several processes take place to preserve our ability to form high contrast
images over a wide range of intensities without sacrificing acuity. The overall aim of this project is
to study the functional role of inhibitory synaptic activity in setting the sensitivity of the retina to
different mean light levels and to investigate the modulatory effect of these inhibitory synapses on
ganglion cell excitability under different physiological conditions.
The specific aims are:
1. To investigate the role of spontaneous inhibitory synaptic input in modulating the
excitability properties of retinal ganglion cells. The presence of tonic inhibitory synaptic input onto
ganglion cells and its role in spike generation will also be investigated. Hypothesis: spontaneous
inhibitory input onto retinal ganglion cells modulates their excitability and their input-output
relation (gain).

2. To examine the extent of inhibitory synaptic input, both phasic and tonic, under different
background light levels. Hypothesis: spontaneous inhibitory input onto retinal ganglion cells
changes with ambient light levels, depends on light-adaptation conditions and contributes to
adaptation.

3. To investigate whether changes in the gain of ganglion cells observed during contrast
adaptation, originate from increased phasic and tonic inhibitory synaptic input. Hypothesis:
exposure to a high contrast stimulus increases ensuing inhibitory synaptic activity and
environmental GABA concentration, thus leading to a reduction in the response to subsequent
stimuli.

The significance of this proposal is that it will contribute to our understanding of how
neuronal output and integration properties are finely tuned by the interplay between excitatory and
inhibitory inputs. It will also shed light onto the mechanisms responsible for a visual plasticity and
psychophysical phenomenon which starts in the retina and appears later at higher cortical stages as
well as at the perceptual level.

Background
Environmental light levels vary over approximately 10 log units, from a starlit night to a
sunny afternoon. Efficient information coding restricts the operational range of retinal ganglion
cells to 2.5 log units. To overcome this mismatch, the visual system is endowed with several
adaptational mechanisms to match the bandwidth of the neural code with that of its natural stimulus.
Adaptation starts in the retina and consists of different processes: 1) use of different neuronal
networks -rod circuit vs. cone circuit-, 2) photoreceptor adaptation, 3) lateral inhibition and 4) gain
control.
The output of retinal ganglion cells depends on the balance between stimulus-elicited
excitatory and inhibitory synaptic input as well as on their membrane potential and spike threshold,
which are in turn influenced by background noise consisting of excitatory and inhibitory
spontaneous activity. Ganglion cells receive excitatory input from bipolar cells, which constitute
47% of all neurons in the inner nuclear layer (INL) and inhibitory input mostly from amacrine cells
which represent 49% of the INL neurons (Masland and Raviola, 2000). Approximately 20% of the
synaptic input onto ganglion cells originates from bipolar cells, which use glutamate as their
neurotransmitter, whilst the remaining 80% derives from amacrine cells, which signal mostly via
GABA and glycine (Ghosh and Grunert, 1999; Jacoby et al., 1996).
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GABAA and glycine receptors have been localised to the somatodendritic compartments of
ganglion cells (Grünert et al., 1993; Grünert and Wässle, 1993;). Functional activation of these
receptors in the form of agonist-induced, spontaneous and light-evoked GABA and glycine currents
has been reported (Flores-Herr et al., 2001; Protti et al., 1997; Tian et al., 1998). Consistent with the
anatomical evidence, ratios of 3:1 and 2:1 between inhibitory and excitatory light-evoked synaptic
currents have been reported for salamander and rabbit ganglion cells respectively (Pang et al., 2002;
Taylor and Vaney, 2002).
In addition to fast acting phasic inhibition, many central nervous system neurons display
persistent tonic inhibitory currents induced by environmental GABA acting on extrasynaptic
receptors (Semyanov et al., 2004). These tonic currents can transfer more that three times the charge
transferred by phasic currents and thus, strongly modulate neuronal gain and shape the excitability
of the neurons (Nusser and Mody, 2002).
Inhibitory input modifies excitability in two
different manners: “simple hyperpolarisation”
interacts in a linearly subtractive manner whilst
“shunting inhibition” interacts in a nonlinear
manner acting as a divisor. In the subtractive model,
different levels of hyperpolarisation shift the input-
output relation by subtracting off a fix amount
proportional to the inhibitory conductance. During
shunting inhibition, membrane potential is
“clamped” to the resting potential, thus shunting
excitatory inputs and attenuating depolarisation. The
consequence of such a division operation results in
changes in the neuronal gain, which is the slope of
the input-output relation (Borg-Graham et al., 1998). Figure 1. Two types of inhibitory mechanisms. A The black
solid trace corresponds to the hypothetical relationship of
These two inhibitory mechanisms are illustrated in neuronal firing rate as a function of injected current. “Simple
Figure 1. Interestingly, tonic inhibitory currents have inhibition” shifts the curve to the right (dashed red line) whilst
excitation shifts it to the left (dashed blue line). “Shunting
been shown to produce shunting inhibition and inhibition” via tonic inhibitory input modifies the slope of the
modulate neuronal gain (Chance et al., 2002; input-output relation (dashed green line). Modified from
Chance et al., 2002; Semyanov et al., 2004. B Changes in
Mitchell and Silver, 2003). In this project we are membrane potential in response to current injection in a retinal
going to investigate whether inhibitory synaptic ganglion cell. Firing rate will be investigated under different
light conditions and in the presence of pharmacological agents
activity and “shunting inhibition” modify the spiking which target GABA receptors.
properties of ganglion cells as shown in Figure 1B.
Our preliminary data show that retinal ganglion cells display an inward current which is
enhanced by the GABA uptake blocker NO-711. The blocking effect of GABA and glycine
receptor antagonists on this current, confirms that it is mediated by inhibitory neurotransmitters (see
Figure 2). In this proposal, we are going to delve into the biophysical and pharmacological
characterisation of this tonic current and investigate whether and how it changes under various light
adaptation conditions.

Figure 2 Preliminary results. Tonic GABAergic current.

Control trace was obtained in Ames medium. In this
condition, inhibitory spontaneous synaptic currents and a
tonic inward current are observed. Addition of NO-711, a
GABA uptake blocker, increased the inward current by
70pA. Further addition of an inhibitory neurotransmitter
blocker cocktail (containing 10 M bicuculline, 100 M
picrotoxin and 1 M strychnine), abolished all IPSCs and
reduced the tonic inward current by 120pA. Note the shifts in
baseline current, first as an inward current and then as an
outward current. Recordings were obtained from a ganglion
cell in a whole-mount mouse retina preparation. Patch-clamp
recordings were performed using a KCl based intracellular
solution. Nguyen and Protti, unpublished results
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Contrast adaptation in the visual system is expressed as a reduction in the neuronal response
to a low contrast stimulus upon exposure to a high contrast stimulus. This adaptive change in the
neuronal gain is used to optimise the limited neuronal output (frequency of action potential) as a
function of the broad range of inputs. Contrast adaptation is observed at the psychophysical level
and its underlying bases have been ascribed to neuronal activity in different areas of the visual
system, from the retina to the cortex. At the retinal level, contrast adaptation has been shown to
occur at the bipolar cell level as well as at the ganglion cell level, and it has been postulated that
amacrine cells are also involved in its generation.
Different cellular mechanisms have been proposed to be responsible for contrast adaptation.
In bipolar cells, glutamate receptor desensitisation as well as Ca2+ and other second messenger
mediated mechanisms have been shown to partly mediate this phenomenon (Rieke, 2001). At the
ganglion cell level, reduced excitability and adaptation have been attributed to depolarisation due to
activation of voltage-gated Na+ channels (Kim and Rieke, 2001). Paradoxically, other data suggest
that gain reduction in ganglion cells results from hyperpolarisation arising from amacrine cell input
(Baccus and Meister, 2002). Taken together, these pieces of evidence suggest that multiple
mechanisms are implicated in the generation of contrast adaptation. Based on the strong inhibitory
input from amacrine cells onto ganglion cells and on the reported changes in amacrine cell activity
under varying contrast conditions (Baccus and Meister, 2002), we propose to investigate whether
increase tonic inhibitory input underlies the decrease in gain observed upon contrast adaptation.

Research Plan, methods and techniques

Preparation, maintenance and recording.
Whole-cell voltage and current-clamp recordings from ganglion cells will be carried out in
mouse retinal whole-mounts. To preserve sensitivity of the retinal tissue to light, the animal will be
dark-adapted before isolation of the retina. The dissection will be performed in oxygenated Ames
medium under infrared (IR) illumination to prevent photoreceptor bleaching and light adaptation.
All subsequent procedures will be carried out under IR illumination.
Retinal whole-mounts are placed photoreceptor side down in a chamber and visualised via an
upright microscope (40x) with an IR-sensitive video camera. To gain access to ganglion cells with a
patch electrode, their soma is exposed by making an opening in the inner-limiting membrane which
covers the ganglion cell layer. Patch pipettes (6-8 M) are filled with KCl and K-Gluconate based
intracellular solutions containing Lucifer Yellow allowing morphological reconstruction of the cells
following recordings.
Aim 1. Role of spontaneous inhibitory synaptic input in the excitability properties of retinal
ganglion cells. Recordings.
To investigate the role of phasic and tonic inhibition in ganglion cell excitability, spontaneous
and current-induced action potentials (as in Figure 1B) will be recorded before and after perfusion
with GABA and glycine receptors antagonists, inhibitors of GABA uptake and GABA agonists. To
assess the existence of tonic inhibitory currents, spontaneous inhibitory synaptic currents and
holding currents will be monitored in control conditions and in the presence of the aforementioned
pharmacological agents (see Figure 2). Fluctuation analysis of the currents in the presence of
different pharmacological agents will be performed to estimate mean conductance levels of the
channels involved in generating tonic currents (as in Gottesman and Miller, 2003).
Aim 2. Light-induced changes in phasic and tonic inhibitory synaptic input.
Spontaneous inhibitory and excitatory currents will be characterised based on their
biophysical and pharmacological properties (Protti et al., 1997). Spontaneous and tonic inhibitory
currents will be measured under different light-adapted conditions. Background light will be
projected onto the retina through the microscope optics using a computer-controlled miniature LCD
display coupled to the microscope. Light intensity will be varied over 7 log units by interposing
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neutral density filters in the optical path. The effects of GABA and glycine signalling modulators on
phasic and tonic membrane currents at different light-adapted levels will be investigated. Spiking
patterns (transient vs. sustained) under different light-adapted conditions will also be investigated.
Aim 3. Role of phasic and tonic inhibitory synaptic input in contrast adaptation and gain
control.
The role of inhibitory synaptic input on stimulus-induced changes in gain will be investigated
by recording light-evoked currents and their ensuing synaptic activity in response to presentation of
cycles of light stimuli that provoke contrast adaptation. Gratings and checkerboards of low and high
contrast will be displayed in cycles consisting of test stimulus (low contrast) - adapting stimulus
(high contrast) - test stimulus (low contrast). The effect of GABA/glycine blockers, agonists and
GABA uptake blockers on contrast adaptation will be investigated. The putative involvement of
signal molecules that act retrogradely to modulate inhibitory synaptic activity (i.e.
endocannabinoids) will also be explored.

Budget Justification

Personnel
Support for one (1) part-time laboratory assistant to carry out general lab tasks, prepare the
lab for the experiments and process tissue for microscopy is requested. They will also be required to
assist with ordering, budget monitoring and some clerical work.
Equipment
Support is requested for the purchase of a light weight, high-contrast DLP projector to display
light stimuli onto the retina at an estimated cost of $ 3,600. This equipment is critical for Aim 3 of
this project, to allow us to display high-contrast stimuli.

An additional amount of $13,140 is required made up of the following costs;

It is estimated that 3 experiments per week will be carried out for 40 weeks of the year, each
experiment using 1 mouse.
120 mice @ $12 each mouse = $ 1,440
Consumables, drugs and chemicals@ $60/expt = $ 7,200
Minor equipment = $ 3,000
Publication costs = $ 1,500
Total (per annum) $13,140

Reference List

Baccus, S. A., and Meister, M. (2002). Fast and slow contrast adaptation in retinal circuitry. Neuron 36, 909-919.
Borg-Graham, L. J., Monier, C., and Fregnac, Y. (1998). Visual input evokes transient and strong shunting inhibition in
visual cortical neurons. Nature 393, 369-373.
Chance, F. S., Abbott, L. F., and Reyes, A. D. (2002). Gain Modulation from Background Synaptic Input. Neuron 35,
773-782.
Flores-Herr, N., Protti, D. A., and Wassle, H. (2001). Synaptic currents generating the inhibitory surround of ganglion
cells in the mammalian retina. J Neurosci 21, 4852-4863.
Ghosh, K. K., and Grunert, U. (1999). Synaptic input to small bistratified (blue-ON) ganglion cells in the retina of a
new world monkey, the marmoset Callithrix jacchus. J Comp Neurol 413, 417-428.
Gottesman, J., and Miller, R. F. (2003). N-methyl-D-aspartate receptors contribute to the baseline noise of retinal
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ganglion cells. J Neurosci 21, 287-299.
Masland, R. H., and Raviola, E. (2000). Confronting complexity: strategies for understanding the microcircuitry of the
retina. Annu Rev Neurosci 23, 249-284.
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Mitchell, S. J., and Silver, R. A. (2003). Shunting inhibition modulates neuronal gain during synaptic excitation. Neuron
38, 433-445.
Nusser, Z., and Mody, I. (2002). Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J
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Protti, D. A., Gerschenfeld, H. M., and Llano, I. (1997). GABAergic and glycinergic IPSCs in ganglion cells of rat
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