Neural circuitry and plasticity in the adult vertebrate inner retina

. Glutamatergic synapses between retinal bipolar cells and amacrine cells ccxlc for transient and sustained events in the visual environment under widely varying conditions of background illumination; i.e. from very light to dark background conditions. 'llle cell types and synaptic mechanisms responsible for coding the transient and sustained information arc relatively well described. Recent studies suggest that these synapses arc highly plastic in response to environmental stimulation, functionally and structurally remodeling during changes in the ambient lighting conditions bathing the retina. Our current studies of the 7.ebrafish retina arc investigating these plastic changes at the bipolar to amacrine cell synapses using a combination of techniques, including patch recording and 2-photon microscopy in the zebrafish retinal slice. We are particularly interested in how the newly discovered endogenous cannahinoid signaling system of the retina controls plasticity at these synapses.


Introduction
Glutamatergic synaptic transm1sston in the inner vertebrate retina occurs at specialized synapses that include synaptic ribbons [I] in the presynaptic bipolar cells, and a variety of glutamate receptors in the postsynaptic amacrine [2] and ganglion cells. In the fish retina, the release of glutamate from the presynaptic bipolar cells has been suggested to be sustained by some [3] and transient by others [4]. In tiger salamander retina, the presynaptic bipolar cells appear to only usc sustain.ed type voltage-gated calcium channels [5] that would support sustained calcium-dependent glutamate release. But in fish retina, the presynaptic bipolar cells come in two types with respect to voltage-gated calcium channels; one type expressing only sustained and the other type expressing only transient voltage-gated calcium channels [6], and thus calcium-dependent glutamate release could he brief at those synapses where the bipolar cell expresses transient calcium current, or prolonged where the bipolar cell expresses sustained calcium current.
Recent evidence suggests that these synapses structurally remodel themselves when they experience different lighting conditions. That is, when the retina experiences conditions where it goes from dark to light, or vice versa, the bipolar cell axon terminals rapidly remodel themselves [7]. In other parts of the brain, cannabinoids have been shown to regulate LTP f8), a form of synaptic plasticity, and to regulate focal adhesions, which control cellular shape and arc thought to be involved in LTP [9].

Synaptic Transmission in Identified Retinal Neurons: Patch Clamp Electrophysiology
Retinal slices arc made according to the procedures described by [6] as adapted from [I 0]. The physiologically recorded cells are also identified using either Lucifer yellow or Calccin dyes to fill the cell which is then made to nuoresce under appropriate illuminating conditions.

Bipolar Cell Voltage-Gated Calcium Currents
Voltage-gated calcium currents in bipolar cells of the tiger salamander are shown in Figure  I A, while those from the zcbrafish retina arc shown in Figure I B. While all tiger salamander retinal bipolar cells exhibit sustained calcium currents, the zcbrafish bipolar cells exhibit either transient (66%) or sustained (34%) calcium currents. Because glutamate release from these bipolar cells is calcium dependent, the kinetics of the calcium currents may very well control, at lea~t partially, the kinetics of glutamate relca~c from the bipolar cells.

Release or Glutamate From llipolar Cells
In salamander retina, the release of glutamate from bipolar cells appears to be sustained. This is inferred from measuring the excitatory postsynaptic currents (EPSCs) in the postsynaptic amacrine cells. Normally the EPSCs are very brief, but in the presence of drugs to remove rapid desensitization of the glutamate receptors in the amacrine cells, the EPSC is sustained ( Figure 2). This suggests that that the relea~e of glutamate from bipolar cells onto amacrine cells is sustained in salamander. Similar experiments in the zebrafish retina are currently being performed.

Imaging or Living Bipolar Cell Axon Terminals
Imaging of living retinal bipolar cell axon terminals is now possible using either fluorescent laser scanning confocal microscopy or 2-photon fluorescent laser scanning microscopy. Figure 5 shows an example of a Oil labeled bipolar cell in the zehrafish retinal slice. The area of interest is within the box and shows two bipolar cell axon terminals nestled together. A putative postsynaptic process impinges on the bipolar cell axon terminal that resides on the left. These are confocal images, and recently we have made similar observations with Drs. Fraser and Potter at CalTech using 2-photon laser scanning microscopy. Two-photon laser scanning microscopy has the advantages of allowing the tissue to be viewed for longer periods of time without bleaching and destruction of the tissue.

Cannabinoids in the Vertebrate Retina
Cannabinoids have been shown to regulate plasticity of the brain within the hippocampus [8]. we have therefore begun a search in the vertebrate retina to determine whether there is an endogenous cannabinoid signaling system in the retina, and whether cannabinoids regulate synaptic plasticity in the inner retina.

Endogenous Cannabinoids
Retinas were removed and prepared for biochemical analysis using mass spectrometry and gas chromatography [8]. Interestingly, 2-arachidonylglycerol and palmitoylethanolamide was found in the retina, but anandamide was not. The retinas were analyzed under light adapted conditions, and it is possible that anandamide production and/or release is regulated by the light adapted state of the retina such that· anandamide is not released in the light. Thus, in light adapted retinas, two of the three endogenous cannabinoids found in other parts of the brain are found in the vertebrate retina.

C81 Cannabinoid Receptors
CB I cannabinoid receptors were detected in the fish retina using fluorescent antibodies. Pronounced staining for CBI was found in the inner plexiform layer of the retina, consistent with CB I receptors being localized to the bipolar cell axon terminals. Future studies using double labeling techniques must demonstrate that CB I receptors are expressed in the axon terminals.

C82 Cannabinoid Receptors
We have also localized the mRNA for CB2 cannabinoid receptors in the retina. CB2 mRNA is present in the inner nuclear layer, but we don't yet know the expression pattern of the CB I receptors. The functional role of CB2 in other parts of the nervous system is not well understood, although it docs appear to regulate cAMP. Because cAMP is involved in synaptic plasticity in the retina, it will he of interest to determine whether CB2 is also involved in the remodeling of bipolar to amacrine cell synapses.

Conclusions
Glutamatergic synaptic transmiSSion between retinal bipolar cell axon terminals and amacrine cells arc capable of generating transient and sustained signals and operate over an extreme range of background lighting conditions with apparent gain changes during the changes in lighting conditions. Part of the process of adapting to the different lighting conditions involves a structural remodeling of these synapses which can now be viewed in living tissue using confocal and 2-photon imaging. We have discovered an endogenous cannabinoid signaling system in retina, thought to regulate synaptic plasticity in other parts of the brain, that we are investigating to determine whether synaptic plasticity at the bipolar to amacrine cell synapse is modulated by activation of CB I and CB2 cannabinoid receptors.