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At the bench: Two-photon microscopy illuminates our understanding of vision

This is the first post in Scientifica’s 'At the bench' series. Articles written by researchers on their area of study and laboratory techniques.

Many organisms rely on visual cues for their survival and well-being. Humans have wondered for aeons how we perceive the visual space. The earliest systematic studies emanate from pioneering work by the Arab polymath Abū ʿAlī al-Ḥasan ibn al-Ḥasan ibn al-Haytham during the early years of the last millennium1. He performed studies on the basic physical properties of light and also put forth a comprehensive theory of vision.

Since then, technological and theoretical advances in different branches of science have allowed us to dig deeper into the mechanisms of how the visual information is processed. We know that the task of detecting and processing visual information is performed first by the neuronal circuits in the retina (see Fig. 1). Although we now know the fundamental aspects of the neurons in the retina - their gross connectivity, molecular identities, and their function - many questions still remain unanswered

The work in the Lagnado lab focuses on the study of synaptic computation in the context of the retina. Particularly, we are interested in the mechanisms controlling neurotransmitter release at synapses; how synapses determine the processing of information by a neural circuit, and various forms of plasticity of the circuit.

Synaptic Function in the Retina

The functional precision of any neural circuit depends on the rapid transfer of signals at synaptic junctions. The efficiency of synaptic transfer is not fixed; the synapse acts as a computational unit, whereby the relation between input and output varies dynamically according to the recent and past activity of the network2,3. For example, the retina is thought to continually adjust its input-output relation in response to changes in the statistics of the visual scene. Elucidating the biophysical mechanisms of plasticity at single synaptic contacts is therefore essential for understanding the information processing in complex synaptic circuits.

To understand how synapses and neurons function, we need to be able to record their activity during ongoing sensory stimulation. There are two aspects to this - one, we need microscopy methods to see what neurons and synapses are doing and two, we need molecular sensor/s that reliably report their activity.

In most cases, the neural circuitry is buried deep inside the tissue; therefore, conventional light microscopy techniques fail to capture the activity of these circuits. To study the retinal circuits in their native condition, we take advantage of two-photon microscopy – where, two low-energy photons cooperate to cause a higher-energy electronic transition in a fluorescent molecule4. An important advantage of two-photon microscopy is that near IR excitation penetrates tissue better than the visible light used in one-photon microscopy because of reduced scattering and absorption by endogenous chromophores. In parallel, to monitor physiologically relevant neuronal activity, we take advantage of genetically-encoded fluorescent molecules that report either calcium concentration, neurotransmitter release, or membrane voltage.

We developed a synaptically-targeted and genetically-encoded calcium indicator by fusing vesicle protein synaptophysin to GCaMP5. This allows for monitoring activity of a population of synapses in a circuit with a resolution of a single synapse. We also employ reporters of synaptic vesicle release such as iGluSnFr6 and SypHy7. We express such fluorescent reporters in defined cell types of the zebrafish retina using specific promoter sequences and we shine a two-photon laser light on the retinal circuitry and scan over the cell population to record fluorescence change in response to ongoing visual stimulation; thus, we use light to study vision.

Synaptic processing in bipolar cells

Using such an approach we have begun to unravel the basic properties of synapses in the retina with a particular focus on the synaptic terminals of bipolar cells8,9,10,11 (Fig. 2). We observed that synapses display luminance sensitivities varying over 104 with a log-normal distribution and that half of the synaptic terminals display triphasic tuning curves allowing for a broader dynamic range over which bipolar cells signal light and improve the efficiency with which luminance information is transmitted. The complexity of visual processing became more evident from our recent work9,10: that is, the conversion of the visual signal from the analog form of graded potentials to the digital form of spikes first occurs in bipolar cells. This was surprising, because hitherto it was thought that spiking initiates first in ganglion cells and amacrine cells in response to graded inputs from bipolar cells. About 65% of the bipolar cell terminals show both spontaneous and light-evoked spiking activity, the large majority in the form of calcium spikes triggered by voltage-dependent calcium channels in the synaptic terminals. Importantly, in the presence of continuous visual contrast, a bipolar cell can switch between graded and spiking signals with a strong dependence on the prevalent membrane voltage.

Recently, we also demonstrated an intriguing strategy for the transfer of visual information. We found that some bipolar synapses adapt following an increase in light stimulus variance and the other population becomes sensitized11. Evidence from our lab and others indicates that the depletion of vesicle pools in response to repetitive stimulation is thought to be a mechanism behind adapting terminals. The converse effect of facilitation occurs via depression in the inhibition of the input from amacrine cells (Fig. 3). A combination of adaptation and sensitisation might help to improve overall information transfer when visual statistics fluctuate in time.

Another basic property of synaptic terminals that shape response kinetics, gain, and adaptation is the size12,13. We discovered that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Smaller terminals generate faster and larger calcium signals to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Even a single bipolar cell can have two terminals varying in size; thus, one bipolar cell is able to multiplex the visual information in separate channels with different temporal filters.

The future of retinal studies

Even though our understanding of the transfer of visual information through the retinal synaptic circuits has gained a lot of ground, many questions still remain unanswered. For example, what are amacrine cells exactly doing to shape the transmission? How do synaptic circuits respond to sudden change in visual statistics, such as motion reversal? What are spikes in bipolar cell terminals important for and how do these calcium spikes alter neurotransmitter release?

The creation of better variants of genetically encoded indicators and their applications using two-photon and light-sheet microscopy will allow not only for a better understanding of retinal circuits but also of neuronal and synaptic circuits in other parts of the brain.


‪1. Alhacen's Theory of Visual Perception: A Critical Edition, with English Translation and Commentary, of the First Three Books of Alhacen's De Aspectibus, the Medieval Latin Version of Ibn Al-Haytham's Kitab Al-Manazir, Volumes 1-2‬ (2001) American Philosophical Society 819 pages‬‬‬‬‬‬‬‬‬‬

2. Abbott L.F., Regehr W.G. Synaptic computation (2004) Nature 431:796-803

3. Silver R.A. Neuronal arithmetic (2010) Nature Reviews Neuroscience 11:474-489.

4. Svoboda K., Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience (2006) Neuron 50, 823–839

5. Dreosti E., Odermatt B., Dorostkar M.M., Lagnado L. A genetically encoded reporter of synaptic activity in vivo (2009) Nature Methods 6, 883–889

6. Marvin J.S., Borghuis B.G., Tian L., Cichon J., Harnett M.T., Akerboom J., Gordus A., Renninger S.L., Chen T.W., Bargmann C.I., Orger M.B., Schreiter E.R., Demb J.B., Gan W.B., Hires S.A., Looger L.L. An optimized fluorescent probe for visualizing glutamate neurotransmission (2013) Nature Methods 10, 162–170

7. Royle S.J., Granseth B., Odermatt B., Derevier A., Lagnado L. Imaging pHluorin-based probes at hippocampal synapses (2008) Methods in Molecular Biology 457, 293-303

8. Odermatt B, Nikolaev A, Lagnado L Encoding of luminance and contrast by linear and nonlinear synapses in the retina (2012) Neuron 73, 758-773.

9. Baden T., Esposti F., Nikolaev A., and Lagnado L. Spikes in retinal bipolar cells phase-lock to visual stimuli with millisecond precision (2011) Current Biology 21, 1859-1869.

10. Dreosti E., Esposti F., Baden T., Lagnado L. In vivo evidence that retinal bipolar cells generate spikes modulated by light (2011) Nature Neuroscience 14, 951-952.

11. Nikolaev A., Leung K.M., Odermatt B., Lagnado L. Synaptic mechanisms of adaptation and sensitization in the retina (2013) Nature Neuroscience 16, 934–941.

12. Baden T., Nikolaev A., Esposti F., Dreosti E., Odermatt B., Lagnado L. A synaptic mechanism for temporal filtering of visual signals (2014) PLoS Biology 12:e1001972

13. Suh B., Baccus S.A. Building Blocks of Temporal Filters in Retinal Synapses (2014) PLoS Biology 12(10): e1001973

14. Esposti F., Johnston J., Rosa J.M., Leung K.M., Lagnado L. Olfactory stimulation selectively modulates the OFF pathway in the retina of zebrafish (2013) Neuron 79, 97–110

About the author:

Dr Nachiket D. Kashikar studied Microbiology as an undergraduate at the University of Pune before moving to Forschungszentrum Jülich (Germany) for his PhD, where he investigated the biophysical principles underlying sperm chemotaxis. After a short stint as a post-doctoral fellow at the Max Planck Institute for Biophysical Chemistry in Göttingen, he moved to Leon Lagnado’s lab. He obtained a Marie Curie post-doctoral fellowship to support his work at the MRC Laboratory of Molecular Biology, Cambridge and then the University of Sussex. His primary research interests are sensory physiology, synaptic transmission, optical imaging, and fluorescent reporters of cellular activity.

Banner image credit: Rozan Vromann (Sussex Neuroscience, University of Sussex)


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