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Sensory integration in the Zebrafish brain, what are the functions of the thalamus and the cerebellum ?

Gilles Vanwalleghem1*, Lucy A. Heap1, Itia Favre-Bulle1, Kevin Schuster1, Andrew Thompson1 and Ethan K. Scott1
  • 1 The University of Queensland, School of Biomedical Sciences, Australia

The integration of sensory information across modalities occurs in all forms of life from single cells to humans. This integration can allow inputs from one modality to modify responses to another, either enhancing or depressing the magnitude of the neural activity that results from a given stimulus [1]. Integration can also reduce the lag between sensory input and motor reactions or the latency of neurons’ responses [2, 3]. In the cat superior colliculus (SC), integration occurs via multisensory neurons receiving converging inputs [4]. Each of these neurons has excitatory receptive fields, one for each modality to which it responds, forming topographic maps of each modality, thus allowing spatial registration of multisensory inputs. However, recent data suggest that multimodal integration may also occur without multisensory neurons. An example can be found in zebrafish larvae, where visual input modulates an auditory escape response. The neurons driving the response, called Mauthner cells, do not receive direct visual input, but the efficacy of the VIIIth nerve afferents were increased by dopaminergic neurons from the hypothalamus [1], allowing for visual modulation of audition. Complex neural circuits such as this one may be responsible for many behavioural responses involving cross-modal integration. The optic tectum (OT) of zebrafish is the anatomical equivalent of the SC; as in the mammals, the superficial layers of the OT receive the retinal afferents and process visual input [5]. This processed information flows to the deeper layers where it is relayed to pre-motor nuclei. Furthermore, it has been shown that multimodal inputs converge on OT neurons in Xenopus tadpoles [6] and that the periventricular neurons of the rainbow trout receive multi-sensory afferents and as such may be responsible for their integration [7]. While the superficial layers of the tectal neuropil have been well characterised, the functions of the deeper layers remain elusive, and the mechanisms by which they may mediate multimodal integration are unknown. The same applies to the thalamus, which is well known as a crossroads for a wide array of sensory information, but which is poorly understood at the level of cellular circuits. Although the cerebellum is generally viewed as a motor processing centre, evidence also suggests functions in sensory integration [8, 9]. Recent work has shown that cerebellar lesions disrupt pitch discrimination in human patients [10], and this has been corroborated by PET imaging showing an increase of activity in the lateral cerebellum during pitch discrimination exercises [11]. In rats, cerebellar Purkinje cells respond to whiskers stimulation and present a topographical map of the inputs [12]. In summary, while numerous brain regions have been shown to process sensory information, and while the interconnectedness of these regions must underlie sensory integration, we still have a poor appreciation for how different modalities are integrated, and how resulting behaviours are produced. The details of these relationships can only be described if the circuits can be characterised at the cellular level, both in terms of their anatomy and their activity. As a transparent animal and with the rise of powerful light-based tools to monitor and manipulate the brain, the larval zebrafish offers a perfect window into functioning neural circuits. The system is particularly promising given the similarities that exist in the key sensory processing centres between zebrafish and mammals. Recent work in the host’s lab has shown cerebellar outputs to the deep layers of the tectal neuropil and to the thalamus [13] and robust projections also exist from the thalamus to the tectal neuropil (Scott lab, manuscript in preparation). This establishes, at least at an anatomical level, the necessary components for complex cross-modal integration, with two important sensory-processing structures sending converging information into a third structure, the OT, which itself has been implicated in sensory integration. It also parallels closely the connectivity of the homologous mammalian brain structures in what may be an evolutionarily conserved integration mechanism. To clarify these relationships, we use transparent zebrafish larvae, in combination with transgenics and optogenetics, to study the functional links among cerebellum, optic tectum and thalamus in a way that has been previously unapproachable. We are using optogenetic neuromodulation [14] of the regions of interest using channelrhodopsin (ChR) by a Spatial Light Modulator (SLM) [15]. The SLM allows us to project holograms to precisely activate selected brain regions. Combined with the genetically encoded calcium sensor GCaMP6 [16] and single plane illumination microscopy (SPIM), we are mapping out the functional connectivity among the thalamus, cerebellum, and tectum, describing the scheme of functional separation and overlap in tectal cells receiving thalamic and cerebellar input. This will reveal the “code” within the tectal circuitry that integrates streams of information for different brain regions and sensory modalities. Outside of the OT, not much is known regarding the brain regions involved in sensory processing in zebrafish and how they are connected. We are using a pan-neuronal GCaMP6 [16] to identify, in an unbiased fashion, other brain regions involved in sensory integration, focusing on visual, auditory and lateral line input. To that end we are imaging the whole brain while we play sensory stimuli of each studied modality. This will allow us to identify the compartments of each brain regions that are responsive to each modalities, and register these patterns to identify how the inputs from different modalities may be combined or kept separate. For the data analysis, we are using the Thunder library [17] running on the NECTAR cluster. We are using simple correlation and non-negative matrix factorization that we found to be more robust and informative than principal component analysis. In our preliminary unbiased screen, we observed visual response in the thalamus and cerebellum, which confirms our interest in those brain regions. Interestingly, the thalamic response appeared tuned to a moving spot moving caudo-rostrally. Using genetically targeted expression of GCaMP in the thalamus, we further investigated the response profile of the thalamic response to changing polarity and size of the spot. Smaller spots or large bright spots on a dark background did not elicit any response. The thalamic response appears tuned to bigger, fast spots known to elicit hunting behaviour. By expressing ChR in either the thalamus or the cerebellum, we were able to use the SLM to specifically activate those brain regions and look for neural responses in the OT. We found that activating the cerebellum triggered responses in the optic tectum. Activating the thalamus, which is inhibitory, triggered rebound firing in the optic tectum. These experiments show that functional connectivity exists between the cerebellum, thalamus and optic tectum. We plan to investigate how those feeback may be involved in the filtering of sensory information to trigger specific behavioural responses. This work will pave the way for further studies into the integration of multimodalities inputs, and the possible mechanisms by which they influence behavioural outputs. As a future direction, hopefully in my own independent research group, I would aim to identify the behavioural relevance of this integration in a stationary rheotaxis preparation. Such a preparation would allow me to present conflicting stimuli (flow and visual, for instance) to study their impacts on integration, and to activate or silence brain regions or circuits implicated in the above work.

References

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Keywords: Zebrafish, GCaMP, optogenetics, Thalamus, optic tectum

Conference: Neuroinformatics 2015, Cairns, Australia, 20 Aug - 22 Aug, 2015.

Presentation Type: Poster, to be considered for oral presentation

Topic: Neuroimaging

Citation: Vanwalleghem G, Heap LA, Favre-Bulle I, Schuster K, Thompson A and Scott EK (2015). Sensory integration in the Zebrafish brain, what are the functions of the thalamus and the cerebellum ?. Front. Neurosci. Conference Abstract: Neuroinformatics 2015. doi: 10.3389/conf.fnins.2015.91.00018

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Received: 30 May 2015; Published Online: 05 Aug 2015.

* Correspondence: Dr. Gilles Vanwalleghem, The University of Queensland, School of Biomedical Sciences, St. Lucia, QLD, 4072, Australia, Gilles.Vanwalleghem@gmail.com