Two-Photon Laser Ablation and In Vivo Wide-Field Imaging of Inferior Olive Neurons Revealed the Recovery of Olivocerebellar Circuits in Zebrafish

Abstract

The cerebellum, a brain region with a high degree of plasticity, is pivotal in motor control, learning, and cognition. The cerebellar reserve is the capacity of the cerebellum to respond and adapt to various disorders via resilience and reversibility. Although structural and functional recovery has been reported in mammals and has attracted attention regarding treatments for cerebellar dysfunction, such as spinocerebellar degeneration, the regulatory mechanisms of the cerebellar reserve are largely unidentified, particularly at the circuit level. Herein, we established an optical approach using zebrafish, an ideal vertebrate model in optical techniques, neuroscience, and developmental biology. By combing two-photon laser ablation of the inferior olive (IO) and long-term non-invasive imaging of whole-brain imaging at a single-cell resolution, we succeeded in visualization of the morphological changes occurring in the IO neuron population and showed at a single-cell level that structural remodeling of the olivocerebellar circuit occurred in a relatively short period. This system, in combination with various functional analyses, represents a novel and powerful approach for uncovering the mechanisms of the cerebellar reserve, and highlights the potential of the zebrafish model to elucidate the organizing principles of neuronal circuits and their homeostasis in health and disease.

A functional schizophrenia-associated genetic variant near the TSNARE1 and ADGRB1 genes

“…Representative GFP expression pattern from F 2 zebrafish embryos suggest that this sequence is functional in three major neuroanatomical regions ( Figures 1 and S1 ): (1) the habenula, which is involved in aversive social responses in zebrafish, 46 including anxiety, 47 fear, 48 and aggression 49 ; (2) the caudal hypothalamus, which is involved in appetite control, 50 but in which dopaminergic (DA) neurons are also involved in audio-sensory functions, including locomotion and sensation of acoustic/vibrational stimuli 51 ; and (3) the inferior olive, which receives DA projections from the caudal hypothalamus, 51 but also provides climbing fibers to Purkinje cells of the cerebellar cortex that are required for motor learning. 52 , 53 The more widespread GPF signal in these fish is consistent with expression in glutamatergic neurons, as well as a few regions with GABAergic neurons ( Figure 1 ). These findings suggest that the SCZ-associated variant, rs4129585, likely lies within a TFBS that elicits its regulatory function in glutamatergic neurons and regions of the brain that have been implicated in neurodevelopment and SCZ pathology.…”

Diving into the zebrafish brain: exploring neuroscience frontiers with genetic tools, imaging techniques, and behavioral insights

“…One of the innovative methods used to study plasticity of neuronal connectivity in the brain is laser ablation of neuronal cells ( Figure 3B ). It may be also employed to study the role of a subpopulation of neurons in behavior ( Muto and Kawakami, 2018 ) or to study single neurons connectivity development within a circuit ( Hiyoshi et al, 2021 ). Laser ablation can be performed using a two-photon laser microscope and is especially well-suited for disrupting the activity of a limited number of specific neurons.…”

“…Expression of fluorescent proteins in the specific population of neurons allows for the ablation of these neurons at desirable locations and timing at a high spatial resolution (single-cell level). Hiyoshi et al (2021) used two-photon laser ablation of the inferior olive combined with long-term live imaging of the whole brain and showed structural remodeling of the olivo-cerebellar circuit at a single-cell resolution ( Figure 3C ). Irreversible ablation of neurons caused severe damage in the inferior olive and enabled live observation of remodeling of the circuit ( Hiyoshi et al, 2021 ).…”

“… Hiyoshi et al (2021) used two-photon laser ablation of the inferior olive combined with long-term live imaging of the whole brain and showed structural remodeling of the olivo-cerebellar circuit at a single-cell resolution ( Figure 3C ). Irreversible ablation of neurons caused severe damage in the inferior olive and enabled live observation of remodeling of the circuit ( Hiyoshi et al, 2021 ).…”

A Functional Schizophrenia-associated genetic variant near theTSNARE1andADGRB1genes

“…Representative GFP expression pattern from F2 zebrafish embryos suggest that this sequence is functional in three major neuroanatomical regions (Figure 1 & Figure S1): (1) the habenula, which is involved in aversive social responses in zebrafish 45 , including anxiety 46 , fear 47 , and aggression 48 ; (2) the caudal hypothalamus, which is involved in appetite control 49 , but in which dopaminergic (DA) neurons are also involved in audio-sensory functions, including locomotion and sensation of acoustic/vibrational stimuli 50 ; and (3) the inferior olive, which receives DA projections from the caudal hypothalamus 50 , but also provides climbing fibres to Purkinje cells of the cerebellar cortex that are required for motor learning 51,52 . The more widespread GPF signal in these fish is consistent with expression in glutamatergic neurons, as well as a few regions with GABAergic neurons (Figure 1).…”