Alyan, Adnan

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Cal Poly Humboldt Digital Commons @ Cal Poly Humboldt IdeaFest 2024 IdeaFest 2024 Comparative Anatomy of Mouse, Zebrafish, and Trout Adnan Alyan aa788@humboldt.edu Follow this and additional works at: https://digitalcommons.humboldt.edu/ideafest2024 Recommended Citation Alyan, Adnan, "Comparative Anatomy of Mouse, Zebrafish, and Trout" (2024). IdeaFest 2024. 6. https://digitalcommons.humboldt.edu/ideafest2024/6 This Article is brought to you for free and open access by the IdeaFest at Digital Commons @ Cal Poly Humboldt. It has been accepted for inclusion in IdeaFest 2024 by an authorized administrator of Digital Commons @ Cal Poly Humboldt. For more information, please contact kyle.morgan@humboldt.edu. Comparative Anatomy of Perineuronal Nets in Mouse, Zebrafish and T.routAdnan Alyan, Ethan GahtanCal Poly Humboldt , Departments of Biology and PsychologyBACKGROUNDPerineuronal nets (PNNs) and perinodal matrices are rigid, protein-saccharide structures that encapsulate certain neurons 1,2,3. PNNs form around fast-firing, parvalbumin-expressing GABAergic neurons, among other neuron types, and have been found in nearly all parts of the mammalian CNS 1,2,4.PNN function. PNNs develop at the end of critical periods of neural plasticity, such as when visual cortex neurons commit to receiving input from either the left or right eye. PNNs may stabilize synapses, protecting them from damage but limiting future plasticity 1,2. For example, enzymatic digestion of PNNs in mature cat brains can re-open occular dominance plasticity. PNN disruption has been shownin Fragile X syndrome, Alzheimer’s Disease, epilepsy5,6 schizophrenia, and bipolar disorder 7,8.PNNs across species - Zebrafish and beyond. Zebrafish are an important model organism with potential advantages for studying the role of PNNs inbrain plasticity. Previous research has identified PNNcomponents in zebrafish 9,10, but their presence has yet to tested using with method of Wisteria FloribundaAgglutinin (WFA), standard in the mammalian PNN research.4,6Current study - cros-species comparision.Taking a comparative approach, we used the same WFA staining method to label PNNs in brain sections from mouse (as a positive control), zebrafish (of interest as a research model), and trout (to assess generalizability across fish, of the same teleostei infraclass). Parvalbumin neurons were also labeled in the same slices to assess their known association. ??PNNs‘C     Early developing brain: PNNs absent ritical period’ learning (plasticity) occursTenascin-RHyaluronanCSPG Link proteins(HAPLN)HAPLN connects CSPG to hyaluronan Mature Brain: PNNs form‘Critical period’ learning endsPNN formation: Upregulation of hyaluronan and proteoglycan binding link protein (HAPLN)Hyaluronan synthase (unbound)Immature parvalbumin-expressing   neuron**WFA labelingof PNNsFigure 1: Formation of PNN during the critical period of neural plasticity. (Above) Parvalbumin-expressing neurons produce hyaluronan, chondroitin-sulfate proteoglycans (CSPGs), and Tenascin-R during early stages of development, but notably lack HAPLN link proteins. Production of CSPG link proteins is hypothesized to trigger PNN formation. (Below) HAPLN link proteins are expressed at the end of critical period of plasticity and PNN structure begins to form. Note HAPLN links CSPGs to hyaluronan and Tenascin-R connects up to three CSPG chains together.METHODSAll mehods were approved by the Cal Poly Humboldt IACUC. Brains from adult mouse (N=1), zebrafish (N=6), and trout (N=3), were processed similarly with these main steps: (1) Fixation in paraformaldehyde; (2) Sectioning in the coronal plane 50-100um using a vibratome after agarembedding; (3) standard immuno-fluorescence labeling procedure for parvalbumin with a primary andsecondary antibody (using red fluorescence); (4) co-labeling with Fluorescein-conjugated WFA, a greenfluorescein lecin which binds to the CSPG components of PNNs; (5) Imaging using a confocal microscope(Olympus FV1000) to document associations of red parvalbumin labeling within neuronal cell bodies withsurrounding densities of green WFA.RESULTSMouseBAAdapted from Lensjø et al.4C DAdapted from Carcellar et al. *Figure 2. Vibratome sections mouse sec-tions imaged via confocal microscope from our study (fig 2A & 2B) compared to images pulled from the literature (fig 2C & 2D). Both our slices and the example slides have been subjected to a similar IHC both con-sisting of PV anti-body with WFA. Dif-fuse PNNs can be visualized (fig 2A & 2B) in line with previ-ous examples from the literature (fig 2C & 2D). On a neuro-nal level, WFA-stain-ing PNN can be found enwrapping the neuron (fig 2D), and to a lesser extent, axonal and dendritic processes.  Trout ZebrafishABEFGC HDFigure 3. Various images of Vibratomed trout and zebrafish brain slices of various locations and magnifi-cations. High rates of colocalization between PV and WFA staining can be found across all images. WFA will typi-cally present in a shape consistent with the out-line of a neuron, the body of this neuron either being empty space or PV+ staining. Closeups of this (fig 2D, 2H) reveals the extent of PV+ and WFA stain-ing concomitance, though PNNs were also commonly observed without association with parvalbumin. Results are highly in line with existing depictions of PNN in the literature. PNNs identifification. We observed bright, net-like WFA labelling surrounding parvalbumin neurons, consistent with our hypothesis that PNNs are present in the brains of both trout and zebrafish (fig 3). We base this claim on the success of our positive control (fig 2A & 2B), which was successful in replicating PNNs as described in the literature. This serves proof that our methods are capable of replicating these results in other PNN positive animals. The reticular nature of the structures we see in our trout and zebrafish images coincides with our mouse control. This is further supported by the similar rates of colocalization with PV+ staining. Closer examination (fig 3D & 3H) reveals WFA is brightest when on the outskirts of a PV+ neuron, consistent with what is known about PNNs. It follows that the results of our IHC and imaging suggest the presence of PNNs in both zebrafish and trout.DISCUSSION & NEXT STEPSThe possibility of PNNs in zebrafish and trout brings with it an abundance of opportunities. Though the litera-ture is currently dominated by mice models, zebrafish possess several advantages that would make them a more advantageous animal model. A transparent embryo, external fertilization, and high reproduction rates make a strong case for zebrafish as a new and novel animal model for PNN. Replicating prior mouse-vali-dated research in zebrafish and other Teleostei could provide a multi-species approach to said research thereby adding further support for current purported PNNs function. This study also serves to provide a foun-dation for future research on cross species comparison of PNNs. Though there exists studies examining PNNs in birds and amphibians, there is a severe lack of both functional and anatomical comparisons of PNNs across species.  Though the current study identified visually similar appearing structures via IHC, it re-mains to be seen if these structures play a similar role in brain function. It follows that the literature could greatly benefit from an experimental design analyzing the functional effects of PNN modification from a cross-species perspective. Similarities in structure but differences in function could point to underlying mech-anisms of a more complex nature than initially thought, perhaps in the form of a “limiting factor” somewhere else in the CNS. It should be noted that that current study is largely qualitative, and lacks any quantitative analysis at time of writing. Despite this, the promising findings of this study have the potential to usher in a new wave of research pursing a deeper level of understanding towards PNNs through a zebrafish model.REFERENCES1. Fawcett, J. W. et al. (2019). The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. Nature Reviews Neuroscience, 20, 451–465. 2. Wang, D., & Fawcett, J. (2012). The perineuronal net and the control of CNS plasticity. Cell and Tissue Research, 349 (1), 147–160. 3. Vo, T. et al. (2013).  The chemore-pulsive axon guidance protein semaphorin3A is a constituent of perineuronal nets in the adult rodent brain. Molecular and Cellular Neuroscience, 56, 186-200.  4. Morris, N.P. & Henderson, Z. (2000) Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/diagonal band complex. Eur J Neu-rosci. 12(3), 828-38. 5. Vorobyov, V. (2013). Effects of digesting chondroitin sulfate proteoglycans on plasticity in cat primary visual cortex. Journal of Neuroscience, 33 (1) 234-243. 6. Lensjø, K. K. et al. (2017). Removal of Perineuronal Nets Unlocks Juvenile Plasticity Through Network Mechanisms of Decreased Inhibition and IncreasedGamma Activity. The Journal of Neuroscience, 37(5), 828-838. 7. Pantazopolos, H. et al. (2014). Aggrecan and chondroitin-6-sulfate abnormalities in schizophrenia and bipolar disorder: a postmordem study on the amygdala. Translational Psychiatry, 5 (1), e496. 8. Wen, T. H. et al. (2018). The Perineuronal ‘Safety’ Net? Perineuronal NetAbnormalities in Neurological Disorders. Frontiers in Molecular Neuroscience, 11, 270. 9. Kanai, T. et al. (2007). Distribution of a brain-specific extracellular matrix protein in developing and adult zebrafish. Brain Research, 1129, 53-62. 10. Arata, N. et al. (2003). A periaxonal net in the zebrafish central nervous system. Brain Research,961(2), 179-189.  10. Carceller H, Gramuntell Y, Klimczak P, Nacher J. Perineuronal Nets: Subtle Structures with Large Implications. The Neuroscientist. 2023;29(5):569-590.

Comparative Anatomy of Mouse, Zebrafish, and Trout

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Comparative Anatomy of Mouse, Zebrafish, and Trout

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