Using claudin-2 to mark mossy fibers in the mouse hippocampus

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Abstract

The purpose of the present study was to test the possibility of identifying hippocampal mossy fiber projections by staining mouse brain sections with antibodies to the claudin-2 protein.

Material and methods: the subjects of the study were 9 male wild mice. Serial frontal sections of the brain were prepared using a cryostat. In order to identify hippocampal mossy fiber projections, sections were immunohistochemically stained with polyclonal antibodies to the claudin-2 protein and analyzed under a fluorescence microscope. The resulting images were processed in ZEN and ImageJ software, then the morphometric parameters were compared with literature data on staining of mossy fibers with antibodies to the zinc transporter ZNT3.

Results: Our results suggest that claudin-2 can be used as a marker of hippocampal mossy fibers in mice. The staining pattern obtained with antibodies to claudin-2 replicates that obtained with Timm staining of mossy fibers or with antibodies to the zinc transporter ZNT3: intense staining of the hilus of the dentate gyrus, with a clear pattern of infra- and suprapyramidal mossy fibers, including in the area of the stratum lucidum. The total area of dorsal hippocampal mossy fibers identified by claudin-2 antibodies was not statistically significantly different from the area measured by ZNT3 staining.

Conclusion: Immunohistochemical staining of mouse brain sections for claudin-2 can be used to identify hippocampal mossy fibers.

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About the authors

Ainazik Ismailova

Immanuel Kant Baltic Federal University

Author for correspondence.
Email: aiismailova@stud.kantiana.ru
ORCID iD: 0009-0002-5162-3884

student, Educational and scientific cluster “Institute of Medicine and Life Sciences (MEDBIO)”, High School of life sciences, Laboratory for Synthetic biology

Russian Federation, ul. Universitetskaya, 2, Kaliningrad, 236041

Ksenia Vladimirovna Ichetkina

Immanuel Kant Baltic Federal University

Email: ksenya.ichetkina@gmail.com
ORCID iD: 0009-0004-8519-3354

student, Educational and scientific cluster “Institute of Medicine and Life Sciences (MEDBIO)”, High School of life sciences, Laboratory for Synthetic biology

Russian Federation, ul. Universitetskaya, 2, Kaliningrad, 236041, Russian Federation

Ekaterina Alexandrovna Kurilova

Immanuel Kant Baltic Federal University

Email: ekaterinakuuurilova@gmail.com
ORCID iD: 0000-0003-0031-116X

PhD student of High School of life sciences, Institute of Medicine and Life Sciences (MEDBIO), Laboratory for Synthetic biology

Russian Federation, ul. Universitetskaya, 2, Kaliningrad, 236041, Russian Federation

Oksana Pavlovna Tuchina

Immanuel Kant Baltic Federal University

Email: otuchina@kantiana.ru
ORCID iD: 0000-0003-1480-1311

head of laboratory for Synthetic biology, Educational and scientific cluster “Institute of Medicine and Life Sciences (MEDBIO)”, High School of life sciences, Laboratory for Synthetic biology, Candidate of Biological Sciences, Associate Professor 

Russian Federation, ul. Universitetskaya, 2, Kaliningrad, 236041

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2. Fig. 1.Identification of hippocampal mossy fibers using antibodies to claudin-2. А–И micrographs of the mouse brain hippocampal frontal slices, in a rostral-caudal orientation Note.CA1, CA2, CA3 – cornu Ammonis 1, 2, 3 hippocampal regions; DG – gyrus dentatus, dentate gyrus; DG-po, DG-mo, DG-gs – polymorphic, molecular and granular layers of the dentate gyrus; Fi – fimbria hippocampi, fimbria of hippocampus; TH – thalamus, thalamus.

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3. Fig. 2.Identification of hippocampal mossy fibers using antibodies to claudin-2. А – И micrographs of the mouse brain hippocampal frontal slices, in a rostral-caudal orientation Note.CA1, CA2, CA3 – cornu Ammonis 1, 2, 3 hippocampal regions; DG – gyrus dentatus, dentate gyrus; Fi – fimbria hippocampi, fimbria of hippocampus. H – hilus, polymorphic layer of the dentate gyrus; SP – suprapyramidal mossy fibers; IP – infrapyramidal mossy fibers; the solid line indicates measurable total area of the mossy fibers of the dorsal hippocampus, the dotted line indicates the measurement of the length of supra- and infrapyramidal fibers.

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4. Fig. 3.Distribution pattern of hippocampal mossy fibers and CA3 pyramidal neurons. А – micrograph of the hippocampus on a frontal section of a mouse brain stained with antibodies to claudin-2 and a schematic representation of granule neurons, mossy fibers and pyramidal neurons of the CA3 region. Б – scheme of the proposed location and interactions of claudin-2 with other synaptic contact proteins Note.CA3 – cornu Ammonis 3 hippocampal region; DG – gyrus dentatus, dentate gyrus; DG-po, DG-mo, DG-gs – polymorphic, molecular and granular layers of the dentate gyrus; sl – stratum lucidum CA3; the arrow shows the contact en passant, the double arrow shows large buds with filopodia; AMPAR, NMDAR – glutamate receptors, Glu – glutamate, ZnT3 – zinc transporter, Claudin-2 – claudin-2, EphrinB-EphB2 – ephrins.

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5. Comparison of morphometric parameters when staining dorsal hippocampal mossy fibers for claudin-2 and zinc transporter ZNT3

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