Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov;25(6):28-45.
doi: 10.21315/mjms2018.25.6.4. Epub 2018 Dec 28.

Trkb-IP3 Pathway Mediating Neuroprotection in Rat Hippocampal Neuronal Cell Culture Following Induction of Kainic Acid

Pei Nei Chong  1 , Muthuraju Sangu  1 , Tee Jong Huat  1 , Faruque Reza  1 , Tahamina Begum  1 , Abdul Aziz Mohamed Yusoff  1 , Hasnan Jaafar  2 , Jafri Malin Abdullah  1   3
Affiliations

Trkb-IP3 Pathway Mediating Neuroprotection in Rat Hippocampal Neuronal Cell Culture Following Induction of Kainic Acid

Pei Nei Chong et al. Malays J Med Sci. 2018 Nov.

Abstract

Background: Following brain injury, development of hippocampal sclerosis often led to the temporal lobe epilepsy which is sometimes resistant to common anti-epileptic drugs. Cellular and molecular changes underlying epileptogenesis in animal models were studied, however, the underlying mechanisms of kainic acid (KA) mediated neuronal damage in rat hippocampal neuron cell culture alone has not been elucidated yet.

Methods: Embryonic day 18 (E-18) rat hippocampus neurons were cultured with poly-L-lysine coated glass coverslips. Following optimisation, KA (0.5 μM), a chemoconvulsant agent, was administered at three different time-points (30, 60 and 90 min) to induce seizure in rat hippocampal neuronal cell culture. We examined cell viability, neurite outgrowth density and immunoreactivity of the hippocampus neuron culture by measuring brain derived neurotrophic factor (BDNF), γ-amino butyric acid A (GABAA) subunit α-1 (GABRA1), tyrosine receptor kinase B (TrkB), and inositol trisphosphate receptor (IP3R/IP3) levels.

Results: The results revealed significantly decreased and increased immunoreactivity changes in TrkB (a BDNF receptor) and IP3R, respectively, at 60 min time point.

Conclusion: The current findings suggest that TrkB and IP3 could have a neuroprotective role which could be a potential pharmacological target for anti-epilepsy drugs.

Keywords: GABAA receptor; IP3 & BDNF-TrkB receptor; epilepsy; kainic acid; rat hippocampal neuron culture.

PubMed Disclaimer

Conflict of interest statement

Conflicts of Interest None.

Figures

Figure 1
Figure 1
Morphological changes of E-18 rat hippocampal neuron culture. (A) Inverted microscopy images of hippocampal neuronal culture on different days-in-vitro (DIV) at 10× magnification using a Dino-Eye camera. A–F Morphological changes of the cell were observed at every 3 to 4 days of DIV 1; DIV-5; DIV 9; DIV-12; DIV 16; DIV 21. Scale bar represents 200 μm; (B) Inverted microscopy images of hippocampal neuronal culture at DIV12 at 10× magnification using a Canon Ds126191 microscope. Morphological changes of the cell were observed and captured in the neuron culture without treatment as a control condition, and culture with KA treatment as the treatment conditions (KA1: 30 min KA treatment; KA2: 60 min KA treatment; KA3: 90 min KA treatment). Scale bar represents 200 μm
Figure 2
Figure 2
(A) Measurement of cell viability in RFU units for the control group and treatment groups (KA1, KA2 and KA3, incubated with 0.05 μm KA for 30, 60 and 90 min, respectively). (B) Neurite outgrowth density in RFU units in the control group and treatment groups (KA1, KA2, and KA3, incubated with 0.05 μm KA for 30, 60 and 90 min, respectively)
Figure 3
Figure 3
(A) Immunofluorescence images of hippocampal neuronal culture stained with primary antibody: anti-BDNF (Abcam) and secondary antibody Alexa Fluor 488 (Invitrogen) co-stained with Dapi (Invitrogen) for control; KA1: hippocampal neuronal culture + KA incubated 30 min; KA2: hippocampal neuronal culture + KA incubated 60 min; KA3 hippocampal neuronal culture + KA incubated 90 min. The scale bar represents 100 μM. (B) Intensity of BDNF stained rat hippocampal neuronal culture with control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation). (C) Number of BDNF stained particles count in rat hippocampal neuronal culture with treatment: control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation)
Figure 4
Figure 4
KA inhibited TrkB expression. (A) Immunofluorescence images of hippocampal neuronal culture stained with primary antibody: Anti-TrkB (Abcam) and secondary antibody Alexa Fluor 488 (Invitrogen) co-stained with Dapi (Invitrogen). (B) Immunofluorescence intensity from control and cell culture with KA treatment; KA1: 30 min KA treated neuron culture; KA2: 60 min KA treated neuron culture; KA3: 90 min KA treated neuron culture. (C) Number of TrkB stained particles count in rat hippocampal neuronal culture with treatment: control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation)
Figure 5
Figure 5
IP3R induced after KA introduction. (A) Immunofluorescence images of hippocampal neuronal culture stained with primary antibody: Anti-IP3R (Abcam) and secondary antibody Alexa Fluor 594 (Invitrogen) (B) Immunofluorescence intensity from control and cell culture with KA treatment; KA1: 30 min KA treated neuron culture; KA2: 60 min KA treated neuron culture; KA3: 90 min KA treated neuron culture. (C) Number of IP3 stained particles count in rat hippocampal neuronal culture with treatment: control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation)
Figure 6
Figure 6
KA did not significantly affect GABAAR α-1. (A) Immunofluorescence images of hippocampal neuronal culture stained with primary antibody: anti-GABRA1 (Abcam) and secondary antibody Alexa Fluor 488 (Invitrogen) Control; KA1: hippocampal neuronal culture + KA incubated for 30 min; KA2: hippocampal neuronal culture + KA incubated for 60 min; KA3: hippocampal neuronal culture + KA incubated for 90 min. The scale bar represents 100 μM. (B) Intensity of GABRA1 stained rat hippocampal neuronal culture in the control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation). (C) Number of GABAAR α-1 stained particles count in rat hippocampal neuronal culture with treatment: control group (without KA), KA1 (30 min KA incubation), KA2 (60 min KA incubation), KA3 (90 min KA incubation)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Lytton WW. Computer modelling of epilepsy. Nat Rev Neurosci. 2008;9(8):626–637. doi: 10.1038/nrn2416. - DOI - PMC - PubMed
    1. Wan L, Liu X, Wu Z, Ren W, Kong S, Dargham RA, et al. Activation of extrasynaptic GABAA receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons. Neurosci Bull. 2014;30(5):866–676. doi: 10.1007/s12264-014-1466-8. - DOI - PMC - PubMed
    1. Treiman DM. GABAergic mechanisms in epilepsy. Epilepsia. 2001;42:8–12. doi: 10.1046/j.1528-1157.2001.042suppl.3008.x. - DOI - PubMed
    1. Thom M. Review: hippocampal sclerosis in epilepsy: a neuropathology review. Neuropathol Appl Neurobiol. 2014;40(5):520–543. doi: 10.1111/nan.12150. - DOI - PMC - PubMed
    1. Soukupová M, Binaschi A, Falcicchia C, Zucchini S, Roncon P, Palma E, et al. Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy. Exp Neurol. 2014;257:39–49. doi: 10.1016/j.expneurol.2014.04.014. - DOI - PubMed

LinkOut - more resources