Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition

Abstract

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

Fig 1. Summary of experiments.

Fig 2. Essential components of B27 supplement.

(A) Low magnification confocal images of Nissl staining in cultures at 8 DIV, conditions are indicated on the left side of images, scale bars, 500 μm. (B) Corresponding neuron counts in CA3c, CA3b and CA1. Statistical differences are indicated for comparisons between B27 group versus other groups. Error bars indicate SD. Statistical significance is indicated as **, representing p < 0.01. (C) Confocal images of Nissl staining in CA3c, CA3b and CA1, scale bars, 50 μm. n = 3 cultures, each condition.

Fig 3. Effects of different concentrations of BSA, selenium, and insulin.

(A-C) Left: brightfield images of cultures maintained in media with indicated concentrations of BSA, selenium or insulin, scale bars represent a distance of 500 μm; Right: time course of LDH and lactate concentration in culture supernatant of cultures; n = 3 cultures, each condition. Error bars indicate SD. Significant statistical differences are indicated for comparison of 0X selenium versus 1X selenium and 0.01X insulin versus 1X insulin, with ***p < 0.001, **p < 0.01.

Fig 4. Insulin enhances neuronal survival.

(A) Low magnification confocal images of Nissl staining in cultures at 8 DIV, conditions are indicated on the left side of images, scale bars, 500 μm. Chart shows effect of insulin on slice area; (B) Left: neuron counts in CA3c, CA3b and CA1; Right: neuron counts corrected by the normalized slice area. n = 3 cultures, each condition. Statistical differences are indicated for comparison between 1X group and other groups. (C) Representative confocal images of Nissl staining in CA3c, CA3b and CA1, scale bars, 50 μm. (D) Left: representative confocal images of neurons in CA3c, scale bars represent a distance of 5 μm; Right: corresponding cell size in CA3c. n = 30 cells, each condition. Significant statistical differences are indicated for comparison of 0X group versus 1X group. Error bars indicate SD. Significant differences are labeled as *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 5. Neurobasal-A simplification.

(A) List of hypothesized noncritical components. (B) Left: brightfield microscope images at 21 DIV, scale bars represent a distance of 500 μm; Right: time course of LDH and lactate concentration in spent culture media from cultures treated with replicated NeurA, or NeurA without set1, set2 or set3; n = 3 cultures, each condition. Significance is shown for replicate group versus–set1 group. (C) Cultures maintained in the presence of different concentrations of essential amino acids. Top: photomicrographs at 7 DIV. Scale bars, 500 μm; n = 3 cultures, each condition. Statistical significance is indicated for 0x group and 0.01x group versus control. (D) Cultures maintained in the presence of reduced and control concentrations of amino acids. Images are from 14 DIV; n = 3 cultures, each condition. (E) Evaluation of the removal of 4 metabolic epilepsy-related essential amino acids (4S: leucine, isoleucine, phenylalanine and valine). Images are from 10 DIV; n = 3 cultures, each condition. Statistical differences are shown for experimental groups versus control. (F) Cultures maintained in the presence of 1x (control) and without vitamins (- vitamins mix) and corresponding greyscale and LDH measurements. Images are from 10 DIV; n = 3 cultures, each condition. Error bars indicate SD. Significances are labeled as ***p < 0.001, **p < 0.01 and *p < 0.05.

Fig 6. Cultures in media with different glycine and serine concentrations.

(A) Left: low magnification confocal images of NeuN staining in cultures at 21 DIV, glycine concentrations are indicated on left side of images, scale bars, 500 μm; Middle: representative confocal images of NeuN staining in CA1, scale bars, 50μm; Right: corresponding CA1 neuron counts; n = 3 cultures, each condition. Statistical differences are indicated for comparisons between 1x group versus other groups. (B) Cultures maintained in different concentrations of serine. Significances are indicated for 0x group versus 1x group; n = 3 cultures, each condition. (C) Lactate and LDH results for cultures treated with combined glycine and serine, n = 3 cultures, each condition. Error bars represent SD. Significant differences are labeled as *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 7. Modification of glucose and electrolyte concentrations affects cell survival and ictal activity.

(A-D) Left: brightfield images of cultures maintained in media with indicated concentrations of glucose, Ca²⁺, Mg²⁺, or K⁺ at 14 DIV, scale bars, 500 μm; Right: time course of LDH and lactate release into culture medium. n = 3 cultures, each condition. Error bars indicate SD. Concentrations of ions that correspond to rat CSF are underlined and indicated by solid symbols. Significant statistical differences are indicated for comparison of 4 mM Ca²⁺, 4 mM Mg²⁺ and 8.5 mM K⁺ versus CSF concentrations of corresponding ions., with **p < 0.01 and ***p < 0.001.

Fig 8. Comparison between NeurA, CST and CBM media.

(A) Brightfield images at 10 DIV. Scale bars, 500 μm. Time course of LDH and lactate concentration in culture supernatant; n = 3 each condition per time point. Error bars indicate SD. Statistical significant differences are indicated for CBM and CST versus NeurA, with **p < 0.01. (B) Representative confocal images of CA1 and CA3 area of NeuN-stained organotypic hippocampal cultures at 10 DIV. Scale bars, 50 μm. Neuron counts in CA1 and CA3 are shown on the right; n = 3 each condition. (C) Representative recordings of electrographic seizure-like events in organotypic cultures on 10 DIV. Seizure detail is shown in lower traces. Red lines indicate the threshold for paroxysmal event detection. (D) Representative recordings on different days in vitro in cultures maintained in NeurA, revealing the time course of epileptogenesis in organotypic hippocampal cultures. (E) Electrographic seizures (ictal events) in NeurA and CBM in electrographic recordings of seizing slices. For 0–3 DIV, 4–7 DIV, 8–10 DIV, 11–14 DIV,n = 17, 9, 8, 10 for NeurA, n = 15, 8, 10, 10 for CBM, respectively. n represents the number of cultures. *p < 0.05. Error bars represent 95% confidence intervals for incidence of ictal activity and SD for other graphs.

Fig 9. Epileptogenesis occurs independently of medium composition.

(A) Left: brightfield images at 14 DIV. Modified medium refers to the medium with lower concentrations of glycine and serine (0.01 mM) and higher concentration of magnesium (2.0 mM) compared to CBM. Scale bars, 500 μm; Right: time course of LDH and lactate concentration; n = 3 cultures, each condition. Error bars indicate SD. *p < 0.05. (B) Representative recordings of electrographic seizure-like events in organotypic cultures maintained in CBM and modified medium on 11 DIV. Seizure detail is shown in lower traces. Red lines indicate the threshold for paroxysmal event detection. (C) Comparison of electrographic seizures in cultures maintained in CBM or modified medium during 10–14 DIV. n = 13 cultures for CBM; n = 14 cultures for modified medium; only cultures with seizures used for quantification of seizure frequency, duration, and time seizing. Error bars represent 95% confidence intervals for incidence of ictal activity and SD for other graphs.