Reactive Astrogliosis Causes the Development of Spontaneous Seizures

Mice with chronic astrogliosis develop spontaneous seizures

Epileptic foci are known to contain reactive astrocytes (D'Ambrosio, 2004; Wetherington et al., 2008; Carmignoto and Haydon, 2012; Coulter and Eid, 2012), yet whether they are a consequence of neuronal dysfunction or an active contributor is currently unknown (Verkhratsky et al., 2012). Since hippocampal neuronal hyperexcitability had been observed following adenoviral-induced astrogliosis (Ortinski et al., 2010), we sought to investigate whether the widespread astrogliosis in β1^−/− mice leads to spontaneous seizure development.

To assess gross cortical activity, we recorded subcranial cortical EEGs from different age groups of β1^−/− mice and controls. EEGs (22–258 h continuous recording) were recorded while simultaneously videotaping each animal (see Table 1 for a list of all β1^−/− animals and respective recording times).

We observed highly abnormal EEG activity in β1^−/− mice compared with their control littermates (Fig. 2a). This activity consisted of tonic–clonic seizures accompanied by a clear convulsive phenotype (Fig. 2b,d) and frequent interictal spiking (Fig. 2c,e). Tonic–clonic seizures were exhibited by 58% of β1^−/− animals (29/50 mice), while seizures were never observed in control littermates (0/48 mice). We recorded EEGs in four different age groups (4 and 5–6 weeks, 2–3 months, and older than 6 months), and found that the seizure incidence was highest in 5- to 6-weeks-old animals (Fig. 2f). However, seizure frequency was not significantly different between age groups (ROUT outlier test, Kruskal—Wallis test, p = 0.297, Dunn's multiple comparison test; Fig. 2g). Given that seizures occurred on average only once every 1–2 d, it is possible that the recording periods were not always long enough to compute an accurate seizure frequency (Table 1). Thus, we might overestimate the number of animals without seizures.

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Figure 2.

Mice with chronic astrogliosis develop spontaneous seizures and interictal spiking. a, EEG recordings reveal abnormal EEG activity in β1^−/− mice when compared with control mice. b, Abnormal activity in β1^−/− mice manifests in spontaneous tonic–clonic seizures or in interictal spiking (c). d, High temporal resolution of the start of a seizure. At the end of the seizure, EEG signal depression is typically observed. e, High temporal resolution of an interictal spike. f, The percentage of seizing animals is lowest at 4 weeks of age and increases in older animals. g, β1^−/− mice experience an average of one to two seizures within 48 h. Seizure frequency is not significantly different between age groups. h, Interictal spiking occurs at a high frequency in all age groups and is significantly increased in β1^−/− mice older than 6 months. Error bars refer to SEM. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

In contrast, we observed age-dependent interictal spiking in almost all β1^−/− mice, which increased dramatically in older mice (ROUT outlier test, one-way ANOVA, p ≤ 0.0001, Tukey's multiple comparison test; Fig. 2h). Only 14% of β1^−/− mice showed none or little interictal spiking, whereas the remaining 86% have considerably higher values (n = 43) when compared with littermate controls (n = 20). Half of the β1^−/− mice (3/6) with little or no interictal spiking were 4 weeks of age, the age group presenting with less severe astrogliosis. This suggests a high penetrance of the phenotype at least with regard to interictal spiking and GFAP expression (7% of the mice analyzed by immunohistochemistry showed GFAP expression comparable to controls while all other animals had elevated GFAP levels in the cortical gray matter, n = 28).

To assess a correlation between GFAP immunohistochemistry and seizure frequency, the Spearman correlation coefficient between these two variables was computed. The coefficient of ρ = 0.37 (p = 0.006) indicates that GFAP levels and seizure frequency tend to increase together.

We were unable to conduct continuous EEG recordings on mice younger than 4 weeks since animals are housed separately after cranial electrode implantation, and therefore they had to be of weaning age for surgery. In addition, we waited ∼5 d after implantation before recording. We observed that β1^−/− mice often weakened and showed weight loss after several days of recording. These animals were removed from further recording.

It can be difficult to place EEG electrodes on the brain surface without them penetrating into the brain or injuring the meninges, which can cause astrocyte and microglia activation. In a subset of control and β1^−/− animals, we found that implantation of the electrodes had caused tissue damage (Fig. 3). To assess how this acute injury affects seizures in control and β1^−/− mice, we identified the group of animals that had incurred additional damage based on GFAP and Cd11b immunohistochemistry. We divided the animals into three groups: animals without damage, animals with mild damage, or animals with severe damage. In animals without tissue damage we did not observe microglia activation as assessed by Cd11b levels (Fig. 3a). Note that the microglia activation and the concomitant increase in Cd11b immunoreactivity caused by electrode damage by far exceeds the mild microglia activation that we normally observe in β1^−/− animals (Robel et al., 2009). This allowed us to identify electrode-injured brain areas despite high GFAP levels in β1^−/− mice (Fig. 3b). In mice with mild injury electrodes had not penetrated the brain tissue but Cd11b (and GFAP in controls) was upregulated when compared with neighboring brain areas. In animals with severe damage, electrodes had penetrated the brain tissue.

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Figure 3.

Tissue damage caused by electrode implantation does not affect the seizure phenotype. Immunohistochemistry for Cd11b as a marker of reactive microglia (a) and GFAP in β1^−/− and control mice that had undergone electrode implantation and EEG recordings (b). c, Quantification of animals without tissue damage or mild and severe tissue damage. β1^−/− mice were divided in two groups based on whether or not tissue damage was detected. Seizure and interictal spiking frequencies were computed and did not differ significantly (seizures: with damage, n = 8; without damage, n = 22; Mann–Whitney, p = 0.86; interictal spikes: with damage, n = 5; without damage, n = 11; Mann–Whitney, p = 0.48; animals >6 months were excluded from the analysis since the frequency of interictal spikes is significantly higher at this age). Box-and-whisker plots display minimum to maximum values, and mean is shown as +.

We then compared seizure and interictal spiking frequencies between animals with and without additional acute tissue damage. We never observed seizures in control animals. More specifically, 10 control animals with acute damage (Fig. 3c, mild and severe) were recorded for a total of 769 h and six of these mice were recorded again 4–6 weeks later for a total of 331 h. The lack of seizures in control mice suggests that inflammation induced by this acute and locally confined injury was not sufficient to trigger epileptogenesis at least during our observation period of up to 8 weeks after electrode implantation.

To ensure that the acute injury and chronic astrogliosis in β1^−/− mice do not have a synergistic effect on seizure and spiking frequency, we compared β1^−/− with and without electrode damage. There was no significant difference in interictal spiking or seizure frequencies between these groups (Fig. 3c).

These data show that chronic astrogliosis in β1^−/− mice is associated with abnormal EEG activity representative of interictal spiking and spontaneous seizures in a majority of mice while focal gliosis resulting from a localized acute injury does not cause epilepsy.

Neuronal hyperexcitability characterizes β1^−/− mice

Epileptic seizures result from abnormal changes in neuronal excitability (Goldberg and Coulter, 2013). To examine whether this was the case in β1^−/− animals, we performed whole-cell patch-clamp recordings on layer II/III cortical pyramidal neurons in acute brain slices of mice 4–6 and 6–8 weeks old. We found that the neuronal RMP was significantly more depolarized in β1^−/− neurons (4–6 weeks: RMP = −63.28 ± 1.77 mV, n = 32; 6–8 weeks: RMP = −68.87 ± 1.38 mV, n = 30) than in controls (4–6 weeks: RMP = −71.83 ± 0.8 mV, n = 35, two-tailed unpaired t test, p < 0.0001; 6–8 weeks: RMP = −73.03 ± 0.77, n = 32, two-tailed unpaired t test, p = 0.0002; Fig. 4a), while the input resistances were higher in 4- to 6-week-old β1^−/− mice (control R_i = 102.6 ± 8.28 MΩ, β1^−/− R_i = 137.9.36 MΩ, two-tailed Mann–Whitney test, p = 0.0014; Fig. 4b) but comparable between both groups at 6–8 weeks of age (control R_i = 78.38 ± 7.97 MΩ, β1^−/− R_i = 84.86 ± 5.55 MΩ, two-tailed Mann–Whitney test, p = 0.1016; Fig. 4b).

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Figure 4.

Layer II/III pyramidal neurons are hyperexcitable in mice with chronic astrogliosis. a, The RMP of Layer II/III pyramidal neurons is significantly depolarized in acute slices of β1^−/− mice compared with controls. b, The input resistance differs at 4 weeks of age but is unchanged in β1^−/− neurons compared with controls. Whiskers in box-and-whisker plots show minimum to maximum values. The middle line is plotted at the median. c, Whole-cell patch-clamp recordings from pyramidal neurons before (ACSF) and after removal of Mg²⁺ (Mg²⁺-free) followed by AP-5 shows a faster onset and longer duration of ictal (>2 s in duration) events in acute slices of β1^−/− mice compared with controls at 6–8 weeks of age. d, Quantification of the latency to onset of ictal events (defined as events of >2 s in length) after removal of Mg²⁺ in β1^−/− mice and littermate controls, as well as in β1^−/− Nex::Cre mice and their littermate controls. e, Quantification of the latency to onset of interictal (defined as events of 500 ms to 2 s length) after removal of Mg²⁺. f, Quantification of the latency to onset of interictal and ictal events after bicuculline application. g, Examples of voltage responses to increasing amplitude current injections, from −100 to +240 pA (in 20 pA steps) in whole-cell, current-clamped pyramidal peritumoral neurons in β1^−/− and controls. h, The average action potential number obtained in response to −20 to 240 pA current pulses was plotted as a function of applied current yielding the input–output curves shown. i, Graphical display of input/output curves of postsynaptic potentials recorded by whole-cell patch-clamp recording from pyramidal cells in superficial cortical layer II/III and the response area as a function of stimulation intensity in b1^−/− and control cortical slices. j, Western blot of the GluR2/3 subunit of the AMPA receptor. Error bars refer to SEM. Box-and-whisker plots display minimum to maximum values, and mean is shown as +. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

To examine whether β1^−/− pyramidal neurons are hyperexcitable, we chose four different approaches. First, we determined the latency of the development of ictal (defined as events longer than 2 s) and of interictal (defined as events with a duration between 500 ms and 2 s) activity following removal of Mg²⁺ from the bath solution allowing for the removal of the Mg²⁺ block from the NMDA receptor as previously described (Buckingham et al., 2011). In control mice, latency to the first interictal event was 23.69 ± 3.25 min at 4–6 weeks (n = 12) and 15.49 ± 2.29 min (n = 14) at 6–8 weeks of age (Fig. 4c–e). The onset of ictal discharges was 58.24 ± 8.69 min (n = 9) and 37.93 ± 2.53 min (n = 9) at 6–8 weeks of age (Fig. 4c–e). Some neurons did not respond with ictal discharges within the recording period of 60–90 min. Nonresponsive neurons were excluded from the analysis since we could not determine the onset time. There were more nonresponding neurons in control brain slices compared with slices obtained from β1^−/− mice, suggesting that the mean value for ictal onset in control animals is likely underestimated. Pyramidal neurons in β1^−/− brain slices showed a significantly shorter latency to onset of interictal (4–6 weeks: 12.04 ± 2.82 min, n = 12, two-tailed unpaired Mann–Whitney test, p = 0.0015; 6–8 weeks: 8.64 ± 0.91 min, n = 10, two-tailed unpaired t test, p = 0.0243) and ictal (4–6 weeks: 21.43 ± 4.94 min, n = 10, two-tailed unpaired Mann–Whitney test, p = 0.0095; 6–8 weeks: 15.27 ± 1.78 min, n = 10, two-tailed unpaired t test, p ≤ 0.0001) activity in both age groups (Fig. 4c–e).

Similarly, we determined the latency of the development of ictal and of interictal activity following application of bicuculline, which is a GABA_A receptor antagonist facilitating excitatory synchronization (Gutnick et al., 1982). Whereas the onset of interictal events was unchanged between controls and β1^−/− at 4–6 weeks of age (Fig. 4f, control; 8.33 ± 1.54 min, n = 10; β1^−/−; 6.1 ± 0.59 min, n = 14, two-tailed unpaired Mann–Whitney test, p = 0.54), the onset of ictal events was significantly faster in β1^−/− pyramidal cells (control: 17.21 ± 3.25 min, n = 8; β1^−/−: 10.27 ± 1.58 min, n = 15, two-tailed unpaired t test, p = 0.041).

Moreover, we quantified the number of action potentials fired in response to current injections. We found that this number was significantly greater in β1^−/− pyramidal cells than in control neurons in response to 180, 200, 220, and 240 pA current injection (Fig. 4g,h), indicating a lowered excitability threshold in β1^−/− neurons of both age groups.

Last, we recorded input/output curves in control and β1^−/− cortical slices. A stimulus electrode was used to deliver incrementally increasing stimuli to deep cortical layer IV, and postsynaptic currents were recorded in the superficial cortical layers (II/III). Input/output curves were constructed of the response area and amplitude as a function of stimulus intensity (Fig. 4i). Recordings from β1^−/− pyramidal cells were significantly larger than controls. Increasing stimulation resulted in greater excitatory response areas in β1^−/− slices compared with controls (Fig. 4i; two-way ANOVA followed by Sidak's multiple comparison test, n = 5 for each genotype).

Fast synaptic excitation relevant to epilepsy is largely mediated by AMPA receptors, and AMPA receptor antagonists can reduce epileptiform activity in animal models and human epilepsy (Rogawski, 2013). To address whether AMPA receptor expression was changed in β1^−/− mice, we performed a Western blot analysis for the GluR2/3 subunit. At 4 and 6 weeks of age, protein levels were not significantly changed between control and β1^−/− mice. In older animals, Glu2/3 levels were slightly reduced (Fig. 4j). This excludes an increase in GluR2/3 protein expression levels as a contributor for seizure generation in β1^−/− mice.

Neuronal deletion of β1-integrins does not induce neuronal hyperexcitability

To exclude the possibility that neurons rather than astrocytes were selectively affected by the β1-integrin deletion, we performed the same experiments in mice where β1-integrin deletion was neuron specific. To mediate recombination of the floxed β1-integrin gene, β1^fl/fl mice were bred to Nex::Cre^+/− mice, which express Cre under the control of regulatory sequences of the transcription factor Nex (Goebbels et al., 2006). This leads to genetic deletion in intermediate progenitors and neurons of the dorsal telencephalon at ∼E11.5 (Goebbels et al., 2006). We previously reported that astrogliosis was absent in these mice, despite the early downregulation of β1-integrin proteins (Robel et al., 2009). However, layer II/III pyramidal neuron whole-cell patch-clamp recordings conducted in acute brain slices from β1-integrin^fl/fl, Nex::Cre^+/− mice and their control littermates (β1-integrin^fl/wt, Nex::Cre^+/−; β1-integrin^fl/fl, Nex::Cre^−/−; β1-integrin^fl/wt, Nex::Cre^−/−) showed that there is a similar latency to epileptiform event onset (longer than 2 s) in all genotypes following the removal of Mg²⁺ (Fig. 4d; control, 34.21 ± 10.51 min, n = 6; β1-integrin^fl/fl, Nex::Cre^+/− 40.0 ± 4.52 min, n = 7, two-tailed unpaired t test, p = 0.6035).

Since neuron-specific β1-integrin deletion did not induce hyperexcitability, we conclude that astrogliosis causes β1^−/− pyramidal cells to become depolarized and hyperexcitable.

Do reactive astrocytes in β1^−/− mice have impaired K⁺ homeostasis?

To elucidate possible mechanisms underlying neuronal hyperexcitability and seizures in β1^−/− mice, we first focused on the well established, astrocytic homeostatic functions. It has been suggested that impairments in astrocytic potassium and glutamate homeostasis can contribute to neuronal hyperexcitability and seizure generation (Seifert et al., 2010). Thus, we used a combination of Western blot, immunohistochemistry, and electrophysiological recordings to assess whether potassium and glutamate uptake is intact in β1^−/− astrocytes.

Whole-cell astrocyte patch-clamp recordings were performed on acute brain slices obtained from β1^−/− or control mice at 6–8 weeks of age. To identify astrocytes, these animals expressed eGFP under the control of the astrocyte-specific Aldh1l1 promoter (Cahoy et al., 2008; Fig. 5a). The mean input resistance (control R_i = 18.45 ± 1.44 MΩ, n = 57; β1^−/− R_i = 22.01 ± 2.84 MΩ, n = 39; two-tailed Mann–Whitney test, p = 0.9985; Fig. 5b) and the mean RMP (control RMP = −78.08 ± 0.47 mV, n = 32; β1^−/− RMP = −78.19 ± 0.50 mV, n = 30; two-tailed t test, p = 0.8713; Fig. 5c), were comparable between β1^−/− and control astrocytes. However, cell capacitance, which correlates with cell size, was significantly increased in β1^−/− astrocytes (control C = 10.54 ± 0.91 pF, n = 45; β1^−/− C = 12.98 ± 0.87 pF, n = 42; two-tailed Mann–Whitney test, p = 0.0158; Fig. 5d), consistent with their hypertrophied phenotype.

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Figure 5.

Potassium homeostasis is moderately impaired in mice with chronic astrogliosis. a, For whole-cell patch-clamp recordings, astrocytes were identified by their eGFP in acute slices of β1^−/− and control mice (6–8 weeks of age) carrying an allele expressing eGFP under the astrocyte-specific Aldh1l1 promoter. Placement of the recording pipette and puff pipette is indicated. b, The input resistance (R_i) and resting RMP (c) of parenchymal astrocytes in the cortical gray matter is unchanged between β1^−/− and control mice. d, The cell capacitance is increased in β1^−/− astrocytes. e, To activate Kir4.1-mediated potassium currents in astrocytes a voltage-step protocol was used in ACSF and after bath application of the Kir4.1 inhibitor BaCl₂. Traces represent a point-by-point subtraction of the corresponding current traces isolating the Ba²⁺-sensitive component of the whole-cell current. Quantification of Kir currents (I_Kir in pA/pF) as the Ba²⁺-sensitive conductance at −140 mV normalized to the whole-cell capacitance revealed significantly reduced Kir4.1 currents in β1^−/− astrocytes. f, Potassium uptake was similar in β1^−/− and control astrocytes after pressure application of 125 mm KCl for 50 ms. Whiskers in box-and-whisker plots show minimum to maximum values. The line in the middle of the box is plotted at the median, + indicates the mean. g, The majority of astrocytes expresses Kir4.1 at lower levels in fine processes only. Few astrocytes express higher Kir4.1 levels in cell bodies and processes. h, Western blot analysis probing for Kir4.1 protein showed similar levels of protein expression and distribution in the cortical gray matter of β1^−/− and control mice at 6 weeks of age, but increased levels in older mice. Error bars in bar graphs refer to SEM. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

Astrocytic potassium (K⁺) conductance and uptake is thought to be mediated by the barium-sensitive potassium channel Kir4.1 (Olsen and Sontheimer, 2008). To activate Kir4.1-mediated currents in these cells, we used a specific voltage-step protocol as previously described (Olsen et al., 2007), and isolated these currents using 100 μm Ba²⁺, which specifically inhibits Kir channels at this concentration (Ransom and Sontheimer, 1995). A point-by-point subtraction of the corresponding whole-cell current was done to isolate the Ba²⁺-sensitive component (Kir current; Fig. 5e). We quantified the Kir current, I_Kir (expressed in pA/pF), as the Ba²⁺-sensitive conductance at −140 mV, normalized to the whole-cell capacitance from these recordings (Fig. 5e). I_Kir was significantly reduced in β1^−/− astrocytes (I_Kir = −195.4 ± 26.09 pA/pF, n = 26) compared with controls (control I_Kir = −257.4 ± 20.7 pA/pF, n = 31; two-tailed Mann–Whitney test, p = 0.0019; Fig. 5e).

To assess whether this reduction in Kir currents impairs K⁺ uptake, we challenged astrocytes in β1^−/− and control acute brain slices with pressure-applied K⁺ (125 mm KCl), which induces an inward current under voltage-clamp conditions. The number of ions that moved across the membrane can be determined from the charge transfer, i.e., the area of the Ba²⁺-sensitive current induced by the puff. Surprisingly, there was no difference between β1^−/− and control astrocytes in the number of K⁺ ions moving across the membrane (control: 1.08 ± 0.24 fmol K⁺, n = 13; β1^−/−: 1.29 ± 0.23, n = 11, two-tailed unpaired t test, p = 0.5438; Fig. 5f).

We complemented these studies with immunohistochemical and Western blot analyses to address whether reduced Kir currents were a result of reduced levels of Kir4.1 protein. However, there were no significant differences in Kir4.1 protein expression (relative expression levels normalized to GAPDH) or distribution between β1^−/− and control animals at 4 and 6 weeks (Fig. 5g,h). There were, however, differences in levels of Kir4.1 expression in control mouse parenchymal gray matter astrocytes. Most astrocytes showed low Kir4.1 levels within the fine processes; however, some astrocytes expressed higher Kir4.1 levels in cell bodies and processes. A similar pattern could be seen in brain slices from β1^−/− mice (Fig. 5g). Kir current reduction seems to occur at a functional level and was not associated with reduced levels of Kir4.1 protein. Interestingly, Kir4.1 protein levels were increased in β1^−/− animals >6 months old (Fig. 5h) possibly reflecting compensatory changes in β1^−/− astrocytes at later stages.

In summary, reactive astrocytes in β1^−/− mice show reduced Kir currents despite normal Kir4.1 protein levels. However, this did not significantly alter the resting membrane potential or K⁺ uptake. The latter may be due to the large, poorly localized K⁺ challenge. A reduced density of Kir channels on fine processes may, nevertheless, show impaired local K⁺ homeostasis.

Do reactive astrocytes in β1^−/− mice show impaired glutamate homeostasis?

To examine the possibility that glutamate uptake may be impaired in β1^−/− mice, we first analyzed expression of the two astrocytic glutamate transporters, Glt-1 (slc1a2) and GLAST (slc1a3), by immunohistochemistry and Western blot. Levels of both proteins were similar at 4 and 6 weeks of age (Fig. 6a–c). In β1^−/− animals older than 6 months, Glt-1 levels were slightly reduced (Fig. 6b), whereas GLAST protein levels remained comparable between controls and β1^−/− mice at this age (Fig. 6b). There was no apparent difference in Glt-1 distribution between the 6-week-old control and β1^−/− mice (Fig. 6a; Lehre et al., 1995) on the differential expression of Glt-1 and GLAST).

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Figure 6.

Glutamate uptake is impaired in mice with chronic astrogliosis. a, Immunohistochemistry against the glutamate transporter Glt-1 and Western blot probing for Glt-1(b) and GLAST in the cortical gray matter of β1^−/− (c) and control mice at different ages (4 and 6 weeks and >6 months). d, Glutamate uptake was assessed after puff application of 200 μm glutamate for 500 ms before and after the application of the glutamate transporter inhibitors TBOA and DHK. ACSF contained the inhibitors (inh.) TTX, AP-5, bicuculline, and CdCl₂. TBOA/DHK-sensitive glutamate uptake was reduced in β1^−/− astrocytes when compared with littermate controls. Whiskers in box-and-whisker plot show minimum to maximum values. The line in the middle of the box is plotted at the median, + indicates the mean. e, Protein expression levels of the astrocytic enzyme glutamine synthetase was unchanged in the cortical gray matter of β1^−/− and control mice at 4 and 6 weeks of age, but reduced in both genotypes in mice older than 6 months. Error bars in bar graphs refer to SEM. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

To assess if glutamate transport is changed, whole-cell patch-clamp recordings from eGFP⁺ astrocytes were performed in acute brain slices from β1^−/− or control mice. High glutamate concentrations (200 μm) were applied using a puff pipette, and uptake currents were recorded in β1^−/− astrocytes and controls while blocking K⁺ uptake with BaCl₂, and neuronal activity with TTX, d- AP5, bicuculline, and CdCl₂. Recordings were repeated after blocking glutamate transporters with the inhibitors TBOA and DHK (Fig. 6d, traces). The amount of glutamate taken up computed as the TBOA/DHK-sensitive charge transfer (area under the curve) and was significantly reduced in β1^−/− astrocytes (Fig. 6d; control 0.0857 ± 0.0123 fmol glutamate, n = 6; β1^−/− 0.0204 ± 0.0120, n = 5, two-tailed unpaired t test, p = 0.0045).

Despite previous reports of a downregulation of the astrocytic enzyme glutamine synthetase in reactive astrocytes (Ortinski et al., 2010), we found comparable levels of this protein in the cortical gray matter of β1^−/− and control brains. We found glutamine synthetase (GS) protein levels to be significantly reduced in older mice of both genotypes (Fig. 6e).

These data suggest that impaired astrocytic uptake of glutamate may cause increased extracellular glutamate concentrations that could contribute to enhanced neuronal excitability.

Chronic gliosis is associated with an immature expression profile of neuronal chloride transporters

Recent studies have suggested that decreased inhibition, either from reduced GABA synthesis (Ortinski et al., 2010) or GABA acting as an excitatory instead of an inhibitory neurotransmitter, can contribute to neuronal hyperexcitability (Lee et al., 2011; Ben-Ari et al., 2012; Rangroo Thrane et al., 2013). The action of GABA on neurons depends on neuronal intracellular chloride concentrations. Mature neurons normally have a low intracellular chloride [Cl⁻]_i concentration resulting in hyperpolarizing GABA responses (Kahle et al., 2008). Neuronal [Cl⁻]_i concentrations are maintained by the balanced expression of the cation-chloride cotransporters (CCC) NKCC1 and KCC2. Immature neurons show high expression of NKCC1 relative to low KCC2 expression, which maintains a high [Cl⁻]_i. A developmental expression level switch occurs during the second postnatal week in rodents. KCC2 levels increase and NKCC1 levels decrease, resulting in low [Cl⁻]_i, and hyperpolarized responses after GABA binds to GABAA receptors (Kahle et al., 2008).

To determine whether CCC expression is changed in mice with chronic astrogliosis, we performed Western blots to detect KCC2 and NKCC1 (Fig. 7a) in β1^−/− and control cortical gray matter (n = 4 for each genotype and age group). We found that levels of KCC2 and NKCC1 protein and the ratio between KCC2 and NKCC1 were unchanged at 4 weeks (Fig. 7a–d). At 6 weeks, KCC2 levels remained unchanged, whereas NKCC1 expression was significantly increased. In mice 6 months and older, KCC2 protein was significantly lower. In mice with chronic reactive astrogliosis, KCC2 to NKCC1 ratios are decreased after they are 6 weeks old (Fig. 7a–d).

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Figure 7.

Bumetanide treatment rescues the seizure phenotype in a subset of mice with chronic astrogliosis. a, Western blot analysis of the potassium-chloride cotransporter (KCC2) and the sodium potassium-chloride cotransporter (NKCC1). b, Quantification of KCC2. c, NKCC1 expression levels after normalization to the loading control GAPDH. d, β1^−/− mice present with an upset ratio of the chloride transporters KCC2 and NKCC1 at 6 weeks and >6 months of age. Immunohistochemistry for NKCC (e) and KCC2 (f) in β1^−/− and control cortical gray matter at 3 months of age. White arrow heads point at neurons with changed NKCC1 or KCC2 expression patterns. Blue arrow heads point at normal, ring-like expression of KCC2 along the periphery of neuronal cell bodies. Error bars refer to SEM. Statistical significance with *p < 0.05, **p < 0.01, and ***p < 0.001.

NKCC1 is also expressed in astrocytes and endothelial cells (Pedersen et al., 2006; Macaulay and Zeuthen, 2012). Hence, we used immunohistochemistry to detect NKCC1 in animals 3 months of age. In controls, NKCC1 was expressed evenly throughout the cortical gray matter sparing only neuronal nuclei. We attempted to colocalize NKCC1 with fine astrocytic processes using Glt-1 (Fig. 6a) as a marker in confocal images. However, the resolution achieved by light microscopy was insufficient to unequivocally demonstrate an overlap between NKCC1 and Glt-1. Nevertheless, in β1^−/− mice, we found increased ring-like NKCC1 labeling within the cell soma of NeuN-labeled neurons (Fig. 7e). We did not observe increased levels of NKCC1 in astrocyte cell bodies or the main processes after labeling with GFAP (Fig. 7e).

Immunohistochemistry of the neuron-specific KCC2 revealed expression of this transporter at the periphery of the soma and within processes as previously reported (Campbell et al., 2015). In β1^−/− animals the KCC2 expression pattern was changed showing a more diffuse labeling not just at the periphery but also within neuronal soma (Fig. 7f).

Together, these data indicate that neuronal NKCC1 and KCC2 protein distribution and levels changed in β1^−/− mice and that their neurons have adopted an immature phenotype.

Epileptogenesis in the absence of blood–brain barrier defects

Blood–brain barrier breach has been reported in epilepsy animal models and in epilepsy patients (Heinemann et al., 2012; Janigro, 2012). Serum factors are deposited in the brain parenchyma after blood–brain barrier breakdown and this alone can generate inflammation and astrogliosis and trigger seizures (Seiffert et al., 2004; Adams et al., 2007; Schachtrup et al., 2010; Heinemann et al., 2012; Janigro, 2012). One effect of albumin deposition is astrocytic TGFRII activation, which can then lead to downregulation of Kir4.1 and Glt-1 through a TGF-β-mediated mechanism (Frigerio et al., 2012). It has been difficult to decipher whether these serum factors impact neuronal excitability directly or indirectly.

In our model, astrogliosis occurs in the absence of blood–brain barrier damage (Robel et al., 2009), reflected by stable levels of Kir4.1 and Glt-1 proteins and mild microglial activation during seizure onset. This suggests that astrogliosis by itself is sufficient to induce epileptogenesis.

Direct changes in reactive astrocytes contribute to epileptogenesis

Astrocytes are essential for normal brain homeostasis. They maintain low concentrations of potassium and glutamate within the extracellular space and supply neurons with glutamine, a necessary precursor for glutamate and GABA synthesis. Yet in human epileptic hippocampi, glutamate levels have been found to be five times higher than in normal hippocampi (Coulter and Eid, 2012).

In β1^−/− mice with chronic astrogliosis, we found functional impairments in the Kir4.1 potassium channel and in glutamate transporters, but their protein levels were unchanged during seizure onset. Glutamate transporter trafficking, required for proper glutamate uptake, is crucially dependent on the cytoskeleton (Sullivan et al., 2007; Sheean et al., 2013). In reactive astrocytes, the cytoskeleton is markedly changed (Robel et al., 2011). Thus, glutamate uptake could be disrupted due to improper localization of the transporters despite normal total protein levels.

During development, immature, proliferating astrocytes do not yet express Kir4.1, and therefore have a depolarized RMP of approximately −50 mV (Ransom and Sontheimer, 1995; Bordey and Sontheimer, 1997). As the brain matures and astrocytic Kir4.1 expression levels increase (Nwaobi et al., 2014), astrocytes acquire their characteristic, very negative, RMP of approximately −85 mV (Olsen and Sontheimer, 2008). At the same time, protoplasmic cortical gray matter astrocytes downregulate typical proteins expressed in a dedifferentiated, immature state, such as GFAP (in mice), nestin, and vimentin or tenascin-C and 473HD, and exit the cell cycle. However, after adult brain injury, reactive astrocytes re-adopt an immature phenotype and can re-enter the cell cycle, coinciding with the loss of Kir4.1 and cell membrane depolarization (MacFarlane and Sontheimer, 2000; Higashimori and Sontheimer, 2007). Mice with chronic astrogliosis show upregulation of some of these markers, but do not re-enter the cell cycle (Robel et al., 2009). Accordingly, we did not observe Kir4.1 protein loss or membrane depolarization. Nonetheless, barium-sensitive Kir4.1-mediated potassium currents were reduced in mice with chronic astrogliosis. This alone may contribute to epileptogenesis since Kir4.1 conditional knock-out mice display stress-induced seizures (Djukic et al., 2007).