Effects of early-life seizures on excitatory and inhibitory markers during auditory cortex development

By: Evan L. Honig

Abstract

Early-life seizures are associated with developmental disorders such as autism and intellectual disability. Previous research in our lab has shown that pentylenetetrazol (PTZ)-induced seizures at an early age prematurely unsilence synapses and cause disruption of thalamocortical critical period plasticity within the auditory cortex, indicative of excitatory-inhibitory (E-I) imbalance. 1 Proteins in both excitatory and inhibitory neurotransmitter systems are vital for regulating critical period plasticity in the developing brain. Ionotropic receptor subunits, inhibitory interneurons, and chloride transporter expression have been shown to be altered as a result of neonatal seizures. 2, 3, 4However, the competitive AMPA receptor antagonist, NBQX, has been shown to reverse various seizure-induced changes. 5 Here, we examined whether neonatal PTZ-induced seizures contribute to E-I imbalance via disruption of the developmental profile of various inhibitory and excitatory proteins in both the cortex and primary auditory cortex (A1), and if treatment with NBQX can rescue these alterations. While there were no significant differences in expression among treatment groups in older animals for these markers in both the cortex and auditory cortex, there were significant differences in cortical and auditory cortical expression of various excitatory and inhibitory proteins between control and PTZ-treated animals earlier in development in both a layer- and region-specific manner along the rostral-caudal axis.

Background

Neonatal seizures have been implicated in developmental disorders such as autistic-like behavior and intellectual disability. Shortly following birth, the brain undergoes dynamic changes in excitatory and inhibitory (E-I) levels during the critical period (CP) of development (Fig. 1). Critical periods of development are windows of heightened activity-dependent synaptic plasticity and excitatory drive, thereby increasing susceptibility to neonatal seizures during this time. Additionally, given that E-I levels are tightly regulated during development, any perturbation such as seizures can have profound and lasting effects. Expression levels of various ionotropic receptor subunits and chloride transporters contribute to the regulation of these E-I levels and the synaptogenesis that occurs during these critical periods. 7 Prior research has shown that neonatal seizures decrease the number of silent synapses--synapses with NMDA receptor (NMDAR) but no AMPA receptors (AMPAR)--by precociously converting NMDAR-only synapses into those that also express AMPARs and disrupting long-term potentiation (LTP) in CA1 hippocampal neurons.

8 More recently, our lab has also shown that pentylenetetrazol (PTZ)-induced seizures in early postnatal development affect the primary auditory cortex. Thalamocortical silent synapses within A1 were prematurely unsilenced and thalamocortical tonotopic plasticity, a key feature in auditory cortex critical periods, was impaired. 1, 9 Furthermore, our lab has demonstrated that early-life seizures cause greater 2 spread of activity in a caudal direction in A1 following activation of thalamic inputs to the region. 3Taken together, these findings are indicative of E-I imbalance and disruption of CP plasticity, possibly in a region-specific manner. As such, these alterations in E-I levels during development have become potential therapeutic targets for antiepileptic drug development.

Although a variety of proteins help regulate E-I levels in the developing brain, dysregulated expression of multiple ionotropic receptor subunits has been implicated in early-life seizures and epilepsy. The differential expression of receptor subunits across development changes receptor kinetics and ion permeability, thereby disrupting normal subunit expression to dysregulate E-I imbalance and alter CP plasticity. Excitatory drive from AMPA and NMDA receptors has been shown to be vital to synaptogenesis--especially during critical periods. However, altered subunit expression of both NMDAR and AMPAR within the hippocampus is known to occur following seizures, with respective increases and decreases in selective subunits known to impart more excitable properties. 2, 10

Alterations in inhibitory drive are also known to occur following early-life seizures, with reduced expression of GABAA receptor subunits observed following early-life seizures. 4 Given that inhibitory GABAA receptor signaling is dependent on the balance of the expression of chloride transporters, Na + -K + -Cl - cotransporter 1 (NKCC1), which mediates chloride influx, and K + -Cl - cotransporter 2 (KCC2), which mediates chloride efflux, the relative expression levels of these transporters are vital for E-I balance. 11 Therefore, preferential expression of NKCC1 renders GABA excitatory, whereas preferential KCC2 expression renders GABA inhibitory (Fig. 2). Upregulation of NKCC1 and concurrent downregulation of KCC2 in the cortex have been shown to contribute to early-life post-traumatic seizures. 11

Cortical interneurons also play a vital role in regulating inhibitory tone and GABAergic signaling during development. At birth, inhibitory GABAergic circuitry is underdeveloped and matures postnatally. Parvalbumin (PV) interneurons are crucial for increasing inhibitory tone and regulating plasticity within the cortex. PV development is first marked by postnatal expression of the PV protein, and increasing PV maturity is further marked by their association with perineuronal nets (PNN), where these PNN-associated PV cells represent the end of the critical period and the plasticity typical during this time (Fig. 3). 13 As interneurons are vital for regulating the spread of activation in A1 following thalamic input, dysregulation of their development may contribute to the greater excitatory spread of activation seen in A1 following seizures (Fig. 4). Furthermore, changes in both PV mRNA and cell expression have been shown to occur within A1 following early-life PTZ seizures in both a region- and layer-specific manner. 3



Despite the profound physiological and protein expression changes following early-life seizures, administration of the competitive AMPAR antagonist, NBQX, soon after seizures has been shown to reverse many of these effects. Multiple NBQX treatments immediately and within 36 hours following early-life seizures reduce premature unsilencing of silent synapses in CA1 and reverse impaired LTP in the hippocampus. 8 Our lab has also shown that NBQX given before, immediately, and 12 hours after seizure induction rescues both behavioral deficits and decreases in specific AMPA receptor subunit expression in the hippocampus that accompany neonatal seizures in mice. 2, 5 Furthermore, NBQX treatment immediately post-seizure restores auditory tonotopic plasticity in A1. Although A1 is vital for language acquisition, few projects have explored protein-level changes that may occur within this region to disrupt CP plasticity following seizures.

Therefore, we examined whether neonatal PTZ-induced seizures contribute to E-I imbalance via disruption of the developmental profile of these various inhibitory and excitatory proteins in both the primary auditory cortex and cortex, and if treatment with NBQX can rescue these alterations.

Materials & Methods

PTZ-induced seizures
Acute seizures are induced in male wild-type C57BL6/J mice with daily IP injections of PTZ (60mg/kg) from P9-11 (Fig. 5). Saline-injected littermates are used as controls. One hour post-PTZ injection, mice that were used for Western blotting are injected with either NBQX (20mg/kg) or vehicle (saline). Mice used for immunohistochemistry did not received NBQX treatment. Mice are video-monitored for 2 hours post-PTZ treatment.



Western Blots
Mice from each treatment group were sacrificed at P13 and P20 (n=4-6/group), and cortex samples were dissected. 14Auditory cortex samples were microdissected using a brain matrix, and auditory cortex samples from two animals of the same treatment group were pooled to obtain sufficient protein concentrations for membrane-bound measures by Western blots. Cortical samples were not pooled. Tissue samples were flash frozen and processed for membrane and whole cell protein preparations. Tissue was homogenized in lysis buffer. Equal amounts of proteins were electrophoresed on 4–20% Tris-HCl gels (Bio-Rad, Hercules, CA, USA) and then transferred to low fluorescence polyvinylidenedifluoride membranes (Bio-Rad). Immunoblots were incubated with respective primary antibodies (Table 1) at 4°C overnight. Membranes were then incubated with the IRDye 800 Goat anti-rabbit (1:15000, LiCor) or IRDye 700 Goat anti-mouse (1:20000, LiCor) IgG secondary antibodies. Protein bands were visualized fluorescently with LiCor Odyssey and measured with the Image Studio program. To control for differences in protein loading, raw values were normalized to corresponding β-actin (Millipore-Sigma) within each immunoblot. Normalized values for each protein were expressed as a percent of the mean expression level of littermate control tissues.



Immunohistochemistry
Mice from both treatment groups (PTZ vs Saline) were perfused at P12 with a 4% PFA solution. Entire brains were collected and post-fixed in 4% PFA solution for 2 hours, followed by a 30% sucrose/PBS solution. 16μm serial coronal sections containing A1 were collected. Sections were stained for PV (Swant) and biotinylated Wisteria floribunda lectin (for PNN, Vector), and NeuN (Millipore).

Statistics
Data was tested for normality using the Shapiro-Wilk normality test. Statistical significance for Western blotting was assessed using a one-way ANOVA with multiple comparisons (Tukey’s). Either student’s t tests or Mann-Whitney (if non-parametric) was used to assess statistical significance for IHC. Data was considered statistically significant if the p value was less than 0.05. All results were expressed as mean ± standard error. Chi Squared test was used to compare differences in survival rates between PTZ treatment groups.


Results

Alterations in modulators of excitatory neurotransmission
Within the excitatory system, various NMDA and AMPA receptor subunits were examined given that particular subunits are preferentially expressed during different times in development. While NR1 is the obligate subunit of the NMDARs, the NR2 receptor subunits are developmentally regulated. There is NR2B-NR2A developmental switch, in which NR2B--predominant in the immature brain--decreases in expression and NR2A increases in expression. 15 Likewise, for the AMPA receptor, GluA2 and GluA1 experience a similar developmental switch, in which GluA1 is highly expressed early in life and GluA2 increases postnatally and is required to make AMPARs impermeable to calcium. 16 Given that both AMPA and NMDA receptors play a vital role in CP plasticity within the developing brain, expression of these receptor subunits was quantified via Western blot and measured relative to Saline+Vehicle treated animals.

Within the auditory cortex, P13 mice showed a trend of decreased expression of both AMPAR and NMDAR subunits following seizures compared to controls. NR2A, NR2B, NR1, GluA1, and GluA2 all exhibited a significant decrease in the auditory cortex in P13 PTZ+NBQX treated animals compared to SAL+VEH controls (Fig. 6C-D, P13 PTZ+NBQX (NR2A): 34.21±10.83%, p<0.05; NR2B: 37.71±12.40%, p<0.01; NR1: 60.55±8.592%, p<0.05; GluA1: 31.96±8.758%, p<0.05; GluA2: 32.99±11.61%, p<0.05; refer to Table 2B for SAL+VEH values). There was also a trend of decreased GluA2 expression relative to GluA1 in PTZ+VEH treated animals (Fig 6D, P13 PTZ+VEH: 83.32±3.524% vs. SAL+VEH: 100±3.12%, p=0.0766). Again, older animals were also examined to determine if these expression level changes remained later in development. Unlike the P13 mice, P20 mice demonstrated no significant differences in expression levels of the NMDA or AMPA receptor subunits across treatment groups in the auditory cortex (Fig. 6A-B).

In the cortex, there was a similar trend of decreased excitatory ionotropic receptor subunit expression in P13 mice post-seizure compared to SAL+VEH. Both NR2A and NR2B exhibited a significant decrease and NR1 demonstrated a non-significant but trending decrease in P13 PTZ+VEH treated animals compared to SAL+VEH (Fig. 7D, P13 PTZ+VEH (NR2A): 41.42±8.067%, p<0.05; NR2B: 59.39±7.779%, p<0.05; NR1: 62.32±9.072%, p=0.0629; refer to Table 2D for SAL+VEH values). Compared to SAL+VEH, P13 PTZ+VEH animals demonstrated a trend of increased expression of the ratio of NR2B to NR2A for (Fig 7D, P13 PTZ+VEH: 158.8±17.21 vs SAL+VEH: 100±4.338%, p=0.0661). P13 cortical samples also showed a decrease in GluA1 and 2 expression and a trend of decreased GluA2:GluA1 expression in PTZ+VEH animals compared to controls (Fig. 7C, P13 PTZ+VEH (GluA1): 53.08±8.178%, p<0.05; P13; GluA2: 46.06±8.548%, p<0.05; A1:A2: 84.55±3.557%, p=0.1441; refer to Table 2D for SAL+VEH values). P13 PTZ+NBQX animals showed similar trends of decreasing GluA1 and GluA2 expression, although not significant (Fig. 7C, P13 PTZ+NBQX (GluA1): 62.21±7.999%, p=0.0981; GluA2: 60.06±9.483%, p=0.1053; refer to Table 2D for SAL+VEH values). Similar to the auditory cortex, P20 animals did not show any significant differences in both AMPAR and NMDAR subunit expression across treatment groups in the cortex (Fig. 7A-B).

Alterations in modulators of inhibitory neurotransmission
During development, there is differential subunit expression of the GABAA receptors that regulate receptor kinetics. GABAA⍺3 is preferentially expressed at younger ages and decreases during development, whereas GABAA⍺1 expression increases as the brain matures. 17The relative expression levels of these subunits often correlate with both the kinetics and function of GABAA receptors to directly affect inhibitory current and additionally reveal whether there is precocious maturation or a more immature state within the GABAergic system. GABAA receptor signaling is also dependent on the balance of the two chloride transporters, NKCC1 and KCC2, as mentioned previously. 12 Therefore, dysregulation of GABAA subunits and chloride transporter expression could be a possible factor contributing to the effects of early-life seizures. As such, expression levels of membrane-bound GABAA⍺1 and GABAA⍺3 and the chloride transporters were quantified via Western blot for both cortical and auditory cortical samples and measured relative to Saline+Vehicle treated control (SAL+VEH).

Within the auditory cortex, P13 mice demonstrated an overall pattern of decreased expression of most GABAA receptor subunits and increased NKCC1 expression following seizures compared to SAL+VEH. P13 PTZ+NBQX mice had a significant decrease of GABAA⍺3 expression (Fig. 8F, P13 PTZ+NBQX: 54.47±6.681% vs SAL+VEH: 100±7.772, p<0.05). Furthermore, both isoforms of KCC2 in PTZ+NBQX at P13 exhibited a trending decrease compared to SAL+VEH (Fig 8D, P13 PTZ+NBQX (140kD): 62.15±7.695%, p=0.1228; 250kD: 53.08±14.08%, p=0.1083; refer to Table 2B for SAL+VEH values). An increase in NKCC1 as well as a decrease in KCC2 point to altered intracellular chloride ion levels that cause depolarizing GABA signaling and contribute to E-I imbalance following seizures. Given that expression of these proteins is so dynamic during development, older animals were examined to see if similar trends persisted later in life. Unlike P13 expression levels, P20 animals showed no significant differences in membrane-bound inhibitory protein expression levels across treatment groups in the auditory cortex, suggesting that the possible dysregulation of chloride transporters and the resulting E-I imbalance earlier in development do not persist later in life (Fig. 8A-C).

Cortical samples were also examined, and there was a similar trend of decreased GABAA receptor subunit expression in PTZ-treated animals compared to controls at P13. GABAA⍺1 exhibited a significant among all treatment groups relative to SAL+VEH controls at P13 (Fig 9D, refer to Table 2D for values). There was also a trend of decreased GABAA⍺3 expression for PTZ+VEH treated animals and a reduced ratio of ⍺1:⍺3 expression among both PTZ+VEH and PTZ+NBQX treated animals (Fig 9D, refer to Table 2D for values). At P20, tGABAA⍺3 and 1 demonstrated no significant differences among treatment groups (Fig. 9A-C). P13 PTZ+NBQX treated animals exhibited a trend of reduced expression of the 140kD KCC2 isoform compared to controls (Fig. 9E, P13 PTZ+NBQX: 66.36±9.859% vs SAL+VEH: 100±8.471%, p=0.1228). Both P13 and P20 animals of all treatment groups did not show any significant differences in remaining chloride transporter expression compared to controls (Fig. 9B-C & E-F).

Alterations in Parvalbumin interneuron development

During development, PV interneuron maturity increases to regulate CP plasticity. Following birth, numbers of PV+ cells increase within the cortex through development, and further maturity of the cells is marked by their association with PNN, which marks the end of the critical period and the plasticity typical during this time. 13 Therefore, disruption of this maturational profile of PV cells helps to explain some of the deleterious effects such as impaired language acquisition following early-life seizures in A1. Furthermore, our lab has found a greater spread of excitatory activity in A1 from thalamic inputs specifically in the caudal direction. Given that PV interneurons in layer 4 are vital for controlling the spread of activation in A1 from thalamocortical circuits, this finding points to possible dysregulation of interneurons in this region in both a layer- and region-specific manner within A1. 3 As such, expression of PV cells, both PV+ only and PV PNN+, following neonatal PTZ seizures was quantified in each cortical layer along the rostral-caudal axis within A1 via IHC and measured relative to Saline controls.

In both PTZ and saline treated animals, there was a difference in PV density across layers of A1, in which layer 4 had the highest density of these cells compared to all other layers--consistent with previous findings in our lab and by others. 3, 18 First, total number of PV cells were examined in order to detect any gross alterations in overall PV expression within A1. In rostral A1, PTZ treated animals did not show any significant decreases in total PV within any layers (Fig. 10A). However, within caudal A1, total PV exhibited a decrease following PTZ seizures at P12 only within layer 2⁄3 (Fig. 10B; p<0.01, refer to table 3 for raw values). Next, we examined the different subsets of PV cells in order to determine more specific changes to PV interneuron development. When subdividing the PV groups into PV+ only vs. PV PNN+, we see alterations in the number of PV+ only cells following seizures. Compared to controls, PTZ treated animals showed a trend of decreasing PV+ only expression within rostral section in layer 2⁄3, and a significant decrease in PV+ only cells within layer 2⁄3 of caudal sections (Fig. 10C-D; rostral layer 2⁄3: p=0.141, caudal layer 2⁄3: p<0.01; refer to Table 3 for raw values).There were no significant differences between PTZ and control animals in PV PNN+ expression within any layers in both caudal and rostral A1 (Fig. 10E-F). And although not significant, there was a pattern of decreasing density of total PV, PV+ only, and PV PNN+ in most layers in both rostral and caudal sections post seizure--especially layer 4.



Survival rates following NBQX treatment

Given NBQX effectiveness at rescuing seizure-induced behavioral and physiological alterations in prior works, the survival rates of PTZ-NBQX and PTZ+VEH treated animals were compared. Given that this dosage of PTZ induced tonic-clonic seizures in these animals, we experienced some death among PTZ-treated animals. Through PTZ administration, PTZ+VEH animals of both ages had a survival rate of 58.1% (n=31). However, PTZ+NBQX treatment significantly increased survival among both P13 and P20 animals to 90.6% (p<0.01, n=32). Additionally, there was no change in survival rate in SAL+NBQX compared to SAL+VEH. Interestingly, despite the decrease in protein expression that occurs in SAL+NBQX animals, there were no adverse effects on growth as observed by comparable weight growths (relative to SAL+VEH).


Discussion

The preliminary focus of this project was to study changes in E-I balance within the cortex and auditory cortex following early-life seizures by examining protein markers of excitatory and inhibitory systems. In order to examine if these alterations persist across development, two timepoints--P13 and P20--were used. The data from this project suggests that earlier in development, there may be dynamic changes within these systems in both brain regions as a result of acute neonatal seizures. The data shows that P13 PTZ-treated animals exhibit decreases in expression of most ionotropic receptor subunits and increases in depolarizing GABA as a function of chloride transporter expression--possibly causing E-I imbalance post-seizure. However, given the limited number of animals in each treatment group, the data is preliminary and some trends seen during this project may not persist when more animals are added. Yet across all treatment groups, P20 animals did not show differences in expression of any of these proteins. This suggests that neonatal seizures may exert a profound effect in these regions on proteins vital for plasticity and E-I balance, but primarily during the time within the auditory cortical critical period window (P12-15). Therefore, even though protein expression returned to control levels later in life, these early alterations in protein expression levels may cause lasting changes associated with neonatal seizures, such as unsilencing synapses and disruption of auditory tonotopic rearing. Furthermore, changes in protein levels during the critical period may lead to persistent physiological changes within the cells themselves, despite their apparent similar protein expression at the older age.

Previous work with NBQX has demonstrated its efficacy at reversing some seizure-induced alterations on behavioral and molecular levels, such as rescue of both social deficits and reduced GluA2 expression in the hippocampus. 2, 5 Therefore, another main focus of this project was to test the effects of the AMPA antagonist, NBQX, following PTZ seizures on the expression of these markers. Given the aforementioned effects, we hypothesized that NBQX treatment would have a rescuing effect on many of the changes in protein expression. However, to our surprise, NBQX did not reverse any of the post-seizure protein expression changes in either brain region at either age, and it even appeared to exacerbate the effects in the P13 auditory cortex. Although NBQX has been reported to reverse the other post-seizure effects mentioned above, our experimental model differs from the paradigm in previous studies that showed the rescuing effects of NBQX. In prior studies, animals were given hypoxic seizures only on one day (P10) and samples were collected 12 hours following seizure induction. Our current project utilized PTZ as the mode of seizure induction that was given for 3 consecutive days, with NBQX treatment 1 hour post-PTZ each day. Furthermore, our samples were collected a full 48 hours following the last PTZ induction rather than the 12 hours used in previous studies. Although this lack of rescued protein expression could be attributed to the low number of samples, another possibility is that NBQX may not potentially be as effective at rescuing those expression changes that still persist beyond the initial seizure as compared to those alterations that occur immediately after seizures as a result of hyperexcitability. 2 Despite the fact that NBQX may not rescue post-seizure changes at the molecular level, it nevertheless demonstrated effectiveness on the whole animal scale. Overall, NBQX administration in PTZ animals significantly increased survival compared to vehicle-treated PTZ mice. Therefore, this data suggests that NBQX may be useful at reducing some of the deleterious effects of early-life seizures, although it may achieve this reduction via rescuing alterations of protein expression of markers not studied within this project.

Given that different cortical areas vary in the time window of their respective critical developmental period, we examined both auditory cortex and the remaining cortex to examine whether our treatment had differential effects depending on the cortical region. To our surprise, the general patterns of protein expression appear to be consistent across both auditory cortex and cortex despite these different maturation rates. P13 auditory cortex and cortex samples both showed decreases in ionotropic protein receptor subunit expression, whereas both P20 auditory cortex and cortex samples did not exhibit changes in expression levels. This data demonstrates that not only is protein expression in the auditory cortex disrupted following P9-11 seizures, but also other cortical regions such as the somatosensory cortex may be affected, even though the somatosensory critical period differs from that of the auditory cortex.

Given the lack of robust results via Western blotting seen in this data, IHC was utilized to assess changes in protein expression. Although there may not be obvious changes using Western blotting, IHC could reveal regional differences within the auditory cortex that would be indistinguishable via Western blotting. As it has been shown that A1 differentially matures along the rostral-caudal axis, we subsequently examined possible regional vulnerabilities in protein expression within the auditory cortex that may not have been apparent when utilizing Western blotting. And since inhibitory PV interneurons, which are vital for regulating both the onset and closure of CP plasticity, have been shown to have altered expression in both a layer and rostro-caudal specific manner, we examined PV expression in the auditory cortex following early-life seizures via IHC to determine if there are region-specific protein expression changes that may disrupt E-I balance and CP plasticity in the developing brain. 3 The data shows that at P12, PTZ animals showed a decreasing pattern in total PV, specifically in PV+ only cells, in layer 2⁄3 of A1. However, this decrease is only significant in caudal sections. While patterns of decreased PV cell numbers occur across all cortical layers, the significant reduction specifically in layer 2⁄3 is consistent with other labs findings of decreased PV expression in upper cortical layers in Autism Spectrum Disorders. 19 Previous findings in our lab have also demonstrated using the same protocol via IHC that this dysregulation does not persist later in life following seizures. Therefore, at the start of the auditory critical period, there is a reduction in total PV expression in layer 2⁄3, specific to PV+ only cells, suggestive of disruption of the onset of PV expression rather than overall maturational profile of PV development following neonatal seizures. Furthermore, the greater dysregulation of caudal sections is consistent with previous findings of greater excitatory spread of activity in the caudal direction post-seizure. 3 As PV interneurons are vital for regulating the spread of activity from thalamic inputs in A1, a decrease in PV following seizures may help to explain this greater caudal excitatory spread. Taken with the fact that caudal sections of A1 mature later than rostral sections, all of these findings are indicative of a possible greater vulnerability to seizures in caudal areas of A1 during early development. 9 This presents PV interneuron development--specific to certain layers and regions--as a possible therapeutic target to combat lasting and deleterious comorbidities that accompany early-life seizures.

References

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Appendix


Natural Science and Mathematics: Menu

1. Activated Glia May Induce Developmentally Regulated Inflammation and Synaptic Remodeling in a TSC Mouse Model of Epilepsy by Karbi L. Choudhury

2. Effects of early-life seizures on excitatory and inhibitory markers during auditory cortex development by Evan L. Honig



About the Author

Evan L. Honig
University of Pennsylvania
ehonig@sas.upenn.edu


Table of Contents

1. Abstract

2. Background

3. Materials & Methods

4. Results

5. Discussion

6. References

7. Appendix