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Prenatal hypoxia down regulates the GABA pathway in newborn mice cerebral cortex; partial protection by MgSO4
Int. J. Devl Neuroscience 26 (2008) 77–85 www.elsevier.com/locate/ijdevneu
Prenatal hypoxia down regulates the GABA pathway in newborn mice cerebral cortex; partial protection by MgSO4 Vered Louzoun-Kaplan a, Michal Zuckerman a, J. Regino Perez-Polo b, Hava M. Golan a,b,* a
Department of Developmental Molecular Genetics, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel b Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Received 27 June 2007; received in revised form 31 August 2007; accepted 4 September 2007
Abstract The fetal and newborn brain is particularly susceptible to hypoxia, which increases the risk for neurodevelopmental deficits, seizures, epilepsy and life-span motor, behavioral and cognitive disabilities. Here, we report that prenatal hypoxia at gestation day 17 in mice caused an immediate decrease in fetal cerebral cortex levels of glutamate decarboxylase, a key proteins in the GABA pathway. While maternal MgSO4 treatment prior to hypoxia did not have an early effect, it did accelerate maturation at a later stage based on the observed protein expression profile. In addition, MgSO4 reversed the hypoxia-induced loss of a subpopulation of inhibitory neurons that express calbindin in cortex at postnatal day 14. In the hippocampus, responses to prenatal hypoxia were also evident 4 days after the hypoxia. However, in contrast to the observations in cerebral cortex, hypoxia stimulated key protein expression in the hippocampus. The hippocampal response to hypoxia was also reversed by maternal MgSO4 treatment. The data presented here suggests that decreased levels of key proteins in the GABA pathway in the cerebral cortex may lead to high susceptibility to seizures and epilepsy in newborns after prenatal or perinatal hypoxia and that maternal MgSO4 treatment can reverse the hypoxiainduced deficits in the GABA pathway. # 2007 Published by Elsevier Ltd on behalf of ISDN. Keywords: Hypoxia; Glutamate decarboxylase; VGAT; Brain injury
1. Introduction Prenatal and perinatal hypoxia are major causes of neurodevelopmental deficits, and the resultant motor, behavioral and cognitive outcomes. Moreover, perinatal hypoxia is frequently associated with epilepsy (Arpino et al., 2001; Toet et al., 2005). In the etiology of epilepsy, the inhibitory tone and the balance between excitation and inhibition are critical factors. In the developing brain, GABA is a major neurotransmitter (Gozlan and Ben-Ari, 2003) that undergoes significant changes during late pregnancy and early postnatal life in both humans and rodents (Herlenius and Lagercrantz, 2004; Conti et al., 2004). In the rodent, the expression of the GABA synthesizing enzyme—glutamate decarboxylase * Corresponding author at: Department of Developmental Molecular Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, BeerSheva 84105, Israel. Tel.: +972 8 647 9974; fax: +972 8 627 6215. E-mail address: [email protected] (H.M. Golan). 0736-5748/$34.00 # 2007 Published by Elsevier Ltd on behalf of ISDN. doi:10.1016/j.ijdevneu.2007.09.002
(GAD) changes from an embryonic isoform (GAD45) to the postnatal GAD65 and GAD67 isoforms; the profile of GABA A and B receptor subunit expression is also regulated during this developmental period (Barker et al., 1998). The GABAergic synaptic potentials mediated by GABA A receptors maintain a majority of somatic inhibition. GABA A synaptic potentials depend on the equilibrium potential of chloride ions dependent on the expression of K/Cl co-transporter (KCC2) (Kaila, 1994; Owens and Kriegstein, 2002; Rivera et al., 1999). Within the 1st and 2nd postnatal weeks of life in mice, KCC2 expression in the central nervous system is complete. The temporal expression of KCC2 is regulated by GABA and brain derived neurotrophic factor (BDNF), among several factors (Ganguly et al., 2001; Aguado et al., 2003). Oligimerization of KCC2 also appears to have a regulatory role (Blaesse et al., 2006). There is a decrease in the density of GABA immuno-reactive (IR) neurons in all areas of the rat hippocampus, with a transient reduction in Parvalbumin (PV)-IR neurons, in response to perinatal anoxia (Dell’Anna et al., 1996). The loss of Calbindin
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(CB)-IR neurons in the cortex and both CB-IR and PV-IR neurons in the striatum was observed 2 months after perinatal asphyxia in the rat (Van de Berg et al., 2003). Long-term consequences of prenatal hypoxia in mice also include a reorganization of PV-IR and CB-IR neurons in the hippocampus with a significant decrease in both neuronal cell types in the cerebral cortex (Gerstein et al., 2005). In human infants, there is a significant decline in GAD67-IR neurons in the sub-plate and white matter after perinatal injury with white matter lesion. The loss of Calretinin (CR)-IR neurons after perinatal brain injury has been reported in the same brain regions, but not the cortex, with cell loss present in neonates with normal white matter (Robinson et al., 2006). The large number of newborns exposed to perinatal hypoxia and the resulting morbidity and mortality have prompted a search for treatments that may attenuate the damage to the developing brain and improve long-term outcomes. Maternal treatment with MgSO4, a compound with known vasodilatation influence, when preeclampsia is diagnosed or as neuroprotective treatment in preterm birth, has been reported to reduce the risk for cerebral palsy (Nelson and Grether, 1995; Crowther et al., 2003; Sibai, 2004). We have shown that MgSO4 treatment has moderate beneficial effects on hippocampal and cerebral GABA pathways in adult mice exposed to prenatal hypoxia (Gerstein et al., 2005). These long-lasting effects may result from changes in brain circuits caused by the primary injury. In order to increase our understanding of the direct effects of MgSO4 treatment on prenatal hypoxia on the GABA pathway, we examined early responses of key components in the GABA pathway to prenatal hypoxia and maternal MgSO4 treatment. Vesicular GABA transporter (VGAT), KCC2, CB, PVand GAD isoforms were measured in the hippocampus and cerebral cortex of fetal and newborn mice.
following the 4-h injection protocol (Hallak et al., 2000). Mice from the control group (S + A) were placed in similar chambers and perfused with air for 2-h periods. After the exposure to hypoxia or air, mice were returned to their cages. The mouse colony was maintained in the animal facility of Ben-Gurion University of the Negev, Beer-Sheva, Israel, in a 12 h light:12 h dark cycle, and food and water were provided ad libitum. All procedures were performed according to guidelines of the Israeli Council on Animal Care and approved by the Animal Care and Use Committee of Ben-Gurion University of the Negev.
2.4. Surgical procedure and brain fixation Twenty-four hours after the hypoxia episode was completed six pregnant mice from each group were anesthetized with Ketamine and concomitantly with Rompun (i.p.). After adequate anesthesia was achieved, fetuses were isolated, brains quickly removed into 4 8C cold saline solution, the following brain regions separated and kept at 80 8C for later analysis: cerebral cortex, hippocampus and cerebellum. The other six pregnant mice from each group completed their pregnancy and gave birth and at postnatal day (P) 1 and 14, part of the newborns from each litter were anesthetized, sacrificed and brain tissues harvested as described above. The other newborns from the same litters were sampled for immunohistochemistry.
The maternal MgSO4 injection protocol involves an i.p. dose of 270 mg/kg followed by 27 mg/kg every 20 min for 4 h; injections were given in a volume of 0.1 ml. A second dose of 270 mg/kg was given at the end of the 4-h period. Control mice were injected with saline with the same volume and schedule. The protocol that was selected followed Hallak et al. (2000). Using this protocol, 30 min after the injection of MgSO4, the MgSO4 values in the mothers’ bloodsamples were double the normal values (Golan et al., 2004), and 126% in fetal forebrain (Hallak and Cotton, 1993).
2.4.1. Immunohistochemistry (IHC) Offspring were anesthetized with Ketamine and concomitantly with Rompun administered (i.p.). After adequate anesthesia, trans-cardiac perfusion with PFA 4% was performed. Offspring brains were removed and maintained overnight in 4% PFA, then washed in PBS, incubated in 10% sucrose for 2 h and 30% sucrose over-night. These brains were dehydrated in increasing concentrations of absolute ethanol: 70% 1, 95% 3, 100% 3, for 1 h each. They were then embedded in paraffin and four micron sagittal sections were made with a microtome (JUNG SM2000R, Leica, Germany). Sagittal sections at 1.2– 1.5 mm from the midline were prepared (the exact position was estimated using ‘‘The mouse brain’’ by Paxinos and Franklin, 2001). The sections were mounted on SuperFrost Plus slides (Menzel-Glaser, Germany) and dried at 37 8C for 72 h. The sections were deparaffinized and rehydrated: xylene, 4 7 min in ethanol (6 dips each); 100% 3, 75% 1; dH2O, 1 5 min PBS, pH 7.4. After deparaffinization and rehydration were performed, antigen retrieval was performed with 6 M urea, 2 5 min in a microwave, and slides were washed (after cooling): dH2O 3, PBS 1 5 min. Non-specific staining was blocked with a serum and albumin-containing solution. Primary antisera were diluted in blocking buffer and applied for 1 h at room temperature in a humid chamber. The slides were washed to remove unbound antibodies with PBS— 3 5 min. Biotinylated secondary antibodies were applied for 1 h at room temperature in a humid chamber (Vector, Burlingame, CA). The slides were washed to remove unbound antibodies with PBS—2 5 min. Endogenous peroxidase was quenched by incubation in 3% H2O2 in 80% methanol for 25 min. Avidin–biotin conjugate (Vectastain Elite ABC Kit-Standard, Vector, Burlingame, CA) was applied for 30 min at room temperature in a humid chamber. The slides were washed in PBS—2 5 min. Staining development was performed with 0.06% DAB + H2O2, with cobalt enhancement (1% CoCl2). The sections were mounted with Eukitt. For a negative control, normal serum replaced the primary antibodies. The following antibodies were used at the following concentrations: mouse anti-Parvalbumin, 1:2000, Sigma–Aldrich, St. Louis, MO; Rabbit anti-Calbindin 1:750, Chemicon International Inc., Temecula CA. Sections were sampled on an Olympus IX-70 microscope at a magnification of 10 and 20 and via a SuperCam video camera (Applitec, Israel) with constant illumination. The density of PV-IR and CB-IR neurons in the hippocampus and M1 was analyzed by ‘particle analysis’. Each ‘region of interest’ within each of these areas was measured in five to nine sections, 20 mm apart.
2.3. Hypoxia induction
2.5. Immunoblot analysis
Following Mg injections, the pregnant mice were placed in a Plexiglass chamber (20 cm 10 cm 10 cm) that was perfused with a gas mixture of 9% oxygen, 3% CO2, and balanced nitrogen, for a 2-h period immediately
Tissue was homogenized in the presence of protease inhibitors (Sigma– Aldrich, St. Louis, MO). Proteins were separated by 6 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit anti-GAD65/
2. Experimental procedures 2.1. Animals Pregnant, C57BL/6J mice at gestation day 17th (The morning after mating was considered as gestation day 1.) were randomly assigned to one of three groups: (1) saline injections (intra-peritoneal; i.p.) in room air exposure (S + A, n = 12); (2) saline injections (i.p.) with hypoxia chamber exposure (S + H, n = 12); and (3) MgSO4 injections (i.p.) with hypoxia chamber exposure (Mg + H, n = 12).
2.2. MgSO4 administration
V. Louzoun-Kaplan et al. / Int. J. Devl Neuroscience 26 (2008) 77–85 67, rabbit anti-KCC2 and mouse anti-actin were purchased from Sigma–Aldrich (St. Louis, MO), rabbit anti-VGAT was purchased from Synaptic Systems (Gottingen, Germany), were used as primary antibodies. Anti-mouse and antirabbit IgG horse-radish peroxidase-conjugate secondary antibody (Upstate, Lake Placid, NY) were used for detection. The signal was visualized with an enhanced chemoluminescence kit (Santa Cruz Biotechnology) and by exposing the membranes to X-ray film. The bands were quantified with TINA software (Raytest, Straubenhardt, Germany). All protein blots were normalized to actin blots for each individual lane, except when mentioned. The results of each trial were normalized to the trial average. The final results are averages of two independent duplicates. The ratio GAD67/GAD65 was calculated from the signal of these two isoforms, in the same lane and with a similar duration of exposure.
2.6. Statistical analysis Statistical analyses were performed using SPSS software. Analysis of variance was used to compare the effects of the treatments. The two-tail Student’s t-test for two populations with unequal variance was used within significance limits 0.05. Data are represented as mean S.E.M.
3. Results 3.1. Effects of MgSO4 treatment on GAD65/67 levels after maternal hypoxia In order to characterize the effect of MgSO4 treatment on the GABA pathway, we measured glutamate decarboxylase (GAD65 and GAD67, the two GABA synthesizing enzyme isoforms) levels in P14 mice cerebral cortex and hippocampus homogenates using Western blot assays (Fig. 1A). The expression of both GAD65 and GAD67 remarkably increased with age, as reported previously by others (Barker et al., 1998; Kaufman et al., 1991). In addition, the level of both enzyme isoforms observed in the cerebral cortex (C) at embryonic day 18 and postnatal day 1 were higher than their levels in the hippocampus (Fig. 1A). Twenty-four hours after maternal hypoxia, the levels of GAD65/67 decreased significantly in fetal cerebral cortices, compared to controls (P < 0.005). Pretreatment with MgSO4 had no effect on hypoxia-induced GAD65/67 decreases as shown in Fig. 1B (P < 0.01). This reflects a change in both GAD isoforms; GAD67/actin in control was 1.32 0.2 and 0.83 0.03 (P < 0.05) in the prenatal hypoxia-treated group; they were 0.87 0.06 (P < 0.05) in the Mg + H group. GAD65/actin in the control was 1.35 0.1, 0.82 0.06 (P < 0.05) in the prenatal hypoxia alone group and 0.7 0.04 (P < 0.01) in the Mg + H group. At P1, 4 days after the insult, GAD65/67 levels in the cerebral cortex were similar to those in the control group (Fig. 1C and D). However, 20 days after the insult, maternal pretreatment with MgSO4 before the exposure to hypoxia induced a significant increase in GAD65/67 levels in the cerebral cortex, compared to controls (Fig. 1D). Hippocampal GAD65/67 levels were not affected 24 h after maternal hypoxia, although there was a delayed increase after hypoxia at P1 and P14, compared to controls (P < 0.05 and P < 0.005, respectively). Hippocampal levels of GAD65/67 in the MgSO4 pretreated group did not differ from controls at both time points (Fig. 1E–G). Thus, maternal MgSO4 pretreatment reversed hypoxia-induced changes in hippocampal GAD65/67 levels at all times assayed.
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3.2. Effects of MgSO4 treatment on VGAT levels after maternal hypoxia Vesicular GABA transporter (VGAT) is expressed in presynaptic terminals of inhibitory neurons, taking up GABA into synaptic vesicles. VGAT levels 24 h after maternal hypoxia with and without MgSO4 pretreatment remained unchanged in the cerebral cortex and the hippocampus (Fig. 2A and C). Given that VGAT and GAD65 co-localize to synaptic vesicles and interact in GABA uptake (Jin et al., 2003), the ratio between these proteins in each sample was determined. While there was no significant change in VGAT levels, the ratio of GAD65/ VGAT was significantly decreased in the cerebral cortex 24 h after hypoxia, possibly due to hypoxia-induced decreases in GAD65 levels (Table 1). There were similar VGAT levels in P14 cerebral cortices after all treatments (Fig. 2B). In contrast, in the hippocampus of P14 newborns prenatally exposed to hypoxia, there was a significant increase in VGAT levels, compared to control newborns (P < 0.05). Maternal pretreatment with MgSO4 reversed the hypoxia-induced VGAT increases (Fig. 2D). 3.3. Effects of MgSO4 treatment on KCC2 levels after maternal hypoxia The K/Cl co-transporter, KCC2, is absent in the fetal brain and its postnatal expression is tightly regulated over time. Moreover, KCC2 oligomerization is likely to be required for co-transporter activation (Blaesse et al., 2006). An example of a KCC2 Western blot is shown in Fig. 3A. The KCC2 monomer was observed in all P14 samples but not the KCC2 dimer, which was rarely detected in control and prenatal hypoxia-treated group samples. Nevertheless, all cerebral cortex samples of P14 newborns to mothers exposed to MgSO4 and hypoxia showed a robust expression of KCC2 dimers. While maternal hypoxia did not influence the levels of KCC2 in the newborn cortex 20 days after the hypoxia event, MgSO4 treatment did increase KCC2 cortical levels of both the monomer (P < 0.05) and dimer (P < 0.01) species compared to controls (Fig. 3B). 3.4. Effects of MgSO4 treatment on Calbindin levels after maternal hypoxia The Ca-binding protein Calbindin (CB) is expressed in a subpopulation of inhibitory cells in the cerebral cortex and the hippocampus, as well as in other brain regions. One day after maternal hypoxia there was no change in CB levels in fetal cerebral cortex and hippocampus. In addition, MgSO4 treatment prior to the hypoxia episode did not have any effect on CB levels at this age (Fig. 4A and C). A delayed decrease in CB levels in response to maternal hypoxia was observed in the cerebral cortex at P14 (P < 0.05), compared to controls, as illustrated in Fig. 4B and D. Maternal pretreatment with MgSO4 reversed the hypoxia effect at 20 days post insult. Changes in CB protein levels may indicate a decrease in CB expression in each cell or a smaller number of cells expressing
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Fig. 1. The effect of prenatal hypoxia and MgSO4 on GAD65/67 levels. An example of GAD65 and GAD67 immunoblot with antibody recognizing both GAD isoforms in fetus cerebral cortex and hippocampus, at the various time points tested; embryonic day 18 (E18), postnatal day 1 (P1) and postnatal day 14 (P14) in the hippocampus (H) and cerebral cortex (C) (A, upper panels). The effect observed 1 day after the hypoxia in the cerebral cortex and hippocampus (A, lower panels). GAD65/67 levels normalized to actin levels in the cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 1 and 14, 4 and 20 days (4D and 20D) after the insults (B–D). GAD67/67 levels in the hippocampus 1D, 4D and 20D after the insult (D–F). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D and 4D: all groups n = 6; 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
CB. When we examined CB-IR neurons in the hippocampus, CB-IR neurons were frequently found in the dendritic fields (stratun oriens, SO, and stratum radiatum, SR) of the CA1-CA3 areas (Fig. 5A). To determine the source of the observed
changes in CB levels in the P14 newborns, CB-IR cell density at P14 was measured. There was a similar density of CB-IR neurons in all subfields of the hippocampus CA1 area and in the subiculum, as depicted in Fig. 5B. However, maternal
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Fig. 2. The effect of prenatal hypoxia and MgSO4 on VGAT levels. VGAT levels in the cerebral cortex were normalized to actin levels. Cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 14, 20 days (20D) after the insults (A and B). VGAT levels in the hippocampus 1D and 20D after the insult (C and D). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
hypoxia decreased CB-IR cell density in cerebral cortical layers 2–3 (P < 0.05), layers where CB-IR cells show the higher density. There was also a decreasing trend in CB-IR neuronal density in layers 4–6, compared to control newborns. Maternal pretreatment with MgSO4 prevented the changes in CB-IR neuron density in the superficial layers. However, there was a significant decrease in the density of these neurons in the deep cortical layers (L5-6, P < 0.05) in P14 newborn exposed prenatally to MgSO4 prior to hypoxia compared to controls.
Effects of MgSO4 treatment on Parvalvumin immunoreactive neurons after maternal hypoxia The Ca-binding protein Parvalbumin (PV), is expressed in a subpopulation of inhibitory neurons in cerebral cortex, hippocampus and other brain regions. Its expression is upregulated during the 2nd postnatal week. Examination of PV-IR neurons in brain sections of control P14 newborns showed that in two out of eight newborns examined PV-IR neurons were present in the hippocampus at this age, while PV-
Table 1 Direction of change in protein levels in prenatal hypoxia with and without magnesium Brain area and treatment
Time
Protein Total GAD
GAD67/GAD65 ratio
VGAT
GAD65/VGAT ratio
KCC2
CB
1 day 20 days
# =
# =
= =
# =
=
= #
1 day 20 days
# "
# =
= =
# =
"
= =#
Hippocampus, S+H
1 day 20 days
= "
=
= "
=
= =
Hippocampus, Mg+H
1 day 20 days
= =
=
= =
=
= =
Cortex, S+H Cortex, Mg+H
The table summarized the direction of change in protein levels for GAD65/67, VGAT, KCC2, and CB and the change in CB and PV in prenatal hypoxia (S + H), and in maternal Mg treatment prior to hypoxia (Mg + H) (both in comparison to control group). No change (=); increase (") and decrease (#), arrows indicating a significant change P < 0.05 or less; 24 h after the insult = 1 day and at postnatal day 14–20 days after the insult.
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treatment prior to hypoxia enhanced the density of PV-IR neurons in the hippocampus, as shown by a trend of higher cell density and a larger number of newborns in which PV-IR neurons were evident. There was a similar trend present in the newborns that were exposed prenatally to hypoxia (Fig. 6B and C). 4. Discussion
Fig. 3. The effect of prenatal hypoxia and MgSO4 on KCC2 levels. An example of KCC2 immunoblot in P14 cerebral cortex (A). KCC2 monomer and dimer levels in the cerebral cortex of newborns at postnatal day 14, 20 days (20D) after the insults were normalized actin (B). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
immuno-reactivity was detected in the same brain sections in Purkinje cells in the cerebellum in all brain sections (not shown). An example of PV-IR neurons in a brain section from the Mg + H group is shown in Fig. 6A. Maternal MgSO4
Prenatal hypoxia produces a significant change in the ontogeny of key components of the GABAergic system in the newborn brain. The response of the GABAergic system to prenatal hypoxia differs in cerebral cortex from hippocampus in both magnitude and time course, as illustrated in Table 1. There was a significant decrease in a subpopulation of inhibitory neurons that express Calbindin 20 days after the hypoxiainduced neuronal loss that was reversed by the MgSO4 treatment in the cortex. In the hippocampus, responses to prenatal hypoxia were also evident 4 days after the hypoxia. However, in contrast to the observations in cerebral cortex, hypoxia stimulated key protein expression in the hippocampus. The hippocampal response to hypoxia was also reversed by maternal MgSO4 treatment. The data presented here suggests that decreased levels of key proteins in the GABA pathway in the cerebral cortex may lead to high susceptibility to seizures and epilepsy in newborns after prenatal or perinatal hypoxia
Fig. 4. The effect of prenatal hypoxia and MgSO4 on CB levels. CB levels in the cerebral cortex were normalized to actin levels. Cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 14, 20 days (20D) after the insults (A and B). CB levels in the hippocampus 1D after the insult (C). An example of CB immunoblot, cerebral cortex of newborn, 20 days after the insult. White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
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Fig. 5. The effect of prenatal hypoxia and MgSO4 on CB-IR cell density. An example of CB-IR neurons in the cerebral cortex of newborn at postnatal day 14 (A and B). (C) CB-IR cell density in CA1 region, stratum oriens (SO), stratum radiatum (SR) and stratum pyramidale (SP) and in the sabiculum (Sub). (D) CBIR neurons in the cerebral cortex layers; layers 2 and 3 (L2-3), layer 4 (L4) and layers 5 and 6 (L5-6). White bars, S + A; gray bars, S + H; black bars, Mg + H. S + A, n = 5; S + H, n = 4; Mg + H, n = 4. Five sections per animal, three fields of 500 mm2 from each section, were analyzed. P-values refer to comparison to S + A group.
and that maternal MgSO4 treatment can reverse the hypoxiainduced deficits in the GABA pathway. In humans, prenatal hypoxia and ischemia are major causes of neurodevelopmental damage. In a large proportion of these cases, as well as those with perinatal asphyxia, the newborn develops seizures and long-term epilepsy (Lindstrom et al., 2006; Arpino et al., 2001; Toet et al., 2005). The observed decreases in GAD levels by 24 h from the onset of hypoxia are expected to significantly decrease GABA levels. The functional coupling between GAD65 and VGAT activity allows uptake of newly synthesized GABA by synaptic vesicles (Jin et al., 2003). The change in GAD65/VGAT ratios observed in the cerebral cortex 1 day after hypoxia may reflect an impairment in GABA uptake by presynaptic vesicles. As implied from the observed differences in cellular localization (Kaufman et al., 1991) and regulation (Martin and Rimvall, 1993; Chen et al., 2003), of GAD67 and GAD65, the two isomers have distinct functions. GAD65 deficient mice are particularly susceptible to seizures and present increased mortality, which is likely to be associated with seizure activity (Kash et al., 1997). All of the above suggests that the decreased GAD levels and GAD65/VGAT
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Fig. 6. The effect of prenatal hypoxia and MgSO4 on PV-IR cell density. An example of PV-IR neurons in the hippocampus of newborn at postnatal day 14 (A and B). (C) PV-IR cell density in the hippocampus. (D) The percent of newborns tested that at least one PV-IR neurons was detected in it hippocampus. White bars, S + A; gray bars, S + H; black bars, Mg + H. S + A, n = 6; S + H, n = 7; Mg + H, n = 5. Four to six sections per animal.
ratios present in the cerebral cortex 24 h after prenatal hypoxia may increase the risk for subsequent seizure activity. The report of a lower frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSC) in newborn rats after hypoxiainduced seizures supports the hypothesis that there are hypoxiainduced deficiencies in presynaptic mechanisms (Sanchez et al., 2007). Although there was no change in the levels of cortical GAD in newborn mice after prenatal hypoxia, there was a significant decline in CB and the number of CB-IR neurons in the superficial cortical layers. Given that a loss of CB-IR neurons in the cortex of hypoxia-exposed mice (Gerstein et al., 2005) and rats (Van de Berg et al., 2003) observed as adults has been reported, it is likely that the changes seen here after prenatal hypoxia in the CB-IR neuron population at P14 persist into adulthood.
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In rodents, the expression and activity of GAD isoforms increases during the 1st postnatal month and the expression and oligomerizarion of KCC2 also increases during the 2nd postnatal week and later (Herlenius and Lagercrantz, 2004; Dzhala et al., 2005; Blaesse et al., 2006). Maternal MgSO4 treatment prior to the hypoxia episode did not prevent the early hypoxia-induced changes in GAD isoforms. Yet, at P14, that is 20 days after the insult, there was an increase in GAD65/67 levels and facilitated maturation of the KCC2 transporter that could augment inhibitory cortical behavior in the affected newborns. It is possible that the increase in these proteins above the control level is due to MgSO4 influence. Indeed, maternal treatment with MgSO4 followed by air exposure, induced in 8month-old offspring remodeling of GAD65/67 in the hippocampus and an increase in its immuno-reactivity in the deep layers of the cortex (Gerstein et al., 2005). Although there was no direct change in hippocampal GABA pathway activity 24 h after hypoxia, indirect or sub-threshold responses to prenatal hypoxia are evident, based on the delayed changes in GAD65/67 and VGAT levels observed at P14. Maternal MgSO4 treatment did not affect the levels of the various proteins measured here at 24 h after hypoxia but it did reverse the later changes documented at P14. The disparity in the responses of the GABA pathway in the cerebral cortex and hippocampus may result from differences in maturational state or susceptibility to hypoxia (McQuillen and Ferriero, 2004). It is possible that the restoration of some of the hypoxic-induced alterations by MgSO4 treatment could have been the result of decreasing the hypoxic insult to the fetal brain by it vasodilator effects. The observation that MgSO4 treatment reversed the hippocampal delayedresponse to hypoxia may suggest an involvement of a different population of NMDA-R subunits that are affected differently by the MgSO4 treatment, as reviewed by Jensen (2006). Overall, the data presented here support the existence of an underlying mechanism to account for an increasing susceptibility to seizures in the developing brain after prenatal hypoxia. It also supports the hypothesis that MgSO4 treatment attenuates the responses of the GABA pathway to hypoxia and may facilitate cerebral maturation. Acknowledgements The study was supported by a grant from the United States– Israel Binational Science Foundation to Drs. H. Golan and J.R. Perez-Polo. References Aguado, F., Carmona, M.A., Pozas, E., Aguilo, A., Martinez-Guijarro, F.J., Alcantara, S., Borrell, V., Yuste, R., Ibanez, C.F., Soriano, E., 2003. BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl co-transporter KCC2. Development 130 (7), 1267–1280. Arpino, C., Domizio, S., Carrieri, M.P., Brescianini, D.S., Sabatino, M.G., Curatolo, P., 2001. Prenatal and perinatal determinants of neonatal seizures occurring in the first week of life. J. Child Neurol. 16 (9), 651–656. Barker, J.L., Behar, T., Li, Y.X., Liu, Q.Y., Ma, W., Maric, D., Maric, I., Schaffner, A.E., Serafini, R., Smith, S.V., Somogyi, R., Vautrin, J.Y., Wen,
X.L., Xian, H., 1998. GABAergic cells and signals in CNS development. Perspect. Dev. Neurobiol. 5 (2–3), 305–322. Blaesse, P., Guillemin, I., Schindler, J., Schweizer, M., Delpire, E., Khiroug, L., Friauf, E., Nothwang, H.G., 2006. Oligomerization of KCC2 correlates with development of inhibitory neurotransmission. J. Neurosci. 26 (41), 10407– 10419. Chen, C.H., Battaglioli, G., Martin, D.L., Hobart, S.A., Colon, W., 2003. Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase. Biochim. Biophys. Acta 1645 (1), 63–71. Conti, F., Minelli, A., Melone, M., 2004. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Res. Brain Res. Rev. 45 (3), 196–212. Crowther, C.A., Hiller, J.E., Doyle, L.W., Haslam, R.R., 2003. Effect of magnesium sulfate given for neuroprotection before preterm birth: a randomized controlled trial. JAMA 290 (20), 2669–2676. Dell’Anna, E., Geloso, M.C., Magarelli, M., Molinari, M., 1996. Development of GABA and calcium binding proteins immunoreactivity in the rat hippocampus following neonatal anoxia. Neurosci. Lett. 211, 93–96. Dzhala, V.I., Talos, D.M., Sdrulla, D.A., Brumback, A.C., Mathews, G.C., Benke, T.A., Delpire, E., Jensen, F.E., Staley, K.J., 2005. NKCC1 transporter facilitates seizures in the developing brain. Nat. Med. 11 (11), 1205– 1213. Ganguly, K., Schinder, A.F., Wong, S.T., Poo, M., 2001. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105 (4), 521–532. Gerstein, M., Huleihel, M., Mane, R., Stilman, M., Kashtuzki, I., Hallak, M., Golan, H., 2005. Remodeling of hippocampal GABAergic system in adult offspring after maternal hypoxia and magnesium sulfate load: immunohisto-chemical study. Exp. Neurol. 196, 18–29. Golan, H., Kashtuzki, I., Hallak, M., Sorokin, Y., Huleihel, M., 2004. Maternal hypoxia during pregnancy induces fetal neurodevelopmental brain damage: partial protection by magnesium sulfate. J. Neurosci. Res. 78, 430–441. Gozlan, H., Ben-Ari, Y., 2003. Interneurons are the source and the targets of the first synapses formed in the rat developing hippocampal circuit. Cereb. Cortex 13 (6), 684–692. Hallak, M., Cotton, D.B., 1993. Transfer of maternally administered magnesium sulfate into the fetal compartment of the rat: assessment of amniotic fluid, blood, and brain concentrations. Am. J. Obstet. Gynecol. 169, 427– 431. Hallak, M., Hotra, J.W., Kupsky, W.J., 2000. Magnesium sulfate protection of fetal rat brain from severe maternal hypoxia. Obstet. Gynecol. 96, 124– 128. Herlenius, E., Lagercrantz, H., 2004. Development of neurotransmitter systems during critical periods. Exp. Neurol. 190 (Suppl. 1), S8–S21. Jensen, F.E., 2006. Developmental factors regulating susceptibility to perinatal brain injury and seizures. Curr. Opin. Pediatr. 18, 628–633. Jin, H., Wu, H., Osterhaus, G., Wei, J., Davis, K., Sha, D., Floor, E., Hsu, C.C., Kopke, R.D., Wu, J.Y., 2003. Demonstration of functional coupling between gamma-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc. Natl. Acad. Sci. U.S.A. 100 (7), 4293– 4298. Kaila, K., 1994. Ionic basis of GABAA receptor channel function in the nervous system. Prog. Neurobiol. 42 (4), 489–537. Kash, S.F., Johnson, R.S., Tecott, L.H., Noebels, J.L., Mayfield, R.D., Hanahan, D., Baekkeskov, S., 1997. Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 94 (25), 14060–14065. Kaufman, D.L., Houser, C.R., Tobin, A.J., 1991. Two forms of the gammaaminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J. Neurochem. 56, 720–723. Lindstrom, K., Lagerroos, P., Gillberg, C., Fernell, E., 2006. Teenage outcome after being born at term with moderate neonatal encephalopathy. Pediatr. Neurol. 35 (4), 268–274. Martin, D.L., Rimvall, K., 1993. Regulation of g-aminobutyric acid synthesis in the brain. J. Neurochem. 60, 395–407. McQuillen, P.S., Ferriero, D.M., 2004. Selective vulnerability in the developing central nervous system. Pediatr. Neurol. 30, 227–235.
V. Louzoun-Kaplan et al. / Int. J. Devl Neuroscience 26 (2008) 77–85 Nelson, K.B., Grether, J.K., 1995. Can magnesium sulfate reduce the risk of cerebral palsy in very low birthweight infants? Pediatrics 95, 263–269. Owens, D.F., Kriegstein, A.R., 2002. Is there more to GABA than synaptic inhibition? Nat. Rev. Neurosci. 3 (9), 715–727. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates, second ed. Academic Press, San Diego. Rivera, C., Voipio, J., Payne, J.A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., Pirvola, U., Saarma, M., Kaila, K., 1999. The K co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397 (6716), 251–255. Robinson, S., Li, Q., DeChant, A., Cohen, M.L., 2006. Neonatal loss of g– aminobutyric acid pathway expression after human perinatal brain injury. J. Neurosurg. 104 (Suppl. 6), 396–408.
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Sanchez, R.M., Justice, J.A., Zhang, K., 2007. Persistently decreased basal synaptic inhibition of hippocampal CA1 pyramidal neurons after neonatal hypoxia-induced seizures. Dev. Neurosci. 29 (1–2), 159–167. Sibai, B.M., 2004. Magnesium sulfate prophylaxis in preeclampsia: lessons learned from recent trials. Am. J. Obstet. Gynecol. 190, 1520– 1526. Toet, M.C., Groenendaal, F., Osredkar, D., van Huffelen, A.C., de Vries, L.S., 2005. Postneonatal epilepsy following amplitude-integrated EEG-detected neonatal seizures. Pediatr. Neurol. 32 (4), 241–247. Van de Berg, W.D., Kwaijtaal, M., de Louw, A.J., Lissone, N.P., Schmitz, C., Faull, R.L., Blokland, A., Blanco, C.E., Steinbusch, H.W., 2003. Impact of perinatal asphyxia on the GABAergic and locomotor system. Neuroscience 117, 83–96.
Prenatal hypoxia down regulates the GABA pathway in newborn mice cerebral cortex; partial protection by MgSO4 Vered Louzoun-Kaplan a, Michal Zuckerman a, J. Regino Perez-Polo b, Hava M. Golan a,b,* a
Department of Developmental Molecular Genetics, Faculty of Health Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel b Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Received 27 June 2007; received in revised form 31 August 2007; accepted 4 September 2007
Abstract The fetal and newborn brain is particularly susceptible to hypoxia, which increases the risk for neurodevelopmental deficits, seizures, epilepsy and life-span motor, behavioral and cognitive disabilities. Here, we report that prenatal hypoxia at gestation day 17 in mice caused an immediate decrease in fetal cerebral cortex levels of glutamate decarboxylase, a key proteins in the GABA pathway. While maternal MgSO4 treatment prior to hypoxia did not have an early effect, it did accelerate maturation at a later stage based on the observed protein expression profile. In addition, MgSO4 reversed the hypoxia-induced loss of a subpopulation of inhibitory neurons that express calbindin in cortex at postnatal day 14. In the hippocampus, responses to prenatal hypoxia were also evident 4 days after the hypoxia. However, in contrast to the observations in cerebral cortex, hypoxia stimulated key protein expression in the hippocampus. The hippocampal response to hypoxia was also reversed by maternal MgSO4 treatment. The data presented here suggests that decreased levels of key proteins in the GABA pathway in the cerebral cortex may lead to high susceptibility to seizures and epilepsy in newborns after prenatal or perinatal hypoxia and that maternal MgSO4 treatment can reverse the hypoxiainduced deficits in the GABA pathway. # 2007 Published by Elsevier Ltd on behalf of ISDN. Keywords: Hypoxia; Glutamate decarboxylase; VGAT; Brain injury
1. Introduction Prenatal and perinatal hypoxia are major causes of neurodevelopmental deficits, and the resultant motor, behavioral and cognitive outcomes. Moreover, perinatal hypoxia is frequently associated with epilepsy (Arpino et al., 2001; Toet et al., 2005). In the etiology of epilepsy, the inhibitory tone and the balance between excitation and inhibition are critical factors. In the developing brain, GABA is a major neurotransmitter (Gozlan and Ben-Ari, 2003) that undergoes significant changes during late pregnancy and early postnatal life in both humans and rodents (Herlenius and Lagercrantz, 2004; Conti et al., 2004). In the rodent, the expression of the GABA synthesizing enzyme—glutamate decarboxylase * Corresponding author at: Department of Developmental Molecular Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, BeerSheva 84105, Israel. Tel.: +972 8 647 9974; fax: +972 8 627 6215. E-mail address: [email protected] (H.M. Golan). 0736-5748/$34.00 # 2007 Published by Elsevier Ltd on behalf of ISDN. doi:10.1016/j.ijdevneu.2007.09.002
(GAD) changes from an embryonic isoform (GAD45) to the postnatal GAD65 and GAD67 isoforms; the profile of GABA A and B receptor subunit expression is also regulated during this developmental period (Barker et al., 1998). The GABAergic synaptic potentials mediated by GABA A receptors maintain a majority of somatic inhibition. GABA A synaptic potentials depend on the equilibrium potential of chloride ions dependent on the expression of K/Cl co-transporter (KCC2) (Kaila, 1994; Owens and Kriegstein, 2002; Rivera et al., 1999). Within the 1st and 2nd postnatal weeks of life in mice, KCC2 expression in the central nervous system is complete. The temporal expression of KCC2 is regulated by GABA and brain derived neurotrophic factor (BDNF), among several factors (Ganguly et al., 2001; Aguado et al., 2003). Oligimerization of KCC2 also appears to have a regulatory role (Blaesse et al., 2006). There is a decrease in the density of GABA immuno-reactive (IR) neurons in all areas of the rat hippocampus, with a transient reduction in Parvalbumin (PV)-IR neurons, in response to perinatal anoxia (Dell’Anna et al., 1996). The loss of Calbindin
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(CB)-IR neurons in the cortex and both CB-IR and PV-IR neurons in the striatum was observed 2 months after perinatal asphyxia in the rat (Van de Berg et al., 2003). Long-term consequences of prenatal hypoxia in mice also include a reorganization of PV-IR and CB-IR neurons in the hippocampus with a significant decrease in both neuronal cell types in the cerebral cortex (Gerstein et al., 2005). In human infants, there is a significant decline in GAD67-IR neurons in the sub-plate and white matter after perinatal injury with white matter lesion. The loss of Calretinin (CR)-IR neurons after perinatal brain injury has been reported in the same brain regions, but not the cortex, with cell loss present in neonates with normal white matter (Robinson et al., 2006). The large number of newborns exposed to perinatal hypoxia and the resulting morbidity and mortality have prompted a search for treatments that may attenuate the damage to the developing brain and improve long-term outcomes. Maternal treatment with MgSO4, a compound with known vasodilatation influence, when preeclampsia is diagnosed or as neuroprotective treatment in preterm birth, has been reported to reduce the risk for cerebral palsy (Nelson and Grether, 1995; Crowther et al., 2003; Sibai, 2004). We have shown that MgSO4 treatment has moderate beneficial effects on hippocampal and cerebral GABA pathways in adult mice exposed to prenatal hypoxia (Gerstein et al., 2005). These long-lasting effects may result from changes in brain circuits caused by the primary injury. In order to increase our understanding of the direct effects of MgSO4 treatment on prenatal hypoxia on the GABA pathway, we examined early responses of key components in the GABA pathway to prenatal hypoxia and maternal MgSO4 treatment. Vesicular GABA transporter (VGAT), KCC2, CB, PVand GAD isoforms were measured in the hippocampus and cerebral cortex of fetal and newborn mice.
following the 4-h injection protocol (Hallak et al., 2000). Mice from the control group (S + A) were placed in similar chambers and perfused with air for 2-h periods. After the exposure to hypoxia or air, mice were returned to their cages. The mouse colony was maintained in the animal facility of Ben-Gurion University of the Negev, Beer-Sheva, Israel, in a 12 h light:12 h dark cycle, and food and water were provided ad libitum. All procedures were performed according to guidelines of the Israeli Council on Animal Care and approved by the Animal Care and Use Committee of Ben-Gurion University of the Negev.
2.4. Surgical procedure and brain fixation Twenty-four hours after the hypoxia episode was completed six pregnant mice from each group were anesthetized with Ketamine and concomitantly with Rompun (i.p.). After adequate anesthesia was achieved, fetuses were isolated, brains quickly removed into 4 8C cold saline solution, the following brain regions separated and kept at 80 8C for later analysis: cerebral cortex, hippocampus and cerebellum. The other six pregnant mice from each group completed their pregnancy and gave birth and at postnatal day (P) 1 and 14, part of the newborns from each litter were anesthetized, sacrificed and brain tissues harvested as described above. The other newborns from the same litters were sampled for immunohistochemistry.
The maternal MgSO4 injection protocol involves an i.p. dose of 270 mg/kg followed by 27 mg/kg every 20 min for 4 h; injections were given in a volume of 0.1 ml. A second dose of 270 mg/kg was given at the end of the 4-h period. Control mice were injected with saline with the same volume and schedule. The protocol that was selected followed Hallak et al. (2000). Using this protocol, 30 min after the injection of MgSO4, the MgSO4 values in the mothers’ bloodsamples were double the normal values (Golan et al., 2004), and 126% in fetal forebrain (Hallak and Cotton, 1993).
2.4.1. Immunohistochemistry (IHC) Offspring were anesthetized with Ketamine and concomitantly with Rompun administered (i.p.). After adequate anesthesia, trans-cardiac perfusion with PFA 4% was performed. Offspring brains were removed and maintained overnight in 4% PFA, then washed in PBS, incubated in 10% sucrose for 2 h and 30% sucrose over-night. These brains were dehydrated in increasing concentrations of absolute ethanol: 70% 1, 95% 3, 100% 3, for 1 h each. They were then embedded in paraffin and four micron sagittal sections were made with a microtome (JUNG SM2000R, Leica, Germany). Sagittal sections at 1.2– 1.5 mm from the midline were prepared (the exact position was estimated using ‘‘The mouse brain’’ by Paxinos and Franklin, 2001). The sections were mounted on SuperFrost Plus slides (Menzel-Glaser, Germany) and dried at 37 8C for 72 h. The sections were deparaffinized and rehydrated: xylene, 4 7 min in ethanol (6 dips each); 100% 3, 75% 1; dH2O, 1 5 min PBS, pH 7.4. After deparaffinization and rehydration were performed, antigen retrieval was performed with 6 M urea, 2 5 min in a microwave, and slides were washed (after cooling): dH2O 3, PBS 1 5 min. Non-specific staining was blocked with a serum and albumin-containing solution. Primary antisera were diluted in blocking buffer and applied for 1 h at room temperature in a humid chamber. The slides were washed to remove unbound antibodies with PBS— 3 5 min. Biotinylated secondary antibodies were applied for 1 h at room temperature in a humid chamber (Vector, Burlingame, CA). The slides were washed to remove unbound antibodies with PBS—2 5 min. Endogenous peroxidase was quenched by incubation in 3% H2O2 in 80% methanol for 25 min. Avidin–biotin conjugate (Vectastain Elite ABC Kit-Standard, Vector, Burlingame, CA) was applied for 30 min at room temperature in a humid chamber. The slides were washed in PBS—2 5 min. Staining development was performed with 0.06% DAB + H2O2, with cobalt enhancement (1% CoCl2). The sections were mounted with Eukitt. For a negative control, normal serum replaced the primary antibodies. The following antibodies were used at the following concentrations: mouse anti-Parvalbumin, 1:2000, Sigma–Aldrich, St. Louis, MO; Rabbit anti-Calbindin 1:750, Chemicon International Inc., Temecula CA. Sections were sampled on an Olympus IX-70 microscope at a magnification of 10 and 20 and via a SuperCam video camera (Applitec, Israel) with constant illumination. The density of PV-IR and CB-IR neurons in the hippocampus and M1 was analyzed by ‘particle analysis’. Each ‘region of interest’ within each of these areas was measured in five to nine sections, 20 mm apart.
2.3. Hypoxia induction
2.5. Immunoblot analysis
Following Mg injections, the pregnant mice were placed in a Plexiglass chamber (20 cm 10 cm 10 cm) that was perfused with a gas mixture of 9% oxygen, 3% CO2, and balanced nitrogen, for a 2-h period immediately
Tissue was homogenized in the presence of protease inhibitors (Sigma– Aldrich, St. Louis, MO). Proteins were separated by 6 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit anti-GAD65/
2. Experimental procedures 2.1. Animals Pregnant, C57BL/6J mice at gestation day 17th (The morning after mating was considered as gestation day 1.) were randomly assigned to one of three groups: (1) saline injections (intra-peritoneal; i.p.) in room air exposure (S + A, n = 12); (2) saline injections (i.p.) with hypoxia chamber exposure (S + H, n = 12); and (3) MgSO4 injections (i.p.) with hypoxia chamber exposure (Mg + H, n = 12).
2.2. MgSO4 administration
V. Louzoun-Kaplan et al. / Int. J. Devl Neuroscience 26 (2008) 77–85 67, rabbit anti-KCC2 and mouse anti-actin were purchased from Sigma–Aldrich (St. Louis, MO), rabbit anti-VGAT was purchased from Synaptic Systems (Gottingen, Germany), were used as primary antibodies. Anti-mouse and antirabbit IgG horse-radish peroxidase-conjugate secondary antibody (Upstate, Lake Placid, NY) were used for detection. The signal was visualized with an enhanced chemoluminescence kit (Santa Cruz Biotechnology) and by exposing the membranes to X-ray film. The bands were quantified with TINA software (Raytest, Straubenhardt, Germany). All protein blots were normalized to actin blots for each individual lane, except when mentioned. The results of each trial were normalized to the trial average. The final results are averages of two independent duplicates. The ratio GAD67/GAD65 was calculated from the signal of these two isoforms, in the same lane and with a similar duration of exposure.
2.6. Statistical analysis Statistical analyses were performed using SPSS software. Analysis of variance was used to compare the effects of the treatments. The two-tail Student’s t-test for two populations with unequal variance was used within significance limits 0.05. Data are represented as mean S.E.M.
3. Results 3.1. Effects of MgSO4 treatment on GAD65/67 levels after maternal hypoxia In order to characterize the effect of MgSO4 treatment on the GABA pathway, we measured glutamate decarboxylase (GAD65 and GAD67, the two GABA synthesizing enzyme isoforms) levels in P14 mice cerebral cortex and hippocampus homogenates using Western blot assays (Fig. 1A). The expression of both GAD65 and GAD67 remarkably increased with age, as reported previously by others (Barker et al., 1998; Kaufman et al., 1991). In addition, the level of both enzyme isoforms observed in the cerebral cortex (C) at embryonic day 18 and postnatal day 1 were higher than their levels in the hippocampus (Fig. 1A). Twenty-four hours after maternal hypoxia, the levels of GAD65/67 decreased significantly in fetal cerebral cortices, compared to controls (P < 0.005). Pretreatment with MgSO4 had no effect on hypoxia-induced GAD65/67 decreases as shown in Fig. 1B (P < 0.01). This reflects a change in both GAD isoforms; GAD67/actin in control was 1.32 0.2 and 0.83 0.03 (P < 0.05) in the prenatal hypoxia-treated group; they were 0.87 0.06 (P < 0.05) in the Mg + H group. GAD65/actin in the control was 1.35 0.1, 0.82 0.06 (P < 0.05) in the prenatal hypoxia alone group and 0.7 0.04 (P < 0.01) in the Mg + H group. At P1, 4 days after the insult, GAD65/67 levels in the cerebral cortex were similar to those in the control group (Fig. 1C and D). However, 20 days after the insult, maternal pretreatment with MgSO4 before the exposure to hypoxia induced a significant increase in GAD65/67 levels in the cerebral cortex, compared to controls (Fig. 1D). Hippocampal GAD65/67 levels were not affected 24 h after maternal hypoxia, although there was a delayed increase after hypoxia at P1 and P14, compared to controls (P < 0.05 and P < 0.005, respectively). Hippocampal levels of GAD65/67 in the MgSO4 pretreated group did not differ from controls at both time points (Fig. 1E–G). Thus, maternal MgSO4 pretreatment reversed hypoxia-induced changes in hippocampal GAD65/67 levels at all times assayed.
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3.2. Effects of MgSO4 treatment on VGAT levels after maternal hypoxia Vesicular GABA transporter (VGAT) is expressed in presynaptic terminals of inhibitory neurons, taking up GABA into synaptic vesicles. VGAT levels 24 h after maternal hypoxia with and without MgSO4 pretreatment remained unchanged in the cerebral cortex and the hippocampus (Fig. 2A and C). Given that VGAT and GAD65 co-localize to synaptic vesicles and interact in GABA uptake (Jin et al., 2003), the ratio between these proteins in each sample was determined. While there was no significant change in VGAT levels, the ratio of GAD65/ VGAT was significantly decreased in the cerebral cortex 24 h after hypoxia, possibly due to hypoxia-induced decreases in GAD65 levels (Table 1). There were similar VGAT levels in P14 cerebral cortices after all treatments (Fig. 2B). In contrast, in the hippocampus of P14 newborns prenatally exposed to hypoxia, there was a significant increase in VGAT levels, compared to control newborns (P < 0.05). Maternal pretreatment with MgSO4 reversed the hypoxia-induced VGAT increases (Fig. 2D). 3.3. Effects of MgSO4 treatment on KCC2 levels after maternal hypoxia The K/Cl co-transporter, KCC2, is absent in the fetal brain and its postnatal expression is tightly regulated over time. Moreover, KCC2 oligomerization is likely to be required for co-transporter activation (Blaesse et al., 2006). An example of a KCC2 Western blot is shown in Fig. 3A. The KCC2 monomer was observed in all P14 samples but not the KCC2 dimer, which was rarely detected in control and prenatal hypoxia-treated group samples. Nevertheless, all cerebral cortex samples of P14 newborns to mothers exposed to MgSO4 and hypoxia showed a robust expression of KCC2 dimers. While maternal hypoxia did not influence the levels of KCC2 in the newborn cortex 20 days after the hypoxia event, MgSO4 treatment did increase KCC2 cortical levels of both the monomer (P < 0.05) and dimer (P < 0.01) species compared to controls (Fig. 3B). 3.4. Effects of MgSO4 treatment on Calbindin levels after maternal hypoxia The Ca-binding protein Calbindin (CB) is expressed in a subpopulation of inhibitory cells in the cerebral cortex and the hippocampus, as well as in other brain regions. One day after maternal hypoxia there was no change in CB levels in fetal cerebral cortex and hippocampus. In addition, MgSO4 treatment prior to the hypoxia episode did not have any effect on CB levels at this age (Fig. 4A and C). A delayed decrease in CB levels in response to maternal hypoxia was observed in the cerebral cortex at P14 (P < 0.05), compared to controls, as illustrated in Fig. 4B and D. Maternal pretreatment with MgSO4 reversed the hypoxia effect at 20 days post insult. Changes in CB protein levels may indicate a decrease in CB expression in each cell or a smaller number of cells expressing
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Fig. 1. The effect of prenatal hypoxia and MgSO4 on GAD65/67 levels. An example of GAD65 and GAD67 immunoblot with antibody recognizing both GAD isoforms in fetus cerebral cortex and hippocampus, at the various time points tested; embryonic day 18 (E18), postnatal day 1 (P1) and postnatal day 14 (P14) in the hippocampus (H) and cerebral cortex (C) (A, upper panels). The effect observed 1 day after the hypoxia in the cerebral cortex and hippocampus (A, lower panels). GAD65/67 levels normalized to actin levels in the cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 1 and 14, 4 and 20 days (4D and 20D) after the insults (B–D). GAD67/67 levels in the hippocampus 1D, 4D and 20D after the insult (D–F). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D and 4D: all groups n = 6; 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
CB. When we examined CB-IR neurons in the hippocampus, CB-IR neurons were frequently found in the dendritic fields (stratun oriens, SO, and stratum radiatum, SR) of the CA1-CA3 areas (Fig. 5A). To determine the source of the observed
changes in CB levels in the P14 newborns, CB-IR cell density at P14 was measured. There was a similar density of CB-IR neurons in all subfields of the hippocampus CA1 area and in the subiculum, as depicted in Fig. 5B. However, maternal
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Fig. 2. The effect of prenatal hypoxia and MgSO4 on VGAT levels. VGAT levels in the cerebral cortex were normalized to actin levels. Cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 14, 20 days (20D) after the insults (A and B). VGAT levels in the hippocampus 1D and 20D after the insult (C and D). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
hypoxia decreased CB-IR cell density in cerebral cortical layers 2–3 (P < 0.05), layers where CB-IR cells show the higher density. There was also a decreasing trend in CB-IR neuronal density in layers 4–6, compared to control newborns. Maternal pretreatment with MgSO4 prevented the changes in CB-IR neuron density in the superficial layers. However, there was a significant decrease in the density of these neurons in the deep cortical layers (L5-6, P < 0.05) in P14 newborn exposed prenatally to MgSO4 prior to hypoxia compared to controls.
Effects of MgSO4 treatment on Parvalvumin immunoreactive neurons after maternal hypoxia The Ca-binding protein Parvalbumin (PV), is expressed in a subpopulation of inhibitory neurons in cerebral cortex, hippocampus and other brain regions. Its expression is upregulated during the 2nd postnatal week. Examination of PV-IR neurons in brain sections of control P14 newborns showed that in two out of eight newborns examined PV-IR neurons were present in the hippocampus at this age, while PV-
Table 1 Direction of change in protein levels in prenatal hypoxia with and without magnesium Brain area and treatment
Time
Protein Total GAD
GAD67/GAD65 ratio
VGAT
GAD65/VGAT ratio
KCC2
CB
1 day 20 days
# =
# =
= =
# =
=
= #
1 day 20 days
# "
# =
= =
# =
"
= =#
Hippocampus, S+H
1 day 20 days
= "
=
= "
=
= =
Hippocampus, Mg+H
1 day 20 days
= =
=
= =
=
= =
Cortex, S+H Cortex, Mg+H
The table summarized the direction of change in protein levels for GAD65/67, VGAT, KCC2, and CB and the change in CB and PV in prenatal hypoxia (S + H), and in maternal Mg treatment prior to hypoxia (Mg + H) (both in comparison to control group). No change (=); increase (") and decrease (#), arrows indicating a significant change P < 0.05 or less; 24 h after the insult = 1 day and at postnatal day 14–20 days after the insult.
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treatment prior to hypoxia enhanced the density of PV-IR neurons in the hippocampus, as shown by a trend of higher cell density and a larger number of newborns in which PV-IR neurons were evident. There was a similar trend present in the newborns that were exposed prenatally to hypoxia (Fig. 6B and C). 4. Discussion
Fig. 3. The effect of prenatal hypoxia and MgSO4 on KCC2 levels. An example of KCC2 immunoblot in P14 cerebral cortex (A). KCC2 monomer and dimer levels in the cerebral cortex of newborns at postnatal day 14, 20 days (20D) after the insults were normalized actin (B). White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
immuno-reactivity was detected in the same brain sections in Purkinje cells in the cerebellum in all brain sections (not shown). An example of PV-IR neurons in a brain section from the Mg + H group is shown in Fig. 6A. Maternal MgSO4
Prenatal hypoxia produces a significant change in the ontogeny of key components of the GABAergic system in the newborn brain. The response of the GABAergic system to prenatal hypoxia differs in cerebral cortex from hippocampus in both magnitude and time course, as illustrated in Table 1. There was a significant decrease in a subpopulation of inhibitory neurons that express Calbindin 20 days after the hypoxiainduced neuronal loss that was reversed by the MgSO4 treatment in the cortex. In the hippocampus, responses to prenatal hypoxia were also evident 4 days after the hypoxia. However, in contrast to the observations in cerebral cortex, hypoxia stimulated key protein expression in the hippocampus. The hippocampal response to hypoxia was also reversed by maternal MgSO4 treatment. The data presented here suggests that decreased levels of key proteins in the GABA pathway in the cerebral cortex may lead to high susceptibility to seizures and epilepsy in newborns after prenatal or perinatal hypoxia
Fig. 4. The effect of prenatal hypoxia and MgSO4 on CB levels. CB levels in the cerebral cortex were normalized to actin levels. Cerebral cortex of fetus 1 day (1D) after the insult and newborns at postnatal day 14, 20 days (20D) after the insults (A and B). CB levels in the hippocampus 1D after the insult (C). An example of CB immunoblot, cerebral cortex of newborn, 20 days after the insult. White bars, S + A; gray bars, S + H; black bars, Mg + H. 1D: all groups n = 6, 20D: S + A, n = 7; S + H, n = 5; Mg + H, n = 6. P-values refer to comparison to S + A group.
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Fig. 5. The effect of prenatal hypoxia and MgSO4 on CB-IR cell density. An example of CB-IR neurons in the cerebral cortex of newborn at postnatal day 14 (A and B). (C) CB-IR cell density in CA1 region, stratum oriens (SO), stratum radiatum (SR) and stratum pyramidale (SP) and in the sabiculum (Sub). (D) CBIR neurons in the cerebral cortex layers; layers 2 and 3 (L2-3), layer 4 (L4) and layers 5 and 6 (L5-6). White bars, S + A; gray bars, S + H; black bars, Mg + H. S + A, n = 5; S + H, n = 4; Mg + H, n = 4. Five sections per animal, three fields of 500 mm2 from each section, were analyzed. P-values refer to comparison to S + A group.
and that maternal MgSO4 treatment can reverse the hypoxiainduced deficits in the GABA pathway. In humans, prenatal hypoxia and ischemia are major causes of neurodevelopmental damage. In a large proportion of these cases, as well as those with perinatal asphyxia, the newborn develops seizures and long-term epilepsy (Lindstrom et al., 2006; Arpino et al., 2001; Toet et al., 2005). The observed decreases in GAD levels by 24 h from the onset of hypoxia are expected to significantly decrease GABA levels. The functional coupling between GAD65 and VGAT activity allows uptake of newly synthesized GABA by synaptic vesicles (Jin et al., 2003). The change in GAD65/VGAT ratios observed in the cerebral cortex 1 day after hypoxia may reflect an impairment in GABA uptake by presynaptic vesicles. As implied from the observed differences in cellular localization (Kaufman et al., 1991) and regulation (Martin and Rimvall, 1993; Chen et al., 2003), of GAD67 and GAD65, the two isomers have distinct functions. GAD65 deficient mice are particularly susceptible to seizures and present increased mortality, which is likely to be associated with seizure activity (Kash et al., 1997). All of the above suggests that the decreased GAD levels and GAD65/VGAT
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Fig. 6. The effect of prenatal hypoxia and MgSO4 on PV-IR cell density. An example of PV-IR neurons in the hippocampus of newborn at postnatal day 14 (A and B). (C) PV-IR cell density in the hippocampus. (D) The percent of newborns tested that at least one PV-IR neurons was detected in it hippocampus. White bars, S + A; gray bars, S + H; black bars, Mg + H. S + A, n = 6; S + H, n = 7; Mg + H, n = 5. Four to six sections per animal.
ratios present in the cerebral cortex 24 h after prenatal hypoxia may increase the risk for subsequent seizure activity. The report of a lower frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSC) in newborn rats after hypoxiainduced seizures supports the hypothesis that there are hypoxiainduced deficiencies in presynaptic mechanisms (Sanchez et al., 2007). Although there was no change in the levels of cortical GAD in newborn mice after prenatal hypoxia, there was a significant decline in CB and the number of CB-IR neurons in the superficial cortical layers. Given that a loss of CB-IR neurons in the cortex of hypoxia-exposed mice (Gerstein et al., 2005) and rats (Van de Berg et al., 2003) observed as adults has been reported, it is likely that the changes seen here after prenatal hypoxia in the CB-IR neuron population at P14 persist into adulthood.
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In rodents, the expression and activity of GAD isoforms increases during the 1st postnatal month and the expression and oligomerizarion of KCC2 also increases during the 2nd postnatal week and later (Herlenius and Lagercrantz, 2004; Dzhala et al., 2005; Blaesse et al., 2006). Maternal MgSO4 treatment prior to the hypoxia episode did not prevent the early hypoxia-induced changes in GAD isoforms. Yet, at P14, that is 20 days after the insult, there was an increase in GAD65/67 levels and facilitated maturation of the KCC2 transporter that could augment inhibitory cortical behavior in the affected newborns. It is possible that the increase in these proteins above the control level is due to MgSO4 influence. Indeed, maternal treatment with MgSO4 followed by air exposure, induced in 8month-old offspring remodeling of GAD65/67 in the hippocampus and an increase in its immuno-reactivity in the deep layers of the cortex (Gerstein et al., 2005). Although there was no direct change in hippocampal GABA pathway activity 24 h after hypoxia, indirect or sub-threshold responses to prenatal hypoxia are evident, based on the delayed changes in GAD65/67 and VGAT levels observed at P14. Maternal MgSO4 treatment did not affect the levels of the various proteins measured here at 24 h after hypoxia but it did reverse the later changes documented at P14. The disparity in the responses of the GABA pathway in the cerebral cortex and hippocampus may result from differences in maturational state or susceptibility to hypoxia (McQuillen and Ferriero, 2004). It is possible that the restoration of some of the hypoxic-induced alterations by MgSO4 treatment could have been the result of decreasing the hypoxic insult to the fetal brain by it vasodilator effects. The observation that MgSO4 treatment reversed the hippocampal delayedresponse to hypoxia may suggest an involvement of a different population of NMDA-R subunits that are affected differently by the MgSO4 treatment, as reviewed by Jensen (2006). Overall, the data presented here support the existence of an underlying mechanism to account for an increasing susceptibility to seizures in the developing brain after prenatal hypoxia. It also supports the hypothesis that MgSO4 treatment attenuates the responses of the GABA pathway to hypoxia and may facilitate cerebral maturation. Acknowledgements The study was supported by a grant from the United States– Israel Binational Science Foundation to Drs. H. Golan and J.R. Perez-Polo. References Aguado, F., Carmona, M.A., Pozas, E., Aguilo, A., Martinez-Guijarro, F.J., Alcantara, S., Borrell, V., Yuste, R., Ibanez, C.F., Soriano, E., 2003. BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl co-transporter KCC2. Development 130 (7), 1267–1280. Arpino, C., Domizio, S., Carrieri, M.P., Brescianini, D.S., Sabatino, M.G., Curatolo, P., 2001. Prenatal and perinatal determinants of neonatal seizures occurring in the first week of life. J. Child Neurol. 16 (9), 651–656. Barker, J.L., Behar, T., Li, Y.X., Liu, Q.Y., Ma, W., Maric, D., Maric, I., Schaffner, A.E., Serafini, R., Smith, S.V., Somogyi, R., Vautrin, J.Y., Wen,
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