A new model for prenatal brain damage

Available online at www.sciencedirect.com R

Experimental Neurology 181 (2003) 258 –269

www.elsevier.com/locate/yexnr

A new model for prenatal brain damage I. GABAA receptor activation induces cell death in developing rat hippocampus Joseph L. Nun˜ez,* Jesse J. Alt, and Margaret M. McCarthy Physiology Department, University of Maryland School of Medicine, Baltimore, MD 21201, USA Received 2 May 2002; revised 29 November 2002; accepted 6 December 2002

Abstract Premature infants are at exceptionally high risk for hypoxic–ischemic insults and other traumatic events that result in permanent brain damage. However, no current models adequately mimic these events. An emerging concept is that the major excitatory drive in immature neurons is derived from depolarizing responses following activation of the ␥-aminobutyric acid (GABA)A receptor, resulting in the opening of voltage-sensitive calcium channels. While calcium-mediated signal transduction is trophic in developing neurons, excessive calcium entry is a major mediator of excitotoxicity. We report that exogenous activation of GABAA receptors by muscimol in newborn rats increases cell death in the hippocampus. The effects are region specific, persistent, and greater in males. Muscimol-induced damage is prevented by pretreatment with diltiazem, an L-type voltage-sensitive calcium channel blocker. Results using hippocampal cultures parallel those observed in vivo, indicating that the effects are mediated directly in the hippocampus. Existing models of pediatric hypoxic–ischemic brain damage focus on the effects of glutamate in the postnatal day 7 rat, because it is considered analogous to the newborn human. This makes the newborn rat analogous to the late gestational human. Ischemia in newborn rats induces GABA release and we propose that treatment with muscimol mimics the cell death cascade induced by hypoxia–ischemia in premature human infants. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Excitotoxicity; GABA; Sex difference

Introduction Premature infants are at high risk for intracranial hemorrhage, asphyxia, and the damaging effects of subsequent hypoxia–ischemia (Holmes et al., 2002; Simon, 1999; Yager, 1999). Yet, little attention has been given to the development of an animal model for human prenatal brain damage. Existing rodent models of neonatal brain damage have focused on the postnatal day 7 rat, which is considered analogous to the newborn human (Holmes and Ben-Ari, 1998; Rice et al., 1981; Stein and Vannucci, 1988). This analogy is largely based on the observation that rat pups younger than postnatal day 7 are relatively impervious to * Corresponding author. Physiology Department, University of Maryland School of Medicine, 5-006 Bressler Research Building, 655 W. Baltimore Street, Baltimore, MD 21201, USA. Fax: ⫹1-410-706-8341. E-mail address: [email protected] (J.L. Nun˜ez).

the deleterious effects of glutamate, an important mediator of ischemia-induced injury (Choi and Rothman, 1990; Khalilov et al., 1999; Marks et al., 1996). The lack of damage induced by glutamate in the early postnatal period is due to the immature state of the hippocampal glutamatergic system at birth: N-methyl-D-aspartate (NMDA), 2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainic acid (KA), and metabotropic glutamate receptors, the three primary subreceptor types of the glutamatergic system, are not fully functional (Campochiaro and Coyle, 1978; Cherubini et al., 1991; Holmes et al., 2002). Moreover, the kinetic properties of the channel only allow for limited activation and accompanying calcium influx at this age (Cheng et al., 1999; Marks et al., 1996, 2000). Prior to the maturation of the hippocampal glutamatergic system, the major excitatory drive in the developing hippocampus is provided by ␥-aminobutyric acid (GABA)

0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4886(03)00053-0

J.L. Nun˜ez et al. / Experimental Neurology 181 (2003) 258 –269

(Cherubini et al., 1991; Leinekugel et al., 1999). This GABA-mediated excitation is a function of the intracellular chloride concentration being higher in immature neurons. Activation of the GABAA receptor and efflux of chloride results in membrane depolarization (Cherubini et al., 1991; Ganguly et al., 2001; Leinekugel et al., 1995). GABAmediated depolarization leads to the opening of voltagesensitive calcium channels, thereby increasing intracellular calcium (Ganguly et al., 2001; Obrietan and van den Pol, 1995). The effects of GABAA receptor activation and endogenous calcium influx are generally considered trophic (Behar et al., 1996). However, excessive calcium influx initiated by glutamate, such as occurs following hypoxia– ischemia, is a major component of excitotoxicity (Choi, 1988, 1992; Marks et al., 1996, 2000; Siejso and Bengtsson, 1989). During an ischemic event, there is rapid release of numerous neurotransmitters (Globus et al., 1991; Hagberg et al., 1985; Wang et al., 2001a). Within 30 min of such an event, tissue levels of GABA increase 15-fold, while extracellular levels of GABA increase 250-fold in the neonatal rat hippocampus (Andine et al., 1991). The observations that excessive release of GABA follows an ischemic event, and that GABAA receptor activation induces calcium influx in the neonatal period, led us to hypothesize that excessive activation of the GABAA receptor early in development would be excitotoxic, and this excitotoxicity would be the result of increased calcium. We report two novel and important findings: (1) excessive activation of GABAA receptors in early postnatal neurons is neurodamaging in a region-specific manner, and (2) this damage appears to be due to excessive calcium entry via the L-type voltage-sensitive calcium channel.

Materials and methods Animals Animals were first-generation descendants of Sprague– Dawley albino rats from Charles River Lab (Wilmington, MA). Female rats were time bred with male breeders in the University of Maryland School of Medicine animal colony. Pregnant females were checked every morning for the presence of pups. Day of birth was designated postnatal day 0. Litters were excluded which did not contain at least four male and four female pups (experiment 1) or six males and four females (experiment 2). All animals were housed under a 12:12 h light/dark cycle, with food and water provided ad libitum. All animal procedures were approved by UMB IACUC. Treatment of neonatal rats Postnatal day 0 male and female rats were removed from the dams and placed on a heated pad (37°C) to maintain

259

normal nest temperature. Animals were administered 5 ␮g/50 ␮l (sc) of muscimol or an equal volume of saline vehicle (experiment 1). Pilot experiments were used to find a treatment regime of muscimol that would lead to hippocampal damage without elevated mortality or gross behavioral and pathological deficits. Work from our lab has documented that a one-time 25-␮g muscimol injection on postnatal day 0 led to increased pCREB levels 30 min after treatment (Auger et al., 2002) but the incidence of mortality was high by 2 h after treatment (A. Auger, personal communication). Wuttke and colleagues have demonstrated that injecting rats with 20 ␮g muscimol per 100 g body wt from postnatal day 0 to 22 leads to reductions in the volume of the sexually dimorphic nucleus of the hypothalamus (Wuttke et al., 1992). Using these two studies as a dose range, and the criteria mentioned above, the dose of 5 ␮g of muscimol two times daily over postnatal days 0 and 1 was used. In a separate set of animals (experiment 2), postnatal day 0 male and female rats were administered 50 ␮g/50 ␮l (sc) of diltiazem, the L-type calcium channel blocker, or sterile water vehicle. Thirty minutes later, animals were administered 5 ␮g/50 ␮l (sc) of muscimol or sterile saline vehicle. In animals from both experiment 1 and experiment 2, muscimol or vehicle was administered again 4 h later, and the entire procedure was repeated the next day. All animals were kept on heated pads during the entire time they were separated from the dams. In order to minimize the impact of maternal separation, the separation time for each injection lasted less than 10 min. Animals were closely monitored following neonatal injection. Each rat displayed stereotypic response following muscimol treatment: a decrease in activity and moderate drop in body temperature, followed by a return to normal activity levels by 3–5 min after injection. The 5-␮g dose of muscimol administered did not lead to seizures. The rats injected with diltiazem displayed no abnormal behavioral or physiological changes following treatment. The injection sites were sealed with cyanoacrylate Vetbond surgical adhesive (3M Animal Care Products, St. Paul, MN). Pups were marked depending upon the manipulation they underwent by injecting India ink into their paws and returned to the dam within 15 min. Pups were sacrificed on postnatal day 7. Rat pups were removed from their mothers and decapitated and their brains were fixed overnight in 4% paraformaldehyde with 2.5% acrolein and then for 24 h in 4% paraformaldehyde. Brain weights were taken prior to fixation. Brains were stored in 30% sucrose in paraformaldehyde for 72 h and then sectioned on a cryostat. Two sets of consecutive 60-␮m sections were made through the entire hippocampus. One set was used for cresyl violet staining and the other for neuronal nuclear antigen immunocytochemistry. Four groups of animals were investigated in experiment

260

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Table 1 Groups in experiment 1 and experiment 2 Group

n

Experiment 1 Sham male Male ⫹ muscimol Sham female Female ⫹ muscimol

6 6 6 6

anterior plane of the hippocampus (similar to Plate 27 in Paxinos and Watson (1986)). The last traced section for every animal was the last plane in which the hippocampal formation was present (see Plate 44, left side in Paxinos and Watson (1986)).

Stereological analysis

Volumetric estimation In order to obtain the volume of each individual subfield (CA1, CA2/3), the dentate gyrus, and the total volume of the hippocampus, we first had to find an estimate of the volume shrinkage factor (Uylings et al., 1986). If the correction for shrinkage is not performed, the volume of the region of interest may be over- or underestimated. One male and one female brain was sectioned fresh frozen on a cryostat. The 60-␮m sections were made through the entire hippocampal formation, and the tissue was immediately mounted onto slides, lightly stained, and then coverslipped. This “unprocessed” tissue was compared to the fixed and immunocytochemically “processed” tissue. While stereology allows for unbiased estimates of particle number, calculation of regional volume using Cavilieri estimation is based upon knowing the actual thickness of each section on the slide (h), such as in the formula V(ref) ⫽ ⌺nAi · h, where V(ref) is the volume of the region of interest and ⌺nAi is the cross-sectional area of the ith plane of the region of interest (for n planes). But because errors inherent to determining h for each section may occur, we used the equation V(ref) ⫽ (SF)v · t · ⌺nAi where (SF)v is the volume shrinkage factor, t is the constant section thickness (as recorded from the cryostat), and n is the number of sections measured (Uylings et al., 1986). As is evident from these two equations, (SF)v · t ⬵ h. We calculated (SF)v using the equation (SF)v ⫽ Vf /Vs, where Vf is the volume of the region of interest in the experimental brain (perfused, stained, and coverslipped), and Vs is the volume of the region of interest in the control (fresh frozen) brain. There were no differences in (SF)v between males and females. In the current experiment, (SF)v ⫽ 0.262 for postnatal day 7 rats, and (SF)v ⫽ 0.45 for postnatal day 25 rats (see Nun˜ ez et al., 2003). These values were used in the equation V(ref) ⫽ (SF)v · t · ⌺nAi, where t ⫽ 60 ␮m and n equals 7 to 8 and Ai is the cross-sectional area of each region in a given plane. Ai was measured using the program Neuroexplorer (Microbrightfield, version 2.01). Using the above formula, the volumes for CA1, CA2/3, and the dentate gyrus were determined.

Using the Neurolucida program package (MicrobrightField version 2.01, Colchester, VT), tracings were made from the anterior through the posterior extent of the hippocampal formation. In the tracings, a distinction was made between the CA1 and CA2/3 fields and the dentate gyrus. A total of seven to eight tracings were made per hippocampal formation per hemisphere in each animal. The traced sections were evenly spaced 240 ␮m apart. The first plane was a randomly chosen section within 180 ␮m of the most

Cell number estimation Using an unbiased stereological technique, the optical disector (Gundersen et al., 1988). NeuN immunoreactive (NeuN-ir) cells, and pyknotic cells were counted within three distinct regions of the hippocampal formation: CA1 field, CA2/3 field, and dentate gyrus. The optical disector technique eliminates bias in counting as a result of cell size and shape. Pyknotic cells were counted in cresyl-violetstained sections and were characterized by the clumps of

Experiment 2 Sham male Male ⫹ muscimol Male ⫹ diltiazem ⫹ muscimol Sham female Female ⫹ muscimol Female ⫹ diltiazem ⫹ muscimol

6 5 5 6 5 5

1 and six groups of animals were investigated in experiment 2 (Table 1). Immunocytochemistry Neuronal nuclear antigen (NeuN) is a protein exclusively expressed in neurons. The tissue labeled with NeuN was used for quantification of neuron number. Free-floating tissue sections were rinsed with 0.1 M phosphate-buffered saline (PBS) and cleared of endogenous phosphatase activity by exposure to sodium borohydride. After sections were rinsed, the monoclonal mouse anti-NeuN antibody (Chemicon, Temecula, CA; 1:70,000 in 0.1 M PBS/0.4% Triton X-100) was applied and the tissue was incubated for 48 h at 4°C. On the third day, tissue was rinsed prior to exposure to biotinylated goat anti-mouse IgG secondary antibody (Vector, Burlingame, CA), followed by rinses and the addition of Vectastain Elite ABC reagents (Vector, Burlingame, CA). The tissue was visualized via addition of nickel-enhanced diaminobenzidine in sodium acetate. After the reaction, the tissue was rinsed, mounted onto gelatin-subbed slides, dehydrated, and coverslipped. The other set of tissue was stained with cresyl violet for quantification of hippocampal volume and pyknotic cell number. Cresyl violet densely stains the condensed clumps of nuclear chromatin that are characteristic of apoptosis.

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269 Table 2 Effect of mucimol treatment on body and brain mass of postnatal day 7 male and female rats Group

Body mass (g)

Brain mass (g)

Sham male Male ⫹ muscimol Sham female Female ⫹ muscimol

16.88 ⫾ 0.41 14.72 ⫾ 0.77 15.51 ⫾ 0.44 13.91 ⫾ 0.78

0.729 ⫾ 0.02 0.651 ⫾ 0.04 0.705 ⫾ 0.02 0.650 ⫾ 0.04

Note. Data represent mean ⫾ SEM values.

condensed chromatin within the nucleus. Using the Neurolucida program package (Microbrightfield version 2.01), coronal sections were imaged onto a computer screen. A counting frame (100 ⫻ 100 ␮m with 60X objective for NeuN immunoreactive cell counts, and 100 ⫻ 100 ␮m with 40X objective for pyknotic cell counts) was utilized. The counting frame was moved in a raster fashion throughout CA1, CA2/3, and the dentate gyrus. A total of 8 –10 counting frames were sampled per region, per hemisphere, in each section. Cell counts were performed through a defined 25-␮m depth of the tissue section. The defined depth allowed for a 5- to 10-␮m border, thus avoiding the issue of lost caps or bottoms. In order to obtain cell number, either NeuN-ir or pyknotic, the area of the counting frame (Aframe) was multiplied by defined depth of the tissue section (25 ␮m) to obtain the volume of the counting frame (Vframe). The cell counts made within this volume (P) were divided by the volume of the counting frame (Vframe) to obtain cell density measures (Nv). In order to determine the total number of cells (N), the cell density (Nv) was multiplied by the total volume of the region of interest (Vref). A subset of the animals in experiment 1 (four from each of the groups) underwent pyknotic cell analysis in the parietal cortex (area Par 1) and ventromedial nucleus of the hypothalamus (VMH). As described above, the optical disector technique was used to obtain pyknotic cell density in both of these regions. Counts were made from two sections spaced 240 ␮m apart, starting from a plane equivalent to Paxinos and Watson (Paxinos and Watson, 1986 Fig. 29). The counting frame was moved throughout Par 1, equally sampling all lamina. Neuronal cell cultures Primary cultures of hippocampal neurons were prepared based on Banker and Goslin (Banker and Goslin, 1998). Briefly, a timed pregnant Sprague–Dawley female was sacrificed, with the embryonic day 18 fetuses removed and placed in a petri dish containing HBSS⫹ (88 ml sterile H2O, 10 ml Hank’s balanced salt solution (Ca2⫹- and Mg2⫹-free) 10⫻, 1 ml Hepes buffer, 1.0 M, pH 7.3, 1 ml antibiotic/ antimycotic 100⫻ liquid). Hippocampi were dissected into a centrifuge tube containing HBSS⫹. After all hippocampi were collected, HBSS⫹ was added to the tube to a volume of 4.5 ml, with 0.5 ml trypsin (2.5%). Cells were incubated

261

for 15 min in a 37°C water bath. The supernatant was removed and washed three times for 5 min with HBSS⫹. Cells were dissociated by pipetting up and down using a Pasteur pipet, and cell number and viability were determined by Trypan blue exclusion. Cells were plated on 18-mm poly-L-lysine-coated cover slips at a density of approximately 300,000 cells per coverslip and placed in 60-mm dishes containing 4 ml plating medium [86 ml MEM, 10 ml horse serum, 3 ml glucose (filter sterilized, 20%) 1 ml pyruvic acid, 100 mM]. Cells were allowed to adhere for 4 h in a 37°C, 5% CO2 incubator. The neuron cultures were removed from the plating dishes and placed in glial feeders neuron-side down, prepared according to Banker and Goslin (Banker and Goslin, 1998). The 60-mm glial dishes contained 4 ml of serumfree, glutamate-free neuronal maintenance medium [86 ml MEM, 10 ml ovalbumin, 1% in MEM, 10 ml N2 solution (97.5 ml MEM, 1 ml putrescine solution 16.1 mg 1 mL H2O, 1 ml selenium dioxide 330 ␮g 100 ml H2O, 0.5 ml insulin solution 10 mg/ml, 100 mg transferrin, (human), 3 ml glucose (filter sterilized, 20%), 1 ml pyruvic acid, 100 mM, 1 ml antibiotic/antimycotic 100⫻ liquid)]. One-third of the maintenance medium was replaced after 7 days. Treatment of hippocampal cultures To investigate the mechanism and time course over which excitatory GABA-induced death occurs, primary cultures of the embryonic day 18 rat hippocampus were treated twice daily on DIV 5 and 6 with 10 ␮M muscimol (GABAA agonist) or Hepes (experiment 3) to mimic the procedure performed in vivo in experiment 1. In a separate experiment (experiment 4), primary cultures from the embryonic day 18 rat hippocampus were treated on DIV 5 with 1 ␮M diltiazem (L-type calcium channel blocker) and then 5 min later with 10 ␮M muscimol. The cultures were treated with muscimol 4 h later, and the entire procedure was repeated on DIV 6. These procedures mimicked those performed in vivo in experiment 2. Enzymatic assay Culture supernatant was collected 2, 24, 48, and 72 h following muscimol treatment (experiment 3) and 2, 8, 24, Table 3 Effect of neonatal muscimol treatment on hippocampal volume in postnatal day 7 male and female rats Group

Volume (mm3) CA1 ⫹ CA2/3

Sham male Male ⫹ muscimol Sham female Female ⫹ muscimol

1.88 1.43 1.73 1.25

⫾ ⫾ ⫾ ⫾

0.041 0.037* 0.036 0.029*

Dentate gyrus 0.590 0.393 0.472 0.380

⫾ ⫾ ⫾ ⫾

0.016 0.010* 0.011 0.012*

Note. Data represent mean ⫾ SEM values. * Significant difference from sham controls of the same sex, P ⬍ 0.01.

262

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Results In order to assess the possibility that muscimol treatment had a general deleterious effect on development, body and brain mass were taken just prior to sacrifice. While there was an overall effect between groups on body mass (P ⬍ 0.02), due to males weighing more than females, there was no significant difference between muscimol-treated and sham males or females on body or brain mass (see Table 2). Experiment 1: neonatal muscimol treatment promotes hippocampal cell death in vivo

Fig. 1. Neonatal muscimol treatment in both males and females induces a loss of hippocampal and dentate gyrus neurons. Stereological estimates of neuron number were obtained from six animals in each group. Tissue was labeled with neuronal nuclear antigen (NeuN). Data are means ⫾ SEM. Columns that share the same letter are significantly different from one another (ANOVA; P ⬍ 0.01, n ⫽ 6/group).

32, 48, 72, and 96 h following diltiazem, muscimol, or the diltiazem followed by muscimol treatment (experiment 4) for lactate dehydrogenase (LDH) assay. LDH is an assay of cellular injury in response to a cytotoxin. Briefly, 200-␮l aliquots of culture supernatant (performed in triplicate) and controls (background control, glia cultured with conditioned medium only; low control, Hepes-treated cultures; high control, cultures treated with 2 ␮l Triton X-100) were obtained and stored at 4°C. Lactate dehydrogenase release was assayed using an ELISA reader and the Cytotoxicity Detection Kit (Roche Biochemicals, Indianapolis, IN). The LDH value obtained from the background control was subtracted from all measures. In order to obtained an accurate assessment of cytotoxicity, the following formula was used: cytotoxicity ⫽

experimental value ⫺ low control ⫻ 100. high control ⫺ low control

A total of three replications of each LDH assay were performed. Statistical analysis One-way analyses of variance were run on measures of brain weight, body weight, hippocampal and dentate gyrus volume, NeuN-ir cell number, and pyknotic cell number. NeuN-ir counts and hippocampal volumetric estimates were made individually from CA1 and CA2/3, with the data reported as the addition of the two areas for clarity. Twoway analyses of variance (treatment, time) were performed on in vitro lactate dehydrogenase data. Post hoc Newman– Keuls and t test comparisons were conducted, and a level of P ⬍ 0.05 was required to obtain statistical significance.

Stereological estimates performed on PND 7 indicated significant overall differences between the groups in neuron number in the hippocampus (P ⬍ 0.0001) and dentate gyrus (P ⬍ 0.0001). Similar results were observed on hippocampal volume (see Table 3). Because the effects observed in CA1 were similar to those in CA2/3 in all cases, the data were collapsed across regions in the hippocampus proper. Therefore, the values for the hippocampus are from the addition of CA1 and CA2/3. Post hoc tests indicated a significant reduction in neuron number in the overall hippocampus and dentate gyrus of muscimol-treated pups (both

Table 4 Effect of neonatal muscimol treatment on neuron number in the PN 7 rat hippocampus Neuron number (⫻105)

Animal

Sham male 1 Sham male 2 Sham male 3 Sham male 4 Sham male 5 Sham male 6 Mean/CV Male ⫹ muscimol 1 Male ⫹ muscimol 2 Male ⫹ muscimol 3 Male ⫹ muscimol 4 Male ⫹ muscimol 5 Male ⫹ muscimol 6 Mean/CV Sham female 1 Sham female 2 Sham female 3 Sham female 4 Sham female 5 Sham female 6 Mean/CV Female ⫹ muscimol Female ⫹ muscimol Female ⫹ muscimol Female ⫹ muscimol Female ⫹ muscimol Female ⫹ muscimol Mean/CV

1 2 3 4 5 6

CA1 ⫹ CA2/3

Dentate gyrus

6.64 6.87 6.59 6.24 6.36 6.66 6.56/0.03 5.13 5.29 4.85 4.75 4.63 4.33 4.83/0.07 5.26 5.17 5.20 5.19 5.09 5.45 5.23/0.02 4.52 4.18 4.09 4.16 4.06 3.99 4.17/0.04

2.41 2.47 2.44 2.54 2.25 2.53 2.44/0.04 1.25 1.41 1.65 1.77 1.73 1.50 1.55/0.13 1.87 1.66 1.65 1.38 1.84 1.85 1.71/0.11 1.61 1.63 1.63 1.72 1.70 1.71 1.67/0.03

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

263

Fig. 2. Representative photomicrographs illustrating NeuN-ir neurons in the CA1 region of the hippocampus in (A) sham males, (B) males ⫹ muscimol, (C) sham females, and (D) females ⫹ muscimol. Both sham males and females have more neurons than their muscimol-treated counterparts. Scale bar ⫽ 100 ␮m.

males and females) compared to their saline-treated controls (P ⬍ 0.01 for each measure; Fig. 1). The total number of neurons, along with the interanimal coefficient of variance (CV ⫽ SD/mean) of the neuron number estimates, is shown in Table 4. Muscimol treatment of males led to a 30% decrease in the number of neurons in the hippocampus (P ⬍ 0.01) and a 46% decrease in the dentate gyrus relative to control males (P ⬍ 0.01). In muscimol-treated female pups there was a 22% reduction in hippocampal neuron number compared to control females (P ⬍ 0.01) and only a modest 3% decrease in the dentate gyrus. For representative photomicrographs of NeuN-ir cells in the CA1 region of the hippocampus in all groups, see Fig. 2A–D. Independent of treatment, there was a sex difference in hippocampal and dentate gyrus neuron number, with control males having more neurons in both regions compared to control females (P ⬍ 0.01 for each region). Complementary to the change in neuron number there were significant differences in the number of pyknotic cells observed in the hippocampus (P ⬍ 0.001) and dentate gyrus (P ⬍ 0.01) on PND 7 (Fig. 3). In both regions of the rat brain, muscimol-treated males had significantly more (⬃30%) pyknotic cells than saline-treated males (P ⬍ 0.01). In female pups, neonatal muscimol treatment led to increased pyknotic cells on PND 7 by only 17% in the hippocampus proper (P ⬍ 0.05), with no observable effect of treatment in the dentate gyrus. For representative photomicrographs of pyknotic cells in the CA1 region of the hippocampus in all groups, see Fig. 4A–D. These data indicate that in the hippocampus and dentate gyrus of males and in

the hippocampus of females, muscimol treatment on PND 0 and 1 leads to the persistence of elevated levels of cell death for a minimum of 6 days after exposure. In order to determine whether the damaging effects of developmental exposure to muscimol was a generalized

Fig. 3. Neonatal muscimol treatment in both males and females leads to an increase in pyknotic cell number in the male rat hippocampus and dentate gyrus. Tissue was labeled with cresyl violet for pyknotic cell analysis. Stereological estimates of pyknotic cell number were obtained from six animals in each group. Data are means ⫾ SEM. Columns that share the same letter are significantly different from one another (ANOVA; P ⬍ 0.01, n ⫽ 6/group).

264

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Fig. 4. Representative photomicrographs illustrating pyknotic cells in the CA1 region of the hippocampus in (A) sham males, (B) males ⫹ muscimol, (C) sham females, and (D) females ⫹ muscimol. Both males and females treated with muscimol have more pyknotic cells than their control counterparts. Arrows denote locations of pyknotic cells. Scale bar ⫽ 10 ␮m.

phenomenon or restricted to the hippocampus, the density of pyknotic cells was also investigated in the parietal cortex (Par 1) and the ventromedial nucleus of the hypothalamus (VMH; see Table 5). There were overall group differences in pyknotic cell density in Par 1 (P ⬍ 0.0001). Neonatal muscimol-treated females had greater pyknotic cell density than control females (P ⬍ 0.05), but males treated with muscimol had decreased pyknotic cell density in Par 1 when compared to control males (P ⬍ 0.01), indicating a mixed effect of muscimol as a function of sex in this brain region. There was also a sex difference in pyknotic cell density in Par 1, with control males having higher pyknotic cell density than control females (P ⬍ 0.01). This is consistent with

previous work from the primary visual cortex (as with Par 1, a sensory region of the cerebral cortex), that on PND 7, males have a greater pyknotic cell density than females (Northington et al., 2001). In the VMH, there was no effect of muscimol treatment upon pyknotic cell density in either males or females. These data demonstrate that the damaging effects of muscimol are region specific, and its ability to preferentially damage the hippocampal formation is not reflective of an overall toxicity.

Table 5 Pyknotic cell density in the parietal cortex and ventromedial nucleus of the hypothalamus (VMH) of muscimol-treated postnatal day 7 male and female rats

Diltiazem is a drug that binds to the BTZ site on the L-type voltage-sensitive calcium channel (VSCC) and blocks calcium entry (Glossman et al., 1984; Striessnig et al., 1998). Diltiazem acts exclusively on the L-type VSCC at concentrations of 1 ␮M and lower. At concentrations greater than 10 ␮M, it also blocks P-type VSCC (Dobrev et al., 1999). In the current experiment, we used diltiazem at a concentration of 1 ␮M, ensuring blockade of only the Ltype VSCC. Estimation of hippocampal volume performed on PND 7 illustrated a significant effect of treatment in both males (P ⬍ 0.0001) and females (P ⬍ 0.03). Similar results were observed in the dentate gyrus of males (P ⬍ 0.0001)

Group

Sham male Male ⫹ muscimol Sham female Female ⫹ muscimol

Density of pyknotic cells (cells/mm3) Par 1

VMH

0.832 ⫾ 0.05 0.501 ⫾ 0.07* 0.271 ⫾ 0.05 0.498 ⫾ 0.09*

0.762 ⫾ 0.08 0.692 ⫾ 0.05 0.803 ⫾ 0.12 0.909 ⫾ 0.09

Note. Data represent mean ⫾ SEM values. * Significant difference from sham controls of the same sex, P ⬍ 0.01.

Experiment 2: blockade of L-type voltage-sensitive calcium channels eliminates subsequent neonatal muscimol-induced damage in vivo

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

265

Experiment 3: neonatal muscimol treatment promotes hippocampal cell death in vitro There was an overall effect of muscimol treatment on LDH release (P ⬍ 0.0001) (Fig. 6). Post hoc t tests indicated that at all time points investigated, muscimol-treated cultures displayed greater release of LDH than controls (P ⬍ 0.01 for each measure). Even as soon as 2 h after muscimol treatment, cytotoxicity was elevated in the treated cultures relative to controls. The elevated levels of LDH release persisted for at least 3 days after treatment, with a peak around 48 h after treatment. Experiment 4: blockade of L-type voltage-sensitive calcium channels eliminates subsequent neonatal muscimol-induced damage in vitro

Fig. 5. Pretreatment with the L-type voltage-sensitive calcium channel blocker diltiazem blocked neonatal muscimol-induced cell loss in vivo in the hippocampus and dentate gyrus of both males (A) and females (B). Basic volume estimation of hippocampal and dentate gyrus volumes were obtained from six animals in each group. Data are means ⫾ SEM. Columns that share the same letter are significantly different from one another (ANOVA; P ⬍ 0.01, n ⫽ 5– 6/group).

(Fig. 5A) and females (P ⬍ 0.02) (Fig. 5B). In all groups, diltiazem treatment blocked the deleterious effects of subsequent muscimol treatment on hippocampal and dentate gyrus volume (P ⬍ 0.05 for each measure). In the hippocampus proper, diltiazem-treated males and females had volumes that were equivalent to controls. However, such was not the case in the dentate gyrus. While diltiazem treatment blocked subsequent muscimol damage in males (P ⬍ 0.01), these animals had dentate gyrus volumes smaller than that of control males (P ⬍ 0.05). Although not significant, the direction was the same in the dentate gyrus of females. These data indicate that while blockade of the L-type voltage-sensitive calcium channel appears to completely reverse muscimol induced damage in the hippocampus proper, it is not sufficient to block all damage in the dentate gyrus.

We documented a significant overall effect of treatment (P ⬍ 0.0001) and time (P ⬍ 0.0001) on lactate dehydrogenase release (Fig. 7). As in experiment 3, muscimol treatment led to elevated LDH release by 2 h, with persistence at all time points, relative to controls (P ⬍ 0.05 for each measure). Peak LDH levels were observed at 24 h following muscimol treatment, with a gradual decline over the next 72 h. Although the decline in LDH levels may represent a loss of muscimol toxicity, it may also indicate a loss of hippocampal neurons, with fewer cells being able to release LDH. So, while an important indicator of death, these data may actually underestimate the total amount of cell loss. As with the in vivo results, treatment with the L-type voltagesensitive calcium channel blocker diltiazem completely abolished muscimol-induced damage. At all time points between 2 and 96 h, diltiazem plus muscimol-treated cultures did not differ from controls. And at all time points except 8 h, the diltiazem plus muscimol-treated cultures displayed LDH release significantly lower than that of the

Fig. 6. Muscimol treatment in neonatal hippocampal cultures is cytotoxic as assessed by the lactate dehydrogenase (LDH) assay. Each LDH time point represents the mean of three replications of three different runs (n ⫽ 3). Data are means ⫾ SEM. The letter “a” represents significant difference from control (baseline measure) at the same time point (ANOVA; P ⬍ 0.01).

266

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Fig. 7. Pretreatment with the L-type voltage-sensitive calcium channel blocker diltiazem completely blocked muscimol-induced cytotoxicity in neonatal hippocampal cultures as assessed by the lactate dehydrogenase (LDH) assay. Each LDH time point represents the mean of three replications of three different runs (n ⫽ 3). Data are means ⫾ SEM. The letter “a” represents significant difference from control (baseline measure at the same time point), and “b” represents significant differences from diltiazem ⫹ muscimol-treated cultures at the same time point (ANOVA; P ⬍ 0.01).

muscimol-treated cultures (P ⬍ 0.01). Diltiazem treatment, while similar to control cultures between 2 and 32 h, displayed elevated levels of LDH release at 48 through 96 h (P ⬍ 0.01), indicating the requirement for endogenous levels of calcium influx through the L-type channel for long-term hippocampal neuron survival in vitro.

Discussion These studies investigated the effect of acute activation of GABAA receptors in the newborn rat on cell death in the developing hippocampus. Muscimol, a selective GABAA receptor agonist, administered over the first 2 days of postnatal life resulted in a substantial loss of hippocampal and dentate gyrus neurons by postnatal day 7, with evidence of continuing cell death. In response to the same insult, males displayed more damage than females, particularly in the dentate gyrus. By the use of primary hippocampal cultures, we observed in vitro results that paralleled those in vivo and argue against a peripherally mediated effect. Finally, the damaging effects of muscimol were either completely prevented or attenuated by pretreatment with the calcium channel blocker diltiazem both in vivo and in vitro. Excessive calcium influx is a major mediator of excitotoxicity and occurs upon activation of glutamatergic receptors (Choi, 1988, 1992). However, in the newborn rat, opening of glutamate receptors induces only minor increases in intracellular calcium (Bichler et al., 1993; Cheng et al., 1999; Marks et al., 1996, 2000) and it is not until the second or third postnatal week that activation of glutamate recep-

tors induces significant elevation in intracellular calcium and neuronal damage (Bickler et al., 1994; Cheng et al., 1999). Thus, around the time of birth, activation of NMDA and AMPA/KA receptors does not produce the same robust neurological or behavioral impairments seen in older animals (Holmes and Ben-Ari, 1998; Marks et al., 1996). At the same age, activation of the GABAA receptor in hippocampal neurons results in membrane depolarization sufficient to promote calcium influx via opening of voltagesensitive calcium channels, most notably the L-type (Cherubini et al., 1991; Ganguly et al., 2001; Leinekugel et al., 1995). This alternate pathway for calcium influx is functional on postnatal day 0, prior to maturation of NMDA and AMPA/KA receptors (Holmes et al., 2002; Leinekugel et al., 1999). Calcium entry via voltage-sensitive calcium channels has been documented to be excitotoxic (Choi, 1988; Siejso, 1990), while blockade of the L-type VSCC following neonatal models of glutamate induced excitotoxicity is partially neuroprotective (Gunn et al., 1989, 1994; Kohling et al., 2000). Peripheral administration of the GABAA receptor agonist diazepam on postnatal day 7 induces hippocampal cell death (Ikonomidou et al., 2000), with similar results observed in primary hippocampal and cortical cultures (Erdo et al., 1991; Honegger et al., 1998; Xu et al., 2000). However, this cell death appears to be the result of inhibitory GABA action suppressing normal glutamatergic activation and/or decreasing the endogenous trophic levels of calcium influx. The damaging effects of GABAA receptor activation on postnatal day 7 are blocked by depolarizing KCl and calcium entry via opening of the L-type calcium channel. Thus, chronic inhibition due to activation of the GABAA receptor can be damaging on postnatal day 7, whereas the effects observed in the present experiment are likely the result of excessive excitation. There is also evidence that antagonism of the GABAA receptor on postnatal day 7, prior to kainic acid administration, completely blocks damage in the rat cerebellum (Chen et al., 1999). This phenomenon appears to occur through a calcium-independent mechanism. The importance of the present findings lies in the excitatory actions of GABA, suggesting an excitotoxic mechanism analogous to that commonly seen following glutamatergic agonist administration. One potential caveat of the present results is that GABA itself induces the switch from a depolarizing to hyperpolarizing effect in neonatal cultured hippocampal neurons (Ganguly et al., 2001). Seven days of muscimol treatment (10 –50 ␮M) led to a 15% decrease in the percentage of neurons that responded with calcium influx following activation of GABAA receptors (Ganguly et al., 2001). We administered 10 ␮M muscimol in vitro for 2 days, with a similar concentration in vivo. The shorter duration and lower concentration of muscimol combined with a protective effect of diltiazem suggest that the cell loss and LDH release we observed are due to continued excitatory actions of GABA.

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Elevated calcium influx has been reported to induce loss of mitochondrial cytochrome c (Zhan et al., 2001), bax translocation (Cao et al., 2001), caspase-3 activation (Brecht et al., 2002; Wang et al., 2001a), and permanent disruption of mitochondrial depolarization and intracellular calcium homeostasis (Vergun et al., 1999), thereby leading to cell death. Along with the total amount of calcium influx leading to cellular injury, the distinct route by which calcium enters the cell influences neurotoxicity (Sattler et al., 1998). While calcium buffering by the mitochondria and calcium buffering proteins are protective against the increase in intracellular calcium accompanying neonatal glutamate receptor activation (Sattler et al., 1998; Vergun et al., 1999), they may not be sufficient to protect against the repetitive activation of the L-type VSCC as in the current paradigm. Future work will investigate potential caspasedependent and -independent mechanisms of muscimol-induced death. Consistent with previous findings in animal models as well as humans, we report that males are more sensitive than females to early brain injury (Lauterbach et al., 2001; Naeye et al., 2000). The same insult produced a slightly greater loss in males of neurons in the hippocampus proper (8% more) and a substantially greater loss of neurons in the dentate gyrus (43% more) compared to females. Evidence for continued cell death in the hippocampus of males indicates that the disparity between males and females may increase as development progresses. When behaviorally tested as juveniles, muscimol-treated males performed more poorly than muscimol-treated females in the first trial block of Morris water maze testing, and both sexes of muscimoltreated animals performed more poorly than their salinetreated counterparts (see accompanying paper, Nun˜ ez et al., 2003). Sex differences in exposure to estradiol in neonatal development may be an important basis for the increased sensitivity in males given that newborn males have significantly higher levels of estradiol in the brain than females (Rhoda et al., 1984). Estradiol doubles the magnitude of calcium influx via L-type calcium channels following GABAA receptor activation in cultured hypothalamic neurons (Perrot-Sinal et al., 2001), and a similar mechanism may be operating in the hippocampus. Regional specificity of the damaging effects of exogenous muscimol was revealed by significant loss of neurons in the hippocampal formation, but not in the ventromedial nucleus of the hypothalamus (VMH). In the parietal cortex, muscimol treatment reduced cell death in males but increased it in females, whereas only males lost a significant percentage of neurons in the dentate gyrus following muscimol treatment. Regional specificity is also observed in the damaging actions of excitatory amino acids. The persistence of cell death for 1 week following exogenous muscimol is also consistent with reports documenting elevated levels of cell death for at least 7 days following neonatal hypoxia–ischemia (Rhoda et al., 1984). Excitotoxic cell death in the neonate occurs over a continuum,

267

including both apoptosis and necrosis (Clarke, 1999; Nicotera et al., 1999; Portera-Calliau et al., 1997; Roy and Sapolsky, 1999). Current work from our lab investigating the in vivo time course of hippocampal cell loss and our present in vitro data highlight an early period of cell death that appears to be necrotic, with persisting death similar to apoptosis (J. Nun˜ ez, personal observation). Also, in the current experiment, cell death following GABAA receptor activation was affected by sex. Although pyknotic cells were observed 6 days after muscimol treatment in both the male and the female rat hippocampus proper, they were only observed at this time in the male dentate gyrus. It is possible that cell death persists for an extended period of time due to the lack of appropriate connections being formed within the developing hippocampus. Based on the current findings, we propose that treatment of newborn rats with muscimol mimics the initiation of a cell death cascade induced by hypoxia or injury in premature infants and is analogous to the accepted method of glutamate administration to the week-old rat pup to model the newborn human. While glutamatergic agonists fail to extensively damage the rat brain prior to PND 7 (Campochiaro and Coyle, 1978; McDonald et al., 1988), excessive GABAA receptor activation at this time leads to decreased hippocampal neuron number in both male and female rats, with damage being more severe in males. Neonatal muscimol-induced damage is blocked by the L-type voltage-sensitive calcium channel blocker diltiazem; thus, the increase in toxic levels of intracellular calcium appears to be the regulator of cell death. Therefore, we propose that the excitatory drive through the GABAA receptor in early development may be an important contributor to some forms of brain damage in premature infants. Acknowledgments This work was supported by NIH Grant R01 MH 52716 to MMM. JLN was supported by a SFN Postdoctoral Minority Neuroscience Fellowship. References Andine, P., Orwar, O., Jacobson, I., Sandberg, M., Hagberg, H., 1991. Changes in extracellular amino acids and spontaneous neuronal activity during ischemia and extended reflow in the CA1 of the rat hippocampus. J. Neurochem. 57, 222–229. Auger, A.P., Perrot-Sinal, T.S., Auger, C.J., Ekas, L.A., Tetel, M.J., McCarthy, M.M., 2002. Expression of the nuclear receptor coactivator, cAMP response-element binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain. Endocrinology 143, 3009 –3016. Banker, G., Goslin, K., 1998. Culturing Nerve Cells. MIT Press, Cambridge, MA. Behar, T.N., Li, X.Y., Tran, H.T., Ma, W., Dunlap, V., Scott, C., Barker, J.L., 1996. GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. J. Neurosci. 16, 1808 –1818.

268

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269

Bickler, P.E., Gallego, S.M., Hansen, B.M., 1993. Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia. J. Cereb. Blood Flow Metab. 13, 811– 819. Brecht, S., Gelderblom, M., Srinivasan, A., Mielke, K., Dityateva, G., Herdegen, T., 2001. Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity. Mol. Brain Res. 94, 25–34. Campochiaro, P., Coyle, J.T., 1978. Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation. Proc. Natl. Acad. Sci. USA 75, 2025–2029. Cao, G., Minami, M., Pei, W., Yan, C., Chen, D., O’Horo, C., Graham, S.H., Chen, J., 2001. Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death. J. Cereb. Blood Flow Metab. 21, 321–333. Chen, Q., Moulder, K., Tenkova, T., Hardy, K., Olney, J.W., Romano, C., 1999. Excitotoxic cell death dependent on inhibitory receptor activation. Exp. Neurol. 160, 215–225. Cheng, C., Fass, D.M., Reynolds, I.J., 1999. Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca2⫹] increases and NMDA receptor mRNA levels. Brain Res. 849, 97–108. Cherubini, E., Gaiarsa, J.L., Ben-Ari, Y., 1991. GABA: an excitatory transmitter in early postnatal life. Trends Neurosci. 14, 515–519. Choi, D.W., 1988. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 11, 465– 469. Choi, D.W., 1992. Excitotoxic cell death. J Neurobiol. 23, 1261–1276. Choi, D.W., Rothman, S.M., 1990. The role of glutamate neurotoxicity in hypoxic–ischemic neuronal death. Annu. Rev. Neurosci. 13, 171–182. Clarke, P.G.H., 1999. Cell Death and Diseases of the Nervous System. Humana Press, Totowa, NJ. Dobrev, D., Milde, A.S., Andreas, K., Ravens, U., 1999. The effects of verapamil and diltiazem on N, P- and Q-type calcium channels mediating dopamine release in rat striatum. Br. J. Pharmacol. 127, 576 –582. Erdo, S.L., Michler, A., Wolff, J.R., 1991. GABA accelerates excitotoxic cell death in cortical cultures: protection by blockers of GABA-gated chloride channels. Brain Res. 542, 254 –258. Ganguly, K., Schnider, A.F., Wong, S.T., Poo, M., 2001. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105, 521–532. Globus, M.Y.T., Busto, R., Martinez, E., Valdes, I., Dietrich, W.D., Ginsberg, M.D., 1991. Comparative effect of transient global ischemia on extracellular levels of glutamate, glycine and gamma-aminobutyric acid in vulnerable and nonvulnerable brain regions in the rat. J. Neurochem. 57, 470 – 478. Glossman, H., Ferry, D.R., Goll, A., Rombusch, M., 1984. Molecular pharmacology of the calcium channel: evidence for subtypes., multiple drug receptor sites., channel subunits and the development of a radioiodinated 1,4-dihydropyridine calcium channel label, [125I]iodipine. J. Cardiovasc. Pharmacol. 6, S608 –S621. Gundersen, H.J.G., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A., West, M.J., 1988. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. Acta Pathol. Microbiol. Immunol. Scand. 96, 857– 881. Gunn, A.J., Mydlar, T., Bennet, L., Faull, R.L.M., Gorter, S., Cook, C., Johnston, B.M., Gluckman, P.D., 1989. The neuroprotective actions of a calcium channel antagonist, flunarizine, in the infant rat. Pediatr. Res. 25, 573–576. Gunn, A.J., Williams, C.E., Millard, C.E., Tan, W.K.M., Gluckman, P.D., 1994. Flunarizine, a calcium channel antagonist, is partially prophylactically neuroprotective in hypoxic–ischemic encephalopathy in the fetal sheep. Pediatr. Res. 35, 657– 663. Hagberg, H., Lehmann, A., Sandberg, M., Nystrom, B., Jacobson, I., Hamberger, A., 1985. Ischemia-induced shift of inhibitory to excitatory

amino acids from intra-to extracellular compartments. J. Cereb. Blood Flow Metab. 5, 413– 419. Holmes, G.L., Ben-Ari, Y., 1998. Seizures in the developing brain: perhaps not so benign after all. Neuron 21, 1231–1234. Holmes, G.L., Khazipov, R., Ben-Ari, Y., 2002. New concepts in neonatal seizures. Neuroreport 13, A3–A8. Honegger, P., Pardo, B., Monnet-Tschudi, F., 1998. Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures. Dev. Brain Res. 105, 219 –225. Ikonomidou, C., Bittigau, P., Ishimaru, M.J., Wozniak, D.F., Koch, C., Genz, K., Price, M.T., Stefovska, V., Horster, F., Tenkova, T., Dikranian, K., Olney, J.W., 2000. Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 287, 1056 –1060. Khalilov, I., Dzhala, V., Medina, I., Leinekugel, X., Melyan, Z., Lamsa, K., Khazipov, R., Ben-Ari, Y., 1999. Maturation of kainite-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro. Eur. J. Neurosci 11, 3468 –3480. Kohling, R., Straub, H., Speckmann, E.J., 2000. Differential involvement of L-type calcium channels in epileptogenesis of rat hippocampal slices during ontogenesis. Neurobiol. Dis. 7, 471– 482. Lauterbach, M.D., Raz, S., Sander, C.J., 2001. Neonatal hypoxic risk in preterm birth infants: the influence of sex and severity of respiratory distress on cognitive recovery. Neuropsychology 15, 411– 420. Leinekugel, X., Tseeb, V., Ben-Ari, Y., Bregestovski, P., 1995. Synaptic GABAA activation induces Ca2⫹ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. J. Physiol. 487, 319 –329. Leinekugel, X., Khalilov, I., McLean, H., Caillard, O., Gaiarsa, J.L., Ben-Ari, Y., Khazipov, R., 1999. GABA is the principal fast-acting excitatory transmitter in the neonatal brain. Adv. Neurol. 79, 189 –201. Marks, J.D., Friedman, J.E., Haddad, G.G., 1996. Vulnerability of CA1 neurons to glutamate is developmentally regulated. Dev. Brain Res. 97, 194 –206. Marks, J.D., Bindokas, V.P., Zhang, X.M., 2000. Maturation vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons. Dev. Brain Res. 124, 101–116. McDonald, J.W., Johnson, M.V., 1990. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev. 15, 41–70. McDonald, J.W., Silverstein, F.S., Johnston, M.V., 1988. Neurotoxicity of N-methyl-D-aspartate is markedly enhanced in developing rat central nervous system. Brain Res. 459, 200 –203. Naeye, R.L., Burt, L.S., Wright, D.L., Blanc, W.A., Tatter, D., 1971. Neonatal mortality, the male disadvantage. Pediatrics 48, 902–906. Nakajima, W., Ishida, A., Lange, M.S., Gabrielson, K.L., Wilson, M.A., Martin, L.J., Blue, M.E., Johnston, M.V., 2000. Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat. J. Neurosci. 20, 7994 – 8004. Nicotera, P., Leist, M., Manzo, L., 1999. Neuronal cell death: a demise with different shapes. Trends Pharm. Sci. 20, 46 –51. Northington, F.J., Ferriero, D.M., Graham, E.M., Traystman, R.J., Martin, L.J., 2001. Early neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal death is apoptosis. Neurobiol. Dis. 8, 207–219. Nun˜ ez, J.L., Lauschke, D.M., Juraska, J.M., 2001. Cell death in the development of the posterior cortex in male and female rats. J. Comp. Neurol. 436, 32– 41. Nun˜ ez, J.L., Alt, J.J., McCarthy, M.M., 2003. A novel model for prenatal brain damage. II. Long-term deficits in hippocampal cell number and hippocampal-dependent behavior following neonatal GABAA receptor activation. Exp. Neurol. 181, 270 –280. Obrietan, K., van den Pol, A.N., 1995. ABA neurotransmission in the hypothalamus: developmental reversal from Ca2⫹ elevating to depressing. J. Neurosci. 15, 065–5077. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney, Australia. Perrot-Sinal, T.S., Davis, A.M., Gregerson, K.A., Kao, J.P.Y., McCarthy, M.M., 2001. Estradiol enhances excitatory gamma-aminobutyric acid

J.L. Nun˜ ez et al. / Experimental Neurology 181 (2003) 258 –269 mediated calcium signaling in neonatal hypothalamic neurons. Endocrinology 142, 2238 –2243. Portera-Calliau, C., Price, D.L., Martin, L.J., 1997. Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum. J. Comp. Neurol. 378, 70 – 87. Rhoda, J., Corbier, P., Roffi, J., 1984. Gonadal steroid concentration in serum and hypothalamus of the rat at birth: aromatization of testosterone to 17beta-estradiol. Endocrinology 114, 1754 –1760. Rice, J.E., Vannucci, R.C., Brierley, J.B., 1981. The influence of immaturity on hypoxic–ischemic brain damage in the rat. Ann. Neurol. 9, 131–141. Roy, M., Sapolsky, R., 1999. Neuronal apoptosis in acute necrotic insult: why is this subject such a mess? Trends Neurosci. 22, 419 – 422. Sattler, R., Charlton, M.P., Hafner, M., Tymianski, M., 1998. Distinct influx pathway, not calcium load, determine neuronal vulnerability to calcium neurotoxicity. J. Neurochem. 71, 2349 –2364. Siesjo, B.K., 1990. Calcium in the brain under physiological and pathological conditions. Eur. Neurol. 30, 3–9. Siesjo, B.K., Bengtsson, F., 1989. Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia and spreading depression: a unifying hypothesis. J. Cereb. Blood Flow Metab. 9, 127–140. Simon, N.P., 1999. Long-term neurodevelopmental outcome of asphyxiated newborns. Clin. Perinatol. 26, 767–778. Stein, D.T., Vannucci, R.C., 1988. Calcium accumulation during the evolution of hypoxic–ischemic brain damage in the immature rat. J. Cereb. Blood Flow Metab. 8, 834 – 842. Striessnig, J., Grabner, M., Mitterdorfer, J., Hering, S., Sinnegger, M.J., Glossman, H., 1998. Structural basis of drug binding to L Ca2⫹ channels. Trends Pharmacol. Sci. 19, 108 –115.

269

Uylings, H.B.M., van Eden, C.G., Hofman, M.A., 1986. Morphometry of size/volume variables and comparison of their bivariate relations in the nervous system under different conditions. J. Neurosci. Methods 18, 19 –37. Vergun, O., Keelan, J., Khodorov, B.I., Duchen, M.R., 1999. Glutamateinduced mitochondrial depolarization and perturbation of calcium homeostasis in cultured rat hippocampal neurons. J. Physiol. 519, 451– 466. Wang, X., Karlsson, J.O., Zhu, C., Bahr, B.A., Hagberg, H., Blomgren, K., 2001a. Caspase-3 activation after neonatal rat cerebral hypoxia–ischemia. Biol. Neonate 79, 172–179. Wang, X., Shimizu-Sasamata, M., Moskowitz, M.A., Newcomb, R., Lo, E.H., 2001b. Profiles of glutamate and GABA efflux in core versus peripheral zones of focal cerebral ischemia in mice. Neurosci. Lett. 313, 121–124. Weiss, J.H., Hartley, D.M., Koh, J., Choi, D.W., 1990. The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity. Science 24, 1474 –1477. Wuttke, W., Bach, F., Flugge, G., 1992. GABAergic influence on the development of the sexually dimorphic nucleus of male and female rats. Brain Res. 573, 341–344. Xu, W., Cormier, R., Fu, T., Covey, D.F., Isenberg, K.E., Zorumski, C.F., Mennerick, S., 2000. Slow death of postnatal hippocampal neurons by GABAA receptor overactivation. J. Neurosci. 20, 3147–3156. Yager, R.G.Y., 1999. Pathophysiology of perinatal brain damage. Brain Res. Rev. 30, 107–134. Zhan, R.Z., Wu, C., Fujihara, H., Taga, K., Qi, S., Naito, M., Shimoji, K., 2001. Both caspase dependent and caspase independent pathways may be involved in hippocampal CA1 neuron death because of loss of cytochrome c from mitochondria in a rat forebrain ischemia model. J. Cereb. Blood Flow Metab. 21, 529 –540.