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1Departments of Pediatrics and 2Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico
Submitted 12 October 2005; accepted in final form 17 April 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Levene et al. 1985; Myers 1972; Thornberg et al. 1995). It has been suggested that an overabundance of excitatory synaptic inputs, excessive release of excitatory amino acids (EAA), and subunit composition of EAA receptors (which favors high-Ca2+ conductance) in the neonatal brain may all contribute to vulnerability to H-I injury (Johnston 1995; Mishra et al. 2001; Stein and Vannucci 1988; Tremblay et al. 1988). Excitotoxic injury in neonatal animal models leads to the selective loss of neurons, including CA1 pyramidal cells of the hippocampus (Towfighi et al. 1995). Less extreme hypoxic insults, although not resulting in appreciable neuronal death, produce a markedly increased seizure susceptibility that extends into later life (Jensen et al. 1991; Volpe 2000). In rat, vulnerability to hypoxic insults is greatest around post natal (PN) days 10–12, comparable insults being ineffective at PN 5 or PN60 (ibid).
It is well established that excessive Ca2+ influx is a critical factor in excitotoxic cell damage (Choi 1995; Siesjo and Bengtsson 1989), but there is relatively little cellular information on EAA-induced disturbances in Ca2+ homeostasis that occur at a developmental period corresponding to birth in humans where H-I insults are common. This period corresponds approximately to post natal (PN) days 7–13 in the rat (Khazipov et al. 2001). In addition to the temporal aspects of excitotoxic Ca2+ loads, recent interest has focused on the spatial aspects of pathologic Ca2+ loads during challenge with excitotoxic agonists. Specifically, it has been found that transient exposures to excitotoxic agonists such as glutamate, kainate, or n-methyl-D-aspartate (NMDA), can result in sustained very high Ca2+ levels in restricted regions of distal dendritic processes (Connor and Cormier 2000; Connor et al. 1988; Randall and Thayer 1992). The fact that these responses persist for long periods after agonist washout, and recovery of Ca2+ levels in the remainder of the cell that did not experience high glutamate levels, has led to these responses being termed "secondary Ca2+ responses" (Connor et al. 1988; Wadman et al. 1992). Importantly, after initiation of these responses in a distal dendrite segment, these secondary responses can propagate slowly throughout neurons, ultimately arriving at the soma and leading to cell death in the acute slice preparation (Shuttleworth and Connor 2001).
Here we have investigated glutamate-evoked primary and secondary Ca2+ responses in CA1 neurons in hippocampal slices from neonatal rat (PN 7–13) and compared them with like responses in neurons from older animals (PN 21–28). A glutamate exposure protocol has been employed instead of oxygen-glucose deprivation (OGD) because response onsets are more rapid and easier to control and most damage is prevented by glutamate receptor antagonists (Ford et al. 1989; Jensen 2002). We report that degenerative secondary responses are much easier to initiate in cells from the younger age group than in the older group. Moreover, the spatial profile of the secondary response was entirely different in the two groups. In the younger neurons, the secondary response was always initiated in small to intermediate-sized dendrites, whereas in the older neurons, the response was more difficult to elicit and started at or near the soma. Regardless of the initiation site or degree of stimulus necessary to initiate it, these secondary responses, after invading the whole cell, resulted in loss of plasmalemma integrity. This shift in sensitivity and site of initiation is, at least in part, brought about by increased importance of GABAergic receptor activation in dendrites over this developmental period. A preliminary report has appeared (Azimi-Zonooz et al. 2003).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All procedures were approved by the Institutional Animal Care and Committee at the University of New Mexico Health Sciences Center and were in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. All pregnant rats were obtained from Harlan Laboratories (Bar Harbor, ME) at E18–E20 stage and housed in standard conditions (12 h/12 h light/dark cycle). Pregnant rats were monitored every 12 h to document time/day of delivery. After delivery, pups from the litter (average litter size: 8–14) were utilized at PN 7–13 and PN 21–28.
Slice preparation
Rat pups (neonatal, PN 7–13) and young adults (PN 21–28) of either sex were killed with a mixture of ketamine and xylazine (85 and 15 mg/ml, respectively; 150 µl sc) and decapitated. Brains were removed and placed in ice-cold cutting solution, containing (in mM) 3 KCl, 1.25 NaH2PO4, 6 MgSO4, 26 NaHCO3, 0.2 CaCl2, 10 glucose, 220 sucrose, and 0.43 ketamine. Coronal sections (350–400 µm) were cut using a Vibratome, and slices were transferred into room-temperature artificial cerebrospinal fluid (ACSF; containing in mM: 126 NaCl, 3 KCl, 1.25 NaHPO4, 1 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 glucose, equilibrated with 95% O2-5% CO2). After warming to 35°C and holding for 1 h, ACSF was changed again, and slices were held at room temperature until used for recording.
Electrophysiological recording
Individual slices were transferred to the recording chamber on a fixed-stage microscope (Zeiss Axioscope, Jena, Germany) and perfused with warmed (34.7–35°C), oxygenated ACSF at 1.7–2 ml/min. Intracellular recordings were made from single CA1 pyramidal neurons (depth <100 µm into the slice) using glass microelectrodes, advanced using a Nanostepper micropositioner (Scientific Precision Instruments, Oppenheim, Germany). Voltage recordings were made using an Axoclamp 2B amplifier and digitized and recorded using Axon Instruments Digidata 1322A (Molecular Devices, Union City, CA). Neurons were accepted for analysis if they had steady resting membrane potentials (RMPs) more negative than –60 mV and generated action potentials >60 mV in response to depolarizing current pulses.
Ca2+ measurements
Individual pyramidal neurons were microinjected with fura-2 (Molecular Probes, Eugene, OR) via the recording microelectrode. The recording/injection microelectrode tips were filled with 10 mM indicator in 0.5M KAc/0.5 M KCl and back-filled with 3M KCl. These microelectrodes had resistances of 
100 M
when filled with 3M KCl, and >150 M initially when filled with injection mixture. After a stable impalement was made, indicator was injected by passing hyperpolarizing current pulses (200 pA) for 10–15 min. After determining spike-train-driven Ca2+ changes, the microelectrode was then carefully removed to permit repetitive glutamate stimulations that were generally accompanied by moderate slice swelling. Only neurons that had stable resting [Ca2+]i levels before and after electrode removal for >20 min were selected for glutamate challenges.
Glutamate was applied as a bolus (50 µl, of 2, 10, or 40 mM stocks) to a small upstream well in the recording chamber. A standard dose of 10 mM glutamate was chosen because stimulating with glutamate at 2 mM produced small or undetectable Ca2+ responses and 40 mM glutamate did not appear to result in any additional increases in soma or dendritic Ca when compared with 10 mM glutamate challenges. GABA (50 µl-40 mM) was bath-applied similar to glutamate. The recording chamber volume was 2 ml. Dye-flow tests indicated that peak concentration of an additive at the slice location was reached 20 s after bolus addition and that 80% clearance required 3 min. The nominal maximum bath concentration following the standard bolus, 50 µl, 10 mM, was 250 µM. It was determined that a bolus of 1/5 of the standard concentration (50 µM in the bath) did not elicit Ca2+ increases. To a first approximation then, the bath concentration of glutamate remained at effective stimulating levels for 2 min. Both of these numbers probably exceed the amplitude and exposure duration for glutamate at neuronal receptors due to strong uptake by glia present in the slice (Barbour and Hausser 1997). Glutamate stimuli were delivered every 20 min until secondary responses were observed. In some experiments, neurons were preexposed to bicuculline (25 µM) for 6–8 min before glutamate bath application.
Neurons were visualized and imaged (0.06–0.33 Hz, 100- to 300-ms exposures) using a water-immersion objective (x40, Zeiss), and a CCD-based imaging system (Photometrics CC200; Roper Scientific, Diluth, GA) run by IPLab software (Scanalytics, Fairfax, VA). Conversion to Ca2+ concentrations were made by ratio measurements of Fura-2 fluorescence (Grynkiewicz et al. 1985) with alternate 350/380 excitation as previously described (Petrozzino et al. 1995). For figure presentation, images were first background-subtracted and then ratioed and pseudocolor images generated. The raw 380-nm fluorescence image was then used as a template image to generate masked images. Conversion to estimated Ca2+ concentrations were done based on in vitro Ca2+ concentration standards (Molecular Probes). Calculated values of Ca2+ that exceed the accurate range of fura-2 (20 nM to 3 µM) are flagged by red ordinates in graphical data. All nonindicator chemicals were obtained from Sigma (St. Louis, MO). Bicuculline stock solution was dissolved in DMSO with final dilution of 1/10000 in ACSF. Group data are presented as means ± SE. Two-sample, independent t-test were used to compare means, with P < 0.05 held as statistically significant.
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RESULTS |
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Figure 1 shows Ca2+ changes elicited by bath application of glutamate (see METHODS) in a neonatal CA1 neuron (PN 12) selected because of its relatively parfocal orientation. The standard stimulus protocol (METHODS) produced large Ca2+ increases in the soma and dendrites during the agonist exposure, which are termed the primary Ca2+ response. Maximum increases occurred 12–15 s after glutamate concentration in the chamber peaked, as estimated from dye flow tests. Color maps are shown in Fig. 1A, b–d. Ca2+ levels recovered near to prestimulus value in 2–3 min. The Ca2+ changes shown are responses to glutamate bolus number 4 in a series where the inter-stimulus interval was 20 min. The increases in Ca2+ for the first three glutamate exposures were similar to the fourth exposure except that poststimulus recovery was more complete. Six neurons in the PN 7–13 age group (5 animals) showed similar peak responses (Soma: 2,494 ± 498 nM, dendritic: 3655 ± 855 nM).
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5 min after the fourth stimulus where Ca2+ had returned to near prestimulus levels except at the location noted by the vertical arrow. Further monitoring of activity, well after glutamate washout, revealed the appearance of a propagating front of high Ca2+ (Fig. 1A, f and g), referred to as the secondary response. Where the initiation site of a secondary response was identifiable, as in Fig. 1A, e, it is clear that propagation of the secondary response was bidirectional. Once the response had invaded all visible regions of distal apical dendrites, the focus was adjusted (Fig. 1, h) to allow clear visualization of invasion of the proximal dendrite and soma (Fig. 1A, I and j). This global Ca2+ increase was irreversible. The time course and the magnitude of glutamate-evoked primary and secondary Ca2+ responses in soma and at one location in the apical dendrite (hollow arrow, panel B) are plotted in Fig. 1B.
In four of the six neurons studied, secondary responses were initiated after the second glutamate stimulus. The neuron of Fig. 1 required four stimuli and the sixth neuron produced a secondary response with a 14-min delay after the first stimulus. Overall, the number of stimuli required was 1.8 ± 0.5 (n = 6). All cells were followed for minimum of 30 min after the secondary response invaded the soma, and none showed Ca2+ recovery in that time interval. Three of the six cells were followed at a low sampling rate for 
60 min. In all three cells, >90% loss of indicator occurred 45–60 min after soma invasion. Indicator loss is indicative of the disruption of plasma membrane integrity and loss of cell viability.
Age differences in secondary responses and vulnerability to glutamate stimulus
The mammalian CNS undergoes substantial maturational changes during the early postnatal period, and it has been suggested that these changes may affect vulnerability of the CNS to HI injury (Bickler and Hansen 1998; Johnston 1995). Figure 2 illustrates responses observed in neurons from PN 21 to 28 day rats to bath-applied glutamate. In contrast to the neonatal cells (PN 7–13, see preceding text), we found that at this later stage, primary responses to the standard glutamate stimulus were restricted to the soma and proximal apical dendrites (initial 50–100 µm, Fig. 2A, b–d) and that secondary Ca2+ responses always (n = 7/7) started in these regions and propagated outward to distal apical dendrites (Fig. 2A, e–g). The key difference in this age group was that the primary response to the glutamate exposure was very small in the apical dendrites >100 µm from the soma even though there were large (>3 µM) Ca2+ increases in the soma and proximal apical dendrites. In the example shown, the third glutamate stimulus produced a response in which Ca2+ levels in peri-somatic region did not return to basal levels and subsequently rose to micromolar levels without recovery. The Ca2+ increase then advanced outward into distal apical dendrites. In Fig. 2A, e—g, arrows indicate the leading edge of the advancing secondary response. The time course and the magnitude of glutamate-evoked primary and secondary Ca2+ responses in the soma and apical dendrites are plotted in Fig. 2, B and C. Figure 2C shows measurements during the development of the secondary response on an expanded time scale. The secondary Ca2+ response spread throughout the neuron and subsequently led to dye loss indicative of plasmalemma breakdown.
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50 µm from the soma. Overall, a greater number of glutamate applications was required to initiate secondary Ca2+ responses in PN 21–27 neurons compared with the younger group (3.7 ± 0.6 n = 7, vs. 1.8 ± 0.5 n = 6, t-test P < 0.02). As with the younger group, one neuron (1/7) generated a secondary response after the first stimulus. The remaining six neurons required three or more stimuli. Similar to the neonatal neurons, stimulus at 1/5th standard concentration produced responses that were <100 nM. To exclude glutamate concentration as a factor limiting secondary response induction in the older age group cells, the concentration of glutamate in the bolus was increased by fourfold. In 2/2 neurons, this increase neither changed spatial profile of Ca2+ increases nor decreased the number applications necessary to initiate secondary responses.
As would be predicted from the data in the preceding text, the difference in primary Ca2+ responses between neonates and young adults is most clearly demonstrated when glutamate-evoked responses are compared >100 µm from the soma in each age group as is done in Fig. 3. In distal apical dendrites of neonatal neurons (PN 8) responding to glutamate, [Ca2+]i rose from 100 nM to several micromolar (Fig. 3c). As illustrated in Fig. 3d (arrows), Ca2+ in portions of the viewable dendritic tree sometimes remained transiently elevated after other regions had recovered. Such events were predictive of a full secondary response developing after the next stimulus. This localized Ca2+ response in tertiary dendrites 250 µm from soma re-emphasizes the role of degenerative calcium signaling in neuronal regions not usually monitored during excitotoxic injury. In contrast, the Ca2+ rise in distal apical dendrites stimulated in normal ACSF was negligible in young adults neurons (illustrated in Fig. 3, b'–c').
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One of the important developmental changes in the hippocampus prior to PN21 is the increasing importance of GABAergic synapses onto pyramidal neurons from glutamate-activated interneurons (see DISCUSSION). We hypothesized that the relative efficacy of GABAergic inputs to the pyramidal neuron dendrites might be a contributing factor to the large differences in response to exogenous glutamate stimulus between the age groups studied here. To address this possibility, glutamate stimulation experiments were performed in the presence of bicuculline (25 µM, 6–8 min preexposure). As hypothesized glutamate stimulation after preexposure to bicuculline led to large and sustained increase in Ca2+ levels in the distal dendrites of neurons from the older animals. This finding is illustrated by showing a dendrite region unresponsive to glutamate in normal ACSF (discussed in the preceding text, Fig. 3, b' and c') giving a large response after bicuculline (Fig. 3, d'). Bicuculline exposure alone did not cause changes in basal Ca2+ levels. In 4/4 neurons (PN 21–27), pretreatment with bicuculline allowed robust glutamate-stimulated Ca2+ increases in distal apical dendrites without the decline with distance from the soma observed without the blocker. Moreover, the standard glutamate stimulation with bicuculline preexposure generated a secondary Ca2+ response in dendrites after a single exposure in all of the neurons. These results show that GABAA-receptor activation during exogenous glutamate exposure severely limits Ca2+ accumulation in distal apical dendrites and, as a consequence, prevents initiation of the secondary response in this compartment.
Other factors
Other likely factors that could change over the developmental period studied and affect vulnerability to glutamate are the intrinsic excitability of the neurons and the coupling of action potential firing to Ca2+ increases. We found no evidence for either being a significant contributor. Depolarizing current of 200 pA elicited 19–21 action potentials/s in neurons of both groups. These action potential trains generated Ca2+ somatic increases of 131 ± 8 and 219 ± 58 nM in the younger and older age groups, respectively. Apical dendrite increases, measured 40–60 µm from the soma where the largest increases occurred, were 609 ± 98 and 644 ± 132 nM in the younger and older groups (n = 3, each group).
An additional set of experiments was done to determine whether GABA receptor activation could induce excitotoxicity during PN 7–13 period of development as it does in the very early period PN 0–1 (Leinekugel et al. 1999; Nunez et al. 2003; Xu et al. 2000). The reversing of the Cl– gradient and the transition from GABA-induced excitation to inhibition occurs after PN 5 (Ben-Ari 2002); however, we considered it to be of some interest to see whether residual effects of GABA on Ca2+ levels remained in the PN 7–13 slices. In six cell tested, bath application of GABA (40 µM) had no effect on Ca2+ levels. In three of the cells, GABA exposure was followed by glutamate exposure (30 min after GABA exposure), which caused Ca2+ increases similar to described in the preceding text (Fig. 1). It would therefore appear that GABA does not produce a Ca2+-mediated excitotoxic response at this later phase of development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) that generates significantly larger responses to physiological stimulation (Hsia et al. 1998). There is also a shift in the glutamatergic synaptic population away from "silent" synapses, those that do not generate post synaptic current at normal resting potential to the classical form in which an initial AMPA receptor activation produces depolarization sufficient to bring NMDA receptors into a conducting state. It is controversial whether the silence is due to the absence of functional AMPA receptors in the postsynaptic membrane (Durand et al. 1996; Petralia et al. 1999; but see Xiao et al. 2004) or to low probability of glutamate release in normal synaptic transmission (Gasparini et al. 2000; Renger et al. 2001), which fails to activate the AMPA receptors. In either model, one might expect greater or unchanged, not lesser, Ca2+ increases due to glutamate stimulation. Also, the developmentally linked shift seems to be largely accomplished before the period studied here (Zhu and Malinow 2002; Zhu et al. 2000).
Due to known changes in GABAergic innervation during development, we hypothesized that a strengthening of GABAergic input would potentially be a factor that would limit depolarization and minimize Ca2+ entry into the dendrites of more developed neurons and thus increase their resistance to EAA toxicity. For example, it has been shown that there is a large increase in the number of GABAergic inputs to each pyramidal cell over the period studied here (PN 7–28) as determined from MEPP analysis (Groc et al. 2003). Additionally there is an increasing expression of slow GABAA inhibitory postsynaptic potentials (IPSPs) (Banks et al. 2002) that appear to be preferentially generated in dendritic locations (Banks et al. 1998). Finally, though glutamic acid decarboxylase (GAD)-containing interneurons are present in the hippocampus during the developmental period studied here, the synaptic ultrastructure undergoes considerable refinement over this period, changing from simple apposition of pre and poststructures to structures displaying adult synapse specializations (Kunkel et al. 1986), suggesting more efficient transmission. All of these factors would be expected to decrease depolarization in pyramidal neuron dendrites during global exposure to glutamate because the trilaminar and bistratified interneurons that project to pyramidal neuron dendrites in areas CA1 and CA3 (Sik et al. 1995) as well as basket cells projecting to stratum pyramidal will be activated concurrently with the pyramidal neurons. The postsynaptic Cl– and K+ conductances thus activated appear to be sufficient to short circuit inward current through AMPA and KA receptors that would normally depolarize the dendrites enough to allow Ca2+ entry through NMDA receptors and voltage-gated channels. The striking effect of bicuculline on dendritic responses in the older neurons (Fig. 4, d'), and the greatly increased vulnerability to glutamate toxicity of neurons from young adult animals is certainly consistent with this hypothesis. The strong effect of bicuculline also argues against intrinsic differences in Ca2+ influx or regulation and more effective uptake of glutamate by glia at the later developmental stage as being the sole differences between age groups. These differences should still prevail in the presence of bicuculline.
We did not observe GABA-induced Ca2+ increases at PN 7–13; this indicates that glutamate is the predominant excitotoxin in this stage of neurodevelopment although GABA release may potentially exacerbate damage caused by neuronal swelling (Allen et al. 2004). Consistent with our observations, muscimol-induced cell loss mainly occurs at PN 0–1 and dramatically decreases by PN 7 (J. Nunez, personal communication). An earlier study (Connor and Cormier 2000) in which glutamate stimulation of CA1 neurons produced large Ca2+ transients and secondary responses in distal dendrites might appear to be at variance with the present results. In that study, however, glutamate was applied very locally by iontophoresis from a microelectrode and would have affected few interneurons since their density is low.
Why do our results show greater damage from glutamate exposure in the neurons from young versus the older animals when it has been recognized for many years that very young animals are more resistant to hypoxic or H-I insults than are older animals (cf. Duffy et al. 1975; Fazekas and Alexander 1941)? First, the difference may not be as extreme as is commonly thought because much of the classical work showing a high degree of tolerance in very young mammals was carried out in newborn rodents and recent work has demonstrated appreciable neurogenesis after early cell loss in the hippocampus. Thus a 20-min hypoxia (100% nitrogen, PN 1) produced a 27% cell loss in area CA1 assessed at 7 days post insult, but this loss was compensated by newly generated neurons so that at PN 21, there was no significant difference in neuronal density (Daval et al. 2004). There are, however, a number of studies assessing acute cell loss showing that hypoxia and H-I are better tolerated at the earliest postnatal ages and becomes progressively more severe with maturation (Cherubini et al. 1989; Ferriero et al. 1988; Kass and Lipton 1989). This increase in vulnerability has been ascribed to the strengthening of the glutamatergic transmission system that occurs over this time span, including increased receptor number and increased releasable glutamate. In slice at least there is both increased glutamate release and larger Ca2+ increases during hypoxia (Bickler and Hansen 1998). In isolated neurons, the response to exogenous glutamate is small at PN 1–3 but increases greatly over the first 3 wk of development (Marks et al. 1996). It has also been reported that the sensitivity of the isolated neurons themselves to death after anoxia increases over development (Friedman and Haddad 1993). Therefore our finding of heightened vulnerability to brief exposures to exogenous glutamate in the PN 8–13 day period does not correlate with developmental changes in cell death patterns seen after what may be more severe insults in either in vivo or in isolated cell models, which show increasing vulnerability after this period.
Instead our findings may relate more closely to at least one nonlethal effect of hypoxia, an increased tendency toward epileptiform discharge at later periods of development. In vivo insults that produce this change when administered in the PN 10–13 window are ineffective at earlier or later times (Jensen et al. 1991, 1992). This maximal sensitivity of CA1 neurons to hypoxic conditions at PN 10–13 is also observed in brain slice (Jensen et al. 1998). Although we have pushed stimulus parameters hard enough here to achieve cell death, because it is a clear endpoint, the more relevant observation may simply be the ability to initiate long-lasting Ca2+ increases in the neuronal dendrites at one developmental stage and not the other. These increases might well recover in vivo, or as shown here sometimes in vitro (Fig. 3D). In an earlier study (Connor and Cormier 2000), glutamate stimuli were deliberately titrated to low levels, and it was demonstrated that recovery could occur after Ca2+ increases lasting 
10 min in the slice preparation. It is also noted that in rat, transient global ischemia (10–15 min) generates very large, but transient Ca2+ increases (Silver and Erecinska 1990), but cell death does not occur until 3–4 days later. In slice, OGD of similar duration, kills CA1 neurons within 1–2 h (Lobner and Lipton 1993; Obeidat and Andrew 1998). Thus a given insult in vitro may produce a more severe effect than in vivo.
With regard to the tendency toward post insult epileptiform discharge, there is basis in the literature for increased excitability in neurons after significant increases in dendritic Ca2+ that should favor this type of activity. Short-term effects include depolarization-induced suppression of inhibition, DSI (Lenz and Alger 1999; Morishita and Alger 2001), and facilitation of L-type Ca2+ channels (Hell et al. 1996; Hudmon et al. 2005; Tsubokawa et al. 2000). The secondary Ca2+ response itself is supported by dendritic cation influx, which, if the response is triggered only in small parts of the dendritic tree, produces depolarizations at the soma of a few millivolts (Connor and Cormier 2000). Long-term potentiation protocols that generate Ca2+ increases lasting only several seconds result in the trafficking of AMPA receptors to the plasmalemma (Bredt and Nicoll 2003) resulting in larger excitatory postsynaptic potentials (EPSPs) for extended periods.
Future efforts will be directed toward better defining the electrophysiological consequences nonlethal secondary Ca2+ responses in the PN 7–13 age group to determine whether they are consistent with an increased tendency to epileptiform activity. It will also be of interest to explore a number of other factors that might also be important in controlling vulnerability to glutamate, such as changes in expression of the many types of K channels in CA1 neurons, some of which have a profound effect on dendritic Ca2+ signals (Petrozzino and Connor 1994), expression of non-voltage-gated Ca2+ channels, e.g., trp family members, the loss of gap junction coupling between pyramidal neurons that also occurs over the developmental time period examined here (Azimi-Zonooz et al. 2003), and increased sensitivity of interneurons to glutamate. It is also of importance to investigate why GABAergic inputs on or near the soma are insufficient to bring about the large reduction of the Ca2+ response seen in the dendrites.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. A Connor, Dept. of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM 87131-0001 (E-mail: jconnor{at}unm.edu)
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REFERENCES |
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). The secondary Ca2+ response then spread toward the distal apical dendrites as well as the soma (f and g). The propagation toward distal apical dendrites occurred faster when compared with spread toward soma. The field of focus was switched to soma level as the secondary response propagated toward and invaded the soma (I and j). Dye loss (>90%) consistent with cell loss occurred 
