Chronic NMDA Exposure Accelerates Development of GABAergic Inhibition in the Superior Colliculus

Sandra M. Aamodt, Jian Shi, Matthew T. Colonnese, Wellington Veras, and Martha Constantine-Paton

Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aamodt, Sandra M., Jian Shi, Matthew T. Colonnese, Wellington Veras, and Martha Constantine-Paton. Chronic NMDA Exposure Accelerates Development of GABAergic Inhibition in the Superior Colliculus. J. Neurophysiol. 83: 1580-1591, 2000. Maturation of excitatory synaptic connections depends on the amount and pattern of their activity, and activity can affect development of inhibitory synapses as well. In the superficial visual layers of the superior colliculus (sSC), developmental increases in the effectiveness of gamma -aminobutyric acid (GABAA) receptor-mediated inhibition may be driven by the maturation of visual inputs. In the rat sSC, GABAA receptor currents significantly jump in amplitude between postnatal days 17 and 18 (P17 and P18), approximately when the effects of cortical inputs are first detected in collicular neurons. We manipulated the development of these currents in vivo by implanting a drug-infused slice of the ethylene-vinyl acetate copolymer Elvax over the superior colliculus of P8 rats to chronically release from this plastic low levels of N-methyl-D-aspartate (NMDA). Sham-treated control animals received a similar implant containing only the solvent for NMDA. To examine the effects of this treatment on the development of GABA-mediated neurotransmission, we used whole cell voltage-clamp recording of spontaneous synaptic currents (sPSCs) from sSC neurons in untreated, NMDA-treated, and sham-treated superior colliculus slices ranging in age from 10 to 20 days postnatal. Both amplitude and frequency of sPSCs were studied at holding potentials of +50 mV in the presence and absence of the GABAA receptor antagonist, bicuculline methiodide (BMI). The normal developmental increase in GABAA receptor currents occurred on schedule (P18) in sham-treated sSC, but NMDA treatment caused premature up-regulation (P12). The average sPSCs in early NMDA-treated neurons were significantly larger than in age-matched sham controls or in age-matched, untreated neurons. No differences in average sPSC amplitudes across treatments or ages were present in BMI-insensitive, predominantly glutamatergic synaptic currents of the same neurons. NMDA treatment also significantly increased levels of glutamate decarboxylase (GAD), measured by quantitative western blotting with staining at P13 and P19. Cell counting using the dissector method for MAP 2 and GAD67 at P13 and P19 indicated that the differences in GABAergic transmission were not due to increases in the proportion of inhibitory to excitatory neurons after NMDA treatment. However, chronic treatments begun at P8 with Elvax containing both NMDA and BMI significantly decreased total neuron density at P19 (~15%), suggesting that the NMDA-induced increase in GABAA receptor currents may protect against excitotoxicity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During the final stages of neural development, the temporal and spatial patterning of synaptic activity can regulate neuron number, synapse position, and synaptic strength, thereby assuring that developing circuitry adapts the growing brain to its environment (Constantine-Paton and Cline 1998). In many systems, N-methyl-D-aspartate (NMDA) receptor activation during a sensitive period is required for appropriate synaptic maturation, because NMDA receptor blockade causes topographically incorrect positioning of synapses (Bear et al. 1990; Cline and Constantine-Paton 1989; Hahm et al. 1991; Rabacchi et al. 1992; Schlaggar et al. 1993; Schnupp et al. 1995; Simon et al. 1992).

In theory, appropriate neuronal responsiveness could develop by adjusting excitation and/or inhibition, based on environmental input, to maintain activity levels within an effective range. This balance is critical because small decreases in GABAergic inhibition can lead to seizures (Chagnac-Amitai and Connors 1989; Kriegstein et al. 1987). For technical reasons, most research on activity-dependent synaptic plasticity has focused on excitatory neurons with extrinsic projections. However, inhibitory neural development is also modulated by activity, as shown in dissociated central neurons (Memo et al. 1991; Rutherford et al. 1997; Seil and Drake-Baumann 1994; Zhu et al. 1995) and by sensory deprivation in vivo (Benevento et al. 1995; Hendry and Carder 1992; Micheva and Beaulieu 1997). Nevertheless, in many CNS regions, excitatory inputs develop before hyperpolarizing or shunting GABA-mediated synaptic events are detected (Kirkwood and Bear 1994; LoTurco et al. 1991; Luhmann and Prince 1990). Consequently it is possible that inhibition in vivo is regulated to balance excitation during the early stages of synapse formation.

In this study, we chronically applied low levels of NMDA to the developing superior colliculus beginning at postnatal day 8 (P8), before functional GABAergic inhibition normally appears. The treatment produced early potentiation of GABAA receptor currents and increased glutamate decarboxylase (GAD) protein levels in the superficial visual layers of the superior colliculus (sSC). The up-regulation of GABAA receptor currents occurred 4 days after the onset of treatment and was not associated with an increase in the ratio of inhibitory to excitatory neurons. However, when bicuculline methiodide (BMI) was applied along with NMDA to P8 colliculi, a significant drop in total neuron number was detected at P19. Thus the NMDA-induced increases in GABAA receptor currents appear to protect against some cell loss that could potentially result from tonic activation of NMDA receptors.

Our observations suggest that the immature GABAA receptor system in the sSC in vivo is poised to dampen potentially damaging overexcitation as the visual inputs develop. Such a mechanism could also moderate afferents that, by chance, arrive earlier or in larger numbers than other inputs destined to contribute to sSC function. Similar latent inhibitory circuitry residing throughout the CNS could play a significant role in buffering the normal adaptive pattern of circuit formation against developmental perturbations in the onset, source or amount of excitatory activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and surgery

Timed pregnant Sprague-Dawley female rats were purchased from Camm, and their litters were used for all experiments. The day of birth was counted as postnatal day 0 (P0). Chronic local drug treatment was begun at P8 by surgically implanting a 180-µm-thick slice of the inert ethylene-vinyl acetate copolymer Elvax directly over the superficial layers of the superior colliculus, as previously described (Simon et al. 1992). Briefly, rat pups were anesthetized under a halothane vaporizer. A small incision was made over the sagittal sinus, and a slit was opened in the skull to allow insertion of a slab of Elvax. Three or four sutures were used to close the incision, antibiotic ointment was applied, and pups were returned to the mother after they recovered from anesthesia.

The Elvax contained a final concentration of 100 µM NMDA in water (NMDA treatment). This concentration was estimated to release molecules of the size of NMDA in the range of hundreds of nanomoles/day (Cline and Constantine-Paton 1989, 1990; Simon et al. 1992; Smith et al. 1995). Elvax was also prepared to contain an equivalent volume (20 µl) of water (sham treatment), 500 µM BMI in 20 µl chloroform (bicuculline treatment) or both (NMDA/bicuculline treatment) in 20 µl chloroform.

Electrophysiology

For electrophysiology, NMDA- or sham-treated pups, ages P10 to P20, were anesthetized with ether and killed by decapitation. The diencephalon and midbrain were rapidly dissected and placed in ice-cold artificial cerebral spinal fluid (ACSF) containing (in mM) 117 NaCl, 3 MgCl2, 4 KCl, 3 CaCl2, 1.2 NaHPO4, 26 NaHCO3, and 16 mM glucose, saturated with 95% O2-5% CO2 to a final pH of 7.4. Parasagittal slices of the midbrain were cut at 300-400 µm on a Vibratome (Oxford) and secured in the recording chamber. The slices were maintained at room temperature (22-24°C) and perfused with ACSF at 4 ml/min. Recording began 2 h later, to allow slices to recover from anesthesia and cutting and to assure wash out of chronically applied NMDA.

Recording procedures have been presented previously (Shi et al. 1997). Briefly, borosilicate glass (World Precision Instruments) patch electrodes with tip resistances of 5-10 MOmega were filled with (in mM) 122.5 Cs-gluconate, 17.5 CsCl, 10 HEPES (CsOH), 0.2 NaEGTA, 2 MgATP, 0.3 NaGTP, 8 NaCl, and 0.2% biocytin at pH 7.3. Amplifier and electrode offsets were zeroed in ACSF before obtaining a patch, and recorded voltages were adjusted for a liquid junction potential offset of 10 mV. The GABAA receptor antagonist BMI (2 µM; Sigma) was bath applied. A complete solution change occurred approximately once per minute.

Whole cell recordings were made from neurons in the stratum griseum superficiale or stratum zonale. All cells studied had resting potentials between -45 and -58 mV, seal resistances of 2-2.5 GOmega , and series resistances of <21 MOmega . We rarely attempted to patch and study more than two neurons per collicular slice, particularly in treated animals where each cell was usually held for 1-2 h to study not only GABAA receptor currents but also the effects of treatment on glutamatergic currents (Shi, Aamodt, and Constantine-Paton, unpublished observations). In general, because of the small size of the young colliculus, only 1 or 2 slices (300-400 µm) were obtained from each animal. Signals were recorded using an Axoclamp ID patch-clamp amplifier, filtered at 5 kHz and interfaced (CED 1401 Plus, Cambridge Electronic Design, Cambridge, UK) with a Pentium-based computer (Gateway 2000) that stored the data and provided on-line display of responses and off-line data analysis. CED patch- and voltage-clamp software was used to acquire and analyze data.

This analysis focused on spontaneous synaptic currents (sPSCs) to facilitate comparisons with earlier data and to eliminate complications produced by changes in the effectiveness of evoked synaptic activation that occur over the P10-P20 interval examined, probably as a result of myelination and presynaptic maturation of collicular inputs. Frequencies of sPSCs for each cell were obtained by selecting intervals of at least 70 s (between 300 and 1,000 events) starting at 2-3 min after setting the holding potential and at least 5 min after a solution change. We counted all single fast currents (rise times <9 ms to peak) with amplitudes greater than twice baseline occurring in that interval. When multiple events superimposed, amplitudes of later events were measured only when they fell after the previous current had returned to less than ~20% of peak value. This criterion was chosen to maximize the accuracy of the amplitude measurements while minimizing the number of events excluded from analysis when the frequency of events was high. The same criteria were applied to superimposed currents when estimating frequency. We analyzed only recordings in which the series resistance and input impedance did not change more than 10% over the course of the experiment. Inhibitory postsynaptic currents were analyzed by holding neurons at +50 mV, well above the chloride equilibrium potential, which is approximately -40 mV with these electrode and bath solutions. Large spontaneous outward currents that reversed near -40 mV were evident with this procedure. Application of BMI (2 µM) at +50 mV eliminated these large currents. The remaining smaller events were presumably glutamatergic. In preliminary experiments, they reversed around 0 mV and were eliminated when both 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) were applied. Also in preliminary experiments, the GABAB receptor antagonist baclofen (20 µM, RBI) had no effect on sPSCs in sSC slices, regardless of whether they had been exposed to NMDA.

Morphological analyses of biocytin-filled neurons

To determine the range of cell types and the location of cells analyzed physiologically, we identified cells by labeling them with biocytin in the recording electrode. Slices were fixed in 4% Formalin overnight, and some were cut at 90 µm on dry ice with a sliding microtome, followed by a 4% Formalin postfix before staining with Texas Red-conjugated streptavidin (Jackson Labs). In later experiments, equally good morphology was obtained with unsectioned slices. Morphology was not analyzed in all experiments. However, the slices examined represented an age-balanced sample from sham- and NMDA-treated animals, as well as untreated slices that included some of the slices used in the experiments on BMI-sensitive currents presented here and other untreated slices in the P10-P20 age range. All cells that were recovered (>160 from over 120 different animals) were localized within the superficial layers of the superior colliculus. Only cells with well-filled processes were used for morphological classification. Cells were classified morphologically with epifluorescence on a standard compound microscope. Detailed morphology of a subset of these cells was examined using a BioRad 60 or a BioRad 1024 confocal microscope. Cell type was categorized by an investigator blind to the treatment the animal had received.

Molecular analyses

For biochemical experiments, rats were killed at P13 or P19 by carbon dioxide followed by cervical dislocation, and the superficial layers of the superior colliculus were rapidly dissected. For protein extraction, tissue was homogenized immediately in 20 volumes of buffer (10 mM phosphate buffer, pH 7.0, 5 mM EGTA, 5 mM EDTA, 1 mM DTT and Complete protease inhibitor, Boehringer Mannheim) and then either fractionated or frozen in liquid nitrogen and stored at -80°C until needed. Freezing did not affect results in preliminary experiments. Crude fractionation was done with a modification of the Yip and Kelly (1989) procedure. Rapidly thawed homogenate was centrifuged for 10 min at 4°C at 16,000 × g, and the supernatant (crude soluble fraction) was collected and placed on ice. The pellet was resuspended in 1/4 volume of 2 mM HEPES (pH 7.2) and centrifuged for 10 min at 4°C at 11,000 × g. The supernatant was discarded, and the pellet was resuspended in 0.5 mM HEPES (pH 7.3) containing 0.32 M sucrose and centrifuged for 8 min at 450 × g. The supernatant from this spin (crude particulate fraction) and the crude soluble fraction were placed in Laemmli buffer, heated to 90°C for 5 min, and then frozen in aliquots at -80°C.

Immunoblotting of proteins was done with primary antibodies to GAD, both the 65-kD isoform found mainly at synapses (GAD65, 1:2,000, Boehringer-Mannheim) and the 67-kD isoform found mainly in cell bodies and proximal dendrites (GAD67, 1:4,000, Chemicon) (Kaufman et al. 1991), and to the two GABA transporters found exclusively in the brain (GAT-1, 1:200; GAT-3, 1:1,000, Chemicon) (Ikegaki et al. 1994). Proteins were run on 6 or 8% polyacrylamide minigels at 5 or 10 µg per lane and then transferred to nitrocellulose by electroblotting (Idea Scientific). Total protein was visualized with Ponceau stain. Blots were blocked with 1% dried milk in 0.1% Tween/0.1 M phosphate-buffered saline (TPBS) for 30 min, then incubated in primary antibody in TPBS for 1 h at room temperature. After four 10-min rinses in milk-TPBS, blots were incubated in secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit, 1:12,000 or goat anti-mouse 1:2,000). Blots were washed 6 × 5 min in TPBS, then reacted with chemiluminescent substrate (Pierce) and exposed to film (Kodak).

Band density on the autoradiographs was measured by densitometry with NIH Image 1.57 and its gel-plotting macros. Pixel intensities were calibrated to optical densities with a density wedge. Measurements were confirmed to be within the linear range of the film by analysis of a dilution series processed with the samples. All data are reported as means ± SE of band optical densities.

Analyses of cell density

Pups were injected intraperitoneally with heparin, anesthetized with ether, and perfused transcardially with a brief saline prewash, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. Pup ages were chosen to coincide with those used for western blotting, and any colliculi with misplaced Elvax or visible necrosis to a sixth or more of one colliculus were eliminated from further analysis. After postfixing overnight, brains were cryoprotected in 30% sucrose PBS, and 30-µm coronal sections were cut on a vibratome. Thirty micrometers was chosen for section thickness after several pilot studies showed reproducible antibody penetration throughout sections of this thickness at P13 and P19. Beginning 90 µm from the rostral edge of the SC, every other pair (i.e., 10, 11, 14, 15...) of free-floating sections was processed for immunohistochemistry with primary antibodies against GAD67 (1:3,000 in 4% horse serum PBS, Chemicon) to label GABAergic cell bodies, or against microtubule-associated protein-2 (MAP-2, 1:2,000 in 4% horse serum, Boehringer Mannheim) to label all neurons. After blocking in 4% horse serum, sections were incubated for 20 h at 4°C in primary antibody, washed, incubated for 1 h in biotinylated secondary antibody, and washed again. They were subsequently incubated in peroxidase streptavidin for 1 h and developed with 0.06% DAB HCl and 0.006% H2O2 in PBS for 5 min (MAP-2) or 15 min (GAD67). Sections were mounted, dehydrated, coverslipped, and coded so that the analyses could be conducted blind to treatment conditions. Neurons were counted on a Leitz microscope with 100 × 1.30 NA oil objective using the optical dissector method (Gundersen 1986). We counted six fields (65 µm square for P13, 75 µm ×100 µm for P19), consistently spaced across the mediolateral extent of the sSC, on each section. Control sections in which the primary antibody was not added showed only very light DAB staining within the neuropil.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrophysiology

The functional effects of chronic NMDA treatment were studied by whole cell patch-clamp recording. We have previously shown that the frequency of spontaneous excitatory postsynaptic currents (EPSCs) is high in the young postnatal rat sSC and that the development of mature GABAergic inhibition is associated with an abrupt reduction in sEPSC frequency after P17, which is sensitive to bath-applied BMI (Shi et al. 1997). GABA is the dominant inhibitory transmitter in the mature sSC, and all GABAergic contacts in these visual layers derive from intrinsic GABAergic interneurons (Mize 1992).

To directly examine the fast synaptic currents through GABAA receptors, we recorded postsynaptic currents in the sSC at a holding potential of +50 mV in the presence of 2 mM Mg2+ in normal, untreated sSC slices (n = 41 neurons from 21 animals) and in sSC slices after in vivo NMDA (n = 38 neurons from 21 animals) or sham treatment (n = 44 neurons from 22 animals).

Recordings were obtained between P10 and P20. However, it is likely that a residual effect of surgery affected most recordings made shortly after Elvax implantation. Neurons in both sham- and NMDA-treated animals were difficult to record from on P10 and had somewhat smaller GABA current amplitudes compared with untreated animals on P10 and P11.

When held at +50 mV, sSC neurons P18 and older had spontaneous currents composed of two types of outward current: larger-amplitude, BMI-sensitive currents that reversed around -40 mV, and smaller-amplitude, BMI-insensitive currents that reversed around 0 mV. The latter population was sensitive to the glutamate receptor antagonists CNQX and AP5 and was detected in neurons from slices of all ages (P10-P20) and treatment groups. The two populations of currents can be seen in Fig. 1 in the traces from P19 neurons from all treatment groups. At P12, BMI-sensitive currents were relatively small in both untreated and sham-treated neurons. Neurons in slices from NMDA-treated colliculi, however, showed large, BMI-sensitive currents as early as P12.



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Fig. 1. Representative traces recorded in postnatal day 12 (P12) and P19 neurons held at +50 mV from untreated, sham-treated, and N-methyl-D-aspartate (NMDA)-treated colliculi, showing the effects of the GABAA receptor antagonist bicuculline methiodide (BMI). At this holding potential, the neuronal membrane potential is ~90 mV away from the chloride equilibrium potential. Large-amplitude, BMI-sensitive spontaneous postsynaptic currents (sPSCs) are apparent in all treatment groups at P19. However, at P12, the amplitude of BMI-sensitive activity is much higher in NMDA-treated neurons than in sham- or untreated neurons.

Changes in BMI-sensitive and BMI-insensitive currents across age and treatment groups were examined quantitatively by measuring the amplitudes (Fig. 2) and frequencies (Fig. 3) of the sPSCs (see METHODS for sPSC selection criteria) for each neuron without BMI. BMI was subsequently applied to each slice, and the amplitudes and the frequencies of the remaining events were measured 6-7 min later. Only neurons for which stable recording conditions were maintained over the course of the experiments were included in this analysis. Data for BMI-insensitive currents were not included if large outward currents were not recovered on wash out of BMI.



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Fig. 2. Quantitative analysis of the amplitudes of sPSCs like those shown in Fig. 1. Left: scatterplots of the average amplitudes of sPSCs in individual neurons of the superficial visual layer of the superior colliculus (sSC) against postnatal age in untreated (A), sham-treated (C), and NMDA-treated (E) tissue. Right: (B, D, and F) averages (±SE) of the data for the same neurons grouped in 2-day bins across the P10-P20 interval. For all data, filled symbols represent sPSCs that include GABAA receptor chloride currents, whereas open symbols represent data taken from the same neurons in the presence of 2 µM BMI to reveal the developmental pattern of the glutamatergic currents. Glutamatergic currents are generally smaller than the GABAA receptor currents in these data because the neurons were voltage clamped ~50 mV above the glutamatergic channel reversal potentials of ~0 mV but ~90 mV above the chloride reversal potential. Both untreated and sham-treated neurons show a significant up-regulation of the GABAA receptor-mediated potentials between P17 and P18. In NMDA-treated neurons, this up-regulation occurs at P12 and remains high. See text for the statistical analyses of these data.



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Fig. 3. Quantitative analysis of the frequencies of sPSCs like those shown in Fig. 1. Data are from the same neurons for which sPSC amplitude data are shown in Fig. 2. There were no statistical differences across treatment groups in these data except for the P18-P19 age interval in the presence of BMI, when frequencies in sham-treated tissue were significantly elevated from untreated tissue (B vs. A).

The scatterplots in Fig. 2, A, C, and E show the average sPSC amplitudes for each of the neurons studied both in the absence and in the presence of BMI. The amplitude and frequency data from these cells was averaged across 2-day intervals, and an ANOVA (mANOVA) was used to examine the effects of age and drug treatment on these averages. Tukey post hoc tests were used to make pair-wise comparisons between ages within treatments and between separate treatment groups at each age. Comparisons were considered significant at P < 0.05.

The two-way ANOVA for average sPSC amplitudes without BMI showed significant differences across both treatments (df = 2; F ratio = 21.5; P < 0.001) and ages (df = 5; F ratio = 16.0; P < 0.001). In normal animals, the amplitudes of sSC neuron sPSCs (Fig. 2, A and B) and their frequencies (Fig. 3A) were relatively low from P10-P17 and then rose significantly between P16-P17 and P18-P19. However, the size and frequency of the BMI-insensitive currents did not change between P10 and P20. These findings are consistent with our earlier study (Shi et al. 1997), which suggested that GABAA receptor currents begin to exert pronounced inhibitory effects in sSC slices only after P17.

The amplitudes of sPSCs from sham-treated sSC were somewhat decreased throughout, although this difference between sham and untreated reached significance only in the P16-P19 age range. Despite this depression in current amplitude, the pattern of sPSC amplitude development in sham-treated sSC neurons was similar to that found in normal neurons (Fig. 2, B vs. D). As in untreated neurons in the absence of BMI, sham sPSC amplitudes were generally low and did not change between P12 and P17, but these amplitudes increased abruptly and significantly at P18-P20. This amplitude increase was not found when GABAA receptor currents were blocked with BMI, indicating that it reflected a selective increase in these events.

The developmental patterning of sPSC amplitudes was different after NMDA treatment, however (Fig. 2, F vs. D). Large-amplitude sPSCs appeared in NMDA-treated neurons at P12, and they maintained this large size throughout the period studied. These amplitudes were significantly higher than in sham-treated neurons in the P12-P13 and the P16-P17 interval and significantly higher than in untreated neurons in the P12-P13 interval (Tukey post hoc test, P = 0.005). As before, the size of the BMI-insensitive currents remained unchanged throughout the period studied.

Frequencies of the spontaneous currents at +50 mV were also compared among untreated, sham- and NMDA-treated neurons (Fig. 3). The two-way ANOVA over all ages and treatments without BMI showed no significant differences between treatment groups (df = 2; F ratio = 2.8; P = 0.067) but some difference between ages (df = 5; F ratio = 12.2; P < 0.001) within treatments. There was a tendency for frequencies to increase in all older slices. Frequencies of sPSCs were significantly higher in the P18-P20 range than in the P10-P17 range in untreated neurons. In sham-treated neurons, sPSC frequency was significantly different at P20 from the frequencies in the P10-P17 age range. In NMDA-treated slices, the rise in sPSC frequency appeared slightly delayed. There were no significant differences in frequency in the P10-P19 range, but frequencies were significantly increased at P20.

For the sPSC frequency data obtained with BMI, which predominantly reflect glutamatergic currents, the ANOVA revealed significant differences both between treatments and across ages within treatment. Thus there was a tendency for sPSC frequency to increase in older neurons in both Elvax-treated groups. However, this was significant relative to normal neurons only for sham-treated neurons in the P18-P19 age interval. Within treatments, normal neurons showed no frequency changes across all ages. However, sham-treated neurons showed a significant frequency increase between the early (P12-P17) and the late (P18-P19) interval, and NMDA-treated neurons did not show a significant frequency increase until P20.

Morphology of biocytin-filled cells

Cells in sham-treated and NMDA-treated colliculi were categorized according to cell body shape and the organization and orientation of dendritic branches, using previous Golgi descriptions of neurons in both developing (Labriola and Laemle 1977) and mature (Langer and Lund 1974) rat sSC. Figure 4 contains confocal z-series projections illustrating this classification with typical biocytin-filled, narrow-field vertical neurons, distinguished by their pyramidal cell body and narrowly arborizing, dorsally directed dendrites, and typical horizontal neurons, distinguished by their large, laterally extending principal dendrites and horizontally oriented elliptical cell bodies. These relatively broad morphological characteristics are distinct in both sham- and NMDA-treated colliculi. Only a few cells failed to meet the criteria of a particular cell class, and these were classified as ambiguous. Complexity or size of dendritic or axon arbors were not measured because these characteristics varied with the efficiency of biocytin filling. However, no qualitative differences across treatment groups were apparent in any of these parameters or in the size of somata within any cell class. Furthermore, there was no correlation between any particular cell type and unusually large or small BMI-sensitive currents.



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Fig. 4. Confocal images of the morphologies of sSC neurons in sham- and NMDA-treated sSC. Neurons were reacted with Texas Red-conjugated streptavidin for biocytin infused during whole cell recordings. Two types of neurons, narrow-field vertical cells (top), and horizontal neurons (bottom) are illustrated. Narrow-field vertical cells were the most common sSC neuronal type encountered in the patch-clamp studies across all treatment groups. Micrographs illustrate the invariant characteristics of their morphology: basal dendrites and narrowly arborizing, apical dendrites. Horizontal neurons are a GABAergic neuronal type in the sSC. These are characterized by pronounced laterally organized dendrites and an elliptical, horizontally oriented soma.

Five of the six major cell categories described in the superficial layers of the colliculus were sampled with our blind whole cell patch-clamping technique (Table 1), the exception being a small inwardly oriented cell, the marginal cell, located in the immediate subpial region. A few cells of this morphology were observed in several slices, but they were classified as ambiguous because they did not appear to have a subpial position. The proportions of the remaining five cell classes differed between untreated, sham-treated and NMDA-treated slices (Pearson chi 2 = 38.88, df = 8), and most of this difference was due to changes in two cell classes. First, wide-field vertical neurons were much more heavily represented in untreated colliculus slices than in either Elvax-treated group. It seems likely, however, that this difference is related to age, not treatment. The excitatory wide-field vertical neurons (Mize et al. 1982) are the largest cells in the sSC, and a retrospective analysis of the age distribution of this cell type indicated that, across all treatment groups, they were more frequently encountered in younger tissue (P14 and below). The blind whole cell patching technique is likely to be biased toward these larger cells, particularly in young slices. The soma size of all sSC neurons increases with age (Labriola and Laemle 1977), which would reduce this bias in older tissue. We analyzed a larger number of cells from younger slices in untreated animals compared with either experimentally treated group (61% between P10-P13 for untreated as compared with 50 and 46% from NMDA- and sham-treated neurons, respectively), which may account for the overrepresentation of wide-field vertical cells in untreated tissue.


                              
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Table 1. Summary of biocytin-filled neurons

The second large difference in cell type frequency was observed in horizontal cells. This morphologically distinctive, superficially located class is one of the major GABAergic cell types in the sSC (Mize 1992; Sterling and Davis 1980). This cell type, represented by 16% of the neurons from NMDA-treated sSC, was less prevalant in untreated (2%) and sham-treated (10%) populations. Horizontal cells have been described in Golgi studies of the sSC as early as the first week (Labriola and Laemle 1977). However, they receive most of their inputs from the cerebral cortex (Mize et al. 1982), whose inhibitory GABAA receptor-mediated effects on collicular neuron receptive fields (Binns and Salt 1997b) are first observed around P18 in the rat (Binns and Salt 1997a). It is tempting to speculate that activation of horizontal cells may accelerate their differentiation and thus make them easier to patch, because eight of the nine NMDA-treated horizontal neurons were from slices P16 or younger. Only two of the five horizontal neurons in sham-treated tissue were found in this young age range, and the only horizontal neuron found in untreated tissue was in a P20 slice. Unfortunately, these numbers are small and complicated by the largely unknown determinants of stable whole cell patch clamping in young tissue. Thus any convincing statement concerning the early NMDA-dependent maturation of a particular inhibitory cell type will require considerably more study.

Biochemistry and immunocytochemistry

Levels of both GAD isoforms (GAD67 and GAD65) and the two neuronal GABA transporters (GAT-1 and GAT-3) rise dramatically during the second postnatal week in normal sSC, providing a biochemical index of GABA differentiation that corresponds closely to the increased effectiveness of BMI in normal neuropil at this age (Shi et al. 1997). Thus we compared levels of both GAD isoforms and GABA transporters between sham- and NMDA-treated tissue by normalizing protein levels in each group to normal age-matched tissue measured from the same gels. This comparison was unambiguous at P19, when relatively high levels of GAD were present in tissues from all treatment groups. Despite our inability to detect differences in GABAA receptor currents between treatment groups in the P18-P19 interval, both GAD isoforms were significantly elevated in NMDA-treated tissue over sham-treated tissue at P19 (P = 0.02 for GAD65, n = 3 animals, 9 immunoblots; P = 0.03 for GAD67, n = 3 animals, 3 immunoblots; paired Student's t-test). No significant difference between NMDA- and sham-treated tissue was found in the levels of the two GABA transporters, GAT-1 and GAT- 3, at this age (Fig. 5). In P13 sSC, when significant physiological changes between treatment groups were observed, quantitative biochemical comparisons were difficult because of the low levels of all the GABA-associated proteins that we measured. Thus GABA transporters could not be examined at P13, and no significant differences in levels of the GAD67 isoform were detectable at this age between NMDA- and sham-treated sSC (P = 0.2, paired Student's t-test, n = 2 sham- and 2 NMDA-treated animals, 3 immunoblots), possibly because of the relatively high levels of background staining. Nevertheless, GAD65 levels in NMDA-treated sSC were increased compared with sham-treated sSC (P = 0.04, paired Student's t-test, n = 5 sham-treated and 5 NMDA-treated animals, 11 immunoblots) at this age.



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Fig. 5. Quantitative immunoblotting of glutamate decarboxylase (GAD) and GABA transporter (GAT) proteins from superficial visual layers of the superior colliculus at P13 or P19 after NMDA (hatched) or sham (black) treatment begun at P8. A: at P13, NMDA treatment increased protein levels for the 65-kD isoform of GAD over sham-treated levels (P = 0.04), but had no significant effect on the 67-kD isoform. B: at P19, NMDA treatment significantly increased protein levels for both GAD isoforms (GAD65, P = 0.02; GAD67, P = 0.03). C: however, this NMDA treatment did not affect levels of GABA transporter-1 or -3 at P19. Averaged optical density measurements are shown. All averages have been normalized to protein levels in untreated tissue of the same age and run on the same gel to facilitate comparison between films. Note that levels of the GAD67 isoform are depressed relative to untreated tissue at both P13 (A) and P19 (B). This difference was significant using a paired sample Student's t statistic on the optical density measurements for sham and untreated sSC from the same gel. Error bars are ±SE.

Biochemical analysis also suggested that levels of the GAD67 enzyme were lower in sham-treated as compared with untreated sSC at both ages examined. The GAD67 levels in sham-treated tissue relative to untreated lanes of the same gels produced values less than one (Fig. 5). Optical density measurements for sham and normal tissue run on the same gel and taken at P13 and P19 were significantly different when specifically tested for such a decrease using a paired-sample Student's t statistic. The decrease in GAD67 level is consistent with the tendency toward decreased GABA function in sham-treated versus untreated sSC that we detected in the physiological analyses (Fig. 2, D and B). Levels of the other isoform, GAD65, were not consistently affected. The stronger effect of sham treatment on GAD67 over GAD65 may reflect differences in the control of the two enzymes by activity. GAD67 is presumed to represent most of the constitutively active GAD in the brain because it is almost entirely saturated with the activating cofactor pyridoxal phosphate (PLP), whereas the GAD65 enzyme is more immediately controlled by activity through transient associations with PLP (Erlander et al. 1991). Thus tonic decreases in GABA synaptic activity might be expected to involve decreases in levels of the GAD67 enzyme, whereas the need to change levels of GAD65 could be minimized by changes in cofactor association.



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Fig. 6. Photomicrographs of adjacent coronal section pairs cut from P19 rat sSC and immunohistochemically stained for GAD67 (1st and 3rd column) or MAP-2 (2nd and 4th column). Chronic exposure to Elvax alone (sham, 1st row), Elvax with NMDA (2nd row), Elvax with BMI (not shown), or Elvax with NMDA and BMI (3rd row) was begun at P8. All sections are from the crown of the colliculus. In the low-power micrographs, all 3 sSC layers [stratum zonale (SZ), stratum griseum superficale (SGS), and stratum opticum (SO)] are visible. The high-power micrographs are from the SGS (pial surface is up), where dissector counts were taken at about this magnification. Scale bars are 50 and 20 µm, respectively.

Cell density analyses

Cell densities were estimated in sham- and drug-treated sSC to answer two questions: did the increase in GABAA receptor currents in NMDA-treated tissue reflect a differential loss of excitatory neurons relative to GABAergic neurons, and could the increase in GABAA receptor currents represent an early onset of inhibition that could protect the neurons of the young sSC from over-excitation? It was important to address the latter question, because some early postnatal GABAergic currents are excitatory (Ben-Ari et al. 1997), and voltage-clamp recordings do not permit identification of chloride currents as either depolarizing or hyperpolarizing.

For cell counting, anti-MAP-2 staining was used to identify all neurons, and GAD immunohistochemistry, as a marker for GABAergic neurons (Houser et al. 1983), was used on adjacent sections. We stained for GAD67, which is primarily localized to cell bodies (Hendrickson et al. 1994), instead of the predominantly synaptic GAD65 in this analysis because the dense local GABAergic innervation in the sSC (Mize 1992; Okada 1992) resulted in high levels of neuropil staining that obscured cell bodies.

Immunohistochemistry for both GAD67 and MAP-2 was qualitatively similar in all treatment conditions, and no histological or cytoarchitectonic differences were observed (Fig. 6). Although light staining of the entire sSC neuropil was present with the GAD67 antibody, GAD-positive somata were easily identified by granular deposits in the cytoplasm (columns 1 and 3, Fig. 6). MAP-2 staining of somata and processes was always pronounced, with no visible background (columns 2 and 4). Neuronal cell bodies in the center of each 30-µm section were discernible with both antibodies. However, unambiguous identification of particular cell types was not possible because of the thinness of the section.

The optical dissector method (Gundersen 1986) was chosen to quantify density, because it is robust to changes in cell size. At P13, when there were pronounced differences in GABAergic function between NMDA- and sham-treated littermates, we found no differences between their cell densities (Fig. 7). Both the total neuron density and the ratio of GABAergic neuron density to total neuron density were the same in both treatment groups (sham, n = 4 animals; NMDA, n = 3 animals).



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Fig. 7. GAD67- and MAP-2-positive cell body densities as determined by optical dissector counting at P13. We observed no significant differences in cell densities between sham- and NMDA-treated groups at this age, when the functional synaptic differences are most pronounced. Error bars are ±SE.

Next, in P19 tissue, we determined whether the early increases in GABAergic currents detected physiologically could be facilitating cell survival in the continuous presence of a glutamate analogue. P19 was chosen to allow a longer interval for treatment-induced changes to appear and because, at this age, sSC neurons were well past their normal period of neuron death, which ends at P11 (Arees and Astrom 1977; Giordano et al. 1980). For this analysis, in addition to the sham- and NMDA-treated groups, the densities of neurons positive for GAD67 and MAP-2 were also examined in sSC where GABAA receptor-mediated responses were chronically antagonized with BMI. Nine litters were each divided into two or three of the four treatment conditions: sham, BMI, NMDA, or NMDA with BMI. Neurons positive for GAD67 and MAP-2 were counted in the sSC of 17 sham-treated, 17 NMDA-treated, 6 BMI-treated, and 7 BMI- and NMDA-treated pups.

Cell densities at P19 were lower than the cell densities at P13 in all treatment groups, probably because of an overall expansion of the sSC neuropil due to axonal, dendritic, and glial maturation. When NMDA and BMI were applied simultaneously, sSC neuron density was reduced by 15% compared with sham, BMI, or NMDA treatment (mANOVA, dF = 3, F ratio = 13.6, P < 0.001 by Tukey post hoc analysis), suggesting that the increases in GABAergic currents detected after NMDA treatment may act to mitigate any increased excitation and therefore protect young sSC neurons from excitotoxic cell death (Choi 1992). In addition, GABAergic neuron density in the NMDA-treated group and in the group treated with both BMI and NMDA was decreased slightly relative to sham-treated sSC (mANOVA, dF =3, F ratio = 5.6, P < 0.05 by Tukey post hoc analysis; Fig. 8). The latter effect suggests a greater sensitivity of the sSC GABAergic interneurons to early over-activation, similar to results in developing hippocampus (Geary et al. 1996; Houser and Esclapez 1996). Nevertheless, decreases in GABAergic neuron number are in the wrong direction and occur at the wrong age (P19) to account for the early (P13) up-regulation of GABAA receptor currents that we observed.



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Fig. 8. GAD67- and MAP-2-positive cell body densities as determined by optical dissector counting at P19 for 4 different treatment groups: sham, BMI, NMDA, and NMDA plus BMI. Double asterisks denote a P value below 0.01 when compared with sham using the Tukey HSD multiple pair-wise comparison, whereas single asterisks denote a P value below 0.05 for the same comparison. Error bars are ±SE. Total cell density is decreased when NMDA treatment is combined with BMI treatment, indicating that the GABAA receptor currents in NMDA-treated sSC are protective. The decreased densities of GABA neurons in both the NMDA- and NMDA- plus BMI-treated group suggest that the GABA neurons may be more sensitive to cell death from chronic NMDA exposure than are the non-GABAergic neurons.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the superficial layers of the superior colliculus, all GABA-mediated inhibition is generated by local interneurons (Mize 1992), and most sSC neurons respond to optic tract stimulation with short-latency excitation followed by GABAA receptor-mediated shunting inhibition. In addition, GABAA receptor-mediated surround inhibition, driven largely by descending input from visual cortex, shapes response properties of these neurons (Binns and Salt 1997b). In a previous study, we found that GABAA receptor-mediated inhibition of excitatory activity within sSC slices appears abruptly at P18 in rats, ~4 days after the eyes open. This timing corresponds closely to the refinement of descending cortical projections (Thong and Dreher 1986) and the onset of visual cortical effects on individual collicular neuron response properties in vivo (Binns and Salt 1997a), suggesting that the differentiation of these inhibitory inputs might be driven by visual cortical activity.

To test the hypothesis that glutamatergic activation of GABAergic interneurons facilitates the differentiation of GABAA receptor-mediated responses in this neuropil, we studied the development of BMI-sensitive currents and GAD expression in the sSC in normal tissue and in tissue treated chronically with low levels of NMDA. The present voltage-clamp analysis of synaptic currents in both normal and sham-treated sSC neurons supports our earlier observation (Shi et al. 1997) of a rapid up-regulation of GABAA receptor current amplitudes between P17 and P18. This up-regulation is not observed in the presence of BMI and is therefore selective for GABAA receptor responses. Furthermore, when sSC neurons were continuously exposed to low levels of NMDA starting at P8, this rapid up-regulation of GABAA receptor currents occurred at P12, 4 days after the initiation of NMDA treatment, and significantly earlier than in normal or sham-treated neurons (Fig. 2). Although it may be coincidental that 4 days elapse between eye-opening and the abrupt increase in inhibition in untreated animals, and between NMDA treatment and the early onset of inhibition in experimental animals, these data do suggest that NMDA receptor activity may lead to GABAergic synaptic maturation in this system. It remains to be determined whether the effect is a direct result of NMDA receptor activation on the GABAergic neurons themselves or an indirect effect of activating other neurons in the sSC, as has been suggested for activity-dependent changes in cortical GABAergic neurons in culture (Rutherford et al. 1997, 1998).

The frequency of synaptic events studied at +50 mV was relatively unchanged by chronic NMDA treatment. During the P10-P20 interval studied, sPSCs frequencies in all three groups increased at, or shortly after, P18-P19. This late frequency increase was observed in the predominantly glutamatergic responses remaining after BMI application from both NMDA- and sham-treated neurons. The late frequency increase was not observed in the BMI-insensitive currents from untreated neurons. These late differences in frequency were small, however, and differences across treatments were only statistically significant between the sham and untreated neurons in the P18-P19 interval (Fig. 3).

In many regions of the mature and developing CNS, changes in GAD levels seem to reflect changes in the amount of activity carried by the particular pathway (Benevento et al. 1995; Hendry and Carder 1992; Hendry and Jones 1988; Micheva and Beaulieu 1997). Our initial study of synaptic differentiation in the sSC suggested a close correlation between developmental increases in GAD65, GAD67, GAT-1, and GAT-3 protein levels and the increased effectiveness of GABAA receptor-mediated inhibition (Shi et al. 1997). Thus we measured these biochemical indices of GABAergic differentiation in sham- and NMDA-treated sSC to determine whether they changed in association with the functional changes in inhibition after NMDA treatment. The present data suggest that the relationship may be more complicated. At P13, when the amplitudes of GABAA receptor currents were significantly different between NMDA-treated and control animals, there were significant increases in GAD65 levels in NMDA-treated sSC, but no significant changes in GAD67 levels. This lack of effect may result from high background staining with the anti-GAD67 antibody and relatively low levels of GAD67 protein at this age, but these concerns do not apply to the P19 data. At P19, when no significant differences in the GABAA receptor currents were apparent among the groups, immunoblotting showed significantly increased levels of both GAD isoforms in NMDA- as compared with sham-treated tissue (Fig. 5).

These observations suggest that parameters other than levels of the GABA synthetic proteins are important in determining the efficacy of GABAergic synapses under conditions of chronic low-level exposure to NMDA. The most likely candidates for such an effect are the GABAA receptors themselves and/or changes in the efficacy of synaptic release. Indeed a number of investigators have documented subunit changes in GABAA receptors during development (Fritschy et al. 1994; Oh et al. 1995), and some GABAA receptor changes are correlated with activity changes early in development (Harris et al. 1995; Kumar and Schliehs 1993). In adults, the potentiation of hippocampal inhibitory synapses during the increased activity associated with kindling involves increased GABAA receptor number (Nusser et al. 1998). Furthermore pre- and postsynaptic GABAergic up-regulation following chronically increased activation have been observed in tissue culture (Memo et al. 1991; Ramakers et al. 1994; Turrigiano et al. 1998; Zhu et al. 1995).

The tendency toward depression of GABAA receptor current amplitude in sham-treated tissue relative to normal (which only reached physiological significance at P18-P19) was paralleled in the biochemical data at both P13 and P19 by a decrease in GAD67 levels (Fig. 5). This was surprising, given the loose association between increased GAD levels and increased synaptic current in our data, but decreases in activity have been correlated with decreased GAD expression by previous investigators (Bear et al. 1985; Benevento et al. 1995; Hendry and Carder 1992; Micheau and Beaulieu 1997). Elvax may produce a pressure blockade of some axons in the young sSC and thus mildly decrease the overall level of activity relative to untreated animals. Evidence consistent with a similar sham effect from Elvax has been previously reported in frog tecta (Hickmott and Constantine-Paton 1997).

When NMDA and BMI were applied simultaneously, sSC neuron density was reduced by 15% compared with sham, BMI, or NMDA treatment. Decreases in cell density with NMDA treatment alone were not observed at P13 (Fig. 7), and at P19 (Fig. 8) they were small and restricted to the GAD-positive neuron population, indicating that the physiologically and biochemically observed increases in GABAergic indexes were not due to an increased proportion of inhibitory to excitatory neurons. In addition, simultaneous exposure to BMI and NMDA did not appear to exacerbate the small density decreases in the GAD-positive population. These observations suggest that the observed GABA currents are associated with a protective effect on young collicular neurons and are therefore likely to shunt or counteract any excitation that is produced by the continuous presence of a glutamate analogue. They also suggest that any protective effects of the early increases in GABA receptor currents are not exerted on the GABA neurons themselves.

Interpretation of the results of chronic treatment of neuropil with neurotransmitter receptor agonists and antagonists can clearly be complicated by the compensatory adjustments of multiple neurotransmitter receptors. It is also difficult to extrapolate the actual level of excitation in intact, chronically treated neuropil from assays of transmitter alterations in a brain slice. Nevertheless, a parallel study of the development of glutamatergic currents in sSC neuropil similarly treated with NMDA suggests that NMDA receptor currents remain exceptionally potent in this tissue, whereas the development of non-NMDA ionotropic currents is retarded. These glutamatergic receptor changes reduce spontaneous activity in slices from NMDA-treated tissue whenever NMDA channels are blocked (Shi et al., unpublished observations). Presumably this condition helps to counteract increased activity in collicular neurons caused by the low levels of NMDA continuously present in the treated animals, and these compensations could account for the relatively small amount of cell loss observed under any of the treatment conditions.

Conclusions

In normal rat sSC development, the onset of pronounced GABAA receptor-mediated inhibition occurs 4 days after eye opening. In this interval, pattern vision helps to organize collicular circuitry (Binns and Salt 1997a), and increased synaptic activity is observed in the sSC (Shi et al. 1997). The establishment of descending inhibition from the cortex onto sSC neurons at this time may therefore serve an important homeostatic function in addition to its role in patterning collicular neuron responses. Our findings indicate that a mechanism previously defined in tissue culture exists in vivo, namely that GABAergic synapses become more potent when excitation is experimentally increased. During normal development of many CNS regions, as in the sSC, inhibition generally appears after excitation (Kirkwood and Bear 1994; LoTurco et al. 1991; Luhman and Prince 1990) and could be similarly facilitated by the increasing effectiveness of excitatory transmission. Thus maturation of GABAA receptor-mediated inhibition by excitation may be an important developmental mechanism for assuring that young, and highly effective, glutamatergic currents are susceptible to tight control even before inhibitory circuitry normally appears.


    ACKNOWLEDGMENTS

The authors thank Dr. Sandra Hill-Felberg, K. Lee, and S. Gian for assistance in the anatomic preparation and analysis of biocytin-filled neurons.

This work was supported by National Institutes of Health Grants NS-32290 and EY-06039 to M. Constantine-Paton and NS-09569 and MH-11535 to S. M. Aamodt.

Present address of M. Constantine-Paton: Dept. of Biology, Massachusetts Institute of Technology, 68-380, 77 Mass Ave., Cambridge, Massachusetts 02139.


    FOOTNOTES

Address for reprint requests: M. Constantine-Paton, Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Bldg. 68-380, Cambridge, MA 02139.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 June 1999; accepted in final form 12 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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