Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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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 -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.
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INTRODUCTION |
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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.
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METHODS |
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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 M
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 G
,
and series resistances of <21 M
. 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.
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RESULTS |
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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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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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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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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
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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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