«Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory ...»
To analyze STP of GABAA PSCs neurons were voltage-clamped at −50 or −60 mV, and only outward currents were used. Trains of five pulses at 10 and 20 Hz were evoked for each neuron, and five sweeps were presented at 0.1 Hz. The sweeps were averaged in Clampfit, and amplitudes were measured for each pulse from the 1 ms prior to the stimulation artifact to the absolute peak deflection. The ratios of the amplitudes of pulse 2 and pulse 5 to pulse 1 were calculated at both frequencies and used for statistical analyses.
4.3.7 Picospritzer response.
The exogenous GABAA receptor agonist muscimol (Sigma-Aldrich) was focally applied to patch-clamped neurons with a picospritzer (Parker Hannifin, Cleveland, OH). After patching, the tip of a second pipette, identical to microelectrodes for patch clamping and filled with a solution of 100 μM muscimol dissolved in regular ACSF, was brought within 15 μm of the soma of the patched neuron. Five responses (0.1 Hz) to 5-ms puffs of muscimol at 5 PSI were recorded in voltage clamp at −50, −60, and −70 mV. The majority of responses were measured in the presence of bath-applied tetrodotoxin (TTX, 1 μM; Tocris), but there was no discernible difference with or without the drug, so data were pooled. Decay time constant was measured from the average response with a single-exponential fit in Clampfit. The reversal potential of the picospritzer response was estimated from the peak of the mean response at each recording voltage by interpolation. The peak conductance was calculated for, and averaged across, the response at each voltage, excluding any recordings made within 5 mV of the estimated reversal potential.
4.3.8 Single-cell and whole tissue RT-PCR.
To perform single-cell RT-PCR, at the end of the patch-clamp recording session the cell cytoplasm was aspirated into the patch recording pipette by applying gentle negative pressure under visual control. Pipettes contained ∼5 μl of RNase-free patch solution. The contents of the patch pipette were expelled into a microcentrifuge tube containing 5 μl of the reverse transcription cocktail (Applied Biosystems, Foster City, CA). The reverse transcription product was amplified in triplicate and screened for 18S rRNA. Only those cell samples positive for 18S rRNA were subjected to amplification with primers. The procedure used to determine mRNA transcript expression in single cells has been described in detail previously (Hazra et al., 2011).
The sequences for the oligonucleotide primers are detailed in Table 4.1. PCR products were visualized by staining with ethidium bromide and separated by electrophoresis in a 1% agarose gel. RT-PCR was also performed on whole BLA of P7 rats, from tissue isolated by microdissection from 300-μm-thick slices. The slices were made with the protocol for electrophysiological recording described above, and RNA isolation and RT-PCR were performed as described previously (Hazra et al., 2011).
All statistical analyses were performed with Prism 4 (GraphPad, LaJolla, CA). All tests for significant effects of age were performed with one-way ANOVAs, except for the effects of age on STP, which was tested with a two-way ANOVA with repeated measures, with the second factor being stimulation frequency. For all ANOVAs and posttests, significance was defined at α = 0.05. To perform pairwise comparisons following significant main effects in ANOVA, Tukey's posttests were generally used. The only exception was the two-way ANOVA, which was followed by Bonferroni posttests comparing all pairs of group means. For the estimation of GABAA reversal potential, one data point in the P7 group was more than 2 standard deviations from the group mean and was therefore excluded from analysis. To perform a one-way ANOVA on the peak conductance of IPSCs, data were log-transformed to correct for heteroscedasticity assessed with Bartlett's test.
4.4 Results 4.4.1 Compound synaptic response to local electrical stimulation.
In total, we made whole cell patch-clamp recordings from 170 BLA principal neurons from 62 rats of ages ranging from P7 to P36. We first investigated whether the response of the local BLA network to electrical stimulation changed during the first postnatal month (Figure 4.2). Here, we characterized the stimulation response in current clamp at three different baseline membrane potentials and found three distinct components (Figure 4.2A). The first, a fast excitatory component, was depolarizing at all ages and was blocked by the AMPA/kainate receptor antagonist DNQX (20 μM, data not shown). There was also a fast inhibitory response (Figure 4.2A, filled arrowheads) that shunted the glutamatergic component at all ages and was blocked by the specific GABAA receptor antagonist SR95531 (5 μM, data not shown). Finally, there was a second, slower inhibitory component (Figure 4.2A, open arrowheads) that was blocked by the specific GABAB receptor antagonist CGP52432 (2 μM, data not shown). With age, there was an apparent hyperpolarization of the reversal potential of the GABAA component and an apparent reduction of the amplitude and duration of the GABAB response. In P7 neurons, the stimulation response had a long duration, typically lasting 1 s (n = 18). By P14, the GABAB component generally had smaller amplitude and duration, lasting 1 s (n = 17).
Responses were highly similar between P21 and P28, with a fast, inhibitory GABAA peak and a GABAB response that terminated within 500 ms of stimulation (n = 11 for P21, 8 for P28).
Between P7 and P28 the input resistance and membrane time constant of BLA principal neurons show 10- and 3-fold reduction, respectively (Ehrlich et al., 2012), which would dramatically alter the waveform of the evoked PSPs. Hence we next measured the properties of evoked synaptic currents in voltage-clamp mode in another set of neurons. By blocking the GABAB component with a cesium-based patch solution, we were able to isolate the GABAA component of the evoked response (Figure 4.2B). Consistent with our current-clamp recordings, stimulation at holding potentials of −50, −60, and −70 mV reliably resulted in a fast excitatory PSC (EPSC) at every time point examined and a GABAA PSC that became faster with age. We measured the duration of the GABAA response, which exhibited a significant, threefold reduction across the first postnatal month (Figure 4.2C; 1-way ANOVA, F3,60 = 5.81, P 0.001).
A majority of the change occurred between P7 and P14, with a significant reduction in duration (mean ± SE) from 466.0 ± 49.1 ms at P7 (n = 18) to 280.9 ± 38.4 ms at P14 (n = 17; Tukey's posttest, P 0.01). The duration was relatively stable from P14 to P21 and P28, with a value of
281.0 ± 36.6 ms at P21 (n = 18) and 219.6 ± 55.1 ms at P28 (n = 11). The stimulation response at P28 matched the biphasic response seen in adult BLA principal neurons, with a fast glutamatergic EPSC that is rapidly shunted by a GABAergic IPSC (see Figure 4.2B, inset). We hypothesized that the slow GABAA response in immature BLA neurons was due to slow individual GABAA PSCs or, alternatively, to the depolarized reversal potential of GABAA receptors removing a brake on network activity. Therefore, we next quantified the kinetics and reversal potential of isolated GABAA receptor-mediated currents in BLA principal neurons across the first postnatal month.
4.4.2 Kinetics of fast synaptic inhibition of BLA principal neurons.
To quantify changes to the kinetics of GABAA currents in BLA principal neurons we recorded spontaneous IPSCs (Figure 4.3). Across the first postnatal month, IPSCs became significantly faster with age until around P21 (Figure 4.3A). In particular, IPSC 10–90% rise time exhibited a significant decrease of more than twofold across the first postnatal month (Figure 4.3B; 1-way ANOVA, F3,36 = 7.80, P 0.001). There was also a significant reduction across this window in the time constant of IPSC decay (Figure 4.3C; 1-way ANOVA, F3,36 = 3.45, P 0.05) and a similar trend in IPSC half-width (Figure 4.3D). Specifically, spontaneous IPSCs of neurons at P7 were relatively slow, with a rise time (mean ± SE) of 2.29 ± 0.34 ms, a decay time constant of 5.76 ± 0.19 ms, and a half-width of 5.86 ± 0.59 ms (n = 6). By P14 rise time was reduced to 1.68 ± 0.14 ms, although decay time constant and half-width were relatively unchanged at 6.13 ± 0.43 ms and 5.70 ± 0.40 ms, respectively (n = 16). At P21 IPSCs were faster, with rise time reduced to 1.15 ± 0.12 ms, decay time constant reduced to 4.93 ± 0.22 ms, and half-width reduced to 4.65 ± 0.19 ms (n= 11). At P28 IPSCs showed kinetics similar to those at P21 and were significantly faster than at younger ages; rise time was 1.10 ± 0.12 ms (Tukey's posttests, P 0.001 vs. P7 and P 0.01 vs. P14), decay time constant was 4.59 ± 0.36 ms (P
0.05 vs. P14), and half-width was 4.51 ± 0.42 ms (not significant; n = 7).
Unlike IPSC kinetics, there was no age-dependent change in the size of spontaneous IPSCs. We found no significant effect of age on peak conductance, although it tended to increase with age (Figure 4.3E; 1-way ANOVA, F3,23 = 0.87, P 0.05; n = 5 or 6). At later time points there was an emergence of large IPSCs not observed at P7, but they were not frequent enough to significantly alter the mean conductance. Moreover, no significant difference was observed in the coefficient of variation of peak conductance for each neuron with age (Figure 4.3F; F3,22 = 1.28, P 0.05; n = 5 or 6). There was a weak positive correlation of peak IPSC conductance and decay time constant (R2 0.2 at each age; data not shown).
To exclude the possibility that the developmental change in IPSC kinetics was due to presynaptic effects, we also measured responses to focal application of the GABAA receptor agonist muscimol (Figure 4.4). As illustrated in Figure 4.4A, the tip of a pipette was placed near a patched BLA principal neuron, and 100 μM muscimol was picospritzed onto the soma. The muscimol response became much larger and faster with age, as seen in Figure 4.4, B and C. There was a significant overall effect of age on the decay time constant of the response, with a significant decrease between P14 and P21 (Figure 4.4D; 1-way ANOVA with Tukey's posttests, F3,53 = 5.57,P 0.01). At P7, the decay time constant of the muscimol response was
407.7 ± 54.2 ms (mean ± SE, n = 12), which increased to 431.1 ± 34.7 ms at P14 (n = 16). The decay time constant then decreased significantly to 291.9 ± 20.3 ms at P21 (P 0.05; n = 12) and
266.3 ± 24.6 ms at P28 (P 0.01; n = 12). The inverse developmental trajectory was observed for the peak conductance of the muscimol response, which increased significantly with age and also transitioned abruptly from P14 to P21 (Figure 4.4E; 1-way ANOVA with Tukey's posttests, F3,43 = 5.67, P 0.01). At P7 the peak conductance was 15.6 ± 4.1 nS (n = 10), which decreased to 13.8 ± 2.6 nS at P14 (n = 14). The peak conductance then increased significantly to
31.6 ± 5.5 nS at P21 (P 0.05; n = 10) and 36.4 ± 7.1 nS at P28 (P 0.01; n = 10).
The kinetics of GABAA receptors are influenced by their subunit composition, which is classically regulated during development. Hence we next used single-cell RT-PCR to identify developmental changes in the expression of GABAA receptor subunit mRNA in BLA principal neurons (Figure 4.5). There was an age-dependent increase in the proportion of neurons with detectable transcripts for 7 of the 13 GABAA receptor subunits tested: namely, α1, α2, α3 and α5, β2 and β3, and γ2 (Figure 4.5A, B; n = 10). No expression was detected for α4, α6, β1, γ3, or δ, and only one neuron at any age was found to express γ1 mRNA. There was no detectable expression of any subunit transcripts at P7; however, because rRNA for the housekeeper gene 18S was present in all cells used for this analysis, we assume this was due to limited sensitivity of the technique for low levels of transcript. As a positive control, we screened whole BLA tissue for mRNA of GABAAreceptor subunits (Figure 4.5C); as early as P7, the whole BLA contained detectable levels of mRNA for every subunit screened (n = 4).
Using single-cell RT PCR, we observed an increase in the expression of the α1 subunit, which confers faster IPSC kinetics, relative to that of α2, which confers slower IPSC kinetics.
Specifically, while 4 of 10 neurons at P14 expressed α2 mRNA, 0 had expression for α1; by P28 the proportions for the subunits were comparable, with 4 and 6 of 10 neurons expressing α1 and α2, respectively. Interestingly, there was a strong clustering effect at P21 and P28, with the β3 subunit being expressed by 11 of 12 neurons with detectable expression of α2 but 0 of 7 with α1.
The β2- and γ2-subunits had the opposite pattern: β2 and γ2 were detected in 5 of 7 neurons with α1 expression, while 0 of 12 neurons with α2 also expressed β2 and only one expressed γ2. We address this clustering, as well as the relationship between the developmental trajectories of IPSC kinetics and subunit expression, in the Discussion.
4.4.3 Depolarized reversal potential of GABAA receptors in immature BLA principal neurons.