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Corresponding with the emergence of inhibitory GABAA potentials was a shortening of the network response to local electrical stimulation. Specifically, in immature neurons lowfrequency stimulation evoked slow, depolarizing GABAA responses and even slower, inhibitory GABAB responses resulting in a compound potential that could last between 1 and 2 s. By P21 the mature phenotype was present, in which stimulation evoked a fast, inhibitory GABAA response followed by a curtailed GABAB response, returning to baseline within 1 s (Rainnie et al., 1991a; Washburn and Moises, 1992). Although we have previously reported that immature neurons have larger input resistances and membrane time constants (Ehrlich et al., 2012), this did not fully account for the slower synaptic potentials. Our voltage-clamp recordings also revealed a prolongation of stimulation-evoked GABAA currents in immature neurons.
The long duration of the network response in immature neurons likely results from the depolarized reversal potential of GABAA receptors. In adult BLA principal neurons, feed-forward activation of inhibitory GABAA receptors provides a fast shunt, limiting the extent of BLA activation (Rainnie et al., 1991a). As we have shown here, in immature BLA principal neurons GABAA is depolarizing, which should enable or even promote feed-forward excitation within the BLA. Without feed-forward GABAA receptor-mediated inhibition, in the immature BLA a different brake is afforded: potent activation of GABAB receptors. However, GABAB acts on a slower timescale than GABAA, which would explain the long-duration network responses observed here. The strong GABAB receptor activation we found at P7 could mitigate the risk of hyperexcitability and excitotoxicity due to GABA release by opposing the depolarizing action of GABAA at this age.
GABAB receptors are thought to be localized extrasynaptically, and in adulthood they are activated when a train of stimuli releases sufficient GABA to spill over into the extrasynaptic compartment (Kim et al., 1997; Fritschy et al., 1999; Scanziani, 2000; Kulik et al., 2002;
Beenhakker and Huguenard, 2010). The strong GABAB response to a single stimulation in the immature BLA suggests there is an age-dependent difference in the accessibility of GABA to GABAB receptors after stimulation. This developmental change must be interpreted in the context of the concurrent increase in input resistance, but the amplitude of GABAB relative to GABAA PSPs decreases considerably with age. This difference could be afforded by agedependent changes in the architecture of GABAergic synapses. To support this notion, GABAB receptors in cerebellar neurons move from dendritic shafts at P7 to spines at P21 (Lujan and Shigemoto, 2006). Moreover, activation of metabotropic receptors with a single stimulation may be a general phenomenon early in development; we observed a muscarinic current that was abolished by atropine (5 μM) and evoked by single stimulation in some neurons at P7 but not at any later time points (unpublished observation).
4.5.3 Faster IPSCs with age.
The function of GABA in the BLA also depends on the kinetics of GABAA receptormediated IPSCs, and we found significant developmental changes to their kinetics. Specifically, there was a nearly twofold reduction in spontaneous IPSC rise time from P7 to P21, when the mature, fast waveform was expressed. Decay time constant and IPSC half-width exhibited a similar trajectory. As with the decay time constant of spontaneous IPSCs, there was an abrupt decrease from P14 to P21 in the kinetics of the response to exogenous muscimol. Interestingly, while the peak conductance of the response to muscimol significantly increased with age, we found no age-dependent change in the peak conductance underlying spontaneous IPSCs. This effect may be due to the proximity of receptor activation to the soma, since the picospritzer pipette was placed close to the soma while IPSCs presumably originate throughout the dendritic arbor. This notion is supported by the fact that dendrites of BLA principal neurons expand greatly throughout the first postnatal month, with the total dendritic length increasing more than threefold as dendrites come to extend more than twice as far from the soma (Chapter 3). Furthermore, this difference may be due to a developmental change in the ratio of synaptic to extrasynaptic GABAAreceptors.
When considered in the context of a nearly threefold reduction in membrane time constant from P7 to P28, GABAA PSPs are likely much faster in the adult BLA (Chapter 2;
Ehrlich et al., 2012). The presence of slow IPSCs early in development has been well documented throughout the brain (Draguhn and Heinemann, 1996; Hollrigel and Soltesz, 1997; Pouzat and Hestrin, 1997; Dunning et al., 1999). Comparable developmental changes were also found in the kinetics of miniature IPSCs in marmoset amygdala principal neurons, albeit on a different time course (Yamada et al., 2012). The kinetics of individual IPSCs should influence their effect on spike timing (Pouille and Scanziani, 2001) and are known to regulate the ability of GABAergic afferents to entrain postsynaptic oscillations (Tamas et al., 2004). Furthermore, faster IPSCs should more precisely control the timing of spikes and membrane potential oscillations due to postinhibitory rebound (Ryan et al., 2012), promoting the viability of temporal coding mechanisms in the adult BLA.
The maturation of IPSC kinetics corresponds with changes to the expression of GABAA receptor subunits in BLA principal neurons. We observed an increase with age in the proportion of neurons expressing seven different GABAA subunits. The subunit mRNA we found in BLA principal neurons at P21 and P28 closely matches protein expression in the adult BLA, aside from the α4-, β1-, and δ-subunits (Sieghart and Sperk, 2002). Expression of α4, β1, and δ is likely found in other cell types. Expression of the α1-subunit, among others, emerged at P21, confirming results found with mRNA from whole BLA (Zhang et al., 1992). This change is well documented throughout the brain and is known to contribute to the faster kinetics observed with age (Hornung and Fritschy, 1996; Dunning et al., 1999; Davis et al., 2000; Okada et al., 2000;
Vicini et al., 2001; Bosman et al., 2002; Mohler et al., 2004; Eyre et al., 2012). Developmental changes in subunit expression are also known to regulate channel localization and drug sensitivity (Nusser et al., 1996; Hevers and Luddens, 2002). Activation of receptors containing the GABAA receptor α1-subunit directly influence critical period onset (Huntsman et al., 1994;
Fagiolini et al., 2004), suggesting that the emergence of α1 expression may trigger other aspects of emotional circuit development. Despite the apparent contribution of postsynaptic changes, identified with exogenous muscimol application, to the development of IPSC kinetics, the observed maturation of IPSC kinetics may be due, in part, to changes in the activity of different subtypes of interneurons. This notion is supported by the fact that interneurons exhibit specificity in the subunit composition of GABAA receptors to which they are apposed. For instance, in the hippocampus synapses formed by parvalbumin-expressing interneurons on pyramidal cell somas preferentially express the GABAA receptor α1-subunit (Klausberger et al., 2002). In the BLA, parvalbumin-expressing interneurons do not emerge until P17 and reach maturity at P30 (Berdel and Morys, 2000), which corresponds with the emergence of α1 expression between P14 and P21 observed here.
We also observed clustering of subunit expression at P21 and P28, with α1-, β2-, and γ2subunits primarily expressed in distinct neurons from α2, α3, α5, and β3. While this result was unexpected and curious considering the homogeneous population of GABAA PSCs we observed, there is some precedent for separation of α1 protein from other α-subunits (Hutcheon et al., 2004). There is an important caveat for interpreting this clustering, namely, the high rate of false negatives. Similarly, while neurons at P7 provided enough RNA to detect 18S and chloride pump expression, the lack of GABAA subunit mRNA detected is not evidence for an absolute lack of GABAA receptors. The presence of GABAA receptor-mediated PSCs and receptor subunit mRNA in whole tissue at P7 clearly refutes this, meaning that the developmental changes we see in subunit transcript expression are not concrete but indicate trends in expression levels. As with all single-cell RT-PCR results, it will be important to extend these findings by quantifying mRNA and protein expression in the developing BLA.
4.5.4 Short-term synaptic depression of GABAA IPSCs in immature BLA.
We also found distinct changes to GABAergic synaptic plasticity during development. At P7, GABAA inputs to BLA principal neurons exhibited robust early and late synaptic depression.
Gradually with age the synaptic depression waned and shifted toward short-term facilitation. By P28, the amplitude of the response was maintained at the second pulse (early STP) and facilitated at the fifth (late STP). Comparable changes on a similar developmental trajectory have been observed for GABAergic and glutamatergic synapses in other brain regions (Pouzat and Hestrin, 1997; Reyes and Sakmann, 1999). For late STP, we observed facilitation at P28 that disappeared by P35; while this trend was not significant, it raises the interesting possibility of a temporary window with short-term facilitation around P28. There was also a significant effect of stimulation frequency on late STP. Short-term depression is classically sensitive to stimulation frequency, likely because of the kinetics of depletion and restoration of releasable neurotransmitter pools (Zucker and Regehr, 2002; Elfant et al., 2008). Interestingly, for late STP there was also a significant interaction effect; the influence of stimulation frequency on STP decreased with age and may therefore be specific to short-term depression.
The developmental change in STP may be explained by several mechanisms, although the simplest involves a change in release probability—from high-release-probability, high-output GABAergic terminals in immature neurons to low release probability, low output in the mature BLA (for review, see Zucker and Regehr, 2002). High GABAergic output would be parsimonious with the robust activation of GABABreceptors we observed in immature BLA principal neurons.
In light of this hypothesis, future studies should address the contribution of parvalbumin expression to STP in the BLA. This calcium-binding protein directly influences STP and presynaptic calcium dynamics (Vreugdenhil et al., 2003; Collin et al., 2005), and its expression in the BLA changes during the first postnatal month (Berdel and Morys, 2000). We can rule out a contribution of presynaptic GABAB receptors to the observed short-term depression in younger animals, because CGP52432 was included in the bath during these experiments; however, presynaptic GABAA receptors can play a similar role (MacDermott et al., 1999). GABAA receptor desensitization is also known to contribute to short-term depression (Overstreet et al., 2000), although there is no precedent for a developmental change in this phenomenon. Finally, shortterm depression of immature GABAergic IPSCs may involve ionic plasticity, a depolarization of the chloride reversal following strong GABAA activation (for review, see Raimondo et al., 2012).
To better understand the maturation of STP we observed, future studies should differentiate the various interneuron subtypes found in the BLA, which play different roles in the network but were grouped in the population response used here.
STP affords synapses with a variety of temporal filtering mechanisms, suggesting that the BLA processes information and communicates using different mechanisms with age (Buonomano, 2000; Fortune and Rose, 2001; Pfister et al., 2010). The role of synaptic filters in tuning the network to specific frequencies is particularly important because BLA oscillations have been implicated in the expression and consolidation of fear memories (Madsen and Rainnie, 2009; Sangha et al., 2009; Popa et al., 2010; Lesting et al., 2011) and BLA inhibition is thought to promote these oscillations (Chapter 6; Ryan et al., 2012). Short-term depression of inhibition, as we observed in the juvenile BLA, promotes high-pass filtering of excitatory input and increases the information transmitted by bursts (Abbott and Regehr, 2004; George et al., 2011).
Therefore, short-term depression may provide salience for high-frequency and bursting activity in the immature BLA, possibly providing compensation for the reduced sensitivity to highfrequency input of immature BLA principal neurons (Ehrlich et al., 2012).
We have shown that synaptic inhibition in the developing BLA is not static but undergoes a number of profound changes that will directly influence BLA physiology and its contribution to emotional processing. The function of GABA receptors and, therefore, of the entire BLA are in flux during the first postnatal month, which likely contributes to the emotional changes observed during this window. Future studies should determine whether development of synaptic transmission in the amygdala contributes to the expression of critical periods that render the brain vulnerable to the pathogenesis of emotional disorders like anxiety, depression, and autism spectrum disorders. To improve our understanding of the etiology of psychiatric disorders, it will be important to characterize how the normative development of the amygdala is influenced by genetic predispositions and risk factors for psychiatric disease (Pine, 2002; Monk, 2008). To this end, in Chapter 5 we describe the effects on BLA development of a risk-factor for neurodevelopmental disorders, prenatal stress.
Table 4.1 PCR primers used in this study.
Table 4.2 IPSC Amplitude Ratios for Short-term Plasticity.
The ratios of IPSC amplitudes following 10 and 20 Hz stimulation are listed for each time-point as mean ± SEM. Early STP corresponds to the ratio of pulse 2 to pulse 1, and late STP to the ratio of pulse 5 to pulse 1.
Statistical tests for significance are described in the Results.
Figure 4.1: Schematic of recording and stimulation sites Figure 4.