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«Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory ...»

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We next examined the effect of age on the GABAA reversal potential in BLA principal neurons (Figure 4.6). Here GABAA receptor-mediated inhibitory PSPs (IPSPs) were elicited with bipolar stimulation of the dorsolateral BLA in the presence of a cocktail of neurotransmitter receptor antagonists for AMPA/kainate, NMDA, and GABAB receptors (see Methods). The residual monosynaptic IPSP evoked in the presence of this cocktail was completely abolished by the GABAAreceptor antagonist SR95531 (5 μM, data not shown). The reversal potential of the GABAA response was estimated with peak IPSP amplitudes from stimulation at three different baseline membrane potentials. This approach corroborated our initial observations, with the GABAA reversal potential exhibiting a significant hyperpolarizing shift with age (Figure 4.6A, B; 1-way ANOVA, F3,21 = 19.91, P 0.0001). For example, the reversal potential was significantly reduced from −54.3 ± 1.0 mV at P7 (n = 8) to −66.6 ± 2.3 mV at P14 (Tukey's posttests, P 0.001; n = 7). From P14 onward, the mean reversal potential did not change significantly but became less variable across neurons, reaching −69.1 ± 2.0 mV at P21 (n = 5) and −69.7 ± 0.9 mV at P28 (n = 5). While the whole cell patch-clamp technique is not ideal for measuring GABAA reversal potentials because of dialysis of the intracellular chloride concentration (which would bias the results for every neuron, regardless of age, toward the Nernst reversal of approximately −33 mV), the observed trend of more hyperpolarizing GABAA responses with age was robust to the bias. Interestingly, despite the depolarized reversal potential of GABAAreceptors at P7, we were unable to elicit action potentials with these evoked, isolated GABAA PSPs in P7 neurons, even when depolarized toward action potential threshold (n = 4, data not shown).

The reversal potential of the GABAA-mediated IPSP is influenced by the intracellular concentration of chloride ions, which is tightly regulated by the activity of two selective ion pumps: NKCC1, which promotes excitatory GABAA receptor-mediated potentials, and KCC2, which facilitates an inhibitory response to GABAAreceptor activation. The expression of these pumps is developmentally regulated, and we were therefore interested in the developmental trajectory of chloride pump expression in BLA principal neurons. Using single-cell RT-PCR, we measured the expression at P7, P14, P21, and P28. Consistent with the depolarized reversal potential we observed in immature neurons, there was a shift in mRNA expression from NKCC1 to KCC2 between P7 and P21 (Figure 4.6D). Specifically, there was comparable expression of the two transcripts at P7, with 9 and 10 of 15 neurons expressing detectable levels of NKCC1 and KCC2, respectively. At P14, NKCC1 was more prominent, expressed by 17 of 20 neurons compared with only 5 of 20 with KCC2 expression. No NKCC1 transcripts were detected at P21 or P28, but the majority of neurons expressed KCC2 (21 of 23 at P21, 5 of 8 at P28).

4.4.4 Short-term plasticity of GABAA IPSCs.

The influence of GABA on BLA principal neuron activity depends not only on the amplitude, kinetics, and valence of individual synaptic events but also on the patterning and plasticity of these events. Therefore, we next characterized the development of STP of GABAA receptor-mediated synaptic transmission. Isolated GABAA receptor-mediated IPSCs were evoked, as before, in voltage-clamped neurons at P7, P14, P21, and P28 with electrical stimulation within the BLA and a cocktail of neurotransmitter antagonists. Trains of five pulses were evoked at 10 and 20 Hz, and the amplitudes of the GABAA IPSCs within each train were measured (Figure 4.7). At both 10- and 20-Hz stimulation, IPSCs at P7 exhibited robust shortterm depression (Figure 4.7A, B). The depression was lost gradually with age. Because of this gradation, we included a group of neurons at P35 to determine whether the trend reached an asymptote at P28.

To quantify the developmental changes, we used two metrics based on IPSC amplitudes:

the early STP, defined as the ratio of the amplitudes of the second and first IPSCs in the train, and the late STP, defined as the ratio of the fifth and first IPSCs in the train (Table 4.2). For early STP, there was a significant main effect of age (2-way ANOVA with repeated measures, F4,23 = 8.53, P 0.001) but no effect of stimulation frequency (Figure 4.7C). All possible pairwise comparisons were made with Bonferroni posttests. For 10-Hz stimulation, early STP exhibited no significant developmental transitions. However, significant individual transitions were found for 20-Hz stimulation. Specifically, early STP for 20-Hz stimulation increased significantly from P7 to P21 (P 0.05), P28 (P 0.001), and P35 (P 0.001). There were also significant increases from P14 to P28 (P 0.05) and P35 (P 0.01) and from P21 to P35 (P 0.01). For both stimulation frequencies the values at P28 and P35 were highly similar, suggesting that the phenotype is stable beyond P28.

Late STP, the ratio of the fifth IPSC to the first, also exhibited robust synaptic depression in immature neurons that transitioned toward synaptic facilitation with age. A two-way ANOVA with repeated measures revealed significant main effects of age (F4,23 = 3.709, P 0.05) and stimulation frequency (F1,23 = 14.58, P 0.001) as well as a significant interaction effect (F4,23 = 3.29, P 0.05; Figure 4.7D). Pulse ratios for 20-Hz stimulation were generally smaller than those for 10 Hz. Late STP for 10-Hz stimulation exhibited an increase with age, although none of the individual transitions was statistically significant. For 20-Hz stimulation, late STP increased significantly from P7 to P28 (P 0.05). For both stimulation frequencies, n = 3 neurons at P7, 5 at P14, 7 at P21, 6 at P28, and 7 at P35.

4.4.5 Spontaneous GABA activity is rhythmically organized throughout the first postnatal month.

Although we observed many differences in the character of GABAergic synaptic transmission with age, it is important to consider these changes in the context of normal GABA function. In slice preparations of the adult BLA, groups of principal neurons simultaneously receive rhythmic, compound synaptic events that consist mainly of GABAA receptor-mediated IPSCs but also include glutamatergic EPSCs. We have observed them not only in BLA slices from adult rats but also in rhesus macaques, where they promote intrinsic membrane potential oscillations and coordinate network activity (Chapter 6; Ryan et al., 2012). Considering the important functional role of these synaptic events, we characterized their expression during postnatal development (Figure 4.8). We observed rhythmic, compound IPSCs as early as P7 and at every time point studied (Figure 4.8A, B). As expected, the rhythmic PSCs were depolarizing from rest at P7 and became consistently hyperpolarizing by P21. The waveform changed with age, as rhythmic IPSCs at P7 were smooth while those at P28 were sharp, a cluster of many distinct release events (Figure 4.8A). Interestingly, the proportion of neurons receiving compound IPSCs was similar at all ages, with rhythmic events spontaneously observed in ∼20– 40% of neurons (6 of 14 neurons at P7, 6 of 28 at P14, 8 of 25 at P21, and 4 of 16 at P28; Figure

4.8B). Consistent with the mature BLA (Ryan et al., 2012), as early as P7 rhythmic IPSCs were perfectly synchronized across BLA principal neurons (Figure 4.8C).

4.5 Discussion In this study, we provided the first evidence that synaptic transmission, in particular GABAergic transmission, undergoes significant change throughout BLA development. Similar to the intrinsic physiology of BLA principal neurons (Ehrlich et al., 2012), we demonstrated that inhibitory synaptic transmission reaches maturity ∼3–4 wk after birth. Specifically, GABAA receptor-mediated PSCs become faster and more hyperpolarizing and lose short-term depression with age until P21–P28, when rats are in late infancy (Quinn, 2005). In addition, these physiological changes correspond with maturation of the expression of genes that influence GABAergic function specifically in BLA principal neurons—in the case of PSC kinetics a change in GABAA receptor subunit expression and in the case of PSC reversal a shift in chloride transporter expression. Considering the critical role of GABAergic transmission in the function of BLA principal neurons and the nucleus in general, these changes likely contribute to the maturation of amygdala function throughout postnatal development.

4.5.1 Shift from depolarizing to hyperpolarizing GABAA transmission.

One of the most profound changes we observed in the developing BLA was a transition in the GABAA reversal potential. The reversal potential in principal neurons became more hyperpolarized with age, shifting from approximately −55 mV at P7 to −70 mV at P21. The magnitude and time course of this change matched those identified in cell-attached recordings of hippocampal pyramidal neurons, which exhibit a similar hyperpolarization of the GABAA reversal potential of ∼15 mV between birth and P17 (Tyzio et al., 2008). The existence of excitatory GABAA receptor-mediated potentials during brain development has been well documented (Ben-Ari, 2002; Owens and Kriegstein, 2002). In BLA principal neurons before P14, GABAA receptor activation caused depolarization, suggesting that these receptors play a different functional role early in development. Depolarizing GABAA receptors should render the BLA more excitable, which may be important for communication between the BLA and other limbic brain regions like the extended amygdala, hippocampus, and prefrontal cortex, before strong connections have been formed (Bouwmeester et al., 2002b). Despite the inability of GABAA receptor activation at P7, to drive action potential firing, GABA may be excitatory at that age through interactions with glutamatergic transmission (Gulledge and Stuart, 2003; Valeeva et al., 2010). One consequence of depolarizing GABAA receptors is that positive allosteric modulators like barbiturates and benzodiazepines could paradoxically facilitate activation of BLA principal neurons during infancy, which could have important clinical ramifications, particularly in light of the role of the BLA as an epileptogenic locus and the incidence of infantile seizures (Racine et al., 1972; White and Price, 1993).

Depolarizing GABAA transmission likely serves an important function in normative brain development: GABAA receptors are thought to fulfill the role AMPA receptors play in the mature brain, providing sufficient postsynaptic depolarization to enable NMDA receptor activation and subsequent synaptic strengthening (Ben-Ari et al., 1997). This phenomenon likely occurs in the immature BLA when glutamate and GABA are simultaneously released during the rhythmic, compound PSCs we described here. Rhythmic, compound PSCs could serve this purpose in the BLA as early as P7, with 43% of neurons recorded at P7 receiving them. Supporting this notion, synaptic events in the immature hippocampus similar to our rhythmic PSCs, termed “giant depolarizing potentials,” have been shown to promote synaptic strengthening (Mohajerani and Cherubini, 2006). This function of rhythmic PSCs is distinct from that proposed in the adult BLA, where rhythmic IPSCs promote intrinsic membrane potential oscillations and synchronize action potentials across BLA principal neurons (Chapter 6; Ryan et al., 2012). Supporting a distinct function for rhythmic PSCs in the immature and mature BLA, a much higher proportion (~80%) of BLA principal neurons receive these events in slices from adult animals (ibid.).

Interestingly, mature oscillatory properties of BLA principal neurons are not present until around P21 (Chapter 2; Ehrlich et al., 2012), supporting convergent developmental timing of inhibitory GABA and membrane potential oscillations. In the mature BLA rhythmic IPSCs are driven by a syncytium of burst-firing parvalbumin interneurons (Rainnie et al., 2006), but parvalbumin expression does not emerge in the BLA until around P14 (Berdel and Morys, 2000); rhythmic PSCs at P7 may be driven by burst-firing interneurons that may later express parvalbumin.

A possible caveat to our measurements of reversal potential is that dialysis of the cytosol by our patch solution could alter the electrochemical gradients of ions passed by GABAA receptors, particularly chloride. However, if dialysis had an effect, it would be to minimize differences across time points, shifting the reversal potential toward the Nernst reversal of chloride, approximately −33 mV for our patch solution and ACSF. The disparity between the observed reversals and the Nernst reversal, as well as the significant effect we found of age on GABAA reversal, suggests that the chloride concentration near GABAA receptors is locally regulated. Interestingly, there is a precedent for chloride reversal being robust to dialysis of the neuronal cytosol (Jarolimek et al., 1999; Gonzalez-Islas et al., 2009). Therefore, we are confident in our observations of a hyperpolarization of the GABAA reversal with age.

Hyperpolarization of the GABAA reversal potential with age is typically thought to result from a change in the expression of chloride pumps from NKCC1, which renders GABAA excitatory by extruding chloride, to KCC2, which renders it inhibitory by accumulating the ion intracellularly (Ben-Ari, 2002). Indeed, our single-cell RT-PCR study indicated that at P7 and P14 a high proportion of principal neurons express mRNA for NKCC1, but expression was undetectable by P21. In contrast, mRNA for KCC2 was prominent at P21 and P28. These changes in chloride pump expression are also consistent with those in other brain regions (Ben-Ari, 2002).

Further experiments will be required to determine whether levels of protein expression follow suit and whether similar changes in chloride pump expression occur in BLA interneurons.

4.5.2 Development of a GABAergic shunt of the network response.

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