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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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Application of NiCl diminished the resonant properties of BLA principal neurons (Figure 6.6D1) and completely blocked the forskolin- and 4-AP-induced MPO in all neurons tested (n = 6) (Figure 6.6D2-3, 6.7F), suggesting that an interaction between IT and voltage-gated K+ channels, most likely IA channels, play a critical role in MPO expression in BLA neurons.

Application of high-micromolar 4-AP, however, can also block other K+ channels, including several that are also sensitive to micromolar concentrations of TEA. Hence, to determine if the effects of 4-AP on the MPOs resulted from a non-selective blockade of K+ channels, we repeated the experiments above in the presence of TEA (500 M). As illustrated in Figure 6.8, application of TEA failed to mimic the 4-AP effect in either the presence or absence of simulated IPSPs. Moreover, concurrent application of forskolin (10 M) and TEA also failed to unmask a significant increase in MPO amplitude over TEA alone (Figure 6.8C, n = 5), suggesting that the forskolin effect may only be observed when IA channel activity is reduced by 4-AP.

To verify that the effects of forskolin were mediated by direct activation of the adenylyl cyclase-cAMP signaling cascade, we then examined the membrane response to application of the inactive forskolin isomer, dideoxy-forskolin (10 M), in the presence of 4-AP. Dideoxy-forskolin failed to mimic the forskolin effect on MPOs in either the presence or absence of artificial IPSPs, suggesting that activation of the adenylyl cyclase-cAMP signaling cascade selectively facilitates IPSP-enhanced MPOs in principal neurons of the BLA (Figure 6.8D, n = 6).

Finally, we examined if modulation of intracellular Ca2+ levels also play a role in regulating the MPO. Here, inclusion of the Ca2+ chelator, BAPTA (5 mM), in the patch solution completely blocked the MPO induced by co-application of 4-AP (500µM) and forskolin (10µM) (Figure 6.9A, n = 6), suggesting that fluctuations in intracellular Ca2+ levels also play an important role in the expression of MPOs in BLA principal neurons. However, this result raised the possibility that the drug-induced MPO may be independent of activation of the cAMP-PKA signaling cascade. To address this question, we included the competitive antagonist of cAMPinduced PKA activation, cAMPs-RP, in the patch solution. Inclusion of cAMPs-RP (25 µM) completely blocked the MPO induced by forskolin (Figure 6.9B, n = 4). Conversely, inclusion of a non-hydrolysable cAMP analogue, 8-Br-cAMP (5-10 µM), in the patch pipette unmasked an MPO in the presence of TTX alone that was similar in magnitude to that induced by forskolin (Figure 6.9C, n = 6). Hence, Ca2+ influx through IT channels, elevation of intracellular Ca2+, and activation of the adenylyl cyclase-cAMP-PKA signaling cascade each play an important role in the expression of MPOs in BLA principal neurons.

The sensitivity of the MPO to modulation by intracellular Ca2+ and activation of the cAMP-PKA signaling cascade suggested that receptors coupled to Gαs would facilitate MPOs, whereas those coupled to Gαi would attenuate MPOs. To test this hypothesis, we examined the effect of prior application of the selective mGluR2/3 agonist, LY379268, on the 4-AP- and forskolin-induced MPOs. Principal neurons of the BLA express high levels of mGluR2/3 receptors (Rainnie et al., 1994; Muly et al., 2007), which couple to Gi/o proteins to inhibit adenylyl cyclase activity (Pin and Duvoisin, 1995), and we reasoned that activation of these receptors would attenuate drug-induced MPOs. As illustrated in Figure 6.9D (n = 10), application of LY379268 (50 µM) completely blocked the MPOs.

6.5 Discussion In the present study, we demonstrate that spontaneous, compound IPSPs function to increase spike-timing precision both within and across BLA principal neurons. Previous studies have shown that these IPSPs are driven by local, burst-firing PV+ neurons (Rainnie, 1999b), which have a high level of connectivity with BLA principal neurons. These data suggest that spontaneous, compound IPSPs would function to synchronize action potentials in a large population of principal neurons. We also show that compound IPSPs promote and entrain a high delta / low theta frequency membrane potential oscillation (MPO) that is uncovered by activation of the cAMP-PKA signaling cascade. The oscillatory nature of BLA principal neurons is also manifested as a modifiable inherent resonance frequency. We propose that the interaction of compound IPSPs with the oscillatory properties of BLA principal neurons is a viable mechanism for synchronizing firing activity in this cell population, promoting network oscillations within the BLA, and enhancing coherent oscillations between the BLA and other brain regions involved in fear.

6.5.1 Synchronized inhibition drives coordinated activity of BLA principal neurons Recent evidence suggests a wide variety of behaviors require synchronized neural activity and network oscillations, both of which are promoted by synaptic inhibition (Soltesz and Deschenes, 1993; Pouille and Scanziani, 2001; Person and Perkel, 2005; Sohal et al., 2006; Szucs et al., 2009). Here, we demonstrate that BLA principal neurons receive highly synchronized, rhythmic inhibition which, in turn, synchronizes firing activity among groups of BLA principal neurons. Importantly, spontaneous activity of interneurons in the prefrontal cortex at theta frequency entrains the firing of principal neurons to an ongoing network theta oscillation (Benchenane et al., 2010). This example from the prefrontal cortex suggests the coordination of principal neuron firing by inhibition is critical for salient output of some neural circuits. Through coordinating the firing of large groups of BLA principal neurons, compound IPSPs should improve salience by promoting summation of output and leading to spike-timing dependent plasticity in both the BLA and its targets.

In order to study the effect of compound IPSPs on spike-timing precision, we used two proxies: artificial IPSPs generated by direct current injection at the soma, and compound IPSPs evoked by direct stimulation of interneurons in the BLA under glutamatergic blockade. We showed that spike-timing precision within single neurons is improved by spontaneous IPSPs, artificial IPSPs, and stimulation-evoked IPSPs, with artificial IPSPs being significantly more effective than evoked IPSPs. Furthermore, artificial IPSPs were able to significantly coordinate firing across neurons, but evoked IPSPs were not, due to the observed variability in the waveform. Spontaneous, compound IPSPs observed across pairs had a highly consistent waveform (evident in a representative pair in Figure 6.1A), likely because they are generated by burst-firing PV+ interneurons, which innervate BLA principal neurons perisomatically and have their activity coordinated through a syncytium. In contrast, stimulation of the BLA to evoke IPSPs probably recruited multiple subtypes of GABAergic interneurons targeting multiple compartments of the principal neurons (McDonald and Betette, 2001; McDonald and Mascagni, 2002; Mascagni and McDonald, 2003) and hence introduced variability across cells in the IPSP waveform. While PV+ interneurons seem uniquely positioned to generate synaptic inhibition that is ideal for interacting with an MPO and coordinating activity of BLA principal neurons, the possibility is not excluded that other inhibitory input, for instance feed-forward inhibition from cortical or thalamic sources (Rainnie et al., 1991b; Szinyei et al., 2000), could exert a similar coordinating influence.

The fact that artificial IPSPs were able to mimic the effects of evoked and spontaneous IPSPs on spike-timing precision without directly influencing the membrane conductance suggests they act primarily via membrane hyperpolarization. This hyperpolarization likely causes activation of IH and de-inactivation of voltage-gated currents including IT, which would contribute to calcium spikes upon rebound (Hutcheon et al., 1994). Because IT is typically inactive near resting membrane potential, the observed effect of compound IPSPs on spike timing is probably more applicable when BLA principal neurons are depolarized from rest. It is also important to consider that compound IPSPs occur amidst ongoing synaptic activity, not in the absence of synaptic input as when tested here. In the in vivo system, compound IPSPs may not produce spikes in the absence of excitatory transmission, but rather interact with ongoing synaptic activity to influence the timing of spikes.

The ability of compound IPSPs to coordinate spiking activity most likely occurs across large groups of BLA principal neurons due to the broad connectivity of PV+ interneurons (Muller et al., 2006), the synchronization of PV+ interneuron firing activity through a syncytium (Muller et al., 2005; Woodruff and Sah, 2007a, b), and, as shown here, the robustness of IPSP coordination of spike timing across principal neurons despite varying intrinsic properties.

Although synchronizing large networks of principal neurons will improve potency of efferent signaling, it could also limit the specificity of signaling. Cortical inputs to the BLA are organized topographically (McDonald et al., 1999), and synchrony throughout the nucleus could weaken the specificity afforded by this topography. A loss of specificity in this circuit through excessive synchronization within the amygdala may lead to generalization of fear learning, which has been implicated in affective disorders such as post-traumatic stress disorder (Rainnie and Ressler, 2009). Furthermore, less than a quarter of BLA neurons appear to be incorporated into the engram for any specific fear memory (Han et al., 2007; Han et al., 2009). If encoding and recall of fear memories depend on network oscillations, there must be a mechanism to preferentially incorporate some neurons while excluding others. Some potential mechanisms include regulation of the extent of the syncytium or of projections from the PV+ interneurons onto principal neurons, or, more interestingly, interactions between variability in the frequency of the network oscillation with variations in preferred resonance frequency of the principal cells.

Considering the prominent role inhibition appears to play in coordinating the activity of BLA principal neurons, it is likely that stimuli altering the frequency of IPSPs in vivo could drastically change the output activity of the BLA. For instance, activation of serotonin 2A or cholecystokinin B receptors, both of which are implicated in emotional learning (Chhatwal et al., 2009), increase the frequency of rhythmic IPSPs in BLA principal neurons through indirect excitation of interneurons (Rainnie, 1999b; Chung and Moore, 2007). A similar effect is observed in the BLA in response to local release of dopamine in mice (Loretan et al., 2004) and primates (Muly et al., 2009). Moreover, the BLA receives dopaminergic input from the ventral tegmental area, which also exhibits a network oscillation at 2-5 Hz during working memory tasks (Fujisawa and Buzsaki, 2011), raising important questions about the nature of the interaction of phasic dopamine release with a BLA circuit that itself generates rhythmic activity.

6.5.2 Resonance frequency and intrinsic membrane oscillations in BLA principal neurons In the present study we have shown that BLA principal neurons in the rat have an intrinsic resonance that was extremely consistent, with nearly all neurons displaying a peak resonance between 4.2 and 4.4 Hz. This intrinsic resonance was insensitive to application of TTX (1 µM), whereas a previous study in guinea pigs reported neurons in the lateral and basolateral nuclei of the amygdala express a TTX-sensitive inherent resonance frequency at 2.5 Hz (Pape and Driesang, 1998). The difference in reported resonance frequencies is likely due to the different model species, as we have also seen differences in peak resonance frequency of principal neurons between rat and primate (unpublished observation). The difference in TTX sensitivity, however, is likely explained by the concentrations of TTX employed. In the study by Pape and colleagues the resonance frequency was abolished by 20 µM TTX, compared to the 1 µM TTX used here. High concentrations of TTX are known to block the persistent Na+ current, and future studies should investigate whether it contributes to resonance in BLA principal neurons, as it does in LA neurons (Pape et al., 1998). Similar to our observations, hippocampal principal neurons also display resonance that is insensitive to 1 µM TTX with a peak at 4.1 Hz (Pike et al., 2000).

In addition to selectively filtering synaptic input in high delta / low theta bands, BLA principal neurons also express high- and low-threshold MPOs in this frequency range, as described by Pape and colleagues (1998). Here we show the presence of an MPO that occurs at the peak resonance frequency of these neurons (~4-5 Hz) and seems to share some mechanisms with both previously described oscillations. Although Pape and colleagues found no effect of specific Ca2+ channel blockers on the high threshold membrane oscillations (Pape and Driesang, 1998), recordings with a BAPTA-containing electrode completely abolished the oscillation. In our hands, bath application of NiCl completely abolished the MPO, suggesting a strong influence of T-type Ca2+ channels. The Pape study also reported that high-threshold membrane oscillations were insensitive to 10 mM 4-AP, suggesting that voltage-gated K+ channels were not involved in that membrane oscillation (1998). We observed, however, that application of 100-500 µM 4-AP significantly enhanced the membrane oscillations, suggesting IA may actively suppress the MPO, acting in opposition to IT. This could also be related to changes in input resistance, but the lack of effect of 500 µM TEA suggests a specific role of IA. A similar relationship between IT and IA has been shown in other systems (Pape et al., 2004; Molineux et al., 2005), and factors that either enhance IT or reduce IA could then unmask the expression of the intrinsic membrane oscillations.

In agreement with Pape and colleagues, we did not find an effect on intrinsic membrane oscillations of blocking IH with ZD7228 (60 µM, data not shown). While this is not an exhaustive pharmacological characterization, we believe we have identified the major currents involved in mediating this MPO. Other currents, including the persistent sodium current and calcium-activated potassium currents may also be involved (Pape and Driesang, 1998), and future study to illuminate their roles in this phenomenon would be valuable.

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