Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

The Membrane Proximal Region of AMPA Receptors in Lateral Amygdala is Essential for Fear Memory Formation.

The membrane proximal region (MPR) of AMPA receptor (AMPAR) is needed for receptor trafficking and synaptic plasticity. However, its roles in long-term memory formation are not known. To assess the possible roles of AMPAR-MPR in rat lateral amygdala (LA) in short- and long-term fear memory formation, we used glutamate receptors (GluAs)-MPR competitive peptides MPR(DD) and MPR(AA). The MPR(DD) peptide is derived from GluA1 MPR and was previously shown to impair synaptic plasticity and to inhibit GluA1 containing AMPAR insertion into the synapse in an activity-dependent manner. The MPR(AA) peptide is derived from GluA2/4 MPR, and this receptor fragment was shown to be essential for GluA4 protein interaction needed for its insertion into the neuronal membrane and synapse. The peptides were linked to a TAT peptide (TAT-MPR(DD) and TAT-MPR(AA)) to facilitate internalization into LA cells. Infusion of the TAT-MPR(DD) peptide into LA 30 min before fear conditioning led to a significant impairment of long-term fear memory formation. Injection of TAT-MPR(DD) peptide into LA 30 min before fear conditioning impaired short-term fear memory formation. The TAT-MPR(DD) peptide had no effect on memory retrieval when injected into LA 30 min before fear memory test. Infusion of the TAT-MPR(AA) peptide into LA 30 min before fear conditioning led to a significant impairment of long-term fear memory formation. In contrast, the TAT-MPR(AA) had no effect on short-term fear memory formation. A TAT-control peptide had no effect on short- or long-term fear memory. These results show that the AMPAR-MPR in LA is needed for fear memory formation and that the MPR region of GluA1 is essential for acquisition of memory, whereas the MPR region of GluA4 is essential for long-term fear memory consolidation.

ABL1 in thalamus is associated with safety but not fear learning.

In auditory fear conditioning a tone is paired with a footshock, establishing long lasting fear memory to the tone. In safety learning these stimuli are presented in an unpaired non-overlapping manner and enduring memories to the tone as a safety signal are formed. Although these paradigms utilize the same sensory stimuli different memories are formed leading to distinct behavioral outcome. In this study we aimed to explore whether fear conditioning and safety learning lead to different molecular changes in thalamic area that receives tone and shock inputs. Toward that end, we used antibody microarrays to detect changes in proteins levels in this brain region. The levels of ABL1, Bog, IL1B, and Tau proteins in thalamus were found to be lower in the group trained for safety learning compared to the fear conditioning group 6 h after training. The levels of these proteins were not different between safety learning and fear conditioning trained groups in auditory cortex. Western blot analysis revealed that the ABL1 protein level in thalamus is reduced specifically by safety learning but not fear conditioning when compared to naïve rats. These results show that safety learning leads to activation of auditory thalamus differently from fear conditioning and to a decrease in the level of ABL1 protein in this brain region. Reduction in ABL1 level in thalamus may affect neuronal processes, such as morphogenesis and synaptic efficacy shown to be intimately regulated by changes in this kinase level.