Perturbation of GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Induces Trans-regional Metaplasticity at the Medial Prefrontal Cortex

Cerebral Cortex, January 2018;28: 395–410 doi: 10.1093/cercor/bhx300 Advance Access Publication Date: 9 November 2017 Original Article ORIGINAL ARTICLE regional Metaplasticity at the Medial Prefrontal Cortex Rinki Saha1, Kuldeep Shrivastava1, Liang Jing4, Rachel Schayek1, Mouna Maroun1, Martin Kriebel2, Hansjürgen Volkmer2 and Gal Richter-Levin1,3,4 1 Sagol Department of Neurobiology, University of Haifa, Haifa 31905, Israel, 2NMI Natural and Medical Sciences Institute, University of Tübingen, 72770 Reutlingen, Germany, 3Department of Psychology, University of Haifa, Haifa 31905, Israel and 4The Institute for the Study of Affective Neuroscience, University of Haif, Haifa 31905, Israel Address correspondence to Prof. Gal Richter-Levin, Sagol Department of Neurobiology, Department of Psychology, University of Haifa, Haifa 31905, Israel. Email: . Rinki Saha and Kuldeep Shrivastava contributed equally to this work Abstract GABAergic synapses in the basolateral amygdala (BLA) play an important role in fear memory generation. We have previously reported that reduction in GABAergic synapses innervating specifically at the axon initial segment (AIS) of principal neurons of BLA, by neurofascin (NF) knockdown, impairs fear extinction. BLA is bidirectionally connected with the medial prefrontal cortex (mPFC), which is a key region involved in extinction of acquired fear memory. Here, we showed that reducing AIS GABAergic synapses within the BLA leads to impairment of synaptic plasticity in the BLA-mPFC pathway, as well as in the ventral subiculum (vSub)-mPFC pathway, which is independent of BLA involvement. The results suggest that the alteration within the BLA subsequently resulted in a form of trans-regional metaplasticity in the mPFC. In support of that notion, we observed that NF knockdown induced a severe deficit in behavioral flexibility as measured by reversal learning. Interestingly, reversal learning similar to extinction learning is an mPFC-dependent behavior. In agreement with that, measurement of the immediate-early gene, c-Fos immunoreactivity after reversal learning was reduced in the mPFC and BLA, supporting further the notion that the BLA GABAergic manipulation resulted in trans-regional metaplastic alterations within the mPFC. Key words: amygdala, medial prefrontal cortex, metaplasticity, neurofascin, reversal learning Introduction Emerging evidence from behavioral and electrophysiological studies on rodents indicate that amygdala plays critical role in orchestrating fear response (Phelps and LeDoux 2005; Pape and Pare 2010). However, amygdala circuits act via dynamic interactions between several other wired brain structures (Pitkänen et al. 2000; Johansen et al. 2012). Consequently, fear memories rely on the interplay between the basolateral amygdala (BLA), © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Perturbation of GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Induces Trans- 396 | Cerebral Cortex, 2018, Vol. 28, No. 1 subsequent alterations in behavioral responses. Here in the present study, we could demonstrate how selective manipulation of GABAergic synapses in the BLA generates trans-regional metaplasticity in mPFC. We have further examined reversal learning in the water maze (WM), which is considered to be another mPFCdependent behavior (de Bruin et al. 1994; Dalley et al. 2004). The result indicated that trans-regional metaplasticity leads to impaired cognitive flexibility in neurofascin knockdown rats. Additionally, we have checked c-Fos immunoreactivity as a proxy for neuronal activation in mPFC, right after the reversal learning. In agreement with the above results, reduced activation of c-Fos in mPFC was found, supporting the notion of trans-regional metaplastic changes induced by BLA within the mPFC. To examine whether this form of trans-regional metaplasticity was selective to the BLA-mPFC pathway or more general, we examined the impact of a similar manipulation within the BLA on dentate gyrus (DG) plasticity and related behavior. Synaptic plasticity in the DG, spatial object recognition (SOR) and contextual fear conditioning were not affected by NF knockdown in the BLA. We thus demonstrate for the first time how a moderate change of a specific GABAergic synapse in one region results in transregional alteration in the subsequent responsiveness of other region at both the behavioral and electrophysiological levels. Materials and Methods Animals Male Sprague–Dawley rats (Harlan Laboratories, Jerusalem, Israel) weighing 200–224 g on arrival were group-housed at room temperature (21 ± 2 °C) on a 12 h light/dark cycle (lights on at 07:00 hours), with water and food pellets ad libitum. Experimental procedures began after 5 days of acclimation to the vivarium. All experiments were conducted in accordance with the NIH guidelines for the care and use of laboratory animals and were approved by the University of Haifa ethical committee. Stereotaxic Lentivirus Injection Rats received bilateral microinjections of either lentiviral vectors (construct represented in Fig. S1A, for more details (Saha et al. 2017)) expressing miRNA directed against NF (NF1707) or the control miRNA (CTR) into the BLA (detailed in vitro and in vivo validation and functional characterization of the constructs are available in Saha et al. (2017)). Deeply anesthetized rats (10% Ketamine, 100 mg/kg, (Richter Pharma, Wells, Austria) and 2% medetomidine, 10 mg/kg (Orion pharma, Espoo, Finland), both i.p.) were mounted on a stereotaxic frame (Stoelting instruments) under control of body temperature and antibiotic (15% Vetrimoxin, 0.2 mL/kg, s.c.; Vetmarket, Petah Tikva, Israel) and analgesic (Calmagine, 0.3 mL/kg, s.c.; Vetmarket) treatments. Body temperature was maintained throughout surgery with an adjustable warming pad. A midline incision was made down the scalp, and craniotomy was made using a dental drill. Next, a 33 G stainless steel cannula mounted on a microsyringe (Nanofil, World Precision Instruments) were lowered bilaterally into the BLA at the following coordinates: −2.8 mm AP, ± 4.7 mm ML from bregma; −7.7 mm DV from brain surface, according to Paxinos and Watson (2006). After 5 min of rest in the target area, 2 μL of viral vector suspension were injected at 0.15 μL/min (UMP3 microsyringe pump and Micro4 controller, World Precision Instruments) and the microsyringe was slowly withdrawn 10 min after the injection. After closing the scalp by suture, Antisedan the hippocampus and the medial prefrontal cortex (mPFC). In this fear memory network, hippocampus modulates contextual aspects of fear learning (...truncated)