Evidence that a defined population of neurons in lateral amygdala is directly involved in auditory fear learning and memory
Introduction
A central concept in memory research is that of the engram, the hypothetical physical change in the brain responsible for storing, or encoding, a memory. Although there has been considerable effort to identify circuitry associated with particular learning and memory models (LeDoux, 2000, Thompson, 2005), no studies have identified all the changes which constitute an engram in vertebrates. A major reason for this is that it has been very difficult to identify the neurons which are directly involved in memory storage in the brain. There have some been real successes in this area in the classic studies of the marine snail, Aplysia (Kandel, 2001) and mollusc, Hermissenda (Blackwell, 2006). However, both of these models involve simple reflex motor behaviour and very simple circuitry, a long way from the complexities of the vertebrate brain.
In vertebrates, one method that enables identification of neurons involved in a certain task employs the immediate early gene c-fos as a marker of neuronal activation (Knapska, Radwanska, Werka et al., 2007). We previously utilized the transgenic fos-tau-LacZ (FTL) mouse to visualise neurons activated by fear conditioning (Butler et al., 2015, Trogrlic et al., 2011, Wilson and Murphy, 2009). The FTL transgene uses the c-fos promoter to drive expression of β-galactosidase into the cell bodies and processes of c-fos activated neurons (Murphy et al., 2007, Wilson and Murphy, 2009). Specific, spatially discrete populations of neurons within amygdala, hypothalamus and lateral septum were identified that were specifically activated following acquisition of conditioned fear, rather than by exposure to other sensory stimuli (Butler et al., 2015, Trogrlic et al., 2011, Wilson and Murphy, 2009). We proposed that these populations of neurons form nodes in a network that encodes fear memory (Butler et al., 2015, Trogrlic et al., 2011). In particular, one of these populations of neurons was found within the ventrolateral (vl) nucleus of the lateral amygdala (LA), a key region involved in fear conditioning (Diaz-Mataix et al., 2014, Izquierdo et al., 2016, Johansen et al., 2011, Josselyn and Frankland, 2018, Kim and Jung, 2006, Maren, 2003).
Other experiments have utilised c-fos regulated expression in transgenic mice to identify neuronal populations in dentate gyrus that are activated by exposure to specific contexts during fear conditioning (Garner et al., 2012, Liu et al., 2012, Ramirez et al., 2013, Reijmers et al., 2007). Subsequent inhibition or activation of these neurons demonstrated that these populations of c-fos+ neurons were required for memory of specific contexts (Garner et al., 2012, Liu et al., 2012, Ramirez et al., 2013). These experiments not only demonstrate that different hippocampal neurons are required in the memory of different contexts, they also validate the approach of using c-fos regulated expression as a marker of neurons involved in learning and memory. Nevertheless, and despite the functional strength of these experiments, the c-fos expressing neurons activated by learning could not be distinguished from those activated by different sensory stimuli during the context fear conditioning process. It could thus not be determined which of these neurons were directly involved in contextual memory formation.
Whereas we have identified discrete populations of neurons activated specifically by fear conditioning (Butler et al., 2015, Trogrlic et al., 2011, Wilson and Murphy, 2009), we do not have clear evidence to establish that these neurons are functionally involved in fear learning and memory. To determine if the FTL expressing neurons in LAvl are required for fear learning and memory, it is theoretically possible to inhibit or lesion these neurons. Whereas there are effective methods for inhibiting or lesioning c-fos expressing neurons (Cruz et al., 2013, Garner et al., 2012, Koya et al., 2016, Liu et al., 2012, Ramirez et al., 2013), these approaches have been used for volumes of brain tissue which were much larger than those containing the spatially discrete populations of neurons we have identified (the volume containing these neurons in LAvl is ~0.1 mm3). Even for the techniques which use stereotaxic injections to target particular brain areas, for the very small fraction of mice which could be accurately and bilaterally targeted to LAvl, it may not be possible to inhibit most of the learning specific neurons and at the same time avoid c-fos expressing neurons in closely neighbouring parts of the brain, such as dorsal or ventromedial LA (LAd, LAvm) or the basolateral amygdala (BLA) subnuclei.
We have used an alternative approach to obtain evidence of functional involvement of neuronal populations in fear conditioning. A number of key signalling pathways required for fear conditioning have been identified, with particular focus on the LA. One such requirement is phosphorylation of Extracellular Signal Regulated Kinase 1 and 2 (ERK1/2), a mitogen activated protein kinase. Inhibition of ERK1/2 phosphorylation in LA in the period following fear conditioning results in attenuation of long term auditory fear memory and ERK1/2 is transiently phosphorylated in rat LA following auditory fear conditioning (Schafe, Atkins, Swank et al., 2000). Thus, phosphorylation of ERK1/2 in LA following fear conditioning is required for the formation of fear memory. This finding not only points to the involvement of ERK1/2 in the memory process, it also means that the cells in LA in which ERK1/2 is phosphorylated following fear conditioning are required for fear memory formation because the essential phosphorylation step is occurring in these cells. It follows that the cells in LA which are directly involved in fear memory can be identified based on their expression of phosphorylated pERK1/2.
Experiments assessing pERK1/2 expression after fear conditioning have generally shown an increase in number of pERK1/2 neurons in LA, but have not identified a clear pattern of learning specific expression (Besnard et al., 2014, Di Benedetto et al., 2009, Schafe et al., 2000). Some of these experiments were not necessarily designed to find learning specific populations of neurons. Thus, there may have been expression of pERK1/2 due to non-specific sensory stimuli or to some context learning in the control groups, which could have masked the identification of a discrete population of learning specific neurons. We modified the traditional auditory fear conditioning protocol to minimise effects of non-specific sensory stimuli and context learning (Butler et al., 2015, Nithianantharajah and Murphy, 2008). Using this refined approach, we successfully identified FTL+ neurons specifically activated by auditory fear conditioning (Butler et al., 2015). Here, we have employed this same protocol of auditory fear conditioning to identify neurons in LA that express pERK1/2 under these specific learning conditions, to provide evidence for their involvement in fear learning and memory.
Section snippets
Animals
All experiments were conducted with approval of the Animal Ethics Committee of the University of Melbourne and in accordance with the guidelines of the National Health and Medical Research Council (Australia). Male FTL+ mice aged 8–12 weeks were obtained from the Biomedical Sciences Animal Facility, University of Melbourne, singly housed in standard 15 cm × 30 cm × 12 cm cages on a 12 h light/dark cycle, and had food and water supplied ad libitum. Two days prior to the commencement of the
Auditory fear conditioning
Pavlovian or classical conditioning involves presentation of a neutral stimulus, the conditioned stimulus (CS), paired with a biologically significant stimulus, the unconditioned stimulus (US), such as pain or food. Once learning has occurred, presentation of CS alone is able to elicit a response, and this learned reaction to the previously neutral CS is known as the conditioned response. To identify neurons involved in auditory fear conditioning, we utilised a method that enables training of
Discussion
It is well established that activation of ERK1/2 by phosphorylation is part of a key signalling pathway in memory formation (Adams & Sweatt, 2002). In studies of fear memory, inhibitors of ERK1/2 phosphorylation infused either systemically (Atkins et al., 1998, Selcher et al., 1999) or locally into LA impaired long term memory consolidation (Di Benedetto et al., 2009, Schafe et al., 2000) and reconsolidation (Duvarci, Nader, & LeDoux, 2005). However, short term memory was unaffected (Schafe et
Acknowledgements
We would like to thank Ellie Hyun-Jung Cho from the Biological Optical Microscopy Platform, University of Melbourne; Maya Kesar, Sarah Taverner and staff of the Biomedical Sciences Animal Facility, University of Melbourne. This research was supported by funding from the National Health and Medical Research Council of Australia and the Department and Anatomy and Neuroscience, University of Melbourne.
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These authors contributed equally to this work.