The dopaminergic VTA - basal amygdala projection contributes to signal aversive state in fear memory

Dopamine released from midbrain neurons is an important neuromodulatory signal during associative learning in many forebrain circuits. One model of associative learning is auditory cued fear learning, during which an innocuous sensory percept like a tone (called conditioned stimulus, CS), acquires an aversive meaning after pairing with an aversive event, like a mild footshock (called unconditioned stimulus, US). The amygdala plays an important role in fear learning in mammals. Nevertheless, our knowledge of the synaptic- or neuromodulatory input(s) which carry information about the US to the amygdala is still limited. Here, I have investigated the hypothesis that dopamine, released from midbrain dopaminergic neurons in the ventral tegmental area (VTA) of mice, might signal aversive sensory events to the amygdala during fear learning. Using viral anterograde tracing approaches in transgenic mice expressing Cre-recombinase under the control of the dopamine transporter promoter (DAT-Cre), we found a dopaminergic projection from VTA to the basal amygdala (BA) and to the central amygdala (CeA). In addition, retrograde tracing approaches showed that tyrosine hydroxylase positive (TH+) dopamine neurons targeting these two areas might be differentially distributed in the VTA. To investigate a role of these projections during fear learning, we silenced the electrical activity of VTA dopamine neurons, or else their projection fibers to the BA or the CeA, by using Cre-dependent expression of a light-driven proton pump Archaerhodopsin (Arch). Silencing VTA neurons specifically during the footshock presentation (US) led to a moderate reduction of fear memory tested one day following the initial US - CS pairing. Silencing the dopaminergic fibers bilaterally in the BA (but not in the CeA) during footshock presentation had a somewhat larger effect on fear memory retrieval, and similarly reduced contextual fear memory. Furthermore, we made recordings of the spiking activity of DAT+ - as well as of non-identified VTA neurons in-vivo in behaving mice using optrodes. We found that about half of all units strongly respond to footshock stimulation (US), and show increasing responses to tones (CS) during the US - CS pairing. To investigate the possible cellular functions of dopamine in the BA during fear learning, we performed in-vitro recordings of genetically identified parvalbumin (PV)-positive interneurons and BA principal neurons that project to the CeA. This showed that bath application of dopamine or of a D1- receptor agonist enhances the evoked firing rate of CeA projectors in the BA. Finally, bath application of dopamine reduces afferent excitatory inputs onto PV-positive interneurons in the BA and in the LA. Taken together, by using complementary optogenetic, electrophysiological and anatomical approaches, this PhD thesis suggests that VTA dopamine neurons facilitate associative learning in the BA by providing a neuromodulatory signal to the BA during the time of the footshock. Furthermore, the VTA might also provide dopamine to the amygdala during fear memory retrieval. Thus, this work contributes to an understanding of the functional role of the dopaminergic VTA to BA projection in auditory-cued fear learning.

A VTA to Basal Amygdala Dopamine Projection Contributes to Signal Salient Somatosensory Events during Fear Learning

Michael Kintscher, Olexiy Kochubey, Ralf Schneggenburger, Wei Tang

The amygdala is a brain area critical for the formation of fear memories. However, the nature of the teaching signal(s) that drive plasticity in the amygdala are still under debate. Here, we use optogenetic methods to investigate the contribution of ventral tegmental area (VTA) dopamine neurons to auditory-cued fear learning in male mice. Using anterograde and retrograde labeling, we found that a sparse and relatively evenly distributed population of VTA neurons projects to the basal amygdala (BA). In vivo optrode recordings in behaving mice showed that many VTA neurons, among them putative dopamine neurons, are excited by footshocks, and acquire a response to auditory stimuli during fear learning. Combined cfos imaging and retrograde labeling in dopamine transporter (DAT) Cre mice revealed that a large majority of BA projectors (>95%) are dopamine neurons, and that BA projectors become activated by the tone-footshock pairing of fear learning protocols. Finally, silencing VTA dopamine neurons, or their axon terminals in the BA during the footshock, reduced the strength of fear memory as tested 1 d later, whereas silencing the VTA-central amygdala (CeA) projection had no effect. Thus, VTA dopamine neurons projecting to the BA contribute to fear memory formation, by coding for the saliency of the footshock event and by signaling such events to the basal amygdala.

2020

An insular cortex - lateral amygdala network in fear learning

Shriya Palchaudhuri

Animals are capable of evaluating sensory cues for possible threats and adapting their behaviours accordingly. Fear learning is an evolutionarily conserved behaviour crucial for animal survival, during which sensory percepts with a negative reinforcing quality, also called unconditioned stimuli (US), influence the valence of initially neutral sensory cues (conditioned stimulus, CS). The lateral amygdala (LA) is thought to integrate information about sensory cues like tones (the CS), and information about primary reinforcers like painful stimuli (the US). Nevertheless, synaptic afferents to the LA and upstream brain areas remain understudied in fear learning. The posterior insular cortex (pInsCx) has been shown to project to the LA and is known to be a multimodal sensory area which processes auditory, somatosensory-nociceptive, and interoceptive information, making it an interesting brain area with a putative role in fear learning. My PhD thesis therefore aimed to investigate a possible connection from the pInsCx to the LA, and the role of this pathway in the fear learning circuitry. I first used anatomical anterograde and retrograde tracing strategies in the mouse to study the afferents to the LA. Besides previously known inputs, I found that the granular (GI) and the dysgranular (DI) areas of the pInsCx strongly project to the anterior LA. With optogenetically-assisted circuit mapping (OACM), I found a glutamatergic monosynaptic connection from the pInsCx to LA principal neurons that, in agreement with the anatomical findings, was localized more to the anterior LA. In comparison, a well-studied auditory thalamic (MGm/PIN) input was localized more to the posterior LA. Despite this regional input specificity, ~30% LA principal neurons received projections from both areas. Thus, in the first part, using anatomical tracing and OACM, I have unambiguously shown a robust functional excitatory connection from the pInsCx (GI/DI) to the LA.In the second part of my thesis, I aimed to characterize the role of the pInsCx and the pInsCx-LA pathway in fear learning. Using archaerhodopsin-mediated optogenetic inhibition, I found that silencing of footshock-driven activity of pInsCx neurons in-vivo, partially blocks auditory-cued fear memory. These results were also supported by chemogenetic silencing of the pInsCx during the training day, which led to a modest impairment of fear retrieval a day later. Next, to investigate the role of the pInsCx-LA projection more specifically, I used a retrograde expression approach to silence LA-projectors during the footshock, which again, suppressed fear memory one day later. In further ex-vivo experiments, I found that EPSCs at the pInsCx-LA connection exhibited an increased AMPA/NMDA ratio following fear learning, indicating that plasticity occurs at this pathway during fear learning. Finally, miniature microscopic Ca2+ imaging of LA neurons combined with halorhodopsin-mediated axon inhibition, showed that the pInsCx-LA excitatory synapse contributes to transmitting footshock information to a sub-population of LA neurons. This study unravels the relationship between two serial nodes, the pInsCx and the LA, in the fear learning network. Thus, the pInsCx-LA connection is a novel pathway in auditory-cued fear memory that is strengthened with learning and that partly provides aversive information to the amygdala.

EPFL2022

Fear Learning: An Evolving Picture for Plasticity at Synaptic Afferents to the Amygdala

Denys Osypenko, Shriya Palchaudhuri, Ralf Schneggenburger

Unraveling the neuronal mechanisms of fear learning might allow neuroscientists to make links between a learned behavior and the underlying plasticity at specific synaptic connections. In fear learning, an innocuous sensory event such as a tone (called the conditioned stimulus, CS) acquires an emotional value when paired with an aversive outcome (unconditioned stimulus, US). Here, we review earlier studies that have shown that synaptic plasticity at thalamic and cortical afferents to the lateral amygdala (LA) is critical for the formation of auditory-cued fear memories. Despite the early progress, it has remained unclear whether there are separate synaptic inputs that carry US information to the LA to act as a teaching signal for plasticity at CS-coding synapses. Recent findings have begun to fill this gap by showing, first, that thalamic and cortical auditory afferents can also carry US information; second, that the release of neuromodulators contributes to US-driven teaching signals; and third, that synaptic plasticity additionally happens at connections up- and downstream of the LA. Together, a picture emerges in which coordinated synaptic plasticity in serial and parallel circuits enables the formation of a finely regulated fear memory.

SAGE PUBLICATIONS INC2022

An insular cortex - lateral amygdala network in fear learning

Shriya Palchaudhuri

EPFL2022