The Ministry of Fear: ‘The Conjuring’ of Fright in the Amygdala by the Raphe
Authors: Praachi Tiwari, Sashaina E. Fanibunda, Vidita A. Vaidya
Journal: Neuron (2019)

In this issue of Neuron, Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar characterize a distinct serotonergic circuit from the dorsal raphe nucleus to the basal amygdala that facilitates fear conditioning and memory. In this issue of Neuron, Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar characterize a distinct serotonergic circuit from the dorsal raphe nucleus to the basal amygdala that facilitates fear conditioning and memory. Many of us have had the living daylights scared out of us and felt shivers running up and down our spines. We should count our lucky stars if this was in front of the silver screen, holding a tub of buttered popcorn. And yet, even when we know what will unfold in a predictable horror film, we respond on cue to a creaking door, chilling visuals, or bloodcurdling scream by jumping out of our skin, thrilling the film director. Fear responses are crucial for our survival and have fixed and flexible components, with flexibility facilitating the optimal modulation of fear responses incorporating context, cues, and experience. The fear conditioning paradigm has been extensively used to uncover how we acquire fear learning and the manner in which we extinguish fear (Craske et al., 2018Craske M.G. Hermans D. Vervliet B. State-of-the-art and future directions for extinction as a translational model for fear and anxiety.Philos Trans R Soc Lond B Biol Sci. 2018; 373Crossref Scopus (172) Google Scholar). The amygdala is a central component of the fear processing neurocircuitry (Pape and Pare, 2010Pape H.-C. Pare D. Plastic synaptic networks of the amygdala for the acquisition, expression, and extinction of conditioned fear.Physiol. Rev. 2010; 90: 419-463Crossref PubMed Scopus (758) Google Scholar). Serotonergic innervation of the amygdala (Steinbusch, 1981Steinbusch H.W. Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals.Neuroscience. 1981; 6: 557-618Crossref PubMed Scopus (2108) Google Scholar) is implicated in modulation of fear, and dysregulation of this pathway contributes to psychopathology (Bauer, 2015Bauer E.P. Serotonin in fear conditioning processes.Behav. Brain Res. 2015; 277: 68-77Crossref PubMed Scopus (93) Google Scholar). Despite substantial evidence that serotonin plays an important role in regulating fear and anxiety, the underlying neurocircuitry mediating the influence of serotonin on the hub of fear, the amygdala, remains poorly delineated (Bocchio et al., 2016Bocchio M. McHugh S.B. Bannerman D.M. Sharp T. Capogna M. Serotonin, amygdala and fear: assembling the puzzle.Front. Neural Circuits. 2016; 10: 24Crossref PubMed Scopus (96) Google Scholar). In this issue of Neuron, Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar employ a diversity of approaches to elegantly characterize the contribution of serotonergic input from the dorsal raphe nucleus (DRN) to the basal nucleus of the amygdala (BA) in the regulation of fear (Figure 1). Using viral-based tracing approaches, they characterize a distinct subpopulation of DRN serotonergic neurons co-immunopositive for VGluT3 that terminate in the BA and are distinct from the DRN projection to the nucleus accumbens (NA). Capitalizing on tracing methods that reveal inputs onto these distinct DRN 5-HT subpopulations, they uncover discrete input pathways with a bias toward inputs from the periaqueductal gray (PAG) and locus coeruleus (LC) onto the BA-projecting 5-HT DRN neurons and inputs from the ventral pallidum (VP) onto NA-projecting DRN neurons. This segregation of 5-HT DRN neurons into separate subpopulations with discrete projections strengthens the building evidence of substantial heterogeneity within the DRN and indicates functional diversity of 5-HT neurons that regulate fear (LC/PAG to DRN to BA) as distinct from those 5-HT neurons that modulate reward (VP to DRN to NA). Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar also tease out the anatomical targets of the BA neurons that receive DRN-5-HT input, indicating widespread projections to extended amygdala circuitry, cortical regions including sensorimotor cortices, PFC and ventral hippocampus, as well as onto midbrain regions of the DRN and PAG. This circuit hence is also ideally poised to tune the outputs from the BA to an extensive fear network, thus shaping fear response. Fiber photometry for GCaMP6 in DRN 5-HT neuron terminals within the BA indicated enhanced calcium signal during fear conditioning, with a strong correlation to the degree of freezing and an increase noted across training sessions. This was selectively noted in the DRN to BA pathway, but not the DRN to NA circuit, and this increase across conditioning is indicative of a role for this pathway in facilitating fear learning. The degree of activity of the DRN-BA pathway served almost like a forecast for the strength of fear memory post-extinction training. These findings set up the hypothesis to test the contribution of this distinct DRN-BA pathway in modulating fear response. To test this hypothesis, Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar used optogenetics to bidirectionally regulate the DRN-BA projection in specific phases of the fear conditioning task. Activation of the DRN-BA neurocircuit during conditioning enhanced both fear learning and retrieval post-extinction training, whereas inhibition attenuated freezing responses during fear memory both on commencement of and post-extinction training. To parse out the contribution of the DRN-BA pathway during fear extinction, which involves discrete as well as overlapping circuitry with fear learning, activity of the DRN-BA pathway was altered during extinction training. While neither activation nor inhibition of the DRN-BA circuit altered fear responses during extinction trials, activation enhanced fear memory whereas inhibition attenuated fear responses during extinction retrieval. These findings indicate that the DRN-BA 5-HT pathway facilitates fear, agnostic to whether this pathway is activated during the conditioning or extinction phase of the fear conditioning task. Since conditioning and extinction are thought to recruit distinct BA neurons, this opens up the key question of the BA neuron subpopulation that responds to 5-HT inputs during fear learning versus extinction training and the contribution of 5-HT receptors that mediate these fear potentiating effects. The role of the DRN-BA circuit was tested with electrophysiology alongside optogenetic activation of the DRN-BA circuit during fear conditioning, with firing responses of BA neurons examined during all stages of the fear conditioning paradigm. In keeping with prior evidence of inhibition of the BA by 5-HT inputs (Bocchio et al., 2015Bocchio M. Fucsina G. Oikonomidis L. McHugh S.B. Bannerman D.M. Sharp T. Capogna M. Increased serotonin transporter expression reduces fear and recruitment of parvalbumin interneurons of the amygdala.Neuropsychopharmacology. 2015; 40: 3015-3026Crossref PubMed Scopus (34) Google Scholar), optogenetic activation during conditioning was linked to a decrease in BA neuron mean firing rate during conditioning. However, as a consequence of DRN-BA circuit activation during conditioning, an enhanced recruitment of BA neuron firing was observed during fear retrieval in the first five sessions of extinction training, with a strong correlation of firing rate to the onset of the freezing response. Interestingly, stimulation of the DRN-BA pathway during conditioning resulted in a robust increase in the firing rate of putative “fear neurons” in the BA during extinction retrieval. This suggests that recruitment of this discrete DRN-BA circuit biases BA neurons toward a robust retention of fear learning and an impairment of fear extinction memory formation, thus facilitating a shift toward stronger encoding of fear responses. Synchronous oscillations of amygdala neurons in the theta range (4–10 Hz) have been linked to fear states (Seidenbecher et al., 2003Seidenbecher T. Laxmi T.R. Stork O. Pape H.-C. Amygdalar and hippocampal theta rhythm synchronization during fear memory retrieval.Science. 2003; 301: 846-850Crossref PubMed Scopus (561) Google Scholar, Pape et al., 2005Pape H.-C. Narayanan R.T. Smid J. Stork O. Seidenbecher T. Theta activity in neurons and networks of the amygdala related to long-term fear memory.Hippocampus. 2005; 15: 874-880Crossref PubMed Scopus (109) Google Scholar). Sengupta and Holmes, 2019Sengupta A. Holmes A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory.Neuron. 2019; 103 (S0896-6273(19)30482-9)Google Scholar show that activating the DRN-BA projection during conditioning enhances theta power, emerging at the end of conditioning and also evident during fear memory retrieval. Further, experiments reveal that DRN-BA stimulation promotes phase locking of BA neuron firing with theta rhythms, commencing at the end of conditioning and also noted in the fear memory retrieval observed on initial conditioned stimulus presentation during extinction training. This implies that activating the DRN-BA pathway during conditioning not only strengthens fear encoding in the BA, but it also aligns the temporal dynamics of neuronal firing to enhance fear states. Given that the serotonergic terminals from the DRN to BA corelease glutamate (Sengupta et al., 2017Sengupta A. Bocchio M. Bannerman D.M. Sharp T. Capogna M. Control of amygdala circuits by 5-HT neurons via 5-HT and glutamate cotransmission.J. Neurosci. 2017; 37: 1785-1796Crossref PubMed Scopus (74) Google Scholar), it is an intriguing possibility that glutamate may also contribute to the modulatory effects on fear evoked by DRN-BA circuit. Infusion of a combination of 5-HT1A and 5-HT2A receptor antagonists into the BA overrode the ability of the DRN-BA circuit to enhance fear responses. A key open question is the precise subpopulations of 5-HT responsive BA “fear” neurons involved during fear encoding and extinction training, as well as the contribution of different 5-HT receptors within these neuronal ensembles (Bocchio et al., 2016Bocchio M. McHugh S.B. Bannerman D.M. Sharp T. Capogna M. Serotonin, amygdala and fear: assembling the puzzle.Front. Neural Circuits. 2016; 10: 24Crossref PubMed Scopus (96) Google Scholar). Further, though the results support a primary influence of 5-HT released by the DRN onto the BA in the attuning of fear, they do not preclude the possibility of an involvement of glutamate co-secreted by these 5-HT neurons in influencing fear. The DRN-BA pathway is a “putative gain control” regulator that could scale other inputs onto the BA, serving to predispose this circuit in favor of fear, promoting fear learning and impeding effective extinction. This opens up a range of questions for future enquiry into the role of the DRN-BA circuit in the etiopathology and treatment of psychiatric disorders. A Discrete Dorsal Raphe to Basal Amygdala 5-HT Circuit Calibrates Aversive MemorySengupta et al.NeuronJune 13, 2019In BriefSengupta and Holmes use in vivo imaging, optogenetic, electrophysiological, and histological approaches to investigate the circuit-specific role of the DRN→BA 5-HT pathway in fear learning. They show this pathway is uniquely positioned, functionally and anatomically, to shape fear memory. Full-Text PDF Open Archive