Abstract
The ventromedial prefrontal cortex (vmPFC; rodent infralimbic cortex (IL)), is posited to be an important locus of fear extinction-facilitating effects of the dopamine (DA) bio-precursor, L-DOPA, but this hypothesis remains to be formally tested. Here, in a model of impaired fear extinction (the 129S1/SvImJ inbred mouse strain; S1), we monitored extracellular DA dynamics via in vivo microdialysis in IL during fear extinction and following L-DOPA administration. Systemic L-DOPA caused sustained elevation of extracellular DA levels in IL and increased neuronal activation in a subpopulation of IL neurons. Systemic L-DOPA enabled extinction learning and promoted extinction retention at one but not ten days after training. Conversely, direct microinfusion of DA into IL produced long-term fear extinction (an effect that was insensitive to ɑ-/ß-adrenoreceptor antagonism). However, intra-IL delivery of a D1-like or D2 receptor agonist did not facilitate extinction. Using ex vivo multi-electrode array IL neuronal recordings, along with ex vivo quantification of immediate early genes and DA receptor signalling markers in mPFC, we found evidence of reduced DA-evoked mPFC network responses in S1 as compared with extinction-competent C57BL/6J mice that were partially driven by D1 receptor activation. Together, our data demonstrate that locally increasing DA in IL is sufficient to produce lasting rescue of impaired extinction. The finding that systemic L-DOPA increased IL DA levels, but had only transient effects on extinction, suggests L-DOPA failed to reach a threshold level of IL DA or produced opposing behavioural effects in other brain regions. Collectively, our findings provide further insight into the neural basis of the extinction-promoting effects of DA and L-DOPA in a clinically relevant animal model, with possible implications for therapeutically targeting the DA system in anxiety and trauma-related disorders.
Similar content being viewed by others
Introduction
Cognitive behavioural therapy involving exposure to fear eliciting cues is an effective treatment for anxiety and trauma-related disorders [1,2,3]. During an extinction session, repeated exposure to a cue (conditioned stimulus, CS) that predicts a previously experienced aversive outcome (unconditioned stimulus, US) produces a reduction in conditioned responses via formation of a CS-no US association that behaviourally competes with the original CS-US memory. Deficits in fear extinction are reported in diverse anxiety- and trauma-associated disorders [e.g. post-traumatic stress disorder (PTSD)], taking the form of impaired extinction learning and/or subsequent cue- or context-driven fear relapse ([4, 5], for review see [6]). Hence, identifying the key loci within the neural circuitry underlying extinction deficits could inform strategies for the augmentation of fear extinction as a treatment for these disorders (for review see [7, 8]).
In recent years, we and other research groups have demonstrated a role of the neurotransmitter dopamine (DA) in fear extinction (for recent reviews see [9,10,11]) as enhancing DA levels by systemic administration of the DA bio-precursor, L-DOPA, DA or the non-selective monoaminergic drug, methylphenidate, facilitates extinction memory consolidation in healthy humans [12,13,14,15] and extinction-competent rodents, such as the C57BL/6 J (BL6) mouse strain [12, 16,17,18]. However, the neural loci of L-DOPA’s effects on extinction are only now beginning to be unravelled [13, 15, 19, 20].
Previous studies have shown that loss of DAergic neurons by intra-mPFC injection of 6-OHDA delays extinction learning and impairs retrieval [21, 22], whereas facilitation of extinction by L-DOPA is accompanied by increased activation in human vmPFC and mouse infralimbic cortex (IL; also area 25, consistent with the human subgenual anterior cingulate cortex [23,24,25]) [12]. Moreover, pharmacological blockade of D2 and D1 receptors specifically within IL has been shown to impair fear extinction learning and retrieval [26,27,28,29]. These observations are particularly notable because the vmPFC and IL—though the exact human analogous subregion of the vmPFC needs to be still clarified [25]—are strongly implicated in fear extinction ([30,31,32,33]; for review see [6, 34,35,36,37]): for example, vmPFC/IL recruitment during early extinction predicts successful extinction consolidation [30, 38,39,40,41,42] and the efficacy of therapeutic interventions [36, 43].
In this context, systemic administration of L-DOPA increases vmPFC activity during extinction consolidation in healthy volunteers in a manner predicting the later strength of extinction memory [13]. In addition, long-term rescue of deficient fear extinction in the 129S1/SvImJ (S1) mouse strain following a dietary, multimodal Zinc-targeting intervention is associated with normalization of aberrant neuronal activity and upregulation of DA receptor genes in mPFC [20, 44]. However, L-DOPA’s ability to rescue fear extinction in S1 mice over time periods longer than 24 h is limited [20]. Along similar lines, a recent study found that L-DOPA’s extinction-facilitating effects and associated vmPFC (re-)activation are attenuated in women with PTSD [19]. Hence, while these findings suggest the vmPFC/IL is a key locus of the fear extinction-promoting effects of L-DOPA [12, 13, 20], the drug’s actions in this region and its associated effects on extinction could be limited in individuals with deficient extinction.
Recent work has shown that, during fear extinction, DA neurons in the ventral tegmental area (VTA) signal a positive prediction error on omission of the expected aversive outcome (footshock) that drives extinction [45,46,47] and safety learning [48]. Indeed, DA levels rise during fear extinction training in the medial prefrontal cortex (mPFC) [49, 50]. However, since VTA DA projections to the mPFC are sparse [51] and seem to be involved in the encoding of aversive events [52,53,54] and in fear extinction impairment, rather than facilitation [45], extinction-related mPFC DA has been suggested to originate from another source, for example locus coeruleus noradrenergic neurons [55].
To address these questions in the current study, we used the S1 strain characterised by insufficient fear extinction—a hallmark of anxiety—and trauma-related disorders including PTSD, panic disorder and generalised anxiety disorder. S1 mice display several other clinical features including deficient safety learning, overgeneralization of fear, lower heart rate variability and, at the neural level, dysfunctions in key brain areas of the extinction neurocircuitry including the mPFC and amygdala, consistent with imaging studies of patients with PTSD [for review see [56]]. We examined the extinction-related effects of L-DOPA and DA in the S1 mice, and combined these behavioural analyses with in vivo measurement of DA (microdialysis) and ex vivo analysis (immediate early gene (IEG) activation, gene expression) of DA-related activity in mPFC/IL. Our findings provide further evidence that IL is a key locus of DAergic facilitation of extinction and show that systemic L-DOPA extinction-promoting effects are attenuated in animals with deficient extinction.
Materials and methods
Animals
All experimental procedures on adult male S1 and BL6 mice were approved by the national ethical committee on animal care and use (Bundesministerium für Bildung, Wissenschaft und Forschung) in compliance with international laws and policies (for further details on this and all other aspects of the Materials and methods, see Supplemental Information).
Stereotaxic surgery
Guide cannulae for microinfusers were implanted above the IL target according to previous protocols [57], focusing on the rostral part of IL because of its strong connections to the basal amygdala, relative to more caudal IL [58]. Microdialysis probes were too large to be restricted exclusively to IL and therefore targeted a larger region encompassing IL and dorsally-neighbouring prelimbic cortex (PL) which we refer to collectively as mPFC. During recovery, single-housed mice received analgesic care.
Extinction of cued conditioned fear
Animals were cue fear conditioned by using three pairings of a tone cue (75 dB, 30 s; CS) with a co-terminating mild, scrambled foot shock (0.6 mA, 2 s; US) in context A. Fear extinction was performed by exposing animals to 16 CS for training and two CS tones for retrieval in a novel context B which differed from context A in visual, olfactory and spatial signals on experimental days 2, 3 and 13, respectively, (ABBB design) according to previous protocols [20, 57].
In vivo microdialysis measurements of extracellular mPFC DA
Microdialysis fractions of 5 and 10 minutes, respectively, were continuously collected in rostral mPFC of freely moving mice, as previously described [59], before, during and after fear extinction sessions (Fig. 1A). DA concentrations in microdialysates were determined by using high-performance liquid chromatography with electrochemical detection according to our published protocol [59].
Systemic and intra-IL drug administration
For systemic administration, L-DOPA (20 mg/kg), benserazide (12.5 mg/kg), the D1-like receptor agonist SKF-81297 (10 mg/kg) and the selective D2 receptor agonist sumanirole (10 mg/kg) were dissolved in saline or saline containing 1% DMSO and 3.3% Tween 80 for systemic (i.p., 10 ml/kg) administration. For intra-IL administration, DA (40 µg/hemisphere), SKF-81297 (0.1 µg/hemisphere), sumanirole (10 µg/hemisphere), phentolamine (20 µg/hemisphere) and timolol (20 µg/hemisphere) were dissolved in artificial cerebrospinal fluid (aCSF).
Multi-electrode array (MEA) mPFC neuronal recordings
Spontaneous action potential discharge activity was recorded in acute mPFC coronal brain slices (300 µm) according to a previous protocol [60] under physiological conditions and in the presence of DAergic ligands.
Quantitative polymerase chain reaction (qPCR)
Reverse qPCR with forward and reverse primers designed against the target of interest was performed on complementary cDNA derived from homogenised frozen tissue punches containing both IL and PL subregions using a commercially available kit (Qiagen Biosciences, USA) in combination with Fast SYBR green master mix (Applied Biosciences, USA).
Quantitative D1-like and D2-like receptor autoradiography
D1-like and D2-like receptors were visualized on frozen tissue sections (12 µm) using [3H]-SCH-23390 (1 nM) and [3H]-nemonapride (1 nM) according to previous protocols [61]. Receptor density was determined in IL and PL, which were identified with reference to a mouse brain atlas, from digitized autoradiograms.
Arc fluorescence in situ hybridization (FISH) in mPFC neurons
mRNA expression of the activity-regulated cytoskeleton-associated protein (Arc) was detected by TSA-amplified FISH using a customized, fluorescein-labelled probe (Exiqon, Denmark) according to a previous protocol [62] and quantified in IL and PL by fluorescent microscopy.
IEG immunohistochemistry of pERK and pCREB in mPFC neurons
Immunohistochemistry was performed on free-floating brain slices (40 µm) using rabbit anti-pCREB and rabbit anti-pERK (Cell Signalling Technology, USA) as primary antibodies and a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, USA). The number of immuno-positive cells was quantified in IL and PL.
Statistical analysis
Data are expressed as mean ± standard error of the mean (sem). Statistical analysis of data was performed using Statistica 13 (StatSoft Europe). The level of statistical significance was set to p < 0.05.
Results
L-DOPA rescues fear extinction and augments extinction-related mPFC DA levels
We first performed in vivo microdialysis to monitor extracellular DA concentrations in mPFC (Fig. 1A–C) of S1 mice during fear extinction training after systemic treatment with a dose of L-DOPA-previously shown to rescue fear extinction in this mouse strain [20]. On experimental day 1, mice were fear conditioned to a tone CS in context A [pairing effect: F(2,34) = 37.41, p < 0.001; Fig. 1D]. On the next day microdialysates were collected in the mPFC of both experimental groups in home cages until stabilized at an average of 0.22 ± 0.01 fmol/sample within the first 100 minutes [time: F(9,135) = 1.30, p = 0.243; time × treatment: F(9,135) = 1.25, p = 0.273; Fig. 1C] reflecting baseline. Then, either L-DOPA (20 mg/kg i.p.) or vehicle was administered i.p. one hour before fear extinction training and mice were placed into context B (differing from context A in visual, olfactory and spatial cues) for microdialysate collection during a further one-hour baseline period followed by extinction training.
In mPFC dynamic changes in extracellular DA concentrations were observed on the day of extinction training [time × treatment: F(27,378) = 4.91, p < 0.001; Fig. 1C]. L-DOPA produced a pronounced and sustained increase in extracellular DA levels, as compared to vehicle, during the context B baseline period. Importantly, extinction training was associated with a further increase in DA levels over context B baseline, to the same extent in both treatment groups, but at an overall higher level in L-DOPA-treated mice than vehicle-treated controls (Fig. 1C). In L-DOPA-treated animals DA levels remained higher relative to vehicle-treated controls for another 50 minutes and above baseline throughout the whole observation period after extinction training (Fig. 1C).
Behaviourally, replicating previous data [20], L-DOPA promoted fear extinction learning in S1 mice [time x treatment interaction: F(7,91) = 4.14, p < 0.001; Fig. 1D] and the formation of a fear extinction memory—as indicated by lower levels of CS-related freezing during extinction training and drug-free retrieval testing the following day as compared with vehicle controls. On extinction retrieval test, however, freezing levels of both L-DOPA and vehicle-treated groups was higher than at the end of the extinction training and is most likely to reflect fear incubation, a phenomenon that has been reported previously in the extinction-impaired S1 strain [63].
These data demonstrate that the extinction-rescuing effect of L-DOPA is associated with an increase in mPFC DA levels in vivo.
L-DOPA alters extinction-related neuronal activity in mPFC
To complement these findings, we examined the effects of systemic L-DOPA on extinction-related neuronal activity in mPFC via expression of the IEG Arc—which has been previously shown to be upregulated following successful fear extinction training [64, 65]. In S1 mice we found that L-DOPA treatment did not affect overall Arc mRNA expression in IL following extinction training [t(12) = 0.176, p = 0.863; data not shown, see also Fig. S1C). However, when we separately analysed Arc mRNA expression in cells co-labelled with calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα; Fig. 1E), a marker primarily, though not solely (e.g., [66, 67]), for excitatory pyramidal neurons [68], we observed an increase in Arc mRNA expression exclusively to CaMKIIα-negative cells in the IL following L-DOPA treatment [t(12) = 2.560, p = 0.025; Fig. 1F]. This effect appeared to be most pronounced for cells expressing Arc mRNA in both cytoplasm and nucleus of CaMKIIα-negative cells (Fig. S1E) marking neurons persistently active throughout training as revealed by cellular compartment FISH (catFISH) analysis. Lastly, we observed no L-DOPA-related effects on Arc labelling in PL [overall: t(12) = 0.655, p = 0.525; CaMKIIα-negative cells: t(12) = 1. 477, p = 0.165; Fig. 1F; see also Fig. S1D–F].
These data indicate that systemic L-DOPA treatment produces an extinction-related increase in neuronal activity in a specific subset of IL, but not PL, neurons.
Direct infusion of DA into IL rescues deficient extinction
Our microdialysis and neuronal activation data indicate a concordance between the effects of systemic L-DOPA on mPFC DA levels, IL neuronal activation and a rescue of extinction in otherwise impaired animals. Given this link, we next asked whether direct administration of DA into IL per se was sufficient to recapitulate the effects of systemic L-DOPA. In fear-conditioned S1 mice [pairing: F(2,34) = 56.89, p < 0.001; pairing x treatment: F(2,34) = 0.758, p = 0.476] intra-IL DA microinfusion (Fig. 2A) prior to extinction training caused decreased freezing levels, relative to vehicle infusion, on late not early, extinction training CS trial-blocks [CS × treatment: F(7,119) = 6.93, p < 0.001; Fig. 2B]. Intra-IL DA-treated animals continued to show lower freezing levels on drug-free extinction retrieval tests, both at one [t(17) = 4.00; p < 0.001] and ten [t(15) = 3.63; p = 0.003) days after extinction training as compared with vehicle-treated controls (Fig. 2B).
Previous studies have implicated D1 and D2 receptors, including those expressed in IL, in fear extinction [26,27,28,29, 69, 70]. This led us test whether the pro-extinction effects of intra-IL DA (Fig. 2B) could be mimicked by activation of D1 or D2 receptors in IL of fear-conditioned S1 mice [pairing: F(2,74) = 87.65, p < 0.001; pairing x treatment: F(6,74) = 1.72, p = 0.129]. Pre-training intra-IL infusion (Fig. 2C) of either a D1-like receptor agonist (SKF-81297) or a D2 receptor agonist (sumanirole), or a combination of the two drugs did not affect, relative to vehicle, freezing levels during extinction training [CS × treatment: F(21,259) = 1.00, p = 0.461] and subsequent one day or ten day post-training extinction retrieval tests [day 3: F(3,37) = 0.84, p = 0.480] and day 13 [F(3,36) = 1.37, p = 0.266; Fig. 2D).
These findings demonstrate that intra-IL delivery of DA, but not D1-like or D2 receptor activation alone, is sufficient to produce long-lasting rescue of impaired extinction.
Adrenoceptor blockade enhances intra-IL DA rescue of deficient extinction
DA can also bind to adrenergic receptors, though with lower affinity than to its preferred DA receptors and, additionally, can be converted to noradrenaline (NA) [71]. Since mPFC NA has been shown to alter fear extinction via α- and β- adrenoreceptors ([72], for review see [73, 74]), it may be speculated that NA contributed to the DA-related rescue of extinction we observed in S1 mice. To address this possibility, we microinfused a mixture of the adrenoceptor blockers, pan-α- (phentolamine) and pan-β (timolol), into IL of fear conditioned S1 mice [pairing: F(3,66) = 61.49, p < 0.001; pairing × treatment: F(6,66) = 0.344, p = 0.911] ten minutes prior to intra-IL DA infusion (Fig. 2E), and then tested for extinction training.
We found—replicating our earlier result—that intra-IL DA reduced freezing levels on late extinction training trial-blocks, relative to vehicle controls. In addition, we observed that pre-treatment with the two adrenoceptor blockers accelerated the extinction-related decrease in freezing levels (i.e., effect evident on intermediate trial-blocks), relative to DA-alone [trial × treatment: F(14,112) = 3.24, p < 0.001; Fig. 2F). Freezing levels were lower in the DA+adrenoceptor blocker and DA-alone groups than vehicle controls on one-day and ten-day extinction retrieval tests, but the treatment groups did not differ from one another [day 3: F(2,21) = 7.32, p = 0.004 and day 13 [F(2,19) = 19.39, p < 0.001; Fig. 2F].
The effects of these adrenoceptor blockers suggest that NA activation of adrenoceptors opposes the extinction rescuing effects of intra-IL administration of DA. Though this effect was only evident as lower freezing in the adrenoceptor blocker group during extinction learning, it may have been occluded by low ‘floor’ levels of freezing during extinction retrieval.
Augmenting L-DOPA-induced DA does not prevent long-term fear relapse
Thus far, our results show that increasing IL DA levels is sufficient to rescue extinction learning and retrieval for up to ten days post-training. However, while we showed here that systemic L-DOPA rescued extinction retrieval one day post-training, we had previously shown that this effect did not persist at the ten-day retrieval timepoint [20]. The transient nature of the L-DOPA effect is of potential clinical importance given the efficacy of L-DOPA as an adjunct for extinction-based therapies assumes its ability to confer long-term protection against fear relapse [19].
This led us to ask whether a second dose of systemic L-DOPA would produce sustained DA increases into the extinction consolidation period and longer-lasting effects on extinction. L-DOPA was administered to fear-conditioned S1 mice [pairing: F(2,54) = 40.18, p < 0.001; pairing × treatment: F(4,54) = 0.674, p = 0.613] either prior to extinction training or both prior to and immediately after extinction training. In this experiment, L-DOPA was co-administered with the decarboxylase inhibitor benserazide as a means to prevent peripheral decarboxylation of L-DOPA to DA [75]. We found that, relative to vehicle-treated controls, systemic L-DOPA administration decreased freezing levels on late extinction training trial-blocks [CS × treatment: F(7,196) = 2.07, p = 0.049] and drug-free extinction retrieval testing the next day [F(2,17) = 3.83, p = 0.042), but not on the ten-day test [F(2,19) = 0.20, p = 0.822, Fig. 2G], irrespective of whether L-DOPA was administered once or twice.
The findings of this experiment replicate the short-term extinction-promoting effect of L-DOPA in S1 mice and extend these data by showing that a second dose of L-DOPA during the extinction consolidation period did not bolster the long-term effects of L-DOPA on extinction.
Abnormal DA-mediated mPFC neuronal responses in extinction-deficient mice
The inability of L-DOPA treatment to protect against post-extinction long-term recovery of fear in S1 mice (Fig. 2G, [20]) could potentially reflect abnormal DAergic signalling in mPFC. To investigate this possibility, S1 and extinction-competent BL6 mice (for previous comparison of these behavioural phenotypes, see e.g., [44, 63]) were fear-conditioned [pairing: F(2,32) = 69.03, p < 0.001; pairing x strain: F(2,36) = 1.28, p = 0.289; Fig. 3D] and mPFC DA dynamics between the two strains were compared the next day using in vivo microdialysis (as described above; Fig. 3A, B). Similar, dynamic changes in extracellular DA concentrations were revealed in S1 and BL6 mice on the day of extinction training [time: F(27,432) = 4.44, p < 0.001; strain × time: F(27,432) = 1.09, p = 0.349; Fig. 3C]. After an acclimation period in the home cage, DA levels were stable at 0.22 ± 0.02 fmol/sample in BL6 and 0.20 ± 0.01 fmol/sample in S1 mice [time: F(9,144) = 1.505, p = 0.151] and did not differ between the strains [strain: F(1,16) = 0.106, p = 0.750], irrespective of time [strain × time: F(9,144) = 0.272, p = 0.981; Fig. 3C].
On transfer to the extinction context B, there was a greater initial increase in mPFC DA levels in S1 than BL6 mice – a response likely due to an effect of handling and/or exposure to the novel context, as this difference resolved prior to extinction training. During extinction training, the strains displayed a similar CS-related increase in DA levels (p < 0.09 + 2.5 min vs. −0.5 min and −15 min; p < 0.05 + 2.5 min vs. −25 min and all baseline timepoints) with a peak increase later in training (p < 0.001 + 7.5 min vs. all timepoints before). On return to the home cage, both strains showed a decrease to similar baseline DA levels within ~20 min. Despite the similarity of DA dynamics during extinction, S1 mice exhibited higher levels of CS-related freezing than BL6 mice on training [strain × time: F(7,98) = 11.43, p < 0.001] and extinction retrieval testing the next day [t(16) = 4.42, p < 0.001; Fig. 3D].
These data show that deficient extinction in S1 mice is not related to abnormal extracellular DA availability in mPFC. This suggests that either DA produces abnormal neuronal responses in S1 mPFC neurons or other brain regions and neurotransmitters, respectively, are responsible. Therefore, to provide a functional readout of DA-mediated effects in mPFC, we performed ex vivo multi-electrode array (MEA) recordings comparing DA-evoked mPFC neuronal responses in S1 and BL6 mice (Fig. 4A). There were no differences between strains in spontaneous mPFC neuronal activity, as measured by firing rate (p = 0.12; Fig. 4B) and the percentage of basally active recording electrodes (p = 0.57; Fig. 4C). In response to bath application of DA, however, S1 mice had a lesser percentage of electrodes exhibiting increased activity than BL6 mice, and a greater percentage showing decreased activity (χ² = 32.44, p < 0.001; Fig. 4D). A similar pattern of strain differences was evident in response to application of the D1-like receptor agonist SKF-81297 (100 µM; χ² = 32.93, p < 0.001; Fig. 4E), whereas D2 receptor agonist sumanirole (100 µM) failed to affect activity in either strain (100 µM; χ² = 2.28, p = 0.24; Fig. 4F).
These results show that S1 and BL6 mice exhibited similar extracellular mPFC DA levels during extinction, but differ in mPFC neuronal responses to DA and D1-like receptor activation in a manner suggestive of a bias towards neuronal inhibition over excitation in S1 mice.
Abnormal DA-mediated mPFC signalling in extinction-deficient mice
We next assessed whether altered DA-mediated network responses in mPFC neurons in S1 mice reflected abnormal DA receptor signalling in IL [76]. Using immunohistochemistry, we measured IL neuronal expression of phosphorylated ERK (pERK; Fig. 4G) and CREB (pCREB; Fig. 4J)—two canonical downstream DA signalling molecules [77, 78]—following systemic administration of L-DOPA or SKF-82197. As mPFC network responses were unaltered following application of sumanirole, we did not include it in this experiment fulfilling the 3Rs (reduction). We observed fewer pERK-positive cells in IL, particularly in layers II-III, in S1 than BL6 mice following administration of SKF-82197, but not L-DOPA [strain: F(1,12) = 6.99, p = 0.021; strain × treatment: F(1,12) = 3.73, p = 0.077; Fig. 4H, Table 1). A similar trend was evident for pCREB expression by strain and treatment [strain: F(1,19) = 3.729, p = 0.069; strain × treatment: F(1,19) = 2.09, p = 0.051], and post-hoc analysis of pCREB expression revealed a significantly lower number of pCREB-positive cells in IL of S1 mice after SKF-81297 administration relative to BL6 mice (Fig. 4K, Table 1]. Notably, these differences in pERK and pCREB were specific to IL, with no differences apparent in PL (pERK: strain × treatment: F(1,12) = 0.260, p = 0.620; pCREB: strain × treatment: F(1,19) = 0.1.336, p = 0.262; Fig. 4I, L, Table 1), cingulate or motor cortex (Supplementary Table S1).
Finally, using FISH, we examined in S1 and BL6 mice the extinction-related mRNA expression of the IEG Arc, which is downstream of several converging signalling pathways including the D1 receptor-ERK/CREB transduction [76, 77, 79]. Relative to BL6 mice, Arc mRNA expression was reduced in IL [strain: F(1,42) = 6.260, p = 0.016, Fig. S1G], but not in PL [strain: F(1,41) = 1.565, p = 0.218, Fig. S1H].
These data show that D1-like receptor-mediated signalling is blunted in IL of S1 mice. This effect likely reflects a functional abnormality, rather than reduced D1 receptor expression, as we found no strain differences in basal IL gene expression or number of receptor binding sites for D1 or other DA receptor subtypes (Table 2).
Discussion
A major aim of the current study was to determine whether the mPFC serves as a key neural locus for the extinction-rescuing effects of L-DOPA in a model of deficient extinction (the S1 inbred mouse strain [44, 63]). Our findings show that the systemic delivery of L-DOPA augments extinction-related DA levels in mPFC and generates an extinction memory in S1 mice that persists for one, but not ten days. Conversely, direct microinfusion of DA (but not D1-like or D2 receptor agonists) into IL produced a robust and long-lasting rescue of extinction. Finally, we found evidence of abnormal DA-mediated neuronal responses in IL of S1 mice, suggesting a possible mechanism to account for the lack of long-term extinction-rescuing effects of L-DOPA.
Stimulation of the vmPFC has been proposed as a therapeutic approach to facilitate fear extinction in individuals with PTSD [80], with promising preliminary results [81]. Cross-species translational studies have shown that a single dose of L-DOPA produces long-term facilitation of fear extinction and associated increases in the activity of the vmPFC/IL in extinction-competent human subjects and rodents [12, 13]. Here, replicating and extending previous data in extinction-competent rats ([49, 50], but see [82]), we found extinction training was associated with increased extracellular mPFC DA availability in S1 mice. Specifically, DA levels were elevated during the first five minutes of training and peaked in the second half of the training session, indicating these effects were unrelated to handling or the novelty of the extinction context [83]. These behaviour-related mPFC DA levels were augmented by systemic L-DOPA administration, in line with a previous finding in rats tested in a cocaine-seeking procedure [55]. These data are notable because although the vmPFC/IL is strongly implicated in fear extinction, prior work had not determined a causal contribution of DA neurotransmission in this brain region to fear extinction (for review see [9, 11]).
Of further importance in this regard, systemic L-DOPA rescued extinction in S1 mice that was evident one day (see also [20]) after training. However, this effect was absent ten days post-training (see also [20]). These findings in mice agree well with recent data showing reduced efficacy of systemic L-DOPA in facilitating fear extinction in female patients with PTSD [19], relative to healthy individuals [12,13,14]. In contrast to the time-limited effects of systemic L-DOPA, we demonstrate that infusing DA into IL is in and of itself sufficient to generate an extinction memory in S1 mice. While the mechanisms underlying this effect remain to be elucidated, we posit that elevated DA levels enhanced IL neuronal excitability to promote extinction [30, 39, 40, 84, 85]. These findings also suggest that, unlike direct DA delivery to IL, systemic L-DOPA may not increase DA levels in this region beyond a threshold level necessary to generate a persistent extinction memory in otherwise compromised subjects. In an attempt to mitigate this putative DA deficit, we gave S1 mice L-DOPA both pre and post extinction training (and even in combination with benserazide, as common prescription in clinical practice, in order to reduce its peripheral degradation), but this treatment regimen was also unable to produce a persistent extinction rescue.
Another possibility is that reduced efficacy of L-DOPA is related to abnormal mPFC DA neurotransmission in extinction-comprised subjects. Indeed, there is an increasing number of reports that central DA markers, including the DA transporter, the DA degrading enzyme COMT, D1 and D2 receptors, are abnormal in patients with an anxiety disorder or PTSD [86,87,88,89,90,91,92,93,94,95,96]. In IL of S1 mice, abnormal DA signalling could arise from deficits in DA release from terminals of VTA and/or locus coeruleus [55] or in synaptic signalling at IL neurons. We were able to demonstrate that, relative to BL6 mice, S1 mice exhibited attenuated electrophysiological mPFC neuronal responses to DA and blunted IL neuron molecular signalling responses (notably including Arc [64, 65]) to L-DOPA and DA receptor agonists.
These analyses also showed that L-DOPA modified the activity of a subset of CaMKIIα-negative neurons (likely primarily interneurons) recruited during extinction training. Since interneurons can either synchronize the output of excitatory pyramidal cells [97, 98], increase cortical output via disinhibition [99] or broadly reduce mPFC neuronal output [100, 101], the consequences of extinction-related interneuron activation are difficult to predict. D1 receptors are expressed on VIP-positive interneurons which enhance action potential firing by influencing excitatory and/or disinhibitory microcircuits in deep and superficial layers of the mPFC, respectively [102]. Thus, D1 receptor dysfunction in S1 mice could result in a hypoactive IL ([44], but see [103]) in response to extracellular DA released during fear extinction training. In this context, social anxiety disorder and obsessive-compulsive disorder are associated with decreased D1 receptor binding [92] and increased D2 receptor in cortical subregions [91], with the latter being lower after successful psychotherapy [104].
Phasic DA release of the type that likely occurs during extinction favours activity at D1 over D2 receptors [105], whereas high levels of mPFC DA increases activation of D2 receptors expressed on pyramidal mPFC neurons [106], which in turn has been shown to increase response-related activity of layer 5 neurons in the PFC [107] via activation of NMDA receptors [108, 109] and recruitment of a Gs protein-coupled signalling pathways. Hence, elevated DA produced by DA or L-DOPA might generate a high neuronal activity DA state that shifts mPFC network responses from D1 receptor dominated mode of transmission to one that recruits other DA receptors [110, 111], potentially promoting extinction-enabling processes such as arousal and flexible behavioural control [112].
While additional work is needed to assess this hypothesis, local agonist activation of mPFC D1-like or D2 receptors did not affect within-session extinction in S1 mice (current study). Together with our finding of D1 receptor dysfunction in S1 mice, we posit that, despite triggering some level of downstream signalling following D1 receptor stimulation, the effect of D1 or D2 agonism is insufficient to rescue fear extinction in S1 mice or, alternatively, that IL D1 receptors are not involved in fear extinction acquisition. This latter possibility is supported by previous studies in extinction-competent subjects [26,27,28,29]. Earlier studies have also found that D2 receptor agonism impairs fear extinction, while D2 blockade facilitates extinction [28, 113], therefore it is unlikely that selectively activating D2 receptors would improve extinction in S1 mice. Instead, these data suggest that other DA receptors, including the D3 and D4 subtypes, or combined actions at multiple DA receptors, underlie the extinction-promoting effects of DA and L-DOPA in S1 mice. Indeed, we have previously found that deficient fear extinction in S1 mice is associated with upregulated D3, D4 and D5 receptor expression in mPFC [20]. Future studies examining the functional role of these receptor subtypes would be valuable in deciphering the cause of L-DOPA’s time-limited effects on fear extinction.
In sum, the current findings demonstrate that delivering DA into IL caused long-lasting rescue of deficient fear extinction in a clinically relevant model organism whereas, despite a pronounced rise in mPFC DA, systemic administration of L-DOPA produced shorter-lasting extinction-rescuing effects. Our results suggest that abnormal neuronal and intracellular signalling responses to DA in IL could account for the reduced efficacy of L-DOPA in the extinction-deficient mouse model employed. By extension, these findings support the idea that the use of systemic L-DOPA as a pharmacological adjunct to exposure-based therapy could be particularly efficacious in individuals with impaired fear extinction, but may be of limited efficacy in patients with severely impaired extinction which relies on similar or even same neurobiological abberations as observed in our mouse model.
Data availability
The corresponding author will make available the relevant data upon request. Additional data are provided in supplementary tables of this manuscript.
Materials availability
Additional information on materials will be made available from the corresponding author upon request.
References
Bandelow B, Lichte T, Rudolf S, Wiltink J, Beutel ME. The German guidelines for the treatment of anxiety disorders. Eur Arch Psychiatry Clin Neurosci. 2015;265:363–73.
McLean CP, Levy HC, Miller ML, Tolin DF. Exposure therapy for PTSD: a meta-analysis. Clin Psychol Rev. 2022;91:102115.
Carpenter JK, Andrews LA, Witcraft SM, Powers MB, Smits JAJ, Hofmann SG. Cognitive behavioral therapy for anxiety and related disorders: a meta-analysis of randomized placebo-controlled trials. Depress Anxiety. 2018;35:502–14.
Taylor S, Abramowitz JS, McKay D. Non-adherence and non-response in the treatment of anxiety disorders. J Anxiety Disord. 2012;26:583–9.
Loerinc AG, Meuret AE, Twohig MP, Rosenfield D, Bluett EJ, Craske MG. Response rates for CBT for anxiety disorders: need for standardized criteria. Clin Psychol Rev. 2015;42:72–82.
Sartori SB, Singewald N. Novel pharmacological targets in drug development for the treatment of anxiety and anxiety-related disorders. Pharm Ther. 2019;204:107402.
Bukalo O, Pinard CR, Holmes A. Mechanisms to medicines: elucidating neural and molecular substrates of fear extinction to identify novel treatments for anxiety disorders. Br J Pharm. 2014;171:4690–718.
Hariri AR, Holmes A. Finding translation in stress research. Nat Neurosci. 2015;18:1347–52.
Salinas-Hernández XI, Duvarci S. Dopamine in fear extinction. Front Synaptic Neurosci. 2021;13:635879.
Singewald N, Sartori SB, Reif A, Holmes A. Alleviating anxiety and taming trauma: novel pharmacotherapeutics for anxiety disorders and posttraumatic stress disorder. Neuropharmacology. 2023;226:109418.
Likhtik E, Johansen JP. Neuromodulation in circuits of aversive emotional learning. Nat Neurosci. 2019;22:1586–97.
Haaker J, Gaburro S, Sah A, Gartmann N, Lonsdorf TB, Meier K, et al. Single dose of L-dopa makes extinction memories context-independent and prevents the return of fear. Proc Natl Acad Sci USA. 2013;110:E2428–2436.
Gerlicher AMV, Tüscher O, Kalisch R. Dopamine-dependent prefrontal reactivations explain long-term benefit of fear extinction. Nat Commun. 2018;9:4294.
Gerlicher AMV, Tüscher O, Kalisch R. L-DOPA improves extinction memory retrieval after successful fear extinction. Psychopharmacology. 2019;236:3401–12. https://doi.org/10.1007/s00213-019-05301-4.
Esser R, Korn CW, Ganzer F, Haaker J. L-DOPA modulates activity in the vmPFC, nucleus accumbens, and VTA during threat extinction learning in humans. Elife. 2021;10:e65280.
Abraham AD, Cunningham CL, Lattal KM. Methylphenidate enhances extinction of contextual fear. Learn Mem. 2012;19:67–72.
de Carvalho, Figueiredo MC, de RM, Coimbra NC, Leite-Panissi CRA, de Souza Silva MA, Huston JP, et al. Intranasal dopamine attenuates fear responses induced by electric shock to the foot and by electrical stimulation of the dorsal periaqueductal gray matter. J Psychopharmacol. 2019;33:1524–32.
de Almeida Silva M, de Toledo TS, de Figueiredo RM, Leite-Panissi CRA, Huston JP, Coimbra NC, et al. The activation of D2-like receptors by intranasal dopamine facilitates the extinction of contextual fear and prevents conditioned fear-induced antinociception. Behav Brain Res. 2022;417:113611.
Cisler JM, Privratsky AA, Sartin-Tarm A, Sellnow K, Ross M, Weaver S, et al. L-DOPA and consolidation of fear extinction learning among women with posttraumatic stress disorder. Transl Psychiatry. 2020;10:287.
Whittle N, Maurer V, Murphy C, Rainer J, Bindreither D, Hauschild M, et al. Enhancing dopaminergic signaling and histone acetylation promotes long-term rescue of deficient fear extinction. Transl Psychiatry. 2016;6:e974.
Fernandez Espejo E. Prefrontocortical dopamine loss in rats delays long-term extinction of contextual conditioned fear, and reduces social interaction without affecting short-term social interaction memory. Neuropsychopharmacology. 2003;28:490–8.
Morrow BA, Elsworth JD, Rasmusson AM, Roth RH. The role of mesoprefrontal dopamine neurons in the acquisition and expression of conditioned fear in the rat. Neuroscience. 1999;92:553–64.
Laubach M, Amarante LM, Swanson K, White SR. What, if anything, is rodent prefrontal cortex? eNeuro 2018;5:ENEURO.0315-18.2018. https://doi.org/10.1523/ENEURO.0315-18.2018.
Heukelum, van S, Mars RB, Guthrie M, Buitelaar JK, Beckmann CF, Tiesinga PHE, et al. Where is cingulate cortex? a cross-species view. Trends Neurosci. 2020;43:285–99.
Alexander L, Wood CM, Roberts AC. The ventromedial prefrontal cortex and emotion regulation: lost in translation? J Physiol. 2023;601:37–50.
Mueller D, Bravo-Rivera C, Quirk GJ. Infralimbic D2 receptors are necessary for fear extinction and extinction-related tone responses. Biol Psychiatry. 2010;68:1055–60.
Dadkhah M, Abdullahi PR, Rashidy-Pour A, Sameni HR, Vafaei AA. Infralimbic dopamine D2 receptors mediate glucocorticoid-induced facilitation of auditory fear memory extinction in rats. Brain Res. 2018;1682:84–92.
Zbukvic IC, Park CHJ, Ganella DE, Lawrence AJ, Kim JH. Prefrontal dopaminergic mechanisms of extinction in adolescence compared to adulthood in rats. Front Behav Neurosci. 2017;11:32.
Hikind N, Maroun M. Microinfusion of the D1 receptor antagonist, SCH23390 into the IL but not the BLA impairs consolidation of extinction of auditory fear conditioning. Neurobiol Learn Mem. 2008;90:217–22.
Milad MR, Quirk GJ. Neurons in medial prefrontal cortex signal memory for fear extinction. Nature. 2002;420:70–74.
Giustino TF, Maren S. The Role of the Medial Prefrontal Cortex in the Conditioning and Extinction of Fear. Front Behav Neurosci. 2015;9:298. https://doi.org/10.3389/fnbeh.2015.00298.
Keller NE, Hennings AC, Leiker EK, Lewis-Peacock JA, Dunsmoor JE. Rewarded extinction increases amygdalar connectivity and stabilizes long-term memory traces in the vmPFC. 2021.
Dunsmoor JE, Kroes MCW, Li J, Daw ND, Simpson HB, Phelps EA. Role of human ventromedial prefrontal cortex in learning and recall of enhanced extinction. J Neurosci. 2019;39:3264–76.
Nett KE, LaLumiere RT. Infralimbic cortex functioning across motivated behaviors: can the differences be reconciled? Neurosci Biobehav Rev. 2021;131:704–21.
Namkung H, Thomas KL, Hall J, Sawa A. Parsing neural circuits of fear learning and extinction across basic and clinical neuroscience: towards better translation. Neurosci Biobehav Rev. 2022.134:104502. https://doi.org/10.1016/j.neubiorev.2021.12.025.
Webler RD, Oathes DJ, van Rooij SJH, Gewirtz JC, Nahas Z, Lissek SM, et al. Causally mapping human threat extinction relevant circuits with depolarizing brain stimulation methods. Neurosci Biobehav Rev. 2023;144:105005.
Tovote P, Fadok JP, Lüthi A. Neuronal circuits for fear and anxiety. Nat Rev Neurosci. 2015;16:317–31.
Bukalo O, Pinard CR, Silverstein S, Brehm C, Hartley ND, Whittle N, et al. Prefrontal inputs to the amygdala instruct fear extinction memory formation. Sci Adv. 2015;1:e1500251. https://doi.org/10.1126/sciadv.1500251.
Do-Monte FH, Manzano-Nieves G, Quiñones-Laracuente K, Ramos-Medina L, Quirk GJ. Revisiting the role of infralimbic cortex in fear extinction with optogenetics. J Neurosci. 2015;35:3607–15.
Maroun M, Kavushansky A, Holmes A, Wellman C, Motanis H. Enhanced extinction of aversive memories by high-frequency stimulation of the rat infralimbic cortex. PLoS ONE. 2012;7:e35853.
Mukherjee A, Caroni P. Infralimbic cortex is required for learning alternatives to prelimbic promoted associations through reciprocal connectivity. Nat Commun. 2018;9:2727.
Bukalo O, Nonaka M, Weinholtz CA, Mendez A, Taylor WW, Holmes A. Effects of optogenetic photoexcitation of infralimbic cortex inputs to the basolateral amygdala on conditioned fear and extinction. Behav Brain Res. 2021;396:112913.
Lange I, Goossens L, Michielse S, Bakker J, Vervliet B, Marcelis M, et al. Neural responses during extinction learning predict exposure therapy outcome in phobia: results from a randomized-controlled trial. Neuropsychopharmacology. 2020;45:534–41.
Whittle N, Hauschild M, Lubec G, Holmes A, Singewald N. Rescue of impaired fear extinction and normalization of cortico-amygdala circuit dysfunction in a genetic mouse model by dietary zinc restriction. J Neurosci. 2010;30:13586–96.
Luo R, Uematsu A, Weitemier A, Aquili L, Koivumaa J, McHugh TJ, et al. A dopaminergic switch for fear to safety transitions. Nat Commun. 2018;9:2483.
Salinas-Hernández XI, Vogel P, Betz S, Kalisch R, Sigurdsson T, Duvarci S. Dopamine neurons drive fear extinction learning by signaling the omission of expected aversive outcomes. Elife. 2018;7:e38818.
Cai LX, Pizano K, Gundersen GW, Hayes CL, Fleming WT, Holt S. et al. Distinct signals in medial and lateral VTA dopamine neurons modulate fear extinction at different times. Elife 2020;9:e54936. https://doi.org/10.7554/eLife.54936.
Yau JO-Y, McNally GP. The activity of ventral tegmental area dopamine neurons during shock omission predicts safety learning. Behav Neurosci. 2022;136:276–84.
Feenstra MG, Vogel M, Botterblom MH, Joosten RN, de Bruin JP. Dopamine and noradrenaline efflux in the rat prefrontal cortex after classical aversive conditioning to an auditory cue. Eur J Neurosci. 2001;13:1051–4.
Hugues S, Garcia R, Léna I. Time course of extracellular catecholamine and glutamate levels in the rat medial prefrontal cortex during and after extinction of conditioned fear. Synapse. 2007;61:933–7.
Huang S, Zhang Z, Gambeta E, Xu SC, Thomas C, Godfrey N, et al. Dopamine inputs from the ventral tegmental area into the medial prefrontal cortex modulate neuropathic pain-associated behaviors in mice. Cell Rep. 2020;31:107812.
Vander Weele CM, Siciliano CA, Matthews GA, Namburi P, Izadmehr EM, Espinel IC, et al. Dopamine enhances signal-to-noise ratio in cortical-brainstem encoding of aversive stimuli. Nature. 2018;563:397–401.
Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye KM, et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature. 2012;491:212–7.
Hitora-Imamura N, Miura Y, Teshirogi C, Ikegaya Y, Matsuki N, Nomura H. Prefrontal dopamine regulates fear reinstatement through the downregulation of extinction circuits. Elife. 2015;4:e08274. https://doi.org/10.7554/eLife.08274.
Devoto P, Fattore L, Antinori S, Saba P, Frau R, Fratta W, et al. Elevated dopamine in the medial prefrontal cortex suppresses cocaine seeking via D1 receptor overstimulation. Addict Biol. 2016;21:61–71.
Singewald N, Holmes A. Rodent models of impaired fear extinction. Psychopharmacology. 2019;236:21–32.
Sartori SB, Maurer V, Murphy C, Schmuckermair C, Muigg P, Neumann ID, et al. Combined neuropeptide S and D-cycloserine augmentation prevents the return of fear in extinction-impaired rodents: advantage of dual versus single drug approaches. Int J Neuropsychopharmacol. 2016;19:1–11.
Vertes RP. Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse. 2004;51:32–58.
Lubec J, Kalaba P, Hussein AM, Feyissa DD, Kotob MH, Mahmmoud RR, et al. Reinstatement of synaptic plasticity in the aging brain through specific dopamine transporter inhibition. Mol Psychiatry. 2021;26:7076–90. https://doi.org/10.1038/s41380-021-01214-x.
Kummer KK, El Rawas R, Kress M, Saria A, Zernig G. Social interaction and cocaine conditioning in mice increase spontaneous spike frequency in the nucleus accumbens or septal nuclei as revealed by multielectrode array recordings. Pharmacology. 2015;95:42–9.
Delis F, Mitsacos A, Giompres P. Dopamine receptor and transporter levels are altered in the brain of Purkinje Cell Degeneration mutant mice. Neuroscience. 2004;125:255–68.
Murphy CP, Li X, Maurer V, Oberhauser M, Gstir R, Wearick-Silva LE, et al. MicroRNA-mediated rescue of fear extinction memory by miR-144-3p in extinction-impaired mice. Biol Psychiatry. 2017;81:979–89.
Camp M, Norcross M, Whittle N, Feyder M, D’Hanis W, Yilmazer-Hanke D, et al. Impaired Pavlovian fear extinction is a common phenotype across genetic lineages of the 129 inbred mouse strain. Genes Brain Behav. 2009;8:744–52.
Lee HJ, Haberman RP, Roquet RF, Monfils M-H. Extinction and retrieval + extinction of conditioned fear differentially activate medial prefrontal cortex and amygdala in rats. Front Behav Neurosci. 2015;9:369.
Zhu L, Zhu L, Huang Y, Shi W, Yu B. Memory reconsolidation and extinction of fear conditioning induced different Arc/Arg3.1 expression. Neuroreport. 2018;29:1036–45.
Rovira-Esteban L, Gunduz-Cinar O, Bukalo O, Limoges A, Brockway E, Müller K, et al. Excitation of Diverse Classes of Cholecystokinin Interneurons in the Basal Amygdala Facilitates Fear Extinction. eNeuro. 2019;6:ENEURO.0220-19.2019.
Keaveney MK, Rahsepar B, Tseng H, Fernandez FR, Mount RA, Ta T, et al. CaMKIIα-positive interneurons identified via a microRNA-based viral gene targeting strategy. J Neurosci. 2020;40:9576–88.
Nicole O, Pacary E. CaMKIIβ in neuronal development and plasticity: an emerging candidate in brain diseases. Int J Mol Sci. 2020;21:7272. https://doi.org/10.3390/ijms21197272.
Abraham AD, Neve KA, Lattal KM. Activation of D1/5 dopamine receptors: a common mechanism for enhancing extinction of fear and reward-seeking behaviors. Neuropsychopharmacology. 2016;41:2072–81.
Rey CD, Lipps J, Shansky RM. Dopamine D1 receptor activation rescues extinction impairments in low-estrogen female rats and induces cortical layer-specific activation changes in prefrontal-amygdala circuits. Neuropsychopharmacology. 2014;39:1282–9.
Ranjbar-Slamloo Y, Fazlali Z. Dopamine and noradrenaline in the brain; overlapping or dissociate functions? Front Mol Neurosci. 2020;12:334. https://doi.org/10.3389/fnmol.2019.00334.
Do-Monte FHM, Kincheski GC, Pavesi E, Sordi R, Assreuy J, Carobrez AP. Role of beta-adrenergic receptors in the ventromedial prefrontal cortex during contextual fear extinction in rats. Neurobiol Learn Mem. 2010;94:318–28.
Giustino TF, Maren S. Noradrenergic modulation of fear conditioning and extinction. Front Behav Neurosci. 2018;12:43.
Perez DM. α1-Adrenergic Receptors in Neurotransmission, Synaptic Plasticity, and Cognition. Front Pharmacol 2020;11:581098. https://doi.org/10.3389/fphar.2020.581098.
Shen H, Kannari K, Yamato H, Arai A, Matsunaga M. Effects of benserazide on L-DOPA-derived extracellular dopamine levels and aromatic L-amino acid decarboxylase activity in the striatum of 6-hydroxydopamine-lesioned rats. Tohoku J Exp Med. 2003;199:149–59.
Ledonne A, Mercuri NB. Current concepts on the physiopathological relevance of dopaminergic receptors. Front Cell Neurosci. 2017;11:27.
Cahill EN, Milton AL. Neurochemical and molecular mechanisms underlying the retrieval-extinction effect. Psychopharmacology. 2019;236:111–32.
Nishi A, Kuroiwa M, Shuto T. Mechanisms for the Modulation of Dopamine D1 Receptor Signaling in Striatal Neurons. Front Neuroanatomy 2011;5:43. https://www.frontiersin.org/articles/10.3389/fnana.2011.00043 (accessed 27 Feb2023).
Cadet JL, Jayanthi S, McCoy MT, Beauvais G, Cai NS. Dopamine D1 receptors, regulation of gene expression in the brain, and neurodegeneration. CNS Neurol Disord Drug Targets. 2010;9:526–38.
Marin M-F, Camprodon JA, Dougherty DD, Milad MR. Device-based brain stimulation to augment fear extinction: implications for PTSD treatment and beyond. Depress Anxiety. 2014;31:269–78.
Raij T, Nummenmaa A, Marin M-F, Porter D, Furtak S, Setsompop K, et al. Prefrontal cortex stimulation enhances fear extinction memory in humans. Biol Psychiatry. 2018;84:129–37.
Wilkinson LS, Humby T, Killcross AS, Torres EM, Everitt BJ, Robbins TW. Dissociations in dopamine release in medial prefrontal cortex and ventral striatum during the acquisition and extinction of classical aversive conditioning in the rat. Eur J Neurosci. 1998;10:1019–26.
Feenstra MG. Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward. Prog Brain Res. 2000;126:133–63.
Milad MR, Vidal-Gonzalez I, Quirk GJ. Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally specific manner. Behav Neurosci. 2004;118:389–94.
Vidal-Gonzalez I, Vidal-Gonzalez B, Rauch SL, Quirk GJ. Microstimulation reveals opposing influences of prelimbic and infralimbic cortex on the expression of conditioned fear. Learn Mem. 2006;13:728–33.
van der Wee NJ, van Veen JF, Stevens H, van Vliet IM, van Rijk PP, Westenberg HG. Increased serotonin and dopamine transporter binding in psychotropic medication-naive patients with generalized social anxiety disorder shown by 123I-beta-(4-iodophenyl)-tropane SPECT. J Nucl Med. 2008;49:757–63.
Bressan RA, Quarantini LC, Andreoli SB, Araújo C, Breen G, Guindalini C, et al. The posttraumatic stress disorder project in Brazil: neuropsychological, structural and molecular neuroimaging studies in victims of urban violence. BMC Psychiatry. 2009;9:30.
Bandelow B, Baldwin D, Abelli M, Altamura C, Dell’Osso B, Domschke K, et al. Biological markers for anxiety disorders, OCD and PTSD - a consensus statement. Part I: Neuroimaging and genetics. World J Biol Psychiatry. 2016;17:321–65.
Lee L-T, Tsai HC, Chi MH, Chang WH, Chen KC, Lee IH, et al. Lower availability of striatal dopamine transporter in generalized anxiety disorder: a preliminary two-ligand SPECT study. Int Clin Psychopharmacol. 2015;30:175–8.
Tiihonen J, Kuikka J, Bergström K, Lepola U, Koponen H, Leinonen E. Dopamine reuptake site densities in patients with social phobia. Am J Psychiatry. 1997;154:239–42.
Olver JS, O’Keefe G, Jones GR, Burrows GD, Tochon-Danguy HJ, Ackermann U, et al. Dopamine D(1) receptor binding in the anterior cingulate cortex of patients with obsessive-compulsive disorder. Psychiatry Res. 2010;183:85–88.
Plavén-Sigray P, Hedman E, Victorsson P, Matheson GJ, Forsberg A, Djurfeldt DR, et al. Extrastriatal dopamine D2-receptor availability in social anxiety disorder. Eur Neuropsychopharmacol. 2017;27:462–9.
Schneier FR, Abi-Dargham A, Martinez D, Slifstein M, Hwang D-R, Liebowitz MR, et al. Dopamine transporters, D2 receptors, and dopamine release in generalized social anxiety disorder. Depress Anxiety. 2009;26:411–8.
Schneier FR, Martinez D, Abi-Dargham A, Zea-Ponce Y, Simpson HB, Liebowitz MR, et al. Striatal dopamine D(2) receptor availability in OCD with and without comorbid social anxiety disorder: preliminary findings. Depress Anxiety. 2008;25:1–7.
Nikolaus S, Antke C, Beu M, Müller H-W. Cortical GABA, striatal dopamine and midbrain serotonin as the key players in compulsive and anxiety disorders–results from in vivo imaging studies. Rev Neurosci. 2010;21:119–39.
Norrholm SD, Jovanovic T, Smith AK, Binder E, Klengel T, Conneely K, et al. Differential genetic and epigenetic regulation of catechol-O-methyltransferase is associated with impaired fear inhibition in posttraumatic stress disorder. Front Behav Neurosci. 2013;7:30.
Somogyi P, Nunzi MG, Gorio A, Smith AD. A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res. 1983;259:137–42.
Cardin JA, Carlén M, Meletis K, Knoblich U, Zhang F, Deisseroth K, et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature. 2009;459:663–7.
Pi H-J, Hangya B, Kvitsiani D, Sanders JI, Huang ZJ, Kepecs A. Cortical interneurons that specialize in disinhibitory control. Nature. 2013;503:521–4.
Fanselow EE, Richardson KA, Connors BW. Selective, state-dependent activation of somatostatin-expressing inhibitory interneurons in mouse neocortex. J Neurophysiol. 2008;100:2640–52.
Kvitsiani D, Ranade S, Hangya B, Taniguchi H, Huang JZ, Kepecs A. Distinct behavioural and network correlates of two interneuron types in prefrontal cortex. Nature. 2013;498:363–6.
Anastasiades PG, Boada C, Carter AG. Cell-type-specific D1 dopamine receptor modulation of projection neurons and interneurons in the prefrontal cortex. Cereb Cortex. 2019;29:3224–42.
Fitzgerald PJ, Whittle N, Flynn SM, Graybeal C, Pinard CR, Gunduz-Cinar O, et al. Prefrontal single-unit firing associated with deficient extinction in mice. Neurobiol Learn Mem. 2014;113:69–81.
Cervenka S, Hedman E, Ikoma Y, Djurfeldt DR, Rück C, Halldin C, et al. Changes in dopamine D2-receptor binding are associated to symptom reduction after psychotherapy in social anxiety disorder. Transl Psychiatry. 2012;2:e120.
Floresco SB, West AR, Ash B, Moore H, Grace AA. Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nat Neurosci. 2003;6:968–73.
Gaykema RPA, Nguyen X-MT, Boehret JM, Lambeth PS, Joy-Gaba J, Warthen DM, et al. Characterization of excitatory and inhibitory neuron activation in the mouse medial prefrontal cortex following palatable food ingestion and food driven exploratory behavior. Front Neuroanat. 2014;8:60.
Wang Y, Goldman-Rakic PS. D2 receptor regulation of synaptic burst firing in prefrontal cortical pyramidal neurons. Proc Natl Acad Sci USA. 2004;101:5093–8.
Gee S, Ellwood I, Patel T, Luongo F, Deisseroth K, Sohal VS. Synaptic activity unmasks dopamine D2 receptor modulation of a specific class of layer V pyramidal neurons in prefrontal cortex. J Neurosci. 2012;32:4959–71.
Robinson SE, Sohal VS. Dopamine D2 receptors modulate pyramidal neurons in mouse medial prefrontal cortex through a stimulatory G-protein pathway. J Neurosci. 2017;37:10063–73.
Ott T, Nieder A. Dopamine and cognitive control in prefrontal cortex. Trends Cogn Sci. 2019;23:213–34.
Papenberg G, Karalija N, Salami A, Rieckmann A, Andersson M, Axelsson J, et al. Balance between transmitter availability and dopamine D2 receptors in prefrontal cortex influences memory functioning. Cereb Cortex. 2020;30:989–1000.
Lohani S, Martig AK, Deisseroth K, Witten IB, Moghaddam B. Dopamine modulation of prefrontal cortex activity is manifold and operates at multiple temporal and spatial scales. Cell Rep. 2019;27:99–114.e6.
Dadkhah M, Rashidy-Pour A, Vafaei AA. Temporary inactivation of the infralimbic cortex impairs while the blockade of its dopamine D2 receptors enhances auditory fear extinction in rats. Pharmacol Biochem Behav. 2021;203:173131.
Acknowledgements
This work was supported by the Austrian Science Fund FWF SFB 4410, DK-SPIN W-1206 and FG 18-B to NS and DK-SPIN W1206 to MK and the NIAAA Intramural Research Program to AH. The authors thank Gospava Stojanovic for excellent technical assistance and Eva Maria Fritz for critical feedback on the manuscript.
Author information
Authors and Affiliations
Contributions
SBS and NS designed the experiments with support from TMVK. SBS, TMVK, CM, OGC and KE performed the experiments, collected and analysed the data. KKK conducted the electrophysiological experiments and interpreted the data together with MK. SBS, AH, and NS wrote the manuscript. All authors provided comments on the final version.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Sartori, S.B., Keil, T.M.V., Kummer, K.K. et al. Fear extinction rescuing effects of dopamine and L-DOPA in the ventromedial prefrontal cortex. Transl Psychiatry 14, 11 (2024). https://doi.org/10.1038/s41398-023-02708-8
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41398-023-02708-8
- Springer Nature Limited