Daniela Popa

AMYGDALA INTERCALATED NEURONS ARE REQUIRED FOR EXPRESSION OF FEAR EXTINCTION

Congruent findings from studies of fear learning in animals and humans indicate that research on the circuits mediating fear constitutes our best hope of understanding human anxiety disorders. In mammals, repeated presentations of a conditioned stimulus that was previously paired to a noxious stimulus leads to the gradual disappearance of conditioned fear responses. Although much evidence suggests that this extinction process depends on plastic events in the amygdala, the underlying mechanisms remain unclear. Intercalated (ITC) amygdala neurons constitute probable mediators of extinction because they receive information about the conditioned stimulus from the basolateral amygdala (BLA), and contribute inhibitory projections to the central nucleus (CEA), the main output station of the amygdala for conditioned fear responses. Thus, after extinction training, ITC cells could reduce the impact of conditioned-stimulus-related BLA inputs to the CEA by means of feed-forward inhibition. Here we test the hypothesis that ITC neurons mediate extinction by lesioning them with a toxin that selectively targets cells expressing µ-opioid receptors (µORs). Electron microscopic observations revealed that the incidence of µOR-immunoreactive synapses is much higher in ITC cell clusters than in the BLA or CEA and that µORs typically have a post-synaptic location in ITC cells. In keeping with this, bilateral infusions of the µOR agonist dermorphin conjugated to the toxin saporin in the vicinity of ITC neurons caused a 34% reduction in the number of ITC cells but no significant cell loss in surrounding nuclei. Moreover, ITC lesions caused a marked deficit in the expression of extinction that correlated negatively with the number of surviving ITC neurons but not CEA cells. Because ITC cells exhibit an unusual pattern of receptor expression, these findings open new avenues for the treatment of anxiety disorders.

Behavioral, neurochemical, and electrophysiological characterization of a genetic mouse model of depression

Depression is a multifactorial illness and genetic factors play a role in its etiology. The understanding of its physiopathology relies on the availability of experimental models potentially mimicking the disease. Here we describe a model built up by selective breeding of mice with strikingly different responses in the tail suspension test, a stress paradigm aimed at screening potential antidepressants. Indeed, “helpless” mice are essentially immobile in the tail suspension test, as well as the Porsolt forced-swim test, and they show reduced consumption of a palatable 2% sucrose solution. In addition, helpless mice exhibit sleep–wakefulness alterations resembling those classically observed in depressed patients, notably a lighter and more fragmented sleep, with an increased pressure of rapid eye movement sleep. Compared with “nonhelpless” mice, they display higher basal seric corticosterone levels and lower serotonin metabolism index in the hippocampus. Remarkably, serotonin1A autoreceptor stimulation induces larger hypothermia and inhibition of serotoninergic neuronal firing in the nucleus raphe dorsalis in helpless than in nonhelpless mice. Thus, helpless mice exhibit a decrease in serotoninergic tone, which evokes that associated with endogenous depression in humans. Finally, both the behavioral impairments and the serotoninergic dysfunction can be improved by chronic treatment with the antidepressant fluoxetine. The helpless line of mice may provide an opportunity to approach genes influencing susceptibility to depression and to investigate neurophysiological and neurochemical substrates underlying antidepressant effects.

Coherent amygdalocortical theta promotes fear memory consolidation during paradoxical sleep

Brain activity in sleep plays a crucial role in memory consolidation, an offline process that determines the long-term strength of memory traces. Consolidation efficacy differs across individuals, but the brain activity dynamics underlying these differences remain unknown. Here, we studied how interindividual variability in fear memory consolidation relates to neural activity in brain structures that participate in Pavlovian fear learning. From the end of training to testing 24 h later, some rats showed increased and others decreased conditioned fear responses. We found that overnight bidirectional changes in fear memory were selectively correlated with modifications in theta coherence between the amygdala, medial prefrontal cortex, and hippocampus during paradoxical sleep. Thus, our results suggest that theta coordination in the limbic system may influence interindividual differences in memory consolidation of aversive experiences.

Lasting Syndrome of Depression Produced by Reduction in Serotonin Uptake during Postnatal Development: Evidence from Sleep, Stress, and Behavior

Dysfunction of the serotonin system is implicated in sleep and emotional disorders. To test whether these impairments could arise during development, we studied the impact of early-life, transient versus genetic, permanent alterations of serotonin reuptake on sleep–wakefulness patterns, depression-related behavior, and associated physiological features. Here, we show that female mice treated neonatally with a highly selective serotonin reuptake inhibitor, escitalopram, exhibited signs of depression in the form of sleep anomalies, anhedonia, increased helplessness reversed by chronic antidepressant treatment, enhanced response to acute stress, and increased serotoninergic autoinhibitory feedback. This syndrome was not reproduced by treatment in naive adults but resembled the phenotype of mutant mice lacking the serotonin transporter, except that these exhibited decreased serotonin autoreceptor sensitivity and additional anxiety-like behavior. Thus, alteration of serotonin reuptake during development, whether induced by external or genetic factors, causes a depressive syndrome lasting into adulthood. Such early-life impairments might predispose individuals to sleep and/or mood disorders.

Contribution of 5-HT2 Receptor Subtypes to Sleep–Wakefulness and Respiratory Control, and Functional Adaptations in Knock-Out Mice Lacking 5-HT2A Receptors

Serotonin (5-hydroxytryptamine; 5-HT) plays key roles in sleep–wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. Here, we investigated the relative contribution of 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT2A+/+) and knock-out (5-HT2A–/–) mice that do not express 5-HT2A receptors. Acute blockade of 5-HT2A receptors induced an increase in NREMS in 5-HT2A+/+ mice, but not 5-HT2A–/– mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT2A+/+ mice, 5-HT2B receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT2A–/– mice, indicating a lower sensitivity of 5-HT2B receptors in mutants, with no change in 5-HT2B mRNA. Blockade of 5-HT2C receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT2A+/+ mice but not in 5-HT2A–/– mice. Whole-body plethysmographic recordings indicated that 5-HT2A receptor blockade in 5-HT2A+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT2A–/– mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. Our results demonstrate that 5-HT exerts a 5-HT2B-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT2A receptors. Moreover, 5-HT2A gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT2A receptors in 5-HT2A+/+ mice.

Coherent gamma oscillations couple the amygdala and striatum during learning

The basolateral amygdala (BLA) mediates the facilitating effects of emotions on memory. The BLAs enhancing influence extends to various types of memories, including striatal-dependent habit formation. To shed light on the underlying mechanisms, we carried out unit and local field potential (LFP) recordings in BLA, striatum, auditory cortex and intralaminar thalamus in cats trained on a stimulus-response task in which the presentation of one of two tones predicted reward delivery. The coherence of BLA, but not of cortical or thalamic, LFPs was highest with striatal gamma activity, and intra-BLA muscimol infusions selectively reduced striatal gamma power. Moreover, coupling of BLA-striatal unit activity increased when LFP gamma power was augmented. Early in training, the rewarded and unrewarded tones elicited a modest increase in coherent BLA-striatal gamma. As learning progressed, this gamma coupling selectively increased in relation to the rewarded tone. Thus, coherent gamma oscillations coordinate amygdalostriatal interactions during learning and might facilitate synaptic plasticity.

CENTRAL AMYGDALA ACTIVITY DURING FEAR CONDITIONING

The central amygdala (Ce), particularly its medial sector (CeM), is the main output station of the amygdala for conditioned fear responses. However, there is uncertainty regarding the nature of CeM control over conditioned fear. The present study aimed to clarify this question using unit recordings in rats. Fear conditioning caused most CeM neurons to increase their conditioned stimulus (CS) responsiveness. The next day, CeM cells responded similarly during the recall test, but these responses disappeared as extinction of conditioned fear progressed. In contrast, the CS elicited no significant average change in central lateral (CeL) firing rates during fear conditioning and a small but significant reduction during the recall test. Yet, cell-by-cell analyses disclosed large but heterogeneous CS-evoked responses in CeL. By the end of fear conditioning, roughly equal proportions of CeL cells exhibited excitatory (CeL+) or inhibitory (CeL−) CS-evoked responses (∼10%). The next day, the proportion of CeL− cells tripled with no change in the incidence of CeL+ cells, suggesting that conditioning leads to overnight synaptic plasticity in an inhibitory input to CeL− cells. As in CeM, extinction training caused the disappearance of CS-evoked activity in CeL. Overall, these findings suggest that conditioned freezing depends on increased CeM responses to the CS. The large increase in the incidence of CeL− but not CeL+ cells from conditioning to recall leads us to propose a model of fear conditioning involving the potentiation of an extrinsic inhibitory input (from the amygdala or elsewhere) to CeL, ultimately leading to disinhibition of CeM neurons.

The fear circuit revisited: contributions of the basal amygdala nuclei to conditioned fear.

The lateral nucleus (LA) is the input station of the amygdala for information about conditioned stimuli (CSs), whereas the medial sector of the central nucleus (CeM) is the output region that contributes most amygdala projections to brainstem fear effectors. However, there are no direct links between LA and CeM. As the main target of LA and with its strong projection to CeM, the basomedial amygdala (BM) constitutes a good candidate to bridge this gap. Consistent with this notion, it was reported that combined posttraining lesions of the basal nuclei [BM plus basolateral nucleus (BL)] abolish conditioned fear responses, whereas selective BL inactivation does not. Thus, we examined the relative contribution of BM and BL to conditioned fear using unit recordings and inactivation with muscimol microinfusions in rats. Approximately 30% of BM and BL neurons acquired robust responses to auditory CSs predicting footshocks. While most BL cells stopped firing at CS offset, BM responses typically outlasted the CS by ≥40 s, paralleling the persistence of conditioned fear after the CS. This observation suggests that BM neurons are not passive relays of rapidly adapting LA inputs about the CS. Surprisingly, independent inactivation of either BM or BL with muscimol did not cause a reduction of conditioned freezing even though an extinction recall deficit was seen the next day. In contrast, combined BL–BM inactivation did. Overall, there results support the notion that the basal nuclei are involved in conditioned fear expression and extinction but that there is functional redundancy between them.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H+-dependent carriers (named VGLUT1–3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT1A-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

Early Life Blockade of 5-Hydroxytryptamine 1A Receptors Normalizes Sleep and Depression-Like Behavior in Adult Knock-Out Mice Lacking the Serotonin Transporter

In serotonin transporter knock-out (5-HTT−/−) mice, extracellular serotonin (5-HT) levels are markedly elevated in the brain, and rapid eye movement sleep (REMS) is enhanced compared with wild-type mice. We hypothesized that such sleep impairment at adulthood results from excessive serotonergic tone during early life. Thus, we assessed whether neonatal treatment with drugs capable of limiting the impact of 5-HT on the brain could normalize sleep patterns in 5-HTT−/− mutants. We found that treatments initiated at postnatal day 5 and continued for 2 weeks with the 5-HT synthesis inhibitor para-chlorophenylalanine, or for 4 weeks with the 5-HT1A receptor (5-HT1AR) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), induced total or partial recovery of REMS, respectively, in 5-HTT−/− mutants. Early life treatment with WAY 100635 also reversed the depression-like behavior otherwise observed in these mutants. Possible adaptive changes in 5-HT1AR after neonatal treatment with WAY 100635 were investigated by measuring 5-HT1A binding sites and 5-HT1A mRNA in various REMS- and/or depression-related brain areas, as well as 5-HT1AR-mediated hypothermia and inhibition of neuronal firing in the dorsal raphe nucleus. None of these characteristics were modified in parallel with REMS recovery, suggesting that 5-HT1ARs involved in wild-type phenotype rescue in 5-HTT−/− mutants are located in other brain areas or in 5-HT1AR-unrelated circuits where they could be transiently expressed during development. The reversal of sleep alterations and depression-like behavior after early life blockade of 5-HT1AR in 5-HTT−/− mutants might open new perspectives regarding preventive care of sleep and mood disorders resulting from serotonin transporter impairments during development.