Nucleus accumbens neurons dynamically respond to appetitive and aversive associative learning.

To survive, individuals must learn to associate cues in the environment with emotionally relevant outcomes. This association is partially mediated by the nucleus accumbens (NAc), a key brain region of the reward circuit that is mainly composed by GABAergic medium spiny neurons (MSNs), that express either dopamine receptor D1 or D2. Recent studies showed that both populations can drive reward and aversion, however, the activity of these neurons during appetitive and aversive Pavlovian conditioning remains to be determined. Here, we investigated the relevance of D1- and D2-neurons in associative learning, by measuring calcium transients with fiber photometry during appetitive and aversive Pavlovian tasks in mice. Sucrose was used as a positive valence unconditioned stimulus (US) and foot shock was used as a negative valence US. We show that during appetitive Pavlovian conditioning, D1- and D2-neurons exhibit a general increase in activity in response to the conditioned stimuli (CS). Interestingly, D1- and D2-neurons present distinct changes in activity after sucrose consumption that dynamically evolve throughout learning. During the aversive Pavlovian conditioning, D1- and D2-neurons present an increase in the activity in response to the CS and to the US (shock). Our data support a model in which D1- and D2-neurons are concurrently activated during appetitive and aversive conditioning.

A novel perspective on the role of nucleus accumbens neurons in encoding associative learning.

The nucleus accumbens (NAc) has been considered a key brain region for encoding reward/aversion and cue-outcome associations. These processes are encoded by medium spiny neurons that express either dopamine receptor D1 (D1-MSNs) or D2 (D2-MSNs). Despite the well-established role of NAc neurons in encoding reward/aversion, the underlying processing by D1-/D2-MSNs remains largely unknown. Recent electrophysiological, optogenetic and calcium imaging studies provided insight on the complex role of D1- and D2-MSNs in these behaviours and helped to clarify their involvement in associative learning. Here, we critically discuss findings supporting an intricate and complementary role of NAc D1- and D2-MSNs in associative learning, emphasizing the need for additional studies in order to fully understand the role of these neurons in behaviour.

Involvement of nucleus accumbens D2-medium spiny neurons projecting to the ventral pallidum in anxiety-like behaviour.

BACKGROUND: The nucleus accumbens (NAcc) is a crucial brain region for emotionally relevant behaviours. The NAcc is mainly composed of medium spiny neurons (MSNs) expressing either dopamine receptor D1 (D1-MSNs) or D2 (D2-MSNs). The D1-MSNs project to the ventral tegmental area (VTA) and the ventral pallidum (VP), whereas the D2-MSNs project only to the VP. The D1- and D2-MSNs have been associated with depression-like behaviours, but their contribution to anxiety remains to be determined. METHODS: We used optogenetic tools to selectively manipulate D1-MSN projections from the NAcc core to the VP or VTA and D2-MSN projections to the VP during validated anxiety-producing behavioural procedures in naive mice. In addition, we assessed the effects of optical stimulation on neuronal activity using in vivo electrophysiologic recordings in anesthetized animals. RESULTS: Optogenetic activation of D1-MSN projections to the VTA or VP did not trigger anxiety-like behaviour. However, optical activation of D2-MSN projections to the VP significantly increased anxiety-like behaviour. This phenotype was associated with a decrease in the neuronal activity of putative GABAergic neurons in the VP. Importantly, pretreating D2-MSN-VP animals with the γ-aminobutyric acid modulator diazepam prevented the optically triggered anxiety-like behaviour. LIMITATIONS: The exclusive use of males in the behavioural tests limits broader interpretation of the findings. Although we used optogenetic conditions that trigger quasi-physiologic changes, there are caveats associated with the artificial manipulation of neuronal activity. CONCLUSION: The D2-MSN-VP projections contributed to the development of anxiety-like behaviour, through modulation of GABAergic activity in the VP.

Prenatal dexamethasone exposure alters effort decision making and triggers nucleus accumbens and anterior cingulate cortex functional changes in male rats.

Daily, individuals select actions based on cost-benefit to allocate resources into goal-directed actions. Different brain regions coordinate this complex decision, including the nucleus accumbens (NAc), anterior cingulate cortex (ACC), and ventral tegmental area (VTA). In utero exposure to synthetic glucocorticoids (iuGC), such as dexamethasone, triggers prominent motivation deficits but the impact of this exposure in the ACC-NAc and/or ACC-VTA circuits is unknown. Here, we show that iuGC exposure causes decreased motivation for natural rewards (food) and impaired effort-based decision-making. Importantly, reduced neuronal activation (number of c-fos+ neurons) was observed in the NAc core and ACC of iuGC rats in comparison to CTR rats after performing the effort-based decision-making task. In addition, iuGC treatment led to increased NAc and ACC basal neuronal activity. Electrophysiological recordings during optogenetic modulation of ACC terminals in the NAc revealed that the ACC-NAc circuit is dysfunctional in iuGC animals. These data suggest that iuGC animals present motivational and effort-based decision-making deficits that can be associated with the observed ACC-NAc dysfunction.

The Duration of Stress Determines Sex Specificities in the Vulnerability to Depression and in the Morphologic Remodeling of Neurons and Microglia.

Stress exposure has been shown to induce a variety of molecular and functional alterations associated with anxiety and depression. Some studies suggest that microglia, the immune cells of the brain, play a significant role in determining neuronal and behavioral responses to chronic stress and also contribute to the development of stress-related psychopathologies. However, little is known about the impact of the duration of stress exposure upon microglia and neurons morphology, particularly considering sex differences. This issue deserves particular investigation, considering that the process of morphologic remodeling of neurons and microglia is usually accompanied by functional changes with behavioral expression. Here, we examine the effects of short and long unpredictable chronic mild stress (uCMS) protocols on behavior, evaluating in parallel microglia and neurons morphology in the dorsal hippocampus (dHIP) and in the nucleus accumbens (NAc), two brain regions involved in the etiology of depression. We report that long-term uCMS induced more behavioral alterations in males, which present anxiety and depression-like phenotypes (anhedonia and helplessness behavior), while females only display anxiety-like behavior. After short-term uCMS, both sexes presented anxiety-like behavior. Microglia cells undergo a process of morphologic adaptation to short-term uCMS, dependent on sex, in the NAc: we observed a hypertrophy in males and an atrophy in females, transient effects that do not persist after long-term uCMS. In the dHIP, the morphologic adaptation of microglia is only observed in females (hypertrophy) and after the protocol of long uCMS. Interestingly, males are more vulnerable to neuronal morphological alterations in a region-specific manner: dendritic atrophy in granule neurons of the dHIP and hypertrophy in the medium spiny neurons of the NAc, both after short- or long-term uCMS. The morphology of neurons in these brain regions were not affected in females. These findings raise the possibility that, by differentially affecting neurons and microglia in dHIP and NAc, chronic stress may contribute for differences in the clinical presentation of stress-related disorders under the control of sex-specific mechanisms.

Distinct role of nucleus accumbens D2-MSN projections to ventral pallidum in different phases of motivated behavior.

The nucleus accumbens (NAc) is a key region in motivated behaviors. NAc medium spiny neurons (MSNs) are divided into those expressing dopamine receptor D1 or D2. Classically, D1- and D2-MSNs have been described as having opposing roles in reinforcement, but recent evidence suggests a more complex role for D2-MSNs. Here, we show that optogenetic modulation of D2-MSN to ventral pallidum (VP) projections during different stages of motivated behavior has contrasting effects in motivation. Activation of D2-MSN-VP projections during a reward-predicting cue results in increased motivational drive, whereas activation at reward delivery decreases motivation; optical inhibition triggers the opposite behavioral effect. In addition, in a free-choice instrumental task, animals prefer the lever that originates one pellet in opposition to pellet plus D2-MSN-VP optogenetic activation and vice versa for optogenetic inhibition. In summary, D2-MSN-VP projections play different, and even opposing, roles in distinct phases of motivated behavior.

Constitutive deficiency of the neurogenic hippocampal modulator AP2γ promotes anxiety-like behavior and cumulative memory deficits in mice from juvenile to adult periods.

The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.

Laterodorsal tegmentum-ventral tegmental area projections encode positive reinforcement signals.

The laterodorsal tegmentum (LDT) is a brainstem nucleus classically involved in REM sleep and attention, and that has recently been associated with reward-related behaviors, as it controls the activity of ventral tegmental area (VTA) dopaminergic neurons, modulating dopamine release in the nucleus accumbens. To further understand the role of LDT-VTA inputs in reinforcement, we optogenetically manipulated these inputs during different behavioral paradigms in male rats. We found that in a two-choice instrumental task, optical activation of LDT-VTA projections shifts and amplifies preference to the laser-paired reward in comparison to an otherwise equal reward; the opposite was observed with inhibition experiments. In a progressive ratio task, LDT-VTA activation boosts motivation, that is, enhances the willingness to work to get the reward associated with LDT-VTA stimulation; and the reverse occurs when inhibiting these inputs. Animals abolished preference if the reward was omitted, suggesting that LDT-VTA stimulation adds/decreases value to the stimulation-paired reward. In addition, we show that LDT-VTA optical activation induces robust preference in the conditioned and real-time place preference tests, while optical inhibition induces aversion. The behavioral findings are supported by electrophysiological recordings and c-fos immunofluorescence correlates in downstream target regions. In LDT-VTA ChR2 animals, we observed an increase in the recruitment of lateral VTA dopamine neurons and D1 neurons from nucleus accumbens core and shell; whereas in LDT-VTA NpHR animals, D2 neurons appear to be preferentially recruited. Collectively, these data show that the LDT-VTA inputs encode positive reinforcement signals and are important for different dimensions of reward-related behaviors.

Fractionating stem cells secretome for Parkinson's disease modeling: Is it the whole better than the sum of its parts?

Human mesenchymal stem cells (hMSCs) secretome has been have been at the forefront of a new wave of possible therapeutic strategies for central nervous system neurodegenerative disorders, as Parkinson's disease (PD). While within its protein fraction, several promising proteins were already identified with therapeutic properties on PD, the potential of hMSCs-secretome vesicular fraction remains to be elucidated. Such highlighting is important, since hMSCs secretome-derived vesicles can act as biological nanoparticles with beneficial effects in different pathological contexts. Therefore, in this work, we have isolated hMSCs secretome vesicular fraction, and assessed their impact on neuronal survival, and differentiation on human neural progenitors' cells (hNPCs), and in a 6-hydroxydopamine (6-OHDA) rat model of PD when compared to hMSCs secretome (as a whole) and its protein derived fraction. From the results, we have found hMSCs vesicular fraction as polydispersity source of vesicles, which when applied in vitro was able to induce hNPCs differentiation at the same levels as the whole secretome, while the protein separated fraction was not able to induce such effect. In the context of PD, although distinct effects were observed, hMSCs secretome and its derived fractions displayed a positive impact on animals' motor and histological performance, thereby indicating that hMSCs secretome and its different fractions may impact different mechanisms and pathways. Overall, we concluded that the use of the secretome collected from hMSCs and its different fractions might be active modulators of different neuroregeneration mechanisms, which could open new therapeutical opportunities for their future use as a treatment for PD.

Resilience to stress and sex-specific remodeling of microglia and neuronal morphology in a rat model of anxiety and anhedonia.

Prenatal exposure to stress or glucocorticoids (GC) is associated with the appearance of psychiatric diseases later in life. Microglia, the immune cells of the brain, are altered in stress-related disorders. Synthetic GC such as dexamethasone (DEX) are commonly prescribed in case of preterm risk labour in order to promote fetal lung maturation. Recently, we reported long-lasting differences in microglia morphology in a model of in utero exposure to DEX (iuDEX), that presents an anxious phenotype. However, it is still unclear if stress differentially affects iuDEX males and females. In this work, we evaluated how iuDEX animals of both sexes cope with chronic mild stress for 2 weeks. We evaluated emotional behavior and microglia and neuronal morphology in the dorsal hippocampus (dHIP) and nucleus accumbens (NAc), two brain regions involved in emotion-related disorders. We report that males and females prenatally exposed to DEX have better performance in anxiety- and depression-related behavioral tests after chronic stress exposure in adulthood than non-exposed animals. Interestingly, iuDEX animals present sex-dependent changes in microglia morphology in the dHIP (hypertrophy in females) and in the NAc (atrophy in females and hypertrophy in males). After chronic stress, these cells undergo sex-specific morphological remodeling. Paralleled to these alterations in cytoarchitecture of microglia, we report inter-regional differences in dendritic morphology in a sex-specific manner. iuDEX females present fewer complex neurons in the NAc, whereas iuDEX males presented less complex neuronal morphology in the dHIP. Interestingly, these alterations were modified by stress exposure. Our work shows that stressful events during pregnancy can exert a preserved sex-specific effect in adulthood. Although the role of the observed cellular remodeling is still unknown, sex-specific differences in microglia plasticity induced by long-term stress exposure may anticipate differences in drug efficacy in the context of stress-induced anxiety- or depression-related behaviors.

Correction: Nucleus accumbens medium spiny neurons subtypes signal both reward and aversion.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Nucleus accumbens medium spiny neurons subtypes signal both reward and aversion.

Deficits in decoding rewarding (and aversive) signals are present in several neuropsychiatric conditions such as depression and addiction, emphasising the importance of studying the underlying neural circuits in detail. One of the key regions of the reward circuit is the nucleus accumbens (NAc). The classical view on the field postulates that NAc dopamine receptor D1-expressing medium spiny neurons (D1-MSNs) convey reward signals, while dopamine receptor D2-expressing MSNs (D2-MSNs) encode aversion. Here, we show that both MSN subpopulations can drive reward and aversion, depending on their neuronal stimulation pattern. Brief D1- or D2-MSN optogenetic stimulation elicited positive reinforcement and enhanced cocaine conditioning. Conversely, prolonged activation induced aversion, and in the case of D2-MSNs, decreased cocaine conditioning. Brief stimulation was associated with increased ventral tegmenta area (VTA) dopaminergic tone either directly (for D1-MSNs) or indirectly via ventral pallidum (VP) (for D1- and D2-MSNs). Importantly, prolonged stimulation of either MSN subpopulation induced remarkably distinct electrophysiological effects in these target regions. We further show that blocking κ-opioid receptors in the VTA (but not in VP) abolishes the behavioral effects induced by D1-MSN prolonged stimulation. In turn, blocking δ-opioid receptors in the VP (but not in VTA) blocks the behavioral effects elicited by D2-MSN prolonged stimulation. Our findings demonstrate that D1- and D2-MSNs can bidirectionally control reward and aversion, explaining the existence of controversial studies in the field, and highlights that the proposed striatal functional opposition needs to be reconsidered.

Role of laterodorsal tegmentum projections to nucleus accumbens in reward-related behaviors.

The laterodorsal tegmentum (LDT) is associated with reward considering that it modulates VTA neuronal activity, but recent anatomical evidence shows that the LDT also directly projects to nucleus accumbens (NAc). We show that the majority of LDT-NAc inputs are cholinergic, but there is also GABAergic and glutamatergic innervation; activation of LDT induces a predominantly excitatory response in the NAc. Non-selective optogenetic activation of LDT-NAc projections in rats enhances motivational drive and shifts preference to an otherwise equal reward; whereas inhibition of these projections induces the opposite. Activation of these projections also induces robust place preference. In mice, specific activation of LDT-NAc cholinergic inputs (but not glutamatergic or GABAergic) is sufficient to shift preference, increase motivation, and drive positive reinforcement in different behavioral paradigms. These results provide evidence that LDT-NAc projections play an important role in motivated behaviors and positive reinforcement, and that distinct neuronal populations differentially contribute for these behaviors.

Mild Prenatal Stress Causes Emotional and Brain Structural Modifications in Rats of Both Sexes.

Stress or high levels of glucocorticoids (GCs) during developmental periods is known to induce persistent effects in the neuroendocrine circuits that control stress response, which may underlie individuals' increased risk for developing neuropsychiatric conditions later in life, such as anxiety or depression. We developed a rat model (Wistar han) of mild exposure to unpredictable prenatal stress (PS), which consists in a 4-h stressor administered three times per week on a random basis; stressors include strobe lights, noise and restrain. Pregnant dams subjected to this protocol present disrupted circadian corticosterone secretion and increased corticosterone secretion upon acute stress exposure. Regarding progeny, both young adult (2 months old) male and female rats present increased levels of circulating corticosterone and hyperactivity of the hypothalamus-pituitary-adrenal axis to acute stress exposure. Both sexes present anxious- and depressive-like behaviors, shown by the decreased time spent in the open arms of the elevated plus maze (EPM) and in the light side of the light-dark box (LDB), and by increased immobility time in the forced swim test, respectively. Interestingly, these results were accompanied by structural modifications of the bed nucleus of stria terminalis (BNST) and hippocampus, as well as decreased norepinephrine and dopamine levels in the BNST, and serotonin levels in the hippocampus. In summary, we characterize a new model of mild PS, and show that stressful events during pregnancy can lead to long-lasting structural and neurochemical effects in the offspring, which affect behavior in adulthood.

Nucleus Accumbens Microcircuit Underlying D2-MSN-Driven Increase in Motivation.

The nucleus accumbens (NAc) plays a central role in reinforcement and motivation. Around 95% of the NAc neurons are medium spiny neurons (MSNs), divided into those expressing dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R). Optogenetic activation of D2-MSNs increased motivation, whereas inhibition of these neurons produced the opposite effect. Yet, it is still unclear how activation of D2-MSNs affects other local neurons/interneurons or input terminals and how this contributes for motivation enhancement. To answer this question, in this work we combined optogenetic modulation of D2-MSNs with in loco pharmacological delivery of specific neurotransmitter antagonists in rats. First, we showed that optogenetic activation of D2-MSNs increases motivation in a progressive ratio (PR) task. We demonstrated that this behavioral effect relies on cholinergic-dependent modulation of dopaminergic signalling of ventral tegmental area (VTA) terminals, which requires D1R and D2R signalling in the NAc. D2-MSN optogenetic activation decreased ventral pallidum (VP) activity, reducing the inhibitory tone to VTA, leading to increased dopaminergic activity. Importantly, optogenetic activation of D2-MSN terminals in the VP was sufficient to recapitulate the motivation enhancement. In summary, our data suggests that optogenetic stimulation of NAc D2-MSNs indirectly modulates VTA dopaminergic activity, contributing for increased motivation. Moreover, both types of dopamine receptors signalling in the NAc are required in order to produce the positive behavioral effects.