Social Defeat Stress Model for Adolescent C57BL/6 Male and Female Mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Gains and Losses: Resilience to Social Defeat Stress in Adolescent Female Mice.

BACKGROUND: Adolescence is a unique period of psychosocial growth during which social adversity can negatively influence mental health trajectories. Understanding how adolescent social stress impacts males and females and why some individuals are particularly affected is becoming increasingly urgent. Social defeat stress models for adolescent male mice have been effective in reproducing some physical/psychological aspects of bullying. Designing a model suitable for females has proven challenging. METHODS: We report a version of the adolescent male accelerated social defeat stress (AcSD) paradigm adapted for females. Early adolescent C57BL/6J female mice (N = 107) were exposed to our modified AcSD procedure twice a day for 4 days and categorized as resilient or susceptible based on a social interaction test 24 hours later. Mice were then assessed for changes in Netrin-1/DCC guidance cue expression in dopamine systems, for inhibitory control in adulthood using the Go/No-Go task, or for alterations in dopamine connectivity organization in the matured prefrontal cortex. RESULTS: Most adolescent females showed protection against stress-induced social avoidance, but in adulthood, these resilient females developed inhibitory control deficits and showed diminution of prefrontal cortex presynaptic dopamine sites. Female mice classified as susceptible were protected against cognitive and dopaminergic alterations. AcSD did not alter Netrin-1/DCC in early adolescent females, contrary to previous findings with males. CONCLUSIONS: Preserving prosocial behavior in adolescent females may be important for survival advantage but seems to come at the price of developing persistent cognitive and dopamine deficiencies. The female AcSD paradigm produced findings comparable to those found in males, allowing mechanistic investigation in both sexes.

Amphetamine disrupts dopamine axon growth in adolescence by a sex-specific mechanism in mice.

Initiating drug use during adolescence increases the risk of developing addiction or other psychopathologies later in life, with long-term outcomes varying according to sex and exact timing of use. The cellular and molecular underpinnings explaining this differential sensitivity to detrimental drug effects remain unexplained. The Netrin-1/DCC guidance cue system segregates cortical and limbic dopamine pathways in adolescence. Here we show that amphetamine, by dysregulating Netrin-1/DCC signaling, triggers ectopic growth of mesolimbic dopamine axons to the prefrontal cortex, only in early-adolescent male mice, underlying a male-specific vulnerability to enduring cognitive deficits. In adolescent females, compensatory changes in Netrin-1 protect against the deleterious consequences of amphetamine on dopamine connectivity and cognitive outcomes. Netrin-1/DCC signaling functions as a molecular switch which can be differentially regulated by the same drug experience as function of an individual's sex and adolescent age, and lead to divergent long-term outcomes associated with vulnerable or resilient phenotypes.

Reduced dopamine release in Dcc haploinsufficiency male mice abolishes the rewarding effects of cocaine but not those of morphine and ethanol.

RATIONALE: The Netrin-1/DCC guidance cue pathway is critically involved in the adolescent organization of the mesocorticolimbic dopamine circuitry. Adult mice heterozygous for Dcc show reduced dopamine release in the nucleus accumbens in response to amphetamine and, in turn, blunted sensitivity to the rewarding effects of this drug. OBJECTIVE: Here, we tested whether the protective effects of Dcc haploinsufficiency are specific to stimulant drugs of abuse or instead extrapolate to opioids and ethanol. METHODS: We used the place preference paradigm to measure the rewarding effects of cocaine (20 mg/kg), morphine (5 or 10 mg/Kg), or ethanol (20%) in adult (PND 75) male Dcc haploinsufficient mice or their wild-type litter mates. In a second experiment, we compared in these two genotypes, in vivo dopamine release in the nucleus accumbens after a single i.p. injection of morphine (10 mg/kg). RESULTS: We found reduced morphine-induced dopamine release in the nucleus accumbens of Dcc haploinsufficient male mice, but, contrary to the effects of stimulant drugs, there is no effect of genotype on morphine-induced conditioned preference. CONCLUSION: These findings show that reduced drug-induced mesolimbic dopamine in Dcc haploinsufficient male mice protects specifically against the rewarding effects of stimulant drugs, but not against the rewarding properties of morphine and ethanol. These results suggest that these drugs exert their rewarding effect via different brain circuits.

Custom-Built Operant Conditioning Setup for Calcium Imaging and Cognitive Testing in Freely Moving Mice.

Operant chambers are widely used in animal research to study cognition, motivation, and learning processes. Paired with the rapidly developing technologies for brain imaging and manipulations of brain activity, operant conditioning chambers are a powerful tool for neuroscience research. The behavioral testing and imaging setups that are commercially available are often quite costly. Here, we present a custom-built operant chamber that can be constructed in a few days by an unexperienced user with relatively inexpensive, widely available materials. The advantages of our operant setup compared with other open-source and closed-source solutions are its relatively low cost, its support of complex behavioral tasks, its user-friendly setup, and its validated functionality with video imaging of behavior and calcium imaging using the UCLA Miniscope. Using this setup, we replicate our previously published findings showing that mice exposed to social defeat stress in adolescence have inhibitory control impairments in the Go/No-Go task when they reach adulthood. We also present calcium imaging data of medial prefrontal cortex (mPFC) neuronal activity acquired during Go/No-Go testing in freely moving mice and show that neuronal population activity increases from day 1 to day 14 of the task. We propose that our operant chamber is a cheaper alternative to its commercially available counterparts and offers a better balance between versatility and user-friendly setup than other open-source alternatives.

Unique effects of social defeat stress in adolescent male mice on the Netrin-1/DCC pathway, prefrontal cortex dopamine and cognition (Social stress in adolescent vs. adult male mice).

For some individuals, social stress is a risk factor for psychiatric disorders characterised by adolescent onset, prefrontal cortex (PFC) dysfunction and cognitive impairments. Social stress may be particularly harmful during adolescence when dopamine (DA) axons are still growing to the PFC, rendering them sensitive to environmental influences. The guidance cue Netrin-1 and its receptor, DCC, coordinate to control mesocorticolimbic DA axon targeting and growth during this age. Here we adapted the accelerated social defeat (AcSD) paradigm to expose male mice to social stress in either adolescence or adulthood and categorised them as "resilient" or "susceptible" based on social avoidance behaviour. We examined whether stress would alter the expression of DCC and Netrin-1 in mesolimbic dopamine regions and would have enduring consequences on PFC dopamine connectivity and cognition. While in adolescence the majority of mice are resilient but exhibit risk-taking behaviour, AcSD in adulthood leads to a majority of susceptible mice without altering anxiety-like traits. In adolescent, but not adult mice, AcSD dysregulates DCC and Netrin-1 expression in mesolimbic DA regions. These molecular changes in adolescent mice are accompanied by changes in PFC DA connectivity. Following AcSD in adulthood, cognitive function remains unaffected, but all mice exposed to AcSD in adolescence show deficits in inhibitory control when they reach adulthood. These findings indicate that exposure to AcSD in adolescence vs. adulthood has substantially different effects on brain and behaviour and that stress-induced social avoidance in adolescence does not predict vulnerability to deficits in cognitive performance.Significance statement During adolescence, dopamine circuitries undergo maturational changes which may render them particularly vulnerable to social stress. While social stress can be detrimental to adolescents and adults, it may engage different mechanisms and impact different domains, depending on age. The accelerated social defeat (AcSD) model implemented here allows exposing adolescent and adult male mice to comparable social stress levels. AcSD in adulthood leads to a majority of socially avoidant mice. However, the predominance of AcSD-exposed adolescent mice does not develop social avoidance, and these resilient mice show risk-taking behaviour. Nonetheless, in adolescence only, AcSD dysregulates Netrin-1/DCC expression in mesolimbic dopamine regions, possibly disrupting mesocortical dopamine and cognition. The unique adolescent responsiveness to stress may explain increased psychopathology risk at this age.

Dopamine Axon Targeting in the Nucleus Accumbens in Adolescence Requires Netrin-1.

The fine arrangement of neuronal connectivity during development involves the coordinated action of guidance cues and their receptors. In adolescence, the dopamine circuitry is still developing, with mesolimbic dopamine axons undergoing target-recognition events in the nucleus accumbens (NAcc), while mesocortical projections continue to grow toward the prefrontal cortex (PFC) until adulthood. This segregation of mesolimbic versus mesocortical dopamine pathways is mediated by the guidance cue receptor DCC, which signals dopamine axons intended to innervate the NAcc to recognize this region as their final target. Whether DCC-dependent mesolimbic dopamine axon targeting in adolescence requires the action of its ligand, Netrin-1, is unknown. Here we combined shRNA strategies, quantitative analysis of pre- and post-synaptic markers of neuronal connectivity, and pharmacological manipulations to address this question. Similar to DCC levels in the ventral tegmental area, Netrin-1 expression in the NAcc is dynamic across postnatal life, transitioning from high to low expression across adolescence. Silencing Netrin-1 in the NAcc in adolescence results in an increase in the expanse of the dopamine input to the PFC in adulthood, with a corresponding increase in the number of presynaptic dopamine sites. This manipulation also results in altered dendritic spine density and morphology of medium spiny neurons in the NAcc in adulthood and in reduced sensitivity to the behavioral activating effects of the stimulant drug of abuse, amphetamine. These cellular and behavioral effects mirror those induced by Dcc haploinsufficiency within dopamine neurons in adolescence. Dopamine targeting in adolescence requires the complementary interaction between DCC receptors in mesolimbic dopamine axons and Netrin-1 in the NAcc. Factors regulating either DCC or Netrin-1 in adolescence can disrupt mesocorticolimbic dopamine development, rendering vulnerability or protection to phenotypes associated with psychiatric disorders.

MiR-218: a molecular switch and potential biomarker of susceptibility to stress.

Low miR-218 expression in the medial prefrontal cortex (mPFC) is a consistent trait of depression. Here we assessed whether miR-218 in the mPFC confers resilience or susceptibility to depression-like behaviors in adult mice, using the chronic social defeat stress (CSDS) model of depression. We also investigated whether stress-induced variations of miR-218 expression in the mPFC can be detected in blood. We find that downregulation of miR-218 in the mPFC increases susceptibility to a single session of social defeat, whereas overexpression of miR-218 selectively in mPFC pyramidal neurons promotes resilience to CSDS and prevents stress-induced morphological alterations to those neurons. After CSDS, susceptible mice have low levels of miR-218 in blood, as compared with control or resilient groups. We show further that upregulation and downregulation of miR-218 levels specifically in the mPFC correlate with miR-218 expression in blood. Our results suggest that miR-218 in the adult mPFC might function as a molecular switch that determines susceptibility vs. resilience to chronic stress, and that stress-induced variations in mPFC levels of miR-218 could be detected in blood. We propose that blood expression of miR-218 might serve as potential readout of vulnerability to stress and as a proxy of mPFC function.

Non-Contingent Exposure to Amphetamine in Adolescence Recruits miR-218 to Regulate Dcc Expression in the VTA.

The development of the dopamine input to the medial prefrontal cortex occurs during adolescence and is a process that is vulnerable to disruption by stimulant drugs such as amphetamine. We have previously linked the amphetamine-induced disruption of dopamine connectivity and prefrontal cortex maturation during adolescence to the downregulation of the Netrin-1 receptor, DCC, in dopamine neurons. However, how DCC expression in dopamine neurons is itself regulated is completely unknown. MicroRNA (miRNA) regulation of mRNA translation and stability is a prominent mechanism linking environmental events to changes in protein expression. Here, using male mice, we show that miR-218 is expressed in dopamine neurons and is a repressor of DCC. Whereas Dcc mRNA levels increase from early adolescence to adulthood, miR-218 exhibits the exact opposite switch, most likely maintaining postnatal Dcc expression. This dynamic regulation appears to be selective to Dcc since the expression of Robo 1, the other guidance cue receptor target of miR-218, does not vary with age. Amphetamine in adolescence, but not in adulthood, increases miR-218 in the VTA and this event is required for drug-induced downregulation of Dcc mRNA and protein expression. This effect seems to be specific to Dcc because amphetamine does not alter Robo1. Furthermore, the upregulation of miR-218 by amphetamine requires dopamine D2 receptor activation. These findings identify miR-218 as regulator of DCC in the VTA both in normal development and after drug exposure in adolescence.

An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.

Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: •Use phosphate buffer (PB) as the blocking solution solvent.•Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

DCC Confers Susceptibility to Depression-like Behaviors in Humans and Mice and Is Regulated by miR-218.

BACKGROUD: Variations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction. METHODS: With the use of postmortem brain tissue, mouse models of defeat stress, and in vitro analysis, we assessed microRNA (miRNA) regulation of DCC and whether changes in DCC levels in the PFC lead to vulnerability to depression-like behaviors. RESULTS: We identified miR-218 as a posttranscriptional repressor of DCC and detected coexpression of DCC and miR-218 in pyramidal neurons of human and mouse PFC. We found that exaggerated expression of DCC and reduced levels of miR-218 in the PFC are consistent traits of mice susceptible to chronic stress and of major depressive disorder in humans. Remarkably, upregulation of Dcc in mouse PFC pyramidal neurons causes vulnerability to stress-induced social avoidance and anhedonia. CONCLUSIONS: These data are the first demonstration of microRNA regulation of DCC and suggest that, by regulating DCC, miR-218 may be a switch of susceptibility versus resilience to stress-related disorders.

Early Development of Parvalbumin-, Somatostatin-, and Cholecystokinin-Expressing Neurons in Rat Brain following Prenatal Immune Activation and Maternal Iron Deficiency.

Prenatal maternal infection and maternal iron deficiency during pregnancy are 2 early environmental insults associated with increased risk for schizophrenia in offspring. Substantial evidence suggests that abnormalities in inhibitory γ-aminobutyric acid (GABA) interneuron function, especially in the parvalbumin subtype of GABA interneuron, both developmentally and in adulthood, may contribute mechanistically to cognitive deficits and psychotic symptoms in schizophrenia. This study used a rat model to test whether prenatal immune activation with lipopolysaccharide (LPS; at gestation days, GD, 15 and 16) or maternal iron deficiency (from GD2 to postnatal day P7) or the combination of both insults alters major subtypes of GABAergic interneurons (parvalbumin, somatostatin, cholecystokinin) in brain regions relevant to schizophrenia (medial and dorsolateral prefrontal cortex [PFC], hippocampal CA1 and dentate gyrus, ventral subiculum) in offspring at P14 or P28. Prenatal LPS treatment significantly increased the density of parvalbumin-immunoreactive neurons at P14 in the medial PFC, dorsolateral PFC, and ventral subiculum of offspring born from iron-sufficient but not iron-deficient dams. Prenatal LPS also increased cholecystokinin neuron density in the medial PFC at P28, under both iron-sufficient and iron-deficient conditions. We observed a large increase in parvalbumin neuron density from P14 to P28 in the medial PFC and subiculum across all birth groups, that was not observed in other brain regions, and significant decreases in somatostatin neuron density from P14 to P28 in all brain regions examined across all birth groups, indicating differential developmental trajectories for parvalbumin and somatostatin neurons in various brain regions during this early postnatal period. Thus, it appears that the medial PFC and ventral subiculum, brain regions involved in circuitry modulating ventral tegmental dopamine and nucleus accumbens activities, may be regions vulnerable to effects of prenatal LPS on specific subpopulations of interneurons. It is known that the timing of maturation and expansion of parvalbumin neurons in early development provides threshold levels of inhibition that trigger critical periods for cortical plasticity, leading to long-term circuit consolidation. Thus, our finding of increased parvalbumin neuron density at early developmental times might suggest a mechanism by which an acute prenatal insult like LPS exposure could produce long-term changes in prefrontal cortical or subicular function.

Maternal Oxytocin Administration Before Birth Influences the Effects of Birth Anoxia on the Neonatal Rat Brain.

Ineffective contractions and prolonged labor are common birth complications in primiparous women, and oxytocin is the most common agent given for induction or augmentation of labor. Clinical studies in humans suggest oxytocin might adversely affect the CNS response to hypoxia at birth. In this study, we used a rat model of global anoxia during Cesarean section birth to test if administering oxytocin to pregnant dams prior to birth affects the acute neonatal CNS response to birth anoxia. Anoxic pups born from dams pre-treated with intravenous injections or infusions of oxytocin before birth showed significantly increased brain lactate, a metabolic indicator of CNS hypoxia, compared to anoxic pups from dams pre-treated with saline. Anoxic pups born from dams given oxytocin before birth also showed decreased brain ATP compared to anoxic pups from saline dams. Direct injection of oxytocin to postnatal day 2 rat pups followed by exposure to anoxia also resulted in increased brain lactate and decreased brain ATP, compared to anoxia exposure alone. Oxytocin pre-treatment of the dam decreased brain malondialdehyde, a marker of lipid peroxidation, as well as protein kinase C activity, both in anoxic pups and controls, suggesting oxytocin may reduce aspects of oxidative stress. Finally, when dams were pretreated with indomethacin, a cyclooxygenase (COX) inhibitor, maternal oxytocin no longer potentiated effects of anoxia on neonatal brain lactate, suggesting this effect of oxytocin may be mediated via prostaglandin production or other COX-derived products. The results indicate that maternal oxytocin administration may have multiple acute effects on CNS metabolic responses to anoxia at birth.

Prenatal exposure to bacterial endotoxin reduces the number of GAD67- and reelin-immunoreactive neurons in the hippocampus of rat offspring.

Epidemiological studies implicate prenatal infection as a risk factor for the development of schizophrenia and autism. Subjects with schizophrenia and autism are reported to exhibit reduced levels of glutamic acid decarboxylase 67 (GAD67), a marker for GABA neurons, in various brain regions. Reduced levels of reelin, a secretory glycoprotein present in a subpopulation of GABA neurons, have also been found in these disorders. To test if prenatal infection can cause abnormalities in GAD67 and reelin in the brains of offspring, this study used a rat model of prenatal exposure to the bacterial endotoxin, lipopolysaccharide (LPS), and assessed numbers of GAD67-immunoreactive (GAD67+) and reelin-immunoreactive (reelin+) neurons in the hippocampus of offspring. In offspring at postnatal day 14 (PD14), GAD67+ cell counts were reduced in the dentate gyrus of the prenatal LPS group compared to prenatal saline controls, while at PD28, GAD67+ cells counts were reduced in the prenatal LPS group in both the dentate gyrus and the CA1. There was a decrease in the number of reelin+ cells in the prenatal LPS offspring compared to controls in the dentate gyrus at PD14. However using Western blotting, no significant effects of prenatal LPS on levels of GAD67 or reelin protein were observed in various brain regions at PD14. These findings support the idea that prenatal infection can cause reductions in postnatal expression of GAD67 and reelin, and in this way, possibly contribute to the pathophysiology of schizophrenia or autism.

Gabapentin actions on Kir3 currents and N-type Ca2+ channels via GABAB receptors in hippocampal pyramidal cells.

Gabapentin is a clinically effective anticonvulsant with an unclear mechanism of action. It was described as a GABA(B(1a,2)) receptor subtype-selective agonist, activating postsynaptic K(+) currents and inhibiting postsynaptic Ca(2+) channels in CA1 pyramidal cells, but without presynaptic actions. These activities appeared controversial and we therefore sought to further clarify gabapentin actions in rat hippocampal slices by characterizing K(+) currents and Ca(2+) channels targeted by gabapentin using whole-cell recording and multiphoton Ca(2+) imaging. 1) We found that gabapentin and baclofen induced inwardly rectifying K(+) currents (K(Gbp) and K(Bac), respectively), sensitive to Ba(2+) and Cs(+). 2) A constitutively active K(IR) current, independent of GABA(B) receptor activation and sensitive to Ba(2+) and Cs(+) was also present. 3) K(Gbp), K(Bac), and K(IR) currents showed some differences in sensitivity to Ba(2+) and Cs(+), indicating the possible activation of distinct Kir3 currents, independent of K(IR), by gabapentin and baclofen. 4) Gabapentin inhibition of Ca(2+) channels was abolished by omega-conotoxin GVIA, but not by omega-agatoxin IVA and nimodipine, indicating a predominant action of gabapentin on N-type Ca(2+) channels. 5) Gabapentin actions were linked to activation of pertussis toxin-sensitive G-proteins since N-ethylmaleimide (NEM) blocked K(Gbp) activation and Ca(2+) channel inhibition by gabapentin. 6) Finally, gabapentin reduced epileptiform discharges in slices via GABA(B) receptor activation. The anticonvulsant actions of gabapentin in hippocampal cells may thus involve GABA(B) receptor coupling to G-proteins and modulation of Kir3 and N-type Ca(2+) channels. Moreover, gabapentin and baclofen activation of GABA(B) receptors may couple to distinct cellular targets.