Sarah J. Bailey

Chronic administration of 13-cis-retinoic acid increases depression-related behavior in mice

Retinoid signaling plays a well-established role in neuronal differentiation, neurite outgrowth, and the patterning of the anteroposterior axis of the developing neural tube. However, there is increasing evidence that nutritional vitamin A status and retinoid signaling play an important role in the function of the adult brain. 13-Cis-retinoic acid (13-cis-RA) (isotretinoin or Accutane), a synthetic retinoid that is an effective oral treatment for severe nodular acne, has been linked with depression and suicide in patients. The purpose of this study was to test the hypothesis that chronic administration of 13-cis-RA would lead to depression-related behaviors in mice. Young, adult male mice received 13-cis-RA (1 mg/kg) by daily intraperitoneal injection for 6 weeks. This treatment paradigm produced plasma levels of 13-cis-RA that are comparable to those reported in human patients taking Accutane. In both the forced swim test and the tail suspension test, we found that 13-cis-RA-treated mice spent significantly more time immobile compared to vehicle-treated controls. In the open field test, there was no change in anxiety-related behavior in 13-cis-RA-treated mice. Furthermore, chronic administration of 13-cis-RA did not impair locomotion in either the open field or the rotarod test. Taken together, these results suggest that administration of 13-cis-RA increases depression-related behaviors in mice.

Retinoid-mediated regulation of mood: Possible cellular mechanisms

Vitamin A and its derivatives, the retinoids, have long been studied for their ability to alter central nervous system (CNS) development. Increasingly, it is recognized that sufficient levels of retinoids may also be required for adult CNS function. However, excess dietary vitamin A, due to the consumption of supplements or foods rich in vitamin A, has been reported to induce psychosis. In addition, 13-cis-retinoic acid (13-cis-RA, isotretinoin), the active ingredient in the acne treatment Accutane, has been reported to cause adverse psychiatric events, including depression and suicidal ideation. Nevertheless, epidemiological studies have reported no consistent link between Accutane use and clinical depression in humans. Using an animal model, we have recently shown that 13-cis-RA induces an increase in depression-related behavior. Impairments in spatial learning and memory have also been demonstrated following 13-cis-RA treatment in mice. This review focuses on the behavioral and possible cellular effects of retinoid deficiency or excess in the adult brain in relation to altered mood. Specifically, we discuss the effect of retinoids on depression-related behaviors and whether norepinephrinergic, dopaminergic, or serotonergic neurotransmitter systems may be impaired. In addition, we consider the evidence that adult neurogenesis, a process implicated in the pathophysiology of depression, is reduced by retinoid signaling. We suggest that 13-cis-RA treatment may induce depression-related behaviors by decreasing adult neurogenesis and/or altering the expression of components of serotonergic neurotransmitter system, thereby leading to impaired serotonin signaling.

13-cis-Retinoic Acid Alters Intracellular Serotonin, Increases 5-HT1A Receptor, and Serotonin Reuptake Transporter Levels In Vitro

In addition to their established role in nervous system development, vitamin A and related retinoids are emerging as regulators of adult brain function. Accutane (13-cis-retinoic acid, isotretinoin) treatment has been reported to increase depression in humans. Recently, we showed that chronic administration of 13-cis-retinoic acid (13-cis-RA) to adolescent male mice increased depression-related behaviors. Here, we have examined whether 13-cis-RA regulates components involved in serotonergic neurotransmission in vitro. We used the RN46A-B14 cell line, derived from rat embryonic raphe nuclei. This cell line synthesizes serotonin (5-hydroxytryptamine, 5-HT) and expresses the 5-HT1A receptor and the serotonin reuptake transporter (SERT). Cells were treated with 0, 2.5, or 10 μM 13-cis-RA for 48 or 96 hrs, and the levels of 5-HT; its metabolite, 5-hydroxyindoleacetic acid (5HIAA); 5-HT1A receptor; and SERT were determined. Treatment with 13-cis-RA for 96 hrs increased the intracellular levels of 5-HT and tended to increase intra-cellular 5HIAA levels. Furthermore, 48 hrs of treatment with 2.5 and 10 μM 13-cis-RA significantly increased 5-HT1A protein to 168.5 ± 20.0% and 148.7 ± 2.2% of control respectively. SERT protein levels were significantly increased to 142.5 ± 11.1% and 119.2 ± 3.6% of control by 48 hrs of treatment with 2.5 and 10 μM of 13-cis-RA respectively. Increases in both 5-HT1A receptor and SERT proteins may lead to decreased serotonin availability at synapses. Such an effect of 13-cis-RA could contribute to the increased depression-related behaviors we have shown in mice.

Variability in the benzodiazepine response of serotonin 5-HT 1A receptor null mice displaying anxiety-lie phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABA A receptors

Benzodiazepines (BZs) acting as modulators of GABAA receptors (GABAARs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABAARs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-Hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABAAR subunits α1 and α2. Here, we show that R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABAAR subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABAAR α subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABAARα subunits. In addition, we show that this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABAAR α subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABAAR expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients.

Combined administration of buprenorphine and naltrexone produces antidepressant-like effects in mice

Opiates have been used historically for the treatment of depression. Renewed interest in the use of opiates as antidepressants has focused on the development of kappa opioid receptor (κ-receptor) antagonists. Buprenorphine acts as a partial µ-opioid receptor agonist and a κ-receptor antagonist. By combining buprenorphine with the opioid antagonist naltrexone, the activation of µ-opioid receptors will be reduced and the κ-antagonist properties enhanced. We have established that a combination dose of buprenorphine (1 mg/kg) with naltrexone (1 mg/kg) functions as a short-acting κ-antagonist in the mouse tail withdrawal test. Furthermore, this dose combination is neither rewarding nor aversive in the conditioned place preference paradigm, and is without significant locomotor effects. We have shown for the first time that systemic co-administration of buprenorphine (1 mg/kg) with naltrexone (1 mg/kg) in CD-1 mice produced an antidepressant-like response in behaviours in both the forced swim test and novelty induced hypophagia task. Behaviours in the elevated plus maze and light dark box were not significantly altered by treatment with buprenorphine alone, or in combination with naltrexone. We propose that the combination of buprenorphine with naltrexone represents a novel, and potentially a readily translatable approach, to the treatment of depression.

Lamina-specific differences in GABAB autoreceptor-mediated regulation of spontaneous GABA release in rat entorhinal cortex

Spontaneous synaptic inhibition plays an important role in regulating the excitability of cortical networks. Here we have investigated the role of GABA(B) autoreceptors in regulating spontaneous GABA release in the entorhinal cortex (EC), a region associated with temporal lobe epilepsies. We have previously shown that the level of spontaneous inhibition in superficial layers of the EC is much greater than that seen in deeper layers. In the present study, using intracellular and whole cell patch clamp recordings in rat EC slices, we have demonstrated that evoked GABA responses are controlled by feedback inhibition via GABA(B) autoreceptors. Furthermore, recordings of spontaneous, activity-independent inhibitory postsynaptic currents in layer II and layer V neurones showed that the GABA(B) receptor agonist, baclofen, reduced the frequency of GABA-mediated currents indicating the presence of presynaptic GABA(B) receptors in both layers. Application of the antagonist, CGP55845, blocked the effects of baclofen and also increased the frequency of GABA-mediated events above baseline, but the latter effect was restricted to layer V. This demonstrates that GABA(B) autoreceptors are tonically activated by synaptically released GABA in layer V, and this may partly explain the lower level of spontaneous GABA release in the deep layer.

Chronic 13-cis-retinoic acid administration disrupts network interactions between the raphe nuclei and the hippocampal system in young adult mice.

Previously, we showed that chronic administration of 13-cis-retinoic acid (13-cis-RA) induces depression-related behaviors in mice and that 13-cis-RA alters components of the serotonergic system in vitro. Work by others has shown that 13-cis-RA reduces hippocampal neurogenesis in mice and orbitofrontal cortex metabolism in humans. In the current study, we measured cytochrome oxidase activity, a metabolic marker that reflects steady state neuronal energy demand, in various regions of the brain to determine the effects of 13-cis-RA on neuronal metabolic activity and network interactions between the raphe nuclei and the hippocampal system. Brain cytochrome oxidase activity in young adult male mice was analyzed following 6 weeks of daily 13-cis-RA (1 mg/kg) or vehicle injection and behavioral testing. Chronic 13-cis-RA administration significantly decreased cytochrome oxidase activity only in the inferior rostral linear nucleus of the raphe. However, covariance analysis of interregional correlations in cytochrome oxidase activity revealed that 13-cis-RA treatment caused a functional uncoupling between the dorsal raphe nuclei and the hippocampus. Furthermore, a path analysis indicated that a network comprising lateral habenula to dorsal raphe to hippocampus was effectively uncoupled in 13-cis-RA treated animals. Finally, cytochrome oxidase activity in the dentate gyrus of 13-cis-RA treated mice was inversely correlated with depression-related behavior. Taken together, these data show that 13-cis-RA alters raphe metabolism and disrupts functional connectivity between the raphe nuclei and the hippocampal formation, which may contribute to the observed increase in depression-related behaviors.

Validation of a refined technique for taking repeated blood samples from juvenile and adult mice

Repeated blood sampling from laboratory animals is desirable in certain experimental designs and also for reducing the number of animals used in research. Biochemical methods for analysing blood samples require only small blood volumes to be collected (typically 20–40 µL). In juvenile mice, the small blood volume of the animals also requires only small samples to be taken. Furthermore, for behavioural studies it is desirable to have a method that does not require anaesthesia or the use of invasive indwelling cannulae. We report the validation of a refined method for repeated blood sampling (up to 3 times at 24 h intervals) in juvenile and adult mice using the tail incision method to sample from the lateral tail vein. This method is not stressful, as assessed by low basal levels of the stress hormone corticosterone. Since repeated blood samples can be collected from the same animal at multiple time points, it is not necessary to increase group size for terminal sample collection. Thus, in addition to being a refined method requiring no warming of the tail, no anaesthesia and only gentle restraint, this method also reduces the numbers of mice used for experiments.

Chronic treatment with 13-cis-retinoic acid changes aggressive behaviours in the resident-intruder paradigm in rats

Retinoids, vitamin A related compounds, have an established role in the development of the nervous system and are increasingly recognized to play a role in adult brain function. The synthetic retinoid, 13-cis-retinoic acid (13-cis-RA, Roaccutane) is widely used to treat severe acne but has been linked to an increased risk of neuropsychiatric side effects, including depression. Here we report that chronic administration with 13-cis-RA (1 mg/kg i.p. daily, 7-14 days) in adult rats reduced aggression- and increased flight-related behaviours in the resident-intruder paradigm. However, in the forced swim, sucrose consumption and open field tests treatment for up to 6 weeks with 13-cis-RA did not modify behaviour in adult or juvenile animals. The behavioural change observed in the resident-intruder paradigm is directly opposite to that observed with chronic antidepressant administration. These findings indicate that when a suitably sensitive behavioural test is employed then chronic administration of 13-cis-RA in adult rats induces behavioural changes consistent with a pro-depressant action.

Repeated daily restraint stress induces adaptive behavioural changes in both adult and juvenile mice

Chronic stress is known to be a risk factor for the development of depression and anxiety, disorders which often begin during adolescence. Restraint stress is a commonly used stressor in adult rodents, however the effects of repeated restraint stress in juvenile mice have not been well characterised. Here we have shown for the first time the behavioural and hormonal effects of repeated restraint stress in both adult and juvenile BALB/c and C57BL/6 mice. Repeated daily restraint stress (2h/day for 3, 7 or 14days) provoked a robust physiological response evident as increased corticosterone levels and decreased body weight after 14days. However, habituation of the stress-response was evident during repeated exposure to the stressor in both adult and juvenile mice. The behavioural changes seen in response to repeated restraint stress were complex. In juvenile mice, repeated restraint stress evoked an increase in exploratory behaviours in the elevated plus maze, a decrease in time spent immobile in the forced swim test and a decrease in sucrose preference. In adult mice fewer behavioural changes were seen. Interestingly BALB/c and C57BL/6 mice showed qualitatively similar response to 3days repeated restraint stress. The behavioural changes we observed, as a result of prior stress exposure, may represent an adaptive stress-coping response or resilience. Both the hormonal and behavioural effects of stress were more pronounced in juvenile mice than in adults. This wider range of behavioural responses seen in juvenile mice might reflect a greater ability to engage in adaptive stress-coping strategies that likely have beneficial effects evident in future stress challenges.