Sex differences in electrophysiological properties and voltage-gated ion channel expression in the paraventricular thalamic nucleus following repeated stress.

BACKGROUND: Habituation to repeated stress refers to a progressive reduction in the stress response following multiple exposures to the same, predictable stressor. We previously demonstrated that the posterior division of the paraventricular thalamic nucleus (pPVT) nucleus regulates habituation to 5 days of repeated restraint stress in male rats. Compared to males, female rats display impaired habituation to 5 days of restraint. To better understand how activity of pPVT neurons is differentially impacted in stressed males and females, we examined the electrophysiological properties of pPVT neurons under baseline conditions or following restraint. METHODS: Adult male and female rats were exposed to no stress (handling only), a single period of 30 min restraint or 5 daily exposures to 30 min restraint. 24 h later, pPVT tissue was prepared for recordings. RESULTS: We report here that spontaneous excitatory post-synaptic current (sEPSC) amplitude was increased in males, but not females, following restraint. Furthermore, resting membrane potential of pPVT neurons was more depolarized in males. This may be partially due to reduced potassium leakage in restrained males as input resistance was increased in male, but not female, rats 24 h following 1 or 5 days of 30-min restraint. Reduced potassium efflux during action potential firing also occurred in males following a single restraint as action potential half-width was increased following a single restraint. Restraint had limited effects on electrophysiological properties in females, although the mRNA for 10 voltage-gated ion channel subunits was altered in the pPVT of female rats. CONCLUSIONS: The results suggest that restraint-induced changes in pPVT activation promote habituation in males. These findings are the first to describe a sexual dimorphism in stress-induced electrophysiological properties and voltage-gated ion channel expression in the pPVT. These results may explain, at least in part, why habituation to 5 days of restraint is disrupted in female rats.

Arc-Mediated Plasticity in the Paraventricular Thalamic Nucleus Promotes Habituation to Stress.

BACKGROUND: Habituation is defined as a progressive decline in response to repeated exposure to a familiar and predictable stimulus and is highly conserved across species. Disrupted habituation is a signature of posttraumatic stress disorder. In rodents, habituation is observed in neural, neuroendocrine, and behavioral responses to repeated exposure to predictable and moderately intense stress or restraint. We previously demonstrated that lesioning the posterior paraventricular thalamic nucleus (pPVT) impairs habituation. However, the underlying molecular mechanisms and specific neural connections among the pPVT and other brain regions that underlie habituation are unknown. METHODS: Behavioral and neuroendocrine habituation was assessed in adult male Sprague Dawley rats using the repeated restraint paradigm. Pan-neuronal and Cre-dependent DREADDs (designer receptors exclusively activated by designer drugs) were used to chemogenetically inhibit the pPVT and the subpopulation of pPVT neurons that project to the medial prefrontal cortex (mPFC), respectively. Activity-regulated cytoskeleton-associated protein (Arc) expression was knocked down in the pPVT using small interfering RNA. Structural plasticity of pPVT neurons was assessed using Golgi staining. Local field potential recordings were used to assess coherent neural activity between the pPVT and mPFC. The attentional set shifting task was used to assess mPFC-dependent behavior. RESULTS: Here, we show that Arc promotes habituation by increasing stress-induced spinogenesis in the pPVT, increasing coherent neural activity with the mPFC, and improving mPFC-mediated cognitive flexibility. CONCLUSIONS: Our results demonstrate that Arc induction in the pPVT regulates habituation and mPFC function. Therapies that improve synaptic plasticity during posttraumatic stress disorder therapy may enhance habituation and the efficacy of posttraumatic stress disorder treatment.

Sex- and Stress-Dependent Effects on Dendritic Morphology and Spine Densities in Putative Orexin Neurons.

We recently found that non-stressed female rats have higher basal prepro-orexin expression and activation of orexinergic neurons compared to non-stressed males, which lead to impaired habituation to repeated restraint stress at the behavioral, neural, and endocrine level. Here, we extended our study of sex differences in the orexin system by examining spine densities and dendritic morphology in putative orexin neurons in adult male and female rats that were exposed to 5 consecutive days of 30-min restraint. Analysis of spine distribution and density indicated that putative orexinergic neurons in control non-stressed females had significantly more dendritic spines than those in control males, and the majority of these were mushroom spines. This morphological finding may suggest more excitatory input onto orexin neurons in female rats. As orexin neurons are known to promote the hypothalamic-pituitary-adrenal response, this morphological change in orexin neurons could underlie the impaired habituation to repeated stress in female rats. Dendritic complexity did not differ between non-stressed males and females, however repeated restraint stress decreased total dendritic length, nodes, and branching primarily in males. Thus, reduced dendritic complexity of putative orexinergic neurons is observed in males but not in females after 5days of repeated restraint stress. This morphological change might be reflective of decreased orexin system function, which may allow males to habituate more fully to repeated restraint than females. These results extend our understanding of the role of orexin neurons in regulating habituation and demonstrate changes in putative orexin cell morphology and spines that may underlie sex differences in habituation.

Age- and sex-dependent impact of repeated social stress on morphology of rat prefrontal cortex pyramidal neurons.

Chronic stress can lead to psychiatric illness characterized by impairments of executive function, implicating the prefrontal cortex as a target of stress-related pathology. Previous studies have shown that various types of chronic stress paradigms reduce dendritic branching, length and spines of medial prefrontal cortex (mPFC) pyramidal neurons. However, these studies largely focused on layer II/III pyramidal neurons in adult male rats with less known about layer V, the site of projection neurons. Because the prefrontal cortex develops throughout adolescence, stress during adolescence may have a greater impact on structure and function than stress occurring during adulthood. Furthermore, females display greater risk of stress-related psychiatric disorders, indicating sex-specific responses to stress. In this study, male and female adolescent (42-48 days old, 4 rats per group) or adult (68-72 days old, 4 rats per group) Sprague-Dawley rats were exposed to 5 days of repeated social stress in the resident-intruder paradigm or control manipulation. We examined dendritic morphology of cells in the mPFC in both layer II/III and Layer V. Repeated social stress resulted in decreased dendritic branching in layer II/III apical dendrites regardless of sex or age. In apical layer V dendrites, stress increased branching in adult males but decreased it in all other groups. Stress resulted in a decrease in dendritic spines in layer V apical dendrites for male adolescents and female adults, and this was mostly due to a decrease in filopodial and mushroom spines for male adolescents, but stubby spines for female adults. In sum, these results suggest that repeated stress reduces complexity and synaptic connectivity in adolescents and female adults in both input and output layers of prelimbic mPFC, but not in male adults. These changes may represent a potential underlying mechanism as to why adolescents and females are more susceptible to the negative cognitive effects of repeated or chronic stress.

Sex Differences in Risk and Resilience: Stress Effects on the Neural Substrates of Emotion and Motivation.

Risk for stress-sensitive psychopathologies differs in men and women, yet little is known about sex-dependent effects of stress on cellular structure and function in corticolimbic regions implicated in these disorders. Determining how stress influences these regions in males and females will deepen our understanding of the mechanisms underlying sex-biased psychopathology. Here, we discuss sex differences in CRF regulation of arousal and cognition, glucocorticoid modulation of amygdalar physiology and alcohol consumption, the age-dependent impact of social stress on prefrontal pyramidal cell excitability, stress effects on the prefrontal parvalbumin system in relation to emotional behaviors, contributions of stress and gonadal hormones to stress effects on prefrontal glia, and alterations in corticolimbic structure and function after cessation of chronic stress. These studies demonstrate that, while sex differences in stress effects may be nuanced, nonuniform, and nonlinear, investigations of these differences are nonetheless critical for developing effective, sex-specific treatments for psychological disorders.

Psychostimulants As Cognitive Enhancers in Adolescents: More Risk than Reward?

Methylphenidate and other psychostimulants, originally developed to treat attention deficit-hyperactivity disorder, are increasingly abused by healthy adolescents and adults seeking an advantage in scholastic performance and work productivity. However, how these drugs may affect cognitive performance, especially in the young brain, remains unclear. Here, we review recent literature and emphasize the risks of abuse of psychostimulants in healthy adolescents and young adults. We conclude that while the desire for cognitive enhancement, particularly with rising costs of education and increasingly competitive nature of scholarship programs, is unlikely to diminish in the near future, it is crucial for the scientific community to thoroughly examine the efficacy and safety of these stimulants in healthy populations across development. The current dearth of knowledge on the dose-response curve, metabolism, and cognitive outcomes in adolescents following methylphenidate or other psychostimulant exposure may be perpetuating a perception of these drugs as "safe" when that might not be true for developing brains.

A Clinically-Relevant Dose of Methylphenidate Enhances Synaptic Inhibition in the Juvenile Rat Prefrontal Cortex.

Methylphenidate (MPH) is perhaps the most commonly prescribed psychoactive substance for young children and adolescents; however, its effects on the immature brain are not well understood. MPH is increasingly abused by adolescents and prescriptions are being issued to increasingly younger children without rigorous psychological testing, raising the potential for misdiagnosis; it is therefore crucial to understand how this drug might impact a healthy, developing brain. Recently, we have shown that a clinically-relevant dose of MPH depresses the activity of pyramidal neurons in the prefrontal cortex of normal juvenile rats, but its effects on inhibitory synaptic transmission remain to be explored. We therefore recorded spontaneous (s), miniature (m), and evoked (e) inhibitory postsynaptic currents (IPSCs) in layer 5 pyramidal neurons in juvenile rat prefrontal cortex. We found a dose-dependent effect of MPH on sIPSC frequency but not amplitude, where 0.3 mg/kg significantly decreased frequency, but 1 mg/kg significantly increased frequency. Moreover, mIPSCs were not affected by either dose of MPH, whereas the amplitudes, as well as paired-pulse ratios and coefficient of variations of evoked IPSCs were significantly increased after MPH treatment, indicating a presynaptic action. Tonic GABA current was also not affected by MPH treatment. Taken together, these results suggest that MPH administration to a healthy juvenile may enhance excitation of GABAergic interneurons; thus shifting the excitation-inhibition balance in the prefrontal cortex towards inhibition, and depressing overall prefrontal cortical activity. Our findings also indicate that the adolescent brain is more sensitive to MPH than previously thought, and dose ranges need to be reconsidered for age as well as size.

Age- and Sex-Dependent Impact of Repeated Social Stress on Intrinsic and Synaptic Excitability of the Rat Prefrontal Cortex.

Stress is implicated in psychiatric illnesses that are characterized by impairments in cognitive functions that are mediated by the medial prefrontal cortex (mPFC). Because sex and age determine stress vulnerability, the effects of repeated social stress occurring during early adolescence, mid-adolescence, or adulthood on the cellular properties of male and female rat mPFC Layer V neurons in vitro were examined. Repeated resident-intruder stress produced age- and sex-specific effects on mPFC intrinsic and synaptic excitability. Mid-adolescents were particularly vulnerable to effects on intrinsic excitability. The maximum number of action potentials (APs) evoked by increasing current intensity was robustly decreased in stressed male and female mid-adolescent rats compared with age-matched controls. These effects were associated with stress-induced changes in AP half-width, amplitude, threshold, and input resistance. Social stress at all ages generally decreased synaptic excitability by decreasing the amplitude of spontaneous excitatory postsynaptic potentials. The results suggest that whereas social stress throughout life can diminish the influence of afferents driving the mPFC, social stress during mid-adolescence additionally affects intrinsic characteristics of mPFC neurons that determine excitability. The depressant effects of social stress on intrinsic and synaptic mPFC neurons may underlie its ability to affect executive functions and emotional responses, particularly during adolescence.

Evolution of the Study of Methylphenidate and Its Actions on the Adult Versus Juvenile Brain.

OBJECTIVE: Methylphenidate (MPH) is the most often prescribed medication for treatment of ADHD. However, many of its specific cellular and molecular mechanisms of action, as well as developmental consequences of treatment, are largely unknown. This review provides an overview of current understanding of MPH efficacy, safety, and dosage in adult and pediatric ADHD patients, as well as adult animal studies and pioneering studies in juvenile animals treated with MPH. METHOD: A thorough review of the current literature on MPH efficacy and safety in children, adults, and animal models was included. Results of studies were compared and contrasted. RESULTS: While MPH is currently considered safe, there is a lack of knowledge of potential developmental consequences of early treatment, as well as differences in drug actions in the developing versus mature brain system. CONCLUSION: This review emphasizes the need for further research into the age-dependent activities and potency of MPH, and a need for tighter control and clinical relevance in future studies.

Transient inactivation of the medial prefrontal cortex impairs performance on a working memory-dependent conditional discrimination task.

The rodent medial prefrontal cortex (mPFC) has been implicated in working memory function; lesions and inactivation of this region have been shown to result in impairments in spatial working memory (WM) tasks. Our laboratory has developed a tactile-visual conditional discrimination (CD) task, which uses floor insert cues to signal the correct goal-arm choice in a T maze. This task can be manipulated by altering the floor insert cues to be present throughout the trial (CDSTANDARD) or to be present only at the beginning of the trial (CDWM), thus making the task either WM-independent or WM-dependent, respectively. This ability to manipulate the working memory demand of the task while holding all other task features constant allows us to rule out the possibility that confounding performance variables contribute to the observed impairment. A previous study from our lab showed that mPFC inactivation did not impair performance on CDSTANDARD, confirming that mPFC inactivation does not induce sensorimotor or motivational deficits that could impact task performance. To examine whether mPFC inactivation impairs CDWM, the current study transiently inactivated the mPFC with bilateral microinfusions of muscimol immediately prior to testing on the CDWM task. As predicted, CDWM task performance was significantly impaired during the muscimol-infusion session compared with the control saline-infusion sessions. Together with our previous demonstration that the mPFC in not required for CDSTANDARD, these results not only confirm that the mPFC is crucial for working memory, but also set the stage for using the task-comparison approach to investigate corticolimbic interactions during working memory.

Performance enhancement at the cost of potential brain plasticity: neural ramifications of nootropic drugs in the healthy developing brain.

Cognitive enhancement is perhaps one of the most intriguing and controversial topics in neuroscience today. Currently, the main classes of drugs used as potential cognitive enhancers include psychostimulants (methylphenidate (MPH), amphetamine), but wakefulness-promoting agents (modafinil) and glutamate activators (ampakine) are also frequently used. Pharmacologically, substances that enhance the components of the memory/learning circuits-dopamine, glutamate (neuronal excitation), and/or norepinephrine-stand to improve brain function in healthy individuals beyond their baseline functioning. In particular, non-medical use of prescription stimulants such as MPH and illicit use of psychostimulants for cognitive enhancement have seen a recent rise among teens and young adults in schools and college campuses. However, this enhancement likely comes with a neuronal, as well as ethical, cost. Altering glutamate function via the use of psychostimulants may impair behavioral flexibility, leading to the development and/or potentiation of addictive behaviors. Furthermore, dopamine and norepinephrine do not display linear effects; instead, their modulation of cognitive and neuronal function maps on an inverted-U curve. Healthy individuals run the risk of pushing themselves beyond optimal levels into hyperdopaminergic and hypernoradrenergic states, thus vitiating the very behaviors they are striving to improve. Finally, recent studies have begun to highlight potential damaging effects of stimulant exposure in healthy juveniles. This review explains how the main classes of cognitive enhancing drugs affect the learning and memory circuits, and highlights the potential risks and concerns in healthy individuals, particularly juveniles and adolescents. We emphasize the performance enhancement at the potential cost of brain plasticity that is associated with the neural ramifications of nootropic drugs in the healthy developing brain.

Methylphenidate and the juvenile brain: enhancement of attention at the expense of cortical plasticity?

Methylphenidate (Ritalin) is the most commonly prescribed psychoactive drug for juveniles and adolescents. Used to treat attention-deficit/hyperactivity disorder (ADHD) and for cognitive enhancement in healthy individuals, it has been regarded as a relatively safe medication for the past several decades. However, a thorough review of the literature reveals that the age-dependent activities of the drug, as well as potential developmental effects, are largely ignored. In addition, the diagnosis of ADHD is subjective, leaving open the possibility of misdiagnosis and excessive prescription of the drug. Recent studies have suggested that early life exposure of healthy rodent models to methylphenidate resulted in altered sleep/wake cycle, heightened stress reactivity, and, in fact, a dosage previously thought of as therapeutic depressed neuronal function in juvenile rats. Furthermore, juvenile rats exposed to low-dose methylphenidate displayed alterations in neural markers of plasticity, indicating that the drug might alter the basic properties of prefrontal cortical circuits. In this review of the current literature, we propose that juvenile exposure to methylphenidate may cause abnormal prefrontal function and impaired plasticity in the healthy brain, strengthening the case for developing a more thorough understanding of methylphenidate's actions on the developing, juvenile brain, as well as better diagnostic measures for ADHD.

Treatment with a clinically-relevant dose of methylphenidate alters NMDA receptor composition and synaptic plasticity in the juvenile rat prefrontal cortex.

Methylphenidate (Ritalin, MPH) is the most commonly prescribed psychoactive drug for children. Used to treat attention-deficit/hyperactivity disorder (ADHD) and for cognitive enhancement in healthy individuals, its cellular mechanisms of action and potential long-term effects are poorly understood. We recently reported that a clinically relevant (1 mg/kg i.p., single injection) dose of MPH significantly decreased neuronal excitability in the juvenile rat prefrontal cortical neurons. Here we further explore the actions of acute treatment with MPH on the level of NMDA receptor subunits and NMDA receptor-mediated short- and long-term synaptic plasticity in the juvenile rat prefrontal cortical neurons. We found that a single dose of MPH treatment (1 mg/kg, intraperitoneal) significantly decreased the surface and total protein levels of NMDA receptor subunits NR1 and NR2B, but not NR2A, in the juvenile prefrontal cortex. In addition, the amplitude, decay time and charge transfer of NMDA receptor-mediated EPSCs were significantly decreased whereas the amplitude and short-term depression of AMPA receptor-mediated EPSCs were significantly increased in the prefrontal neurons. Furthermore, MPH treatment also significantly increased the probability and magnitude of LTP induction, but had only a small effect on LTD induction in juvenile rat prefrontal cortical neurons. Our data thus present a novel mechanism of action of MPH, i.e., changes in glutamatergic receptor-mediated synaptic plasticity following early-life treatment. Furthermore, since a single dosage resulted in significant changes in NMDA receptors, off-label usage by healthy individuals, especially children and adolescents, may result in altered potential for plastic learning.

Distinct age-dependent effects of methylphenidate on developing and adult prefrontal neurons.

BACKGROUND: Methylphenidate (MPH) has long been used to treat attention-deficit/hyperactivity disorder (ADHD); however, its cellular mechanisms of action and potential effects on prefrontal cortical circuitry are not well understood, particularly in the developing brain system. A clinically relevant dose range for rodents has been established in the adult animal; however, how this range will translate to juvenile animals has not been established. METHODS: Juvenile (postnatal day [PD] 15) and adult (PD90) Sprague Dawley rats were treated with MPH or saline. Whole-cell patch clamp recording was used to examine the neuronal excitability and synaptic transmission in pyramidal neurons of prefrontal cortex. Recovery from MPH treatment was also examined at 1, 5, and 10 weeks following drug cessation. RESULTS: A dose of 1 mg/kg intraperitoneal MPH, either single dose or chronic treatment (well within the accepted therapeutic range for adults), produced significant depressive effects on pyramidal neurons by increasing hyperpolarization-activated currents in juvenile rat prefrontal cortex, while exerting excitatory effects in adult rats. Minimum clinically-relevant doses (.03 to .3 mg/kg) also produced depressive effects in juvenile rats, in a linear dose-dependent manner. Function recovered within 1 week from chronic 1 mg/kg treatment, chronic treatment with 3 and 9 mg/kg resulted in depression of prefrontal neurons lasting 10 weeks and beyond. CONCLUSIONS: These results suggest that the juvenile prefrontal cortex is supersensitive to methylphenidate, and the accepted therapeutic range for adults is an overshoot. Juvenile treatment with MPH may result in long-lasting, potentially permanent, changes to excitatory neuron function in the prefrontal cortex of juvenile rats.

A cembranoid from tobacco prevents the expression of nicotine-induced withdrawal behavior in planarian worms.

Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 microM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 microM nicotine for 24 h in the presence of 1 microM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence.

Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones.

Here we report the prevention and reversal of cocaine-induced behaviors in planarian worms by parthenolide and two related cyclic sesquiterpene lactones (SL), costunolide and santonin. Using established protocols, we studied two cocaine-induced behavioral effects in planaria; the induction of motility decrease and the induction of C-like hyperkinesia. Cocaine, parthenolide, costunolide, santonin, and a lactone-less cyclic sesquiterpene, beta-eudesmol, decreased planarian motility in a concentration-dependent manner. Only cocaine induced C-like hyperkinesia. At concentrations that did not show any motility decrease, parthenolide, costunolide and santonin, but not beta-eudesmol, significantly reduced the cocaine-induced motility decrease and C-like hyperkinesia, in a concentration-dependent manner. Furthermore, parthenolide, costunolide and santonin were able to rescue planaria from C-like hyperkinesia, after the worms were exposed to cocaine. Conversely, cocaine at a concentration that did not show any measurable effects (10 microM), was able to alleviate the SL-, but not the beta-eudesmol-induced motility decrease. Liquid Chromatography/Mass Spectrometry experiments demonstrated that cocaine does not interact directly with any of the cyclic sesquiterpenoids, which suggests specific biochemical targets for these compounds in planarians. Our data suggests a common binding site for cocaine and the sesquiterpene lactones in planarians.

Toxicity and behavioral effects of dimethylsulfoxide in planaria.

In this work, we describe aspects of the toxicity and behavioral effects of dimethylsulfoxide (DMSO) in planaria. Planarian worms have traditionally been a favored animal model in developmental biology. More recently, this organism is being recognized as an animal model in neuropharmacology research. DMSO is often used in cell and tissue culture as a cryoprotectant agent and is also commonly used to enhance the solubility of hydrophobic drugs in aqueous solutions. This compound can elicit various physiological effects in both vertebrates and invertebrates. Many drugs and drug candidates are hydrophobic, needing solvents like DMSO to be able to reach their physiological targets. As planaria becomes increasingly popular in neuropharmacology research, a description of the DMSO effects in this organism is essential. We found that DMSO is toxic to planarians at concentrations above 5% (705 mM), with an LD(50) of 10% (1.4M) at exposure times above 5 min. At sub-toxic concentrations, DMSO decreases planarian exploratory behavior in a concentration-dependent manner. This reduction in locomotor behavior is reversible and preincubation-independent. DMSO at a concentration of 0.1% (14.1 mM), which is usually enough to solubilize hydrophobic substances in aqueous solutions, did not display any toxic or behavioral effects in planaria. Therefore, in this animal model, DMSO concentrations above 0.1% should be avoided in order to be able to reliably observe any behavioral or toxic effects of hydrophobic drugs.