Sign- and goal-tracking score does not correlate with addiction-like behavior following prolonged cocaine self-administration.

RATIONALE: In classical conditioning, sign-tracking reflects behavior directed toward a conditioned stimulus (CS) in expectation of a reward (unconditioned stimulus, US); in contrast, goal-tracking describes behavior directed toward the location of delivery of a US. As cues previously paired with drugs of abuse promote drug-seeking and drug-taking behavior in both animals and humans and thus contribute to the severity of substance abuse, sign-tracking may represent a maladaptive cue-focused behavior that may increase addiction vulnerability as compared to goal-tracking. Recent studies do, in fact, support this possibility. Previous work in this area has focused primarily on paradigms using relatively limited exposure to drug rather than extended drug intake. OBJECTIVES: Here, we used the DSM-IV-based 3-criteria (3-CRIT) model and examined whether a relationship exists between sign- or goal-tracking phenotypes and the prevalence of criteria associated with addiction-like behavior following extended cocaine self-administration as measured in this model. METHODS: Forty-six male Sprague Dawley rats underwent a Pavlovian conditioned approach (PCA) procedure and were characterized along a continuum as goal-trackers (GTs), intermediates (INTs), or sign-trackers (STs). The animals were subsequently trained to intravenous self-administer cocaine during 45 self-administration (SA) sessions and characterized for the 3 criteria outlined in the model: persistence of drug-seeking, motivation for cocaine-taking, and resistance to punishment. RESULTS: We performed correlational analyses on the traits measured, finding no relationships between PCA score and addiction-like characteristics measured using the 3-CRIT model of addiction. However, STs showed significantly greater resistance to punishment than GTs. CONCLUSIONS: Phenotyping along a continuum of PCA scores may not be a valid predictor for identifying vulnerability to the addiction-like behaviors examined using the 3-CRIT model. However, PCA phenotype may predict a single feature of the 3-CRIT model, resistance to punishment, among those rats classified as either STs or GTs.

Pianp deficiency links GABAB receptor signaling and hippocampal and cerebellar neuronal cell composition to autism-like behavior.

Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.

Pavlovian to Instrumental Transfer Responses Do Not Correlate With Addiction-Like Behavior in Rats.

Pavlovian learning plays a prominent role in the etiology of addiction. The influence of Pavlovian conditioning on the expression of an instrumental response can be studied using the Pavlovian-to-instrumental transfer (PIT) paradigm. This paradigm consists of independent Pavlovian conditioning and instrumental training prior to the combination of both during the test. During this test, the reward is not available, and an increase in the instrumental responding during conditioned stimuli presentation is a measure of PIT. Recent studies have reported a higher PIT in alcohol and nicotine dependent patients, suggesting that enhanced PIT might be a marker for dependence vulnerability. However, these studies did not use standard PIT procedures, and a clear correlation between an enhanced PIT and drug-related and addictive behaviors has so far not been demonstrated. For a systematic evaluation rats were trained in a cocaine addiction model. Addicted-like and non-addicted-like rats were subsequently assessed in the PIT paradigm. In a further experiment, rats were first tested in the PIT paradigm and thereafter subjected to cocaine self-administration (CSA) training. Our results revealed that addicted-like rats did not differ from non-addicted-like in their performance in the PIT test. However, CSA behavior showed a positive correlation with PIT. This data suggests that stronger PIT may predict higher motivational impact of conditioned stimuli on drug self-administration and improved learning of drug-cue association rather than the risk to develop addiction as such.

Sign- and goal-tracking rats show differences in various executive functions: Authors.

Sign tracking (ST) is a complex Pavlovian trait that is known to impact instrumental behaviour. Recent work suggests that this trait also correlates with altered top-down executive control relative to goal tracking (GT) rats. This raises the question as to the extent to which both phenotypes differ in executive functions. Moreover, it is unclear which cognitive processes might cause potential differences between ST and GT rats. We therefore compared the behaviour of ST and GT rats in several assays, such as outcome devaluation, attentional set shifting and reversal learning, conditional responding, as well as delayed alternation to measure different aspects of executive functioning. Goal-directed behaviour per se was not different between ST and GT rats in the outcome devaluation task. ST rats performed slightly better than GT rats in one condition of the set shifting task (place->cue shift) and the delayed alternation task, but did not perform as well in the conditional responding task. Thus, differential behavioural performance between ST and GT rats was dependent on the specific task context. Further, we found evidence that the differences in executive functions are likely related to increased incentive salience attribution and impulsive action in ST rats.

Behavioral Effects of Acute Systemic Low-Dose Clozapine in Wild-Type Rats: Implications for the Use of DREADDs in Behavioral Neuroscience.

Designer receptors exclusively activated by designer drugs (DREADDs) are popular tools used to manipulate the activity of defined groups of neurons. Recent work has shown that DREADD effects in the brain are most likely not mediated by the proposed ligand clozapine-N-oxide (CNO) but its metabolite clozapine (CLOZ). However, it is not known whether low doses of CLOZ required to activate DREADDs already have DREADD-independent effects on behavior as described for higher CLOZ doses used in previous preclinical studies. To close this gap, we compared effects of acute systemic (i.p.) CLOZ treatment vs. vehicle (VEH) in a wide range of behavioral tests in male wild-type rats. We found that CLOZ doses as low as 0.05-0.1 mg/kg significantly affected locomotion, anxiety and cognitive flexibility but had no effect on working memory or social interaction. These results highlight the need for careful controls in future chemogenetic experiments and show that previous results in studies lacking CNO/CLOZ controls may require critical re-evaluation.

Forebrain-specific, conditional silencing of Staufen2 alters synaptic plasticity, learning, and memory in rats.

BACKGROUND: Dendritic messenger RNA (mRNA) localization and subsequent local translation in dendrites critically contributes to synaptic plasticity and learning and memory. Little is known, however, about the contribution of RNA-binding proteins (RBPs) to these processes in vivo. RESULTS: To delineate the role of the double-stranded RBP Staufen2 (Stau2), we generate a transgenic rat model, in which Stau2 expression is conditionally silenced by Cre-inducible expression of a microRNA (miRNA) targeting Stau2 mRNA in adult forebrain neurons. Known physiological mRNA targets for Stau2, such as RhoA, Complexin 1, and Rgs4 mRNAs, are found to be dysregulated in brains of Stau2-deficient rats. In vivo electrophysiological recordings reveal synaptic strengthening upon stimulation, showing a shift in the frequency-response function of hippocampal synaptic plasticity to favor long-term potentiation and impair long-term depression in Stau2-deficient rats. These observations are accompanied by deficits in hippocampal spatial working memory, spatial novelty detection, and in tasks investigating associative learning and memory. CONCLUSIONS: Together, these experiments reveal a critical contribution of Stau2 to various forms of synaptic plasticity including spatial working memory and cognitive management of new environmental information. These findings might contribute to the development of treatments for conditions associated with learning and memory deficits.

Towards trans-diagnostic mechanisms in psychiatry: neurobehavioral profile of rats with a loss-of-function point mutation in the dopamine transporter gene.

The research domain criteria (RDoC) matrix has been developed to reorient psychiatric research towards measurable behavioral dimensions and underlying mechanisms. Here, we used a new genetic rat model with a loss-of-function point mutation in the dopamine transporter (DAT) gene (Slc6a3_N157K) to systematically study the RDoC matrix. First, we examined the impact of the Slc6a3_N157K mutation on monoaminergic signaling. We then performed behavioral tests representing each of the five RDoC domains: negative and positive valence systems, cognitive, social and arousal/regulatory systems. The use of RDoC may be particularly helpful for drug development. We studied the effects of a novel pharmacological approach metabotropic glutamate receptor mGluR2/3 antagonism, in DAT mutants in a comparative way with standard medications. Loss of DAT functionality in mutant rats not only elevated subcortical extracellular dopamine concentration but also altered the balance of monoaminergic transmission. DAT mutant rats showed deficits in all five RDoC domains. Thus, mutant rats failed to show conditioned fear responses, were anhedonic, were unable to learn stimulus-reward associations, showed impaired cognition and social behavior, and were hyperactive. Hyperactivity in mutant rats was reduced by amphetamine and atomoxetine, which are well-established medications to reduce hyperactivity in humans. The mGluR2/3 antagonist LY341495 also normalized hyperactivity in DAT mutant rats without affecting extracellular dopamine levels. We systematically characterized an altered dopamine system within the context of the RDoC matrix and studied mGluR2/3 antagonism as a new pharmacological strategy to treat mental disorders with underlying subcortical dopaminergic hyperactivity.

Dopamine antagonism does not impair learning of Pavlovian conditioned approach to manipulable or non-manipulable cues but biases responding towards goal tracking.

Dopamine's (DA) role in reward-processing is currently discussed as either providing a teaching signal to guide learning or mediating the transfer of incentive salience (i.e. motivational aspects) from unconditioned stimuli (US) to conditioned stimuli (CS). We used a Pavlovian conditioned approach (PCA) procedure to further investigate DAs contribution to these processes. Experiment 1 assessed the acquisition of PCA to a manipulable lever cue for 7days under DA-blockade with Flupenthixol (FLU; 225µg/kg) or Saline (SAL) treatment, followed by 6-days off-drug testing. FLU decreased the number of conditioned responses (CR) during the treatment phase, but cessation of treatment resulted in an immediate increase in CR to levels comparable to SAL controls; notably, CR in FLU-treated rats were restricted to goal tracking behaviour. During continued off-drug testing, rats from the FLU group developed sign tracking with a similar temporal pattern as controls. In experiment 2, acquisition of PCA to a non-manipulable auditory cue was investigated. FLU reduced the number of CR during treatment, and removing DA antagonism resulted in a similar rapid increase of CR as seen in experiment 1. These data complement other reports by demonstrating that, independently from the physical properties of the CS, DA is not required for learning predictive aspects of a CS-US relationship but for the development of behaviour (namely sign tracking) which is based on the motivational aspects of a CS-US relationship.

Reduced expression of nogo-a leads to motivational deficits in rats.

Nogo-A is an important neurite growth-regulatory protein in the adult and developing nervous system. Mice lacking Nogo-A, or rats with neuronal Nogo-A deficiency, exhibit behavioral abnormalities such as impaired short-term memory, decreased pre-pulse inhibition, and behavioral inflexibility. In the current study, we extended the behavioral profile of the Nogo-A deficient rat line with respect to reward sensitivity and motivation, and determined the concentrations of the monoamines dopamine and serotonin in the prefrontal cortex (PFC), dorsal striatum (dSTR), and nucleus accumbens (NAcc). Using a limited access consumption task, we found similar intake of a sweet condensed milk solution following ad libitum or restricted feeding in wild-type and Nogo-A deficient rats, indicating normal reward sensitivity and translation of hunger into feeding behavior. When tested for motivation in a spontaneous progressive ratio task, Nogo-A deficient rats exhibited lower break points and tended to have lower "highest completed ratios." Further, under extinction conditions responding ceased substantially earlier in these rats. Finally, in the PFC we found increased tissue levels of serotonin, while dopamine was unaltered. Dopamine and serotonin levels were also unaltered in the dSTR and the NAcc. In summary, these results suggest a role for Nogo-A regulated processes in motivated behavior and related neurochemistry. The behavioral pattern observed resembles aspects of the negative symptomatology of schizophrenia.

Synthetic microRNA-mediated downregulation of Nogo-A in transgenic rats reveals its role as regulator of synaptic plasticity and cognitive function.

We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.

Differential effects of subchronic phencyclidine on anxiety in the light-enhanced startle-, light/dark exploration- and open field tests.

Subchronic treatment with the N-methyl-d-aspartate receptor antagonist phencyclidine (PCP) is a valuable approach to model the symptomatology of schizophrenia, a multi-facetted psychiatric disorder, in rodents. We addressed the question whether subchronic PCP (scPCP) treatment (5 mg/kg bidaily for 7 days) would affect anxiety in rats, since contradictory findings have been reported so far. Anxiety-like behaviour was assessed using the light-enhanced startle paradigm (LES), a method which measures the effect of the natural aversion to light on the startle reflex and does not depend on motivated behaviour or exploratory drive. For comparison, anxiety-like behaviour was measured in the light-dark exploration test (LDT) and in an open field environment (OFT). The scPCP-treatment did not affect baseline startle reactivity or light-enhanced startle, suggesting normal anxiety levels in treated animals. Further, normal anxiety-like behaviour was also found in the OFT. In the LDT, scPCP treated rats displayed shorter latencies to enter the lit compartment and shuttled more between the dark and lit compartments, behaviours indicative of decreased anxiety and/or increased exploratory activity. Our findings therefore suggest that the effects of scPCP-treatment on anxiety-like behaviour are task-dependent and recommend the additional use of tests independent from exploratory drive or other motivated behaviours, such as the LES paradigm.

Stress triggers anhedonia in rats bred for learned helplessness.

Congenitally helpless (cLH) rats, a well-accepted model for depression, show reduced consumption of sweet solutions only under single-housing conditions, indicating anhedonia under stress. We investigated if anhedonic-like behaviour, measured by a reduction of sweetened-condensed milk (SCM) intake and the pleasure-attenuated startle response (PAS), could be induced by an electric foot-shock stress challenge in group-housed rats. After foot-shock stress, reduced SCM intake was observed in cLH rats compared to non-helpless (cNLH) rats. Furthermore, cLH rats also showed a decreased PAS, indicating deficient reward perception. In summary, we demonstrate that a predisposition for learned helplessness interacts with stress to trigger anhedonic-like behaviour in cLH rats. These findings further add to the validity of congenitally learned helplessness as an animal model of depression, since gene-environment interactions are considered to play a role in the etiology of this disorder.

Ambiguous-cue interpretation is biased under stress- and depression-like states in rats.

Negative cognitive bias-the tendency to interpret ambiguous situations pessimistically-is a central feature of stress-related disorders such as depression. The underlying neurobiology of this bias, however, remains unclear, not least because of a lack of translational tools. We established a new ambiguous-cue interpretation paradigm and, with respect to the etiology of depression, evaluated if environmental and genetic factors contribute to a negative bias. Rats were trained to press a lever to receive a food reward contingent to one tone and to press another lever in response to a different tone to avoid punishment by electric foot-shock. In the ambiguous-cue test, the lever-press responses to tones with frequencies intermediate to the trained tones were taken as indicators for the rats' expectation of a positive or negative event. A negative response bias because of decreased positive and increased negative responding was found in congenitally helpless rats, a genetic animal model of depression. Moreover, treatment with a combined noradrenergic-glucocorticoid challenge, mimicking stress-related changes in endogenous neuromodulation, biased rats away from positive responding. This response shift was accompanied by neuronal activation in dentate gyrus and amygdala. Thus, environmental and genetic risk factors for depression induce a response bias, which resembles the pessimistic bias of patients suffering from depression. The behavioral paradigm described constitutes a useful tool to study the neuronal basis of decision making under ambiguous conditions and may promote innovative pharmaco- and psychotherapy for depression.

Chronic corticosterone treatment impairs trace conditioning in rats with a neonatal medial prefrontal cortex lesion.

In contrast to adult lesions, which usually result in immediate functional deficits, neonatal lesions of the medial prefrontal cortex (PFC) in rats can be compensated to some extent by functional tissue during ontogeny with sparing of function in many aspects. In this study, we examined the effects of a neonatal ibotenic acid lesion of the medial PFC on trace conditioning in adult rats, a form of classical conditioning. Since the PFC is also part of the neuroendocrine stress regulation, we additionally investigated the effects of chronic pubertal corticosterone treatment on trace conditioning to test the hypothesis that early lesions render the brain vulnerable to stress-like conditions in later life. Although there was considerable regeneration of much of the lesioned tissue in the medial PFC, a disruption of cortical layering and cortical thinning was observed in the adult brain. The chronic corticosterone treatment led to a decrease of corticosterone blood serum levels and reduced adrenal weights. Neither the neonatal lesion nor the corticosterone treatment alone affected trace conditioning; however, in rats that had received both treatments trace conditioning was impaired, suggesting synergistic effects between the lesion and the glucocorticoid treatment. An interaction between two adverse events that occur at different life stages complies with neurodevelopmental theories for some psychiatric diseases like schizophrenia.

Neonatal medial prefrontal cortex lesion enhances the sensitivity of the mesoaccumbal dopamine system.

Neurodevelopmental models of schizophrenia posit that early brain damage leads to dys- or misconnection effects possibly altering synaptic transmission in brain sites distal of the lesion. We tested the hypothesis that neonatal medial prefrontal cortex (mPFC) lesions affect the sensitivity of the mesoaccumbal dopamine (DA) system. Using extracellular single-unit recordings combined with systemic application of the DA agonist apomorphine, followed by the D2 receptor antagonist haloperidol or the D1 receptor antagonist SCH23390, we compared electrophysiological properties of nucleus accumbens core and shell neurons after bilateral excitotoxic lesions of mPFC induced at postnatal day 7 or in adult rats. Whereas animals with adult mPFC lesions showed an altered discharge pattern within the core region, neonatal mPFC lesions altered the discharge pattern within the shell region. Subcutaneous administration of apomorphine (4 mg/kg) reduced accumbal firing rate in 77% of all neurons. Onset and magnitude of apomorphine-induced inhibition of neuronal activity was faster and stronger in rats with neonatal but not adult mPFC lesions in both core and shell regions. Apomorphine-induced inhibition was partially reversed by 0.1 mg/kg haloperidol only in core region of neonatal lesioned rats. Apomorphine-induced excitation of neuronal activity (in 21% of all neurons) was reversed by the D1 receptor antagonist SCH23390 (0.1 mg/kg) in all excited neurons. These data support the hypothesis that neonatal but not adult lesions of mPFC alter cortico-striatal networks and suggest that disturbance of mPFC development leads to neurodevelopmental changes in mesoaccumbal DA system during adulthood.

Effects of neonatal lesions of the medial prefrontal cortex on adult rat behaviour.

While prefrontal lesions in rodents serve as models for frontal lobe syndromes, neonatal lesions are considered as models for disconnection syndromes, such as schizophrenia. We investigated the effect of neonatal lesions of the rat medial prefrontal cortex (mPFC) together with pubertal dexamethasone-challenge on adult rat behaviour and on apomorphine-induced behavioural changes. Adult lesions were used as controls. Rats with neonatal (postnatal day 7) or adult excitotoxic lesions or sham-lesions of the mPFC were tested 9 weeks after surgery. At postnatal day 49 one group of neonatal operated rats were systemically injected with the glucocorticoid receptor agonist dexamethasone (20 mg/kg), in order to simulate stress-induced glucocorticoid receptor activation. Working memory and perseveration was tested in T-maze tasks (continuous delayed alternation and reversal learning). Additionally, locomotor activity and prepulse inhibition (PPI) of startle was tested with and without apomorphine-treatment. Brain tissue damage was assessed using Nissl-staining and parvalbumine-immunocytochemistry. Pronounced thinning of the prelimbic-infralimbic subregion of the mPFC accompanied by altered cytoarchitecture and reduced number of parvalbumine-immunopositive neurones was found after neonatal lesions while adult lesions resulted in loss of neurones accompanied by gliosis. Neonatal lesions increased perseveration in the T-maze tasks and enhanced PPI, while adult lesions induced a working memory deficit. This differential behavioural outcome presumably reflects neurodevelopmentally induced alterations in neuronal circuits after neonatal lesions versus damage to mPFC alone after adult lesions. Dexamethasone-injection at day 49 did not alter behaviour in these tasks. Motor activity was not affected by neonatal or adult lesions but dexamethasone reduced apomorphine-induced hyperlocomotion.