Optogenetic and chemogenetic approaches reveal differences in neuronal circuits that mediate initiation and maintenance of social interaction.

For social interaction to be successful, two conditions must be met: the motivation to initiate it and the ability to maintain it. This study uses both optogenetic and chemogenetic approaches to reveal the specific neural pathways that selectively influence those two social interaction components.

Acute stress modulates noradrenergic signaling in the ventral tegmental area-amygdalar circuit.

Noradrenergic neurotransmission is a critical mediator of stress responses. In turn, exposure to stress induces noradrenergic system adaptations, some of which are implicated in the etiology of stress-related disorders. Adrenergic receptors (ARs) in the ventral tegmental area (VTA) have been demonstrated to regulate phasic dopamine (DA) release in the forebrain, necessary for behavioral responses to conditional cues. However, the impact of stress on noradrenergic modulation of the VTA has not been previously explored. We demonstrate that ARs in the VTA regulate dopaminergic activity in the VTA-BLA (basolateral amygdala) circuit, a key system for processing stress-related stimuli; and that such control is altered by acute stress. We utilized fast-scan cyclic voltammetry to assess the effects of intra-VTA microinfusion of α1 -AR and α2 -AR antagonists (terazosin and RX-821002, respectively), on electrically evoked phasic DA release in the BLA in stress-naïve and stressed (unavoidable electric shocks - UES) anesthetized male Sprague-Dawley rats. In addition, we used western blotting to explore UES-induced alterations in AR protein level in the VTA. Intra-VTA terazosin or RX-821002 dose-dependently attenuated DA release in the BLA. Interestingly, UES decreased the effects of intra-VTA α2 -AR blockade on DA release (24 h but not 7 days after stress), while the effects of terazosin were unchanged. Despite changes in α2 -AR physiological function in the VTA, UES did not alter α2 -AR protein levels in either intracellular or membrane fractions. These findings demonstrate that NA-ergic modulation of the VTA-BLA circuit undergoes significant alterations in response to acute stress, with α2 -AR signaling indicated as a key target.

Recruitment of inhibitory neuronal pathways regulating dopaminergic activity for the control of cocaine seeking.

Drug seeking is associated with the ventral tegmental area (VTA) dopaminergic (DA) activity. Previously, we have shown that brief optogenetic inhibition of VTA DA neurons with 1 s pulses delivered every 9 s attenuates cocaine seeking under extinction conditions in rats without producing overt signs of dysphoria or locomotor sedation. Whether recruitment of neuronal pathways inhibiting VTA neuronal activity would suppress drug seeking remains unknown. Here, we asked if optogenetic stimulation of the lateral habenula (LHb) efferents in the rostromedial tegmental nucleus (RMTg) as well as RMTg efferents in VTA would reduce drug seeking. To investigate this, we measured how recruitment of elements of this inhibitory pathway affects cocaine seeking in male rats under extinction conditions. The effectiveness of brief optogenetic manipulations was confirmed electrophysiologically at the level of electrical activity of VTA DA neurons. Real-time conditioned place aversion (RT-CPA) and open field tests were performed to control for potential dysphoric/sedating effects of brief optogenetic stimulation of LHb-RMTg-VTA circuitry. Optogenetic stimulation of either RMTg or LHb inhibited VTA DAergic neuron firing, whereas similar stimulation of RMTg efferents in VTA or LHb efferents in RMTg reduced cocaine seeking under extinction conditions. Moreover, stimulation of LHb-RMTg efferents produced an effect that was maintained 24 h later, during cocaine seeking test without stimulation. This effect was specific, as brief optogenetic stimulation did not affect locomotor activity and was not aversive. Our results indicate that defined inhibitory pathways can be recruited to inhibit cocaine seeking, providing potential new targets for non-pharmacological treatment of drug craving.

Changes of EEG spectra in rat brains with different patterns of dysplasia in response to pilocarpine-induced seizures.

Disorders of neurogenesis at early developmental stages lead to irreversible structural and functional impairments of the brain. As further their consequences, increases in brain excitability and the development of drug-resistant epilepsy can frequently be observed in clinical cases. Mechanisms underlying these phenomena can also be examined on animal models of brain dysplasia. This study was conducted on rats with four degrees of brain dysplasia following exposure to gamma radiation on days 13, 15, 17, or 19 of prenatal development. When reached adulthood, the rats received electroencephalographic (EEG) transmitter implantation. Thereafter, pilocarpine was administered, and significant differences in susceptibility to seizures were detected depending on the degree of brain dysplasia. Before, during, and after the seizures, EEG was recorded in free moving animals. Additionally, the intensity of seizure behavioral symptoms was assessed. Strong and moderate correlations were found between the intensity of seizure behavioral symptoms, the power of particular EEG bands, and volumes of dysplastic brains and their regions. The data drew particular attention to correlations between variations in EEG spectra and changes in the midbrain and pons volumes. The results point to possible significant roles of these regions in the observed changes of susceptibility to seizures. Consequently, the frequently used experimental model was considered here not only as representing cases of cortical dysplasia but also of generalized, diffuse dysplasia of the whole brain.

Effects of brief inhibition of the ventral tegmental area dopamine neurons on the cocaine seeking during abstinence.

Preclinical studies strongly suggest that cocaine seeking depends on the neuronal activity of the ventral tegmental area (VTA) and phasic dopaminergic (DA) signaling. Notably, VTA pharmacological inactivation or dopamine receptor blockade in the forebrain may induce behavioral inhibition in general and acute aversive states in particular, thus reducing cocaine seeking indirectly. Such artifacts hinder successful translation of these findings in clinical studies and practice. Here, we aimed to evaluate if dynamic VTA manipulations effectively reduce cocaine seeking. We used male tyrosine hydroxylase (TH) IRES-Cre+ rats along with optogenetic tools to inhibit directly and briefly VTA DA neurons during conditioned stimulus (CS)-induced cocaine seeking under extinction conditions. The behavioral effects of optogenetic inhibition were also assessed in the real-time dynamic place aversion, conditioned place aversion, and CS-induced food-seeking tests. We found that brief and nondysphoric/nonsedative pulses of VTA photo-inhibition (1 s every 9 s, ie, for 10% of time) attenuated CS-induced cocaine seeking under extinction conditions in rats expressing archaerhodopsin selectively on the TH+ neurons. Furthermore, direct inhibition of the VTA DA activity reduced CS-induced cocaine seeking 24 hours after photo-modulation. Importantly, such effect appears to be selective for cocaine seeking as similar inhibition of the VTA DA activity had no effect on CS-induced food seeking. Thus, briefly inhibiting VTA DA activity during CS-induced cocaine seeking drastically and selectively reduces seeking without behavioral artifacts such as sedation or dysphoria. Our results point to the therapeutic possibilities of coupling nonpharmacologic treatments with extinction training in reducing cocaine addiction.

Differential regulation of phasic dopamine release in the forebrain by the VTA noradrenergic receptor signaling.

Phasic dopamine (DA) release from the ventral tegmental area (VTA) into forebrain structures is implicated in associative learning and conditional stimulus (CS)-evoked behavioral responses. Mounting evidence points to noradrenaline signaling in the VTA as an important regulatory input. Accordingly, adrenergic receptor (AR) blockade in the VTA has been shown to modulate CS-dependent behaviors. Here, we hypothesized that α1 - and α2 -AR (but not ß-AR) activity preferentially modulates phasic, in contrast to tonic, DA release. In addition, these effects could differ between forebrain targets. We used fast-scan cyclic voltammetric measurements in rats to assess the effects of intra-VTA microinfusion of terazosin, a selective α1 -AR antagonist, on electrically evoked phasic DA release in the nucleus accumbens (NAc) core and medial prefrontal cortex (mPFC). Terazosin dose-dependently attenuated phasic, but not tonic, DA release in the NAc core, but not in the mPFC. Next, we measured the effects of intra-VTA administration of the α2 -AR selective antagonist RX-821002 on evoked DA in the NAc core. Similar to the effects of α1 -AR blockade, intra-VTA α2 -AR blockade with RX-0821002 strongly and dose-dependently attenuated phasic, but not tonic, DA release. In contrast, no regulation by RX-821002 was observed in the mPFC. This effect was sensitive to intra-VTA blockade of D2 receptors with raclopride. Finally, the ß-AR antagonist propranolol ineffectively modulated DA release in the NAc core. These findings revealed both α1 - and α2 -ARs in the VTA as selective regulators of phasic DA release. Importantly, we demonstrated that AR blockade modulated mesolimbic, in contrast to mesocortical, DA release in previously unstudied heterogeneity in AR regulation of forebrain phasic DA.

Long-Term Consumption of High-Fat Diet in Rats: Effects on Microglial and Astrocytic Morphology and Neuronal Nitric Oxide Synthase Expression.

Obesity in humans is associated with cognitive decline and elevated risk of neurodegenerative diseases of old age. Variations of high-fat diet are often used to model these effects in animal studies. However, we previously reported improvements in markers of memory and learning, as well as larger hippocampi and higher metabolite concentrations in Wistar rats fed high-fat, high-carbohydrate diet (HFCD, 60 % energy from fat, 28 % from carbohydrates) for 1 year; this diet leads to mild ketonemia (Setkowicz et al. in PLoS One 10:e0139987, 2015). In the present study, we follow up on this cohort to assess glial morphology and expression of markers related to gliosis. Twenty-five male Wistar rats were kept on HFCD and twenty-five on normal chow. At 12 months of age, the animals were sacrificed and processed for immunohistochemical staining for astrocytic (glial fibrillary acidic protein), microglial (Iba1), and neuronal (neuronal nitric oxide synthetase, nNOS) markers in the hippocampus. We have found changes in immunopositive area fraction and cellular complexity, as studied by a simplified Sholl procedure. To our knowledge, this study is the first to apply this methodology to the study of glial cells in HFCD animals. GFAP and Iba1 immunoreactive area fraction in the hippocampi of HFCD-fed rats were decreased, while the mean number of intersections (an indirect measure of cell complexity) was decreased in GFAP-positive astrocytes, but not in Iba1-expressing microglia. At the same time, nNOS expression was lowered after HFCD in both the cortex and the hippocampus.

Fiber photometry in neuroscience research: principles, applications, and future directions.

In recent years, fluorescent sensors are enjoying a surge of popularity in the field of neuroscience. Through the development of novel genetically encoded sensors as well as improved methods of detection and analysis, fluorescent sensing has risen as a new major technique in neuroscience alongside molecular, electrophysiological, and imaging methods, opening up new avenues for research. Combined with multiphoton microscopy and fiber photometry, these sensors offer unique advantages in terms of cellular specificity, access to multiple targets - from calcium dynamics to neurotransmitter release to intracellular processes - as well as high capability for in vivo interrogation of neurobiological mechanisms underpinning behavior. Here, we provide a brief overview of the method, present examples of its integration with other tools in recent studies ranging from cellular to systems neuroscience, and discuss some of its principles and limitations, with the aim of introducing new potential users to this rapidly developing and potent technique.

Maternal fluoxetine impairs synaptic transmission and plasticity in the medial prefrontal cortex and alters the structure and function of dorsal raphe nucleus neurons in offspring mice.

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are commonly prescribed to women during pregnancy and breastfeeding despite posing a risk of adverse cognitive outcomes and affective disorders for the child. The consequences of SSRI-induced excess of 5-HT during development for the brain neuromodulatory 5-HT system remain largely unexplored. In this study, an SSRI - fluoxetine (FLX) - was administered to C57BL/6 J mouse dams during pregnancy and lactation to assess its effects on the offspring. We found that maternal FLX decreased field potentials, impaired long-term potentiation, facilitated long-term depression and tended to increase the density of 5-HTergic fibers in the medial prefrontal cortex (mPFC) of female but not male adolescent offspring. These effects were accompanied by deteriorated performance in the temporal order memory task and reduced sucrose preference with no change in marble burying behavior in FLX-exposed female offspring. We also found that maternal FLX reduced the axodendritic tree complexity of 5-HT dorsal raphe nucleus (DRN) neurons in female but not male offspring, with no changes in the excitability of DRN neurons of either sex. While no effects of maternal FLX on inhibitory postsynaptic currents (sIPSCs) in DRN neurons were found, we observed a significant influence of FLX exposure on kinetics of spontaneous excitatory postsynaptic currents (sEPSCs) in DRN neurons. Finally, we report that no changes in field potentials and synaptic plasticity were evident in the mPFC of the offspring after maternal exposure during pregnancy and lactation to a new antidepressant, vortioxetine. These findings show that in contrast to the mPFC, long-term consequences of maternal FLX exposure on the structure and function of DRN 5-HT neurons are mild and suggest a sex-dependent, distinct sensitivity of cortical and brainstem neurons to FLX exposure in early life. Vortioxetine appears to exert fewer side effects with regards to the mPFC when compared with FLX.

Structural changes in the neocortex as correlates of variations in EEG spectra and seizure susceptibility in rat brains with different degrees of dysplasia.

Disturbances of the early stages of neurogenesis lead to irreversible changes in the structure of the mature brain and its functional impairment, including increased excitability, which may be the basis for drug-resistant epilepsy. The range of possible clinical symptoms is as wide as the different stages of disturbed neurogenesis may be. In this study, we used a quadruple model of brain dysplasia by comparing structural and functional disorders in animals whose neurogenesis was disturbed with a single dose of 1 Gy of gamma rays at one of the four stages of neurogenesis, that is, on days 13, 15, 17, or 19 of prenatal development. When reached adulthood, the prenatally irradiated rats received EEG teletransmitter implantation. Thereafter, pilocarpine was administered and significant differences in susceptibility to seizure behavioral symptoms were detected depending on the degree of brain dysplasia. Before, during, and after the seizures significant correlations were found between the density of parvalbumin-immunopositive neurons located in the cerebral cortex and the intensity of behavioral seizure symptoms or increases in the power of particular EEG bands. Neurons expressing calretinin or NPY showed also dysplasia-related increases without, however, correlations with parameters of seizure intensity. The results point to significant roles of parvalbumin-expressing interneurons, and also to expression of NPY-an endogenous anticonvulsant and neuroprotectant reducing susceptibility to seizures and supporting neuronal survival.

Alpha1-adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning.

Activity of the alpha1-adrenergic receptor (α1-AR) in the ventral tegmental area (VTA) modulates dopaminergic activity, implying its modulatory role in the behavioral functions of the dopamine (DA) system. Indeed, intra-VTA α1-AR blockade attenuates conditioned stimulus dependent behaviors such as drug seeking responses signifying a role of the noradrenergic signaling in the VTA in conditioned behaviors. Importantly, the role of the VTA α1-AR activity in Pavlovian associative learning with positive outcomes remains unknown. Here, we aimed to examine how intra-VTA α1-AR blockade affects acquisition of cocaine-induced Pavlovian associative learning in the conditioned place preference (CPP) paradigm. The impact of α1-AR blockade on cocaine-reinforced operant responding and cocaine-evoked ultrasonic vocalizations (USVs) was also studied. In addition, both α1-AR immunoreactivity in the VTA and its role in phasic DA release in the nucleus accumbens (NAc) were assessed. We demonstrated cellular localization of α1-AR expression in the VTA, providing a neuroanatomical substrate for the α1-AR mechanism. We showed that prazosin (α1-AR selective antagonist; 1 µg/0.5 µl) microinfusion attenuated electrically evoked DA transients in the NAc and dose-dependently (0.1-1 µg/0.5 µl) prevented the acquisition of cocaine CPP but did not affect cocaine-reinforced operant responding nor cocaine-induced positive affective state (measured as USVs). We propose that the VTA α1-AR signaling is necessary for the acquisition of Pavlovian associative learning but does not encode hedonic value. Thus, α1-AR signaling in the VTA might underlie salience encoding of environmental stimuli and reflect an ability of alerting/orienting functions, originating from bottom-up information processing to guide behaviors.

Regulation of cocaine seeking behavior by locus coeruleus noradrenergic activity in the ventral tegmental area is time- and contingency-dependent.

Substance use disorder is linked to impairments in the ventral tegmental area (VTA) dopamine (DA) reward system. Noradrenergic (NA) inputs from locus coeruleus (LC) into VTA have been shown to modulate VTA neuronal activity, and are implicated in psychostimulant effects. Phasic LC activity controls time- and context-sensitive processes: decision making, cognitive flexibility, motivation and attention. However, it is not yet known how such temporally-distinct LC activity contributes to cocaine seeking. In a previous study we demonstrated that pharmacological inhibition of NA signaling in VTA specifically attenuates cocaine-seeking. Here, we used virally-delivered opsins to target LC neurons for inhibition or excitation, delivered onto afferents in VTA of male rats seeking cocaine under extinction conditions. Optogenetic stimulation or inhibition was delivered in distinct conditions: upon active lever press, contingently with discreet cues; or non-contingently, i.e., throughout the cocaine seeking session. Non-contingent inhibition of LC noradrenergic terminals in VTA attenuated cocaine seeking under extinction conditions. In contrast, contingent inhibition increased, while contingent stimulation reduced cocaine seeking. These findings were specific for cocaine, but not natural reward (food) seeking. Our results show that NA release in VTA drives behavior depending on timing and contingency between stimuli - context, discreet conditioned cues and reinforcer availability. We show that, depending on those factors, noradrenergic signaling in VTA has opposing roles, either driving CS-induced drug seeking, or contributing to behavioral flexibility and thus extinction.

Regulation of the immediate-early genes arc and zif268 in a mouse operant model of cocaine seeking reinstatement.

Reinstatement of extinguished operant responding for drug is an appropriate model of relapse to drug abuse. Due to the difficulty of implementing in mice the procedure of instrumental intravenous self-administration, mechanisms of reinstatement have so far been studied almost exclusively in rats. A mouse model of reinstatement of cocaine seeking has recently been characterized (Soria et al. 2008). The aim of the present study was to assess regional brain activation, as measured by induction of the immediate early genes (IEG) arc and zif268, during priming- or cue-elicited reinstatement of cocaine seeking using this new mouse model and the in situ hybridization technique. We have demonstrated that cue-elicited reinstatement of cocaine seeking was associated with induction of the IEG in the medial prefrontal cortex (prelimbic and infralimbic) and basolateral amygdala. Priming-induced reinstatement produced a more widespread up-regulation of those genes in forebrain regions including medial prefrontal, orbitofrontal and motor cortex, dorsal striatum and basolateral amygdala. These patterns of IEG expression are in agreement with previous results obtained in rats and thus indicate that the new mouse model of reinstatement is functionally equivalent to rat models. That comparability adds to the usefulness of the mouse model as a tool for addressing neurobiological mechanisms of addiction.

Alpha1-adrenergic receptor blockade in the ventral tegmental area modulates conditional stimulus-induced cocaine seeking.

Exposure to drug-associated cues evokes drug-craving and upregulates noradrenaline (NA) and dopamine (DA) system activity. Importantly, conditional stimulus-induced drug-seeking behavior depends particularly on phasic DA signaling downstream from the ventral tegmental area (VTA), a midbrain structure key for the regulation of cocaine seeking. In particular, the activity of the alpha1-adrenergic receptor (α1-AR), which has recently been hypothesized to modulate salience encoding, is capable of bidirectional regulation of VTA dopaminergic activity. Thus, the impact of the conditional stimuli (CSs) on drug-seeking behavior might involve α1-AR signaling in the VTA. To date, the role of VTA α1-ARs in regulating CS-induced cocaine seeking has not been studied. In male Sprague-Dawley rats, we found that intra-VTA terazosin, a selective α1-AR antagonist, attenuated CS-induced cocaine seeking in a novel context and under extinction conditions, as well as CS-induced reinstatement of cocaine seeking. In contrast, terazosin microinfusion in a dose that attenuated CS-induced cocaine seeking had no effects on CS-induced food seeking or stress (2 mg/kg yohimbine)-evoked reinstatement of cocaine seeking. The potential nonspecific effects (sedative, anxiogenic) of α1-AR blockade of the VTA were also measured in the open-field test. Finally, using immunostaining, we demonstrated dopamine ß-hydroxylase (DBH)-positive afferents in the VTA of cocaine-abstinent rats, providing a neuroanatomical substrate for the α1-AR mechanism. These results demonstrated for the first time that NAergic signaling via VTA α1-ARs potently and selectively regulates CS-induced cocaine seeking. Our findings provide new neuronal mechanisms that regulate cocaine craving.

Profiles of gene expression in the hippocampal formation of rats with experimentally-induced brain dysplasia.

Malformations of cortical development (MCD) are a common cause of intractable seizures in humans. Among these, focal cortical dysplasia (FCD) poses an outstanding challenge. There are several subtypes of FCD that show significant variation in pathology and clinical presentation. All types exhibit disturbed cortical cytoarchitecture and increased propensity for seizures. The etiology is likely heterogenous, with mutations, specifically in genes related to mammalian target of rapamycin (mTOR), identified in only a subset of cases. A more complex mechanism, in which underlying genetic background interacts with early, pre- or perinatal injury or stress, has been proposed. Here, we used a well-established animal model of developmental malformations similar to MCD, induced by prenatal gamma irradiation. Previously, a significant variation between times of treatment has been shown, resulting in distinct and lasting patterns of dysplasia and differentially altered seizure propensity. We set out to describe the molecular background of these patterns by performing microarray analyses of hippocampal samples obtained from adult rats previously irradiated at distinct time points during gestation: E13, E15, E17 or E19 as well as controls. The analysis was performed in three conditions: naïve, during latent phase after pilocarpine-induced status epilepticus and after 21 days of transauricular electric shocks. A set of 22 transcripts, some with known functions related to brain development, epilepsy and reaction to injury, was found to be altered between these groups across all treatments. We discuss the functional implication of these molecular differences, in an attempt to provide broader temporal and developmental context. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 718-735, 2018.

Altered Electroencephalography Spectral Profiles in Rats with Different Patterns of Experimental Brain Dysplasia.

BACKGROUND: Malformations of cortical development, such as focal cortical dysplasia, are commonly associated with intractable epilepsy. Multiple animal models were created in attempts to recapitulate features of human malformations of cortical development. These manipulations give rise to various focal or diffuse anatomical abnormalities, accompanied by altered susceptibility to epileptic seizures. Both in humans and in models of dysplasia, the question of timing of the initiating insult is important. METHODS: Here, we used a rat model of cerebral dysplasia elicited by prenatal irradiation at gestational days (E) 13, 15, 17, or 19. Previous results suggest these animals are characterized by different patterns of dysplasia as well as different reactivity to seizurogenic stimuli in several seizure models. Rats were implanted with telemetric electroencephalography (EEG) transmitters. We compared EEG data from freely moving animals with anatomical observations obtained with MRI, as well as Western blotting and immunohistochemistry. RESULTS: We performed spectral analyses of EEG signal, revealing differential regulation of specific bands (including delta, theta, alpha-beta, gamma) in animals with different patterns of dysplasia. Relative contribution of low-frequency activity in delta band is the lowest in E15 and the highest in E19. Conversely, higher frequency bands, corresponding to alpha/beta and gamma components, are reduced in E15 versus E19. This is accompanied by altered expression of glial markers in the E19 group. CONCLUSION: To our knowledge, this constitutes the first quantitative description of power spectral properties in this experimental model, providing insight into mechanisms underlying the anatomical and electrophysiological abnormalities associated with brain dysplasia of different types. Birth Defects Research, 110:303-316, 2018. © 2017 Wiley Periodicals, Inc.