Optogenetic activation of dorsal raphe serotonin neurons induces brain-wide activation.

Serotonin is a neuromodulator that affects multiple behavioral and cognitive functions. Nonetheless, how serotonin causes such a variety of effects via brain-wide projections and various receptors remains unclear. Here we measured brain-wide responses to optogenetic stimulation of serotonin neurons in the dorsal raphe nucleus (DRN) of the male mouse brain using functional MRI with an 11.7 T scanner and a cryoprobe. Transient activation of DRN serotonin neurons caused brain-wide activation, including the medial prefrontal cortex, the striatum, and the ventral tegmental area. The same stimulation under anesthesia with isoflurane decreased brain-wide activation, including the hippocampal complex. These brain-wide response patterns can be explained by DRN serotonergic projection topography and serotonin receptor expression profiles, with enhanced weights on 5-HT1 receptors. Together, these results provide insight into the DR serotonergic system, which is consistent with recent discoveries of its functions in adaptive behaviors.

Brain-derived neurotrophic factor from microglia regulates neuronal development in the medial prefrontal cortex and its associated social behavior.

Microglia and brain-derived neurotrophic factor (BDNF) are essential for the neuroplasticity that characterizes critical developmental periods. The experience-dependent development of social behaviors-associated with the medial prefrontal cortex (mPFC)-has a critical period during the juvenile period in mice. However, whether microglia and BDNF affect social development remains unclear. Herein, we aimed to elucidate the effects of microglia-derived BDNF on social behaviors and mPFC development. Mice that underwent social isolation during p21-p35 had increased Bdnf in the microglia accompanied by reduced adulthood sociability. Additionally, transgenic mice overexpressing microglial Bdnf-regulated using doxycycline at different time points-underwent behavioral, electrophysiological, and gene expression analyses. In these mice, long-term overexpression of microglial BDNF impaired sociability and excessive mPFC inhibitory neuronal circuit activity. However, administering doxycycline to normalize BDNF from p21 normalized sociability and electrophysiological function in the mPFC, whereas normalizing BDNF from later ages (p45-p50) did not normalize electrophysiological abnormalities in the mPFC, despite the improved sociability. To evaluate the possible role of BDNF in human sociability, we analyzed the relationship between adverse childhood experiences and BDNF expression in human macrophages, a possible proxy for microglia. Results show that adverse childhood experiences positively correlated with BDNF expression in M2 but not M1 macrophages. In summary, our study demonstrated the influence of microglial BDNF on the development of experience-dependent social behaviors in mice, emphasizing its specific impact on the maturation of mPFC function, particularly during the juvenile period. Furthermore, our results propose a translational implication by suggesting a potential link between BDNF secretion from macrophages and childhood experiences in humans.

Neurobiological mechanisms of electroconvulsive therapy for depression: Insights into hippocampal volumetric increases from clinical and preclinical studies.

Depression is a highly prevalent and disabling psychiatric disorder. The hippocampus, which plays a central role in mood regulation and memory, has received considerable attention in depression research. Electroconvulsive therapy (ECT) is the most effective treatment for severe pharmacotherapy-resistant depression. Although the working mechanism of ECT remains unclear, recent magnetic resonance imaging (MRI) studies have consistently reported increased hippocampal volumes following ECT. The clinical implications of these volumetric increases and the specific cellular and molecular significance are not yet fully understood. This narrative review brings together evidence from animal models and human studies to provide a detailed examination of hippocampal volumetric increases following ECT. In particular, our preclinical MRI research using a mouse model is consistent with human findings, demonstrating a marked increase in hippocampal volume following ECT. Notable changes were observed in the ventral hippocampal CA1 region, including dendritic growth and increased synaptic density at excitatory synapses. Interestingly, inhibition of neurogenesis did not affect the ECT-related hippocampal volumetric increases detected on MRI. However, it remains unclear whether these histological and volumetric changes would be correlated with the clinical effect of ECT. Hence, future research on the relationships between cellular changes, ECT-related brain volumetric changes, and antidepressant effect could benefit from a bidirectional translational approach that integrates human and animal models. Such translational research may provide important insights into the mechanisms and potential biomarkers associated with ECT-induced hippocampal volumetric changes, thereby advancing our understanding of ECT for the treatment of depression.

Neurogenesis-independent mechanisms of MRI-detectable hippocampal volume increase following electroconvulsive stimulation.

Electroconvulsive therapy (ECT) is one of the most effective psychiatric treatments but the underlying mechanisms are still unclear. In vivo human magnetic resonance imaging (MRI) studies have consistently reported ECT-induced transient hippocampal volume increases, and an animal model of ECT (electroconvulsive stimulation: ECS) was shown to increase neurogenesis. However, a causal relationship between neurogenesis and MRI-detectable hippocampal volume increases following ECT has not been verified. In this study, mice were randomly allocated into four groups, each undergoing a different number of ECS sessions (e.g., 0, 3, 6, 9). T2-weighted images were acquired using 11.7-tesla MRI. A whole brain voxel-based morphometry analysis was conducted to identify any ECS-induced brain volume changes. Additionally, a histological examination with super-resolution microscopy was conducted to investigate microstructural changes in the brain regions that showed volume changes following ECS. Furthermore, parallel experiments were performed on X-ray-irradiated mice to investigate the causal relationship between neurogenesis and ECS-related volume changes. As a result, we revealed for the first time that ECS induced MRI-detectable, dose-dependent hippocampal volume increase in mice. Furthermore, increased hippocampal volumes following ECS were seen even in mice lacking neurogenesis, suggesting that neurogenesis is not required for the increase. The comprehensive histological analyses identified an increase in excitatory synaptic density in the ventral CA1 as the major contributor to the observed hippocampal volume increase following ECS. Our findings demonstrate that modification of synaptic structures rather than neurogenesis may be the underlying biological mechanism of ECT/ECS-induced hippocampal volume increase.

Dexamethasone induces sleep disturbance in a dose-dependent manner in mice.

Synthetic corticosteroids, the most well-known anti-inflammatory drugs globally, are effective against inflammatory diseases despite their adverse effects that decrease a patient's quality of life (QOL). One of these effects is sleep disturbance, which causes other health issues and further diminishes the QOL. However, the acute effects of steroid drugs on sleep-wake issues are not fully understood and must be clarified in detail using experimental animals. Therefore, this study examines the dose-dependent effect of dexamethasone (DXM), one of the strongest steroid drugs, on the sleep-wake architecture of mice. We conducted acute DXM administration at multiple doses and 24-hour EEG/EMG recordings. Our results revealed that DXM increased the time spent in arousal and decreased that of NREM sleep, even at very low doses. These results imply that steroid-induced sleep disturbance must be addressed at any dosage.

Dopamine Receptor Type 2-Expressing Medium Spiny Neurons in the Ventral Lateral Striatum Have a Non-REM Sleep-Induce Function.

Dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) in the medial part of the ventral striatum (VS) induce non-REM (NREM) sleep from the wake state in animals. However, it is unclear whether D2-MSNs in the lateral part of the VS (VLS), which is anatomically and functionally different from the medial part of the VS, contribute to sleep-wake regulation. This study aims to clarify whether and how D2-MSNs in the VLS are involved in sleep-wake regulation. Our study found that specifically removing D2-MSNs in the VLS led to an increase in wakefulness time in mice during the dark phase using a diphtheria toxin-mediated cell ablation/dysfunction technique. D2-MSN ablation throughout the VS further increased dark phase wakefulness time. These findings suggest that VLS D2-MSNs may induce sleep during the dark phase with the medial part of the VS. Next, our fiber photometric recordings revealed that the population intracellular calcium (Ca2+) signal in the VLS D2-MSNs increased during the transition from wake to NREM sleep. The mean Ca2+ signal level of VLS D2-MSNs was higher during NREM and REM sleep than during the wake state, supporting their sleep-inducing role. Finally, optogenetic activation of the VLS D2-MSNs during the wake state always induced NREM sleep, demonstrating the causality of VLS D2-MSNs activity with sleep induction. Additionally, activation of the VLS D1-MSNs, counterparts of D2-MSNs, always induced wake from NREM sleep, indicating a wake-promoting role. In conclusion, VLS D2-MSNs could have an NREM sleep-inducing function in coordination with those in the medial VS.

Prostaglandin E2 Induces Long-Lasting Inhibition of Noradrenergic Neurons in the Locus Coeruleus and Moderates the Behavioral Response to Stressors.

Neuronal activity is modulated not only by inputs from other neurons but also by various factors, such as bioactive substances. Noradrenergic (NA) neurons in the locus coeruleus (LC-NA neurons) are involved in diverse physiological functions, including sleep/wakefulness and stress responses. Previous studies have identified various substances and receptors that modulate LC-NA neuronal activity through techniques including electrophysiology, calcium imaging, and single-cell RNA sequencing. However, many substances with unknown physiological significance have been overlooked. Here, we established an efficient screening method for identifying substances that modulate LC-NA neuronal activity through intracellular calcium ([Ca2+]i) imaging using brain slices. Using both sexes of mice, we screened 53 bioactive substances, and identified five novel substances: gastrin-releasing peptide, neuromedin U, and angiotensin II, which increase [Ca2+]i, and pancreatic polypeptide and prostaglandin D2, which decrease [Ca2+]i Among them, neuromedin U induced the greatest response in female mice. In terms of the duration of [Ca2+]i change, we focused on prostaglandin E2 (PGE2), since it induces a long-lasting decrease in [Ca2+]i via the EP3 receptor. Conditional knock-out of the receptor in LC-NA neurons resulted in increased depression-like behavior, prolonged wakefulness in the dark period, and increased [Ca2+]i after stress exposure. Our results demonstrate the effectiveness of our screening method for identifying substances that modulate a specific neuronal population in an unbiased manner and suggest that stress-induced prostaglandin E2 can suppress LC-NA neuronal activity to moderate the behavioral response to stressors. Our screening method will contribute to uncovering previously unknown physiological functions of uncharacterized bioactive substances in specific neuronal populations.SIGNIFICANCE STATEMENT Bioactive substances modulate the activity of specific neuronal populations. However, since only a limited number of substances with predicted effects have been investigated, many substances that may modulate neuronal activity have gone unrecognized. Here, we established an unbiased method for identifying modulatory substances by measuring the intracellular calcium signal, which reflects neuronal activity. We examined noradrenergic (NA) neurons in the locus coeruleus (LC-NA neurons), which are involved in diverse physiological functions. We identified five novel substances that modulate LC-NA neuronal activity. We also found that stress-induced prostaglandin E2 (PGE2) may suppress LC-NA neuronal activity and influence behavioral outcomes. Our screening method will help uncover previously overlooked functions of bioactive substances and provide insight into unrecognized roles of specific neuronal populations.

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia.

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

Brain Response to Interferential Current Compared with Alternating Current Stimulation.

Temporal interference (TI) stimulation, which utilizes multiple external electric fields with amplitude modulation for neural modulation, has emerged as a potential noninvasive brain stimulation methodology. However, the clinical application of TI stimulation is inhibited by its uncertain fundamental mechanisms, and research has previously been restricted to numerical simulations and immunohistology without considering the acute in vivo response of the neural circuit. To address the characterization and understanding of the mechanisms underlying the approach, we investigated instantaneous brainwide activation patterns in response to invasive interferential current (IFC) stimulation compared with low-frequency alternative current stimulation (ACS). Results demonstrated that IFC stimulation is capable of inducing regional neural responses and modulating brain networks; however, the activation threshold for significantly recruiting a neural response using IFC was higher (at least twofold) than stimulation via alternating current, and the spatial distribution of the activation signal was restricted. A distinct blood oxygenation level-dependent (BOLD) response pattern was observed, which could be accounted for by the activation of distinct types of cells, such as inhibitory cells, by IFC. These results suggest that IFC stimulation might not be as efficient as conventional brain modulation methods, especially when considering TI stimulation as a potential alternative for stimulating subcortical brain areas. Therefore, we argue that a future transcranial application of TI on human subjects should take these implications into account and consider other stimulation effects using this technique.

Metabolites for monitoring symptoms and predicting remission in patients with depression who received electroconvulsive therapy: a pilot study.

The lack of biomarkers to monitor and predict the efficacy of electroconvulsive therapy (ECT) has hindered its optimal use. To establish metabolomic markers for monitoring and predicting the treatment efficacy of ECT, we comprehensively evaluated metabolite levels in patients with major depressive disorder (MDD) by performing targeted and non-targeted metabolomic analyses using plasma samples before and after the first, third, and final ECT sessions, and 3-7 days after the final session. We compared the plasma metabolomes of age- and sex-matched healthy controls (HCs). Thirteen hospitalized patients with MDD and their corresponding HCs were included in this study. We observed that patients with MDD exhibited lower levels of amino acids, including gamma-aminobutyric acid (GABA), and metabolites involved in tryptophan metabolism and the kynurenine pathway, and higher levels of cortisol at baseline. Furthermore, we investigated the relationship between metabolite levels and depression severity across seven measurement timepoints along with one correlation analysis and found that amino acids, including GABA and tryptophan catabolites, were significantly correlated with the severity of depression. Despite the exploratory nature of this study due to the limited sample size necessitating further validation, our findings suggest that the blood metabolic profile has potential as a biomarker for ECT.

The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis.

The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

Inhibition of the dorsomedial striatal direct pathway is essential for the execution of action sequences.

Contrary to the previous notion that the dorsomedial striatum (DMS) is crucial for acquiring new learning, accumulated evidence has suggested that the DMS also plays a role in the execution of already learned action sequences. Here, we examined how the direct and indirect pathways in the DMS regulate action sequences using a task that requires animals to press a lever consecutively. Cell-type-specific bulk Ca2+ recording revealed that the direct pathway was inhibited at the time of sequence execution. The sequence-related response was blunted in trials where the sequential behaviors were disrupted. Optogenetic activation at the sequence start caused distraction of action sequences without affecting motor function or memory of the task structure. By contrast with the direct pathway, the indirect pathway was slightly activated at the start of the sequence, but the optogenetic suppression of such sequence-related signaling did not impact the behaviors. These results suggest that the inhibition of the DMS direct pathway promotes sequence execution potentially by suppressing the formation of a new association.

Starburst amacrine cells amplify optogenetic visual restoration through gap junctions.

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. There are limits to obtaining efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs) and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase or improve the visual restorative effect.

Acceleration of the Development of Microcirculation Embolism in the Brain due to Capillary Narrowing.

BACKGROUND: Cerebral microvascular obstruction is critically involved in recurrent stroke and decreased cerebral blood flow with age. The obstruction must occur in the capillary with a greater resistance to perfusion pressure through the microvascular networks. However, little is known about the relationship between capillary size and embolism formation. This study aimed to determine whether the capillary lumen space contributes to the development of microcirculation embolism. METHODS: To spatiotemporally manipulate capillary diameters in vivo, transgenic mice expressing the light-gated cation channel protein ChR2 (channelrhodopsin-2) in mural cells were used. The spatiotemporal changes in the regional cerebral blood flow in response to the photoactivation of ChR2 mural cells were first characterized using laser speckle flowgraphy. Capillary responses to optimized photostimulation were then examined in vivo using 2-photon microscopy. Finally, microcirculation embolism due to intravenously injected fluorescent microbeads was compared under conditions with or without photoactivation of ChR2 mural cells. RESULTS: Following transcranial photostimulation, the stimulation intensity-dependent decrease in cerebral blood flow centered at the irradiation was observed (14%-49% decreases relative to the baseline). The cerebrovascular response to photostimulation showed significant constriction of the cerebral arteries and capillaries but not of the veins. As a result of vasoconstriction, a temporal stall of red blood cell flow occurred in the capillaries of the venous sides. The 2-photon excitation of a single ChR2 pericyte demonstrated the partial shrinkage of capillaries (7% relative to the baseline) around the stimulated cell. With the intravenous injection of microbeads, the occurrence of microcirculation embolism was significantly enhanced (11% increases compared to the control) with photostimulation. CONCLUSIONS: Capillary narrowing increases the risk of developing microcirculation embolism in the venous sides of the cerebral capillaries.

Mesolimbic dopamine release precedes actively sought aversive stimuli in mice.

In some models, animals approach aversive stimuli more than those housed in an enriched environment. Here, we found that male mice in an impoverished and unstimulating (i.e., boring) chamber without toys sought aversive air puffs more often than those in an enriched chamber. Using this animal model, we identified the insular cortex as a regulator of aversion-seeking behavior. Activation and inhibition of the insular cortex increased and decreased the frequencies of air-puff self-stimulation, respectively, and the firing patterns of insular neuron ensembles predicted the self-stimulation timing. Dopamine levels in the ventrolateral striatum decreased with passive air puffs but increased with actively sought puffs. Around 20% of mice developed intense self-stimulation despite being offered toys, which was prevented by administering opioid receptor antagonists. This study establishes a basis for comprehending the neural underpinnings of usually avoided stimulus-seeking behaviors.

Slow-rising and fast-falling dopaminergic dynamics jointly adjust negative prediction error in the ventral striatum.

The greater the reward expectations are, the more different the brain's physiological response will be. Although it is well-documented that better-than-expected outcomes are encoded quantitatively via midbrain dopaminergic (DA) activity, it has been less addressed experimentally whether worse-than-expected outcomes are expressed quantitatively as well. We show that larger reward expectations upon unexpected reward omissions are associated with the preceding slower rise and following larger decrease (DA dip) in the DA concentration at the ventral striatum of mice. We set up a lever press task on a fixed ratio (FR) schedule requiring five lever presses as an effort for a food reward (FR5). The mice occasionally checked the food magazine without a reward before completing the task. The percentage of this premature magazine entry (PME) increased as the number of lever presses approached five, showing rising expectations with increasing proximity to task completion, and hence greater reward expectations. Fibre photometry of extracellular DA dynamics in the ventral striatum using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m ) revealed that the slow increase and fast decrease in DA levels around PMEs were correlated with the PME percentage, demonstrating a monotonic relationship between the DA dip amplitude and degree of expectations. Computational modelling of the lever press task implementing temporal difference errors and state transitions replicated the observed correlation between the PME frequency and DA dip amplitude in the FR5 task. Taken together, these findings indicate that the DA dip amplitude represents the degree of reward expectations monotonically, which may guide behavioural adjustment.

Pupil Dynamics-derived Sleep Stage Classification of a Head-fixed Mouse Using a Recurrent Neural Network.

The standard method for sleep state classification is thresholding the amplitudes of electroencephalography (EEG) and electromyography (EMG) data, followed by manual correction by an expert. Although popular, this method has some shortcomings: (1) the time-consuming manual correction by human experts is sometimes a bottleneck hindering sleep studies, (2) EEG electrodes on the skull interfere with wide-field imaging of the cortical activity of a head-fixed mouse under a microscope, (3) invasive surgery to fix the electrodes on the thin mouse skull risks brain tissue injury, and (4) metal electrodes for EEG and EMG recording are difficult to apply to some experimental apparatus such as that for functional magnetic resonance imaging. To overcome these shortcomings, we propose a pupil dynamics-based vigilance state classification method for a head-fixed mouse using a long short-term memory (LSTM) model, a variant of a recurrent neural network, for multi-class labeling of NREM, REM, and WAKE states. For supervisory hypnography, EEG and EMG recording were performed on head-fixed mice. This setup was combined with left eye pupillometry using a USB camera and a markerless tracking toolbox, DeepLabCut. Our open-source LSTM model with feature inputs of pupil diameter, pupil location, pupil velocity, and eyelid opening for 10 s at a 10 Hz sampling rate achieved vigilance state estimation with a higher classification performance (macro F1 score, 0.77; accuracy, 86%) than a feed-forward neural network. Findings from a diverse range of pupillary dynamics implied possible subdivision of the vigilance states defined by EEG and EMG. Pupil dynamics-based hypnography can expand the scope of alternatives for sleep stage scoring of head-fixed mice.

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle.

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter.

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

A reinforcement learning model with choice traces for a progressive ratio schedule.

The progressive ratio (PR) lever-press task serves as a benchmark for assessing goal-oriented motivation. However, a well-recognized limitation of the PR task is that only a single data point, known as the breakpoint, is obtained from an entire session as a barometer of motivation. Because the breakpoint is defined as the final ratio of responses achieved in a PR session, variations in choice behavior during the PR task cannot be captured. We addressed this limitation by constructing four reinforcement learning models: a simple Q-learning model, an asymmetric model with two learning rates, a perseverance model with choice traces, and a perseverance model without learning. These models incorporated three behavioral choices: reinforced and non-reinforced lever presses and void magazine nosepokes, because we noticed that male mice performed frequent magazine nosepokes during PR tasks. The best model was the perseverance model, which predicted a gradual reduction in amplitudes of reward prediction errors (RPEs) upon void magazine nosepokes. We confirmed the prediction experimentally with fiber photometry of extracellular dopamine (DA) dynamics in the ventral striatum of male mice using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m). We verified application of the model by acute intraperitoneal injection of low-dose methamphetamine (METH) before a PR task, which increased the frequency of magazine nosepokes during the PR session without changing the breakpoint. The perseverance model captured behavioral modulation as a result of increased initial action values, which are customarily set to zero and disregarded in reinforcement learning analysis. Our findings suggest that the perseverance model reveals the effects of psychoactive drugs on choice behaviors during PR tasks.