Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Midbrain organoids for Parkinson's disease (PD) - A powerful tool to understand the disease pathogenesis.

Brain Organiods (BOs) are a promising technique for researching disease progression in the human brain. These organoids, which are produced from human induced pluripotent stem cells (HiPSCs), can construct themselves into structured frameworks. In the context of Parkinson's disease (PD), recent advancements have been made in the development of Midbrain organoids (MBOs) models that consider key pathophysiological mechanisms such as alpha-synuclein (α-Syn), Lewy bodies, dopamine loss, and microglia activation. However, there are limitations to the current use of BOs in disease modelling and drug discovery, such as the lack of vascularization, long-term differentiation, and absence of glial cells. To address these limitations, researchers have proposed the use of spinning bioreactors to improve oxygen and nutrient perfusion. Modelling PD utilising modern experimental in vitro models is a valuable tool for studying disease mechanisms and elucidating previously unknown features of PD. In this paper, we exclusively review the unique methods available for cultivating MBOs using a pumping system that mimics the circulatory system. This mechanism may aid in delivering the required amount of oxygen and nutrients to all areas of the organoids, preventing cell death, and allowing for long-term culture and using co-culturing techniques for developing glial cell in BOs. Furthermore, we emphasise some of the significant discoveries about the BOs and the potential challenges of using BOs will be discussed.

Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model.

Midbrain dopamine (mDA) neurons comprise diverse cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson's disease, while those in the ventral tegmental area (VTA) are relatively spared. Here, we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several 'territories' and 'neighborhoods' within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

Wasp sting-induced bilateral thalamic and midbrain infarction.

A male patient in his early 40s presented to the emergency department with an acute onset of respiratory distress and facial oedema, indicative of anaphylaxis. These symptoms emerged 2 hours subsequent to a wasp sting on the left side of his face. Despite initial stabilisation, the patient's state deteriorated into somnolence and disorientation. Notably, he denied any history of seizures, sensory or motor deficits, or bowel/bladder complications. Physical examination unveiled no focal neurological deficits. Routine laboratory tests and drug screening yielded no significant findings. Subsequent brain MRI with angiography exposed bilateral thalami diffusion restriction, strongly implying an acute infarction within the artery of Percheron territory, an atypical vascular variant. The sequence of events, alongside the absence of other conclusive aetiologies, indicated a wasp sting-induced thalamic infarction driven by vasogenic and thrombogenic effects of inflammatory substances.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

Delayed levodopa-responsive parkinsonism following acute midbrain injury.

Acute midbrain injury may cause both hyperkinetic movement disorders and parkinsonism. The temporal interval between the insult and the emergence of hyperkinetic disorders can last years. A delayed appearance of parkinsonism, on the other hand, was rarely described. We present three cases of male patients (50-, 58- and 28-year-old) who developed levodopa-responsive parkinsonism 20, 8 and two years, respectively, after acute brain insult involving the midbrain. Insults included subcortical intracerebral hemorrhage dissecting into the midbrain, embolic basilar occlusion and trauma. A fluorodopa scan, performed in two cases, revealed reduced striatal uptake. All individuals improved on low doses of levodopa and developed motor fluctuations shortly after levodopa was introduced. We conclude that delayed, levodopa-responsive parkinsonism following midbrain injury should be recognized in the relevant clinical setup. Possible mechanisms include age-related loss of dopaminergic neurons superimposed on acute injury and secondary neurodegeneration.

A midbrain GABAergic circuit constrains wakefulness in a mouse model of stress.

Enhancement of wakefulness is a prerequisite for adaptive behaviors to cope with acute stress, but hyperarousal is associated with impaired behavioral performance. Although the neural circuitries promoting wakefulness in acute stress conditions have been extensively identified, less is known about the circuit mechanisms constraining wakefulness to prevent hyperarousal. Here, we found that chemogenetic or optogenetic activation of GAD2-positive GABAergic neurons in the midbrain dorsal raphe nucleus (DRNGAD2) decreased wakefulness, while inhibition or ablation of these neurons produced an increase in wakefulness along with hyperactivity. Surprisingly, DRNGAD2 neurons were paradoxically wakefulness-active and were further activated by acute stress. Bidirectional manipulations revealed that DRNGAD2 neurons constrained the increase of wakefulness and arousal level in a mouse model of stress. Circuit-specific investigations demonstrated that DRNGAD2 neurons constrained wakefulness via inhibition of the wakefulness-promoting paraventricular thalamus. Therefore, the present study identified a wakefulness-constraining role DRNGAD2 neurons in acute stress conditions.

The Mouse Inferior Colliculus Responds Preferentially to Non-Ultrasonic Vocalizations.

The inferior colliculus (IC), the midbrain auditory integration center, analyzes information about social vocalizations and provides substrates for higher level processing of vocal signals. We used multichannel recordings to characterize and localize responses to social vocalizations and synthetic stimuli within the IC of female and male mice, both urethane anesthetized and unanesthetized. We compared responses to ultrasonic vocalizations (USVs) with other vocalizations in the mouse repertoire and related vocal responses to frequency tuning, IC subdivisions, and sex. Responses to lower frequency, broadband social vocalizations were widespread in IC, well represented throughout the tonotopic axis, across subdivisions, and in both sexes. Responses to USVs were much more limited. Although we observed some differences in tonal and vocal responses by sex and subdivision, representations of vocal responses by sex and subdivision were largely the same. For most units, responses to vocal signals occurred only when frequency response areas overlapped with spectra of the vocal signals. Since tuning to frequencies contained within the highest frequency USVs is limited (<15% of IC units), responses to these vocalizations are correspondingly limited (<5% of sound-responsive units). These results highlight a paradox of USV processing in some rodents: although USVs are the most abundant social vocalization, their representation and the representation of corresponding frequencies are less than lower frequency social vocalizations. We interpret this paradox in light of observations suggesting that USVs with lower frequency elements (<50 kHz) are associated with increased emotional intensity and engage a larger population of neurons in the mouse auditory system.

The people behind the papers - Andy Yang and Stephane Angers.

Activation of the Wnt signalling pathway is vital in the anterior-posterior patterning during neural development. In a new study, Stephane Angers and colleagues leverage previously developed selective antibodies against Frizzled receptors of the Wnt pathway to stimulate midbrain progenitor differentiation in human pluripotent stem cells. We caught up with first author Andy Yang and corresponding author Stephane Angers, Professor at the University of Toronto, to learn more about the story behind the paper.

A single-nuclei paired multiomic analysis of the human midbrain reveals age- and Parkinson's disease-associated glial changes.

Age is the primary risk factor for Parkinson's disease (PD), but how aging changes the expression and regulatory landscape of the brain remains unclear. Here we present a single-nuclei multiomic study profiling shared gene expression and chromatin accessibility of young, aged and PD postmortem midbrain samples. Combined multiomic analysis along a pseudopathogenesis trajectory reveals that all glial cell types are affected by age, but microglia and oligodendrocytes are further altered in PD. We present evidence for a disease-associated oligodendrocyte subtype and identify genes lost over the aging and disease process, including CARNS1, that may predispose healthy cells to develop a disease-associated phenotype. Surprisingly, we found that chromatin accessibility changed little over aging or PD within the same cell types. Peak-gene association patterns, however, are substantially altered during aging and PD, identifying cell-type-specific chromosomal loci that contain PD-associated single-nucleotide polymorphisms. Our study suggests a previously undescribed role for oligodendrocytes in aging and PD.

[Rho kinase inhibitor Y27632 promotes survival of human induced pluripotent stem cells during differentiation into functional midbrain dopaminergic progenitor cells in vitro].

OBJECTIVE: To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells (hiPSCs-NECs) into functional midbrain dopaminergic progenitor cells (DAPs). METHODS: HiPSCs were cultured in mTeSRTM medium containing DMH1 (10 µmol/L), SB431542 (10 µmol/L), SHH (200 ng/mL), FGF8 (100 ng/mL), purmorphamine (2 µmol/L), CHIR99021 (3 µmol/L), and N2 (1%) for 12 days to induce their differentiation into primitive neuroepithelial cells (NECs). The hiPSCs-NECs were digested with collagenase Ⅳ and then cultured in neurobasal medium supplemented with 1% N2, 2% B27-A, BDNF (10 ng/mL), GDNF (10 ng/mL), AA, TGF-ß, cAMP, and 1% GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632, and the culture medium was changed the next day to remove Y27632. Continuous induction was performed until day 28 to obtain DAPs. RESULTS: Human iPSCs expressed the pluripotency markers OCT4, SOX2, Nanog, and SSEA1 and were positive for alkaline phosphatase staining. The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2, nestin, and PAX6. In digested hiPSCs-NECs, the addition of 5 µmol/L Y27632 significantly promoted survival of the adherent cells, increased cell viability and the proportion of S-phase cells (P < 0.01), and reduced the rate of apoptotic cells (P < 0.05). On day 28 of induction, the obtained cells highly expressed the specific markers of DAPS (TH, FOXA2, NURR1, and Tuj1). CONCLUSION: Treatment with Y27632 (5 µmol/L) for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation, which indirectly enhances the efficiency of cell differentiation.

Optical Intracranial Self-Stimulation (oICSS): A New Behavioral Model for Studying Drug Reward and Aversion in Rodents.

Brain-stimulation reward, also known as intracranial self-stimulation (ICSS), is a commonly used procedure for studying brain reward function and drug reward. In electrical ICSS (eICSS), an electrode is surgically implanted into the medial forebrain bundle (MFB) in the lateral hypothalamus or the ventral tegmental area (VTA) in the midbrain. Operant lever responding leads to the delivery of electrical pulse stimulation. The alteration in the stimulation frequency-lever response curve is used to evaluate the impact of pharmacological agents on brain reward function. If a test drug induces a leftward or upward shift in the eICSS response curve, it implies a reward-enhancing or abuse-like effect. Conversely, if a drug causes a rightward or downward shift in the functional response curve, it suggests a reward-attenuating or aversive effect. A significant drawback of eICSS is the lack of cellular selectivity in understanding the neural substrates underlying this behavior. Excitingly, recent advancements in optical ICSS (oICSS) have facilitated the development of at least three cell type-specific oICSS models-dopamine-, glutamate-, and GABA-dependent oICSS. In these new models, a comparable stimulation frequency-lever response curve has been established and employed to study the substrate-specific mechanisms underlying brain reward function and a drug's rewarding versus aversive effects. In this review article, we summarize recent progress in this exciting research area. The findings in oICSS have not only increased our understanding of the neural mechanisms underlying drug reward and addiction but have also introduced a novel behavioral model in preclinical medication development for treating substance use disorders.

A novel method of neurophysiological brainstem mapping in neurosurgery.

BACKGROUND: Brainstem mapping with electrical stimulation allows functional identification of neural structures during resection of deep lesions. Single pulses or train of pulses are delivered to map cranial nerves and corticospinal tracts, respectively. NEW METHOD: We introduce a hybrid stimulation technique for mapping the brainstem. The stimulus consists of an electrical single pulse followed by a short train of 3-5 pulses at 500 Hz, at an interval of 60-75 ms. The responses to this stimulation pattern are recorded from appropriate cranial and limb muscles. RESULTS: Both the single pulse and the short train elicit electromyographic responses when motor fibers or motor nuclei of the cranial nerves are stimulated. Responses to the train but not to the preceding single pulse indicate activation of the descending motor tracts, in the mesencephalon and the pons. Conversely, in the medulla, limb responses to stimulation of the corticospinal tracts are elicited by a single pulse. Identification of the extra and intra-axial courses of the trigeminal motor and sensory fibers is possible by recording responses from the masseter and the tongue muscles. COMPARISON WITH EXISTING METHOD(S): To date, either a pulse or a train is delivered during brainstem mapping, switching from one to the other modality according to the expected target structure. This procedure can be time-consuming and may even lead to false negative responses to the stimulation, eventually leading to inaccurate neurosurgical procedures. CONCLUSIONS: The novel hybrid pulse-train technique enhances the advantage of brainstem mapping procedure, minimizing pitfalls and improving patient safety.

Midbrain FA initiates neuroinflammation and depression onset in both acute and chronic LPS-induced depressive model mice.

Both exogenous gaseous and liquid forms of formaldehyde (FA) can induce depressive-like behaviors in both animals and humans. Stress and neuronal excitation can elicit brain FA generation. However, whether endogenous FA participates in depression occurrence remains largely unknown. In this study, we report that midbrain FA derived from lipopolysaccharide (LPS) is a direct trigger of depression. Using an acute depressive model in mice, we found that one-week intraperitoneal injection (i.p.) of LPS activated semicarbazide-sensitive amine oxidase (SSAO) leading to FA production from the midbrain vascular endothelium. In both in vitro and in vivo experiments, FA stimulated the production of cytokines such as IL-1ß, IL-6, and TNF-α. Strikingly, one-week microinfusion of FA as well as LPS into the midbrain dorsal raphe nucleus (DRN, a 5-HT-nergic nucleus) induced depressive-like behaviors and concurrent neuroinflammation. Conversely, NaHSO3 (a FA scavenger), improved depressive symptoms associated with a reduction in the levels of midbrain FA and cytokines. Moreover, the chronic depressive model of mice injected with four-week i.p. LPS exhibited a marked elevation in the levels of midbrain LPS accompanied by a substantial increase in the levels of FA and cytokines. Notably, four-week i.p. injection of FA as well as LPS elicited cytokine storm in the midbrain and disrupted the blood-brain barrier (BBB) by activating microglia and reducing the expression of claudin 5 (CLDN5, a protein with tight junctions in the BBB). However, the administration of 30 nm nano-packed coenzyme-Q10 (Q10, an endogenous FA scavenger), phototherapy (PT) utilizing 630-nm red light to degrade FA, and the combination of PT and Q10, reduced FA accumulation and neuroinflammation in the midbrain. Moreover, the combined therapy exhibited superior therapeutic efficacy in attenuating depressive symptoms compared to individual treatments. Thus, LPS-derived FA directly initiates depression onset, thereby suggesting that scavenging FA represents a promising strategy for depression treatment.

Effects of uterine Doppler on midbrain growth and cortical development in late onset fetal growth restricted fetuses: a prospective cross-sectional study.

OBJECTIVE: To investigate midbrain growth, including corpus callusum (CC), cerebellar vermis (CV) and cortical development in late fetal growth restriction (FGR) depending on uterine artery (UtA) Pulsatility Index (PI) values. METHODS: This was a prospective study including singleton fetuses with late FGR characterized by abnormal cerebral placental ratio (CPR). According to UtA PI values, the FGR fetuses were subdivided into normal ≤95th centile) and abnormal (>95th centile). Neurosonography was performed at 33-44 weeks of gestations to assess CC and CV lengths and the depth of Sylvian fissure (SF), parieto-occipital (POF) and calcarine fissures (CF). Neurosonographic variables were normalized for fetal head circumference size. RESULTS: The study cohort included 60 fetuses with late FGR, 39 with normal UtA PI and 21 with abnormal PI values. The latter group showed significant differences in CC (median (interquartile range) normal 35.9 (28.49-45.53) vs abnormal UtA PI 25.31(19.76-35.13) mm; p < 0.0022), CV (normal 25.78 (18.19-29.35) abnormal UtA PI 17.03 (14.07-24.16)mm; p = 0.0067); SF (normal 10.58 (8.99-11.97)vs abnormal UtA PI 7.44 (6.23-8.46) mm; p < 0.0001), POF (normal 6.85 (6.35-8.14) vs abnormal UtA PI 4.82 (3.46-7.75) mm; p < = 0.0184) and CF (normal 04.157 (2.85-5.41) vs abnormal UtA PI 2.33 (2.49-4.01)); p < 0.0382). CONCLUSIONS: Late onset FGR fetuses with abnormal UtA PI showed shorter CC and CV length and delayed cortical development compared to those with normal uterine PI. These findings support the existence of a link between abnormal brain development and changes in utero placental circulation.

Bilateral thalamic and brainstem anaplastic astrocytoma: A case report.

RATIONALE: Bilateral thalamic glioma is extremely rare and characterized by strictly limited involvement of bilateral thalami. To investigate its clinical and neuroimaging features, we herein reported a rare case of anaplastic astrocytoma (AA) involving both thalami and the brainstem and reviewed the literature. PATIENT CONCERNS: A-33-year-old Chinese woman was referred to our department owing to persistent headache and nausea and vomiting. Neurological examination showed mild cognitive impairment and positive Kernig sign. DIAGNOSIS: Brain magnetic resonance imaging (MRI) demonstrated asymmetrical and swollen lesions involving both thalami, midbrain and pontine tegmentum, without restricted diffusion or enhancement. On day 7 after admission, she was transferred to the department of neurosurgery and underwent a stereotactic brain biopsy of the right thalamic lesion. Histopathological features and immunohistochemistry were consistent with AA, IDH wild-type, World Health Organization grade III. INTERVENTIONS: She was administrated with mannitol and glycerin fructose for decreasing intracranial pressure. OUTCOMES: In spite of receiving chemotherapy, she died on 2-month after her initial diagnosis. LESSONS: AA involving in both thalami and brainstem is a rare entity with poor prognosis. The clinicians and radiologists should deepen their awareness of the specific MRI feature of bilateral thalamic involvement. When MRI alone is insufficient, the utility of stereotactic biopsy is essential for making a definitive diagnosis.

Heterogeneity of mesencephalic dopaminergic neurons: From molecular classifications, electrophysiological properties to functional connectivity.

The mesencephalic dopamine (DA) system is composed of neuronal subtypes that are molecularly and functionally distinct, are responsible for specific behaviors, and are closely associated with numerous brain disorders. Existing research has made significant advances in identifying the heterogeneity of mesencephalic DA neurons, which is necessary for understanding their diverse physiological functions and disease susceptibility. Moreover, there is a conflict regarding the electrophysiological properties of the distinct subsets of midbrain DA neurons. This review aimed to elucidate recent developments in the heterogeneity of midbrain DA neurons, including subpopulation categorization, electrophysiological characteristics, and functional connectivity to provide new strategies for accurately identifying distinct subtypes of midbrain DA neurons and investigating the underlying mechanisms of these neurons in various diseases.

Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

Alpha-synuclein pathology is associated with astrocyte senescence in a midbrain organoid model of familial Parkinson's disease.

Parkinson's disease (PD) is a complex, progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta in the midbrain. Despite extensive research efforts, the molecular and cellular changes that precede neurodegeneration in PD are poorly understood. To address this, here we describe the use of patient specific human midbrain organoids harboring the SNCA triplication to investigate mechanisms underlying dopaminergic degeneration. Our midbrain organoid model recapitulates key pathological hallmarks of PD, including the aggregation of α-synuclein and the progressive loss of dopaminergic neurons. We found that these pathological hallmarks are associated with an increase in senescence associated cellular phenotypes in astrocytes including nuclear lamina defects, the presence of senescence associated heterochromatin foci, and the upregulation of cell cycle arrest genes. These results suggest a role of pathological α-synuclein in inducing astrosenescence which may, in turn, increase the vulnerability of dopaminergic neurons to degeneration.

Midbrain signaling of identity prediction errors depends on orbitofrontal cortex networks.

Outcome-guided behavior requires knowledge about the identity of future rewards. Previous work across species has shown that the dopaminergic midbrain responds to violations in expected reward identity and that the lateral orbitofrontal cortex (OFC) represents reward identity expectations. Here we used network-targeted transcranial magnetic stimulation (TMS) and functional magnetic resonance imaging (fMRI) during a trans-reinforcer reversal learning task to test the hypothesis that outcome expectations in the lateral OFC contribute to the computation of identity prediction errors (iPE) in the midbrain. Network-targeted TMS aiming at lateral OFC reduced the global connectedness of the lateral OFC and impaired reward identity learning in the first block of trials. Critically, TMS disrupted neural representations of expected reward identity in the OFC and modulated iPE responses in the midbrain. These results support the idea that iPE signals in the dopaminergic midbrain are computed based on outcome expectations represented in the lateral OFC.