Topographical anatomy of the subthalamic region with special interest in the human medial forebrain bundle.

OBJECTIVE: The medial forebrain bundle (MFB) is a novel promising deep brain stimulation (DBS) target in severe affective disorders that courses through the subthalamic region according to tractography studies. Its potential therapeutic role arose in connection with the development of hypomania during stimulation of the subthalamic nucleus (STN) in Parkinson's disease, offering an alternative explanation for the occurrence of this side effect. However, until now its course exclusively described by tractography had not yet been confirmed by any anatomical method. The aim of this study was to fill this gap as well as to provide a detailed description of the fiber tracts surrounding the STN to facilitate a better understanding of the background of side effects occurring during STN DBS. METHODS: Ten human cadaveric brains (20 hemispheres) and 100 healthy subjects (200 hemispheres) from the S500 Release of the Human Connectome Project were involved in this study. Nineteen hemispheres were dissected according to Klingler's method. One additional hemisphere was prepared for histological examinations to validate the macroscopical results and stained with neurofibril silver impregnation according to Krutsay. The authors also aimed to reconstruct the MFB using tractography and correlated the results with their dissections and histological findings. RESULTS: The white matter connections coursing through the subthalamic region were successfully dissected. The ansa lenticularis, lenticular fasciculus, thalamic fasciculus, ipsi- and contralateral cerebellar fibers, and medial lemniscus were revealed as closely related fiber tracts to the STN. However, the existence of a distinct fiber bundle corresponding to the MFB described by tractography could not be identified. Using tractography, the authors showed that the depiction of the streamlines representing the MFB was also strongly dependent on the threshold parameters. CONCLUSIONS: According to this study's findings, the streamlines of the MFB described by tractography arise from the limitations of the diffusion-weighted MRI fiber tracking method and actually correspond to subthalamic fiber bundles, especially the ansa lenticularis and lenticular fasciculus, which erroneously continue in the anterior limb of the internal capsule, toward the prefrontal cortex.

A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Context-Dependent Role of Glucocorticoid Receptor Alpha and Beta in Breast Cancer Cell Behaviour.

Background. The dual role of GCs has been observed in breast cancer; however, due to many concomitant factors, GR action in cancer biology is still ambiguous. In this study, we aimed to unravel the context-dependent action of GR in breast cancer. Methods. GR expression was characterized in multiple cohorts: (1) 24,256 breast cancer specimens on the RNA level, 220 samples on the protein level and correlated with clinicopathological data; (2) oestrogen receptor (ER)-positive and -negative cell lines were used to test for the presence of ER and ligand, and the effect of the GRß isoform following GRα and GRß overexpression on GR action, by in vitro functional assays. Results. We found that GR expression was higher in ER- breast cancer cells compared to ER+ ones, and GR-transactivated genes were implicated mainly in cell migration. Immunohistochemistry showed mostly cytoplasmic but heterogenous staining irrespective of ER status. GRα increased cell proliferation, viability, and the migration of ER- cells. GRß had a similar effect on breast cancer cell viability, proliferation, and migration. However, the GRß isoform had the opposite effect depending on the presence of ER: an increased dead cell ratio was found in ER+ breast cancer cells compared to ER- ones. Interestingly, GRα and GRß action did not depend on the presence of the ligand, suggesting the role of the "intrinsic", ligand-independent action of GR in breast cancer. Conclusions. Staining differences using different GR antibodies may be the reason behind controversial findings in the literature regarding the expression of GR protein and clinicopathological data. Therefore, caution in the interpretation of immunohistochemistry should be applied. By dissecting the effects of GRα and GRß, we found that the presence of the GR in the context of ER had a different effect on cancer cell behaviour, but independently of ligand availability. Additionally, GR-transactivated genes are mostly involved in cell migration, which raises GR's importance in disease progression.

A radioanatomical study of 3rd segment terminal branches of the maxillary artery in the pterygopalatine fossa.

This study describes the clinical anatomical topography and relationship of the terminal branches of the maxillary artery to the bony wall of the maxillary sinus in the pterygopalatine fossa (PPF) to estimate the bleeding risk during surgical interventions. Using contrasted computer tomography records, (i) the route of the maxillary artery in the infratemporal fossa, (ii) the number of the arteries in the critical PPF surgery plane, (iii) the diameter of the largest artery in the area and (iv) its relation to the posterior wall of the maxillary sinus were examined. Furthermore, measurements were extended with (v) the minerality of the bony posterior wall of the maxillary sinus on bone-window images. For statistical analyses Student's t- and Fisher-test were applied. 50 patients (n = 50, 100 cases including both sides) were examined in this study. The maxillary artery reached the pterygomaxillary fissure on the lateral side of the lateral pterygoid muscle in 56% of the cases (n = 32), in 37% (n = 23) on its medial side and in 7% (n = 4) on both sides. The number of arteries at the level of the Vidian canal in the PPF varied between 1 and 4 with a median of 2. The diameter of the biggest branch was 1.2-4.7 mm, the median diameter was 1.90 mm. In 41% (n = 30) of the cases the biggest artery directly contacted the posterior wall of the maxillary sinus, and the mineral density of the posterior wall was decreased in 14.3% (n = 12) of all investigated cases. The present description and statistical analysis of the vasculature of the PPF optimizes operative planning-like clip size or the type and direction of the surgical approach-in this hidden and deep head/neck region.

Regional redistribution of CB1 cannabinoid receptors in human foetal brains with Down's syndrome and their functional modifications in Ts65Dn+/+ mice.

AIMS: The endocannabinoid system with its type 1 cannabinoid receptor (CB1 R) expressed in postmitotic neuroblasts is a critical chemotropic guidance module with its actions cascading across neurogenic commitment, neuronal polarisation and synaptogenesis in vertebrates. Here, we present the systematic analysis of regional CB1 R expression in the developing human brain from gestational week 14 until birth. In parallel, we diagrammed differences in CB1 R development in Down syndrome foetuses and identified altered CB1 R signalling. METHODS: Foetal brains with normal development or with Down's syndrome were analysed using standard immunohistochemistry, digitalised light microscopy and image analysis (NanoZoomer). CB1 R function was investigated by in vitro neuropharmacology from neonatal Ts65Dn transgenic mice brains carrying an additional copy of ~90 conserved protein-coding gene orthologues of the human chromosome 21. RESULTS: We detected a meshwork of fine-calibre, often varicose processes between the subventricular and intermediate zones of the cortical plate in the late first trimester, when telencephalic fibre tracts develop. The density of CB1 Rs gradually decreased during the second and third trimesters in the neocortex. In contrast, CB1 R density was maintained, or even increased, in the hippocampus. We found the onset of CB1 R expression being delayed by ≥1 month in age-matched foetal brains with Down's syndrome. In vitro, CB1 R excitation induced excess microtubule stabilisation and, consequently, reduced neurite outgrowth. CONCLUSIONS: We suggest that neuroarchitectural impairments in Down's syndrome brains involve the delayed development and errant functions of the endocannabinoid system, with a particular impact on endocannabinoids modulating axonal wiring.

A hypothalamic dopamine locus for psychostimulant-induced hyperlocomotion in mice.

The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain's clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th+/Dat1+ cells from its anterior subdivision innervate the LS in mice. These dopamine neurons receive dense neuropeptidergic innervation from the SCN. Reciprocal viral tracing in combination with optogenetic stimulation ex vivo identified somatostatin-containing neurons in the LS as preferred synaptic targets of extrahypothalamic A14 efferents. In vivo chemogenetic manipulation of anterior A14 neurons impacted locomotion. Moreover, chemogenetic inhibition of dopamine output from the anterior PeVN normalized amphetamine-induced hyperlocomotion, particularly during sedentary periods. Cumulatively, our findings identify a hypothalamic locus for the diurnal control of locomotion and pinpoint a midbrain-independent cellular target of psychostimulants.

The Role of Extracellular Matrix in Human Neurodegenerative Diseases.

The dense neuropil of the central nervous system leaves only limited space for extracellular substances free. The advent of immunohistochemistry, soon followed by advanced diagnostic tools, enabled us to explore the biochemical heterogeneity and compartmentalization of the brain extracellular matrix in exploratory and clinical research alike. The composition of the extracellular matrix is critical to shape neuronal function; changes in its assembly trigger or reflect brain/spinal cord malfunction. In this study, we focus on extracellular matrix changes in neurodegenerative disorders. We summarize its phenotypic appearance and biochemical characteristics, as well as the major enzymes which regulate and remodel matrix establishment in disease. The specifically built basement membrane of the central nervous system, perineuronal nets and perisynaptic axonal coats can protect neurons from toxic agents, and biochemical analysis revealed how the individual glycosaminoglycan and proteoglycan components interact with these molecules. Depending on the site, type and progress of the disease, select matrix components can either proactively trigger the formation of disease-specific harmful products, or reactively accumulate, likely to reduce tissue breakdown and neuronal loss. We review the diagnostic use and the increasing importance of medical screening of extracellular matrix components, especially enzymes, which informs us about disease status and, better yet, allows us to forecast illness.

Formulation of acyclovir (core)-dexpanthenol (sheath) nanofibrous patches for the treatment of herpes labialis.

Topically applied antiviral creams and patches are the commercially available options for the treatment of herpes labialis. The nanofibrous patches could be a new direction in the formulation. The project aimed to formulate core-shell type nanofibrous scaffolds loaded with dexpanthenol (shell) and acyclovir (core). To achieve the fast dissolution of the antiviral agent, hydroxypropyl-beta-cyclodextrin was used as a complexation agent. The further aim was to study the prepared electrospun scaffolds' morphological- and physicochemical properties and antiviral activity. The fibrous samples were prepared by electrospinning using polyvinylpyrrolidone (PVP) as a shell, hypromellose (HPMC), and poly(ethylene oxide)(PEO) composite or poly(vinyl alcohol) (PVA) as a core polymer. The morphology of the prepared sample was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy. The SEM photos showed that fibrous structures were obtained. In the case of the PVA/PVP composition, the desired structure was obtained. While when HPMC-PEO was used as a core, the core-shell structure could not be observed. The Raman measurements revealed the mixed fibre structure of this sample. All of the fibrous samples released about 100% of acyclovir and also the dexpanthenol within 20 min. Coaxially electrospun fibres of different compositions were successfully prepared with various structural homogeneities, furthermore, a better antiviral activity could be achieved compared to the commercially available Zovirax cream.

Neuropathology of the Brainstem to Mechanistically Understand and to Treat Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder as yet without effective therapy. Symptoms of this disorder typically reflect cortical malfunction with local neurohistopathology, which biased investigators to search for focal triggers and molecular mechanisms. Cortex, however, receives massive afferents from caudal brain structures, which do not only convey specific information but powerfully tune ensemble activity. Moreover, there is evidence that the start of AD is subcortical. The brainstem harbors monoamine systems, which establish a dense innervation in both allo- and neocortex. Monoaminergic synapses can co-release neuropeptides either by precisely terminating on cortical neurons or, when being "en passant", can instigate local volume transmission. Especially due to its early damage, malfunction of the ascending monoaminergic system emerges as an early sign and possible trigger of AD. This review summarizes the involvement and cascaded impairment of brainstem monoaminergic neurons in AD and discusses cellular mechanisms that lead to their dysfunction. We highlight the significance and therapeutic challenges of transmitter co-release in ascending activating system, describe the role and changes of local connections and distant afferents of brainstem nuclei in AD, and summon the rapidly increasing diagnostic window during the last few years.

Secretagogin marks amygdaloid PKCδ interneurons and modulates NMDA receptor availability.

The perception of and response to danger is critical for an individual's survival and is encoded by subcortical neurocircuits. The amygdaloid complex is the primary neuronal site that initiates bodily reactions upon external threat with local-circuit interneurons scaling output to effector pathways. Here, we categorize central amygdala neurons that express secretagogin (Scgn), a Ca2+-sensor protein, as a subset of protein kinase Cδ (PKCδ)+ interneurons, likely "off cells." Chemogenetic inactivation of Scgn+/PKCδ+ cells augmented conditioned response to perceived danger in vivo. While Ca2+-sensor proteins are typically implicated in shaping neurotransmitter release presynaptically, Scgn instead localized to postsynaptic compartments. Characterizing its role in the postsynapse, we found that Scgn regulates the cell-surface availability of NMDA receptor 2B subunits (GluN2B) with its genetic deletion leading to reduced cell membrane delivery of GluN2B, at least in vitro. Conclusively, we describe a select cell population, which gates danger avoidance behavior with secretagogin being both a selective marker and regulatory protein in their excitatory postsynaptic machinery.

Chondroitin sulfate proteoglycan-5 forms perisynaptic matrix assemblies in the adult rat cortex.

Composition of the brain extracellular matrix changes in time as maturation proceeds. Chondroitin sulfate proteoglycan 5 (CSPG-5), also known as neuroglycan C, has been previously associated to differentiation since it shapes neurite growth and synapse forming. Here, we show that this proteoglycan persists in the postnatal rat brain, and its expression is higher in cortical regions with plastic properties, including hippocampus and the medial prefrontal cortex at the end of the second postnatal week. Progressively accumulating after birth, CSPG-5 typically concentrates around glutamatergic and GABAergic terminals in twelve-week old rat hippocampus. CSPG-5-containing perisynaptic matrix rings often appear at the peripheral margin of perineuronal nets. Electron microscopy and analysis of synaptosomal fraction showed that CSPG-5 accumulates around, and is associated to synapses, respectively. In vitro analyses suggest that neurons, but less so astrocytes, express CSPG-5 in rat primary neocortical cultures, and CSPG-5 produced by transfected neuroblastoma cells appear at endings and contact points of neurites. In human subjects, CSPG-5 expression shifts in brain areas of the default mode network of suicide victims, which may reflect an impact in the pathogenesis of psychiatric diseases or support diagnostic power.

N,N-dimethyltryptamine reduces infarct size and improves functional recovery following transient focal brain ischemia in rats.

BACKGROUND AND PURPOSE: N,N-dimethyltryptamine (DMT) is an endogenous ligand of the Sigma 1 receptor (Sig-1R) with documented in vitro cytoprotective properties against hypoxia. Our aim was to demonstrate the in vivo neuroprotective effect of DMT following ischemia-reperfusion injury in the rat brain. METHODS: Transient middle cerebral occlusion (MCAO) was induced for 60 min in male Wistar rats using the filament occlusion model under general anaesthesia. Before the removal of the filament the treatment group (n = 10) received an intra-peritoneal (IP) bolus of 1 mg/kg-body weight (bw) DMT dissolved in 1 ml 7% ethanol/saline vehicle, followed by a maintenance dose of 2 mg/Kg-bw/h delivered over 24 h via osmotic minipumps. Controls (n = 10) received a vehicle bolus only. A third group (n = 10) received a Sig-1R antagonist (BD1063, 1 mg/kg-bw bolus +2 mg/kg-bw/h maintenance) in parallel with the DMT. Lesion volume was measured by MRI 24 h following the MCAO. Shortly after imaging the animals were terminated, and the native brains and sera were removed. Four rats were perfusion fixed. Functional recovery was studied in two separate group of pre-trained animals (n = 8-8) using the staircase method for 30 days. The expression levels of proteins involved in apoptosis, neuroplasticity and inflammatory regulation were assessed by real-time qPCR and ELISA. RESULTS: DMT treated rats were characterized by lower ischemic lesion volume (p = .0373), and better functional recovery (p = .0084) compared to the controls. Sig-1R was expressed both in neurons and in microglia in the peri-infarct cortex, and the DMT induced change in the lesion volume was hindered by BD1063. Lower APAF1 expression (mRNA and protein) and higher BNDF levels were documented on DTM, while decreased TNF-α, IL1-ß, IL-6 and increased IL-10 expressions indicated the compound's anti-inflammatory potential. CONCLUSION: Our results indicate a Sig-1R dependent reduction of the ischemic brain injury following exogenous DMT administration in rats, presumably through a combined anti-apoptotic, pro-neurotrophic and anti-inflammatory treatment effect.

Distribution and classification of the extracellular matrix in the olfactory bulb.

Extracellular matrix (ECM) became an important player over the last few decades when studying the plasticity and regeneration of the central nervous system. In spite of the established role of ECM in these processes throughout the central nervous system (CNS), only few papers were published on the ECM of the olfactory system, which shows a lifelong plasticity, synaptic remodeling and postnatal neurogenesis. In the present study, we have described the localization and organization of major ECM molecules, the hyaluronan, the lecticans, tenascin-R and HAPLN1 link protein in the olfactory bulb (OB) of the rat. We detected all of these molecules in the OB showing differences in the molecular composition, staining intensity, and organization of ECM between the layers and in some cases within a single layer. One of the striking features of ECM staining pattern in the OB was that the reactions are shown dominantly in the neuropil, the PNNs were found rarely and they exhibited thin or diffuse appearance Similar organization was shown in human and mice samples. As the PNN limits the neural plasticity, its rare appearance may be related to the high degree of plasticity in the OB.

Brain-wide genetic mapping identifies the indusium griseum as a prenatal target of pharmacologically unrelated psychostimulants.

Psychostimulant use is an ever-increasing socioeconomic burden, including a dramatic rise during pregnancy. Nevertheless, brain-wide effects of psychostimulant exposure are incompletely understood. Here, we performed Fos-CreERT2-based activity mapping, correlated for pregnant mouse dams and their fetuses with amphetamine, nicotine, and caffeine applied acutely during midgestation. While light-sheet microscopy-assisted intact tissue imaging revealed drug- and age-specific neuronal activation, the indusium griseum (IG) appeared indiscriminately affected. By using GAD67gfp/+ mice we subdivided the IG into a dorsolateral domain populated by γ-aminobutyric acidergic interneurons and a ventromedial segment containing glutamatergic neurons, many showing drug-induced activation and sequentially expressing Pou3f3/Brn1 and secretagogin (Scgn) during differentiation. We then combined Patch-seq and circuit mapping to show that the ventromedial IG is a quasi-continuum of glutamatergic neurons (IG-Vglut1+) reminiscent of dentate granule cells in both rodents and humans, whose dendrites emanate perpendicularly toward while their axons course parallel with the superior longitudinal fissure. IG-Vglut1+ neurons receive VGLUT1+ and VGLUT2+ excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the olfactory bulb, thus being integral to a multisynaptic circuit that could feed information antiparallel to the olfactory-cortical pathway. In IG-Vglut1+ neurons, prenatal psychostimulant exposure delayed the onset of Scgn expression. Genetic ablation of Scgn was then found to sensitize adult mice toward methamphetamine-induced epilepsy. Overall, our study identifies brain-wide targets of the most common psychostimulants, among which Scgn+/Vglut1+ neurons of the IG link limbic and olfactory circuits.

Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer's disease.

Calcium-binding proteins are widely used to distinguish neuronal subsets in the brain. This study focuses on secretagogin, an EF-hand calcium sensor, to identify distinct neuronal populations in the brainstem of several vertebrate species. By using neural tube whole mounts of mouse embryos, we show that secretagogin is already expressed during the early ontogeny of brainstem noradrenaline cells. In adults, secretagogin-expressing neurons typically populate relay centres of special senses and vegetative regulatory centres of the medulla oblongata, pons and midbrain. Notably, secretagogin expression overlapped with the brainstem column of noradrenergic cell bodies, including the locus coeruleus (A6) and the A1, A5 and A7 fields. Secretagogin expression in avian, mouse, rat and human samples showed quasi-equivalent patterns, suggesting conservation throughout vertebrate phylogeny. We found reduced secretagogin expression in locus coeruleus from subjects with Alzheimer's disease, and this reduction paralleled the loss of tyrosine hydroxylase, the enzyme rate limiting noradrenaline synthesis. Residual secretagogin immunoreactivity was confined to small submembrane domains associated with initial aberrant tau phosphorylation. In conclusion, we provide evidence that secretagogin is a useful marker to distinguish neuronal subsets in the brainstem, conserved throughout several species, and its altered expression may reflect cellular dysfunction of locus coeruleus neurons in Alzheimer's disease.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Hypothalamic cell diversity: non-neuronal codes for long-distance volume transmission by neuropeptides.

Volume transmission is a mode of intercellular communication using cerebral liquor to deliver signal molecules over long distances and allow their action for extended periods. For hypothalamic neuropeptides, nerve endings amongst ependymal cells are seen as a site of release into the cerebrospinal fluid. Recent single-cell RNA-seq data identify tanycytes and ventricular ependyma as alternative sources by being unexpectedly rich in neuroactive substances. This notion, coupled with circuit analysis showing regionalized innervation of periventricular ependyma by intrahypothalamic neurons, could allow for the integration of hypothalamic neuronal activity patterns with brain-wide activity changes upon metabolic challenges through phasic volume transmission primed by neuron-ependyma coupling. Here, we discuss emerging data for an ependymal interface and its breaches in neuropsychiatric disease.