The Mouse Brain In Stereotaxic Coordinates
... The skull was exposed, and a small hole was drilled at the desired injection site. The following stereotaxic coordinates were used to target the left medial forebrain bundle (MFB): 1 mm posterior (from Bregma), 1.1 mm lateral and 4.8 mm ventral from dura (Paxinos and Franklin, 2001). 1 µl was injected in adult mice (P90-110) at a rate of 0.5 µl/min using a 300 µm diameter cannula attached to a 25 µl Hamilton syringe controlled by a motorized pump (Bioanalytical Systems, USA). The injection cannula was left in place for six additional minutes before slowly retracting it. ...
... Anesthesia and surgical procedures were as indicated for the nigrostriatal lesions. A CaMKII-driven ChR2-EYFP expressing adeno-associated virus (rAAV2/CaMKII-hChR2(H134R)-EYFP-WPRE-PA) was injected in the thalamus at the following coordinates: -1.3 mm posterior from Bregma, 0.8 mm lateral and -3 mm ventral from dura (Paxinos and Franklin, 2001). 0.5 μl of viral vector was delivered using a glass micropipette (World Precision Instruments) pulled with a vertical glass puller (Narishige). ...
Striatal cholinergic interneurons (SCIN) exhibit pause responses conveying information about rewarding events, but the mechanisms underlying them remain elusive. Thalamic inputs induce a pause mediated by intrinsic mechanisms and regulated by dopamine D2 receptors, though the underlying membrane currents are unknown. Moreover, the role of D5 receptors (D5R) has not been addressed so far. We show that glutamate released by thalamic inputs in the dorsolateral striatum induces a burst in SCIN, followed by the activation of a Kv1-dependent delayed rectifier current responsible for the pause. Endogenous dopamine promotes the pause through D2R stimulation, while pharmacological stimulation of D5R suppresses it. Remarkably, the pause response is absent in parkinsonian mice rendered dyskinetic by chronic L-DOPA treatment but can be reinstated acutely by the inverse D5R agonist clozapine. Blocking the Kv1 current eliminates the pause reinstated by the D5R inverse agonist. In conclusion, the pause response is mediated by delayed rectifier Kv1 channels, which are tonically blocked in dyskinetic mice by a mechanism depending on D5R ligand-independent activity. Targeting these alterations may have therapeutic value in Parkinson’s disease.
Highlights
Thalamostriatal input triggers a burst followed by a pause in SCIN.
Kv1, but not Kv7 or Kir2.2 channels, are necessary for the expression of the pause.
D2R stimulation promotes, and D5R stimulation inhibits the pause.
Thalamic bursts are not followed by a pause in SCIN from dyskinetic mice.
D5R inverse agonism restores a Kv1-dependent pause response in dyskinetic mice.
... Brain blocks were embedded in OCT compound, and a series of 16-µm-thick coronal sections containing the NAc were prepared with a cryostat (CM1850, Leica Biosystems, Tokyo, Japan). The sections collected were equivalent to the rostrocaudal level from bregma 1.70 mm to 0.86 mm in the Mouse Brain Atlas [40]. Brain sections were stored at − 80 °C for up to 2 days. ...
... Defining planes Three anterior-posterior sections from the rostral, intermed (abbreviated as "intermed"), and caudal NAc were defined (Fig. S3) based on the atlases by Franklin and Paxinos [40] and the Allen Institute [41]. The rostral plane represented (relative to the bregma) 1.78, 1.70, and 1.54 mm coronal sections, the intermed plane, 1.42, 1.34, and 1.18 mm sections, and the caudal plane, 1.10, 0.98, and 0.86 mm sections (Fig. S3E). ...
Itch is a protective/defensive function with divalent motivational drives. Itch itself elicits an unpleasant experience, which triggers the urge to scratch, relieving the itchiness. Still, it can also result in dissatisfaction when the scratch is too intense and painful or unsatisfactory due to insufficient scratch effect. Therefore, it is likely that the balance between the unpleasantness/pleasure and satisfaction/unsatisfaction associated with itch sensation and scratching behavior is determined by complex brain mechanisms. The physiological/pathological mechanisms underlying this balance remain largely elusive. To address this issue, we targeted the "reward center" of the brain, the nucleus accumbens (NAc), in which itch-responsive neurons have been found in rodents. We examined how neurons in the NAc are activated or suppressed during histamine-induced scratching behaviors in mice. The mice received an intradermal injection of histamine or saline at the neck, and the scratching number was analyzed by recording the movement of the bilateral hind limbs for about 45 min after injection. To experimentally manipulate the scratch efficacy in these histamine models, we compared histamine's behavioral and neuronal effects between mice with intact and clipped nails on the hind paws. As expected, the clipping of the hind limb nail increased the number of scratches after the histamine injection. In the brains of mice exhibiting scratching behaviors, we analyzed the expression of the c-fos gene (Fos) as a readout of an immediate activation of neurons during itch/scratch and dopamine receptors (Drd1 and Drd2) using multiplex single-molecule fluorescence in situ hybridization (RNAscope) in the NAc and surrounding structures. We performed a model-free analysis of gene expression in geometrically divided NAc subregions without assuming the conventional core–shell divisions. The results indicated that even within the NAc, multiple subregions responded differentially to various itch/scratch conditions. We also found different clusters with neurons showing similar or opposite changes in Fos expression and the correlation between scratch number and Fos expression in different itch/scratch conditions. These regional differences and clusters would provide a basis for the complex role of the NAc and surrounding structures in encoding the outcomes of scratching behavior and itchy sensations.
... Stable adhesion of the probe onto the cranium was achieved as described previously 42 . The coordinates were set to inject into the nucleus accumbens (NAc) core (at the border with the lateral shell) at bregma anterior-posterior (AP) +1.10 mm, medial-lateral (ML) ±1.50 mm, dorsal-ventral (DV) -4.60 mm, according to a mouse brain atlas 43 . These coordinates resulted in minimal injection into the anterior commissure. ...
... Using an epi uorescence microscope (Axio Observer.Z.1, Zeiss), mounting medium allowed for localization of GRAB DA or EGFP expression, and Nissl staining allowed for localization of the optic bre placement. Using a mouse brain atlas 43 ...
Whilst reward pathologies e.g., anhedonia and apathy, are major and common in stress-related neuropsychiatric disorders, their neurobiological bases and therefore treatment are poorly understood. Functional imaging studies in humans with reward pathology indicate that attenuated BOLD activity in nucleus accumbens (NAc) occurs during reward anticipation/expectancy but not reinforcement; potentially, this is dopamine (DA) related. In mice, chronic social stress (CSS) leads to reduced reward learning and effortful motivation and, here, DA-sensor fibre photometry was used to investigate whether these behavioural deficits co-occur with altered NAc DA activity during reward anticipation and/or reinforcement. In CSS mice relative to controls: (1) Reduced discriminative learning of the sequence, tone-on + appetitive behaviour = tone-on + sucrose reinforcement, co-occurred with attenuated NAc DA activity throughout tone-on and sucrose reinforcement. (2) Reduced effortful motivation during the sequence, operant behaviour = tone-on + sucrose delivery + tone-off / appetitive behaviour = sucrose reinforcement, co-occurred with attenuated NAc DA activity at tone-on and typical activity at sucrose reinforcement. (3) Reduced effortful motivation during the sequence, operant behaviour = appetitive behaviour + sociosexual reinforcement co-occurred with typical NAc DA activity at female reinforcement. Therefore, in CSS mice attenuated NAc DA activity is specific to reward anticipation and as such potentially causal to deficits in learning and motivation. CSS did not impact on the transcriptome of ventral tegmentum DA neurons, suggesting that its stimulus-specific effects on NAc DA activity originate elsewhere in the neural circuitry of reward processing.
... A Nikon ECLIPSE 80i fluorescence microscope was used to capture 4× and/or 10× objective images of whole coronal sections that were digitally stitched together using NIS-element software. Brain regions were identified using a stereotaxic mouse brain atlas [7]. To determine the average fluorophore density within specific brain regions, a ninepoint qualitative scale was used (based on the reported scale to assess Rxfp3 mRNA [27]: (-) no fluorophore; (+/-) very low; (+) low; (+/++) low to moderate; (++) moderate; (++/+++) moderate to high; (+++) high; (+++/++++) high to very high; (++++) very high density of fluorophore-positive cells (i.e., the highest density recorded, such as within the suprachiasmatic nucleus; all other areas were scored relative to this). ...
... Schematic summary of fluorophore distributions (averaged from RXFP3-Cre/YFP and RXFP3-Cre/tdTomato mouse brain), compared to previously published RXFP3 mRNA distribution. Coronal images (A-P) from a mouse brain atlas[7] with overlaid summary of in situ hybridisation mapping of RXFP3 mRNA distribution (blue dots), adapted from a published figure(Smith et al., 2010). This figure was modified by removing RXFP3 binding site and relaxin-3 immunohistochemical data, and by adding the density of fluorophores averaged from RXFP3-Cre/YFP and RXFP3-Cre/tdTomato mice, represented by shaded green areas. ...
... The drilling spots for the epidural EEG electrodes were marked stereotactically and craniotomies were performed using a dental micro driller (Ø 600 μm, Henry Schein Dental, Germany). The electrodes were placed above the prefrontal (PFC; 3.1 mm anterior to bregma, 1.5 mm lateral to midline), primary motor (MC; 1.5 mm anterior to bregma, 2 mm lateral to midline), sensory (SC; 0.9 mm posterior to bregma, 3 mm lateral to midline), and visual cortex (VC; 2.9 mm posterior to bregma, 2.3 mm lateral to midline) [31]. Additional holes were added to insert a ground electrode (frontal) and two jeweler's screws for stability (parietal). ...
... In sub-anesthetic concentrations ketamine is primarily blocking NMDA receptors of GABAergic, inhibitory interneurons [78]. The inhibition of inhibitory interneurons results in a disinhibition of excitatory neurons leading to an increased power in the beta and low gamma band (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) in humans and rodents [11,19,79]. Further increase of ketamine concentration during anesthesia leads to an inhibition of excitatory neurons and subsequently induced unconsciousness which is characterized by a gamma burst, transitioning into stable beta-gamma oscillations [11,19]. ...
The exact mechanisms and the neural circuits involved in anesthesia induced unconsciousness are still not fully understood. To elucidate them valid animal models are necessary. Since the most commonly used species in neuroscience are mice, we established a murine model for commonly used anesthetics/sedatives and evaluated the epidural electroencephalographic (EEG) patterns during slow anesthesia induction and emergence. Forty-four mice underwent surgery in which we inserted a central venous catheter and implanted nine intracranial electrodes above the prefrontal, motor, sensory, and visual cortex. After at least one week of recovery, mice were anesthetized either by inhalational sevoflurane or intravenous propofol, ketamine, or dexmedetomidine. We evaluated the loss and return of righting reflex (LORR/RORR) and recorded the electrocorticogram. For spectral analysis we focused on the prefrontal and visual cortex. In addition to analyzing the power spectral density at specific time points we evaluated the changes in the spectral power distribution longitudinally. The median time to LORR after start anesthesia ranged from 1080 [1st quartile: 960; 3rd quartile: 1080]s under sevoflurane anesthesia to 1541 [1455; 1890]s with ketamine. Around LORR sevoflurane as well as propofol induced a decrease in the theta/alpha band and an increase in the beta/gamma band. Dexmedetomidine infusion resulted in a shift towards lower frequencies with an increase in the delta range. Ketamine induced stronger activity in the higher frequencies. Our results showed substance-specific changes in EEG patterns during slow anesthesia induction. These patterns were partially identical to previous observations in humans, but also included significant differences, especially in the low frequencies. Our study emphasizes strengths and limitations of murine models in neuroscience and provides an important basis for future studies investigating complex neurophysiological mechanisms.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12871-024-02552-3.
... The principal sensory nucleus (Pr5) of the spinal trigeminal tract and locus coeruleus (LC) were microdissected by Palkovit's technique. A mouse brain stereotaxic atlas was used as a reference (Paxinos and Franklin, 2019). ...
... To confirm this, we co-stained for HA and OLIG2, and in a distinct mouse, co-stained for HA and NeuN, which marks the nucleus of most neurons. We attempted to identify structures by comparing our sections to a mouse brain atlas [43]. Note that in the low-magnification overview pictures, the scattered HApositive cells that are presumably OPCs are difficult to see because they generally have lower signal intensity in comparison to neurons that express HA-C1QL1. ...
C1QL1 is expressed in several organs including brain, but research progress has been slowed by a dearth of available antibodies. Therefore, we created a novel mouse line in which an epitope tag is inserted into the endogenous C1ql1 locus. This tool, along with improved methods, allowed a careful examination of endogenous protein expression locations, including subcellular immunolocalization to the synaptic cleft.
... Following placement in a stereotaxic frame, mice were administered lidocaine topically around the incision site. Holes were drilled bilaterally above the LA (Anterior/Posterior (AP) -1.2 mm, Medial/Lateral (ML) ± 3.4 mm, Dorsal/Ventral (DV) -4.7 mm relative to bregma) [51]. Bilateral microinjections of herpes simplex viral (HSV) vectors (1.0 μl per side) were performed with glass micropipettes over 10 min. ...
Memories are thought to be stored within sparse collections of neurons known as engram ensembles. Neurons active during a training episode are allocated to an engram ensemble (‘engram neurons’). Memory retrieval is initiated by external sensory or internal cues present at the time of training reactivating engram neurons. Interestingly, optogenetic reactivation of engram ensemble neurons alone in the absence of external sensory cues is sufficient to induce behaviour consistent with memory retrieval in mice. However, there may exist differences between the behaviours induced by natural retrieval cues or artificial engram reactivation. Here, we compared two defensive behaviours (freezing and the syllable structure of ultrasonic vocalizations, USVs) induced by sensory cues present at training (natural memory retrieval) and optogenetic engram ensemble reactivation (artificial memory retrieval) in a threat conditioning paradigm in the same mice. During natural memory recall, we observed a strong positive correlation between freezing levels and distinct USV syllable features (characterized by an unsupervised algorithm, MUPET (Mouse Ultrasonic Profile ExTraction)). Moreover, we observed strikingly similar behavioural profiles in terms of freezing and USV characteristics between natural memory recall and artificial memory recall in the absence of sensory retrieval cues. Although our analysis focused on two behavioural measures of threat memory (freezing and USV characteristics), these results underscore the similarities between threat memory recall triggered naturally and through optogenetic reactivation of engram ensembles.
This article is part of a discussion meeting issue ‘Long-term potentiation: 50 years on’.
... The experimental endpoint for mRNA and protein analysis was one or two months post- 69,7071,72 The guide cannula is closed using a dummy (PlasticsOne, #C235DCS) and secured using a dust cap (PlasticsOne, #303DC). The mice were also implanted with two EEG screw electrodes, one parietal (mid-distance between bregma and lambda and 1 mm lateral from the mid-line) and one on the cerebellum (6.5 mm caudal, 0 mm lateral from bregma, reference electrode) electrodes (Pinnacle Technology Inc., Catalog #8403) and . ...
Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.
... The brains were sectioned coronally at 30-µm thickness (Sakura Finetek). The sections of the mPFC (1.70 mm anterior to the bregma) were chosen using a stereomicroscope (Fig. 2a), according to a mouse brain atlas [35]. After washing with PBS, the sections were treated with 0.1% Triton X-100 in PBS (TPBS) for 1 h at room temperature and blocked with 1% bovine serum albumin (FUJIFILM WAKO, Osaka, Japan) in TPBS for 1 h. ...
The prevalence of depression in women increases during the postpartum period. We previously reported that subchronic exposure to social stress decreased passive coping in postpartum female mice. This study aimed to investigate whether noradrenaline regulation might regulate coping styles in mice. We first determined whether a different type of stress, subchronic physical stress, decreases passive coping in postpartum females. Postpartum female, virgin female, and male mice were exposed to subchronic restraint stress (restraint stress for 4 h for 5 consecutive days). Subchronic restraint stress decreased passive coping in postpartum females but not in virgin females and males in the forced swim and tail suspension tests. We next examined the neuronal mechanism by which subchronic stress decreases passive coping in postpartum female mice. Neuronal activity and expression of noradrenergic receptors in the medial prefrontal cortex (mPFC) were analyzed using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction, respectively. The mPFC was manipulated using chemogenetics, knockdown, or an α2A adrenergic receptor (AR) antagonist. Immunohistochemistry revealed that subchronic restraint stress increased glutamatergic neuron activation in the mPFC via forced swim stress and decreased α2A AR expression in postpartum females. Chemogenetic activation of glutamatergic neurons in the mPFC, knockdown of α2AAR in the mPFC, and the α2A AR receptor antagonist atipamezole treatment decreased passive coping in postpartum females. Subchronic restraint stress decreased passive coping in postpartum females by increasing glutamatergic neuron activity in the mPFC through α2A AR attenuation. The noradrenergic regulation of the mPFC may be a new target for treating postpartum depression.
... In what follows, and for each mouse strain, we first list the viral construct injected or device implanted, then the relevant stereotaxic coordinates are described. Stereotaxic coordinates are shown with respect to bregma, according to a standardized atlas of the mouse brain56 .Mouse strain ChAT-ires-Cre Injection Location DMV Viral construct AAV1 hSyn FLEx mGFP-2A-Synaptophysin-mRuby (300nL unilateral) Or AAV9-Ef1a-DIO EYFP (300nL each, bilateral) Coordinates: AP:-7.5mm, ML: ±0.3mm, DV -5.5 ~-5.3mm. ...
Psychological states can regulate intestinal mucosal immunity by altering the gut microbiome. However, the link between the brain and microbiome composition remains elusive. We show that Brunner's glands in the duodenal submucosa couple brain activity to intestinal bacterial homeostasis. Brunner's glands mediated the enrichment of gut probiotic species in response to stimulation of abdominal vagal fibers. Cell-specific ablation of the glands triggered a transmissible dysbiosis associated with an immunodeficiency syndrome that led to mortality upon gut infection with pathogens. The syndrome could be largely prevented by oral or intra-intestinal administration of probiotics. In the forebrain, we identified a vagally-mediated, polysynaptic circuit connecting the glands of Brunner to the central nucleus of the amygdala. Intra-vital imaging revealed that excitation of central amygdala neurons activated Brunner's glands and promoted the growth of probiotic populations. Our findings unveil a vagal-glandular neuroimmune circuitry that may be targeted for the modulation of the gut microbiome.
... Regions of interest (ACC, BNST, BLA, and CeA) were isolated by a brain punching technique from the whole-mount brain in the cryostat Leica CM3050 S (Leica Biosystems, Nussloch, Germany) at −20˚C using biopsy pens EMS Rapid-Core (Electron Microscopy Sciences, Hatfield, United States of America). To localise the subregions of interest in the brain, punching was carried out by referencing toluidine blue staining of brain slices [79]. ACC was sampled approximately from 1.34 to 0.5 mm (from bregma) using a 1 mm pen. ...
Trait anxiety is a major risk factor for stress-induced and anxiety disorders in humans. However, animal models accounting for the interindividual variability in stress vulnerability are largely lacking. Moreover, the pervasive bias of using mostly male animals in preclinical studies poorly reflects the increased prevalence of psychiatric disorders in women. Using the threat imminence continuum theory, we designed and validated an auditory aversive conditioning-based pipeline in both female and male mice. We operationalised trait anxiety by harnessing the naturally occurring variability of defensive freezing responses combined with a model-based clustering strategy. While sustained freezing during prolonged retrieval sessions was identified as an anxiety-endophenotype behavioral marker in both sexes, females were consistently associated with an increased freezing response. RNA-sequencing of CeA, BLA, ACC, and BNST revealed massive differences in phasic and sustained responders’ transcriptomes, correlating with transcriptomic signatures of psychiatric disorders, particularly post-traumatic stress disorder (PTSD). Moreover, we detected significant alterations in the excitation/inhibition balance of principal neurons in the lateral amygdala. These findings provide compelling evidence that trait anxiety in inbred mice can be leveraged to develop translationally relevant preclinical models to investigate mechanisms of stress susceptibility in a sex-specific manner.
... The cerebellum of mice aged 3, 6, 9, and 12 months was cryosectioned into 10 μm thick sequential sagittal sections and mounted onto Vectabond ® -coated (Vector laboratories, Newark, USA) slides. Five consecutive sections corresponding to plate 106 of the Paxinos's atlas [27] were selected for analysis. Both Nissl and immunohistochemical staining were analyzed by representative photographs acquired with an upright microscope (Nikon 90i, Nikon, Tokyo, Japan) and a DXM1200F camera. ...
Friedreich’s ataxia is a hereditary neurodegenerative disorder resulting from reduced levels of the protein frataxin due to an expanded GAA repeat in the FXN gene. This deficiency causes progressive degeneration of specific neuronal populations in the cerebellum and the consequent loss of movement coordination and equilibrium, some of the main symptoms observed in affected individuals. Similar to other neurodegenerative diseases, previous studies suggest that glial cells could be involved in the neurodegenerative process and disease progression in Friedreich’s ataxia.
In this work, we have followed and characterized the progression of changes in the cerebellar cortex of the latest Friedreich’s ataxia humanized mouse model, the YG8-800 (Fxn null :YG8s(GAA)>800), which carries a human FXN transgene containing more than 800 GAA repeats.
Comparative analyses of behavioral, histopathological, and biochemical parameters were conducted between Y47R control and YG8-800 mice at different time points. Our findings revealed that the YG8-800 mice display an ataxic phenotype, characterized by poor motor coordination, lower body weight, cerebellar atrophy, neuronal loss, and changes in synaptic proteins. Additionally, early activation of glial cells, predominantly astrocytes and microglia, was observed preceding neuronal degeneration along with an increased expression of key pro-inflammatory cytokines and downregulation of neurotrophic factors.
Together, our results show how the YG8-800 mouse model exhibits a stronger phenotype than previous experimental murine models, reliably recapitulating some of the features observed in the human condition. Accordingly, this humanized model could represent a valuable tool to study Friedreich’s ataxia molecular disease mechanisms and for preclinical evaluation of possible therapies.
... For the interneuron counting in Figure 7, the jointed images for entire brain sections were obtained using a Keyence BZ-X700 microscope with a 10× objective lens, and the counting was performed by naked eye observations of persons who were blinded to the genotype. Four to six coronal sections [AP, −1.34 to −2.06 mm from the Bregma sections (Paxinos and Franklin, 2001)] were analyzed to obtain a mean value for a mouse. ...
The Slitrk family consists of six synaptic adhesion molecules, some of which are associated with neuropsychiatric disorders. In this study, we aimed to investigate the physiological role of Slitrk4 by analyzing Slitrk4 knockout (KO) mice. The Slitrk4 protein was widely detected in the brain and was abundant in the olfactory bulb and amygdala. In a systematic behavioral analysis, male Slitrk4 KO mice exhibited an enhanced fear memory acquisition in a cued test for classical fear conditioning, and social behavior deficits in reciprocal social interaction tests. In an electrophysiological analysis using amygdala slices, Slitrk4 KO mice showed enhanced long-term potentiation in the thalamo-amygdala afferents and reduced feedback inhibition. In the molecular marker analysis of Slitrk4 KO brains, the number of calretinin (CR)-positive interneurons was decreased in the anterior part of the lateral amygdala nuclei at the adult stage. In in vitro experiments for neuronal differentiation, Slitrk4-deficient embryonic stem cells were defective in inducing GABAergic interneurons with an altered response to sonic hedgehog signaling activation that was involved in the generation of GABAergic interneuron subsets. These results indicate that Slitrk4 function is related to the development of inhibitory neurons in the fear memory circuit and would contribute to a better understanding of osttraumatic stress disorder, in which an altered expression of Slitrk4 has been reported.
... Slices were imaged using light microscopy (Zeiss Axio Scope A2, Germany). Areas assessed were found using the following stereotaxic coordinates [57]: Bregma anterior-posterior +0.5 mm (frontal motor cortex). Image analysis was carried out with Golgi microscope (Zeiss AxioImager M2, Germany) and Neurolucida software (MicroBrightField, Williston, VT). ...
The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 −/− mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer’s disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.
... An indirect immunohistochemistry protocol was used with the primary antibody for TH (F-11, human, mouse monoclonal IG 2a, Santa Cruz Biotechnology, CA, USA) followed by the secondary biotinylated antibody (Santa Cruz Biotechnology, CA, USA). The quanti cation of TH + cells in the SNpc was carefully delimited according to the brain atlas of Paximos et al. [13], and cells were counted in 12 elds of vision at 1000X magni cation covering the entire area of the SNpc. ...
Alterations of the microbiota-gut-brain axis has been associated with intestinal and neuronal inflammation in Parkinson’s disease (PD). The aim of this work was to study some mechanisms associated with the neuroprotective effect of a combination (MIX) of lactic acid bacteria (LAB) composed by Lactiplantibacillus plantarum CRL2130 (riboflavin overproducing strain), Streptococcus thermophilus ( St. ) CRL808 (folate producer strain), and St. CRL807 (immunomodulatory strain) in a chronic model of parkinsonism induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in aged mice, and under levodopa-benserazide treatment. Motor skills, tyrosine hydrolase (TH) in brain and cytokine concentrations in serum and in brain were evaluated. The study of the faecal microbiota and the histology of the small intestine was also performed. The results showed that the neuroprotective effect associated with LAB MIX administration did not interfere with levodopa-benserazide treatment and was associated with the significant improvement in the motor tests and a higher number of TH + cells in the brain. In addition, LAB MIX administration was associated with modulation of the immune response. LAB administration decreased intestinal damage with an increase in the villus length /crypt depth ratio. Finally, the administration of the LAB MIX in combination with levodopa-benserazide treatment was able to partially revert the intestinal dysbiosis observed in the model, showing greater similarity to the profiles of healthy controls, and highlighting the increase in the Lactobacillaceae family. Different mechanisms of action would be related to the protective effect of the selected LAB combination which has the potential to be evaluated as an adjuvant for conventional PD therapies.
... Prior work using conventional anterograde and retrograde tracing has shown that the PB Calca/CGRP neurons and the external lateral PB receive inputs from a variety of CNS regions, including the spinal cord, nucleus of the solitary tract (NTS), reticular formation, periaqueductal gray (PAG), superior and inferior colliculi, arcuate nucleus (Arc), lateral dorsal bed nucleus of the stria terminalis (BSTLD), and central nucleus of the amygdala (CeA) (Palmiter, 2018;Kang et al., 2022;Kirouac et al., 2022) ( Table 4 provides a list of all abbreviations which follow Paxinos (Paxinos and Franklin, 2004) except for some abbreviations from Swanson (Swanson, 2004)). The PB Calca/CGRP neurons receive inhibitory inputs from agouti-related protein (AgRP) neurons of the arcuate nucleus (Arc) (Campos et al., 2016), but the mapping and characterization of other inputs remain incomplete. ...
The parabrachial nucleus (PB), located in the dorsolateral pons, contains primarily glutamatergic neurons which regulate responses to a variety of interoceptive and cutaneous sensory signals. The lateral PB subpopulation expressing the Calca gene which produces the neuropeptide calcitonin gene-related peptide (CGRP) relays signals related to threatening stimuli such as hypercarbia, pain, and nausea, yet the afferents to these neurons are only partially understood. We mapped the afferent projections to the lateral part of the PB in mice using conventional cholera toxin B subunit (CTb) retrograde tracing, and then used conditional rabies virus retrograde tracing to map monosynaptic inputs specifically targeting the PBCalca/CGRP neurons. Using vesicular GABA (vGAT) and glutamate (vGLUT2) transporter reporter mice, we found that lateral PB neurons receive GABAergic afferents from regions such as the lateral part of the central nucleus of the amygdala, lateral dorsal subnucleus of the bed nucleus of the stria terminalis, substantia innominata, and the ventrolateral periaqueductal gray. Additionally, they receive glutamatergic afferents from the infralimbic and insular cortex, paraventricular nucleus, parasubthalamic nucleus, trigeminal complex, medullary reticular nucleus, and nucleus of the solitary tract. Using anterograde tracing and confocal microscopy, we then identified close axonal appositions between these afferents and PBCalca/CGRP neurons. Finally, we used channelrhodopsin-assisted circuit mapping to test whether some of these inputs directly synapse upon the PBCalca/CGRP neurons. These findings provide a comprehensive neuroanatomical framework for understanding the afferent projections regulating the PBCalca/CGRP neurons.
... According to The Mouse Brain in Stereotaxic Coordinates map [17], immunohistochemical photomicrographs containing principal part of the PVN (Plates 25-26) and SON (Plates 22-24) were identi ed using a LAS Image Analysis System (Leica, Wetzlar, Germany). Assessment of the number of VP pro le was obtained from the observation of every fourth section of eight mice in the principal region of the SON and PVN in each group. ...
Background Under hypertensive conditions, vitamin D has a protective effect on the brain. Our previous research showed that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] negatively regulates hypertension and central renin–angiotensin system activation partly through a central antioxidative mechanism in 1α-hydroxylase knockout [1α(OH)ase–/–] mice. To further confirm whether the endogenous 1,25(OH)2D3 deficiency and exogenous 1,25(OH)2D3 supplementation alter cerebrovascular function and vasopressin expression through antioxidation, we provided 1α(OH)ase–/– mice and their wild-type littermates with normal diet; a high-calcium, high-phosphorus rescue diet with N-acetyl-l-cysteine supplementation; or 1,25(OH)2D3 subcutaneous injection. We analysed and compared the changes in arterial blood pressure, brain microvessel reactivity, cerebral blood flow, expression of hypothalamic vasopressin, and brain/blood oxidation and antioxidative indices using caudal artery plethysmography, isolated microvessel pressure myographs, laser Doppler flowmetry, immunohistochemistry, western blot and biochemistry.
Results Compared with their wild-type littermates, the hypertension phenotype was present in the 1α(OH)ase–/– mice, hypothalamic paraventricular nucleus and supraoptic nucleus vasopressin expression was significantly upregulated, and the posterior cerebral artery reaction to the vasodilatory effect of acetylcholine and vasoconstrictive effect of the nitric oxide synthase inhibitor L-nitro-arginine was significantly decreased. Brain/blood oxidative stress was increased, but the antioxidative parameters were decreased. These pathologic changes were corrected by 1,25(OH)2D3 or N-acetyl-l-cysteine plus rescue diet.
Conclusions our findings indicate that 1,25(OH)2D3 has an inhibitory effect on vasopressin expression and cerebrovascular dysfunction. 1,25(OH)2D3 may be a promising protective intervention to reduce brain impaired induced by oxidative stress in the hypertension phenotype of 1α(OH)ase–/– mice.
... A programmable microsyringe pump was used to deliver bilaterally 4 µL (8 μL per intact brain) of Atsttrin (Atreaon, Inc.; Newton, CA, USA) dissolved in a sterile 0.9% saline solution (NaCl) with doses of 0.1 μg (0.025 μg/ μL), 0.5 μg (0.125 μg/μL), 1 μg (0.25 μg/μL), 2 μg (0.5 μg/ μL), and 5 μg (1.25 μg/μL) into ST at the following stereotactic coordinates-AP (y), + 0.62; ML (x), ± 1.75 relative to the bregma; and DV (z), − 3.5 mm relative to the dura (Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates, 2nd Edition; Academic Press, 2001, San Diego, CA, USA) with automatic flow rate estimated as 0.5 μL/min [35]. After the injection, the microneedle was kept in the brain for 3 min to minimize the risk of reflux of the injected substance along the intracranial path of the withdrawn cannula. ...
Parkinson’s disease is one of the most common neurodegenerative disorders characterized by a multitude of motor and non-motor clinical symptoms resulting from the progressive and long-lasting abnormal loss of nigrostriatal dopaminergic neurons. Currently, the available treatments for patients with Parkinson’s disease are limited and exert only symptomatic effects, without adequate signs of delaying or stopping the progression of the disease. Atsttrin constitutes the bioengineered protein which ultrastructure is based on the polypeptide chain frame of the progranulin (PGRN), which exerts anti-inflammatory effects through the inhibition of TNFα. The conducted preclinical studies suggest that the therapeutic implementation of Atsttrin may be potentially effective in the treatment of neurodegenerative diseases that are associated with the occurrence of neuroinflammatory processes. The aim of the proposed study was to investigate the effect of direct bilateral intracerebral administration of Atsttrin using stereotactic methods in the preclinical C57BL/6 mouse model of Parkinson’s disease inducted by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. The analysis of the dose dependency effects of the increasing doses of Atsttrin has covered a number of parameters and markers regarding neurodegenerative processes and inflammatory responses including IL-1α, TNFα, IL-6, TH, and TG2 mRNA expressions. Accordingly, the evaluation of the changes in the neurochemical profile included DA, DOPAC, 3-MT, HVA, NA, MHPG, 5-HT, and 5-HIAA concentration levels. The intracerebral administration of Atsttrin into the striatum effectively attenuated the neuroinflammatory reaction in evaluated neuroanatomical structures. Furthermore, the partial restoration of monoamine content and its metabolic turnover were observed. In this case, taking into account the previously described pharmacokinetic profile and extrapolated bioavailability as well as the stability characteristics of Atsttrin, an attempt was made to describe as precisely as possible the quantitative and qualitative effects of increasing doses of the compound within the brain tissue microenvironment in the presented preclinical model of the disease. Collectively, this findings demonstrated that the intracerebral administration of Atsttrin may represent a potential novel therapeutic method for the treatment of Parkinson’s disease.
... Moreover, to assess bilaterality, counts were analysed as the total number of cFos + nuclei per 1000 μm 3 for both sides of the NTS combined, and by right and left subdivisions. That said, exceptions were made for the AP and SolC, which in rodents and lagomorphs are midline structures [11,45] and were, therefore, only analysed as a whole. ...
Functional magnetic resonance imaging (fMRI) suggests that the hypoxic ventilatory response is facilitated by the AMP-activated protein kinase (AMPK), not at the carotid bodies, but within a subnucleus (Bregma -7.5 to -7.1 mm) of the nucleus tractus solitarius that exhibits right-sided bilateral asymmetry. Here, we map this subnucleus using cFos expression as a surrogate for neuronal activation and mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic cells by Cre expression via the tyrosine hydroxylase promoter. Comparative analysis of brainstem sections, relative to controls, revealed that AMPK-α1/α2 deletion inhibited, with right-sided bilateral asymmetry, cFos expression in and thus activation of a neuronal cluster that partially spanned three interconnected anatomical nuclei adjacent to the area postrema: SolDL (Bregma -7.44 mm to -7.48 mm), SolDM (Bregma -7.44 mm to -7.48 mm) and SubP (Bregma -7.48 mm to -7.56 mm). This approximates the volume identified by fMRI. Moreover, these nuclei are known to be in receipt of carotid body afferent inputs, and catecholaminergic neurons of SubP and SolDL innervate aspects of the ventrolateral medulla responsible for respiratory rhythmogenesis. Accordingly, AMPK-α1/α2 deletion attenuated hypoxia-evoked increases in minute ventilation (normalised to metabolism), reductions in expiration time, and increases sigh frequency, but increased apnoea frequency during hypoxia. The metabolic response to hypoxia in AMPK-α1/α2 knockout mice and the brainstem and spinal cord catecholamine levels were equivalent to controls. We conclude that within the brainstem an AMPK-dependent, hypoxia-responsive subnucleus partially spans SubP, SolDM and SolDL, namely SubSol-HIe, and is critical to coordination of active expiration, the hypoxic ventilatory response and defence against apnoea.
... Mice were anaesthetised throughout the experiment with urethane (initial dose 1 g/kg i.p., supplemented as required) and maintained at 36 ± 1°C using a homeothermic blanket. A microdialysis probe (2 mm, Microbiotech MAB4) was stereotaxically implanted into the ventral hippocampus (AP −3.0 mm, ML ± 2.9 mm, DV −4.0 mm relative to bregma and the dura surface; [26]) and perfused (2 µl/min) with artificial cerebrospinal fluid (in mM: 140 NaCl, 4 KCl, 1.2 Na 2 HPO 4 , 0.27 NaH 2 PO 4 , 1 MgCl 2 , 2.4 CaCl 2 and 7.2 glucose). After a 60 min post-implantation period, samples were collected every 20 min for 120 min. ...
Cessation of therapy with a selective serotonin (5-HT) reuptake inhibitor (SSRI) is often associated with an early onset and disabling discontinuation syndrome, the mechanism of which is surprisingly little investigated. Here we determined the effect on 5-HT neurochemistry of discontinuation from the SSRI paroxetine. Paroxetine was administered repeatedly to mice (once daily, 12 days versus saline controls) and then either continued or discontinued for up to 5 days. Whereas brain tissue levels of 5-HT and/or its metabolite 5-HIAA tended to decrease during continuous paroxetine, levels increased above controls after discontinuation, notably in hippocampus. In microdialysis experiments continuous paroxetine elevated hippocampal extracellular 5-HT and this effect fell to saline control levels on discontinuation. However, depolarisation (high potassium)-evoked 5-HT release was reduced by continuous paroxetine but increased above controls post-discontinuation. Extracellular hippocampal 5-HIAA also decreased during continuous paroxetine and increased above controls post-discontinuation. Next, immunohistochemistry experiments found that paroxetine discontinuation increased c-Fos expression in midbrain 5-HT (TPH2 positive) neurons, adding further evidence for a hyperexcitable 5-HT system. The latter effect was recapitulated by 5-HT 1A receptor antagonist administration although gene expression analysis could not confirm altered expression of 5-HT 1A autoreceptors following paroxetine discontinuation. Finally, in behavioural experiments paroxetine discontinuation increased anxiety-like behaviour, which partially correlated in time with the measures of increased 5-HT function. In summary, this study reports evidence that, across a range of experiments, SSRI discontinuation triggers a rebound activation of 5-HT neurons. This effect is reminiscent of neural changes associated with various psychotropic drug withdrawal states, suggesting a common unifying mechanism.
... In the mouse, the entorhinal region is in the caudal and inferior pole of the cortex. Mouse brain atlases were used to identify brain regions based on relative location to prominent landmarks [84]. Four brain sections were labeled and imaged for each mouse, two Iba1 (rostral + caudal sections) and two GFAP (rostral + caudal sections). ...
Background
Neuropsychiatric lupus (NPSLE) describes the cognitive, memory, and affective emotional burdens faced by many lupus patients. While NPSLE’s pathogenesis has not been fully elucidated, clinical imaging studies and cerebrospinal fluid (CSF) findings, namely elevated interleukin-6 (IL-6) levels, point to ongoing neuroinflammation in affected patients. Not only linked to systemic autoimmunity, IL-6 can also activate neurotoxic glial cells the brain. A prior pre-clinical study demonstrated that IL-6 can acutely induce a loss of sucrose preference; the present study sought to assess the necessity of chronic IL-6 exposure in the NPSLE-like disease of MRL/lpr lupus mice.
Methods
We quantified 1308 proteins in individual serum or pooled CSF samples from MRL/lpr and control MRL/mpj mice using protein microarrays. Serum IL-6 levels were plotted against characteristic NPSLE neurobehavioral deficits. Next, IL-6 knockout MRL/lpr (IL-6 KO; n = 15) and IL-6 wildtype MRL/lpr mice (IL-6 WT; n = 15) underwent behavioral testing, focusing on murine correlates of learning and memory deficits, depression, and anxiety. Using qPCR, we quantified the expression of inflammatory genes in the cortex and hippocampus of MRL/lpr IL-6 KO and WT mice. Immunofluorescent staining was performed to quantify numbers of microglia (Iba1 +) and astrocytes (GFAP +) in multiple cortical regions, the hippocampus, and the amygdala.
Results
MRL/lpr CSF analyses revealed increases in IL-17, MCP-1, TNF-α, and IL-6 (a priori p-value < 0.1). Serum levels of IL-6 correlated with learning and memory performance (R² = 0.58; p = 0.03), but not motivated behavior, in MRL/lpr mice. Compared to MRL/lpr IL-6 WT, IL-6 KO mice exhibited improved novelty preference on object placement (45.4% vs 60.2%, p < 0.0001) and object recognition (48.9% vs 67.9%, p = 0.002) but equivalent performance in tests for anxiety-like disease and depression-like behavior. IL-6 KO mice displayed decreased cortical expression of aif1 (microglia; p = 0.049) and gfap (astrocytes; p = 0.044). Correspondingly, IL-6 KO mice exhibited decreased density of GFAP + cells compared to IL-6 WT in the entorhinal cortex (89 vs 148 cells/mm², p = 0.037), an area vital to memory.
Conclusions
The inflammatory composition of MRL/lpr CSF resembles that of human NPSLE patients. Increased in the CNS, IL-6 is necessary to the development of learning and memory deficits in the MRL/lpr model of NPSLE. Furthermore, the stimulation of entorhinal astrocytosis appears to be a key mechanism by which IL-6 promotes these behavioral deficits.
... and nucleus accumbens core (Acb-core: bregma 1.33-1.09) (Franklin and Paxinos, 2008), by independent observers unaware of genotype and LI performance. Neuronal activation in each region was subjected to statistical analyses. ...
The neuronal cell adhesion molecule (NrCAM) is widely expressed and has important physiological functions in the nervous system across the lifespan, from axonal growth and guidance to spine and synaptic pruning, to organization of proteins at the nodes of Ranvier. NrCAM lies at the core of a functional protein network where multiple targets (including NrCAM itself) have been associated with schizophrenia. Here we investigated the effects of chronic unpredictable stress on latent inhibition, a measure of selective attention and learning which shows alterations in schizophrenia, in NrCAM knockout (KO) mice and their wild-type littermate controls (WT). Under baseline experimental conditions both NrCAM KO and WT mice expressed robust latent inhibition (p = 0.001). However, following chronic unpredictable stress, WT mice (p = 0.002), but not NrCAM KO mice (F < 1), expressed latent inhibition. Analyses of neuronal activation (c-Fos positive counts) in key brain regions relevant to latent inhibition indicated four types of effects: a single hit by genotype in IL cortex (p = 0.0001), a single hit by stress in Acb-shell (p = 0.031), a dual hit stress x genotype in mOFC (p = 0.008), vOFC (p = 0.020), and Acb-core (p = 0.032), and no effect in PrL cortex (p > 0.141). These results indicating a pattern of differential effects of genotype and stress support a complex stress × genotype interaction model and a role for NrCAM in stress-induced pathological behaviors relevant to schizophrenia and other psychiatric disorders.
... Images of six sections of each animal's brain were acquired with a 10x objective lens in the region between -1.70 mm to 2.46 mm, starting from bregma [24]. Quantification was performed in that region using imageJ software 1.51j8 (National Institutes of Health, Bethesda, MD, United States). ...
Alzheimer’s disease (AD) is the most common neuronal disorder that leads to the development of dementia. Until nowadays, some therapies may alleviate the symptoms, but there is no pharmacological treatment. Microdosing lithium has been used to modify the pathological characteristics of the disease, with effects in both experimental and clinical conditions. The present work aimed to analyze the effects of this treatment on spatial memory, anxiety, and molecular mechanisms related to long-term memory formation during the aging process of a mouse model of accelerated aging (SAMP-8). Female SAMP-8 showed learning and memory impairments together with disruption of memory mechanisms, neuronal loss, and increased density of senile plaques compared to their natural control strain, the senescence-accelerated mouse resistant (SAMR-1). Chronic treatment with lithium promoted memory maintenance, reduction in anxiety, and maintenance of proteins related to memory formation and neuronal density. The density of senile plaques was also reduced. An increase in the density of gamma-aminobutyric acid A (GABA A ) and α7 nicotinic cholinergic receptors was also observed and related to neuroprotection and anxiety reduction. In addition, this microdose of lithium inhibited the activation of glycogen synthase kinase-3beta (GSK-3β), the classical mechanism of lithium cell effects, which could contribute to the preservation of the memory mechanism and reduction in senile plaque formation. This work shows that lithium effects in neuroprotection along the aging process are not related to a unique cellular mechanism but produce multiple effects that slowly protect the brain along the aging process.
... The brain areas with more than one mCherry(+) cell or the brain areas with mCherry(+) cells in more than one mouse are shown in the figure. The coloring of the dots represents the different mice and the schematic images were acquired from The Mouse Brain Atlas in Stereotaxic Coordinates (Paxinos and Franklin, 2001). Traced cells were also detected in the brain, suggesting that the lumbar Glra3-Cre(+) neurons receive distant descending input. ...
Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, and the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 ( Glra3 ) subunit-expressing neurons in Glra3 -Cre female and male mice. Glra3 -Cre(+) neurons express Glra3 , are located mainly in laminae III‒VI, and respond to glycine. Chemogenetic activation of spinal Glra3 -Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses, but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3 , further supporting the phenotype. Retrograde tracing revealed that spinal Glra3 -Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3 (+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.
Significance Statement Spinal glycinergic neurons regulate itch (pruriception), suggesting that components of the glycinergic system have great potential as drug targets to treat pruritus. Nonetheless, thus far, the pruriceptive roles of any of the glycine receptor (GLR) subunits have not been evaluated. Here, we successfully linked the Glra3 -Cre populations to a pro-pruriceptive role in itch, indicating that GLRA3-expressing neurons may be a potential novel target for itch treatment. The spontaneous stomping and guarding behaviors observed from activating the Glra3 -Cre populations are indicative of a role in sensory hypersensitivity and hence, raises questions regarding the hypersensitivity involvement of these populations for future investigations.
The present study investigated the neural health benefit of beta-sitosterol (BSS) against trimethyltin (TMT)-induced neurodegeneration in mice. Forty male ICR mice were randomly divided into Sham-veh, TMT-veh, TMT-BSS50, and TMT-BSS100. A one-time intraperitoneal injection of 2.6 mg/kg of TMT was given to mice in TMT groups. Vehicle (veh), BSS 50 mg/kg or BSS 100 mg/kg were orally given for 2 weeks. Spatial learning and memory were evaluated. Brain oxidative status, hippocampal neuropathology, and reactive astrocytes were done. White matter pathology was also evaluated. The results indicated the massy effect of TMT on induced motor ability and spatial memory deficits in accordance with increased neuronal degeneration in CA1, CA3, and DG and internal capsule white matter damage. TMT also induced the reduction of reactive astrocytes in CA1 and DG. Brain’s catalase activity was significantly reduced by TMT, but not in mice with BSS treatments. Both doses of BSS treatment exhibited improvement in motor ability and spatial memory deficits in accordance with the activation of reactive astrocytes in CA1, CA3, and DG. However, they successfully prevented the increase of neuronal degeneration in CA1 found only with the BSS dose of 100 mg/kg, and it was indicated as the effective dose for neuroprotection in the vulnerable brain area. This study demonstrated mitigative effects of BSS against motor ability and memory deficits with neural health benefits, including a protective effect against CA1 neurodegeneration and a nurturing effect on hippocampal reactive astrocytes.
INTRODUCTION
Amyloid beta (Aβ) impairs the cerebral blood flow (CBF) increase induced by neural activity (functional hyperemia). Tissue plasminogen activator (tPA) is required for functional hyperemia, and in mouse models of Aβ accumulation tPA deficiency contributes to neurovascular and cognitive impairment. However, it remains unknown if tPA supplementation can rescue Aβ‐induced neurovascular and cognitive dysfunction.
METHODS
Tg2576 mice and wild‐type littermates received intranasal tPA (0.8 mg/kg/day) or vehicle 5 days a week starting at 11 to 12 months of age and were assessed 3 months later.
RESULTS
Treatment of Tg2576 mice with tPA restored resting CBF, prevented the attenuation in functional hyperemia, and improved nesting behavior. These effects were associated with reduced cerebral atrophy and cerebral amyloid angiopathy, but not parenchymal amyloid.
DISCUSSION
These findings highlight the key role of tPA deficiency in the neurovascular and cognitive dysfunction associated with amyloid pathology, and suggest potential therapeutic strategies involving tPA reconstitution.
Highlights
Amyloid beta (Aβ) induces neurovascular dysfunction and impairs the increase of cerebral blood flow induced by neural activity (functional hyperemia).
Tissue plasminogen activator (tPA) deficiency contributes to the neurovascular and cognitive dysfunction caused by Aβ.
In mice with florid amyloid pathology intranasal administration of tPA rescues the neurovascular and cognitive dysfunction and reduces brain atrophy and cerebral amyloid angiopathy.
tPA deficiency plays a crucial role in neurovascular and cognitive dysfunction induced by Aβ and tPA reconstitution may be of therapeutic value.
Advances in high-resolution microscopy and cell staining techniques allow for quantifying well-stained cells and other microstructures in anatomically defined regions of interest (ROIs) in ex vivo brains from humans and animal models of human disease. The current best practice is the optical disector, an unbiased stereology method for counting individual cells or other tissue deposits while manually focusing a thin focal plane through a known z-axis volume (optical disector). By eliminating all known sources of methodological bias, the optical disector method ensures that the ex vivo count of the total number of neurons (∑NCells) converges on true in vivo values. A current limitation with all computer-assisted methods is the time, labor, and effort for counting (clicking) on a hundred or more cells as the counting process repeats at 100–200 x-y locations through each ROI. Faster data collection methods include semiquantitative approaches for sampling and counting 2D neuron profiles (∑NProf), though these methods can include unknown and unknowable amounts of systematic error (bias). Here we compare accuracy, reproducibility, and efficiency of counting total number of NeuN-immunostained neurons using the manual optical disector (gold standard) versus three techniques for counts of NeuN profiles. All counts of ∑NCells and ∑NProf were done to the same sampling intensity in the same high-power (100×) stacks of z-axis images (i.e., disector stacks) collected in a systematic-random manner through the entire neocortex (NCTX) in six mouse brains. The findings showed no statistical differences between ∑NCells by the gold standard and ∑NProf counts using three methods (fully manual, semiautomatic, fully automatic). Reproducibility (inter-rater error) by two similarly trained data collectors ranged from 0% for fully automatic counts of ∑NProf to ~5% for ∑NCells counts by gold standard with average data collection times between <1 min per case to ~12 min per case, respectively. Notably, all three methods for counting ∑NProf required a significant amount of unsupervised time, i.e., from 30 to 90 min per case, for preprocessing image stacks prior to data collection. Estimates of ∑NCells by the manual optical disector required no preprocessing. In summary, the manual optical disector remains the best practice due to the avoidance of all known sources of methodological bias, low inter-rater error, and moderate-to-high throughput. These results provide a baseline for comparisons with other methods for quantifying stained cells in tissue sections, including novel deep learning approaches for automatic counts of well-stained cells and other microstructures in the brain.
The isotropic virtual planes (IVP) method is a global spatial sampling approach for length estimation. Thanks to computer-assisted microscopy, IVP can be applied to thick sections oriented in any particular direction, as the stereological probe is randomly rotated inside the section and, therefore, avoids the possible bias that might occur when structures with a given preferred orientation are analyzed. We have applied this stereological tool to estimate axonal length in two different experimental/methodological approaches: (1) axons from populations of projection neurons and (2) axons from single projection neurons, visualized in mice or human tissues. Our results demonstrate that the IVP is an accurate stereological tool that can be used to estimate the axonal length of single or groups of neurons. In the case of single neurons, the IVP allows collecting these quantitative data efficiently, preventing the need to reconstruct the entire cell’s structure manually. The fact that this method is valid to analyze groups of neurons, whose axons provide terminal arborizations that can be either scattered in broad brain regions or restricted to specific brain areas, makes the IVP a suitable tool to be applied in connectomic experiments performed in either small brains, as in rodents, or primates, where the reconstruction of the entire axon is a tedious and time-consuming task. The analysis of the axonal length is of great importance since it is proportional to the number of synapses the axon establishes with its postsynaptic targets, and it is affected by some pathological conditions.
Respiratory infections are one of the most common causes of illness and morbidity in neonates worldwide. In the acute phase infections are known to cause wide-spread peripheral inflammation. However, the inflammatory consequences to the critical neural control centres for respiration have not been explored. Utilising a well characterised model of neonatal respiratory infection, we investigated acute responses within the medulla oblongata which contains key respiratory regions. Neonatal mice were intranasally inoculated within 24 h of birth, with either Chlamydia muridarum or sham-infected, and tissue collected on postnatal day 15, the peak of peripheral inflammation. A key finding of this study is that, while the periphery appeared to show no sex-specific effects of a neonatal respiratory infection, sex had a significant impact on the inflammatory response of the medulla oblongata. There was a distinct sex-specific response in the medulla coincident with peak of peripheral inflammation, with females demonstrating an upregulation of anti-inflammatory cytokines and males showing very few changes. Microglia also demonstrated sex-specificity with the morphology of females and males differing based upon the nuclei. Astrocytes showed limited changes during the acute response to neonatal infection. These data highlight the strong sex-specific impact of a respiratory infection can have on the medulla in the acute inflammatory phase.
A key to motor control is the motor thalamus, where several inputs converge. One excitatory input originates from layer 5 of primary motor cortex (M1 L5 ), while another arises from the deep cerebellar nuclei (Cb). M1 L5 terminals distribute throughout the motor thalamus and overlap with GABAergic inputs from the basal ganglia output nuclei, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr). In contrast, it is thought that Cb and basal ganglia inputs are segregated. Therefore, we hypothesized that one potential function of the GABAergic inputs from basal ganglia is to selectively inhibit, or gate, excitatory signals from M1 L5 in the motor thalamus. Here, we tested this possibility and determined the circuit organization of mouse (both sexes) motor thalamus using an optogenetic strategy in acute slices. First, we demonstrated the presence of a feedforward transthalamic pathway from M1 L5 through motor thalamus. Importantly, we discovered that GABAergic inputs from the GPi and SNr converge onto single motor thalamic cells with excitatory synapses from M1 L5 . Separately, we also demonstrate that, perhaps unexpectedly, GABAergic GPi and SNr inputs converge with those from the Cb. We interpret these results to indicate that a role of the basal ganglia is to gate the thalamic transmission of M1 L5 and Cb information to cortex.
Alzheimer's disease (AD) is a progressive and degenerative disorder accompanied by emotional disturbance, especially anxiety and depression. More and more evidence shows that the imbalance of mitochondrial Ca 2+ (mCa 2+) homeostasis has a close connection with the pathogenesis of anxiety and depression. The Mitochondrial Calcium Uniporter (MCU), a key channel of mCa 2+ uptake, induces the imbalance of mCa 2+ homeostasis and may be a therapeutic target for anxiety and depression of AD. In the present study, we revealed for the first time that MCU knockdown in hippocampal neurons alleviated anxious and depressive behaviors of APP/PS1/tau mice through elevated plus-maze (EPM), elevated zero maze (EZM), sucrose preference test (SPT) and tail suspension test (TST). Western blot analysis results demonstrated that MCU knockdown in hippocampal neurons increased levels of glutamate decarboxylase 67 (GAD67), vesicular GABA transporter (vGAT) and GABAA receptor a1 (GABRA1) and activated the PKA-CREB-BDNF signaling pathway. This study indicates that MCU inhibition has the potential to be developed as a novel therapy for anxiety and depression in AD.
Rationale
Exercise attenuates addictive behavior; however, little is known about the contribution of exercise duration to this positive effect. The Renin Angiotensin System (RAS) has been implicated both in addictive responses and in the beneficial effects of exercise; though, its role in the advantageous effects of exercise on toluene-induced addictive responses has not been explored.
Objectives
To evaluate the impact of different exercise regimens in mitigating the expression of toluene-induced locomotor sensitization and to analyze changes in RAS elements’ expression at the mesocorticolimbic system after repeated toluene exposure and following voluntary wheel running in toluene-sensitized animals.
Methods
Toluene-induced addictive-like response was evaluated with a locomotor sensitization model in mice. Toluene-sensitized animals had access to running wheels 1, 2, 4 or 24 h/day for 4 weeks; thereafter, locomotor sensitization expression was evaluated after a toluene challenge. RAS elements (ACE and ACE2 enzymes; AT1, AT2 and Mas receptors) expression was determined by Western blot in the VTA, NAc and PFCx of toluene-sensitized mice with and without exercise.
Results
Individual differences in toluene-induced locomotor sensitization development were observed. Access to wheel running 1 and 2 h/day reduced but 4 and 24 h/day completely blocked locomotor sensitization expression. Repeated toluene exposure changed RAS elements’ expression in the VTA, NAc and PFCx, while exercise mainly modified ACE and AT1 in air-exposed and toluene-sensitized mice.
Conclusions
Inhalant-exposed animals show different sensitization phenotypes. Exercise duration determined its efficacy to attenuate the addictive-like response. Toluene exposure and exercise each modified RAS, the latter also modifying toluene-induced changes.
Previous studies on germ‐free (GF) animals have described altered anxiety‐like and social behaviors together with dysregulations in brain serotonin (5‐HT) metabolism. Alterations in circulating 5‐HT levels and gut 5‐HT metabolism have also been reported in GF mice. In this study, we conducted an integrative analysis of various behaviors as well as markers of 5‐HT metabolism in the brain and along the GI tract of GF male mice compared with conventional (CV) ones. We found a strong decrease in locomotor activity, accompanied by some signs of increased anxiety‐like behavior in GF mice compared with CV mice. Brain gene expression analysis showed no differences in HTR1A and TPH2 genes. In the gut, we found decreased TPH1 expression in the colon of GF mice, while it was increased in the cecum. HTR1A expression was dramatically decreased in the colon, while HTR4 expression was increased both in the cecum and colon of GF mice compared with CV mice. Finally, SLC6A4 expression was increased in the ileum and colon of GF mice compared with CV mice. Our results add to the evidence that the microbiota is involved in regulation of behavior, although heterogeneity among studies suggests a strong impact of genetic and environmental factors on this microbiota‐mediated regulation. While no impact of GF status on brain 5‐HT was observed, substantial differences in gut 5‐HT metabolism were noted, with tissue‐dependent results indicating a varying role of microbiota along the GI tract.
Rationale
Parenting experiences with caregivers play a key role in neurodevelopment. We recently reported that adolescents reared by a single-mother (SM) display an anxiety-prone phenotype and drink more alcohol, compared to peers derived from a biparental (BP) rearing condition.
Objectives
To investigate if SM and BP offspring infant mice exhibit differential sensitivity to ethanol-induced locomotor activity and differential activity patterns in brain areas related to anxiety response. We also analyzed anxiety response and ethanol-induced anxiolysis in SM and BP adolescents.
Methods
Mice reared in SM or BP conditions were assessed for (a) ethanol-induced locomotor activity at infancy, (b) central expression of Fos-like proteins (likely represented mostly by FosB, a transcription factor that accumulates after chronic stimuli exposure and serves as a molecular marker of neural plasticity) and cathecolaminergic activity, and (c) anxiety-like behavior and ethanol-induced anxiolysis in adolescence.
Results
Infant mice were sensitive to the stimulating effects of 2.0 g/kg alcohol, regardless parenting structure. SM mice exhibited, relative to BP mice, a significantly greater number of Fos-like positive cells in the central amygdala and basolateral amygdala nuclei. Ethanol treatment, but not parenting condition, induced greater activation of dopaminergic neurons in ventral tegmental area. SM, but not BP, adolescent mice were sensitive to ethanol-induced anxiolysis.
Conclusions
These results highlight the complex relationship between parenting experiences and neurodevelopment. The SM parenting may result in greater neural activation patterns in brain areas associated with anxiety response, potentially contributing to increased basal anxiety and alcohol sensitivity.
Background
Many neuropsychiatric disorders show sex differences in prevalence and presentation. For example, Tourette’s Syndrome (TS) is diagnosed 3-5 times more often in males. Dopamine modulation of the basal ganglia is implicated in numerous neuropsychiatric conditions, including TS. Motivated by an unexpected genetic finding in a family with TS, we previously characterized the modulation of striatal dopamine by histamine.
Methods
We used microdialysis to analyze striatal dopamine response to the targeted infusion of histamine and histamine agonists. siRNA knockdown of histamine receptors was used to identify the cellular mediators of observed effects.
Results
Intracerebroventricular histamine reduced striatal dopamine in male mice, replicating previous work. Unexpectedly, histamine increased striatal dopamine in females. Targeted infusion of selected agonists revealed that the effect in males depends on H2R receptors in the substantia nigra pars compacta (SNc). Knockdown of H2R in SNc GABAergic neurons abrogated the effect, identifying these cells as a key locus of histamine’s regulation of dopamine in males. In females, in contrast, H2R had no role; instead, H3R agonists in the striatum increased striatal dopamine. Strikingly, the effect of histamine on dopamine in females was modulated by the estrous cycle, appearing in estrus/proestrus but not in metestrus/diestrus.
Conclusions
These findings confirm the regulation of striatal dopamine by histamine but identify marked sexual dimorphism in and estrous modulation of this effect. These findings may shed light on the mechanistic underpinnings of other sex differences in the striatal circuitry, perhaps including the marked sex differences seen in TS and related neuropsychiatric conditions.
Ilex kudingcha C.J. Tseng is a nootropic used throughout Asia that shares a number of metabolites with Ilex paraguariensis used throughout South America. Our previous study using a Drosophila melanogaster rugose model of autism spectrum disorders (ASD) showed that consumption of an Ilex kudingcha extract (IKE) mitigates phenotypic characteristics of ASD and in normal mice, alters gene expression involved in cognition, metabolism, and protein synthesis. This study investigated the effects of IKE on prenatal sodium valproate (VPA) treatment-induced ASD core behavioral deficits and ASD associated behaviors, neurochemical markers and histological changes. IKE administration significantly mitigated these behavioral deficits and damaged Purkinje cells, PTEN expression and oxidative stress and resembled treatment with methylphenidate in its effect upon social behavior. These findings extend our previous study with D. melanogaster and together, indicate that IKE consumption ameliorates ASD-like properties in two animal models of ASD with different etiologies. Potential mechanisms involve reduction of oxidative stress, increased PTEN expression and cerebral Purkinje cell health. These data support further studies of IKE and related species for treatment of ASD.
Recently, we and others have shown that manipulating the activity of cholinergic interneurons (CIN) present in the NAc can modulate binge alcohol consumption. The present study is designed to examine the relationship between binge alcohol consumption and the activity of the CIN in real time by using an in vivo microendoscopic technique. We hypothesized that mice exposed to Drinking in the Dark (DID)—a recognized mouse model for binge drinking—would exhibit increased activity in the accumbal shell region (NAcSh). To test this hypothesis, male mice expressing Cre-recombinase in the cholinergic neurons were exposed to binge alcohol consumption (alcohol group), employing the DID method, and utilized in vivo calcium imaging to observe CIN activity in real time during alcohol consumption. The control (sucrose) group was exposed to 10% (w/v) sucrose. As compared to sucrose, mice in the alcohol group displayed a significant increase in the frequency and amplitude of discharge activity, which was measured using calcium transients in the CIN present in the NAcSh. In summary, our findings suggest that the activity of CIN in the NAcSh plays a crucial role in alcohol self-administration. These results emphasize the potential significance of targeting CIN activity as a therapeutic approach for addressing AUD.
Background
An initial neuropathological hallmark of Alzheimer’s disease (AD) is the hippocampal dysfunction caused by amyloid- β (A β ) peptides accumulation. Soluble oligomeric forms of A β shift synaptic plasticity induction threshold leading to memory deficits in male and female mice in early amyloidosis models. Some protein changes underlying those deficits have been previously studied, but the spatial distribution within the hippocampus, as well as the potential sex differences, remain unknown. Since each hippocampal region (dorsal vs . ventral) has clearly distinct functionality and connectivity, we postulated that some protein changes may be unique to each and might also be sex-dependent.
Methods
An innovative spatial proteomics study was performed to map whole hippocampal proteome distribution using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, which allows protein detection with spatial resolution directly on tissue sections. Brains from sixteen adult male and female mice intracerebroventricularly injected with A β 1-42 oligomers or vehicle were sectioned. MALDI imaging was performed using a RapifleXTM MALDI TissuetyperTM TOF/TOF mass spectrometer followed by protein identification by traditional tandem mass spectrometry (MS/MS) directly on the tissue. To precisely delineate both dorsal and ventral hippocampus, a Nissl staining was performed on succeeding tissue sections.
Results
Of the 234 detected peptides, significant differences in expression levels were found in 34 proteins, due to treatment, sex, or hippocampal location. Moreover, a significant protein-protein interaction (PPI) was observed, showing a relationship to long-term potentiation (LTP), the functional basis of memory. Accordingly, 14 proteins related to synaptic plasticity and/or AD were selected to further study. Results showed many of the altered protein to modulate glycogen synthase kinase-3 β (GSK-3 β ), a protein widely involved in the regulation of synaptic plasticity induction threshold. In fact, hippocampal GSK-3 β was found overactivated suggesting a facilitated long-term depression (LTD) instead of LTP in AD models.
Conclusions
This study offers for the first time the specific protein changes in dorsal/ventral hippocampus in both male and female mice, that modulate GSK-3 β activity, providing new insight in the pathogenesis of early AD and valuable potential biomarkers for early diagnosis and therapeutic targets.
Background
Synthetic cathinones (SC) constitute the second most frequently abused class of new psychoactive substances. They serve as an alternative to classic psychostimulatory drugs of abuse, such as methamphetamine, cocaine, or 3,4-methylenedioxymethamphetamine (MDMA). Despite the worldwide prevalence of SC, little is known about their long-term impact on the central nervous system. Here, we examined the effects of repeated exposure of mice during infancy, to 3,4-methylenedioxypyrovalerone (MDPV), a SC potently enhancing dopaminergic neurotransmission, on learning and memory in young adult mice.
Methods
All experiments were performed on C57BL/6J male and female mice. Animals were injected with MDPV (10 or 20 mg/kg) and BrdU (bromodeoxyuridine, 25 mg/kg) during postnatal days 11–20, which is a crucial period for the development of their hippocampus. At the age of 12 weeks, mice underwent an assessment of various types of memory using a battery of behavioral tests. Afterward, their brains were removed for detection of BrdU-positive cells in the dentate gyrus of the hippocampal formation with immunohistochemistry, and for measurement of the expression of synaptic proteins, such as synaptophysin and PSD95, in the hippocampus using Western blot.
Results
Exposure to MDPV resulted in impairment of spatial working memory assessed with Y-maze spontaneous alternation test, and of object recognition memory. However, no deficits in hippocampus-dependent spatial learning and memory were found using the Morris water maze paradigm. Consistently, hippocampal neurogenesis and synaptogenesis were not interrupted. All observed MDPV effects were sex-independent.
Conclusions
MDPV administered repeatedly to mice during infancy causes learning and memory deficits that persist into adulthood but are not related to aberrant hippocampal development.
Attending to salient sensory attributes of food, such as tastes that are new, displeasing, or unexpected, allows the procurement of nutrients without food poisoning. Exposure to new tastes is known to increase norepinephrine (NE) release in taste processing forebrain areas, yet the central source for this release is unknown. Locus ceruleus norepinephrine neurons (LC-NE) emerge as a candidate in signaling salient information about taste, as other salient sensory stimuli (e.g., visual, auditory, somatosensation) are known to activate LC neurons. To determine if LC neurons are sensitive to features of taste novelty, we used fiber photometry to record LC-NE activity in water-restricted mice that voluntarily licked either novel or familiar substances of differential palatability (saccharine, citric acid). We observed that LC-NE activity was suppressed during lick bursts and transiently activated upon the termination of licking and that these dynamics were independent of the familiarity of the substance consumed. We next recorded LC dynamics during brief and unexpected consumption of tastants and found no increase in LC-NE activity, despite their responsiveness to visual and auditory stimuli, revealing selectivity in LC's responses to salient sensory information. Our findings suggest that LC activity during licking is not influenced by taste novelty, implicating a possible role for non-LC noradrenergic nuclei in signaling critical information about taste.
Parkinson’s disease (PD) is a progressive age-related neurodegenerative pathology of the central nervous system, characterized by a selective loss of dopaminergic neurons of the nigrostriatal pathway and by the presence of specific inclusions (Lewy bodies) in dopamine neurons. To study the mechanisms of this pathology and to search for possible ways to correct it, genetic models of PD in mice have been created. Transgenic mice of the B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J strain (referred as B6.Cg-Tg further in the text) represent a model of PD, have the A53T mutation in the human alpha-synuclein gene. The aim of this work was to study the locomotor activity and the level of anxiety, as well as the density of neurons in the brain of male B6.Cg-Tg mice at the age of six months. Wild type C57BL/6J mice of the same sex and age were used as controls. The results of the current study demonstrate that B6.Cg-Tg mice are characterized by the high locomotor activity and the low anxiety. Besides, a selective decrease in the density of neurons in the subventricular zone, the substantia nigra, as well as the CA1, CA3, CA4 zones and the granular layer of the dentate gyrus of the hippocampus was observed in these mice. Thus, mice of the B6.Cg-Tg strain at the age of six months only partially correspond to the main pathophysiological signs of PD. Decrease in the density of neurons in the substantia nigra, as well as in the CA1 and CA3 zones of the hippocampus of B6.Cg-Tg mice resemble similar changes in PD. However, these mice demonstrated neither bradykinesia nor high level of anxiety.
Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro , optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (PACAP) neurons firing activity. GABA-A receptor antagonist or genetic deletion of VGAT in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking VGAT resulted in excitation of PACAP neurons and hypothermia. Mice lacking VGAT expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5 °C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic PACAP neurons. Taken together our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic PACAP neurons is the cellular mechanism that triggers this response.
Many biological molecules in the brain interstitial fluid are involved in neuronal functions. Therefore, measuring the levels of these molecules in the extracellular fluid would provide deep insights into the physiological/pathological mechanisms underlying brain functions/disorders. In vivo microdialysis is a powerful technique used to examine the extracellular levels of various molecules in the brains of living animals. In neuroscience research, this technique has been widely used to investigate relatively small molecules including neurotransmitters and amino acids. However, recent advances in technology have made it possible to assess large molecules in the brain interstitial fluid, such as signaling peptides and proteins, using microdialysis probes with high-molecular-weight cutoff membranes. This chapter describes an in vivo microdialysis method to collect and measure the levels of large biological molecules in the extracellular fluid of the brains of freely moving mice.
This study aimed to investigate the mechanism of Dexmedetomidine (DEX) on sleep homeostasis. We will focus on the effect of DEX on the activity and the signal transmission of oxytocin-expressing (Oxytocin, OXT) neurons in the paraventricular nucleus (PVN) of the hypothalamus (PVN OXT ) in the regulation of sleep-wakefulness cycle. In this study, the guide cannula was implanted to microinject DEX into PVN. PVN OXT neurons were specifically activated by the chemogenetic method. Differences in sleep-wakefulness states were monitored by electroencephalogram (EEG)/ electromyogram (EMG) recording. In addition, the effect of DEX on the electrophysiological activity of PVN OXT neurons was examined by whole-cell patch-clamp technique. EEG/EMG results showed that microinjection of DEX in PVN significantly increased the duration of nonrapid eye movement (NREM) sleep in mice. Chemogenetic activation of PVN OXT neurons after internal cannula injection of DEX to PVN increased the amount of wake. Electrophysiological results show that DEX could inhibit the frequency of action potential (AP) and the spontaneous excitatory postsynaptic current (sEPSC) in PVN OXT neurons through α 2 -adrenoceptors. DEX maintained sleep homeostasis by inhibiting excitatory synaptic signaling in PVN OXT neurons through α 2 -adrenoceptors.
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.
Neurodegenerative disorders pose a significant risk to the well-being of individuals. The incidence of age-related disorders is on the rise, partially attributed to the recent growth in the elderly demographic. Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and spinocerebellar ataxias, are commonly observed in clinical practice. The pathophysiology of these diseases is heterogeneous, as some of them lead to cognitive and memory deficits, while others impact an individual’s respiratory, speech, and motor functions. There is a pressing need for efficacious treatments, which can only be achieved by comprehensively comprehending the aetiology and mechanisms underlying each ailment. Animal models are significant resources for assessing novel therapeutic approaches for treating human ailments and investigating the pathological mechanisms implicated in disease progression. Schizophrenia is a heterogeneous severe psychiatric mental illness with a devastating effect on patients and their caretakers and imposes enormous costs on healthcare systems. The aetiology of schizophrenia is still poorly understood and the biochemical focus has been on antipsychotic drugs that mitigate the symptoms of schizophrenia, the response rate to these drugs is lower than desired; they are slow-acting and produce serious adverse side effects. Preclinical models allow more rapid monitoring of disease progression than is possible in humans, permit invasive studies of structural and molecular changes across the development of the disorder, and identify new drug targets to provide more effective treatment in the future. This chapter discusses in detail the significance of animal models pertaining to Alzheimer’s disease, Parkinson’s disease, and schizophrenia.
Background
The neurovascular unit (NVU) represents the structural and functional relationship between the neural tissue and the blood. Neurovascular dysfunction has been highlighted in neuropsychiatric afflictions, but whether it is a cause or a consequence of the pathology remains to be elucidated. Thus, to elucidate the role of the NVU on the emergence of emotional-cognitive dysfunction, it is necessary to study how its individual components associate. This study therefore aims at investigating whether the development of depressive-related loss of motivation is grounded on NVU adjustments impacting the permeability of the blood-brain barrier (BBB) and in particular, of the structural scaffolding of microvessels.
Methods
Adult male C57BL/6jRj mice chronically treated with corticosterone (CORT) and showing severe motivational deficits in an operant progressive ratio (PR) schedule of reinforcement task, presented altered neural activation assessed through FosB expression in key brain regions involved in motivational processing (anterior insular cortex, basolateral amygdala, bed nucleus of the stria terminalis and ventral tegmental area). We evaluated NVU modifications through immunofluorescence staining targeting specific markers of microglia (IBA-1), endothelial tight junctions (ZO-1) and astrocytes (GFAP). The effect of chronic CORT administration on mice BBB permeability was evaluated through in vivo perfusion of fluorescent 40 kDa Dextran.
Results
Our results highlight that where sustained neuronal activation failed, NVU modifications predict behavioural deficits in CORT-treated animals. Notably, our analyses show that NVU modifications within the ventral tegmental area are essential to understand effort-based related behavioural performance in mice, and most particularly, that the key element of microvessels’ tight junctions ZO-1 plays a pivotal role on motivation-related behavioural output.
Conclusions
Our results confirm a direct role of neurovascular adaptations on emotional and cognitive behavioural performance in mice, and therefore place the NVU in a key position in the research of the biological substrate at the origin of neuropsychiatric disorders.
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