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Welcome to OXION, Universities of Oxford, Cambridge, London and MRC Harwell
Measuring glymphatic function: assessing the toolkit.
Glymphatic flow has been proposed to clear brain waste while we sleep. Cerebrospinal fluid moves from periarterial to perivenous spaces through the parenchyma, with subsequent cerebrospinal fluid drainage to dural lymphatics. Glymphatic disruption is associated with neurological conditions such as Alzheimer's disease and traumatic brain injury. Therefore, investigating its structure and function may improve understanding of pathophysiology. The recent controversy on whether glymphatic flow increases or decreases during sleep demonstrates that the glymphatic hypothesis remains contentious. However, discrepancies between different studies could be due to limitations of the specific techniques used and confounding factors. Here, we review the methods used to study glymphatic function and provide a toolkit from which researchers can choose. We conclude that tracer analysis has been useful, ex vivo techniques are unreliable, and in vivo imaging is still limited. Finally, we explore the potential for future methods and highlight the need for in vitro models, such as microfluidic devices, which may address technique limitations and enable progression of the field.
Ablation of CCL17-positive hippocampal neurons induces inflammation-dependent epilepsy.
OBJECTIVE: Neuronal cell death and neuroinflammation are characteristic features of epilepsy, but it remains unclear whether neuronal cell death as such is causative for the development of epileptic seizures. To test this hypothesis, we established a novel mouse line permitting inducible ablation of pyramidal neurons by inserting simian diphtheria toxin (DT) receptor (DTR) cDNA into the Ccl17 locus. The chemokine CCL17 is expressed in pyramidal CA1 neurons in adult mice controlling microglial quiescence. METHODS: Seizure activity in CCL17-DTR mice was analyzed by electroencephalographic recordings following treatment with DT for 3 consecutive days. Neuroinflammation and neuronal cell death were evaluated by (immuno)histochemistry. Pharmacological inhibition of TNFR1 signaling was achieved by treatment with XPro1595, a dominant-negative inhibitor of soluble tumor necrosis factor. RESULTS: Neuronal cell death was detectable 7 days (d7) after the first DT injection in heterozygous CCL17-DTR mice. Spontaneous epileptic seizures were observed in the vast majority of mice, often with an initial peak at d6-9, followed by a period of reduced activity and a gradual increase during the 1-month observation period. Microglial reactivity was overt from d5 after DT administration not only in the CA1 region but also in the CA2/CA3 area, shortly followed by astrogliosis. Reactive microgliosis and astrogliosis persisted until d30 and, together with neuronal loss and stratum radiatum shrinkage, reflected important features of human hippocampal sclerosis. Granule cell dispersion was detectable only 3 months after DT treatment. Application of XPro1595 significantly reduced chronic seizure burden without affecting the development of hippocampal sclerosis. SIGNIFICANCE: In conclusion, our data demonstrate that sterile pyramidal neuronal death is sufficient to cause epilepsy in the absence of other pathological processes. The CCL17-DTR mouse line may thus be a valuable model for further mechanistic studies on epilepsy and assessment of antiseizure medication.
Deficient synaptic neurotransmission results in a persistent sleep-like cortical activity across vigilance states in mice.
Growing evidence suggests that brain activity during sleep, as well as sleep regulation, are tightly linked with synaptic function and network excitability at the local and global levels. We previously reported that a mutation in synaptobrevin 2 (Vamp2) in restless (rlss) mice results in a marked increase of wakefulness and suppression of sleep, in particular REM sleep (REMS), as well as increased consolidation of sleep and wakefulness. In this study, using finer-scale in vivo electrophysiology recordings, we report that spontaneous cortical activity in rlss mice during NREM sleep (NREMS) is characterized by an occurrence of abnormally prolonged periods of complete neuronal silence (OFF-periods), often lasting several seconds, similar to the burst suppression pattern typically seen under deep anesthesia. Increased incidence of prolonged network OFF-periods was not specific to NREMS but also present in REMS and wake in rlss mice. Slow-wave activity (SWA) was generally increased in rlss mice relative to controls, while higher frequencies, including theta-frequency activity, were decreased, further resulting in diminished differences between vigilance states. The relative increase in SWA after sleep deprivation was attenuated in rlss mice, suggesting either that rlss mice experience persistently elevated sleep pressure or, alternatively, that the intrusion of sleep-like patterns of activity into the wake state attenuates the accumulation of sleep drive. We propose that a deficit in global synaptic neurotransmitter release leads to "state inertia," reflected in an abnormal propensity of brain networks to enter and remain in a persistent "default state" resembling coma or deep anesthesia.
Morphine-Induced Antinociception Is Potentiated and Dopamine Elevations Are Inhibited by the Biased Kappa Opioid Receptor Agonist Triazole 1.1.
Traditional analgesic opioid compounds, which act through μ opioid receptors (MORs), engender a high risk for misuse and dependence. κ opioid receptor (KOR) activation, a potential target for pain treatment, produces antinociception without euphoric side effects but results in dysphoria and aversion. Triazole 1.1 is a KOR agonist biased toward G-protein coupled signaling, potentially promoting antinociception without dysphoria. We tested whether triazole 1.1 could provide antinociception and its effects in combination with morphine. We employed a lactic acid abdominal pain model, which induced acute pain behaviors, decreased basal dopamine levels in the nucleus accumbens (NAc), and increased KOR function. We administered several interventions including triazole 1.1 (30 mg/kg) and morphine (12 or 24 mg/kg), individually and in combination. Triazole 1.1 alone reduced the pain behavioral response and changes to KOR function but did not prevent the reduction in basal dopamine levels. Morphine not only dose-dependently prevented behavioral pain responses but also elevated NAc dopamine and did not prevent the pain-induced increase in KOR function. However, combining low-dose morphine with triazole 1.1 prevents behavioral pain responses, changes to NAc dopamine levels, and changes to KOR function. Therefore, we present triazole 1.1 as a dose-sparing pain treatment to be used in combination with a lower dose of morphine, thus reducing the potential for opioid misuse.
Adding a Twist to Lateral Flow Immunoassays: A Direct Replacement of Antibodies with Helical Affibodies, from Selection to Application.
Immunoreagents, most commonly antibodies, are integral components of lateral flow immunoassays. However, the use of antibodies comes with limitations, particularly relating to their reproducible production, and poor thermal and chemical stability. Here, we employ phage display to develop affibodies, a class of nonimmunoglobulin affinity proteins based on a small three-helix bundle scaffold, against SARS-CoV-2 Spike protein. Subsequently, we demonstrate the utility and viability of affibodies to directly replace antibodies in lateral flow immunoassays. In addition, we highlight several physiochemical advantages of affibodies, including their ability to withstand exposure to high temperature and humidity while maintaining superior performance compared to their antibody counterparts. Furthermore, we investigate the adsorption mechanism of affibodies to the surface of gold nanoparticle probes via a His6-tag, introduced to also facilitate recombinant purification. Through molecular dynamics simulations, we elucidate the structural and physical characteristics of affibody dimers which result in high-performing detection probes when immobilized on nanoparticle surfaces. This work demonstrates that affibodies can be used as direct replacements to antibodies in immunoassays and should be further considered as alternatives owing to their improved physiochemical properties and modular design.
GABA uptake transporters support dopamine release in dorsal striatum with maladaptive downregulation in a parkinsonism model.
Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.
Axo-glial interactions between midbrain dopamine neurons and oligodendrocyte lineage cells in the anterior corpus callosum.
Oligodendrocyte progenitor cells (OPCs) receive synaptic innervation from glutamatergic and GABAergic axons and can be dynamically regulated by neural activity, resulting in activity-dependent changes in patterns of axon myelination. However, it remains unclear to what extent other types of neurons may innervate OPCs. Here, we provide evidence implicating midbrain dopamine neurons in the innervation of oligodendrocyte lineage cells in the anterior corpus callosum and nearby white matter tracts of male and female adult mice. Dopaminergic axon terminals were identified in the corpus callosum of DAT-Cre mice after injection of an eYFP reporter virus into the midbrain. Furthermore, fast-scan cyclic voltammetry revealed monoaminergic transients in the anterior corpus callosum, consistent with the anatomical findings. Using RNAscope, we further demonstrate that ~ 40% of Olig2 + /Pdfgra + cells and ~ 20% of Olig2 + /Pdgfra- cells in the anterior corpus callosum express Drd1 and Drd2 transcripts. These results suggest that oligodendrocyte lineage cells may respond to dopamine released from midbrain dopamine axons, which could affect myelination. Together, this work broadens our understanding of neuron-glia interactions with important implications for myelin plasticity by identifying midbrain dopamine axons as a potential regulator of corpus callosal oligodendrocyte lineage cells.
Transcription factors FOXA1 and FOXA2 maintain dopaminergic neuronal properties and control feeding behavior in adult mice.
Midbrain dopaminergic (mDA) neurons are implicated in cognitive functions, neuropsychiatric disorders, and pathological conditions; hence understanding genes regulating their homeostasis has medical relevance. Transcription factors FOXA1 and FOXA2 (FOXA1/2) are key determinants of mDA neuronal identity during development, but their roles in adult mDA neurons are unknown. We used a conditional knockout strategy to specifically ablate FOXA1/2 in mDA neurons of adult mice. We show that deletion of Foxa1/2 results in down-regulation of tyrosine hydroxylase, the rate-limiting enzyme of dopamine (DA) biosynthesis, specifically in dopaminergic neurons of the substantia nigra pars compacta (SNc). In addition, DA synthesis and striatal DA transmission were reduced after Foxa1/2 deletion. Furthermore, the burst-firing activity characteristic of SNc mDA neurons was drastically reduced in the absence of FOXA1/2. These molecular and functional alterations lead to a severe feeding deficit in adult Foxa1/2 mutant mice, independently of motor control, which could be rescued by L-DOPA treatment. FOXA1/2 therefore control the maintenance of molecular and physiological properties of SNc mDA neurons and impact on feeding behavior in adult mice.
LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function.
Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.
Striatal dopamine neurotransmission: regulation of release and uptake.
Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients.
Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism.
Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates to movement and how this activity is deciphered in target structures such as the striatum. By recording and labeling individual neurons in behaving mice, we show that the representation of brief spontaneous movements in the firing of identified midbrain dopaminergic neurons is cell-type selective. Most dopaminergic neurons in the substantia nigra pars compacta (SNc), but not in ventral tegmental area or substantia nigra pars lateralis, consistently represented the onset of spontaneous movements with a pause in their firing. Computational modeling revealed that the movement-related firing of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types differentially encode spontaneous movement and elucidate how dysregulation of their firing in early Parkinsonism can impair their effector circuits.
Cortical Control of Striatal Dopamine Transmission via Striatal Cholinergic Interneurons.
Corticostriatal regulation of striatal dopamine (DA) transmission has long been postulated, but ionotropic glutamate receptors have not been localized directly to DA axons. Striatal cholinergic interneurons (ChIs) are emerging as major players in striatal function, and can govern DA transmission by activating nicotinic receptors (nAChRs) on DA axons. Cortical inputs to ChIs have historically been perceived as sparse, but recent evidence indicates that they strongly activate ChIs. We explored whether activation of M1/M2 corticostriatal inputs can consequently gate DA transmission, via ChIs. We reveal that optogenetic activation of channelrhodopsin-expressing corticostriatal axons can drive striatal DA release detected with fast-scan cyclic voltammetry and requires activation of nAChRs on DA axons and AMPA receptors on ChIs that promote short-latency action potentials. By contrast, DA release driven by optogenetic activation of intralaminar thalamostriatal inputs involves additional activation of NMDA receptors on ChIs and action potential generation over longer timescales. Therefore, cortical and thalamic glutamate inputs can modulate DA transmission by regulating ChIs as gatekeepers, through ionotropic glutamate receptors. The different use of AMPA and NMDA receptors by cortical versus thalamic inputs might lead to distinct input integration strategies by ChIs and distinct modulation of the function of DA and striatum.
Dopamine receptors--physiological understanding to therapeutic intervention potential.
There are two families of dopamine (DA) receptors, called D1 and D2, respectively. The D1 family consists of D1- and D5-receptor subtypes and the D2 family consists of D2-, D3-, and D4-receptor subtypes. The amino acid sequences of these receptors show that they all belong to a large superfamily of receptors with seven transmembrane domains, which are coupled to their intracellular signal transduction systems by G-proteins. The implications of DA receptors in neuropsychiatry and cardiovascular and renal diseases are discussed. Neuropsychiatry indications include Parkinson's disease, schizophrenia, migraine, drug dependence, mania and depression, and Gilles de la Tourette syndrome. The underlying dysfunction of dopaminergic systems and the potential benefits of dopaminergic therapy in these different indications are critically examined. With respect to the pharmacological treatment of Parkinson's disease, a range of DA agonists are in various stages of preclinical and clinical development. D2-receptor agonist activity is predominant in most effective antiparkinsonian DA agonists. However, in practice, it is difficult to treat patients for several years with DA agonists alone; therapeutic benefit is not sustained. Rather, the use of a combination of DA agonists and levodopa is considered preferable. Reports of the efficacy of DA partial agonists await confirmation, and recent clinical investigations also suggest the potential of D1 receptor agonists as antiparkinson drugs. Regarding migraine pathogenesis, clinical and pharmacological evidence suggests that DA is involved in this disorder. Most prodromal and accompanying symptoms may be related to dopaminergic activation. Several drugs acting on DA receptors are effective in migraine treatment. Furthermore, migraine patients show a higher incidence of dopaminergic symptoms following acute DA agonist administration, when compared with normal controls. In cardiology, the therapeutic benefits of DA agonists are noted in the treatment of heart failure. Low doses of DA are widely used for its specific dopaminergic effects on renal function, which are suggested to be beneficial, and for its alpha- and beta-adrenergic-mediated responses that occur with higher doses. However, studies have been unable to demonstrate that DA can prevent acute renal failure or reduce mortality. It appears that the significant progress that is being made in the molecular understanding of DA receptors will continue to have a tremendous impact in the pharmacological treatment of neuropsychiatric, cardiovascular, and renal diseases.
The Striosome and Matrix Compartments of the Striatum: A Path through the Labyrinth from Neurochemistry toward Function.
The striatum is a heterogeneous structure with a diverse range of neuron types and neuromodulators. Three decades of anatomical and biochemical studies have established that the neurochemical organization of striatum is not uniformly heterogeneous, but rather, can be differentiated into neurochemically discrete compartments known as striosomes (also known as patches) and matrix. These compartments are well understood to differ in their expression of neurochemical markers, with some differences in afferent and efferent connectivity and have also been suggested to have different involvement in a range of neurological diseases. However, the functional outcomes of striosome-matrix organization are poorly understood. Now, recent findings and new experimental tools are beginning to reveal that the distinctions between striosomes and matrix have distinct consequences for striatal synapse function. Here, we review recent findings that suggest there can be distinct regulation of neural function in striosome versus matrix compartments, particularly compartment-specific neurochemical interactions. We highlight that new transgenic and viral tools are becoming available that should now accelerate the pace of advances in understanding of these long-mysterious striatal compartments.
Regulation of Extracellular Dopamine: Release and Uptake
Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the noradrenaline transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients.
Impaired dopamine release in Parkinson's disease.
Parkinson's disease is the second most common neurodegenerative disease and yet the early pathophysiological events of the condition and sequences of dysfunction remain unclear. The loss of dopaminergic neurons and reduced levels of striatal dopamine are descriptions used interchangeably as underlying the motor deficits in Parkinson's disease. However, decades of research suggest that dopamine release deficits in Parkinson's disease do not occur only after cell death, but that there is dysfunction or dysregulation of axonal dopamine release before cell loss. Here we review the evidence for dopamine release deficits prior to neurodegeneration in Parkinson's disease, drawn from a large and emerging range of Parkinson's disease models, and the mechanisms by which these release deficits occur. The evidence indicates that impaired dopamine release can result from disruption to a diverse range of Parkinson's disease-associated genetic and molecular disturbances, and can be considered as a potential pathophysiological hallmark of Parkinson's disease.
Rapid modulation of striatal cholinergic interneurons and dopamine release by satellite astrocytes.
Astrocytes are increasingly appreciated to possess underestimated and important roles in modulating neuronal circuits. Astrocytes in striatum can regulate dopamine transmission by governing the extracellular tone of axonal neuromodulators, including GABA and adenosine. However, here we reveal that striatal astrocytes occupy a cell type-specific anatomical and functional relationship with cholinergic interneurons (ChIs), through which they rapidly excite ChIs and govern dopamine release via nicotinic acetylcholine receptors on subsecond timescales. We identify that ChI somata are in unexpectedly close proximity to astrocyte somata, in mouse and human, forming a "soma-to-soma" satellite-like configuration not typically observed for other striatal neurons. We find that transient depolarization of astrocytes in mouse striatum reversibly regulates ChI excitability by decreasing extracellular calcium. These findings reveal a privileged satellite astrocyte-interneuron interaction for striatal ChIs operating on subsecond timescales via regulation of extracellular calcium dynamics to shape downstream striatal circuit activity and dopamine signaling.
