stereotaxic-surgery-in-infant-mice-and-postoperative-care

Stereotaxic Surgery in Infant Mice and Postoperative Care

Understanding the developmental, cellular, and circuit processes across the postnatal period is crucial for elucidating the complexities of brain ...

Research Triangle Park

Neuromodulation of neural networks, whereby a selected circuit is regulated by a particular modulator, plays a critical role in learning and memory. Among neuromodulators, acetylcholine (ACh) plays a critical role in hippocampus-dependent memory and has been shown to modulate neuronal circuits in the hippocampus. However, it has remained unknown how ACh modulates hippocampal output. Here, using in vitro and in vivo approaches, we show that ACh, by activating oriens lacunosum moleculare (OLM) interneurons and therefore augmenting the negative-feedback regulation to the CA1 pyramidal neurons, suppresses the circuit from the hippocampal area CA1 to the deep-layer entorhinal cortex (EC). We also demonstrate, using mouse behavior studies, that the ablation of OLM interneurons specifically impairs hippocampus-dependent but not hippocampus-independent learning. These data suggest that ACh plays an important role in regulating hippocampal output to the EC by activating OLM interneurons, which is critical for the formation of hippocampus-dependent memory.

Septal cholinergic neurons gate hippocampal output to entorhinal cortex via oriens lacunosum moleculare interneurons.

To achieve simultaneous measurement of multiple cellular events in molecularly defined groups of neurons in vivo, we designed a spectrometer-based fiber photometry system that allows for spectral unmixing of multiple fluorescence signals recorded from deep brain structures in behaving animals. Using green and red Ca indicators differentially expressed in striatal direct- and indirect-pathway neurons, we were able to simultaneously monitor the neural activity in these two pathways in freely moving animals. We found that the activities were highly synchronized between the direct and indirect pathways within one hemisphere and were desynchronized between the two hemispheres. We further analyzed the relationship between the movement patterns and the magnitude of activation in direct- and indirect-pathway neurons and found that the striatal direct and indirect pathways coordinately control the dynamics and fate of movement. VIDEO ABSTRACT.

Spectrally Resolved Fiber Photometry for Multi-component Analysis of Brain Circuits.

Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRAB) sensors that enable these measurements. In response to extracellular DA, GRAB sensors exhibit large fluorescence increases (ΔF/F ∼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRAB sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRAB sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRAB sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice.

Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRAB) sensors with a 230% peak ΔF/F response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral- or transgenic-mediated expression of GRAB sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRAB sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes.

A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine.

Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent G-protein-coupled receptor-activation-based DA (GRAB) sensors and optimized versions of green fluorescent GRAB sensors. In response to extracellular DA, both the red and green GRAB sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics and nanomolar-to-submicromolar affinity. Moreover, the GRAB sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Coexpressing red GRAB with either green GRAB or the calcium indicator GCaMP6s allows tracking of dopaminergic signaling and neuronal activity in distinct circuits in vivo.

Next-generation GRAB sensors for monitoring dopaminergic activity in vivo.

Diagnosing Parkinson's disease (PD) before the clinical onset proves difficult because the hallmark PD symptoms do not manifest until more than 60% of dopamine neurons in the substantia nigra pars compacta have been lost. Here we show that, by evoking a transient dopamine release and subsequently measuring the levels of dopamine metabolites in the cerebrospinal fluid and plasma, a hypodopaminergic state can be revealed when less than 30% of dopamine neurons are lost in mouse PD models. These findings may lead to sensitive and practical screening and diagnostic tests for detecting early PD in the high-risk population.

Dopamine Neuron Challenge Test for early detection of Parkinson's disease.

The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

Chemogenetic stimulation of tonic locus coeruleus activity strengthens the default mode network.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase β (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

Deficiency in endocannabinoid synthase DAGLB contributes to early onset Parkinsonism and murine nigral dopaminergic neuron dysfunction.

We recently reported that the "Dopamine Neuron Challenge Test" (DNC Test), a diagnostic method that measures the levels of dopamine metabolites in cerebrospinal fluid (CSF) and plasma samples after pharmacologically inducing a transient dopamine release, can detect early-stage Parkinson's disease (PD) with high sensitivity and selectivity in mouse models. The use of haloperidol in the original DNC test to challenge dopamine neurons was less than ideal, as it may cause extrapyramidal motor symptoms. Here we report an improved DNC Test, in which the original challenging agents, haloperidol and methylphenidate, are replaced by a single challenging agent, a dopamine autoreceptor preferring antagonist AJ76 or UH232. We show that the improved DNC Test can achieve the same level of sensitivity and selectivity in detecting early PD in a mouse model without causing motor side effects. These findings significantly improve the practicality of using the DNC Test as a screening or diagnostic test for detecting early-stage PD in the high-risk population in humans.

AJ76 and UH232 as potential agents for diagnosing early-stage Parkinson's disease.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRAB and GRAB) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRAB sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

Monitoring norepinephrine release in vivo using next-generation GRAB sensors.

Protein/protein interactions (PPI) play crucial roles in neuronal functions. Yet, their potential as drug targets for brain disorders remains underexplored. The fibroblast growth factor 14 (FGF14)/voltage-gated Na channel 1.6 (Na1.6) complex regulates excitability of medium spiny neurons (MSN) of the nucleus accumbens (NAc), a central hub of reward circuitry that controls motivated behaviors. Here, we identified compound 1028 (IUPAC: ethyl 3-(2-(3-(hydroxymethyl)-1H-indol-1-yl)acetamido)benzoate), a brain-permeable small molecule that targets FGF14, a critical residue located within a druggable pocket at the FGF14/Na1.6 PPI interface. We found that 1028 modulates FGF14/Na1.6 complex assembly and depolarizes the voltage-dependence of Na1.6 channel inactivation with nanomolar potency by modulating the intramolecular interaction between the III-IV linker and C-terminal domain of the Na1.6 channel. Consistent with the compound's effects on Na1.6 channel inactivation, 1028 enhances MSN excitability ex vivo and accumbal neuron firing rate in vivo in murine models. Systemic administration of 1028 maintains behavioral motivation preferentially during motivationally deficient conditions in murine models. These behavioral effects were abrogated by in vivo gene silencing of Fgf14 in the NAc and were accompanied by a selective reduction in accumbal dopamine levels during reward consumption in murine models. These findings underscore the potential to selectively regulate complex behaviors associated with neuropsychiatric disorders through targeting of PPIs in neurons.

Enhanced motivated behavior mediated by pharmacological targeting of the FGF14/Na1.6 complex in nucleus accumbens neurons.

Deep brain stimulation (DBS) effectively treats drug-resistant neurological and psychiatric disorders, yet its mechanisms remain unclear. Here we show that high-frequency DBS of the subthalamic nucleus (STN), a common target for Parkinson's disease (PD), activates afferent axons while inhibiting STN neurons. These contrasting presynaptic and postsynaptic effects arise from a decrease in local neurotransmitter release with a larger decrease in glutamate than GABA, shifting the excitation/inhibition balance toward inhibition. Chemogenetic inhibition, but not excitation, of STN neurons mimics the therapeutic effects of DBS in 6-OHDA-lesioned PD mice. Acute and chronic bilateral chemogenetic STN inhibition restores motor function in a progressive PD mouse model. These findings suggest that inhibition of STN, caused by differential depression of glutamatergic and GABAergic synapses, is a key mechanism of therapeutic DBS. 'Chemogenetic DBS', direct chemogenetic inhibition of postsynaptic neurons, may offer a less invasive and more affordable alternative to electrical DBS for PD and other neurological disorders.

Article	Year
Septal cholinergic neurons gate hippocampal output to entorhinal cortex via oriens lacunosum moleculare interneurons. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29437952	2018
Spectrally Resolved Fiber Photometry for Multi-component Analysis of Brain Circuits. Neuron\| PubMed ID: 29731250	2018
A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice. Cell\| PubMed ID: 30007419	2018
A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine. Neuron\| PubMed ID: 30922875	2019
Next-generation GRAB sensors for monitoring dopaminergic activity in vivo. Nature methods\| PubMed ID: 33087905	2020
Dopamine Neuron Challenge Test for early detection of Parkinson's disease. NPJ Parkinson's disease\| PubMed ID: 34916526	2021
Chemogenetic stimulation of tonic locus coeruleus activity strengthens the default mode network. Science advances\| PubMed ID: 35486721	2022
Deficiency in endocannabinoid synthase DAGLB contributes to early onset Parkinsonism and murine nigral dopaminergic neuron dysfunction. Nature communications\| PubMed ID: 35715418	2022
AJ76 and UH232 as potential agents for diagnosing early-stage Parkinson's disease. Neuropharmacology\| PubMed ID: 36623805	2023
Monitoring norepinephrine release in vivo using next-generation GRAB sensors. Neuron\| PubMed ID: 38547869	2024
Enhanced motivated behavior mediated by pharmacological targeting of the FGF14/Na1.6 complex in nucleus accumbens neurons. Nature communications\| PubMed ID: 39747162	2025
Differential synaptic depression mediates the therapeutic effect of deep brain stimulation. Nature neuroscience\| PubMed ID: 41102541	2025

Jingheng Zhou

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