Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. METHODS: We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. RESULTS: APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid ß (Aß) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. CONCLUSIONS: In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aß by microglia in an AD mouse model.

Optogenetic targeting of astrocytes restores slow brain rhythm function and slows Alzheimer's disease pathology.

Patients with Alzheimer's disease (AD) exhibit non-rapid eye movement (NREM) sleep disturbances in addition to memory deficits. Disruption of NREM slow waves occurs early in the disease progression and is recapitulated in transgenic mouse models of beta-amyloidosis. However, the mechanisms underlying slow-wave disruptions remain unknown. Because astrocytes contribute to slow-wave activity, we used multiphoton microscopy and optogenetics to investigate whether they contribute to slow-wave disruptions in APP/PS1 mice. The power but not the frequency of astrocytic calcium transients was reduced in APP/PS1 mice compared to nontransgenic controls. Optogenetic activation of astrocytes at the endogenous frequency of slow waves restored slow-wave power, reduced amyloid deposition, prevented neuronal calcium elevations, and improved memory performance. Our findings revealed malfunction of the astrocytic network driving slow-wave disruptions. Thus, targeting astrocytes to restore circuit activity underlying sleep and memory disruptions in AD could ameliorate disease progression.

Calcium sensor Yellow Cameleon 3.6 as a tool to support the calcium hypothesis of Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a neurodegenerative disease with increasing relevance as dementia cases rise. The etiology of AD is widely debated. The Calcium Hypothesis of Alzheimer's disease and brain aging states that the dysfunction of calcium signaling is the final common pathway leading to neurodegeneration. When the Calcium Hypothesis was originally coined, the technology did not exist to test it, but with the advent of Yellow Cameleon 3.6 (YC3.6) we are able to test its validity. METHODS: Here we review use of YC3.6 in studying Alzheimer's disease using mouse models and discuss whether these studies support or refute the Calcium Hypothesis. RESULTS: YC3.6 studies showed that amyloidosis preceded dysfunction in neuronal calcium signaling and changes in synapse structure. This evidence supports the Calcium Hypothesis. DISCUSSION: In vivo YC3.6 studies point to calcium signaling as a promising therapeutic target; however, additional work is necessary to translate these findings to humans.

Optogenetic Targeting of Astrocytes Restores Slow Brain Rhythm Function and Slows Alzheimer's Disease Pathology.

Patients with Alzheimer's disease (AD) exhibit non-rapid eye movement (NREM) sleep disturbances in addition to memory deficits. Disruption of NREM slow waves occurs early in the disease progression and is recapitulated in transgenic mouse models of beta-amyloidosis. However, the mechanisms underlying slow-wave disruptions remain unknown. Because astrocytes contribute to slow-wave activity, we used multiphoton microscopy and optogenetics to investigate whether they contribute to slow-wave disruptions in APP mice. The power but not the frequency of astrocytic calcium transients was reduced in APP mice compared to nontransgenic controls. Optogenetic activation of astrocytes at the endogenous frequency of slow waves restored slow-wave power, reduced amyloid deposition, prevented neuronal calcium elevations, and improved memory performance. Our findings revealed malfunction of the astrocytic network driving slow-wave disruptions. Thus, targeting astrocytes to restore circuit activity underlying sleep and memory disruptions in AD could ameliorate disease progression.

Novel Botanical Therapeutic NB-02 Effectively Treats Alzheimer's Neuropathophysiology in an APP/PS1 Mouse Model.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and a major cause of dementia. Some of the hallmarks of AD include presence of amyloid plaques in brain parenchyma, calcium dysregulation within individual neurons, and neuroinflammation. A promising therapeutic would reverse or stymie these pathophysiologies in an animal model of AD. We tested the effect of NB-02, previously known as DA-9803, a novel multimodal therapeutic, on amyloid deposition, neuronal calcium regulation and neuroinflammation in 8- to 10-month-old APP/PS1 mice, an animal model of AD. In vivo multiphoton microscopy revealed that two-month-long administration of NB-02 halted amyloid plaque deposition and cleared amyloid in the cortex. Postmortem analysis verified NB-02-dependent decrease in plaque deposition in the cortex as well as hippocampus. Furthermore, drug treatment reversed neuronal calcium elevations, thus restoring neuronal function. Finally, NB-02 restored spine density and transformed the morphology of astrocytes as well as microglia to a more phagocytic state, affecting neuroinflammation. NB-02 was effective at reversing AD neuropathophysiology in an animal model. Therefore, in addition to serving as a promising preventative agent, NB-02 holds potential as a treatment for AD in the clinic.

Slow Wave Sleep Is a Promising Intervention Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the major cause of dementia, characterized by the presence of amyloid-beta plaques and neurofibrillary tau tangles. Plaques and tangles are associated with sleep-wake cycle disruptions, including the disruptions in non-rapid eye movement (NREM) slow wave sleep (SWS). Alzheimer's patients spend less time in NREM sleep and exhibit decreased slow wave activity (SWA). Consistent with the critical role of SWS in memory consolidation, reduced SWA is associated with impaired memory consolidation in AD patients. The aberrant SWA can be modeled in transgenic mouse models of amyloidosis and tauopathy. Animal models exhibited slow wave impairments early in the disease progression, prior to the deposition of amyloid-beta plaques, however, in the presence of abundant oligomeric amyloid-beta. Optogenetic rescue of SWA successfully halted the amyloid accumulation and restored intraneuronal calcium levels in mice. On the other hand, optogenetic acceleration of slow wave frequency exacerbated amyloid deposition and disrupted neuronal calcium homeostasis. In this review, we summarize the evidence and the mechanisms underlying the existence of a positive feedback loop between amyloid/tau pathology and SWA disruptions that lead to further accumulations of amyloid and tau in AD. Moreover, since SWA disruptions occur prior to the plaque deposition, SWA disruptions may provide an early biomarker for AD. Finally, we propose that therapeutic targeting of SWA in AD might lead to an effective treatment for Alzheimer's patients.

Frequency-dependent exacerbation of Alzheimer's disease neuropathophysiology.

Neuronal activity patterns are disrupted in neurodegenerative disorders, including Alzheimer's disease (AD). One example is disruption of corticothalamic slow oscillations responsible for sleep-dependent memory consolidation. Slow waves are periodic oscillations in neuronal activity occurring at frequencies of <1 Hz. The power, but not the frequency of slow oscillations is altered in a mouse model of AD. Optogenetic rescue of slow oscillations by increasing activity in cortical pyramidal neurons at the frequency of slow waves restores slow wave power, halts deposition of amyloid plaques and prevents neuronal calcium dysregulation. Here we determined whether driving this circuit at an increased rate would exacerbate the amyloid-dependent calcium dyshomeostasis in transgenic mice. Doubling the frequency of slow waves for one month with optogenetics resulted in increased amyloid beta - dependent disruptions in neuronal calcium homeostasis and loss of synaptic spines. Therefore, while restoration of physiological circuit dynamics is sufficient to abrogate the progression of Alzheimer's disease pathology and should be considered an avenue for clinical treatment of AD patients with sleep disorders, pathophysiological stimulation of neuronal circuits leads to activity - dependent acceleration of amyloid production, aggregation and downstream neuronal dysfunction.

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

Reproductive experience modified dendritic spines on cortical pyramidal neurons to enhance sensory perception and spatial learning in rats.

Behavioral adaptations during motherhood are aimed at increasing reproductive success. Alterations of hormones during motherhood could trigger brain morphological changes to underlie behavioral alterations. Here we investigated whether motherhood changes a rat's sensory perception and spatial memory in conjunction with cortical neuronal structural changes. Female rats of different statuses, including virgin, pregnant, lactating, and primiparous rats were studied. Behavioral test showed that the lactating rats were most sensitive to heat, while rats with motherhood and reproduction experience outperformed virgin rats in a water maze task. By intracellular dye injection and computer-assisted 3-dimensional reconstruction, the dendritic arbors and spines of the layer III and V pyramidal neurons of the somatosensory cortex and CA1 hippocampal pyramidal neurons were revealed for closer analysis. The results showed that motherhood and reproductive experience increased dendritic spines but not arbors or the lengths of the layer III and V pyramidal neurons of the somatosensory cortex and CA1 hippocampal pyramidal neurons. In addition, lactating rats had a higher incidence of spines than pregnant or primiparous rats. The increase of dendritic spines was coupled with increased expression of the glutamatergic postsynaptic marker protein (PSD-95), especially in lactating rats. On the basis of the present results, it is concluded that motherhood enhanced rat sensory perception and spatial memory and was accompanied by increases in dendritic spines on output neurons of the somatosensory cortex and CA1 hippocampus. The effect was sustained for at least 6 weeks after the weaning of the pups.

High-intensity focused ultrasound attenuates neural responses of sciatic nerves isolated from normal or neuropathic rats.

Patients with diabetic neuropathy often have neuropathic pain. The purpose of our work was to investigate the effects of high-intensity focused ultrasound (HIFU) on the conduction block of normal and neuropathic nerves for soothing pain. Adult male Sprague-Dawley rats were used, and diabetes was induced by streptozotocin injection. Diabetic neuropathy was evaluated with animal behavior tests. Sciatic nerves of both control and neuropathic rats were dissected from the starting point of the sciatic nerve to the point where the sural nerve ends near the ankle. The nerves were stored in Ringer's solution. The in vitro nerve was placed on a self-developed experimental platform for HIFU exposure. Stimulation and recording of the compound action potentials (CAPs) and sensory action potentials (SAPs) were performed. Control and neuropathic nerves exposed or not exposed to HIFU were submitted to histologic analysis. For the control and neuropathic nerves, suppression of CAPs and SAPs started 2 min post-HIFU treatment. Maximum suppression of SAPs was 34.4 ± 3.2% for the control rats and 11.6 ± 2.0% and 9.8 ± 3.0% for rats 4 wk post-injection and 8 wk post-injection, respectively. Time to full recovery was 25, 70 and 80 min, respectively. Histologic analysis revealed that the nerves in which CAPs and SAPs did not fully recover were damaged thermally or mechanically by HIFU. It is feasible to reversibly block nerves with appropriate HIFU treatment. Diabetic nerves were less suppressed by HIFU and were more vulnerable to permanent damage.

Temporal course of streptozotocin-induced diabetic polyneuropathy in rats.

The temporal course of diabetic polyneuropathy in a rat model plays a critical role in studies on diabetic polyneuropathy treatment. In this study, the temporal course of neuropathic symptoms was investigated in diabetic rats induced by streptozotocin and evaluated by nerve conduction velocity and behavioral assays, including the von Frey test for mechanical allodynia and the hot plate test for hyperalgesia. The results revealed that both mechanical allodynia and heat hyperalgesia started on the 2nd week, while nerve conduction velocity significantly decreased from the 1st week. In addition, the severity of allodynia did not change after the 3rd week. Hyperalgesia and nerve conduction velocity progressively aggravated even to the 8th week. Transmission electron microscopy showed that loss of unmyelinated axons, loosening of the myelin structure, and thickening of the perineurium layer were visible from the 4th week and worsened on the 8th week. Differences in the temporal course of neuropathic symptoms are discussed.

Measurement of ultrasonic attenuation in diabetic neuropathic sciatic nerves for diagnostic and therapeutic applications.

Measurements of ultrasonic attenuation in the sciatic nerves of rats were performed to verify the feasibility of ultrasound diagnosis of peripheral neuropathy and to avoid damage to the nerves caused by overheating in pain management applications. A rat model of diabetic peripheral neuropathy was established. The proximal-segment and middle-segment sciatic nerves of control and neuropathic rats were dissected for the attenuation measurement. Two commercial ultrasound transducers and a self-developed experimental platform were used in the measurements. Using H&E staining and transmission electron (TE) microscopy, morphological analysis of the control and neuropathic nerves was performed to determine the relationship between attenuation and the histology of the nerves. The experimental results showed that the attenuation coefficients of the control, second-week, fourth-week, and eighth-week neuropathic nerves were -6.68 ± 0.50, -5.61 ± 0.34, -6.27 ± 0.40, and -7.10 ± 0.35 dB/cm at 2.68 MHz, respectively. The respective values at 7.50 MHz were -14.96 ± 0.79, -12.65 ± 0.28, -13.98 ± 1.07, and -16.00 ± 0.54 dB/cm. The changes in the attenuation coefficients were significantly different among the second-week, fourth-week, and eighth-week DN nerves. Additionally, the ultrasonic attenuation coefficient of the rat sciatic nerve was fourfold that of the cat brain and cow liver and twofold that of human muscle.

The distribution of muscles fibers and their types in the female rat urethra: cytoarchitecture and three-dimensional reconstruction.

An attempt to explore urethral cytoarchitecture including the distribution of smooth muscles and fast and slow striated muscles of adult female Sprague Dawley rat--a popular model in studying lower urinary tract function. Histological and immunohistochemical stainings were carried out to investigate the distribution of urethral muscle fibers and motor end plates. The urethral sphincter was furthermore three-dimensionally reconstructed from serial histological sections. The mucosa at the distal urethra was significantly thicker than that of other segments. A prominent inner longitudinal and outer circular layer of smooth muscles covered the proximal end of urethra. Thick circular smooth muscles of the bladder neck region (urethral portion) decreased significantly distalward and longitudinal smooth muscles became 2- to 3-fold thicker in the rest of the urethra. An additional layer of striated muscles appeared externally after neck region (urethra) and in association with motor end plates ran throughout the remaining urethra as the striated sphincter layer. Most striated muscles were fast fibers while relatively fewer slow fibers often concentrated at the periphery. A pair of extraneous striated muscles, resembling the human urethrovaginal sphincter muscles, connected both sides of mainly the distal vagina to the dorsal striated muscles in the wall of the middle urethra. The tension provided by this pair of muscles, and in conjunction with the striated sphincter of the urethral wall, was likely to function to suspend the middle urethra and facilitates its closure. Comprehensive morphological data of urethral sphincter offers solid basis for researchers conducting studies on dysfunction of bladder outlet.