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Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice

Neuroscientists use miniature microscopes (miniscopes) to observe neuronal activity in freely behaving animals. The University of California, Los Angeles ...

National Taiwan University

Avian animals are visually inclined, which has caused them to attract increasing attention for visual neurophysiology or electrophysiology studies, including the study of the visual evoked potential (VEP). VEP has developed into an investigative tool for understanding the physiology and the pathology of the visual pathway. Chemical restraint is a common method to minimize motion artifacts in animals when acquiring VEP data, but little is known about its influence on the signal in an avian animal. In addition, it is difficult to make comparisons between conscious state data when the animals are ultimately under anesthesia. Therefore, finding drugs and developing protocols that have an acceptable effect is valuable. We compared the local field potentials of physically and chemically restrained zebra finches (), a small avian species, to simulate a relatively challenging recording condition. Finches were sedated with midazolam-butorphanol, and anesthesia was maintained by isoflurane. Electrodes were implanted into the left nucleus rotundus, which is a visual nucleus in birds. The VEPs of the control group ( = 3) were recorded after they fully recovered and were restrained by towels. The other birds ( = 3) were recorded under anesthesia. The results show that without the visual stimuli, anesthesia generally suppressed the overall power of field potentials. However, by focusing on the spectra during VEPs, visual stimuli still triggered significant VEPs in frequencies below 30.8 Hz, which were even stronger than towel-restrained birds. The drugs also prolonged the latency of the VEP, increased the duration of the VEP when compared to towel-restrained birds. As regard to towel-restrained zebra finches, the field potentials were less synchronized and may need data preprocessing to have clear VEPs. In conclusion, the current study presents evidence of basic VEP for zebra finch under midazolam-butorphanol-isoflurane anesthesia with a protocol that is a safe and feasible anesthetic combination for chemical restraint, which is particularly useful for small animals when obtaining evoked potentials.

Delayed evoked potentials in zebra finch () under midazolam-butorphanol-isoflurane anesthesia.

Daytime somnolence is common in patients with Parkinson's disease (PD); however there is a lack of understanding of the cellular mechanisms involved in mediating these effects. It has been hypothesized that microglial activation and the subsequent increase of pro-inflammatory cytokines play an important role in the pathogenesis of PD. Because some cytokines are involved in the regulation of sleep, this study was designed to determine if tumor necrosis factor (TNF) and interleukin-1beta (IL-1beta), mediate daytime somnolence in the proteasome inhibitor (MG-132)-induced hemiparkinsonian rat model. Our results indicated that microglial activation caused the loss of dopaminergic neurons in the substantia nigra, and the expression of TNF-alpha, but not IL-1beta, increased in the midbrain and hypothalamus in MG-132-induced hemiparkinsonian rats. Slow-wave sleep (SWS) increased after the induction of hemiparkinsonism, but rapid eye movement (REM) sleep was not consistently altered. Application of the TNF receptor fragment (TNFRF) blocked hemiparkinsonism-induced SWS alteration, whereas the IL-1 receptor antagonist (IL-1ra) exhibited no effect. Increased nuclear translocation of NF-kappaB in the midbrain, and the blockade of SWS enhancement in MG-132-induced hemiparkinsonian rats by an inhibitor of NF-kappaB activation indicate that the TNF-NF-kappaB cascade is a critical mediator of MG-132 hemiparkinsonian-induced sleep alteration. This observation suggests potential therapeutic interventions to target the excessive daytime somnolence in patients with PD.

TNF-NF-kappaB signaling mediates excessive somnolence in hemiparkinsonian rats.

Baicalin is an active compound originating from the root of Scutellaria baicalensis Georgi, which has been used for anti-inflammation, anti-bacteria, anti-hypertension, anti-allergy and sedation since ancient China, though the neuronal mechanisms involved in the sedative effect is still unclear. Baicalin possesses the ability to decrease the expression of pro-inflammatory cytokines and nuclear factor (NF)-κB activity. Furthermore, baicalin has demonstrated an anxiolytic-like effect via activation of γ-aminobutyric acid-A (GABA(A)) receptors. Pro-inflammatory cytokines (e.g. interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α) and the GABAergic system promote sleep. This study was designed to determine whether the GABA(A) receptor activation and/or the suppression of pro-inflammatory cytokines mediate(s) baicalin-induced sleep alterations.

Biphasic effects of baicalin, an active constituent of Scutellaria baicalensis Georgi, in the spontaneous sleep-wake regulation.

Patients with post-traumatic stress disorder (PTSD) frequently complain of having sleep disturbances, such as insomnia and rapid eye movement (REM) sleep abnormality. Cannabidiol (CBD), a psycho-inactive constituent of marijuana, reduces physiological non-REM (NREM) sleep and REM sleep in normal rats, in addition to generating its anxiolytic effect. However, the effects of CBD on anxiety-induced sleep disturbances remain unclear. Because anxiety progression is caused by persistent stress for a period of time, we employed the repeated combination tests (RCT) consisting of a 50-min open field (OF) and a subsequent 10-min elevated plus-maze (EPM) for four consecutive days to simulate the development of anxiety. Time spent in the centre arena of OF and during open arms of the EPM was substantially decreased in latter days of RCT, suggesting the habituation, which potentially lessens anxiety-mediated behavioural responses, was not observed in current tests. CBD microinjected into the central nucleus of amygdala (CeA) significantly enhanced time spent in centre arena of OF, increased time during the open arms and decreased frequency of entry to the enclosed arms of EPM, further confirming its anxiolytic effect. The decrease of NREM sleep during the first hour and the suppression of REM sleep during hours 4-10 after the RCT represent the similar clinical observations (e.g. insomnia and REM sleep interruption) in PTSD patients. CBD efficiently blocked anxiety-induced REM sleep suppression, but had little effect on the alteration of NREM sleep. Conclusively, CBD may block anxiety-induced REM sleep alteration via its anxiolytic effect, rather than via sleep regulation per se. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Effect of cannabidiol on sleep disruption induced by the repeated combination tests consisting of open field and elevated plus-maze in rats.

The frequency of electroencephalograms (EEGs) is predominant in theta rhythm during stress (e.g., footshock) in rats. Median raphe nucleus (MRN) desynchronizes hippocampal theta waves via activation of GABAergic neurons in the medial septum-diagonal band of Broca (MS-DBB), a theta rhythm pacemaker. Increased hypocretin mediates stress responses in addition to the maintenance of wakefulness. Hypocretin receptors are abundant in the MRN, suggesting a possible role of hypocretin in modulating stress-induced theta rhythm. Our results indicated that the intensity of theta waves was enhanced by footshock and that a hypocretin receptor antagonist (TCS1102) suppressed the footshock-induced theta waves. Administration of hypocretin-1 (1 and 10 μg) and hypocretin-2 (10 μg) directly into the MRN simulated the effect of footshock and significantly increased theta waves. Co-administration of GABA(A) receptor antagonist, bicuculline, into the MRN blocked the increase of theta waves induced by hypocretins or footshock. These results suggested that stress enhances the release of hypocretins, activates GABAergic neurons in the MRN, blocks the ability of MRN to desynchronize theta waves, and subsequently increases the intensity of theta rhythm.

Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats.

Theta rhythms generated in the hippocampus are controlled by the pacemaker in the medial septum-diagonal band of Broca (MS-DBB). The median raphe nucleus (MRN) transmits serotonergic signals to the MS-DBB, which suppresses the septo-hippocampus-produced theta waves, whereas GABAergic interneurons in the MRN facilitate the generation of theta oscillations. Animal studies have indicated that fear increases theta oscillations. Moreover, anxiolytics reduce reticular formation-elicited theta rhythms and theta blockade decreases anxiety. In this study, we hypothesized that the MRN mediates anxiety reduction caused by the theta blockade. Our results demonstrated that inescapable-footshock stimulation significantly increased the power of low-frequency theta oscillations (4-7 Hz) in rats. Both the electrical stimulation of MRN and administration of bicuculline into the MRN successfully desynchronized footshock-induced theta oscillations. Compared to the naïve rats, inescapable-footshock stimulation diminished the entry percentage and time spent in the open arms of the elevated plus maze (EPM), behavioral indicators of anxiety. Rats treated with either MRN stimulation or bicuculline administration to desynchronize theta oscillations reduced anxiety caused by the inescapable-footshock stimulation. Our results demonstrated that the electrical stimulation of MRN or blockade of the GABAergic pathways in the MRN interferes with theta oscillations and reduces anxiety, implicating the role of MRN.

Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats.

Via an assay using an Amino Acid Analyzer, pepsin-digested chicken liver hydrolysates (CLHs) contain taurine (365.57 ± 39.04 mg/100 g), carnosine (14.03 ± 1.98 mg/100 g), and anserine (151.58 ± 27.82 mg/100 g). This study aimed to evaluate whether CLHs could alleviate thioacetamide (TAA)-induced fibrosis. A dose of 100 mg TAA/kg BW significantly increased serum liver damage indices and liver cytokine contents. Cell infiltration and monocytes/macrophages in livers of TAA-treated rats were illustrated by the H&E staining and immunohistochemical analysis of cluster of differentiation 68 (CD68, ED1), respectively. A significantly increased hepatic collagen accumulation was also observed and quantified under TAA treatment. A significant up-regulation of transforming growth factor-beta (TGF-β) and SMAD family member 4 (SMAD4) caused by TAA treatment further enhanced alpha smooth muscle actin (αSMA) gene and protein expressions. The liver antioxidant effects under TAA treatment were significantly amended by 200 and 600 mg CLHs/kg BW. Hence, the ameliorative effects of CLHs on liver fibrogenesis could be attributed by antioxidation and anti-inflmmation.

Protective Effects of Functional Chicken Liver Hydrolysates against Liver Fibrogenesis: Antioxidation, Anti-inflammation, and Antifibrosis.

Stress is one of major factors that cause sleep problems. Hypocretin represents a stress-related neuropeptide and is well known in maintaining physiological wakefulness. The hypocretinergic neurons originate in the lateral hypothalamic area (LHA) and transmit to several brain regions, including the median raphe nuclei (MRNs). The MRNs modulate both fear responses and sleep-wake activity; however, it remains unclear whether stress alters the levels of hypocretin to regulate MRNs and consequently disrupt sleep. In this paper, we employed the inescapable footshock stimuli (IFS) as a stressor and hypothesized that the IFS-induced sleep disruption is mediated by increased hypocretins in the MRNs. Our results demonstrate that the concentrations of hypocretin in the hypothalamus increased after IFS. Rapid eye movement (REM) sleep was reduced after footshock, and microinjection of non-selective hypocretin receptor antagonist TCS-1102 into the MRNs blocked the IFS-induced decrease of REM sleep. Furthermore, administration of hypocretins into the MRNs mimicked the IFS-induced REM sleep reduction. These results conclude that the increased levels of hypocretins in the MRNs mediate the IFS-induced REM sleep reduction.

Hypocretin in median raphe nucleus modulates footshock stimuli-induced REM sleep alteration.

The endocannabinoid system modulates many brain functions, including episodic memories, which contain memories of time and places. Most studies have focused on the involvement of the endocannabinoid system in spatial memory; however, its role in temporal memory is not well understood. Few studies have tested whether the unilateral endocannabinoid system is sufficient to modulate memory retrieval. Here, we tested whether type 1 cannabinoid receptors in the right hippocampal cornu ammonis area 1 region are enough to modulate the retrieval of episodic memories, specifically their spatial and temporal components. Because rats have innate preferences for displaced or old familiar objects, we changed the locations of "old familiar" and "recent familiar" objects in an open field and measured the rats' exploration times to evaluate spatial and temporal memory. To address the influence of the type 1 cannabinoid receptors on the retrieval of episodic-like memories, two doses of arachidonylcyclopropylamide, a selective type 1 cannabinoid receptor agonist, were infused into the cornu ammonis area 1 of rats ten minutes before the discrimination trials. We observed that rats injected with a low dose of arachidonylcyclopropylamide spent less time investigating displaced objects, suggesting spatial memory impairment, whereas those receiving a high dose explored old familiar objects less frequently, suggesting temporal memory impairment. This indicates that unilateral activation of type 1 cannabinoid receptors in the cornu ammonis area 1 impairs the spatial and temporal aspects of episodic memories. This research mimics the influence of marijuana intoxication effects in humans, such as spatial and temporal disintegration.

Activation of cannabinoid receptor type 1 impairs spatial and temporal aspects of episodic-like memories in rats.

Marijuana intoxication impairs neurocognitive functions. Common side effects of consuming cannabis include time distortion and memory loss. However, the underlying neurophysiological mechanisms involved in these effects remain unclear. We hypothesized that communication between the hippocampal CA1 region and medial entorhinal cortex (MEC) is essential for the transmission of temporal-associated information. We used a differential-reinforcement-of-low-rate (DRL) task, which requires subjects to press a lever at an optimal time point, to correlate the distributions of interresponse time (IRT) with local field potentials (LFPs) recorded in the CA1 and MEC under the effects of a cannabinoid type 1 (CB1) receptor agonist. We used a DRL 10-s schedule and trained the rats to withhold for 10 s before pressing a lever. Our data showed that the percentage of 12.4- to 14-s IRT events rose after activation of CB1 receptors in the MEC. In addition, gamma amplitude synchronization and CA1 theta phase-MEC gamma amplitude coupling decreased during the 6- to 14-s IRT events. These results suggest that activation of CB1 receptors in the MEC disrupt the functional connectivity between the CA1 and the MEC. This inefficient communication may result in increased IRT during a DRL schedule. Overall, we postulate that marijuana intoxication impairs the communication between the CA1 and MEC and influences behavioral performances that require precise timing ability.

Activation of cannabinoid type 1 receptors decreases the synchronization of local field potential oscillations in the hippocampus and entorhinal cortex and prolongs the interresponse time during a differential-reinforcement-of-low-rate task.

We previously demonstrated that seizure occurrences at different zeitgeber times alter sleep and circadian rhythm differently. On the other hand, the synchronized delta wave of electroencephalogram (EEG) during non-rapid eye movement (NREM) sleep facilitates seizure, while the desynchronized EEG of rapid eye movement (REM) sleep suppresses it. We also elucidated that unilateral deep brain stimulation (DBS) of the anterior nucleus of thalamus (ANT) suppresses seizure recurrence. In the present study, we intraperitoneally injected pentylenetetrazol (PTZ, 40 mg/kg) for 14 consecutive days (PTZ kindling) to induce spontaneous seizure in rats, and a 30-min (delivered 10 min before each PTZ injection) or a 3-h DBS of unilateral ANT (delivered 1 h before each PTZ injection) was applied to suppress seizure. The frequency of DBS stimulation was 200 Hz and the electrical current consisted of biphasic square pulses with 50-μA intensity, 100-μs pulse width, and 4.1-ms stimulation interval. Our results found that PTZ-induced spontaneous seizure did not cause a significant change in the quantity of NREM sleep but suppressed the amount of REM sleep. Unilateral ANT DBS prolonged the onset latency of ictal seizure, decreased the spontaneous seizure duration, and increased the survival rate but did not change the amplitude of epileptiform EEGs during ictal period. Unilateral ANT DBS did not significantly alter NREM sleep but increased the amount of REM sleep. An analysis of the spectrograms of fast Fourier transform indicated that the intensities of all frequencies were enhanced during the PTZ-induced ictal period and the subsequent spontaneous seizure. Thirty minutes of unilateral ANT DBS suppressed the augmentation of low-frequency (<10 Hz) intensities during the spontaneous seizure induced by PTZ kindling. We further found that consecutive injections of PTZ progressively increased the enhancement of the delta powers during NREM sleep, whereas unilateral ANT DBS inhibited this progressive enhancement. It was also noticed that 30 min of ANT DBS exhibited a better efficacy in epilepsy suppression than 3 h of ANT DBS. These results elucidated that unilateral ANT DBS enhanced the seizure threshold by increasing the amount of REM sleep and decreasing the progressive enhancement of delta power during NREM sleep to suppress spontaneous seizure recurrences in PTZ kindling-induced epileptic rats.

Deep Brain Stimulation Increases Seizure Threshold by Altering REM Sleep and Delta Powers During NREM Sleep.

Researchers demonstrated an elegant ability for red discrimination in zebra finches. It is interested to understand whether red activates exhibit much stronger response than other colors in neural network levels. To reveal the question, local field potentials (LFPs) was recorded and analyzed in two visual pathways, the thalamofugal and the tectofugal pathways, of zebra finches. Human studies demonstrate visual associated telencephalons communicate with higher order brain areas such as prefrontal cortex. The present study determined whether a comparable transmission occurs in zebra finches. Telencephalic regions of the thalamofugal (the visual Wulst) and the tectofugal pathway (the entopallium) with their higher order telencephalon, nidopallium caudolateral (NCL) were simultaneously recorded. LFPs of relay nuclei (the nucleus rotundus, ROT) of tectofugal pathway were also acquired. We demonstrated that LFP powers in the tectofugal pathway were higher than those in the thalamofugal pathway when illuminating blue lights. In addition, the LFP synchronization was stronger between the entopallium and NCL. LFPs also revealed a higher Granger causality from the direction of entopallium to NCL and from ROT to entopallium. These results suggest that zebra finches' tectofugal pathway predominately processing color information from ROT to NCL, relayed by entopallium, and blue could trigger the strongest response.

Article	Year
Delayed evoked potentials in zebra finch () under midazolam-butorphanol-isoflurane anesthesia. PeerJ\| PubMed ID: 31660277	2019
TNF-NF-kappaB signaling mediates excessive somnolence in hemiparkinsonian rats. Behavioural brain research\| PubMed ID: 20043954	2010
Biphasic effects of baicalin, an active constituent of Scutellaria baicalensis Georgi, in the spontaneous sleep-wake regulation. Journal of ethnopharmacology\| PubMed ID: 21419210	2011
Effect of cannabidiol on sleep disruption induced by the repeated combination tests consisting of open field and elevated plus-maze in rats. Neuropharmacology\| PubMed ID: 21867717	2012
Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats. Behavioural brain research\| PubMed ID: 22579972	2012
Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats. Behavioural brain research\| PubMed ID: 23542088	2013
Protective Effects of Functional Chicken Liver Hydrolysates against Liver Fibrogenesis: Antioxidation, Anti-inflammation, and Antifibrosis. Journal of agricultural and food chemistry\| PubMed ID: 28561587	2017
Hypocretin in median raphe nucleus modulates footshock stimuli-induced REM sleep alteration. Scientific reports\| PubMed ID: 31160650	2019
Activation of cannabinoid receptor type 1 impairs spatial and temporal aspects of episodic-like memories in rats. Journal of integrative neuroscience\| PubMed ID: 32259882	2020
Activation of cannabinoid type 1 receptors decreases the synchronization of local field potential oscillations in the hippocampus and entorhinal cortex and prolongs the interresponse time du... The European journal of neuroscience\| PubMed ID: 32510690	2020
Deep Brain Stimulation Increases Seizure Threshold by Altering REM Sleep and Delta Powers During NREM Sleep. Frontiers in neurology\| PubMed ID: 32903424	2020
Connectivity between nidopallium caudolateral and visual pathways in color perception of zebra finches. Scientific reports\| PubMed ID: 33168854	2020

Yi-Tse Hsiao

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