Name: an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation
Uploaded: 2018-06-02T14:45:00
Description: Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

Supported by the National Natural Science Foundation of China Grants 31522029, 31770929 and 31371149 (to Haitao Wu), Program 973 (2014CB542203) from the State Key Development Program for Basic Research of China (to Haitao Wu), and Grant Z161100000216154 from the Beijing Municipal Science and Technology Commission (to Haitao Wu). The authors thank all the members of the Haitao Wu laboratory for their encouragement and discussions. The authors are extremely grateful to Zhenwei Liu for his help with debugging the apparatus.

Animal experimental procedures followed the institutional guidelines of the Beijing Institute of Basic Medical Sciences (Beijing, China) and the Chinese governmental regulations for the Care and Use of Laboratory Animals. The mice (adult male, 26 ~ 30 g) were housed and kept at a constant temperature of 23 &#176;C, with water and food ad libitum, under a 12-h light/12-h dark cycle (lights on at 7:00 a.m.). All experimental procedures were performed during the light cycle.
1. Surgical Pr...

<ol>
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The HF-DBS technique has been widely used as a powerful tool for the treatment of many neurological disorders since the 1990s. So far, the landmark work of HF-DBS is for the treatment of Parkinson&#39;s disease and essential tremor, which has attracted much attention and interest both in the clinic and scientific community. There are various types of ongoing HF-DBS studies by many groups for HF-DBS&#39;s therapeutic application in certain neurological and psychiatric disorders32,<sup cl...

HF-DBS is a neurosurgical technology for electrical stimulation in the brain, which has been developed since the 1870s1. In the late 1980s, HFS was first used as a potential therapeutic intervention for Parkinson&#39;s disease and other movement disorders2. In the past few decades, HF-DBS has been more and more widely used in the treatment of brain disorders which are currently untreatable by a traditional therapeutic strategy. Particularly, due to the accuracy improvement of the HFS electrode, the highly effective outcomes, and minimal side effects, the number of brain disorders treated by HF-DBS has significantly increased over the past decades3,4,5. For example, HF-DBS has been approved by the US Food and Drug Administration (FDA) for the treatment of Parkinson&#39;s disease (PD), Alzheimer&#39;s type dementia, essential tremor, and other types of movement disorders2,6,7. In PD patients, the dopaminergic medication is reduced up to 50% during HF-DBS8. In addition to the successful treatment of movement disorders, HF-DBS has also demonstrated its powerful effects in the treatment of psychiatric diseases in the clinic, and for cognitive augmentation as well2,9,10,11. It should be noted that the research of HFS for the treatment of other psychiatric disorders are in various stages, offering much promise to patients12.
Although many studies have demonstrated that a focal HFS has both local and remote effects throughout the brain13, the neurological and molecular mechanisms of the effects remain elusive2,14. In the clinic, therapeutic HF-DBS is usually applied in a long-term manner for the treatment of Parkinson&#39;s disease and chronic pain, etc. Many opinions are raised to explain the improvement generated by an HF-DBS treatment, among which one possibility that the HFS current modulates the neuronal network activity, probably by a repetitive depolarization of the axons in the vicinity of the implanted HFS electrode. Or, HF-DBS may change the discharge rate of the output neurons and the projected targets. Also, HF-DBS may lead to long-term synaptic changes, including long-term potentiation (LTP) and long-term depression (LTD), which may contribute to a symptomatic improvement. So far, it is still unclear whether HFS in&#64258;uences the key molecular events that regulate cellular processes such as adult neurogenesis in vivo. Several lines of studies have demonstrated that HFS in rodents could mimic similar neural responses of clinically applied DBS15,16. To understand the underlying cellular mechanisms of HF-DBS, in this study, we first set up an in vivo HFS methodology in mice in an acute (one day) or chronic (five days) manner. Secondly, we set up an activation analysis methodology to determine the alteration of the neuronal activity and neurogenesis after an HF-DBS delivery.
Given that the neuronal production from neural stem cells is abundant during the embryonic development but continues throughout adult life, the hippocampal subgranular zone is one of the major areas where the neurogenesis occurs. The process of neurogenesis is influenced by many physiological and pathological factors. In certain epileptic cases, the hippocampal neurogenesis is dramatically decreased17,18. In addition, a single electroconvulsive therapy could significantly increase the neuronal production in the dentate gyrus19. These observations suggest that the electrophysiological activity plays a critical role in the regulation of adult neurogenesis and synaptic plasticity in hippocampal neurons. Therefore, to further demonstrate the effects of HF-DBS on neuronal activity and neurogenesis, we first carry out an immunostaining assay of the immediate early gene (IEG) c-fos which is a well-known marker of short-term neuronal activity resulting from experience20. Notch1 signaling is also detected to monitor the signaling activation after the HFS delivery21,22. Moreover, we also detect the neuronal production by a BrdU labeling analysis after the HF-DBS induction in various manners, though BrdU staining can also be a marker for gliogenesis.
In the present study, two HFS methodologies are adapted to target the activation of the hippocampal DG directly and indirectly. The electrode is implanted into the DG directly or implanted into the medial perforant path (PP) which sends projections to activate the DG neurons. For the HF-DBS induction, a programmable stimulator is presented for a continuous stimulation via the fixed electrode onto the mouse head. To determine the effects of HFS on neuronal activation and neurogenesis, we detect the expression of c-fos and Notch1 by immunofluorescent staining and the number of BrdU-incorporated positive neurons in the hippocampal DG region, respectively, after the HFS treatment. Particularly, the effects of the HF-DBS on the neurogenesis in the DG are compared between an acute and a chronic stimulation manner, or between a direct and an indirect stimulation manner, respectively.

<table border="0" cellpadding="0" cellspacing="0" style="width:975px;" width="975">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">Brain stereotaxic instrument</td>
			<td style="width:215px;">Stoelting</td>
			<td style="width:199px;">51730D</td>
			<td style="width:307px;">Stereotactic intracranial implantation for mouse</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Stimulator</td>
			<td style="width:215px;">A-M systems</td>
			<td style="width:199px;">Model 3800</td>
			<td>MultiStim 8-Channel programmable stimulator</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Dental driller</td>
			<td style="width:215px;">Saeshin Precision Co., Ltd</td>
			<td style="width:199px;">STRONG 90</td>
			<td>For drilling and crainiotomy&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Burr</td>
			<td style="width:215px;">Meisinger</td>
			<td style="width:199px;">HM1 005#</td>
			<td>For drilling and crainiotomy&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Digidata 1550 Digitizer</td>
			<td style="width:215px;">Molecular Devices</td>
			<td style="width:199px;">AXON 1550</td>
			<td>High-resolution data acquisition</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Cryotome</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">Thermo Cryotome FSE</td>
			<td>Cutting frozen sections of specimens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Confocal microscope</td>
			<td style="width:215px;">Olympus</td>
			<td style="width:199px;">FV-1200</td>
			<td>Japan, with 20x Objective (NA 0.45)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">Mouse surgery tools</td>
			<td style="width:215px;">F.S.T.</td>
			<td style="width:199px;">14084-08,11254-20,16109-14</td>
			<td style="width:307px;">Scissors, forceps, bone cutter, holders etc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pentobarbital sodium</td>
			<td style="width:215px;">R&#38;D systems</td>
			<td style="width:199px;">4579</td>
			<td>20-50mg/kg for i.p. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin G&#160;</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">P3032</td>
			<td>75,000 U for i.m. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Carprofen</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">SML1713</td>
			<td>5-10mg/kg, for s.c. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">4% Paraformaldehyde (PFA)</td>
			<td style="width:215px;">Beijing Solarbio Sci-Tech Co.&#160;</td>
			<td style="width:199px;">P1110</td>
			<td>stocking solution for tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate buffer (PBS)</td>
			<td style="width:215px;">Invitrogen</td>
			<td style="width:199px;">10010023</td>
			<td>pH7.4, 500ml in stocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:215px;">Sakura</td>
			<td style="width:199px;">4583</td>
			<td>Formulation of water-soluble glycols and resins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-BrdU antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab6326</td>
			<td>Dilutions:1/800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-c-fos antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab209794</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:255px;">Goat Anti-Rabbit IgG (Alexa Fluor 568)</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">A11036</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Donkey Anti-Rat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Jackson ImmunoResearch</td>
			<td style="width:199px;">712-546-150</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Antifade mounting medium with DAPI</td>
			<td style="width:215px;">Vector Laboratories</td>
			<td style="width:199px;">H-1200</td>
			<td>Counterstaining with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-Notch1 antibody (C-20)</td>
			<td style="width:215px;">Santa Cruz Biotech</td>
			<td style="width:199px;">sc-6014</td>
			<td>Dilutions:1/50</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Donkey Anti-Goat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab150073</td>
			<td>Dilutions:1/1000</td>
		</tr>
	</tbody>
</table>

an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

Following the HF-DBS stimulation to the hippocampal DG subregion directly or the PP subregion to activate the DG indirectly via inserted electrodes using the stereotactic adjustments, the rodents were anesthetized with pentobarbital and sampled 3 h after the last HF-DBS stimulation for the c-fos and Notch1 immunostaining. For the BrdU staining, 36 h after the last BrdU injection after 1 day or 5 days of HF-DBS stimulation, the rodents were anesthetized with pentobarbital...

Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

The overall goal of this experiment is to observe the effect of high-frequency stimulation on neuronal activity in neurogenesis. This method can help answer key questions in an aligned cellular and molecular mechanisms of high-frequency deep brain stimulation, such as the induction of neuron activity and the neurogenesis in hippocampus. The advantage of this technique is that it demonstrates the powerful effect of high-frequency TBS on neuron activity and the neurogenesis in the adult brain.Though this method can provide insight into the effects of high-frequency TBS on the mouse dentate gyrus, it can also be applied to other systems such as the basal ganglia and the subthalamic regions for the brain disorders in the clinic. To begin this procedure, place an anesthetized mouse onto the stereotactic frame. Align the ear bands with its head, and gently tighten them to support the centered head.Grasp the skin anterior to its ears and remove about one square centimeter of it over the skull. Then remove the muscle and other attachment overlying the skull with a cotton swab to expose the skull surface. Next, apply hydrogen peroxide solution to dry the skull in order to identify the sagittal and lambda suture lines.Then, identify the bregma and lambda. The bregma point will be used as the original point to set the nucleus position. After that, level the skull on the horizontal plane using these two points.Make sure that the heights of these two points are almost the same in the dorsal ventral plane. In addition, make sure that the skull is leveled in the medial lateral plane. Next, mount a homemade two channel copper micro wire electrode into the rotating electrode holder on the stereotactic surgical frame.Place the electrode directly above the bregma and set it as original site. Then, move the electrode gently to the correct position above the skull, without touching it. Use the digital display on the stereotactic surgical frame to indicate the anterior posterior, medial lateral, and dorsoventral positions.When the desired site is identified for the electrode insertion mark the position with a black marker. Afterward, use the dorsoventral stereotactic adjustments to raise the electrode holder. Then, sterilize the drill bit with 70%ethanol.Use a handheld micromanipulator assisted drill, make a burr hole precisely at the marked position. Use a sterile cotton swab to stop any bleeding during the operation. Keep drilling until dura is exposed.Use a bent needle to remove any bone scraps that are generated during the drilling, and make a pinhole on the dura without damaging the underlying soft brain tissue. To confirm that the burr hole is made properly at the desired position, lower the electrode to make sure that it inserts through the burr hole smoothly without touching any obstacle. If no resistance is detected, slowly insert the electrode to the target depth from the surface of the skull.Then, apply dental cement using a small spatula to hold the electrode in place. As the cement thickens, mold it around the inserted electrode and other bone screws to form a nice, smooth cap. Next, disengage the electrode from the electrode holder.Remove the mouse from the stereotactic frame, and return it to a warm cage. Let the mouse move freely and allow it to recover for two days in the cage. To deliver stimulation, connect the implanted electrode to a programmable stimulator via leads.Set up the software interface. Then, set the HFS parameters as six series of six trains of six pulses at 400 Hertz, and the stimulation intensity of 200 milliamps. Deliver HFS for the desired period of time as set up.For the control group, implant electrodes in the mice without delivering any stimulation. For CFAS staining, stimulate the experimental mice two times for one day. For BrdU labeling, apply appropriate stimulations to both the acute stimulation group and the chronic stimulation group.During the course of the stimulation, make sure the mouse is awake. Remember to insure the leads are not twisted. When the stimulation is done, carefully disconnect the electrode pin from the electrode holder in the connector of the recording system.For the CFAS and notch one staining, about three hours after the last HFDBS, anesthetize the mice by injecting pentobarbital to achieve a surgical plane of anesthesia. For BrdU labeling, about 12 hours after the last HFDBS stimulation, give six injects of BrdU to the control and the stimulated mice at two hour intervals. 36 hours after the last injection, deeply anesthetize the mice with pentobarbital and transcardially profuse it with 50 milliliters of cold PBS followed by 50 milliliters of cold 4%paraformaldehyde.Remove its head and make an incision on the skin to expose the skill. Using forceps, chip off the skull slightly and carefully remove the whole brain. Then, post fix the brain is 4%paraformaldehyde for an additional two days, and transfer it to 30%sucrose solution at four degrees Celsius overnight.Using a cryostat, slice the brain into 20 micron coronal sections. Transfer and mount the sections to the glass slide with a brush, and store them a minus 20 degrees Celsius. Afterward, transfer the slides into normal PBS containing no fixative.Wash the sections with PBS three to four times for five minutes each to remove any excess fixative. For the BrdU labeled group, incubate the sections in two molar HCL for 30 minutes at 37 degree Celsius, and rinse them in a 0.1 molar borate buffer. After 10 minutes, wash the sections two times in PBS for five minutes each time.Subsequently, incubate the sections in blocking solution for one hour at room temperature. Then, incubate the sections with anti-CFAS polyclonal antibody, anti-notch one antibody, or anti-BrdU antibody in a blocking solution at four degrees Celsius overnight. Was the sections three times in PBS for 10 minutes each time, then incubate them with Alexa Fluor 568 conjugated anti-rabid, Alexa Fluor 488 conjugated anti-goat, or Alexa Fluor 488 conjugated anti-rat secondary antibody for one hour at room temperature.Was the sections three times in PBS for 10 minutes each time. Subsequently, cover the immuno labeled brain sections using an antifade reagent mounting medium containing DAPI. Let the sections air dry, and store them protected from light at four degrees Celsius.Shown here is an experimental paradigm for the HFS treatment in the generation of BrdU labeled proliferating neurons in the DG.The animals were divided into an acute and a chronic group to receive different patterns of stimulations. Afterward, the animals in both groups were injected with BrdU on day six. On day eight, the animals were anesthetized and profused for the preparation of cryostat sections.Here are the images of the BrdU immunofluorescent staining post-HFDBS in the DG.A quantitative analysis of the neuronal production indicated that the acute stimulation in the DG could not enhance neurogenesis. While the chronic stimulation in the DG significantly up regulated the neurogenesis. Compared to the control group, the number of BrdU labeling cells increased significantly in the chronic group, both in the ipsilateral and contralateral hemisphere.These are the images of the BrdU immunofluorescent staining post-HFDBS in the PP sub-region. A quantitative analysis of the neuronal production indicated that either the acute or chronic stimulation in the PP could not enhance the neurogenesis significantly. The number of BrdU labeling cells was relatively stable with or without the HFS treatment.While attempting this procedure, it's important to remember to locate the electrode properly, and to deliver the stimulation successfully. After it's development, this technique paved the way for researchers in the field neuroscience high-frequency stimulation enrolled in the models. After watching this video, you should have a good understanding of how to deliver high-frequency stimulation to deep brain nucleus.

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Research

JoVE Journal

Neuroscience

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Generation of Local CA1 &amp;#947; Oscillations by Tetanic Stimulation

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...