The work was supported by NIH 1 R21NS057718-01.

1. This is a non-survival surgery. The first step is implantation of the electrodes. In this example, electrodes will be implanted in the forepaw regions of primary somatosensory cortex of both hemispheres.
<ol>
	<li>Anesthetize the rat (male SD rat, 200-300 g) with 2% isoflurane and fix into place on a stereotactic surgical system. Before beginning surgery, ensure that the animal is well anesthetized and exhibits no response to a toe pinch. Remove the fur before opening the scalp. Separate the muscle and other tissue above the skull and block any bleeding on the bone surface using a cauterizer.</li>
	<li>Prepare a pier on the skull surface (near the midline anterior V-shaped junction) as a fixation point for the shaft of the implanted electrode, using dental cement. Setting a small nylon screw set into the bone before applying dental cement may increase stability. The size of the pier shaped with cement should be approximately 5 mm high and 3 x 5 mm2 in area at the base (see Figure 1).</li>
	<li>Using a fine tipped electrical drill, carefully open the skull and expose the dura over the forepaw representation in primary somatosensory cortex of each hemisphere. The diameter of each hole should be around 1 mm, positioned 1 mm anterior to and 4 mm lateral from bregma. Under a microscope, cut a tiny opening in the dura using a syringe needle tip, being careful to avoid any vessel damage.</li>
	<li>Before inserting each electrode, make sure that no bleeding or exudation is present near the incisions. The glass microelectrodes should be prepared before surgery with approximately 3~4 cm shaft-length and impedance of 1~5 M&omega;. Fill the capillary of the electrode with artificial CSF (ACSF) and insert each electrode obliquely (~45&deg;, from posterior to anterior) into the brain ~0.4 mm from the opened dura using the stereotactic arm. Before fixing into place, check the electrical signal. One end of a chloridized silver wire should be dipped into the ACSF and the other end connected to the input leads to the amplifier. A silver wire, attached subcutaneously at the back of the opened skin, serves as reference electrode.</li>
	<li>Before electrode fixation, double check the surgical area and make sure that no bleeding or exudation occurs, and then apply toothpaste to replace the removed skin and muscle on the skull. The use of the toothpaste improves the MR image quality by reducing the susceptibility mismatch at the skull/air interface. Attach the electrode shaft to the prepared pier with dental cement (see Figure 1).</li>
	<li>After the dental cement cures, transfer the animal to the MRI cradle and fix in place. Monitor the rat s physiological condition for the remainder of the study, including body temperature, respiration rate, SPO2 and cardiac rate.</li>
	<li>Position a surface coil (transmit/receive) over the head, with the electrodes protruding from the center of the coil. An additional arch-shaped hard cover that sits atop the cradle serves as a support for fixation of the electrode leads in order to avoid motion caused by the animal breathing. The leads used for simultaneous imaging and recording extend to ~5 m (the amplifier is located just outside the magnet room) and are covered with conductive plastic which serves as a passive shield.</li>
	<li>Anesthesia can be switched from isoflurane to medetomidine to reduce suppression of neural activity if desired. Examine the electrical signal a final time before transferring the animal into the magnet. In our studies, the recording parameters were as follows: x 1000 amplified, 0.1Hz~5 K Hz bandpass-filtered, 60 Hz notch-filtered, 12 kHz sample rate for analog to digital conversion.</li>
</ol>
2. At this point, the animal is inserted into the MRI scanner for simultaneous imaging and recording. The animals are anesthetized throughout the imaging procedure.
<ol>
	<li>A 9.4 T small animal MRI system (Bruker, Germany) was used in our studies. Prior to recording, imaging parameters must be established. A three plane scout image is used to position the fMRI scans. To improve the homogeneity of the magnetic field, the volume of interest is shimmed using FASTMAP1. For fMRI studies, a coronal imaging slice was selected, which included bilateral forepaw primary somatosensory areas, in which the electrodes were implanted. The EPI imaging parameters were FOV, 1.92 x 1.92 cm2; matrix size, 64 x 64; in-plane resolution, 0.3 x 0.3 mm2; slice thickness, 2 mm; TR/TE, 500/15 ms.</li>
	<li>After the imaging setup is complete, simultaneous recording and fMRI can begin. Figure 2 shows a representative EPI image and raw recordings during imaging. The rapid switching of the gradients during image acquisition results in saturated recordings, which persist for only a small portion of each scan cycle (22/500 ms). After image acquisition, the electrical signal returns to baseline with a form of non-saturated oscillation (see Figure 3). Combined fMRI and recording may be conducted during the resting state (as demonstrated in this study) or during stimulation. For stimulus studies, the imaging parameters are the same as for the resting state study, with electrical stimulation of the forepaw supplied using 9 Hz, 1~4 mA current. The rat is euthanized after the final scan.</li>
</ol>
3. After simultaneous imaging and recording, the data must be pre-processed prior to final analysis.
<ol>
	<li>We begin with the removal of the gradient artifacts from the electrophysiological recordings (see Figure 3).
	<ol>
 	<li>The noise structure due to scanning can be extracted by averaging all ~500 ms (TR) sections, each of which corresponds to the interval between two consecutive fMRI images.</li>
 		<li>Subtract the average noise structure from the original recordings. This method corrects only the unsaturated recording segments.</li>
 		<li>Each saturated segment corresponding to gradient alternation during image acquisition is replaced by a line, which passes between the time point before and the time point after gradient-induced saturation.</li>
 </ol>
 </li>
	<li>The denoised recordings of local field potentials (LFPs) are then converted to power time courses, which will have the same temporal resolution as the fMRI time course. The average power within a 2-second bin is used for calculating the lowest frequencies (delta band, 1~4 Hz), with a 1 second bin for theta band (4~8 Hz) frequencies, and a bin of ~0.5 s between neighboring saturated signals for higher frequencies (&gt; 8 Hz, alpha to gamma bands). The sliding window for all frequency bands was moved in 0.5 s increments, matching the TR of the fMRI data.</li>
	<li>For image data, standard fMRI preprocessing is performed, including head motion correction, image smoothing with 0.5 mm FWHM, and linear drift removal.</li>
	<li>Cross correlation analysis is conducted between the LFP power time courses and the time course from each voxel of imaging data. Varying time lags allow the examination of time-dependent correlation (see Figure 4).</li>
</ol>
Representative Results:
As an example, this technique may be used to investigate the relationship between spontaneous neural activity and BOLD fluctuations. Figure 4 shows the correlation maps between LFP power and the BOLD signal at time lags between -2.5 and 9.5 s from one rat. The low frequency BOLD fluctuations (&lt; 0.1 Hz) from cortical areas near the electrode tip are correlated with LFP power changes (&lt; 0.1 Hz) at a delay of 2~6 s.
<img src="/files/ftp_upload/1901/1901fig1.jpg" alt="Figure 1" /> Figure 1. Schematic configuration of electrode implantation and imaging region with a surface coil.
<img src="/files/ftp_upload/1901/1901fig2.jpg" alt="Figure 2" /> Figure 2. A representative coronal EPI image, including the electrode tips, is shown in the left panel. The right panel shows raw electrophysiological recordings before and during imaging.
<img src="/files/ftp_upload/1901/1901fig3.jpg" alt="Figure 3" /> Figure 3. By zooming in on one scan cycle, it can be seen that the artifact (green) during imaging may be removed from original recordings (blue). The denoised time courses (red) were used for further analysis.
<img src="/files/ftp_upload/1901/1901fig4.jpg" alt="Figure 4" /> Figure 4. Coronal maps (from one typical rat) of correlation between the power of spontaneous delta band activity from one electrode and the resting-state BOLD signal at time lags from -2.5 to 9.5 s. Maximum correlation is observed in bilateral SI at approximately 4~5 s in the isoflurane-anesthetized rat. Color bar represents Pearson r.

<ol>
	<li>Gruetter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic,+localized+in+vivo+adjustment+of+all+first-+and+second-order+shim+coils.">Automatic, localized in vivo adjustment of all first- and second-order shim coils.</a> Magn Reson Med. 29 (6), 804-804 (1993).</li><li>Logothetis, N. K., Pauls, J., Augath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+investigation+of+the+basis+of+the+fMRI+signal.">Neurophysiological investigation of the basis of the fMRI signal.</a> Nature. 412 (6843), 150-150 (2001).</li><li>Nir, Y., Fisch, L., Mukamel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+neuronal+firing+rate,+gamma+LFP,+and+BOLD+fMRI+is+related+to+interneuronal+correlations.">Coupling between neuronal firing rate, gamma LFP, and BOLD fMRI is related to interneuronal correlations.</a> Curr Biol. 17 (15), 1275-1275 (2007).</li></ol>

Both electrophysiological recording and BOLD fMRI are separately well-developed techniques. However, simultaneously recording and imaging is challenging due to the mutual interference2 of the two modalities. Here we provide a possible solution for combined experiments in the rodent. The modified method of electrode implantation minimizes influence on the image quality, and the artifact removal for the electrical recordings is necessary to remove the noise induced by image acquisition. Simultaneous imaging and ...

simultaneous fmri and electrophysiology in the rodent brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

Watch this Scientific Journal Video about Simultaneous fMRI and Electrophysiology in the Rodent Brain at JoVE.com

Simultaneous fMRI and Electrophysiology in the Rodent Brain

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

simultaneous-fmri-and-electrophysiology-in-the-rodent-brain

Research

JoVE Journal

Neuroscience

13.3K Views.  Emory University. פיתחנו שיטת הדמיה בתהודה מגנטית תפקודית זמנית הקלטה אלקטרו במוח מכרסם, המספקת פלטפורמה חקירת הקשר בין הפעילות העצבית ואת רמת חמצון הדם תלוי אות (BOLD) MRI.

Video: Simultaneous fMRI and Electrophysiology in the Rodent Brain

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...