Name: fabrication of fine electrodes on the tip of hypodermic needle using photoresist spray coating and flexible photomask for biomedical applications
Uploaded: 2017-11-28T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications at JoVE.com

This work was supported by the &#34;Biomedical Integrated Technology Research&#34; project through a grant provided by GIST in 2017.

1. Electrical Insulation of Hypodermic Needle
NOTE: A transparent heat shrink tube (HST) is employed for the electrical insulation of a hypodermic needle that is 720 &#181;m in diameter and 32 mm in length. The HST is made of polyethylene terephthalate (PET), which shows good chemical resistance to most acids and bases, excellent mechanical durability, and biocompatibility. The initial inner diameter and wall thickness of the HST are 840 &#181;m and 25 &#181;m, respectively. The diameter of the ...

<ol>
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We demonstrated that photolithography using spray coating and a film photomask is a feasible method to fabricate fine IDEs on the curved surface of a hypodermic needle with a small diameter of less than 1 mm. Both the width and the gap of the IDEs are as low as 20 &#181;m, and the fabrication margin from the tip is as small as 680 &#181;m. Within the protocol, the alignment process, including wedge error removal, is a critical step. The production yield was over 90% when the EoN was manufactured individually through a ri...

Hypodermic needles are widely utilized in hospitals for biopsies and drug delivery because they are inexpensive and easy to use. They also have excellent mechanical properties despite their thin diameter and a sharp-edged structure suitable for invasion. During a biopsy, the target tissues are sampled in the hollow of the hypodermic needle with ultrasonography guidance1. Although ultrasonography is free of radiation, safe for fetuses and pregnant women, and provides real-time imaging, it is difficult to see organs that are deep within the body, especially in the case of obese patients because ultrasonic waves cannot penetrate air or fat tissues2. In addition, a surgeon cannot acquire depth information from the two-dimensional ultrasonography that is conventionally utilized in the majority of hospitals, resulting in the need for multiple biopsies if physicians lack skill or experience. In drug delivery for spinal anesthesia, physicians determine that the needle has reached the spinal space if the cerebrospinal fluid (CSF) flows backward into the syringe while carefully inserting the needle into the patient&#39;s back. After confirming the reflux of CSF, the anesthesia drug is injected into the spinal space3. However, physicians risk penetrating or cutting off nerve fibers in the spinal space, causing severe pain to patients and even paraplegia4,5. Thus, this procedure also requires a skillful physician. One solution to overcome and mitigate the aforementioned difficulties is to add a navigation function to the hypodermic needle so that objective information on the needle&#39;s position can be provided. This would help a physician readily perform a biopsy, drug delivery, and even a surgery without relying on their empirical judgment only.
In order to electrically localize the target tissues in the body, a hypodermic needle incorporating an electrical impedance spectroscopy (EIS) sensor has been introduced as EIS-on-a-needle (EoN)6. The EIS sensor is currently utilized in the field of biomedical engineering for applications such as DNA detection7,8,9, bacteria/virus detection10,11,12, and analysis on cells/tissues13,14,15,16,17,18,19,20,21,22. The EoN can discriminate between dissimilar materials in a frequency domain based on their electrical conductivity and permittivity. The discrimination capability of the EoN was verified for various concentration levels of phosphate buffered saline (PBS)23, porcine fat/muscle tissues6,23, and even human renal normal/cancer tissues24,25. This capability of the EoN is expected to considerably increase the biopsy accuracy by locating the target tissues based on the differences in electrical impedance between the target lesion tissues and the neighboring normal tissues. In a similar manner, investigating differences in the electrical impedance between the drug injection space (spinal or epidural space) and surrounding tissues can help physicians deliver an anesthesia drug at the exact target location. Furthermore, the EoN can be utilized to electrically stimulate the brain/muscle as well as to determine an optimal surgical margin during surgeries that involve the partial resection of a tumor, such as partial nephrectomy, to preserve as much normal tissue as possible.
One of the biggest challenges in the realization of the EoN is the fabrication of electrodes on the curved surface of a hypodermic needle having a small radius of curvature. Direct metal patterning using a conventional photolithography process has been regarded as unsuitable for the fabrication of micro-sized electrodes on a curved substrate with a diameter of several millimeters or less. So far, various methods, including conformal printing26, flexible dry film photoresist27, the microfluidic method28, nanoimprint lithography29, and substrate-rotating lithography30, have been introduced to fabricate metal/polymer patterns on a curved surface. However, there are still limitations due to the EoN requirements, such as the required substrate with a diameter of less than 1 mm, total electrode length of 20 mm or more, width and gap of electrodes ranging in tens of micrometers, and high volume production.
In the present study, direct metal patterning by employing photoresist spray coating and a flexible film photomask is proposed to realize micro-sized electrodes on the curved surface of a hypodermic needle. The diameter of the needle is as small as 720 &#181;m (22-gauge), which is widely used for biopsies and drug delivery in hospitals. The production yield of the proposed fabrication method is also evaluated to determine the feasibility of bulk production at an affordable price.

<table>
	<tbody>
		<tr>
			<td>Heat shrink tube</td>
			<td>VENTION MEDICAL, Inc.</td>
			<td>103-0655</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (22G)</td>
			<td>HWAJIN MEDICAL co. ltd</td>
			<td>-</td>
			<td>http://www.hwajinmedical.com</td>
		</tr>
		<tr>
			<td>Heat gun</td>
			<td>Weller</td>
			<td>WHA600</td>
			<td>http://www.weller-tools.com/en/Home.html</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>HWASHIN INSTRUMENT CO, LTD.</td>
			<td>POWERSONIC 620-</td>
			<td>http://www.hwashin.net</td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>AS ONE Corporation</td>
			<td>006560</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputtering</td>
			<td>A-Tech System. Ltd.</td>
			<td>ATS/SPT/0208F</td>
			<td>http://www.atechsystem.co.kr</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>1000412</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray coater</td>
			<td>LITHOTEK</td>
			<td>LSC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>AZ electronic materials</td>
			<td>GXR 601</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Developer (solution)</td>
			<td>AZ electronic materials</td>
			<td>MIF 300</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Aligner</td>
			<td>MIDAS SYSTEM CO.,Ltd.</td>
			<td>MDA-400M</td>
			<td>http://www.midas-system.com</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON Corporation</td>
			<td>L200</td>
			<td>http://www.nikonmetrology.com</td>
		</tr>
		<tr>
			<td>Au wet etchant</td>
			<td>TRANSENE COMPANY, Inc.</td>
			<td>Au etchant type TFA</td>
			<td>http://transene.com</td>
		</tr>
		<tr>
			<td>Cr wet etchant</td>
			<td>KMG Electronic. Chemicals, Inc.</td>
			<td>CR-7</td>
			<td>http://kmgchemicals.com</td>
		</tr>
		<tr>
			<td>Au target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Cr target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Argon gas (99.999%)</td>
			<td>SINIL Gas Co.Ltd</td>
			<td>-</td>
			<td>http://www.sigas.kr</td>
		</tr>
		<tr>
			<td>Acetone solution</td>
			<td>OCI Company Ltd</td>
			<td>-</td>
			<td>http://www.ocicorp.co.kr/company/index.asp</td>
		</tr>
		<tr>
			<td>Impedance analyzer</td>
			<td>Gamry Instruments Inc</td>
			<td>Reference 600</td>
			<td>https://www.gamry.com</td>
		</tr>
		<tr>
			<td>Height Controller</td>
			<td>Mitutoyo Corporation</td>
			<td>192-613</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies Corporation</td>
			<td>10010023</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of fine electrodes on the tip of hypodermic needle using photoresist spray coating and flexible photomask for biomedical applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The interdigitated electrodes (IDEs), as shown in Figure 2, result in a larger effective sensing area on a limited surface compared to other shapes of electrodes. The overall length of the IDEs is designed to be 860 &#181;m to detect and analyze the impedance changes at less than 1 mm intervals in the biotissues, which will provide a high locating accuracy in biopsy and drug delivery procedures. The total width of the IDEs is 400 &#181;m, which is a geometric...

Watch this Scientific Journal Video about Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications at JoVE.com

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

The overall goal of this protocol is to fabricate fine interdigitated electrodes on the tip of a hypodermic needle with the diameter of less than one millimeter for biomedical applications. This method can achieve the dialects metal patterning on the rounded tip of the hypodermic needle by using spray coating of photoresist and photolithography process with a flexible film for a mask. The fabricated micro diode sensor can be widely used for biomedical applications, such as drug delivery, biopsy and even the surgery with high precision by cauterizing electrophotometer targetatious.To begin this procedure, cut a three centimeter length of heat shrink tube adjusting the length if needed based on the penetration depth of the hypodermic needle. Insert the hypodermic needle into the cut heat shrink tube. Using a heat gun, set to 150 degrees Celsius and shrink the tube.After this, separate the hypodermic needle from its hub. Clean the needle in a deionized water bath with ultrasonic agitation. Then, use a hot plate set to 105 degrees to dehydrate the insulated needle for 10 minutes.Next, apply double-sided tape to a clean glass slide. Arrange the clean insulated needle side by side on exposed side of the double-sided tape. Using sputtering equipment, deposit chromium and gold onto the needles as outlined in the text protocol.First, use double-sided tape to fix one of the gold sputtered needles onto a glass slide. Heat the chuck of the spray coater to 100 degrees Celsius. Place the slide on the heated chuck.Allow the needle to heat up for two to three minutes. Then, spray the photoresist onto the gold sputtered needle while still heating using the condition shown here. After this, leave the glass slide on the chuck to heat at 100 degrees Celsius for three minutes for soft baking.Using a microscope set to 100x magnification, inspect the needle to determine if the photoresist is uniformly coated. First, slowly lift a freely moveable sample holding plate until it fully contacts the fixed photomask holding plate. Using a pneumatic pump, fix the sample holding plate.Next, place the photoresist coated needle onto the sample holding plate. Align two of the boundary lines from the projected image with two parallel alignment lines from the photomask using the CCD display monitor to monitor the process in real time. Slowly lift the needle to bring the needle into contact with the fixed flexible photomask.Expose the needle to UV light for 30 seconds at an intensity of 15 millijoules per centimeter squared. Then carry out the developing process for three minutes. After this, use deionized water to rinse the developer out of the sample.Using a microscope set to 200 times magnification, determine if the photoresist is clearly patterned. If the exposed photoresist is not perfectly removed, repeat the developing process at 30 second intervals. Using a pair of tweezers, detach the sample from the glass slide.Immerse in gold wet etchant for one minute. Then, use deionized water to rinse the etchant out. Using a microscope set to 200x magnification, inspect the result.If any gold to be removed still remains, repeat the etching process at 10 second intervals. Next, immerse the needle in chromium etchant for 30 seconds. Use deionized water to rinse the etchant out when done.Immerse the etched needle into an acetone solution for one minute. After this, rinse with deionized water. Transfer the sample to a hot plate heated to 105 degrees Celsius to dehydrate for 10 minutes.Then, cut the shrink tube so that it is two to three millimeters longer than the electrode. Using a heat gun, raise the temperature of the heat shrink tube to 150 degrees to tightly passivate the needle. In this study, interdigitated electrodes are fabricated on the tips of hypodermic needles using photoresist spray coating and a flexible photomask.The electrodes are successfully fabricated with a diameter of 820 micrometers while the fabrication margin from the needle's tip is as small as 680 micrometers. The photomask's width and gap, which are 25 micrometers and 15 micrometers respectively, are designed to resolve UV dose imbalance which arise from imperfect contact between the outer portion of the photomask and the curved surface of the needle. These changes allow for both the width and gap of the electrodes to be successfully fabricated at 20 micrometers by optimizing UV exposure time.The discrimination capability of the electrical impedance spectroscopy on a needle is evaluated using various concentrations of PBS. Because the PBS is diluted serially with deionized water, the electrical conductivity decreases as the concentration of PBS decreases. Thus, the magnitude of impedance increases as the concentration level of PBS decreases.Based on this discrimination capability, depth profiling is performed at a frequency of one megahertz using four layered porcine tissue. According to the penetration depth of the EoN, the magnitude of the impedance and fat tissue, is clearly discriminated from that of muscle tissue. After watching this video, you should understand how to fabricate microelectrodes on a rounded surfaced tray to read a diameter of less than one millimeter.Currently, we are better find the clinical applicability of the needles sensors for derogatory for the spinal anesthesia, thyroid biopsy, and even surgeries such as laparoscopic surgery and passion apronectomy.

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Research

JoVE Journal

Neuroscience

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Automated Assessment of Fine Motor Performance in Mice Using the Erasmus Ladder

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for sustaining life would be possible without the ability to transition from one position to another. Unfortunately, motor skills can be compromised in a wide array of diseases. Therefore, investigating the mechanisms of motor functions at the cellular, molecular, and circuit levels, as well as understanding the symptoms, causes, and progression of motor disorders, is crucial for developing effective treatments. Mouse models are frequently employed for this purpose.
This article describes a protocol that allows the monitoring of various aspects of motor performance and learning in mice using an automated tool called the Erasmus Ladder. The assay involves two phases: an initial phase where mice are trained to navigate a horizontal ladder built of irregular rungs (&#34;fine motor learning&#34;), and a second phase where an obstacle is presented in the path of the moving animal. The perturbation can be unexpected (&#34;challenged motor learning&#34;) or preceded by an auditory tone (&#34;associative motor learning&#34;). The task is easy to conduct and is fully supported by automated software.
This report shows how different readouts from the test, when analyzed with sensitive statistical methods, allow fine monitoring of mouse motor skills using a small cohort of mice. We propose that the method will be highly sensitive to evaluate motor adaptations driven by environmental modifications as well as early-stage subtle motor deficits in mutant mice with compromised motor functions.

Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees.

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for ...