אנו מודים Jos Burgert על עזרה עם כמה מהדמויות. אנו מודים לאינגו Selbach (והקבוצה &quot;Edelkrebsprojekt NRW&quot;) על מאמציו לספק את המעבדה עם חיות ניסוי. אנו מודים לאנה ג שניידר להגהת גרסאות הראשונות של כתב היד. מחקר זה נתמך על ידי SM אמי Noether DFG מענק 206 / 3-1 ומענק הפעלה של אוניברסיטת קלן לסגל נשי.

<ol>
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The authors declare that they have no competing financial interests.

<html> האנטומיה של סרטנים וגרעיני הבטן שלהם כבר תיארו בעבר 5, 18, ​​19, 20, ומומלץ להכיר אותם לפני הנתיחה על מנת להימנע מחיתוך של עצבים חשובים. זה קריטי כדי לשמור על ההכנה בטמפרטורות מתחת 23 ° C כדי למנוע השפלה של חוט העצב הבודד. זו יכ?...

Swimmerets של הסרטנים לשרת פונקציה בשליטה על יציבה והכה בקצב שבו החיות לשחות קדימה, לאוורר המחילות או הנקבות שלהם לאוורר את ביציהן 5, 6. Swimmerets של סרטני האות, leniusculus Pacifastacus, מתרחש בזוגות משניים לחמישית קטע בטן, עם איבר אחד בכל צד של 7 הבטן. מערכת העצבים המרכזית מייצרת בלהג שלו הקצבי המנוע אשר מניע את תנועת swimmeret בחיה שלמה, כמו גם בהכנת חוט עצב בודדת. כאשר אין משוב תחושתי או יורדים קלט נוכחי דפוס מנוע הקצבי מיוצר נקרא תנועה פיקטיבית 1, 2. במערכת swimmeret דפוס מנוע זה אינו שונה בכל פרמטר מהפעילות של swimmerets נמדד בבעלי החיים ללא פגע. התנועה של כל swimmeret היא מונעת על ידי microcircuit שנמצא ובמוגבל לג אחדorresponding hemiganglion 1 -. 3 בכל microcircuit יש גרעין דפוס יצירה שמורכב מחמש interneurons spiking שאינו מזוהה. הם יכולים להיות מאופיינים מבחינה תפקודית כמונעים או של שבץ כוח (IPS) או מונעי של שבץ שבות (IRS) 8. שב&quot;ס אלה וinterneurons IRS אינם מתנדים אנדוגני, ולא הפעילות לסירוגין היא מונע על ידי עיכוב הדדי 9. בגלל interneurons אלה מעכבת את הנוירונים המוטוריים swimmeret ישירות, תנועת PS-RS לסירוגין נוצרה 10. תנועה עם זאת, לא רק דורשת הדור של פעילות, אלא גם תיאום של השבבים העצמאיים השונים. במערכת swimmeret תיאום כזה היא הוקם על ידי microcircuit התיאום אשר מבטיח כי איברים פעילים בזמנים נכונים. microcircuit זה נבנה על ידי שלושה נוירונים שזוהו בכל מגזר 11-15. פרוטוקול זה מספק עבור ההדואר הפעם הראשונה מדריך לנתיחה צעד-אחר-צעד לבודד את השרשרת של הגרעינים (T4 לA6, איור 1). אנו מראים כיצד להצמיד חוט עצב הבטן המבודד וdesheathe כל גנגליון. בהכנת מערכת עצבים מבודדת זה, תאי העצב אחראים לתנועת swimmeret מוכנים לשימוש בניסויי אלקטרו ומורפולוגי. בחלקו השני של פרוטוקול זה מדגים את התכונות העיקריות של הדפוס המוטורי swimmeret. זה כולל מדריך צעד-אחר-צעד לשיא extracellularly הפעילות של הנוירונים מוטוריים swimmeret. האקסונים של תאי עצב מוטורי RS להקרין באמצעות הסניף הקדמי של N1 עצב, בעוד האקסונים של תאי עצב מוטוריים PS להקרין באמצעות הסניף האחורי של אותו העצב (איור 1) 4. לכן הפעילות שלהם ניתן להקליט מענפים אלה עם אלקטרודות סיכת ההפרש. <img alt="איור 1" fo:content-width="2in" src="/files/ftp_upload/52109/52109fig1highres.jpg" width="200" /><br/&gt; איור 1: מערכת עצבים מבודדת מגנגליון חזה 4 (T4) לגנגליון בטן 6 (A6) ותרשים סכמטי שלו T4:. גנגליון חזה 4; T5: גנגליון חזה 5; A1, A2 ... A6 הגנגליון בטן 1, הגנגליון בטן 2 ... הגנגליון בטן 6; N1: עצב N1; N2: עצב N2; N3: עצב N3; PS: שבץ-כוח; RS: שבץ-שיבה. קיצורים כיוונית: = קדמי; P = אחורי. הליך זה לנתיחה וטכניקת אלקטרו הפגינה נוחים לסטודנטים לתואר ראשון ועשוי להשלים קורסים מעשיים סטודנט בפיזיולוגיה. השרשרת בודדת של הגרעינים נעשתה שימוש במספר הניסויים כדי ללמוד לתפקד במערכת עצבים, תיאום, או אפנון של שבבי swimmeret 6, כמו גם שליטה עצבית של התנהגות מסתגלת בתנועה 16, 17. מערכת swimmeret הסרטנים וכך מספקת כמות עצומה הוראה או לא מענייניםגשם הזדמנויות שכל להתחיל עם דיסקציה של חוט עצב הגחון של סרטנים והקלטה תאית של הדפוס המוטורי הפיקטיבי.

<table><tbody><tr><td>4-channel extracellular amplifier: MA 102&amp;nbsp;</td><td>Amplifier</td><td>Elektroniklabor, Zoologie, Universit&amp;auml;t zu K&amp;ouml;ln, Germany</td><td></td></tr><tr><td>air-table</td><td></td><td>Technical Manufacturing Corporation&lt;br/&gt; (TMC) a unit of AMETEK Ultra Precision Technologies, Peabody, MA, USA</td><td>63-534</td></tr><tr><td>Axon Digidata 1440A</td><td>Digitizer</td><td>Axon Instruments, Molecular Devices Design, Union City, CA</td><td>DD1440A</td></tr><tr><td>big bucket&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Clampex &amp;amp; Clampfit</td><td>pClamp 10, recording and analysis software</td><td>Molecular Devices Design, Union City, CA</td><td>pClamps 10 Standard</td></tr><tr><td>cold lamp source</td><td>with flexible light guide (fiber optic bundle)&amp;nbsp;</td><td>Euromex microscopes holland, Arnhem, BD</td><td>LE.5211 &amp;amp; LE.5235</td></tr><tr><td>computer and monitor</td><td>equipped with recording software</td><td></td><td></td></tr><tr><td>container and pipette for liquid waste&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>crayfish saline&amp;nbsp;</td><td>contains (in mM): 5.4 KCl, 2.6 MgCl2, 13.5 CaCl2, and 195 NaCl, buffered with 10mM Tris base and 4.7mM maleic acid; aerated for 3 hours. Adjust at pH of 7.4.&amp;nbsp;</td><td></td><td></td></tr><tr><td>dextran, Texas Red (3000MW, lysine fixable)</td><td>fluorescent dye, lysine fixable</td><td>Life Technologies GmbH, Darmstadt, Germany</td><td>D3328</td></tr><tr><td>dissection dish&amp;nbsp;</td><td>(l x w x h) 15x7x5 cm; linned with black silicone</td><td></td><td></td></tr><tr><td>faraday cage</td><td></td><td></td><td></td></tr><tr><td>fixing pins</td><td></td><td></td><td></td></tr><tr><td>forceps (biology, Dumont #5)</td><td>Forceps: Biology, tip 0.05 x 0.02 mm, length 11cm, INOX</td><td>Fine Science Tools (FST), Germany</td><td>11252-20</td></tr><tr><td>forceps (biology, Dumont #55)</td><td>Forceps: Biology, tip 0.05 x 0.02 mm, length 11cm, INOX</td><td>Fine Science Tools (FST), Germany</td><td>11255-20</td></tr><tr><td>forceps (electronic, Dumont #5)</td><td>Forceps: Standard, tip 0.1 x 0.06 mm, length 11cm, INOX</td><td>Fine Science Tools (FST), Germany</td><td>11251-20</td></tr><tr><td>intracellular electrode</td><td>Borosilicate glass capillaries (outer/inner diameter: 1mm/0.5mm), with filament</td><td>Sutter Instruments, Novato, CA</td><td>BF100-50-10</td></tr><tr><td>Leica S8 Apo StereoZoom</td><td>Dissection Microscope&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Zoom 1x - 8x</td><td>Leica, Germany</td><td>10446298</td></tr><tr><td>microscope table</td><td></td><td></td><td></td></tr><tr><td>mirror</td><td>to illuminate preparation from below</td><td></td><td></td></tr><tr><td>modeling clay</td><td></td><td></td><td></td></tr><tr><td>Olympus SZ61</td><td>Dissection Microscope&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Zoom 0.67x - 4.5x</td><td>Olympus, Germany</td><td></td></tr><tr><td>petri dish&amp;nbsp;</td><td>94 x 16 mm; lined with clear silicone</td><td>Greiner bio-one, Germany</td><td>633180</td></tr><tr><td>ring scissors</td><td>ThoughCut, cutting edge: sharp/blunt, straight: 13cm</td><td>Fine Science Tools (FST), Germany</td><td>14054-13</td></tr><tr><td>spring scissors or alternative: Vannas spring scissors</td><td>cutting edge: 8 mm, tip diameter: 0.2mm, straight: 10cm or cutting edge 2.5 mm, tip diameter 0.075 mm, straight: 8cm</td><td>Fine Science Tools (FST), Germany</td><td>15024-10 or&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 15000-08</td></tr><tr><td>student Vannas&amp;nbsp; spring scissors or alternative:&amp;nbsp; Moria Spring Scissors</td><td>cutting edge: 5mm, tip diameter: 0.35mm, straight: 9cm or cutting edge: 5mm, tip diameter 0,1 mm, straight: 8 cm</td><td>Fine Science Tools (FST), Germany</td><td>91500-09 or&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 15396-00</td></tr><tr><td>sylgard</td><td>184 Silicone Elastomer Base and Curing Agent; for black sylgard add activated carbon</td><td>Dow Corning, Midland, MI, USA</td><td></td></tr><tr><td>syringe filled with petroleum jelly and equipped with a 20 gauche needle with rounded tip</td><td></td><td></td><td></td></tr></tbody></table>

the swimmeret system of crayfish: a practical guide for the dissection of the nerve cord and extracellular recordings of the motor pattern

כאן אנו מדגימים את הנתיחה של חוט עצב בטן הסרטנים. ההכנה כוללת את הגרעינים שעבר שני חזה (T4, T5) ושרשרת של גרעיני בטן (A1 לA6). שרשרת זו של הגרעינים כוללת את החלק של מערכת העצבים המרכזית (CNS) שמניעה את התנועה מתואמת של pleopods (swimmerets): מערכת swimmeret. זה ידוע במשך למעלה מחמישה עשורים שבסרטנים כל swimmeret הוא מונע על ידי הליבה שלה עצמאי דפוס יצירה שיוצרת פעילות לסירוגין קצבי 1-3. הנוירונים המוטוריים innervating השרירים של כל swimmeret מהווים שני אנטומית ותפקודי אוכלוסיות נפרדות 4. אחת מהן הוא אחראי לביטול (שבץ הכח, PS) של swimmeret. האחרים מניע את ההימשכות (מכה החוזרת, RS) של swimmeret. הנוירונים מוטוריים של מערכת swimmeret מסוגלים לייצר באופן ספונטני דפוס מוטורי פיקטיווי, שהוא זהה לדפוס שנרשם בvivo </em&gt; 1. מטרת דו&quot;ח זה היא להציג את מערכת מודל מעניינת ונוחה ללימוד רשתות מניבת קצב ותיאום של שבבים עצמאיים לקורסי המעבדה המעשיים של התלמידים. הפרוטוקול סיפק כולל הוראות שלב-אחר-צעד לנתיחה של חוט עצב הבטן של הסרטנים, מצמיד של השרשרת בודדת של הגרעינים, גרעיני desheathing והקלטת הדפוס המוטורי הפיקטיבי swimmerets extracellularly ממערכת העצבים המבודדים. בנוסף, אנו יכולים לפקח על הפעילות של תאי עצב swimmeret נרשמו intracellularly מהדנדריטים. כאן אנו גם מתארים בקצרה את הטכניקות הללו ולספק כמה דוגמאות. יתר על כן, המורפולוגיה של תאי עצב swimmeret ניתן להעריך באמצעות טכניקות צביעה שונות. כאן אנו מספקים דוגמאות של תאית (על ידי iontophoresis) נוירונים צבע מלא וbackfills של בריכות של הנוירונים מוטוריים swimmeret. במעבדה שלנואנו משתמשים בתכשיר זה ללמוד פונקציות בסיסיות של תנועה פיקטיבית, את ההשפעה של משוב תחושתי בפעילות של מערכת העצבים המרכזית, ותיאום בין שבבים ברמה תאית.

עם ההקלטות תאי בו-זמנית מRS וPS, הנוירונים מוטוריים של גנגליון אחד, הפעילות לסירוגין של בריכות הנוירון מוטורי אלה, יכול להיות במעקב (איור 18), המייצגת את דפוס התנועה הפיקטיבי. <img alt="איור 18" src="/files/ftp_upload/52109/52109fig18highres.jpg" /> <stron...

Watch this Scientific Journal Video about The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern at JoVE.com

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

מערכת Swimmeret של סרטן הנהרות: מדריך מעשי לDissection של חוט העצב ותאי הקלטות של התבנית מוטורית

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

the-swimmeret-system-crayfish-practical-guide-for-dissection-nerve

Research

JoVE Journal

Neuroscience

12.0K Views.  University of Cologne. Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons. 

Video: The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

הקלטות פיזיולוגיים של NMJs פלט גבוה ונמוך על שרירים ברגליים סרטן הנהרות extensor

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

ממברנה פוטנציאלים, התגובות Synaptic, המעגלים הנוירונים, Neuromodulation ו היסטולוגיה שרירים שימוש סרטן הנהרות: מעבדה תרגילים סטודנטים

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception 

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

קולטן שרירים איברים בבטן סרטן הנהרות: מעבדה הסטודנטים תרגיל הפרופריוספציה

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

שיטות אלקטרו עבור הקלטה פוטנציאלים Synaptic מן NMJ של הזחלים תסיסנית

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biology

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

הביתור, הקלטה מ צומת C. elegans Neuromuscular

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

הקלטות של הפעלת מעגלים עצביים מתנהגת בחופשיות בעלי חיים

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracellularly זיהוי תאי עצב מוטורי לשריר בברכת מוטור<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

עצבית במעגל הקלטה ממערכת עצבים מקק שלמה

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

צפה היסטורי הפגנה לפיזיולוגיה NMJ של השריר פותחן סרטן הנהרות

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

רצפטורים פרופריוספציה והמתח בסרטן איברים: תרגילי מעבדה הסטודנטים

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...