This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.
Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preBötzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.
The respiratory neural network of the brainstem provides a fertile domain for understanding the general characteristics of rhythmic neural networks. In particular, the interest is in the development of neonatal rodent breathing and understanding how the breathing rhythm develops. This may be done using a multi-level approach, including in vivo whole animal plethysmography, in vitro en bloc nerve recordings, and in vitro slice recordings that contain the breathing rhythm generator. Reductionist in vitro en bloc and slice recordings are an advantageous method to use when interrogating the mechanisms behind respiratory rhythmogenesis and neural circuitry in the brainstem-spinal cord region of developing rodents. The developing respiratory system includes approximately 40 cell types, characterized by firing pattern, including those of the central respiratory1,2. The central respiratory network includes a group of rhythmically active neurons located in the rostral ventrolateral medulla1,3. Mammalian respiratory rhythmogenesis is generated from an autorhythmic interneuron network dubbed the preBötzinger complex (pBC), which has been localized experimentally via both slice and en bloc preparations of neonatal mammalian brainstem-spinal cords3,4,5,6,7,8. This region serves a similar function to the sinoatrial node (SA) in the heart and generates an inspiratory timing system to drive respiration. From the pBC, the inspiratory rhythm is carried to other regions of the brainstem (including the hypoglossal motor nucleus) and spinal motor pools (such as the phrenic motor neurons that drive the diaphragm)9.
Rhythmic activity may be obtained using brainstem spinal cord en bloc preparations or slices from a variety of cell populations, including C3-C5 nerve rootlets, XII nerve rootlets, hypoglossal motor nucleus (XII MN), hypoglossal premotor neurons (XII pMN), and the pBC3,10,11,12. While these methods of data collection have been successful across a handful of laboratories, many of the protocols are not presented in a way that is fully reproducible for new researchers entering the field. Obtaining viable and rhythmically active en bloc and slice preparations requires an acute attention to detail through all steps of the dissection and slice cutting protocol. Previous protocols extensively describe the various recording procedures and electrophysiology, yet lack detail in the most critical part of obtaining a viable tissue preparation: performing the brainstem-spinal cord dissection and slice procedure.
Efficiently obtaining a rhythmically-active and viable en bloc or slice preparation brainstem-spinal cord electrophysiology recordings requires that all steps be performed correctly, carefully, and swiftly (typically, the whole procedure related here can be performed in approximately 30 min). Critical points of the brainstem-spinal cord electrophysiology protocol that have not been previously well described include the dissection of nerve rootlets and the slicing procedure on the vibratome. This protocol is the first to stepwise visually communicate the brainstem-spinal cord dissection for both new researchers and experts in the field. This protocol also thoroughly explains surgical techniques, landmarks, and other procedures to assist future researchers in standardizing slices and en bloc preparations to contain the exact circuitry desired in each experiment. The procedures presented here can be used in both rat and mouse neonatal pups.
The following protocol has been accepted and approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University. NIH guidelines for the ethical treatment of animals are followed in all animal experiments performed in the laboratory. All ethical standards were upheld by individuals performing this protocol.
1. Solutions
2. Preparation of Dissection and Vibratome Rig
3. Dissection and Isolation of the Neuraxis
4. Slice Protocol
5. Recording Procedures
The method presented here allows a researcher interested in obtaining rhythmically active slices of brainstem to reproducibly and reliably cut a viable, robust slice that will allow recording of fictive motor output for many hours. All of the minimally necessary neural circuit elements for generating and transmitting inspiratory rhythm can be captured in a thin slice using this method. These elements include: the preBötzinger Complex, premotor neurons projecting to the hypoglossal motor neurons (pXII MNs) and the hypoglossal motor neurons (XII MNs), and hypoglossal nerve rootlets. The pBC, XIIn, and C4 nerve rootlets are commonly used for inspiratory rhythm recordings, as illustrated in Figure 7.
Successful use of this procedure will produce a viable and rhythmically active en bloc preparation in 10 – 15 min, or a rhythmically-active slice in <30 min. After isolation of the en bloc brainstem-spinal cord or a thin slice, 15 min of equilibration in the recording chamber is sufficient for the production of fictive motor output. In the case of the slice, increasing the extracellular [K+] to approximately 8 – 9 mM will produce robust neural drive that can last for 24 to 36 h in this laboratory's experience. The preparation should be continuously superfused with carbogenated (95% O2/5% CO2) and heated aCSF (~ 27 °C). Successful dissections are performed quickly and avoid pinching, stretching, or damaging nerve rootlets used for recordings. For optimal results, all steps in the procedure are performed swiftly and the tissue must be bathed or continuously perfused with carbogenated aCSF when performing the dissection and tissue isolation (as described above). For extensive details concerning the neuroanatomy and precise atlases of the brainstem, please see the work of Ruangkittisakul et al.13,14, Ballanyi15 and colleagues. The protocol presented here is one method that has proven reliable in the senior author's laboratory and this report provides a graphical, step-by-step method for generating these slices relying only upon surface landmarks visible on the brainstem or within the brainstem as detailed here.
Figure 1: Initial dissection for the brainstem-spinal cord extraction. (A) Anesthetized pup pinned for dissection. Dashed lines indicate incision guidelines for sagittal incision into skin and transverse cuts caudal to eyes and below the diaphragm. (B) Guide for removing skin and forelimbs from animal. (C) Dorsal aspect of skinned rodent. Dashed lines indicate incision guidelines for skull-flap "clamshell" exposure. Please click here to view a larger version of this figure.
Figure 2: Stages of the dorsal laminectomy. (A) Dashed lines indicate incision guidelines for removing the cerebrum. (B) Result after removing tissue. Dashed lines indicate incision guidelines for dorsal laminectomy. (C) Exposed brainstem-spinal cord following dorsal laminectomy. Please click here to view a larger version of this figure.
Figure 3: Stages of initial ventral dissection. (A) Ventral side of a skinned rodent with severed forebrain and abdomen. Dashed lines indicate incision guidelines for removing the xyphoid process and opening the rib cage to subsequently remove abdominal and thoracic cavity organs. (B) Abdominal and thoracic cavity organs removed. (C) Oropharyngeal structures removed. Dashed lines indicate incision guidelines for hard palate removal. Please click here to view a larger version of this figure.
Figure 4: Stages of the ventral laminectomy. (A) Hard palate and remaining skull parts removed. Incision guidelines indicate how to remove remaining tissue surrounding the brainstem-spinal cord. (B) First cervical vertebra (C1) exposed. (C) Demonstration on lifting C1 and making a transverse cut to the vertebral body to remove it. Nerves are carefully cut flush to the dorsal side of the vertebral body before removing it. This is the most critical step in the dissection. Please click here to view a larger version of this figure.
Figure 5: Preparation of brainstem-spinal cord for slicing. (A) C1-C3 removed from spinal cord. Incision guidelines indicate where to sever the brainstem-spinal cord from the caudal spinal cord. (B) Ventral side of brainstem-spinal cord with labeled nerve rootlets. Incision guidelines indicate recommended transection or remaining rostral structures at the ponto-medullary before recording from the brainstem-spinal cord region. (C) Paraffin slab setup for slicing on the vibratome. The slicing region includes tissue between cranial nerves IX and XII. Do not place micro-pins into the slicing region. Please click here to view a larger version of this figure.
Figure 6: Landmarks used for obtaining slices with an intact hypoglossal motor nucleus and PreBötzinger Complex. (A) Transverse view of brainstem-spinal cord after initial cut has been made rostral to the glossopharyngeal (IX) rootlets. Incision guideline indicates transection level just caudal to the hypoglossal (XII) rootlets. (B) Orientation of paraffin block to blade before cutting a slice containing the pBC and the hypoglossal motor nucleus. Creating a trapezoidal "wedge" shape cut maximizes the likelihood of capturing enough inspiratory neurons in the slice to obtain rhythmic activity during recording. The wedge will include the pBC, the hypoglossal premotor neurons, and the hypoglossal motor neurons, which collectively provide the necessary circuitry to transmit rhythmic drive, which is an index of respiratory rhythmogenesis. Please click here to view a larger version of this figure.
Figure 7: Representative recordings from en bloc or slice preparations. (A) Integrated trace from XII rootlet. (B) Integrated trace from the pBC. Recording from the pBC using an en bloc preparation will require a blind patch clamp. (C) Integrated trace from the C4 nerve rootlet. Please click here to view a larger version of this figure.
Adapting the protocol presented here into an en bloc or slice workflow is advantageous for laboratories and studies that would like to utilize either en bloc brainstem-spinal cord and/or thin slice preparations for electrophysiology recordings. The dissection and slice method presented, combined with methods previously reported by others17,18,19, will allow reproducible preparation of robust and viable tissue that is widely adaptable to a range of experiments using the rodent hindbrain or spinal cord. The dissection presented is highly detailed and includes dorsal and ventral laminectomy as well as extensive detail regarding orientation of the brainstem-spinal cord when preparing to cut slices. The detailed dissection and slice protocol presented allows the researcher to include all circuitry necessary for the generation and transmission of inspiratory rhythm. The main neural populations/structures that can be captured using this method include: the hypoglossal premotor neurons, the hypoglossal motor nucleus, and the preBötzinger complex. Note that regions of the brainstem that contain central CO2 sensors or expiratory rhythm-generating circuits (RTN/pFRG) are not included in the preparation detailed here20,21. Once the en bloc preparation or slice has been obtained, rhythmic activity may be obtained using suction electrodes, surface electrodes, or single-cell recording methods such as extracellular unit or patch-clamping methods3,10,11.
The goal of this protocol is to provide a clear, easy-to-follow method for generating either a brainstem-spinal cord preparation or a thin, rhythmically-active slice. This protocol should be valuable to both experienced and new researchers interested in incorporating rhythmic brainstem/slice preparations in their armamentarium, as there are several critical steps detailed within the procedures that facilitate capture of robust, reproducible, and long-lasting rhythmically active slices.
Once the researcher has become comfortable with identification of the anatomical landmarks and the skills necessary for dissection, the speed of the dissection will increase, and the procedures can be optimized for each individual investigator. The protocol detailed here was taught to the senior author (CGW) during his post-doctoral work with Jeffrey Smith, the originator of the rhythmic brainstem slice preparation3. To ensure rhythmic activity and viability in both the slices and en bloc preparations, all procedures to generate a slice should be finished within approximately 30 min from beginning of the procedure to a slice in the recording chamber. With continuous perfusion, there is very little impact on tissue viability but minimizing the time for dissection allows for longer recording and data acquisition. For respiratory studies, a slice that is as thin as approximately 280 µm thick3 will have rhythmic, robust inspiratory activity, but slices may range up to 550 µm17,18,19,22. Careful practice is required to develop the necessary skills to perform this procedure and minimize the variability in thickness and rostral-caudal position of each slice. Including or excluding certain cell populations will influence the quality and robustness of rhythmic activity obtained in later recording as both excitatory and inhibitory nuclei in the brainstem can impact the "quality" of the rhythm recorded from the slice3.
Finally, it is also critical that aCSF is prepared exactly according to protocol, and is at a standardized pH20, temperature, and oxygenation level. Anoxic preparations will not be rhythmically active and will affect data collection21.
There are several key steps in the procedure that facilitate an investigator capture of viable and rhythmically active preparations. During the ventral laminectomy, keeping the dorsal rib cage intact allows extra leverage and provides more tissue for securing the preparation while performing the relatively delicate isolation of the brainstem-spinal cord from the vertebral column. The dissection steps detailed here allow the investigator to easily produce en bloc preparations or thin slices so, by necessity, extra steps are detailed that can be omitted to obtain just a thin slice. Other useful tips include, while performing the ventral laminectomy, it is important to gain a firm grip on the vertebral body to lift it upwards while severing the nerve rootlets. Once the brainstem-spinal cord has been dissected and extracted, the dura mater and remaining vasculature must be dissected off of the surface of the neural tissue. Blood vessels and dura are tough and can prevent the vibratome blade from cutting a clean slice. Dissection of the dura is not vital for the en bloc preparation, but is recommended to optimize nerve-suction electrode coupling. Finally, careful and methodical practice in the use of the vibratome is necessary for cutting a clean, reproducible slice. This protocol provides a very comprehensive list of detailed steps for the dissection and cutting procedures. The emphasis here is to provide a step-by-step detailed list for an investigator to use as a reliable foundation and training tool from which they can modify their laboratory's procedures as needed.
As a whole, this methodology represents a thirty-year evolution of methods of in vitro electrophysiology. The scope of this paper focuses on the standardization of en bloc and slice brainstem-spinal cord recordings that are typically used for studying inspiratory activity in P0-P5 rats and mice22. However, this dissection process may be utilized across a range of animal sizes, ages, and species for a wide variety of studies, including E18 rats/mice, neonatal rats and mice (postnatal days 0 to 6), and adult mice. Future studies may use the methods presented to streamline protocols across species and ages, allowing studies to interrogate mechanisms in rhythm generation or other physiological process across both species and ages. Ultimately, changing the orientation, thickness, or location of the slice or en bloc preparation will influence rhythmic activity23. Extensive literature has been published in the past concerning the neural populations captured by slice procedures and there are stereotaxic rat and mouse atlases available to provide more detail concerning the neural circuitry discussed here24,25,26. The procedures presented here provide extensive detail with clear illustrations that allow a new investigator to learn to cut rhythmic slices from landmarks readily visible under a laboratory dissecting scope.
The authors have nothing to disclose.
S.B.P is a recipient of a Loma Linda University Summer Undergraduate Research Fellowship.
NaCl | Fisher Scientific | S271-500 | |
KCl | Sigma Aldrich | P5405-1KG | |
NaHCO3 | Fisher Scientific | BP328-1 | |
NaH2PO4 •H2O | Sigma Aldrch | S9638-25G | |
CaCl2•2H2O | Sigma Aldrich | C7902-500G | |
MgSO4•7H2O | Sigma Aldrich | M7774-500G | |
D-Glucose | Sigma Aldrich | G8270-1KG | |
Cold-Light source Halogen lamp 150W | AmScope | H2L50-AY | |
Dissection Microscope | Leica | M-60 | |
Vibratome 1000 Plus | Vibratome | W3 69-0353 | |
Magnetic Base | Kanetic | MB-B-DG6C | |
Isoflurane, USP | Patterson Veterinary | NDC 14043-704-06 | |
Sword Classic Double Edge Blades | Wilkinson | 97573 | |
Histoclear | Sigma-Aldrich | H2779 | |
Dumont #5 Fine Forceps | Fine Science Tools | 11254-20 | |
Dumont #5/45 Forcep | Fine Science Tools | 11251-35 | |
Scalpel Blades #10 | Fine Science Tools | 10010-00 | |
Scalpel Handel #3 | Fine Science Tools | 10003-12 | |
Spring Scissors Straight | Fine Science Tools | 15024-10 | |
Narrow Pattern Forcep Serrated/straight | Fine Science Tools | 11002-12 | |
Castroviejo Micro Dissecting Spring Scissors; Straight | Roboz | RS-5650 | |
Vannas Scissors 3" Curved | Roboz | RS-5621 | |
Insect pins, 0 | Fine Science Tools/8840604 | 26000-35 | |
Insect pins, 0, SS | Fine Science Tools | 26001-35 | |
Insect pins, 00 | Fine Science Tools | 26000-30 | |
Insect pins, 00, SS | Fine Science Tools | 26001-30 | |
Insect pins, 000 | Fine Science Tools | 26000-25 | |
Insect pins, 000, SS | Fine Science Tools | 26001-25 | |
Minutien pins, 0.10 mm | Fine Science Tools | 26002-10 | |
Minutien pins, 0.15 mm | Fine Science Tools | 26002-15 | |
Minutien pins, 0.2 mm | Fine Science Tools | 26002-20 | |
Fisher Tissue prep Parafin | fisher | T56-5 | |
Graphite | fisher | G67-500 | |
Delrin Plastic | Grainger | 3HMT2 | |
18 Gauge Hypodermic Needle | BD | 305195 |