We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).
Prior to beginning this protocol the stomach of the lobster must be removed from the animal and prepared as described in the companion JoVE article (Bierman & Tobin, 2009). The majority of the steps outlined below are to be performed under a dissection microscope.
Part I- Isolating the Commissural Ganglia
Part II- Isolating the Stomatogastric Ganglion (STG) and remaining nerves.
Abbreviations: | |
STNS | Stomatogastric nervous system |
STG | Stomatogastric ganglion |
CoG | Commissural ganglion |
stn | stomatogastric nerve |
mvn | medial ventricular nerve |
ion | inferior oesophageal nerve |
son | superior oesophageal nerve |
agn | anterior gastric nerve |
dvn | dorsal ventricular nerve |
lvn | lateral ventricular nerve |
psn | posterior stomach nerve |
vpln | ventral posterior lateral nerve |
pyn | pyloric nerve |
pdn | pyloric dilator nerve |
The anatomy of lobster stomach and STNS has been well described previously5,6, and variations of this dissection procedure have been utilized in numerous studies. We recommend becoming familiar with the anatomy of the stomach5-7and STNS5,8 prior to beginning dissections. Such familiarity will decrease the risk of accidental damage to nerves and tissue.
To maintain the health of the preparation, it is critical to maintain the preparation at a cool temperature. This is easily achieved by exchanging the saline every 30 mins with 4˚C saline kept in a refrigerator. When immersed in cold saline, the isolated STNS preparation is viable for many hours. Recordings in related Homarus gammarus have demonstrated stable rhythmic activity from the STG throughout 5 days in vitro9; similar robustness has been seen in H. americanus (pers. obs.).
Modulatory input to the STG from the commissural ganglia is required to preserve the ongoing motor patterns. Therefore, to maintain a normal rhythmic activity these ganglia and the ion, son, and stn nerves must be kept intact. The dissection we describe preserves several motor nerves (containing motor neuron axons): the agns (containing Gastric Mill (GM) axons), the mvns (containing Lateral Gastric (LG) and Inferior Cardiac (IC) axons), the pyns (containing Pyloric neuron (PY) axons), the pdns (containing Pyloric Dilator (PD) axons), and the lvns (containing Lateral Pyloric (LP), Medial Gastric (MG), LPG axons as well as those from GM, PD and PY). By dissecting additional (or different) nerves, other motor neuron axons may be recorded. Each individual type of motor neuron axon may be recorded individually by dissecting and recording from the nerve innervating the muscle to which that type of neuron projects.
This preparation may be used for a variety of studies focused on nervous system function. The activity of the neurons may be recorded either using intracellular, sharp electrode recordings from somata or axons, or by recording electrical impulses extracellularly from the nerves. Many ion channels in the STG neurons have been partially cloned and mRNA can be measured from manually pulled somata10,11.
We would like to acknowledge our advisor, Dr. Eve Marder. This research was supported by National Institute of Health grants NS17813 to Eve Marder and NS059255 to A-E T.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Fine Tungsten Wire (0.001 in. diameter) | Tool | California Fine Wire | Cut into 1-2mm pieces to use as fine pins. | |
Forceps #5 | Tool | FST | 91150-20 | |
Minuten Pins | Tool | FST | 26002-10 | |
Moria Spring Scissors (7mm blades) | Tool | FST | 15370-52 | These are the top of the line. There are more economical spring scissors available from the same company. |
Petri Dish (100x15mm) | Tool | Fischer | 08-757-12 | Fill 2-4mm with Sylgard 182. |
Sylgard 182 | 其他 | Dow Corning | 182 | |
Pin Holder | Tool | FST | 26018-17 | Optional. Desheathing can be done with scissors and fine forceps alone. |
Super fine forceps #5SF | Tool | FST | 11252-00 | |
Tungsten Needles | Tool | FST | 10130-05 | Optional. Desheathing can be done with scissors or with pins cut from fine tungsten wire. |
Dissection microscope |