Response Properties of a Photoreceptive Interneuron in the Ventral Nerve
Cord of Crayfish
Bruce R. Johnson1, Robert A. Wyttenbach2, and Ronald R. Hoy1
1Department
of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853.
& Behavioral Biology Program, Emory University, Atlanta, GA 30322
2Neuroscience
Introduction
The crayfish nervous system consists of a brain, subesophageal, thoracic, and
abdominal ganglia (Fig. 1; see accompanying pdf file). The caudal photoreceptors
(CPRs) are two bilaterally paired, non-visual photosensitive interneurons with cell
bodies in the 6th (last, A6) crayfish abdominal ganglion and an axon each projecting
rostrally to the crayfish brain (Fig. 2). These interneurons are intrinsically sensitive
to light and receive mechano-sensitive input from hairs on the crayfish tail. CPR
sensitivity to light and its potential role in crayfish behavior was first described by
Welsh (1934) and Prosser (1934). A large body of literature describes their
physiology, anatomy, and behavioral significance. See Galeano (1976), Wilkens
(1988) and Rodriguez-Sosa et al. (2012) for reviews summarizing this work.
Our goal today is to introduce you to the crayfish CPR preparation by guiding
you though the dissection of the crayfish abdominal nerve cord (ANC) and the
recording procedure to observe the CPR response to white light. From these
beginning steps a variety of student lab exercises and projects can be developed.
Methods
Dissection
Procedures for crayfish dissection and extracellular recording of nerve activity
are from the Crawdad Lab manual (Wyttenbach et al., 2014).
Video procedures for preparation of the crayfish isolated tail and the general
procedure for exposing the ANC are shown in the dissection videos of Crawdad Lab
3, especially Video 3.2. The dissection shown in Video 3.2 is extended to expose the
entire ANC from the anterior abdominal end to A6 (Fig 4). Before starting your
dissection, observe these videos and the methylene blue prep provided.
Recordings
The suction electrode tip can be placed on the midline between any of the
abdominal ganglia, where the CPR axons run close to the ventral ANC surface (Figs.
3 and 5). Suck up a small bubble of nerve cord into the recording tip. We start with
electrode placement between A2 and A3 but since the CPR axons extend from A6 to
the head, any midline recording position will do (Fig. 5).
Equipment and Supplies (From Wyttenbach et al., 2014)
 Computer with A/D (optional if oscilloscope present, but extremely desirable).
We use the LabChart software for data acquisition and analysis.
 Oscilloscope (optional if computer is present; storage optional but useful if no
computer).
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Dissecting microscope (6 to 50× is a good range).
Microscope light (fiber-optic with two arms is most flexible).
Faraday cage (metal or wood frame with metal window screen on sides, top, and
back; approximately 32w × 32h × 24d inches).
BNC cables (2 to 3 feet long, about six per rig).
BNC T-connectors (minimum three per rig).
Alligator clip cables (2 to 3 feet long, minimum four per rig).
Ground wire (chlorided silver wire or Ag-AgCl pellet).
AC amplifier capable of 1000× gain; 60 Hz notch filter desirable. We use an A-M
Systems extracellular amplifier.
Suction electrode (for construction details see Johnson et al., 2007).
Coarse micromanipulator for positioning the suction electrode.
Preparation dishes (Pyrex 90×50 mm, filled 20 mm deep with Sylgard).
Pins to hold the tail in the dish (regular common pins will do).
Normal crayfish saline (in mM: NaCl, 5.4; KCl 205; CaCl2·2H2O, 13.5; MgCl2·6H2O,
2.6; NaHCO3, 2.3; dextrose, 2.0). Four liters is sufficient for five lab groups.
Methylene blue (saturated solution in saline) for demonstration preparation.
Note: Methylene blue powder can be added directly to the dish after dissection.
Large scissors to remove the tail.
Scalpel handles with a supply of fresh #11 blades. It is best to start the dissection
with a fresh scalpel blade.
Forceps (needn’t be fine).
Small paint brushes or coffee stirrers to brush uropod hairs.
Small LED flashlight or other light source
Medium to large crayfish
Results and Discussion
An example of the SPR response to bright light from a blue LED flashlight is
shown in Fig. 6. The AP analysis in Figure 7 shows two distinct units responding to
blue light. They show typical CPR response characteristics: (1) spontaneous activity
in the dark, (2) response latency of seconds, (3) little adaptation during light
stimulation, and (4) continued strong firing after light stimulation stops. In addition,
CPRs are very sensitive to water movements, even subtle ones from small table
vibrations. Response recovery from a light flash may take several minutes. We see
strong light responses between 15 and 20 °C. Preparations below 10 °C show weak
light responses.
Typically freshly dissected preps will have a high level of spontaneous activity,
which may make it difficult to see CPR APs. One- to 2-day old preps kept in the
refrigerator will have less spontaneous activity, and the CPR response may be more
obvious.
We won’t have time in this introductory session to explore more of the CPR
response properties. We suggest the following student explorations. See Galeano
(1976), Wilkens (1988), and Rodriguez-Sosa et al. (2012) for references for more
detail on these and other suggestions for student exploration.
Physiology of the CPRs:
 Locus of photoreception—cover different ganglia and record light responses, cut
A6 input nerves and record light responses. This should also reduce
mechanoreceptor input and demonstrate pacemaker activity in the CPR.
 Light intensity/response relationship
 Response intensity/duration relationship
 CPR refractory times
 Spectral sensitivity of CPR using different color LEDs (Peak response is around
500 nm, Fig. 6)
 Receptive field of the CPR to mechanosensory input—Stimulate hairs on
ipsilateral and contralateral sides of the uropod and examine effects on
spontaneous CPR activity.
 Mechanosensory influences on light response
 With two suction electrodes: (1) determine direction of AP travel along cord to
show it initiates in A6 and (2) determine AP conduction velocity of CPR.
 Circadian rhythms of CPR activity—CPRs may be more sensitive at night
 Amine modulation of spontaneous CPR activity and light response. Only the
effects of serotonin and dopamine have been examined.
 Effect of CPR activation on postural motor programs
Role of CPRs in Crayfish Behavior:
CPRs are thought to initiate backward walking and other abdominal motor
patterns (Simon and Edwards, 1990), and thus contribute to a crayfish’s tendency to
hide in dark, protected spaces. In addition, they may contribute to circadian activity
(Rodriguez-Sosa et al. 2012). Through non-invasive (light shields) and invasive
(lesions) blocks of light reaching the eyes and the ANV, or block of light responses
ascending to higher ganglia, students could explore the behavioral importance of
these non-visual photoreceptors.
Summary
The crayfish CPRs are multimodal interneurons responding to tactile and photic
stimulation. They have attracted the attention of neurobiologists for over 80 years.
Their non-visual photoreception appears to control important aspects of crayfish
behavior, and the position of their axons in the ventral nerve cord allows easy
access for students to monitor their activity. There is a rich background literature on
these neurons that could inspire a broad range of laboratory exercises and student
projects in sensory physiology, neuronal integration, neuronal excitability, circadian
rhythms and the control of behavior by identified neurons. In addition, aspects of
their physiology, such as neuromodulation, have not been studied in depth, and thus
the CPRs offer opportunities for original student research.
References (text and figures):
Cattaert, D. and D. LeRay. (2001). Adaptive motor control in crayfish. Prog.
Neurobiol. 63: 199-240.
Galeano, C. (1976). The caudal photoreceptor of crayfish. A review. Acta Physiol.
Latino- Am. 26: 13-29.
Herman, H.T. (1972). Analysis of the properties of the crayfish caudal photoreceptor
(PRU-photoreceptor unit). In G.A. Kerkut, editor. Experiments in Physiology and
Biochemistry, Vol. 3. Academic Press, London, pp. 155–192.
Johnson, B. R., Hauptman, S. and R. Bonow. (2007). Construction of a simple suction
electrode for extracellular recording of nerve and muscle action potentials. J.
Undergrad. Neurosci. Ed. 6: A21-A26.
Larimer, J.L. (1967). The effects of temperature on the activity of the caudal
photoreceptor. Comp. Biochem. Physiol. 3:683-700.
Rodriguez-Sosa, L., Calderon-Rosete, G., Anaya, V. and T.G. Flores. (2012). The caudal
photoreceptor in the crayfish: An overiew. In: E. Akutagawa and K. Ozaki (eds.),
Photoreceptors: Physiology, Types and Abnormalities. Nova Science Publishers,
Inc. Chapter 3, pp 59- 77.
Prosser, C.L. (1934). Action potentials in the nervous system of the crayfish. II.
Responses to illumination of the eye and caudal ganglion. J. cell. Comp. Physiol. 4:
363-377.
Simon, T. E. and D.H. Edwards (1990). Light evoked walking in crayfish: Behavioral
and neuronal response evoked by the caudal photoreceptor. J. Comp. Physiol. A
166: 745-755.
Welsh, J.H. (1934). The caudal photoreceptor and responses of the crayfish to light. J.
cell. Comp. Physiol. 4: 379-388.
Wilkens, L.A., and J.L. Larimer (1972). The CNS photoreceptor of crayfish:
morphology and synaptic activity. J. Comp. Physiol 80: 389-407.
Wilkens, L.A. (1988). The crayfish caudal photoreceptor: advances and questions
after the first half century. Comp. Biochem.Physiol. Col. 91C: 61-68.
Wyttenbach, R.A., B.R. Johnson, and R.R. Hoy (2014). Crawdad: A lab manual for
neurophysiology. Sinauer Associates, Sunderland, MA.