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Pharmacopeia

Instructor’s Supplement

Introduction

The lab exercises refer to neurotransmitters, neuromodulators, and toxins that can address mechanisms of neural function. This page brings all of that information together in one place, with instructions for making stock solutions and then diluting them to the proper active concentrations. For standard salines, use the Saline calculator.

Compounds

(Click a category title to show its table.)

Dopamine
 10−3 MIncreases activity in all axons in crayfish superficial nerve 3 (Murphy and Larimer, 1991)
10−6 to 10−4 MMay reduce PSP size at the crayfish NMJ (Miller et al., 1985)
10−4 MStarts Lymnaea buccal rhythm, increases its frequency, and changes its phasing (Kyriakides et al., 1989)
10−8 to 10−4 MStarts Helisoma feeding motor pattern and alters it in a concentration-dependent manner (Trimble and Barker, 1983; Quinlan et al., 1997)
Serotonin
 10−7 to 10−5 MMay increase PSP size at the crayfish NMJ (Glusman and Kravitz, 1982; Djokaj et al., 1981)
10−7 to 10−5 MIncreases crayfish MRO responses, especially the tonic unit (Pasztor and Macmillan, 1990)
10−4 MStops Lymnaea feeding rhythm but may increase tonic activity (Kyriakides et al., 1989)
10−6 MStarts Helisoma feeding motor pattern (Trimble and Barker, 1983; Quinlan et al., 1997)
Octopamine
 10−6 to 10−4 MIn crayfish, may increase PSP size at the NMJ (Djokaj et al., 1981)
10−7 to 10−5 MIncreases crayfish MRO responses, especially the phasic unit (Pasztor and Macmillan, 1990)
10−4 MStops Lymnaea feeding rhythm but may increase tonic activity (Kyriakides et al., 1989)
10−5 to 10−4 MIncreases sensitivity of Lymnaea buccal neurons (Vehovszky et al., 2005)
Glutamate
 Neurotransmitter, excitatory on ionotropic receptors, may be inhibitory via metabotropic receptors
10−5 to 10−4 MMay depolarize crayfish muscle fibers
10−5 to 10−4 MMay desensitize crayfish NMJ receptors (Dudel, 1977a)
10−5 to 10−3 MIn Lymnaea, depolarizes some buccal feeding rhythm cells and hyperpolarizes others (Nesic et al., 1996; Brierley et al., 1997)
GABA
 Inhibitory neurotransmitter
10−5 to 10−4 MMay hyperpolarize crayfish muscle fibers and affect PSP size
10−3 MHas slight inhibitory effects on crayfish superficial nerve 3 (Murphy and Larimer, 1991)
Glycine
 Inhibitory neurotransmitter and co-factor on NMDA receptors
≥ 10−1 MMay cause spontaneous IPSPs in crayfish NMJ and suppress excitatory synapses (Finger, 1983)
 
Acetylcholine
 Neurotransmitter with excitatory and inhibitory effects
2×10−4 to 1×10−3 MExcitatory and inhibitory effects in Lymnaea (Elliot et al., 1992)
10−4 MStops or reduces frequency of Lymnaea buccal rhythm; may induce tonic activity (Kyriakides et al., 1989)
Carbachol
 Nicotinic and muscarinic acetylcholine receptor agonist
10−4 to 10−3 MIncreases activity in crayfish superficial nerve 3 (Murphy and Larimer, 1991)
1×10−5 to 6×10−5 MStarts and accelerates crayfish swimmeret patterns (Braun and Mulloney, 1993; Olivo, 2015, 2016)
10−4 MIn our experience with Lymnaea and Helisoma, slows or stops buccal rhythms
Pilocarpine
 Muscarinic acetylcholine receptor agonist (toxic if swallowed)
10−4 to 10−3 MIncreases activity in crayfish nerve 3, especially the inhibitor (Murphy and Larimer, 1991)
10−5 to 10−4 MInitiates crayfish swimmeret motor pattern and modulates burst frequency (Braun and Mulloney, 1993)
Nicotine
 Nicotinic acetylcholine receptor agonist (toxic if swallowed)
10−5 to 10−4 MIncreases activity in crayfish nerve 3, especially the inhibitor (Murphy and Larimer, 1991)
2×10−7 to 4×10−6 MIncreases burst frequency in an active swimmeret preparation but does not initiate rhythmic activity (Braun and Mulloney, 1993)
Atropine
 Muscarinic acetylcholine antagonist (fatal if swallowed or inhaled)
10−3 MDecreases activity of two axons in crayfish superficial nerve 3 while increasing activity of another (Murphy and Larimer, 1991)
5×10−4 MBlocks excitatory response in Lymnaea (Elliot et al. 1992)
Succinylcholine
 Nicotinic receptor antagonist (toxic if swallowed)
2 to 5×10−4 MUsed as anesthetic for Helix and other snails at 0.01-0.02% with 2% MgCl2 (Beeman, 1968)
10−5 to 10−3 MMay help distinguish between nicotinic and muscarinic activity
 
 
Verapamil
 Blocks voltage-gated Ca2+ channels (toxic if swallowed or inhaled)
10−5 to 10−4Should block chemical synapses
(slow to dissolve in saline, needs much vortexing)
CsCl
 Cs+ blocks h-current when applied extracellularly (harmful if swallowed)
10−4 to 10−3 MShould reduce or eliminate post-inhibitory rebound in silent cells
4-AP
 4-aminopyridine blocks voltage-gated K+ channels (irritant, fatal if swallowed)
5×10−3 MMay increase activity of crayfish MROs (Purali and Rydqvist, 1992)
5×10−5 to 5×10−3 MBroadens action potentials and thus increases PSP size
TEA
 Tetraethylammonium blocks voltage- and Ca2+-gated K+ channels when applied extracellularly (irritant)
5×10−2 MMay increase activity of crayfish MROs (Purali and Rydqvist, 1992)
5×10−4 MBroadens action potentials and thus increases PSP size
TEA/CsCl
 Blocks voltage-gated K+ channels when applied intracellularly (irritant, harmful if swallowed)
2 MFill electrode with 2 M TEA/CsCl and inject into the cell with depolarizing current
 

Stock solutions

Stock solutions can be made in deionized water and used with crayfish, snails, or Chara. The small volume of stock that is added to the preparation dish will alter ion concentrations by less than 1%. If your class uses only one preparation, make stocks in the appropriate saline.

Stock concentrationFWStock volumeStock volumeHazards and notes
Acetylcholine 10−2 M181.70.018 g in 10 ml  
4-aminopyridine 5×10−2 M  94.10.235 g in 50 ml0.047 g in 10 ml▵‡
Atropine 10−2 M289.40.029 g in 10 ml ‡
Carbachol 10−2 M182.70.0018 g in 10 ml0.0027 g in 15 ml 
CsCl 5×10−2 M168.40.421 g in 50 ml0.084 g in 10 ml†
Dopamine 10−2 M189.60.019 g in 10 ml0.002 g in 1 mlkeep aliquots frozen
GABA 10−2 M103.10.010 g in 10 ml  
L-glutamic acid 10−2 M147.10.074 g in 50 ml0.015 g in 10 ml 
Glycine 1 M  75.13.75 g in 50 ml (need 10 ml per dish)mix in crayfish saline
Octopamine 10−2 M189.70.095 g in 50 ml0.019 g in 10 ml†
Pilocarpine 10−2 M244.70.025 g in 10 ml †
Serotonin 10−3 M212.70.011 g in 50 ml0.002 g in 10 mlstore powder at 2-8°C
Succinylcholine 10−2 M397.30.040 g in 10 ml †
Sulpiride 10−2 M341.4 0.034 g in 10 ml †
Tetraethylammonium 5×10−2 M165.70.414 g in 50 ml0.083 g in 10 ml▵
Tetraethylammonium/CsCl 2 M---33.7 g CsCl and 33.1 g TEA in 100 ml H2O▵†
Verapamil 10−2 M454.60.00227 g in 5 ml0.00045 g in 1 ml† vortex to dissolve
Custom: formula weight = g/M, volume = ml, concentration = M ⇒ mass = .
Hazards (as identified by the manufacturer): ▵ irritant, † harmful if swallowed, ‡ fatal if swallowed.

Dilution

To produce any concentration

       [stock volume to add] = [dish volume] × [desired concentration] / [stock concentration].
This calculation is approximate because dish volumes vary and adding stock changes the volume. However, we are generally only interested in large (2×, 5×, 10×) changes in concentration, so this variation is unimportant. The list below shows the volumes of commonly used preparation dishes. Appendix D, Pharmacopeia, includes a calculator for students to use with preparation dishes of known volume.

Volumes

References