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Pharmacopeia

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 diluting stock solutions to the proper active concentrations. For standard salines, use the Saline calculator.

Dilution

Most neuroactive substances are effective at very low concentrations. Instead of mixing them at the desired strength and then changing all the saline in a dish, we make concentrated stocks and dilute them. To achieve the desired concentration, add a small amount of stock to your preparation dish. In general, we look at the effects of large (2×, 5×, 10×) changes in concentration, so you do not need to be very accurate with volumes. To calculate the amount of stock to add to your preparation, use the table below. If your preparation dish does not match one of the listed diameters, use the last row and enter the actual volume of saline in your dish.

DishVolumeStock conc.Desired conc.Add
Crayfish tail dish (90 mm, 17 mm saline)100 ml M M
Snail dish (60 mm, 12 mm saline)35 ml M M
Nerve cord dish (55 mm, 5 mm saline)15 ml M M
Ganglion dish (35 mm, 5 mm saline)5 ml M M
Chara chamber (20 mm, 5 mm saline)1.8 ml M M
Other (enter the dish volume) ml M M

To add small volumes of stock to your dish, use a micropipette if available. Otherwise, use a transfer pipette or dropper. A drop from most of these is 50 µl (0.05 ml); your instructor will tell you if your droppers are different.

Common dilutions

Concentrations are cumulative. For example, if you add 2 drops of a 10−2 M stock to a crayfish tail dish, the concentration in the dish is 10−5 M. Add 2 more drops and the concentration is 2×10−5 M. Add 6 more for 5×10−5 M. Another 10 drops brings it up to 10−4 M. In this way, you can start low and test the effects of different concentrations.

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
 

References