Criteria for Assigning a Neurotransmitter Function in C. elegans
By Curtis M. Loer, Department of Biology, University of San Diego

For detailed information on specific neurotransmitters see: The Evidence for Classical Neurotransmitters in C. elegans Neurons

Traditional neurotransmitter criteria
Neurotransmitter criteria for C. elegans
Definition and use of the suffix "-ergic"

Traditional Criteria:

1. Presence of the chemical within the cell. The chemical is either synthesized by the neuron or is taken up from other cells that release it.²
   
2. Stimulus-dependent release. It is released in appropriate quantities by the neuron upon stimulation.
3. Action on postsynaptic cell. Exogenous application of the substance in appropriate amounts mimics the action of the endogenously-released substance on the postsynaptic cell or organ.
4. Mechanism for removal. [Note, not always included as a criterion] A specific mechanism exists to remove the substance from the synaptic cleft, i.e., by degradation or reuptake.

¹ For one version of the traditional criteria, see Criteria That Define a Neurotransmitter, in Neuroscience by Purves et al., 2nd Ed., 2001, Sinauer Associates  (NCBI Bookshelf link)

²Although most cells that release a neurotransmitter also synthesize the neurotransmitter, this criterion has recently been expanded beyond synthesis alone, to include less common cells that do not synthesize, but instead take up the neurotransmitter from the vicinity and then re-release it. We refer to these neurons as 'uptake-dependent.'

For technical reasons, the traditional criteria cannot be fully met for putative neurotransmitters in C. elegans (especially criteria 2 and 3). The advent of new microfluidic devices coupled with optical stimulation may soon overcome the difficulties currently encountered in the worm for isolation and analysis of substances following stimulated release (i.e., traditional physiological techniques to identify a chemical as a neurotransmitter). Nevertheless, a large body of 'circumstantial' evidence indicates that various neurotransmitters are used by specific neurons in the worm. From the available evidence, we may propose a modified set of criteria that will suffice to identify the neurotransmitter(s) used by a particular neuron in the worm, despite the inability to fulfill all the traditional criteria.

Reasonable criteria for identifying a neurotransmitter as functioning in a C. elegans neuron:

1. Localization to the neuron of:
   1. The putative neurotransmitter by:
      1. Immunocytochemistry or
      2. Other histological technique (e.g., formaldehyde-induced flourescence)
      Localization by such techniques should be corroborated by identification of the genuine substance in the whole animal, e.g., by HPLC. (Advances in single cell isolation, identification and chemical analysis techniques may ultimately allow direct chemical identification of the actual neurotransmitter in individual cells in the worm.)
   2. Biosynthetic, transport (vesicular or reuptake) or catabolic enzymes typically associated with the neurotransmitter by:
      1. Immunocytochemistry or other histological technique
      2. Expression of genes encoding such proteins as seen by:
         1. Reporter genes
         2. In situ RNA hybrization (not commonly done in C. elegans)
            
      Similar to (A) above, localization should be corroborated by identification of the enzymatic activities in whole worms, and loss of such activity in appropriate mutants. A cloned version of the gene may also be expressed in vitro or in heterologous cells (e.g., transporters) to demonstrate enzymatic function and test substrate specificity.

2. Behavioral effects, consistent with known connectivity, of:
   1. Neurotransmitter, agonists, or antagonists
   2. Inhibitors of biosynthetic, transport or catabolic enzymes
   3. Mutants with altered NT expression

3. Localization of neurotransmitter receptors to known or putative postsynaptic cells
   To be reliable as evidence for specific neurotransmitter function, the specificity of the receptor should have been determined physiologically, and not simply inferred from sequence.

4. Ancillary supportive evidence can come from other nematodes with clearly homologous neurons (e.g., using the large parasitic nematode Ascaris, in which traditional electrophysiological and pharmacological techniques can be applied.

Definition and use of the suffix '-ergic'

Neurons that use a given neurotransmitter, fulfilling the above criteria, are referred to as 'neurotransmitter-ergic.' For example, neurons using glutamate are 'glutamatergic.' [See the table below for more examples.] We maintain that the suffix '-ergic' indicates a neuron that functionally uses or 'works with' the neurotransmitter. [2. exhibiting or stimulating activity esp. of (such) a neurotransmitter substance <adrenergic> <dopaminergic> - Merriam-Webster Medical Dictionary definition]. This fits with the etymology of -ergy, work [from Latin -ergia, work; from Greek, ergon work], such that -ergic means to 'work with' a neurotransmitter [see also here].

This would also include neurons that do not synthesize the transmitter, but take it up, package and release the transmitter to affect postsynaptic cells. Such neurons may be called 'uptake-dependent' -ergic neurons. In C. elegans, the hermaphrodite neurons VC4 and VC5 may not make their own serotonin (serotonin-synthetic enzyme genes are not expressed there), but take it up, package it and release it to modulate egg laying (see note³³ here). These cells are therefore 'uptake-dependent' serotonergic neurons.

Note that the adjectival form is also sometimes used to describe receptors that bind and are activated by the neurotransmitter ('β2 adrenergic receptor'), for specific synapses at which the transmitter is used ('cholinergic synapse'), and for circuits, pathways or systems in the brain or nervous system ('dopaminergic pathways') that use a given neurotransmitter. Within such pathways or systems, a neuron may be referred to as 'dopaminergic' based simply on the presence of the transmitter in the cell, on a presumption of its function.

Neurotransmitter or class	Designation
Acetylcholine	cholinergic
Adenosine/ATP/nucleotides	purinergic
Biogenic amine	aminergic
Catecholamine	catecholaminergic
Dopamine	dopaminergic
Histamine	histaminergic
GABA	GABAergic
Glycine	glycinergic
Glutamate	glutamatergic
Monoamine	aminergic
Neuropeptide	peptidergic
Norepinephrine	adrenergic/noradrenergic
Octopamine	octopaminergic
Serotonin	serotonergic
Tyramine	tyraminergic

This section should be cited as: Loer, C. M.^§ 2022. Criteria for Assigning a Neurotransmitter Function in C. elegans, in WormAtlas. doi:10.3908/wormatlas.5.201
^§To whom correspondence should be addressed. Curtis Loer: cloer@sandiego.edu