The Neurotransmitter Toolkit

We’ve just seen that many preadaptations for the evolution of neurons were in place. Calcium toolkit for secretion of neurotransmitters? Check. Post-synaptic scaffold? Check. Circuitry for neurogenesis? Check. But what about the neurotransmitters themselves?

Now, in principle, just about any molecule could serve as a neurotransmitter, as you simply need something that specifically binds to a receptor that triggers the opening of an ion channel. In fact, some amino acids, like glutamate, can moonlight as neurotransmitters.

Nevertheless, I decided to check up on two important neurotransmitters – acetylcholine and epinephrine. These neurotransmitters play key roles in your autonomic nervous system, the system that maintains homeostasis through the regulation of all your body’s organs. What’s more, acetylcholine is the neurotransmitter used to stimulate muscle contraction.

The hypothesis of front-loading neurons would lead us to expect that these neurotransmitters exist in organisms that do no possess neurons. And sure enough, they do.

It turns out that acetylcholine is ubiquitous, found in everything from plants to fungi to protozoa to even bacteria. For example, in the study “Ubiquitous expression of acetylcholine and its biological functions in life forms without nervous systems” (Kawashima K, Misawa H, Moriwaki Y, Fujii YX, Fujii T, Horiuchi Y, Yamada T, Imanaka T, Kamekura M. Life Sci. 2007 May 30;80(24-25):2206-9.) the researchers note:

Taken together, these findings demonstrate the ubiquitous expression of ACh and ACh-synthesizing activity among life forms without nervous systems, and support the notion that ACh has been expressed and may be active as a local mediator and modulator of physiological functions since the early beginning of life.

Even more significant is that they discuss how acetylcholine functions to control water homeostasis and photosynthesis in plants. The reason this is significant is because in mammals, the same molecule is also used as a means to control cellular processes. Thus, not only does this neurotransmitter date back to the first life forms, it is playing the same general role.

And what about epinephrine? Feast your eyes on this:

Enteric amoebae of the genus Entamoeba travel from host to host in an encysted form. We previously showed that in vitro cyst development of Entamoeba invadens requires the addition of defined amounts of multivalent galactose-terminated molecules, such as mucin, to the cultures. The amoeba surface lectin that binds mucin is presumed to convey transmembrane signals when clustered by the ligand, but the signaling molecules that function downstream of the lectin are not known. We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium. Micromolar amounts of both epinephrine and norepinephrine induced encystation.

From Coppi A, Merali S, Eichinger D. 2002. Ubiquitous expression of acetylcholine and its biological functions in life forms without nervous systems. Life Sci. 80:2206-9.

And it gets even better:

Entamoeba trophozoites therefore appear to contain G-protein-regulated adenylyl cyclase that functions downstream of an adrenergic ligand receptor.

From Frederick J, Eichinger D. 2004. Entamoeba invadens contains the components of a classical adrenergic signaling system. Mol Biochem Parasitol. 137:339-43.

In other words, not only do these little single-celled amoeba respond to epinephrine, they have basically the same circuitry that your cells have. And I suppose I should mention that if single-celled organisms are expressing and reacting to both acetylcholine and epinephrine, then it stands to reason they have receptors for these molecules.

What’s even more cool is this. When the amoeba form cysts, it is typically in response to environmental stress. It’s the cell’s way to pushing back against outside forces that threaten its survival.

Well, guess what? Your autonomic nervous system, mentioned above, uses the same molecule to respond to environmental stress. Ever hear of adrenalin? That’s the common name for epinephrine and it triggers the “fight or flight” response you have when you feel your life is in danger.

Looking more closely

When all these preadpatations are considered, it is not unreasonable to suppose that the evolution of neurons was in the cards. In fact, let’s use this hypothesis and dig a little deeper.

Our new player will be the humble paramecium. You may remember looking at these little critters in a high school biology course, but if not, you can watch them in the video below.

These single-celled organisms not only synthesize acetylcholine, but they also make the enzyme acetylcholinesterase (AChE). This enzyme breaks down acetylcholine, meaning that paramecia can both make and selectively destroy acetylcholine. The ability to break down acetylcholine is key, because if release of acetylcholine is like flipping the switch ON, eliminating acetylcholine is like flipping the switch OFF. What’s especially neat is that the paramecium AChE looks much like human AChE:

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BuChE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes.

From Delmonte Corrado MU, Politi H, Trielli F, Angelini C, Falugi C. 1999. Evidence for the presence of a mammalian-like cholinesterase in Paramecium primaurelia (Protista, Ciliophora) developmental cycle. J Exp Zool. 283:102-5.

But it gets better, as paramecia have the complete circuit and employ it in the complex process of conjugation:

We recently discovered, in mating-competent Paramecium primaurelia, the presence of functionally related molecules of the cholinergic system: the neurotransmitter acetylcholine (ACh), both its nicotinic and muscarinic receptors and its lytic enzyme acetylcholinesterase (AChE). Our results on the inhibition of mating-cell pairing in vivo in mating-competent cells treated with cholinomimetic drugs support the hypothesis that the cholinergic system plays a role in cell-to-cell adhesion.

From: Delmonte Corrado MU, Politi H, Ognibene M, Angelini C, Trielli F, Ballarini P, Falugi C. 2001. Synthesis of the signal molecule acetylcholine during the developmental cycle of Paramecium primaurelia (Protista, Ciliophora) and its possible function in conjugation. J Exp Biol. 204:1901-7. .

Oh, and if you would like to see two paramecia hooked up in the act of sex, you can watch the video below (yes, it is safe for work):

The circuitry extends even deeper.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized.

From: Amaroli A, Ognibene M, Trielli F, Trombino S, Falugi C, Delmonte Corrado MU. 2006. Detection of NADPH-diaphorase activity in Paramecium primaurelia. Eur J Protistol. 42:201-8.

So paramecia not only possess acetylcholine and the enzymes needed to synthesize and break it down, but they have an enzyme that can apparently produce nitric oxide, something known to regulate release of acetylcholine (oh, and nitric oxide plays a significant role in the mammalian circulatory system too, but that’s another story).

But why stop here? Paramecia also have a GABA-cicruit:

Gamma-aminobutyric acid (GABA)-related molecules were identified in Paramecium primaurelia by immunocytochemical methods, and GABA(A) receptors by their histochemical BODIPY-binding sites. Confocal microscope analysis showed different localizations according to the stages of the developmental cycle.

From: Delmonte Corrado MU, Ognibene M, Trielli F, Politi H, Passalacqua M, Falugi C. 2002. Detection of molecules related to the GABAergic system in a single-cell eukaryote, Paramecium primaurelia. Neurosci Lett. 329(1):65-8.

You might ask, what’s GABA? It only happens to be the major inhibitory neurotransmitter in your brain. Check it out.

So what’s it doing in paramecia? Controlling swimming behavior:

The presence in Paramecium of gamma-aminobutyric acid A-type receptors (GABA(A)) and the capability of the protozoon to synthesize and release the GABA neurotransmitter into the environment have already been demonstrated. This study investigates the involvement of the GABA(A) complex in the swimming control of the ciliated protozoon. The GABA(A) receptors were pharmacologically activated by the selective agonist muscimol and the effect on Paramecium primaurelia swimming behavior was analyzed. Paramecium normally swims forward, but the activation of GABA(A) receptors induced a peculiar response, characterized by alternate periods of whirling and forward swim.

From: Bucci G, Ramoino P, Diaspro A, Usai C 2005. A role for GABAA receptors in the modulation of Paramecium swimming behavior. Neurosci Lett. 386:179-83.

It will be interesting to watch as more and more of the molecular circuitry involved in neurotransmission is uncovered in unicellular life forms (as we would expect from front-loading). Thus, let me propose that what we have here is at least part of the neurotransmitter toolkit. Couple this to the existence of an endocrine toolkit in Tetrahymena and a calcium signaling toolkit in choanoflagellates, and it becomes more clear that multiple, independent preadaptations were in place to facilitate the evolution of metazoa.


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