The Journal of Undergraduate Neuroscience Education (JUNE), Spring 2021, 19(2):R28-R30

AMAZING PAPERS IN NEUROSCIENCE Getting Graded: Teaching Principles of Chemical Synaptic Transmission Without Action Potentials

Laura Strathern School of Psychology and Neuroscience, University of St Andrews, St Andrews, UK KY16 9JP.

The study of synaptic transmission is foundational for in presynaptic . This was one of the undergraduate neuroscience students. Chemical synaptic first papers to clearly demonstrate that both graded and transmitter release is usually presented as evoked by action spike-mediated chemical synaptic transmission could occur potentials, but it can also be caused by subthreshold, concurrently at an identified synapse. Discussion of this ‘graded’ changes in presynaptic . In a work in undergraduate classrooms can encourage students 1980 publication in the Proceedings of the National to think in greater depth about the diversity of transmission Academy of Sciences, Graubard and colleagues measured mechanisms available to neurons. synaptic activity in the crustacean stomatogastric ganglion; they found that postsynaptic voltage changes occur in Key words: ; graded chemical synaptic response to both action potentials and to graded transmission; stomatogastric ganglion; electrophysiology

Chemical synaptic transmission between neurons requires Therefore, teaching this type of synaptic transmission to the release of molecules from a undergraduate neuroscience students can expand presynaptic into a synaptic cleft. Once released, understanding of the diversity of synaptic transmission bind to receptors on the membranes of mechanisms. postsynaptic neurons. Action-potential-evoked release is common within the nervous system. In this mode, an action RESEARCH SUMMARY potential is generated and travels along the axon to the Graubard et al. (1980) used intracellular recordings of the presynaptic terminal. At the presynaptic terminal, it causes motor neurons of the pyloric network, a central pattern a brief , which results in the opening of generator network that is part of the crustacean voltage-gated calcium channels. The resulting influx of stomatogastric ganglion. In this network, a triphasic motor calcium into the presynaptic terminal triggers fusion of pattern is generated by synaptic connections (Marder and neurotransmitter vesicles to the presynaptic membrane so Bucher, 2007) between a single pacemaker interneuron that neurotransmitter can be released (Siegel and Sapru, (anterior burster, AB) and three motor neurons (pyloric, PY; 2015). lateral pyloric, LP; and pyloric dilator, PD). Graded chemical synaptic transmission is an alternative Graubard et al. (1980) demonstrated that at chemical mode in which transmitter release is dependent on inhibitory synapses in the pyloric network of the lobster presynaptic membrane fluctuations, even when an action stomatogastric ganglion, the same neurons that release potential does not occur in the presynaptic neuron. In 1980, neurotransmitter in response to action potentials also do so Graubard, Raper and Hartline published a paper in the in response to subthreshold, graded voltages. They showed Proceedings of The National Academy of Sciences that was that both mechanisms of release could happen together and part of the early work using electrophysiology techniques to occurred as part of the standard communication between explore graded chemical synaptic transmission in the the network neurons. They compared two recordings of stomatogastric ganglion of the lobster, Panulirus interruptus. depolarization in a presynaptic neuron and resulting The paper offers a unique opportunity for educators, as it inhibitory postsynaptic potentials (IPSPs) in a postsynaptic demonstrates graded chemical synaptic transmission in neuron. For both recordings, the experimenters used the direct comparison to the action-potential-evoked same depolarizing presynaptic current, which hovered near transmission that occurs between the same neurons, so the threshold of action potential generation, sometimes students gain an understanding of how to measure both eliciting an action potential and other times not. Comparison types of chemical synaptic transmission. of the presynaptic recordings show the same initial In comparison to action-potential-evoked transmission, depolarization, resulting in identical IPSPs. After a which is taught in detail to undergraduate neuroscience maintained depolarization, one presynaptic recording students, graded chemical synaptic transmission is often not showed a small action potential occurring, while the other thoroughly discussed as a pervasive mechanism. Graded did not. The corresponding postsynaptic recording showed synaptic transmission is common in a variety of neural a small increase in the IPSP amplitude occurring at the time networks, including the retina (Werblin and Dowling, 1969), of the presynaptic spike, not seen in the other recording the crustacean stomatogastric ganglion (Graubard et al., where there was no presynaptic spike. The IPSP amplitude 1980), and the olfactory bulb (Castro and Urban, 2009). increase was clearly seen in response to the action

JUNE is a publication of Faculty for Undergraduate Neuroscience (FUN) www.funjournal.org Strathern Teaching Principles of Chemical Synaptic Transmission without Action Potentials R29 potential, while the initial IPSP was in response to the evoked and graded transmission, but graded synaptic graded neurotransmitter release that occurred before the transmission is the only means of transmission at chemical action potential. These recordings showed that although a synapses for rod and cone photoreceptors in the retina presynaptic action potential evoked a response in the (Siegel and Sapru, 2015). These photoreceptors are postsynaptic neuron, the graded depolarizations of the primary sensory neurons that signal onto bipolar cells. presynaptic cell actually resulted in a larger Neuroscience textbooks such as Essential Neuroscience hyperpolarization in the postsynaptic cell than the action (Siegel and Sapru, 2015) can be used to facilitate discussion potential did. on how the cells of the retina compare to those of the Results from further recordings also showed that stomatogastric ganglion. This textbook describes the neurotransmitter was being released from the presynaptic response of rods and cones to light as graded membrane neuron even at relatively hyperpolarized ‘resting’ membrane responses, and that their release of glutamate is constant in potential (-55mV). This observation has important the absence of light. Reading Graubard et al. (1980) gives implications; it means that information is being transmitted more depth to this explanation. Stomatogastric ganglion at these types of synapses over a wide range of presynaptic cells, like the cells in the retina, are continuously releasing voltages, which in turn enables fine tuning of postsynaptic neurotransmitter due to voltage fluctuations, except when responses. hyperpolarized. However, while neurons of the stomatogastric ganglion have neurites that are both VALUE presynaptic and postsynaptic, the photoreceptors of the Graubard et al. (1980) provides an introduction for retina absorb light at their outer segments and release undergraduate neuroscience students to a more detailed neurotransmitter from their axon terminals. Comparison of understanding of graded chemical synaptic transmission. It these two systems provides a platform for discussing is not the first paper to demonstrate graded chemical multiple aspects of synaptic transmission. synaptic transmission; however, it is short, easily Graubard et al. (1980) may also help students engage accessible, and has figures that provide a clear illustration with even earlier papers on graded synaptic transmission. of key findings. The paper also highlights the advantages of Some of the first studies to discover graded synaptic using small, well understood invertebrate neural networks. transmission in neurons include Werblin & Dowling (1969), Because the stomatogastric ganglion is a system of only who studied the retina of the vertebrate mudpuppy. roughly 30 neurons, with a defined connectome, it is an ideal Although their paper is much longer and more extensive place to study principles of basic synaptic physiology and than Graubard et al. (1980), highly motivated students could neuromodulation. Understanding these principles is critical gain further insight from Werblin and Dowling (1969) into as they fundamentally govern how neurons communicate how graded synaptic transmission was first discovered in with one another. the vertebrate retina. At the conclusion of their paper, Graubard et al. (1980) Graded transmission is sometimes thought of as less discuss the neuronal qualities that support graded common than action-potential-evoked transmission, but this transmission at chemical synapses. They argue that key may not be the case. Roberts and Bush, in their 1981 book features are: 1) closely situated sites for input and output; Neurones Without Impulses provide a compilation of early and 2) a transmitter release threshold that is suitably work showing how common this type of signaling is in different from the threshold for action-potential-evoked nervous systems. Examples of graded synaptic transmission. In their study, each neurite was both receiving transmission continue to be documented in more recent and releasing neurotransmitter, as opposed to classic work. For example, Castro and Urban (2009) discovered neuronal polarization, as seen in the vertebrate motor that mitral cells in the olfactory bulb of the mouse release neuron for example, with release sites at axon terminals, glutamate at dendrodendritic synapses in response to both and input sites at and the soma. Input and output graded signals and action potentials. They suggest this sites must be close together as graded signals will diminish provides neurons with options for qualitatively different with distance, unlike action potentials which are outputs. Additionally, neurons in other sensory systems, regenerative. While these neurons may not have typical such as in the vertebrate hair cells in the inner ear, also use polarity of neurite function, Graubard et al. (1980) point out graded chemical synaptic transmission (reviewed in Juusola that the stomatogastric neurons are representative of other et al., 1996). Graubard et al. (1980) is an excellent neuron types. For example, they share a structural similarity introduction to this modern work and can be used for with vertebrate neurons found in close packed systems that comparison and discussion of the prevalence of graded have dendrodendritic synapses in addition to standard axon- chemical synaptic transmission. synapses, such as olfactory bulb cells (Castro and Pinel and Barnes (2018) in their textbook Biopsychology Urban, 2009). Therefore, if these structurally similar cells remark that an understanding of some of the exceptional also have the appropriate release threshold, they conclude abilities of some cerebral neurons, with graded chemical that their findings could represent a “common and important” synaptic transmission being only one example, can help neuronal mechanism. students put their essential learning of the foundations of This conclusion to the paper lends readily to discussion neuronal signal transmission in a wider perspective, and exploring whether graded transmission can be said to be a appreciate the remarkable diversity of cerebral neurons. common and/or important mode of neuronal computation. Graubard et al. (1980) can provide an ideal platform to Neurons in the stomatogastric ganglion produce both spike- discuss the importance of graded and action-potential- The Journal of Undergraduate Neuroscience Education (JUNE), Spring 2021, 19(2):R28-R30 R30 evoked transmission acting separately or in concert at presynaptic voltage that occur at subthreshold levels. The synapses. constant release of neurotransmitter from these neurons is increased or decreased as a function of these presynaptic AUDIENCE voltage changes and results in corresponding graded After students have been taught principles of action- changes in voltage in the postsynaptic cell. This graded potential-evoked synaptic transmission, Graubard et al. synaptic transmission had a consequential impact on the (1980) can be taught to 3rd and 4th year undergraduates to circuit and was important for establishing the rhythmic solidify the difference between action-potential-evoked pattern of this motor system. Graubard et al. (1980) not only transmission and graded transmission. The methodology presents important research but also provides an excellent will already be familiar to students who have learned about teaching tool for undergraduate neuroscience students to chemical synaptic transmission and the use of expand their understanding of a prevalent but often electrophysiology to study action potentials. This paper overlooked mechanism of chemical neurotransmission. could also be taught in a final year neuroscience course on synaptic transmission, where students who have long REFERENCES understood the standard functioning of action-potential- Castro JB, Urban NN (2009) subthreshold glutamate release from evoked chemical synaptic transmission will have the mitral cell dendrites. J Neurosci 29:7023-7030. opportunity to explore an interesting alternative. To provide Graubard K, Raper J, Hartline D (1980) Graded synaptic students with background information on the mechanisms of transmission between spiking neurons. Proc Natl Acad Sci U S the stomatogastric ganglion in the lobster there is a useful A 77(6):3733-3735. video on YouTube (iBiology, 2013) that teaches these iBiology (2013) Eve Marder (Brandeis University) Part 1: introduction to central pattern generators. YouTube, January 17, mechanisms including the neuronal circuits described in Available at Graubard et al. (1980) and compliments the learning from https://www.youtube.com/watch?v=dGRQmndrUzA. their paper. Juusola M, French AS, Uusitalo RO, Weckström M (1996) Multiple student activities could be planned around Information processing by graded-potential transmission through Graubard et al. (1980). For example, students could discuss tonically active synapses. Trends Neurosci 19(7):292-297. in groups which of the figures they think are most useful for Marder E, Bucher D (2007) Understanding circuit dynamics using clearly depicting the findings of the paper and explain their the stomatogastric nervous system of lobsters and crabs. Annu reasoning to the class. Some of the relevant findings of the Rev Physiol 69:291-316. more complex Werblin and Dowling (1969) and Castro and Pinel J, Barnes S (2018) Biopsychology. 10th edition. London, UK: Pearson Education. Urban (2009) papers could be explained in lecture or tutorial Roberts A, Bush BMH, eds (1981) Neurones without impulses. by the instructor and used to build discussion points on Cambridge, MA: Cambridge University Press. synaptic transmission and where current research Siegel A, Sapru H (2015) Essential Neuroscience 3rd edition. investigating graded transmission could lead. The relevant Philadelphia, PA: Wolters Kluwer. sections of textbook chapters, for example Siegel and Sapru Werblin F, Dowling J (1969) Organization of the retina of the (2015) Chapter 15 on the visual system can be taught in mudpuppy, Necturus maculosus. II. intracellular recording. J class or given as reading for class preparation. Finally, Neurophysiology 32(3):339-355. Graubard et al.’s (1980) paper and the resulting discussions can supplement understanding of the importance and Received July 15, 2020; accepted August 18, 2020. significance of action potentials, and help students understand when graded transmission could actually be The author thanks Dr. Stefan Pulver, Dr. Bruce Johnson, and the University more advantageous than action-potential-evoked of St Andrews’ MRes Neuroscience class of ’20 for sharing ideas and providing feedback on the manuscript. transmission. Address correspondence to: Laura Strathern, School of Psychology and Neuroscience, St Mary’s Quad, South Street, St Andrews, Fife, KY16 9JP. CONCLUSION Email: [email protected] Graubard et al. (1980) discovered that spiking neurons in the pyloric network of the lobster stomatogastric ganglion Copyright © 2021 Faculty for Undergraduate Neuroscience also inhibit each other through increases and decreases in www.funjournal.org