Aplysia R15 neuron
|Fred H. Sieling and Robert Butera (2011), Scholarpedia, 6(10):4181.||doi:10.4249/scholarpedia.4181||revision #126753 [link to/cite this article]|
Neuron R15, located in the abdominal ganglion of the gastropod mollusc Aplysia Californica, is the first intrinsically bursting neuron to receive extensive study using single cell electrophysiology techniques. Much of the investigation of the nature of electrical bursting of single neurons was motivated by the initial study of this neuron, and it was later one of the first to receive extensive study of the role of neuromodulation in biasing single neuron burst dynamics.
Arvanitaki (1941) had recorded electrical activity extracellularly from various neuronal somata that were distinct and visually identifiable. Soon thereafter, the technique of intracellular recording had been invented and was adopted as a tool for investigating the electrical dynamics of visually identifiable neurons in Aplysia. Arvanitaki and Chalazonitis (1955) and simultaneously Tauc (1954) published the first reports of a distinct visually identifiable neuron in the abdominal ganglion of Aplysia, now called R15 (Cogeshall et al., 1966; Frazier et al., 1967).
The study of R15 was accelerated when Eric Kandel adopted Aplysia as his animal model for studying the cellular basis of behavior. Kandel and his colleagues named many of the visually identifiable neurons in the ganglia of Aplysia and mapped out many of Aplysia's neuron circuits. Much of this work continues today.
Electrophysiological Properties of Bursting
Early studies of R15 could find no evidence of rhythmic synaptic input or synchronous activity in connected neurons, leading to the conclusion that bursting in R15 was endogenous. It was shown that isolated somata could continue to burst after ligature (Alving, 1968) or isolated dissection (Chen et al., 1971). The bursting rhythm could also be phase-shifted by well-timed inputs to the soma (Pinsker, 1977), further supporting the idea that the rhythm was intrinsic to the neuron itself. R15 has been referred to as a parabolic burster (Strumwasser, 1968) due to parabolic rise and fall of the interspike interval over the time-course of the burst.
The mechanism of bursting in neuron R15 received extensive study in the 1970s and 1980s. These works are reviewed quite comprehensively in “The generation and modulation of endogenous rhythmicity in the Aplysia bursting pacemaker neurone R15” (Adams and Benson, 1985). In fact, very little experimental work on the ionic basis of bursting in R15 has been published since their comprehensive review article. Early investigators quickly identified several key features associated with the bursting rhythm, features that have now become recognized as common to many intrinsically bursting neurons. These features include:
- the existence and importance of a "negative slope region" in the current-voltage (I-V) curve;
- the recognition that the onset, offset, and progression of the burst were associated with ionic conductances that changed slowly during the burst; and
- the role of an inward cationic current in initiating the burst.
Nevertheless, the literature on this topic is a “chaos of conflicting observations and interpretations” (Adams and Benson, 1985). Some investigators found that a subthreshold oscillation persisted in the presence of tetrodotoxin (TTX), while others did not. Some investigators criticized many of the published studies, as they involved significant changes in the concentrations of Na+ or Ca2+ which significantly altered membrane properties. One laboratory reported that "freshly opened" TTX blocked the subthreshold oscillations, but not if the vial of TTX had been opened for one day. What is agreed upon is that a cationic current is responsible for the negative slope region and the initiation of the burst.
A key point of dispute is the identification of an ionic mechanism that changes slowly during the burst and can account for the termination of the burst. Ideally, this would involve the progressive inactivation of the negative-slope-region (NSR) current responsible for the initiation of the burst or the slow activation of an outward current to counteract this current. Proposed mechanisms have included the slow activation and subsequent inactivation of an outward K+ current (Junge and Stephens, 1973), voltage-activation and Ca2+-inactivation of the NSR current (Adams and Levitan, 1985; Kramer and Zucker, 1985), and a hyperpolarization activated current that summates throughout the burst (Adams, 1985).
While many models of bursting in R15 exist largely based on Adams and Benson's proposed model, exactly how the neuron bursts has never been conclusively resolved. Research on R15's ionic mechanisms largely stopped in the late 1980s. Furthermore, subthreshold currents are difficult to quantify due to their low amplitude compared to other ionic currents in the cell membrane.
Neuromodulation of Bursting
Substances that have been shown to affect the activity of R15 include acetylcholine (Ach), dopamine (DA), Aplysia egg-laying hormone (ELH), Phe-Met-Arg-Phe-amide (FM-RFamide), \(\gamma\)-aminobutyric acid (GABA), serotonin (5-HT), cyclic-adenosine monophosphate (cAMP), and cyclic-guanosine monophosphate (cGMP) (Carpenter et al., 1978; Drummond et al., 1980; Kramer et al., 1988a; Levitan et al., 1987; Levitan and Norman, 1980). Neuromodulators such as DA, 5-HT, ELH, and FM-RFamide have received the most attention, especially 5-HT and DA. DA can bias the cell into a hyperpolarized silent state (Gospe and Wilson, 1980). 5-HT has a more complex effect. At low concentrations, it silences bursting, while at high concentrations it causes elongated bursting and then tonic spiking. These effects occur by activating an inwardly rectifying K+ current at low 5-HT concentrations (Drummond et al., 1980; Benson and Levitan, 1983) and an inward cationic current at higher concentrations of 5-HT (Levitan and Levitan, 1988). 5-HT and DA have generally opposing effects on burst excitability (Lotshaw and Levitan, 1988). Cyclic AMP is the second messenger mediating 5-HT's effects (Lotshaw et al., 1986; Levitan and Levitan, 1988). In addition, there exist dynamic interactions between Ca2+ and cAMP (Kramer et al., 1988). Many of these modulatory effects are mediated intracellularly by cAMP and cGMP.
Computational Models of R15 Burst Dynamics and Modulation
Since the mid 70s, several models of R15 have surfaced with varying levels of quantitative and qualitative accuracy. Most models consist of an array of Hodgkin-Huxley (H-H) type ion channels fit to voltage clamp data derived from preparations of the isolated abdominal ganglion bathed in standard ASW or exotic pharmaceutical cocktails. Voltage time-series and steady state current-voltage (I-V) curves obtained under the above conditions were fit and analyzed.
There are several trends in this modeling work. Newer models increasingly stress the relevance of model components to electrophysiological data. As the sophistication of electrophysiology and molecular biology techniques improved, models were able to incorporate more biophysical measurements. From the 70s to the mid-90s, interest peaked in the mechanism of both R15 burst dynamics (Bertram, 1993; Canavier et al. 1991) and mechanisms of neuromodulation by DA and 5-HT (Bertram, 1993; Butera et al., 1995). In all models, there has been an emphasis on applying non-linear systems theory to neurons, e.g., 2-dimensional nullcline analysis in the phase plane (Butera et al., 1997), bifurcation analysis (Butera et al., 1996), and slow-fast timescale decomposition (Rinzel and Lee, 1985; Bertram, 1994; Butera et al., 1996). Rinzel and Lee (1985) showed that using bifurcation analysis, they were able to identify the parameter space where parabolic bursting occurred in their model. These techniques are part of today’s growing literature in dynamical systems theory (Izhikevich 2007). Other interesting avenues opened via R15 models include geometrical analysis of phase response curves (Demir et al. 1997) and the idea of multistability as an information storage mechanism (Canavier et al. 1993, Canavier et al. 1994, Butera 1998, Newman and Butera 2010; see also Multi-stability in neuronal models).
|Plant model (Plant 1976)||Early biophysical R15 model reproduced many behaviors including parabolic bursting; first H-H type model|
|Rinzel and Lee 1985||Slow-fast decomposition and bifurcation analysis|
|Adams and Benson 1985||Synthesis of confusing array of empirical data|
|Canavier et al. 1990||Demonstrated a period doubling route to chaos in the parametric transition between bursting and spiking|
|Canavier et al. 1991||Added intracellular Ca2+ balance|
|Bertram 1993, 1994||Incorporated detailed effects of 5-HT|
|Canavier et al. 1993, 1994||Showed multistability, 7 modes of activity at one set of parameters|
|Butera et al. 1995||Incorporated intracellular cAMP and PK pathways; explained effects of DA and 5-HT|
|Yu et al. 2004||Added Ca2+-calmodulin interactions with cAMP pathway; made dynamical argument for cAMP oscillations|
Physiological Function of R15
In the first 30 years of work on R15, little attention was paid to its physiological function; however, in a set of 3 companion papers and two other papers Alevizos et al. (1991abcde) produced convincing and surprising data showing that (1) R15 is likely involved in the minutes-long process of egg laying and (2) although in vitro R15 was the best studied bursting neuron through the 1980s, in vivo it actually does not burst most of the time (1991a). It is hypothesized that R15 bursts during egg laying, but a bout of egg laying was never recorded in vivo. Due to the limitations of the reel-to-reel technology used for electrophysiological recordings at the time, Alevizos et al. were restricted to three hours of intermittent recordings per day. Using modern digital technology and by recording in vivo for 24 hours per day, it is possible that this could be determined conclusively through 24 hour/day continuous monitoring.
Relevance to the Modern Study of Bursting Neurons
- R15 was arguably the best studied bursting neuron of the 1970s and 1980s. However, research waned due to its apparent lack of participation in any well-studied motor central pattern generating circuit. The bursting neurons of the stomatogastric ganglion (STG) are arguably the best studied since the 1990s, and participate in central pattern-generating circuits.
- Dynamical neuroscience mechanisms. In the study of bursting neurons, the ubiquitous negative slope region (NSR), which is usually thought of as dependent on activation of inward cationic currents, is equally dependent on de- or in-activation of outward K+ current (Butera 1995). This effect is well understood (Izhikevich, 2007), but R15 is the first and best known example.
- Many examples exist today of well-studied bursting neurons (and other cell types) whose study was heavily informed by these earlier works on R15.
- Dopamine neurons (Amini et al., 1999)
- preBötzinger Complex (Butera et al., 1999; Del Negro et al., 2001; Toporikova and Butera, 2010; Dunmyre et al., 2010)
- Thalamo-cortical relay cells (Huguenard and McCormick, 1992; Wang et al., 1991, 1995)
- pancreatic \(\beta\) cells (Sherman, 1996; Bertram et al., 2000)
- neurons of the stomatogastric ganglion (Guckenheimer et al. 1993; Liu et al., 1998; Soto-Trevino et al., 2005)
Even given the above mountain of research, the mechanism of bursting in R15 is arguably still unresolved, e.g., is the current responsible for bursting ICa, IK, ICaN, or the mysterious INSR? Since the last experimental analysis of R15 bursting mechanisms in the mid-80s, new molecular and genetic tools have emerged that could put this question to rest. The Aplysia Genome Project may reveal the genetic identities of ion channels that sum or combine to form previously described currents. Digital electrophysiology recording technology allows for full 24-hour recordings of R15 in vivo, which could unambiguously discern its putative role in egg-laying. In short, modern tools and technologies and ability for large scale and long term observability could re-empower all of the questions posed above to be revisited with more conclusive results.
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