Bag cell neurons

The bag cell neurons  are located in the abdominal ganglion of the marine mollusk Aplysia. Their function is to control the onset of egg-laying behavior. They have been widely used as a model system to investigate the mechanisms by which neuropeptides are synthesized, processed and secreted from neurons. Because they respond to brief stimulation with a series of changes in their electrical properties that lasts for up to 18 hrs, they have also been used to investigate the mechanisms by which modulation of ion channels produces long-lasting changes in excitability and controls animal behavior.

## Contents

* 1 Introduction
* 2 Role of ion channels in the control of the bag cell secretory afterdischarge
* 2.1 Voltage-dependent potassium channels
* 2.2 Calcium and sodium-dependent potassium channels
* 2.3 A voltage-gated non-selective cation channel is activated by association with PKC and calmodulin at the onset of afterdischarge
* 2.4 Insertion of calcium channels at neuropeptide release sites
* 2.5 Other sources of calcium for neuropeptide release
* 2.6 Summary
* 3 References
* 4 Recommended reading
* 5 External links
* 6 See also

## [edit] Introduction

The bag cell neurons are located in two clusters of ~200-400 cells each at the rostral end of the abdominal ganglion of Aplysia (Kupferman, 1967; Kupfermann & Kandel, 1970; Pinsker & Dudek, 1977). Their primary function is to act as a master switch for a series of reproductive behaviors that culminates in egg laying. The electrophysiological state of these neurons determines whether egg-laying behaviors will occur in response to environmental stimuli.

The series of reproductive behaviors is triggered by the release of several neuropeptides (Arch & Smock, 1977; Pinsker & Dudek, 1977). Their major neurotransmitter is egg-laying hormone (ELH), a 36-amino acid peptide that, like many other neuropeptides, is amidated at its C-terminus, conferring resistance to extracellular proteases (Chiu et al., 1979; Scheller et al., 1983). ELH has actions on numerous other neurons in the abdominal ganglion (Branton et al., 1978), as well as on peripheral targets, particularly the ovotestis (Rothman et al., 1983a). ELH is synthesized as part of a larger precursor (pro-ELH, Figure 1), which also encodes several smaller neuroactive peptides, that are termed \\(\alpha\\)-, \\(\beta\\)- and \\(\gamma\\)-BCP (bag cell peptides). These smaller BCPs are also secreted locally to alter the firing patterns of neurons in the abdominal ganglion, but they do not undergo any posttranslational modification and their actions are rapidly terminated by proteases in the extracellular space (Rothman et al., 1983b; Mayeri et al., 1985; Pulst et al., 1986 & 1987; Sigvardt et al., 1986; Kauer et al., 1987; Fisher et al., 1988; Nagle et al., 1990). Several other peptides, including \\(\delta\\)-BCP are also secreted during afterdischarge (Hatcher & Sweedler, 2008), but their functions are not yet understood. Interestingly, different combinations of neuropeptides are packaged into different secretory granules and are targeted to different branches of the bag cell neurons (Sossin et al., 1990; Li et al., 1998), presumably in order to coordinate the multiple actions of these neurons on both peripheral and central targets.

Figure 1: Organization of pro-ELH, the precursor to ELH and the small BCPs.

Secretion of ELH and the BCPs from the bag cell neurons normally occurs in response to a prolonged burst of action potentials, termed an afterdischarge. In the absence of stimulation, the cells maintain negative resting potentials and display no spontaneous electrical activity. Brief electrical stimulation (5-15 sec) of an afferent input from the head ganglia causes the cells to depolarize by 15-20 mV and to generate an afterdischarge that lasts for about thirty minutes ( Figure 2). The nature of the synaptic input is poorly understood, but it has been suggested that only a few neurons receive direct afferent input and that excitation subsequently spreads to the remainder of the electrically coupled cells (Mayeri et al., 1979). In addition, once the afterdischarge is underway, peptide release occurs in response to both \\(Ca^{2+}\\) influx from the extracellular space and \\(Ca^{2+}\\) release from intracellular stores (Arch, 1972; Stuart et al., 1980; Loechner et al., 1990; Wayne, 1995; Wayne et al .,1998; Michel & Wayne, 2002).

Figure 2: Pattern of firing and of neuropeptide secretion in response to brief stimulation of the input to bag cell neurons.

The series of behaviors that is triggered by neuropeptide release during the afterdischarge lasts about 3-5 hrs. At the end of the afterdischarge, the bag cell neurons enter a prolonged (~18 hr) refractory state during which stimulation evokes action potentials but fails to trigger long-lasting afterdischarges or neuropeptide secretion. This refractory state is believed to prevent the re-initiation of the behavioral sequence once it has begun and to allow for the maturation of a new clutch of eggs in the ovotestis.

The transformation of the electrical properties of the bag cell neurons from their resting state to the afterdischarge and subsequent refractory state can be attributed to the action of second messenger systems on the characteristics of several ion channels that regulate resting potential and the height and width of action potentials. At least two major second messenger systems are activated on stimulation of the input to the bag cell neurons. Cyclic AMP levels in these neurons rise within two minutes of the onset of stimulation, and the activation of protein kinase A (PKA) results in the activation of a tyrosine phosphatase (Kaczmarek et al., 1978, 1980, & 1982; Loechner & Kaczmarek, 1990; Wilson & Kaczmarek, 1993). Stimulation of the input simultaneously triggers activation of protein kinase C (PKC), and formation of inositol trisphosphate (Fink et al., 1988; Wayne et al., 1999). Many of the ion channels that trigger the afterdischarge, or the refractory state, have been identified and their properties are described below.

## [edit] Role of ion channels in the control of the bag cell secretory afterdischarge

The brief stimulus train that triggers neuropeptide secretion in the bag cell neurons causes a major change in their electrical properties. Table 1 lists some of the ion channels that modulate the properties of these neurons. The resting membrane potential of these neurons prior to stimulation is usually between -55 mV and -65 mV but, after the onset of afterdischarge, the inter-spike voltage is typically between -30 mV and -40 mV.

Table 1: Ion channels that regulate excitability of bag cell neurons.  Channel  |  Characteristic  |  Role in neuropeptide secretion

|
|

Ik1  |  V-dependent \\(K^+\\) channel  |  Action potential repolarization
Kv2  |  V-dependent \\(K^+\\) channel  |  Action potential broadening
Slo  |  \\(Ca^{2+} \\)-activated \\(K^+\\) channel  |  Setting state of excitability
I\\(_{CAT}\\)(V-dep)  |  Non-selective V-dependent cation channel  |  Maintains depolarization during afterdischarge and peptide secretion
I\\(_{CAT}\\)(V-indep)  |  Non-selective V-independent cation channel  |  Contributes to depolarization at soma during afterdischarge
Cav1  |  L-type \\(Ca^{2+}\\) channel  |  Upstroke of soma \\(Ca^{2+}\\) action potential (constitutive)
Cav2  |  P/Q/N-type \\(Ca^{2+}\\) channel  |  Neuropeptide secretion
Slack  |  \\(Na^+\\)-activated \\(K^+\\) channel  |  Under investigation

### [edit] Voltage-dependent potassium channels

Over the first 2-3 minutes of afterdischarge there is a progressive increase in the width of evoked action potentials. This increase can be mimicked by elevations of cyclic AMP or the catalytic subunit of PKA, and is prevented by peptide pseudosubstrate inhibitors of PKA (Kaczmarek et al., 1978 & 1980; Conn & Kaczmarek, 1989). Two distinct \\(K^+\\) currents, originally termed Ik1 and Ik2 (Strong & Kaczmarek, 1986) regulate the width of action potentials. The molecular basis of Ik1, which undergoes relatively little inactivation during sustained depolarizations, has not yet been investigated. In contrast, the gene encoding the Ik2 channel, which undergoes cumulative inactivation during repeated depolarizations, has been identified as Kv2.1, the Aplysia homolog of the Drosophila Shab gene (Quattrocki et al., 1994.)

The voltage-dependent Kv2.1 channels play a major role in spike broadening (Quattrocki et al., 1994). Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge (Quattrocki et al., 1994). Moreover elevations of cyclic AMP such as occur during the afterdischarge reduce the amplitude of the voltage-dependent \\(K^+\\) channel, producing further broadening of action potentials (Kaczmarek & Strumwasser, 1984). Kv2.1 channels also undergo rapid clustering and unclustering during afterdischarges (Zhang et al., 2008a). As in mammalian neurons (Misonou et al., 2004), Kv2.1 channels in bag cell neurons are restricted to ring-like clusters in the plasma membrane of the soma and proximal dendrites (Zhang et al., 2008a). Elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhances formation of these clusters on the somata, and the formation of these clusters is associated with a decrease in current amplitude (Zhang et al., 2008a).

### [edit] Calcium and sodium-dependent potassium channels

Large-conductance \\(Ca^{2+}\\)-activated \\(K^+\\) (BK) channels are encoded by the Slo gene. In bag cell neurons, these set the overall level of excitability, and a decrease in the activity of these channels may also, in part, contribute to PKA-dependent broadening of action potentials following stimulation (Zhang et al., 2004). Potent but transient activation of these channels can be observed in response to injection of the second messenger IP3, which releases \\(Ca^{2+}\\) from intracellular stores in bag cell neurons (Fink et al., 1988) and such IP3-dependent activation is thought to produce the brief hyperpolarization that occurs immediately before the onset of afterdischarge. A high density of \\(Ca^{2+}\\)-activated \\(K^+\\) (BK) current normally inhibits the onset of afterdischarge (Nick et al., 1996) and an increase in BK current follows an afterdischarge in mature animals, contributing to the subsequent refractory state that limits further ELH secretion (Zhang et al., 2002).

There are two alternative transcripts of the Slo gene expressed in bag cell neurons. These two isoforms differ in the presence (Slo-a) or absence (Slo-b) of a consensus phosphorylation site for PKA (Zhang et al., 2004). The isoforms differ, predictably, in their response to application of the catalytic subunit of PKA. Activation of PKA reduces the open probability of Slo-a, an effect that is reversed by a PKA inhibitor. By contrast, PKA had no effect on Slo-b. The PKA-regulated Slo-a subunit is present in adult, but not juvenile, bag cell neurons. In juvenile animals, afterdischarges are inhibited by a high density of BK channels (Nick et al., 1996) <vida supra> . Patch clamp recordings from adult and juvenile neurons have confirmed that PKA decreases BK channel activity only in adults (Zhang et al., 2004).

Recent work has also demonstrated a prominent \\(K^+\\) channel that is activated by intracellular \\(Na^+\\) in the bag cell neurons. This large-conductance channel is encoded by the Aplysia Slack gene and its function is currently under investigation (Chen et al., 2007, Zhang et al., 2008c).

### [edit] A voltage-gated non-selective cation channel is activated by association with PKC and calmodulin at the onset of afterdischarge

A non-selective cation channel that is permeable to \\(Na^+\ ,\\) \\(K^+\\) and \\(Ca^{2+}\\)ions provides the prolonged depolarization that drives afterdischarge (Wilson & Kaczmarek, 1993; Wilson et al., 1996; Magoski & Kaczmarek, 2005, Magoski & Geiger, 2006). Activation of this channel requires a physical association between the channel and both calmodulin and PKC (Wilson et al., 1998; Lupinsky & Magoski 2006). Calmodulin acts as a transducer for the stimulatory effects of \\(Ca^{2+}\ ,\\) which is elevated during the afterdischarge (Lupinsky & Magoski, 2006; Fisher et al., 1994).

The association between this cation channel and PKC is dependent on src homology 3 domain protein-protein interactions (Magoski et al., 2002). Moreover, this channel-PKC association is plastic and regulated by src tyrosine kinase itself (Magoski, 2004; Magoski & Kaczmarek, 2005). This physical association/dissociation of the ion channel-enzyme complex appears to represent the major mechanism that controls the transition from afterdischarge to the refractory state (see Figure 2). Entry of the bag cell neurons into the prolonged refractory period that follows an afterdischarge includes dissociation of the channel/PKC complex and an elevation of phosphotyrosine levels (Magoski, 2004; Magoski & Kaczmarek, 2005). Thus, the loss of the channel-PKC association, in association with the increase in BK current described above, represents a molecular basis for the prolonged refractory state. Interestingly it is \\(Ca^{2+}\\)influx through the cation channel that appears selectively to initiate the biochemical pathway that ultimately renders the neurons refractory (Kaczmarek & Kauer, 1983; Magoski et al., 2000).

### [edit] Insertion of calcium channels at neuropeptide release sites

Activation of PKC at the onset of an afterdischarge causes the recruitment of new voltage-dependent \\(Ca^{2+}\\) channels to the plasma membrane. In channel recordings at the soma, the increase in current produced by activation of PKC is associated with the appearance of a new 24 pS conductance (Strong et al., 1987). Fura-2 \\(Ca^{2+}\\) imaging <here and subsequently Ca has become monovalent, needs correction> shows that activation of PKC produces new sites of \\(Ca^+\\) entry at the distal tips of bag cell neurites (Knox et al., 1992), where neuropeptide secretion is believed to occur (Frazier et al., 1967).

There are two voltage-dependent calcium channel genes expressed in bag cell neurons \\(Ca_v1\\) and \\(Ca_v2\\) (White & Kaczmarek, 1997; Zhang et al., 2008b).A variety of evidence indicates that the PKC-regulated channels correspond to the \\(Ca_v2 \alpha\\) subunit (White & Kaczmarek, 1997; White et al., 1998; Zhang et al., 2008b), which is normally confined to the membranes of granules found throughout the soma and in the central region of growth cones (White and Kaczmarek, 1997). Following activation of PKC the organelles containing \\(Ca_v2 \alpha\\) subunit undergo rapid trafficking to the distal edge of the growth cone where they are inserted into the plasma membrane (Zhang et al., 2008b). The insertion of these channel could represent the rapid assembly of new sites for the secretion of neuropeptides.

### [edit] Other sources of calcium for neuropeptide release

In addition to the voltage-dependent \\(Ca^{2+}\\) and non-selective cation channels described above, there exist one or more voltage-independent non-selective cation channels in the plasma membrane of bag cell neurons (Knox et al., 1996; Whim & Kaczmarek, 1998; Hung & Magoski, 2007; Gardam et al., 2008). While the function and molecular identity of these channels in not fully understood it is possible that they play a role in the filling of intracellular stores that release \\(Ca^{2+}\\) in response to stimulation, thereby maintaining neuropeptide release after the onset of discharge.

\\(Ca^{2+}\\) ions are known to be released from several independent intracellular stores in the bag cell neurons. In common with all other neurons, these include mitochondria and the endoplasmic reticulum (Knox et al., 1996; Jonas et al., 1997; Geiger & Magoski, 2008). Of particular significance for neuropeptide release, however, is a non-endoplasmic reticulum, non-mitochondrial store that is activated by insulin and causes ELH secretion (Jonas et al., 1997). In contrast to the endoplasmic reticulum, this store is present at the distal tips of neurites,where secretion occurs, and may represent the secretory granules themselves. This hypothesis is supported by the findings of Kachoei et al. (2006), who demonstrated the presence of an acidic store in bag cell neurons, with properties similar to that of the insulin-sensitive store.

Other \\(Ca^+\\)-release mechanisms include a store-operated Ca2+ influx pathway (Knox et al., 1996; Kachoei et al., 2006), which could also contribute to secretion. Finally, \\(Ca^{2+}\\) influx-induced \\(Ca^{2+}\\) release from both the endoplasmic reticulum and mitochondria represents yet another possible mechanism for promoting elevations of internal \\(Ca^{2+}\\) (Fisher et al., 1994; Geiger & Magoski, 2008).

### [edit] Summary

Very brief stimulation of an input to the bag cell neurons triggers long-lasting changes in their intrinsic electrical properties, and in their ability to secrete neuropeptides. Because of the reproducible stereotypical nature of the stimulation-induced changes in their cellular properties, and because the biological function of these neurons is relatively well understood, they have served as a model system for investigations of prolonged changes in neuronal excitability, as well as of neuropeptide synthesis, processing and release.

## [edit] References

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Internal references

* Valentino Braitenberg (2007) Brain. Scholarpedia, 2(11):2918.

* Eugene M. Izhikevich (2006) Bursting. Scholarpedia, 1(3):1300.

* Paul R. Benjamin (2008) Lymnaea. Scholarpedia, 3(1):4124.

## [edit] Recommended reading

* Arch S, Berry RW (1989) Molecular and cellular regulation of neuropeptide expression: the bag cell model system. Brain Research - Brain Research Reviews. 14:181-201

* Conn PJ, Kaczmarek LK (1989) The bag cell neurons of Aplysia: a model for the study of the molecular mechanisms involved in the control of prolonged animal behaviors, Molecular Neurobiology 3:237-273, 1989

* Nagle GT, Painter SD, Blankenship JE (1989) Post-translational processing in model neuroendocrine systems: precursors and products that coordinate reproductive activity in Aplysia and Lymnaea. J Neuroscience Res 23:359-370, 1989

* Levitan IB, Kaczmarek LK (2002)The Neuron; Cell and Molecular Biology, (3rd Edition) Oxford University Press

## [edit] External links

* Leonard Kaczmarek’s webpage

## [edit] See also