Free bulk D-Asp is abundant in the CNS of both vertebrates and invertebrates. D-Asp has been proposed to have a role in neural development, based on its abundance in neonatal rat brain that declines dramatically after birth (Sakai et al., 1998
), yet persists in the adult (Schell et al., 1997
). In adults, D-Asp appears to have an endocrine role, stimulating the release of hormones from the hypothalamus, pituitary, and testes (D’Aniello, 2007
). Although D-Asp has not been tracked in larval or early juvenile Aplysia, free D-Asp is present in the CNS of adult Aplysia species (Zhao and Liu, 2001
; Miao et al., 2005; Spinelli et al., 2006
We have demonstrated a subpopulation of neurons in Aplysia characterized by D-Asp-activated channels that are insensitive to L-Glu. D-Asp-induced whole cell currents have been documented in Aplysia (Miao et al., 2005) and in other species (Brown et al., 2007
; Gong et al., 2005
); responses to L-Asp in Aplysia californica
and A. dactylomela
also have been documented (Carpenter et al., 1977
). D-Asp has been hypothesized to act as a partial or full agonist at L-Glu receptors of the NMDA, kainate, or AMPA receptor subtypes, depending on the preparation. In support of the ability of D-Asp to act at specific Glu receptors, the relative potency of D-Asp at NMDA-like receptors has been documented (Verdoom and Dingledine, 1988). D-Asp had additional actions opposite that of an excitatory agonist, acting as an antagonist at AMPA receptors (Gong et al., 2005
), and activating a Cl−
conductance as a by-product of uptake by the Glu transporter (Carpenter et al., 1995
; Huang et al., 2004
). The idea of a receptor-channel activated by D-Asp but not L-Glu is untested thus far.
D-Asp is known to activate NMDA-like receptors (Verdoom and Dingledine, 1988), and the electrophysiological characteristics of NMDA-like receptors have been described in molluscan species (Moroz et al., 1993
). Certain characteristics of the D-Asp current documented here share similarities with molluscan NMDA-like receptor currents. The D-Asp-activated current was inward at −70 mV in cultured Aplysia BSC and PVC neurons. This voltage is hyperpolarized compared to the resting potential of −40 to −55 mV observed in PVC and BSC cells in reduced preparations in which the synapse was intact (Walters et al., 1983a
; Walters et al., 2004
). At this voltage, current through NMDA receptors of vertebrates would be negligible due to Mg2+
block of the channel (Mayer and Westbrook, 1987
). In contrast, molluscan NMDA-like receptors in both marine and freshwater species are free of constitutive block by Mg2+
(Moroz et al., 1993
), and open at hyperpolarized potentials. While this may be crucial to NMDA receptor function in marine mollusks, in which the Mg2+
concentration of hemolymph is high (> 10 mM), in freshwater species the requirement for Mg2+
independent function is not as clear, since in these species hemolymph Mg2+
is lower (≤ 1 mM; Gustafson et al., 2005
; Shakhmatova et al., 2006
). An apparent independence from constitutive block of the channel by Mg2+
ions at the resting potential allies the channel studied here with NMDA channels in other molluscs such as Lymnaea, but sets it apart from mammalian NMDA receptors (Dingledine et al., 1999
). The observation that half of Aplysia neurons from pleural ganglia had D-Asp induced currents unresponsive to L-Glu suggests that the D-Asp channel observed in many PVC cells may be unique, but closely related to molluscan NMDA channels.
Both NMDA-like and AMPA-like receptors, termed ApNR1 and ApGluR1 and ApGluR2, respectively, have been cloned from Aplysia (Ha et al., 2006
; Li et al., 2009
), and named for homology with vertebrate receptors in the pore forming regions (Sprengel et al., 2001
). These receptors share with mammalian homologues 95% identity of the pore regions important for ion conduction and 80% identity of the ligand binding domains, suggesting they will show biophysical similarities to vertebrate receptors in these properties. In situ hybridization localized ApNR1 to neurons within most Aplysia ganglia, including PVC and BSC neurons (Ha et al., 2006
). Thus it is possible that the D-Asp receptor-channel studied here is ApNR1. Because successful expression and physiological characterization of the cloned receptors has not yet occurred, due possibly to heteromultimeric composition of the native receptors (Hutton et al., 1991
), characterization of native receptors in intact neurons may be the best way to identify them physiologically. A detailed characterization of D-Asp induced currents in BSC cells is in progress (Carlson and Fieber in preparation).
The tail withdrawal reflex of Aplysia consists of monosynaptic sensory-motoneuron pairs in which the sensory neuron mechanoefferent located in the PVC is believed to be directly excited by tail touch or shock (Walters et al., 1983a
). This tail stimulus is transmitted to effector motoneurons in the nearby pedal ganglion that directly contract the tail. L-Glu released at the synapse causes excitation of the motoneuron. L-Glu is the only hormone known to be involved in the unmodified circuit (Antzoulatos and Byrne, 2004
), because the sensory neuron is activated by electrical stimulation at the tail, and synaptic connections to either other sensory neurons in the PVC or interneurons are unlikely (Walters et al., 1983b
); in this regard, PVC cells are regarded as a homogeneous population. The sensory-motoneuron synapse is, however, capable of being modulated by hormones whose actions result in facilitation (serotonin, Walters et al., 1983b
) or synaptic depression of the reflex (NMDA, Walters et al., 1983b
; L-Glu, FMRFamide, Xu et al., 1994
; Aplysia Mytilus
inhibitory peptide-related peptide, McDearmid et al., 2002
), and it is possible that D-Asp-induced excitatory currents in PVC cells such as those documented here also play a modulatory role.
Without additional functional information about the synapses involving PVC cells, the higher resting potential of senescent PVC cells suggests only that these cells were healthy at the time of experiments.
Although much less is known about the function and role of BSC neurons, they are also mechanosensory cells whose axons travel to the periphery through the buccal nerves, and appear to innervate the buccal mass and perioral area (Fiore and Geppeti, 1981
; Walters et al., 2004
). BSC cells respond to strong physical stimulation with long-duration impulses characterized by large afterhyperpolarizations, and, sometimes, afterdischarges (Walters et al., 2004
). The requirement for chemical neurotransmission of the peripheral mechanical stimulus to the BSC terminals, as well as the identity of any neurotransmitter or modulating hormone acting on sensory BSC cells is unknown. The role of the D-Asp response in BSC cells remains to be elucidated, but it may serve to modulate BSC cell function. Dopamine is the excitatory neurotransmitter of the consummatory neural feeding circuit of Aplysia (Baxter and Byrne 2006
), to which BSC cells contribute based on these prior studies. Modulation of this circuit includes synaptic facilitation and depression, leaving open the possibility that neurotransmitters such as those described for modulation of tail withdrawal, and possibly, D-Asp, contribute.
Certain significant changes observed in senescent PVC and BSC neurons suggest they may be associated with a functional deficit with age, such as the decrease in D-Asp-induced current density and decreased current frequency of PVC cells derived from senescent animals. A decrease in an excitatory current like the D-Asp current could imply a decline in excitatory modulation of PVC cells with age, or a relief from inhibitory modulation for which D-Asp currents provide negative feedback. Either scenario may contribute to a change in reflexive movement in aged Aplysia. Aged Aplysia have impaired righting and tail withdrawal reflexes (Kempsell and Fieber, unpublished data), suggesting that aging-related behavioral changes may reflect changes in nervous system function and not merely muscle wasting.
The absence of changes in D-Asp-induced currents in BSC neurons with aging suggest either that no such changes occur in BSC cells, or that these cells are aging at a different rate than PVC cells (Moroz and Kohn, in press
). Other changes likely had no significance for the aging phenomenon. Thus senescent BSC cells were significantly larger probably because molluscan neurons grow as the animal grows (Croll and Chiasson, 1989
), but may not shrink with aging as the body does. Mature animals’ cells were measured before the growth peak, while the senescent animals’ cells were measured after the peak. No such difference in capacitance with aging was observed in PVC cells. Despite an apparently larger cell body in senescent cells, the dearth of cell processes in aged PVC cells, which ordinarily contribute to the capacitance measurement, may explain the lack of significant difference in size.
Studies have shown that brain glutamatergic receptors or their agonist affinities declined during aging in humans and rodents, and were correlated with motor deficits (Segovia et al., 2001
). Although the role of D-Asp in ionotropic Glu receptor physiology and pathology is unknown, D-Asp-induced ion currents declined with aging in the Aplysia model system. Given the ease of the manipulation of the aging process in Aplysia and the large background on its nervous system function, Aplysia should be a useful model for studies on aging-related changes in brain neurotransmitter-activated ion currents.