Neural mechanisms of magnetic orientation in Tritonia diomedea
Despite our growing understanding of how animals use the Earth's magnetic field, little is known about the neural mechanisms that underlie magnetic orientation behavior (Deutschlander et al., 1999
; Lohmann and Johnsen, 2000). One factor that has complicated such analyses is that most magnetic orientation research has focused on vertebrate animals (Phillips, 1986; Beason and Semm, 1987; Lohmann, 1991; Wiltschko and Wiltschko, 1991, 1995a; Able and Able, 1995). Although several vertebrates have proven to be excellent subjects for behavioral experiments, the complexity of the vertebrate nervous system makes cellular-level investigations of neural circuitry challenging.
One animal model system that appears particularly promising for studies of the neural circuitry underlying magnetic orientation behavior is Tritonia diomedea, a nudibranch mollusc. Behavioral experiments have demonstrated that this animal orients to the Earth's magnetic field (Lohmann and Willows, 1987) in the lab, and field displacement experiments suggest that they use magnetic orientation to guide themselves between shallow and deeper areas (Willows, 1999). In addition, the central nervous system is relatively simple, consisting of approximately 7,000 neurons in six fused ganglia (Boyle et al., 1983). Many of these neurons can be identified individually on the basis of color, size, and location within the central ganglia (Fig. 3a). Moreover, the nervous system is readily accessible for electrophysiological recordings both in semi-intact and isolated brain preparations (Willows et al., 1973).
Intracellular electrophysiological recordings have demonstrated that three bilaterally symmetric pairs of identifiable neurons respond with altered electrical activity to changes in earth-strength magnetic fields (Lohmann
et al., 1991
; Wang
et al., 2003
, 2004
). Two of these pairs, known as the Pd5 and Pd6 neurons, are excited by changes in direction of the ambient magnetic field (Lohmann
et al., 1997
; Popescu and Willows, 1999
; Wang
et al., 2003
). The third pair, known as the Pd7 neurons, is inhibited by the same magnetic stimuli that excite Pd5 and Pd6 (Wang
et al., 2004
). All six of the magnetically responsive cells (LPd5, RPd5, LPd6, RPd6, LPd7, and RPd7) presumably function in the neural circuitry underlying magnetic orientation behavior.
The function of the Pd5 and Pd6 neurons
Recent anatomical, electrophysiological, and immunochemical analyses have provided insight into the roles that some of these neurons are likely to play (Popescu and Willows, 1999; Wang et al., 2003; Cain et al., in review). Both Pd5 and Pd6 have neurites that branch extensively within the pedal ganglia, as well as peripheral branches (axons) of the primary neurite that enter ipsilateral pedal nerves (Lohmann et al., 1991; Wang et al., 2003). These peripheral axons appear to innervate parts of the ipsilateral foot epithelium (Fig. 3b; Wang et al., 2003; Cain et al., in review). Action potentials carried by these axons propagate from the CNS to the peripheral tissues (Wang et al., 2003; Cain et al., in review).
Pd5 and Pd6 are large cells (often >400 µm) and appear white under epi-illumination. Both of these features are characteristic of neurons that produce peptide neurotransmitters (Snow, 1982). A previously unknown group of three neuropeptides (TPeps) has been isolated from the cell bodies of Pd5 and Pd6 using HPLC (Fig. 4a; Lloyd et al., 1996). These peptides localize to the foot tissues innervated by the nerves containing axons of Pd5 and/or Pd6 (Fig. 4b; Willows et al., 1997). Moreover, these peptides are found in dense-cored synaptic vesicles within neurites throughout the foot epithelium (Cain et al., in review).
These findings indicate that the Pd5 and Pd6 are peptidergic, efferent neurons that are likely to function in generating or modulating the motor output of the magnetic orientation circuitry. Although the precise role of these cells has not been determined, three major types of effector cells in the periphery of
Tritonia might plausibly be affected by TPeps released from the Pd5 and Pd6 cells: muscles, cilia, and mucus glands.
The muscles of the foot generate dorsal and ventral flexions during the escape swim and also help turn the animal during mucociliary crawling (Willows et al., 1973). At the onset of swimming, both the Pd5 and the Pd6 neurons burst briefly, but then remain inactive for the duration of the swim (Fig. 4c). This pattern of activity implies that these cells are not directly involved in producing the dorsal and ventral flexions that comprise swimming. Similarly, neither intracellular stimulation of Pd5 or Pd6 nor direct application of TPeps to isolated foot patches results in discernable muscle contraction (Willows et al., 1973; S.D.C., unpublished). Thus, at present, no evidence exists to suggest that the Pd5 or Pd6 neurons control muscle contraction.
The Pd5 and Pd6 cells do, however, appear to control or modulate the activity of ciliated cells (Popescu and Willows, 1999; Wang et al., 2003; Cain et al., in review). A dense field of cilia covers the pedal epithelium and propels the animal forward during muco-ciliary crawling, the primary mode of locomotion in this animal (Audesirk, 1978a, b). Application of TPeps to isolated foot patches or to isolated ciliated cells results in increased ciliary activity (Willows et al., 1997). In addition, increases in electrical activity in the Pd5 cells are correlated with increases in muco-ciliary transport across the foot (Popescu and Willows, 1999). These finding suggest that the role of the Pd5 and Pd6 neurons is cilio-motor in nature.
The precise mechanism by which TPeps increase ciliary beating and ciliary transport rate is not known. Both processes are tightly coupled to mucus secretion, as mucus provides the viscous environment in which the cilia beat (Denny, 1981). Thus, whereas one possibility is that TPeps act directly on cilia to increase their beat frequency, another is that Pd5 and Pd6 influence crawling by altering the amount or types of mucus being secreted. The two possibilities are not mutually exclusive, and clear evidence for either has not yet been obtained.
The function of the Pd7 neurons
Despite some morphological similarities between the Pd7 neurons and the Pd5/Pd6 (i.e., large size, whitish cell body, location in the pedal ganglia), the function of the Pd7 cells may differ from that of the other two pairs of magnetically responsive neurons (Figs. 3a and 4a; Wang et al., 2004). Unlike the Pd5 and Pd6 neurons, the Pd7 cells are inhibited by rotations of the ambient magnetic field. The neurites of Pd7 extend to the cerebral ganglia and one large axon projects from the brain to the anterior tissues through cerebral nerve 3. Action potentials in the Pd7 neuron propagate from the central ganglia toward the periphery (Wang et al., 2004). The target cells to which the Pd7 project have not yet been determined, but the nerves that contain Pd7 axons innervate areas near the mouth, oral veil, and rhinophores (Willows et al., 1973).
Although the function of the Pd7 neurons is not known, one possibility is that these cells control or modulate some subtle aspect of turning or locomotion that occurs during magnetic orientation behavior. For example, the cells might play a role in controlling movements of the oral veil, rhinophores, or other anterior structures as the animal alters or maintains its heading.
An alternative possibility is that the Pd7 neurons function in suppressing behavior that might otherwise impede orientation or locomotion. It is noteworthy that, during the period immediately after an escape swim when Tritonia normally crawls vigorously (Audesirk and Audesirk, 1980), spiking in the Pd7 neurons is greatly reduced relative to pre-swim levels (Fig. 4c). In contrast, spiking in the Pd5 and Pd6 increases after an escape swim (Fig. 4c). An interesting speculation is that the Pd7 neurons might modulate cilia that line the esophagus and are involved in feeding, and that feeding is suppressed during magnetic orientation and after swimming. At present, however, no evidence exists to support or refute this scenario, and additional research will be needed to determine the function of the Pd7 cells.
In summary, a reasonable working hypothesis is that the Pd5 and Pd6 neurons are involved in controlling or modulating the motor output of the magnetic orientation circuitry. These neurons appear likely to influence the ciliary beat rate of the foot epithelium and may therefore play a role in helping the animal align itself with a particular magnetic direction, crawl along a particular heading, or both. The function of the Pd7 neurons is less clear, but a possible role in suppressing behavior incompatible with orientation or locomotion is presently suspected.
As the search for neurons involved in the magnetic orientation circuitry expands, some logical targets for future investigation are neurons presynaptic to the Pd5, Pd6, and Pd7. Among these are neurons that have previously been identified as part of the Tritonia swim central pattern generator (DSIs, VSIs, C2), which also appear to mediate post-swim crawling by influencing Pd5 and Pd6 (Popescu and Frost, 2002). Therefore, this set of neurons might represent a multifunctional swim/crawl network as proposed by Popescu and Frost (2002). The relative simplicity of the Tritonia nervous system provides reason to hope that careful, sustained investigation will eventually lead to a thorough understanding of the neural circuitry underlying magnetic orientation behavior in this neuroethological model animal.
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