Relationship between developmental synaptic ...

Acta Biologica Hungarica 59 (Suppl.), pp. 97–100 (2008) DOI: 10.1556/ABiol.59.2008.Suppl.15

RELATIONSHIP BETWEEN DEVELOPMENTAL SYNAPTIC MODULATION AND CONDITIONING-INDUCED SYNAPTIC CHANGE IN LYMNAEA* SHORT COMMUNICATION T. KARASAWA,1 NAO SATO,2 T. HORIKOSHI2 and M. SAKAKIBARA2** 1 Course of Biological Science and Technology, Graduate School of High-Technology for Human Welfare and 2 Department of Biological Science and Technology, School of High-Technology for Human Welfare, Tokai University, 317 Nishino, Numazu 410-0321, Shizuoka, Japan

(Received: October 22, 2007; accepted: December 7, 2007)

Though adult Lymnaea are bimodal breathers, young animals breathe mainly through the skin and adults through the lung. Operant conditioning changes adult breathing behavior from aerial to cutaneous. We hypothesized that this behavioral change is caused by alterations in the neuronal circuit during both development and conditioning. We focused our study on whether the synaptic connection between RPeD1 and RPA6 neurons is modulated during development and conditioning. Our findings indicated that the RPeD1 has an excitatory monosynaptic contact with the RPA6 in young naïve and operantlyconditioned adult animals. The relationship of this contact was well correlated with their respiratory behavior. Keywords: Conditioning – synaptic modulation – respiratory behavior – Lymnaea

Lymnaea respiratory behavior changes during development; young animals breathe mainly through the skin, whereas adults breathe mainly through the lung [9]. Lukowiak et al. demonstrated that operant conditioning modulates respiratory behavior in adult animals; i.e. prior to conditioning, adult animals show pulmonary respiration whereas after operant conditioning they breathe via the skin [2]. We hypothesized that this behavioral change is due to alterations in the neuronal circuit during both development and conditioning. Respiration in Lymnaea is controlled by three neuron groups; sensory neurons, central pattern generators (CPG), and motor neurons [4, 8]. The right pedal dorsal 1 (RPeD1) is a CPG and neurons in the right pari-

* Presented during the 11th ISIN Symposium on Invertebrate Neurobiology, 25–29 August, 2007, Tihany, Hungary ** Corresponding author; e-mail: [email protected] 0236-5383/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

98

T. KARASAWA et al.

etal A (RPA) group are the motoneurons that control aerial respiratory muscles [7, 8]. Among the RPA neurons, RPA6 has a monosynaptic contact with RPeD1 [1]. We examined whether the synaptic connection between the RPeD1 and RPA6 neurons is modulated during development and/or conditioning. Three experimental groups were analyzed behaviorally and electrophysiologically; naïve young (shell length: > 15 mm) control, adult (shell length; < 20 mm) control, and operantly-conditioned adult. The animals were described previously [6]. The procedure for operant conditioning was performed according to Lukowiak et al. [2]. To select for animals according to respiratory behavior, each animal was placed into a 500-ml flask of hypoxic water (bubbled with N2 for 15 min beforehand in a 1000-ml glass beaker). Young and adult animals that reached the surface of the hypoxic water fewer than 3 times over a 15min period were not included in the study. Four sessions of operant conditioning session were performed for 15 min every 1 h in 1 day. In each session, when the animal reached the water surface, a tactile stimulus was delivered to the pneumostome area with a hand-held Plexiglas rod each time the animal attempted to open its pneumostome. The tactile stimulation to the pneumostome area always evoked a wholebody withdrawal response, which caused the pneumostome to close immediately. The number of tactile stimulations to the pneumostome was recorded for each animal. Memory retention tests were performed 24 h later. Naïve Young

Operant Conditioned Adult

RPA6 20mV

25mV

25ms

Normal Saline

RPeD1 50mV

100mV

25mV

1 2 3 4 5 6

Hi? D | iv. Saline

100mV

Fig. 1. Synaptically-induced excitatory effects of RPeD1 on RPA6. In both cases, excitation in RPeD1 induced a monosynaptic excitatory effect in RPA6. Inset shows serotonin-positive neurons, named RPA1 to RPA6, in the right parietal ganglion

Acta Biologica Hungarica 59, 2008

99

Operant conditioning in Lymnaea

Within a few days after the conditioning, electrophysiologic examination was performed. To assess the synaptic connection between the RPeD1 and RPA6 neurons, simultaneous intracellular recordings were made from the RPeD1 and RPA6 neurons with an isolated central nervous system preparation. The preparation and methods of electrophysiology were described previously [6]. The operantly-conditioned adult animals breathed significantly less often through the lung compared to naïve adult animals, as previously demonstrated [3]. Because RPA group neurons comprising 7 to 13 cells are serotonergic [10], we immunohistologically identified each cell from RPA1 to RPA6, based on its relative position in the right parietal ganglion. Among these neurons, we identified the right most caudal neuron as RPA6, which had a diameter of approximately 50 µm (Fig. 1). Spontaneous impulse discharges in RPeD1 occur more frequently in young animals than in adult animals [5]. In young naïve animals, cutaneous breathers, there was a clear excitatory monosynaptic connection between RPeD1 and RPA6 when the postsynaptic elements were hyperpolarized so as to generate less impulse activity in response to excitation of the RPeD1 neuron, which was induced by injecting depolarizing current. The same excitatory monosynaptic connection was observed in the operantly-conditioned adult preparation, i.e., depolarization of RPeD1 induced an excitatory postsynaptic potential in RPA6, even in high divalent cation-containing saline (24 mM Mg2+; 12 mM Ca2+; Fig. 1). On the other hand, we did not detect any excitatory effect at this synapse in the naïve adult preparation. These electrophysiologic findings were well correlated with the respiratory behavior. Because we do not know the physiologic role of RPA6 in respiratory behavior, we cannot refer to these synaptic changes as direct conditioning-induced synaptic modifications. Previous studies, however, demonstrated that RPA group neurons are motoneurons that control the mantle cavity muscle or pneumostome opener muscle [7, 8]. The present findings indicate that the RPeD1 to RPA6 synapse mediates respiratory information related to aerial respiration. REFERENCES 1. Abe, Y. (2007) The characteristics on synaptic contact with RPeD1 and RPA group neurons in Lymnaea stagnalis. (MS thesis). Numazu, Tokai University. 2. Lukowiak, K., Adatia, N., Krygier, D., Syed, N. (2000) Operant conditioning in Lymnaea: evidence for intermediate- and long-term memory. Learn. Mem. 7, 140–150. 3. Lukowiak, K., Sangha, S., Scheibenstock, A., Parvez, K., McComb, C., Rosenegger, D., Varshney, N., Sadamoto, H. (2003) A molluscan model system in the search for the engram. J. Physiol. Paris 97, 69–76. 4. Lukowiak, K., Syed, N. (1999) Learning, memory and a respiratory central pattern generator. Comp. Biochem. Physiol. A Mol Integr. Physiol. 124, 265–274. 5. McComb, C., Meems, R., Syed, N., Lukowiak, K. (2003) Electrophysiological differences in the CpG aerial respiratory behavior between juvenile and adult Lymnaea. J. Neurophysiol. 90, 983–992. 6. Sakakibara, M., Aritaka, T., Iizuka, A., Suzuki, H., Horikoshi, T., Lukowiak, K. (2005) Electrophysiological responses to light of neurons in the eye and statocyst of Lymnaea stagnalis. J. Neurophysiol. 93, 493–507.


100

T. KARASAWA et al.

7. Syed, N. I., Harrison, D., Winlow, W. (1991) Respiratory behavior in the pond snail Lymnaea stagnalis. J. Comp. Physiol. A 169, 541–555. 8. Syed, N. I., Winlow, W. (1991) Respiratory behavior in the pond snail Lynmaea stagnalis. J. Comp. Physiol. A 169, 557–568. 9. Taylor, B. E., Lukowiak, K. (2000) The respiratory central pattern generator of Lymnaea: a model, measured and malleable. Respir. Physiol. 122, 197–207. 10. Winlow, W., Benjamin, P. R. (1977) Postsynaptic effects of a multiaction giant interneurone on identified snail neurones. Nature 268, 263–265.
