Ultradian Rhythms, Sleep and Sensory Deprivation in

Victor Kuznetsov Georgy Slivko-Koltchik Yuri Panchin IITP, RAS; FBB MSU IITP, RAS; FBB MSU IITP, RAS [email protected] [email protected] [email protected]

Abstract CPGs [7, 9]. Yu. I. Arshavsky et.al. mapped the distribution of all cells in the pedal ganglion by The marine mollusk is a convenient intracellular records during rhythmical wing activity [8]. model organism for electrophysiological studies. The Such locomotor activity is not continuous in freely patterns of activity of different neurons involved in swimming Clione. Periods of activity, lasting minutes behavior like locomotion, hunting etc. are well known. In alternate with periods of inactivity during which the our experiments, the central nervous system was isolated sinks passively [13] Together with behavior like tail and electrical activity was recorded from 1A and 2A bending this results in vertical migrations [12]. locomotor motoneurons. The use of L15 culture medium Periodic alternation of the locomotor activity change the and a liquid lid method allowed keeping isolated brain deeps of the animal’s position in the water column, and alive for several days. One of the main issues in full results in Clione vertical migrations. Reasons of such sensory deprivation experiments is an elimination of all behavior could be different: light, temperature, salinity, sensory input signals. That is why Clione is a very good food searching, and predator avoidance etc. Daily vertical model. All sensory organs like eyes, statocysts and migrations are well known, but Clione has a period of 10- olfactory ganglia that are directly attached to the isolated 30 mins [7]. Not only pedal ganglion [7, 10], but fully brain could be specifically removed. Slow endogenous isolated brain without any sensory inputs (eyes, statocysts activities, found in Clione isolated brain could be viewed as and olfactory ganglia) could live and work for many days related to sleep mechanism in other and this type and be continuously recorded. Without sensory inputs of preparation can contribute to the full sensory Clione is sustained on the full sensory deprivation, but still deprivation studies. generates signals for locomotor processes.

2. Materials and Methods 1. Introduction All electrophysiological experiments were made on Clione limacina, also known as common Clione, sea Clione limacina isolated brain preparations. Eyes could be angel or is a cold-water mollusk that removed or saved, that depends on a type of experiment. predominantly lives in the Arctic Ocean and close regions. Preparations were hold in a modified L15 culture medium. It swims during the whole life. First, sea butterfly was A method of liquid lid was used in a long-time described by Friderich Martens in 1675 [1] and later experiments. The liquid lid method consists of using thin became an interesting popular biology object [2 - 6]. Body layer of oil over the medium in Petri dish. That prevents length of adult specie is about 4 centimeters and can reach drying of preparation for several days. Up to three 8-9 centimeters [7]. As other , Clione has electrodes were injected into neuronal cells. Pedal ganglion big neural cells that promoted detail neurobiology studies has two groups of interneurons (7 and 8) that control of its brain [7-12]. several groups of efferent motoneurons [14]. Clione’s nervous system contains five pairs of ganglia. For our electrophysiology studies we used the largest [7]. Patterns of activity of some neurons during locomotion, efferent neurons that control rhythmical wing activity and hunting, escape reaction, etc. of the sea angel are well- located on the dorsal surface of the pedal ganglion. 1A and known. The central pattern generator (CPG) located in the 2A neurons and sometimes one interneuron were recorded pedal ganglia seems to comprise all the basic neuronal from left or right pedal ganglion [8, 15]. The acquired mechanisms needed for producing the rhythmic efferent signal was passed through amplifier and processed by PC pattern for swimming without participation of other ganglia via ADC and LGaph2 program. All recorded data was

490 analyzed by AWK and SED scripts and Microsoft Office Excel. Vertical migrations were captured and recorded by a webcam. Acquired video was converted to the jpeg picture files and analyzed by ImageJ program.

3. Results

Living and free swimming Clione has a vertical migration behavior during all its life. Changes of animal location in a tall aquarium were recorded for many hours and are shown on the time laps figure. Isolated Clione brain cells are not silenced and do not perform continuous monotone signal, as it could be expected. They are performing rhythmical episodes of activity similar to that in actual swimming. In all experiments periods of Activity alternate with periods of Figure 2. Alternation of Active and Not Active periods with rest-like pauses or Not Active periods. Such oscillations different zoom always exist without any incoming sensory signals for several days in isolated brain. Changes of membrane potential were recorded by using microelectrode technique and analyzed via PC. We measured the ratio of Active and Not Active behavior, basing on this electrophysiological data. On the long term experiments, fully isolated or eye- saved preparation showed ratio that vary from 1 to 3.5. Clione has eyes, but their role was not studied. For mollusc light and temperature could be a signal that allow to measure animal’s depth in the water. We found a difference between light and dark phase activity in series of experiments where dark periods alternated with light periods in forty minutes sequence. During the dark phase Active as well as Not Active periods last longer. But Active periods extend more. That leads to increased Active to Not Active ratio. Increased Active phase during dark phase Figure 3. Long term experiment Active/Not Active ratio allow Clione to lift and leave unlit deep water column regions.

Figure 1. ImageJ time laps of Clione vertical migration Figure 4. The difference between average Active/Not Active behavior in a tall aquarium ratio in dark and light experiments.

491 4. Acknowledgments [8] Yu. I. Arshavsky. I. N. Beloozerova, G.N. Orlovsky, Yu. V. Panchin, G. N. Pavlova, Control of locomotion in marine This work was supported by the Russian Foundation for mollusc Clione limacina. II. Rhythmic neurons of pedal Basic Research [grant number 15-04-06148] ganglia (1985), Exp. Brain Res. 58, 263-272. [9] Yu. I. Arshavsky. I. N. Beloozerova, G.N. Orlovsky, Yu. V. References mollusc Clione limacina. III. On the origin of locomotory rhythm (1985), Exp. Brain Res. 58, 273-284. [10] Yu. I. Arshavsky. T. G. Deliagina, G.N. Orlovsky, Yu. [1] Friderich Martens vom Hamburg, Spitzbergische oder V. Panchin, G. N. Pavlova, L. B. Popova, Control of groenlandische Reise Beschreibung gethan im Jahr 1671 locomotion in marine mollusc Clione limacina. VI. Activity of (1675), p. 189, p1. P. fig. f. isolated neurons of pedal ganglia (1985), Exp. Brain Res. 63, [2] N.P. Vagner, Invertebrates in the White Sea (in Russian) 106-l 12. (1885). [11] Yu. V. Panchin, L. B. Popova, T. G. Deliagina, G.N. [3] M.V. Lebour, Clione limacina in Plymouth waters (1931), J Orlovsky, Yu. I. Arshavsky. Control of locomotion in marine Mar Biol. Ass UK 17:785-795. mollusc Clione limacina. VIII. Cerebropedal Neurons (1995), [4] B.P. Manteifel, On the biology of the pteropodial mollusk J Neurophysiol 73(5), 1912-1923. Clione limacina (Phipps) (1937), Bull Moscow Naturalist Soc, [12] Yu. V. Panchin, Yu. I. Arshavsky. T. G. Deliagina, L. Biol. Sect 46:25-35. B. Popova, G.N. Orlovsky, Control of locomotion in marine [5] J.E. Morton, Observations on the Gymnosomatous pteropod mollusc Clione limacina. IX. Neuronal Mechanisms of Spatial Clione limacina (Phipps) (1958), J Mar. Biol. Ass UK 37:287- Orientation (1995), J Neurophysiol 73(5), 1924-1937. 297. [13] N. M. Litvinova, G. N. Orlovsky, Feeding behavior of [6] C.M. Lalli, Structure and function of the buccal apparatus of Clione limacina (Pterapoda) (1985), Proc. Moscow Biol. Sot. CIione limacina (Phipps) with a review of feeding in 90: 73-77, 1985. gymnosomatous pteropods (1970), J Exp. Mar Biol. Ecol. [14] Yu. I. Arshavsky. T. G. Deliagina, G.N. Orlovsky, Yu. 4:101-118. V. Panchin, L. B. Popova, Short communication interneurons [7] Yu. I. Arshavsky. I. N. Beloozerova, G.N. Orlovsky, Yu. V. mediating the escape reaction of the marine mollusc Clione Panchin, G. N. Pavlova, Control of locomotion in marine limacina (1992), J. exp. Biol. 164, 307-31. mollusc Clione limacina. I. Efferent activity during actual and [15] D. A. Sakharov, On rhythmic activity of pedal ganglia fictitious swimming (1985), Exp. Brain Res. 58, 255-262. in the pteropodial mollusc Clione limacina (1960), Biol. Sci. 3:60-62.

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