Whole-Body Imaging of Neural and Muscle Activity during Behavior in Hydra vulgaris: Effect of Osmolarity on Contraction Bursts

Visual Abstract


Introduction
Calcium imaging of neuronal circuits (Yuste and Katz, 1991) has enabled recent investigations of the circuit basis of animal behavior in a number of transparent organisms such as Caenorhabditis elegans, Drosophila larvae, and zebrafish embryos (Nagel et al., 2005;Liewald et al., 2008;Honjo et al., 2012;Cong et al., 2017;Kim et al., 2017). While these studies have focused on particular parts of the nervous system, to systematically understand the neural code, i.e., the relation between the activity of a nervous system and behavior, it would be ideal to measure the activity of the entire nervous system and the entire muscular tissue during the entire behavioral repertoire of an animal. This is now possible with the transparent fresh-water cnidarian Hydra vulgaris, using transgenic strains that express calcium indicators in every neuron (Dupre and Yuste, 2017) and every muscle cell of the body (Szymanski and Yuste, 2019), and applying machine learning to systematically analyze its behavior (Han et al., 2018). Hydra has a simple body consisting of ectoderm and endoderm myoepithelial cells. Muscular processes, myonemes, run longitudinally in the ectoderm and radially in the endoderm. Thus, each myoepithelial layer can have distinct functions in different behaviors, but can also become coactive during sustained contractions (Szymanski and Yuste, 2019).
Hydra has one of the simplest nervous system in evolution, with several hundreds to a few thousand neurons, depending on the size of the animal (Hadzi, 1909;Parker, 1919;Westfall et al., 1991). The simplicity of Hydra's system gives hope that systematic measurements of the neural and muscular activity of behaving Hydra could be used to decipher the mechanisms of behavior. Hydra neurons are believed to be multifunctional. A sensory neuron with sensory cilia also synapses with epithelial cells as a motor neuron (Westfall, 1973). These neurons are organized in two independent nerve nets, in the ectoderm and endoderm (Dupre and Yuste, 2017). Hydra's nerve nets are distributed throughout the body of the animal, without any cephalization (Epp and Tardent, 1978). Several independent neuronal circuits, interspersed within the nerve nets, are active synchronously in an oscillating manner. The

Significance Statement
We imaged whole-body muscle and neuronal activity in Hydra in response to different physiological and environmental conditions. Osmolarity bidirectionally altered Hydra contractile behavior in a reflexive fashion. These changes were accompanied by specific changes in the activity of one neuronal circuit and one set of muscles. By providing neurobiological mechanisms for a reflex in a cnidarian, this work is a step toward comprehensive deciphering of the mechanisms of animal behavior by measuring the activity of all neurons and muscle cells. main ones named contraction burst (CB) and rhythmic potential (RP)1 circuits, involve independent groups of ectoderm neurons, whereas a third circuit, the RP2 circuit, involves endodermal cells (Dupre and Yuste, 2017). These three circuits are associated with three different motor behaviors: CBs (CB circuit), elongation (RP1), and egestion (RP2; Dupre and Yuste, 2017).
Hydra is a fresh-water animal living in ponds, lakes and streams. Because of this, Hydra experiences fluctuations in temperature and osmolarity as well as the amount of food available, which determines its body size. Previous research has described Hydra responses to changes in environmental and physiological conditions. Those include decreases in contractions with increased osmolarity (Benos and Prusch, 1973) and after feeding (Grosvenor et al., 1996;Rushforth and Hofman, 1972) and necrosis after acute increases in temperature (Bosch et al., 1988). These past studies show that external modification of Hydra behavior is possible.
Motivated by this work, we explored systematically how different environmental conditions affect Hydra behavior, focusing on body contractions. Do do so, we performed measurements of Hydra behavior under standard conditions in mounted and freely behaving animals and used calcium imaging to measure how neurons and muscular cells responds to physiological and environmental conditions important for their survival. Experimental conditions included high or low osmolarity (control, 50 mM sucrose or diH 2 O), temperature (23°C or 30°C), food (zero, one, and four shrimp per day for a week), and body size (mature vs newly released buds). In each of these conditions, we measured the number of contractions and foot detachments in behavior assays, the ectodermal and endodermal muscle activity, and the activity of the CB and RP1 neuronal circuits.
We expected to see major changes in behavior, neuronal, and muscle activity, as the chosen conditions are essential to Hydra survival. But surprisingly, in mounted preparations, we only found robust effects due to osmolarity. Increased osmolarity decreased contractions frequency, consistent with Benos and Prusch (1973), decreased foot detachments and also decreased the activity of CB neurons and ectodermal muscle cells, whereas decreased osmolarity had opposite effects, as a reflex. Our results indicate that Hydra's CB circuit senses osmolarity to control ectodermal muscle and generate contractile behaviors, revealing a specific neuro-muscular reflex that probably evolved for osmoprotection.

Materials
Sucrose and sea salt were purchased from Sigma. Brine shrimp, Artemia nauplii, were obtained from Brine Shrimp Direct. We used transgenic Hydra expressing GCaMP6s in neurons (Dupre and Yuste, 2017) or in ectoderm/endoderm muscle cells (Szymanski and Yuste, 2019).

Hydra culture
Hydra were maintained in media composed of 1.3 mM CaCl 2 , 0.02 mM MgCl 2 , 0.03 mM KNO 3 , 0.5 mM NaHCO 3 , and 0.08 mM MgSO 4 in an 18°C incubator. Hydra were fed brine shrimp three times a week and were starved for 2 d before an experiment.

Environmental or physiological conditions
The following conditions were used. (1) Food: Hydra were fed zero, one, or four shrimp every day for a week. Hydra were starved for 1 d before an experiment.

Calcium imaging
Wide-field calcium imaging of Hydra was conducted at 2 Hz using a fluorescence dissecting microscope (Leica M165) equipped with a long-pass GFP filter set (Leica filter set ET GFP M205FA/M165FC), 1.63Â Plan Apo objective, and a sCMOS camera (Hamamatsu ORCA-Flash 4.0). A mercury arc lamp was used to illuminate the sample. Hydra were mounted between coverslips with 100-to 200-mm spacers, depending on animal thickness. All imaging was conducted at a room temperature ;23°C unless indicated.

Behavior analysis
The number of contractions and foot detachments were manually scored from calcium imaging movies (mounted Hydra between coverslips) or movies of freely moving Hydra in glass-bottom dishes (MatTek). Five animals were placed per well (depth is 700-750 mm) for 1-h recordings.

Analysis of neural and muscular activity
Values for whole-body fluorescent intensity in each frame over time were obtained with ImageJ and used to detect CB and RP1 pulses using a semi-automated program in MATLAB. Whole-body muscle activity was analyzed in the same manner.

Analysis of body column width
Hydra were imaged at 0.5 Hz using a dissecting microscope (Leica M165), 1.63Â Plan Apo objective, and sCMOS camera (Hamamatsu ORCA-Flash 4.0). Hydra were mounted between coverslips with around 200-mm spacer in control media or in high-osmolarity solution (50 mM sucrose). To measure width, the body column of Hydra was fitted into ellipse using a program written by MATLAB. The lowest values from each cycle were used to calculate average width at the end of the elongation.

Statistical methods
Data are shown as average 6 SEM in figures and in the text. Two-tailed unpaired Student's t test or one-way  ANOVA with Tukey's multiple comparison test were conducted in GraphPad Prism software (Table 1).

Code accessibility
All code is available as Extended Data 1. The MATLAB code was used to analyze neural and muscular activity in Figs. 2-4.

Hydra's contractile behavior affected by media osmolarity
Hydra has a small repertoire of highly stereotypical behaviors (Han et al., 2018). One of the most noticeable ones are spontaneous periodic contractions, known as "contraction bursts" (Wagner, 1905;Reis and Pierro, 1955;Passano and Mccullough, 1964). Possible roles of contractions by Hydra include foraging, protection by retraction (Miglietta et al., 2000;Swain et al., 2015), food digestion (Shimizu and Fujisawa, 2003), and excreting excess water from the body (Macklin et al., 1973). Another common behavior of Hydra is locomotion, i.e., translocation of the foot from one place to another. This is initiated by "foot detachment," where the basal disk detaches from a substrate's surface (Rodrigues et al., 2016).
We first tested how these two simple behaviors of Hydra were affected by various physiological and environmental conditions. Conditions chosen included amount of food, osmolarity or temperature of media, and the size of an animal. For the amount of food, Hydra was starved for 1 d before an experiment. For each condition, the frequency and duration of contractions and foot detachments were measured. In mounted preparations, where specimens are place in a microscope chamber with a spacer, osmolarity or body size robustly changed the frequency of contractions ( Fig. 1A-C; see Materials and Methods). High-osmolarity media significantly decreased the frequency of contractions compared with control ( Fig.  1B, p = 0.0380) or low-osmolarity conditions (Fig. 1B, p = 0.0367). Similarly, high-osmolarity media significantly decreased the number of foot detachments compared with control (Fig. 1C, p = 0.0003) or low-osmolarity conditions (Fig. 1C, p , 0.0001). Also, smaller size Hydra had more contractions (Fig. 1B, p = 0.0008) but fewer foot detachments (Fig. 1C, p = 0.0378).
As mounting restricts Hydra behavior, because of compression of body between glass coverslips, we also imaged freely moving Hydra under widefield illumination in the same conditions (Movie 1). Consistent with results in mounted preparations (Fig. 1B,C), in free moving animals, high osmolarity also decreased the number of contractions compared with low osmolarity (Fig. 1E, p = 0.0100) and the number of foot detachments, compared with control (Fig. 1F, p = 0.0134) or low-osmolarity conditions (Fig.  1F, p , 0.0001). But, unlike mounted preparations, wellfed (four shrimp per day) Hydra did not show any difference in behavior, comparing with control conditions. (Fig.  1B, p = 0.8506 for contractions; Fig. 1C, p = 0.8980 for detachments). Also, in well-fed freely moving Hydra, the number of contractions decreased (Fig. 1E, p = 0.0164), while the number of foot detachments increased (Fig. 1F, p = 0.0014). High temperature also increased contractions (Fig. 1E, p , 0.0001) and foot detachments (Fig. 1F, p , 0.0001) in freely moving animals. Overall, osmolarity was the only parameter that robustly changed behavior in both freely moving and mounted specimens. As motor behaviors must be generated as a result of contractile force derived from muscle, we next assessed how these changes in behaviors are accounted for the activity of muscle cells. For these experiments, we used exclusively mounted preparation, as it is yet not feasible to image and reconstruct the activity of neurons and muscle cells in freely moving animals.

Bidirectional effects of osmolarity on ectodermal muscle activity
Hydra's body is composed of two layers of cells: ectodermal and endodermal epitheliomuscular tissues. Both epithelia are separated by an extracellular matrix called mesoglea. Inside these epithelial layers, there is a gastrovascular cavity that functions as a both gut and vasculature and carries nutrients to the entire body (Shimizu and Fujisawa, 2003). Both ectoderm and endoderm epitheliomuscular tissues generate action potentials (Dupre and Yuste, 2017; Szymanski and Yuste, 2019), which likely propagate through gap junctions (Westfall et al., 1980). These muscle cells contract in a calcium-dependent manner through myonemes, intracellular muscle processes that run longitudinally along the ectoderm and radially in