**3. Electrophysiology method**

Human heartbeats were recorded using a PowerLab system (AD Instruments, Australia). A set of three, ready-made Ag–AgCl electrodes (Vitrode V, Nihonkoden Co. Ltd. Tokyo) were used for EKG monitoring. Permanently mounted metal electrodes were glued on the crusta‐ cean carapace for EKG recordings. These EKG signals were transferred to the PowerLab system and PC. All subjects were treated according to the ethical regulations of Tokyo Metropolitan University.

#### **4. Stress hormone measurement**

ceased their impulse discharge [1]. During the CI burst period, the heart rate disappeared or decreased significantly, although a rapid restitution of heartbeats occurred within one second. These brief cessations of heartbeats occurred intermittently and somewhat regularly during "stress-free" periods, for example, when the animal was hiding in a shelter. In contrast, the stress-free behavior was not exhibited if the lobsters or crabs were approached by humans. This response can be likened to the way cicadas stop singing when humans are in close proximity. These findings suggest that crustaceans are incredible specimens because their

Crustacean and human hearts strongly resemble each other in structure and function. It is known that homologous genes (e.g., Nkx2-5, the NK2 homeobox gene) function to form the developing heart of all animals: [6]. In crustaceans and humans, both CA and CI connect with the cardiac pacemaker cells, but CA further proceed to the ventricular muscles beyond the pacemaker cells. Why do CA control the entire heart? The answer is that CA–muscle connections can implement direct modulation of contractile force, whereas CI merely sup‐ press rhythm [5]. The resemblance between crustacean and human indicates that some knowledge obtained from crustacean should be applicable to human. See Recent report by

(1) P. Fossat et al. "Anxiety-like behavior in crayfish is controlled by serotonin." Science 344,

(2) P. FOSSAT et al. "Anxiety-like behavior in crayfish is controlled by serotonin." Society for Neuroscience, Poster 655.18/UU43 - Motivation and Emotions: Rodent Anxiety Models

I studied electrocardiograms (EKG) of both animal models and humans and used modified Detrended Fluctuation Analysis (mDFA) to calculate the scaling exponent (SI) (originally, Peng et al., [7]). In this article, I show that the SI numerically is capable of distinguishing between healthy and unhealthy hearts, and between "stressed" and "relaxed" hearts. Further, I propose the mDFA to be a viable potential method for health/stress checking if incorporated into a

Human heartbeats were recorded using a PowerLab system (AD Instruments, Australia). A set of three, ready-made Ag–AgCl electrodes (Vitrode V, Nihonkoden Co. Ltd. Tokyo) were used for EKG monitoring. Permanently mounted metal electrodes were glued on the crusta‐ cean carapace for EKG recordings. These EKG signals were transferred to the PowerLab system and PC. All subjects were treated according to the ethical regulations of Tokyo Metropolitan

(3) OUTSIDE JEB. The Journal of Experimental Biology (2014) 217, 3389-3391

stress can be detected by electrocardiograms.

Tue, Nov 18, 1:00 - 5:00 PM 2014 Washington DC.

device that can quantify stress through EKGs.

**3. Electrophysiology method**

Fossat et al.

University.

1293–1297 (2014).

362 Advances in Bioengineering

The stress response of the lobster to approach by a human (Figure 3) may involve the contri‐ bution of stress hormones, but combined hormone and EKG measurements have not been conducted. Therefore, I conducted micro-dialysis blood hormone measurements to determine whether hormones are involved in crustacean stress responses. I prepared four different HPLC-ECD (high performance liquid chromatography with electrochemical detection) machines. These machines were respectively set up to separate biogenic monoamines (dopa‐ mine, adrenaline, serotonin and their breakdown products), acetylcholine and related substances, amino acid neurotransmitters (glutamate, GABA, etc.), and peptide hormones. Blood samples were collected using a micro-dialysis probe for all HPLC analyses (Figure 4).

**Figure 4.** The micro-dialysis (MD) probe placed on the dorsal carapace of lobsters (left panel). The tip of the MD probe placed in the blood stream (right panel). H represents the heart chamber and arrows indicate the direction of flow.

**Figure 5.** HPLC chart indicating the detection of DOPAC (a breakdown product of dopamine) using the HPLC system in a blood sample collected from a crayfish (*Procambarus clarkii*) specimen.

**Figure 6.** HPLC chart indicating the detection of adrenaline (A) in a Japanese lobster (*Panulirus japonicus*) specimen*.* Adrenaline release was induced by stress from human handling. Very little adrenaline was detected 20 min and 1.5 hour after the stress stimulus. Therefore, the adrenaline content measured at these times indicates the basal adrenaline content in the blood. Note: noradrenaline (NA) was not detected.

**Figure 7.** EKG recording and MD experiment in a Japanese spiny lobster (*Panulirus japonicus*). A, a human approached the lobster tank and conducted an MD experiment. B, continuous recording from A (note: the time scale is different).

Micro-dialysis HPLC (MD-HPLC) analysis detected a few substances in the blood samples before, during, and after stress stimulation. EKGs were continuously recorded to check stress responses. Figure 7 shows a lobster's response to a human, observed through EKG recordings. Significant features are evident in Figure 7. First, approach by a human interrupted the slow, repetitive heartbeat pattern (Figure 7A). Second, the micro-dialysis experiment resulted in continuous stress to the animal (MD period in Figure 7A). Third, the stressful reaction lasted for approximately one day, the period shown by a white arrow (Figure 7B).

**Figure 8.** Micro-dialysis HPLC detection of dopamine in a Japanese spiny lobster (*Panulirus japonicus*) specimen. The bar indicates a stimulation period of 15 min.

Figure 8 shows the stress-induced release of dopamine. Blood samples were collected for 5 min and collection vials were exchanged every 5 min by an automatic solution-sampling machine. A human entered the room containing the lobster for only a short time period (15 min); this period is indicated by the black bar. The human presence evidently irritated the lobster, triggering a sharp increase in dopamine concentration, but this was followed by a rapid decrease even though the stimulus was maintained for 15 min. The same stimulus also induced adrenaline release, shown in Figure 6. The detection of stress-related hormones was confined to dopamine and adrenaline and the breakdown product DOPAC. No other hormones that directly correlated with stress behavior were detected—e.g., serotonin, noradrenaline, amino acid neurotransmitters, and peptide hormones. Further details about these experiments can be found in [8].

A characteristic feature revealed by the MD-HPLC experiments was that increased levels of stress hormones are not maintained at a consistently high level even though the stress stimulation was steadily maintained for 15 min (Figure 8). In conclusion, acute stress is governed mainly by the functioning of the nervous system, whereas chronic stress more likely relates to hormonal systems. Hormone release seems to be able to trigger a chain reaction in biochemical pathways that results in a state of chronic stress.

Based on the results of the MD-HPLC examinations, I suggest that stress can be quantified by measuring heartbeat rather than hormones. Stress can be determined using heartbeat fluctu‐ ation analysis; the method used was modified DFA (mDFA).
