Dopamine synergizes with caffeine to increase the heart rate of Daphnia

Daphnia culture

Daphnia were isolated from Chitti Vai river of Punjab. For the isolation of Daphnia, 0.5–2.0 litres of river water was collected and transported to laboratory. Adult Daphnia were manually identified as per the standard identification features¹⁵, and filtered out using muslin cloth. These adults were cultured in 300 ml glass jars containing river water that was filtered with muslin cloth. Daphnia culture was supplemented with 0.5% yeast culture, added every third day. Yeast culture, in this case, was used as a food for Daphnia. Algae, yeast or bacteria are preferred food for Daphnia. Although, many workers use river water for Daphnia culture presuming that it would have better mineral composition, in our case, we were also able to culture Daphnia in aged tap water in the similar manner. Cultures were routinely monitored to ensure production of healthy Daphnia.

Counting of Daphnia‘s heart rate

To investigate the effect of certain agents on the heart rate of Daphnia, real-time monitoring of changes in the heart rate of Daphnia is required. We used a microscope equipped with computer-aided real-time imaging capability (Magnus Live usb camera viewer, version 2.0, Magnus Analytics, New Delhi-110044, India), and for each reading heart rate was initially counted without any treatment (control). Subsequently, changes in the heart rate was monitored after the addition of selected agents. Each Daphnia was placed on the glass slide with 100 ul of water. The slide was observed in real time under the microscope at 40x or 100x magnification, and heart rate was counted. To avoid the effect of temperature or other environmental factors, counting was done after five seconds of starting the microscope. Subsequently, the microscope was switched off for five seconds, cardiovascular agents were added (see Table 1), and heart rate was counted again.

Statistical analysis

A paired t test analysis was done to compare changes in heart rates upon treatment with different agents. Statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA). P<0.05 was considered significant.

Dopamine, like caffeine, increases the heart rate of Daphnia in a dose-dependent manner

Dopamine’s effects on Daphnia’s heart has not been reported previously. We hereby report that dopamine instantly increases the heart rate of Daphnia in a dose-dependent manner, and a significant increase (25.7%) in the heart rate was observed, even at a low dose of 0.8 mg/ml (Figure 1). Caffeine showed a similar effect on Daphnia’s heart rate at a 10-times lower concentration than dopamine (28.5% increase at 0.08 mg/ml, Figure 2). Dopamine is the precursor of norepinephrine, and has been shown to augment heart activity by affecting beta-adrenergic receptors, in the case of a canine model¹⁶. Furthermore, dopamine can cause both relaxation and contraction of vascular smooth muscle. Dopamine is also known to augment heart activity, pulmonary pressure, and cardiac index in the case of normal and hypertensive individuals¹⁷.

Figure 1. Dopamine increases the heart rate of Daphnia in a dose-dependent manner.

This experiment was performed two times, and a paired t test analysis vs control indicated the following P values: 0.0070 (for 0.8 mg/ml), 0.0255 (1.6 mg/ml), 0.0424 (2.4 mg/ml), and 0.0344 (3.2 mg/ml). These values are statistically significant.

Figure 2. Caffeine increases the heart rate of Daphnia in a dose-dependent manner.

This experiment was done two times, and a paired t test analysis vs control revealed the following P values: 0.0406 (0.08 mg/ml), 0.0263 (0.16 mg/ml), 0.0367 (0.24 mg/ml), and 0.0189 (0.32 mg/ml). These values are statistically significant.

Dopamine synergizes with caffeine to increase the heart rate of Daphnia

Caffeine, in combination with dopamine, increased Daphnia’s heart rate more than when the agents were administered alone, which suggests a synergistic activity (Figure 3). Dopamine has also been previously reported to play a role in the responses of Drosophila to cocaine, nicotine or ethanol¹⁸.

Figure 3. Dopamine synergizes with caffeine to increase the heart rate of Daphnia.

Daphnia’s heart rate was measured upon treatment with dopamine alone (red) or a combination of dopamine and caffeine (green). The concentration of caffeine (in combination with dopamine) was (A) 40 ug/ml and (B) 120 ug/ml. This experiment was performed two times, and a paired t test analysis vs control indicated the following P values: 0.0374 (0.8 mg/ml dopamine) and 0.0230 (1.6 mg/ml dopamine). These values are statistically significant.

Dopamine overcomes an ethanol-induced decrease of the heart rate of Daphnia

To see the effect on the heart rate of Daphnia, ethanol was used at a concentration ranging from 2–8%, and was found to decrease the heart rate of Daphnia in a dose-dependent manner (Figure 4).

Figure 4. Effect of ethanol on the heart rate of Daphnia.

This experiment was done two times, and a paired t test analysis vs control indicate the following P values: 0.0152 (2% ethanol), 0.0059 (4% ethanol), 0.0130 (6% ethanol), and 0.0280 (8% ethanol). These values are statistically significant.

We observed that dopamine was able to rescue the ethanol-induced decrease in the heart rate of Daphnia, even at a concentration of 0.4 mg/ml (Figure 5).

Figure 5. Dopamine overcomes the ethanol-induced decrease on the heart rate of Daphnia.

At 2% ethanol, dopamine-induced increase in the heart rate was 62.5% compared to control, and 84.8% compared to ethanol-induced heart rate. At 4% ethanol, dopamine-induced increase in the heart rate was 4.3% compared to control, and 33.7% compared to ethanol-induced heart rate.