2.1.

Animal Preparation

After anesthetizing Aplysia californica (250 to 350 g, Marinus Scientific, Long Beach, California) with magnesium chloride (333 mM, 50% body weight), the right pleural abdominal (PA) nerves were dissected out and suctioned into extracellular recording electrodes to record their electrophysiological response. The nerves were maintained in room temperature ($T\sim 20°\mathrm{C}$) Aplysia saline (460 mM NaCl, 10 mM KCl, 22 mM ${\mathrm{MgCl}}_2$, 33 mM ${\mathrm{MgSO}}_4$, 10 mM ${\mathrm{CaCl}}_2$, 10 mM glucose, 10 mM HEPES, pH 7.6), which was modified depending on the type of experiment (see below). Measurements using a calibrated microscope done in the laboratory show that the diameter of the Aplysia PA nerve is $253.09\pm 144.70\mu \mathrm{m}$ in diameter ($N=12$, for animals ranging from 250 to 300 g in weight).

2.2.

Experimental Setup

Nerve-recording electrodes were made by hand-pulling polyethylene tubing (1.27-mm outer diameter, 0.86-mm inner diameter) over a flame and cutting them to the desired diameter. Recording electrodes were suction-filled with Aplysia saline prior to suctioning the nerve. Nerve signals were amplified ($\times \mathrm{10,000}$) and bandpass filtered (300 to 500 Hz) using an AC-coupled differential amplifier (A-M Systems, Sequim, Washington), digitized using an Axon Digidata 1440A digitizer (Molecular Devices, San Jose, California), and recorded using Clampex computer software (Molecular Devices, San Jose, California). Monophasic stimulation current was supplied by a WPI A365 stimulus isolator (World Precision Instruments, Sarasota, Florida) for all experiments (stimulation parameters: $200\mu \mathrm{A}$ current, ${\tau }_p=2\mathrm{ms}$, 1 Hz). The IR block was produced by a tunable diode laser (Capella Neurostimulator; Lockheed-Martin-Aculight, Bothell, Washington) with a wavelength of $\lambda =1875\mathrm{nm}$ coupled to a $400\text{-}\mu \mathrm{m}$-diameter multimode optical fiber (Ocean Optics, Dunedin, Florida). The optical fiber was secured in place using micromanipulators. The laser was triggered at 200 Hz with $200\text{-}\mu \mathrm{s}$ pulses for all experiments. Radiant exposures per pulse at the fiber output were calculated after measuring the power output using a power meter (Molectron EPM 2000, Coherent, Santa Clara, California). The optical fiber was maintained in contact with the nerve surface for all irradiation experiments [Fig. 1(a)]. The energy range used in the study was $215.29\pm 19.55\mathrm{mJ}/{\mathrm{cm}}^2/\text{pulse}$.

Fig. 1

Experimental setup. (a) Schematic of experimental setup, which consists of an Aplysia PA connective nerve in a 3-D chamber that consists of three sections (1, 2, and 3). The sections at the ends (shown in blue) contain normal saline. The section in the middle (2, colored red) contains saline solution with or without ion channel blockers. The nerve is irradiated with IR laser radiation delivered via a fiber optic in the middle section. Suction electrodes at the ends of the nerve allow for stimulation and recording of CAPs ($\mathrm{\Delta }x=2.5$ to 3 mm). (b) A schematic showing the layout of the nerve chamber with dimensions.

A specially designed chamber was fabricated for our experiments. The chamber [Fig. 1(b)] consisted of three sections and was three-dimensional (3-D) printed on a Form 2 printer using black Formlabs Standard Resin (Formlabs Inc., Somerville, Massachusetts, $50\text{-}\mu \mathrm{m}$ layer height). The sections within the chamber were separated using 3-D printed partitions (made of Hewlett-Packard 3-D high reusability polycyclic aromatic 12 to provide finer detail). Resin was used for printing since it provided a good balance between quality and cost. For the experiments described in this paper, the width of the middle section ($\mathrm{\Delta }x$, Fig. 1) was maintained between 2.5 and 3 mm. This width is small enough to allow passive currents to propagate past the middle chamber and reinitiate action potentials, even in the presence of voltage-gated sodium ion channel blockers. The width was also large enough to allow reliable placement of the optical fiber in contact with the nerve. Leaking was minimized by brushing the edges of the partitions with biocompatible Vaseline (Unilever, London/Rotterdam) petroleum jelly to provide a good seal.

2.3.

Channel Blockers and Inhibitors

To test hypotheses derived from computational modeling studies,¹⁸ we used a blocker of voltage-gated potassium ion channels [tetraethylammonium (TEA) chloride] and a blocker of voltage-gated sodium ion channels [tetrodotoxin (TTX)]. The formulation for TEA saline was 410 mM NaCl, 50 mM TEA, 10 mM KCl, 22 mM ${\mathrm{MgCl}}_2$, 33 mM ${\mathrm{MgSO}}_4$, 10 mM ${\mathrm{CaCl}}_2$, 10 mM glucose, 10 mM HEPES, pH 7.6.^19,20 Prior studies^19,20 have shown that TEA blocks both voltage-dependent and ${\mathrm{Ca}}^{+2}$-mediated potassium channels and disrupt potassium currents in Aplysia neurons. A solution of $60\mu \mathrm{M}$ of TTX saline solution (TTX, Enzo Life Sciences Farmingdale, New York) was prepared by adding $600\mu \mathrm{l}$ of 1 mM TTX normal Aplysia saline solution to make a final volume of 10 ml. The concentration of TTX in saline was chosen to be consistent with previously published experimental studies in Aplysia.^21–23 In these studies,^21–23 it has been observed that use of TTX blocks inward currents and decreases spiking activity in Aplysia pacemaking and buccal neurons. The effect of all ion channel blockers was reversible, which was confirmed by performing recordings after washing out the ion channel blocker solutions. The effect of TEA was reversed after 2 to 4 washouts over $\sim 30\min$. The effect of TTX was reversed after 2 to 3 washouts over $\sim 15\min$. The pH of all the saline solutions was adjusted to 7.6, which is the same as that of normal Aplysia saline. All solutions were brought to room temperature ($T\sim 20°\mathrm{C}$) prior to infusion into the chamber.

2.4.

Experimental Protocol

The nerve recording protocol consisted of multiple recordings of 20-s intervals. The nerve was electrically stimulated at a frequency of 1 Hz. Each recording consisted of three phases: (1) the time when the laser was off ($t=0$ to 5 s), (2) the time when the laser was on ($t=5$ to 15 s), and (3) the time when the laser was again off [$t=15$ to 20 s; Fig. 2(a)]. Phase 3 of the recording allowed the nerve to recover from the thermal block induced by the IR laser irradiation during the second phase of the recording. In each study, the power of the laser was adjusted to obtain a complete block of the compound action potential (CAP) within the first 5 s after the laser turned on. Once the value of radiant exposure was determined, it was kept constant for the entire duration of a trial. Each trial consisted of three experimental conditions. The trials were conducted in a series of recordings in A-B-A-C-A format. Recording A refers to the observation of nerve activity in the control saline solution (with IR irradiation), before any ion channel blocker is introduced. Recording B refers to the observation of nerve activity in the presence of TEA (with IR irradiation), and recording C refers to the observation of nerve activity in the presence of TTX (with IR irradiation). Experiments were repeated in six different animals ($N=6$).

Fig. 2

Typical IR block. (a) Recording showing IR laser block obtained when the nerve is in normal Aplysia saline. The laser is switched on from $t=5$ to 15 s and switched off for the last 5 s in each recording. (b)–(d) Signals from each phase of the recording are shown in high temporal resolution [(b) preirradiation, (c) during irradiation, and (d) postirradiation]. In each of the high-resolution signals shown, the electrical recording artifacts are removed. (Laser parameters $\lambda =1875\mathrm{nm}$, $\text{optical fiber size}=400\mu \mathrm{m}$, $f=200\mathrm{Hz}$, ${\tau }_p=200\mu \mathrm{s}$, and $H_o=183\mathrm{mJ}/{\mathrm{cm}}^2$.)