Effect of continuous and modulated ultrasound on fish neurons.

Pashovkin Timofei Nikolaevich ORCID: 0000-0001-9697-9230 Doctor of Biology Leading Researcher; Institute of Cell Biophysics of the Russian Academy of Sciences - a separate division of the FITC PNCBI RAS 29 Nauki ave., 48 sq., Pushchino, Moscow region, 142290, Russia pashovkin@mail.ru Other publications by this author

Sadikova Diana Gablel'fartovna PhD in Physics and Mathematics Researcher; Institute of Cell Biophysics of the Russian Academy of Sciences - a separate enterprise of the FITC PNCBI RAS 142290, Russia, Pushchino, Studentskiy lane, 16, sq. 54 sdg7@list.ru

Introduction.

Any effects that lead to changes in the electrical activity of neurons are central to basic research and are important for the clinical treatment of neurological disorders. In model animals and humans, visualization of changes in the activity of both individual neurons and neural activity on the scale of the whole brain is the main way to develop neurobiological technologies. The available electrical and optical methods, as a rule, do not work at this scale due to their inherent physical limitations. An alternative to these methods is the use of ultrasound, which interacts with brain tissues with a fundamental resolution of about 100 microns and a time resolution of 1 ms. Transcranial ultrasound stimulation (TUS) is currently being intensively developed as a new, non-invasive neuromodulation method ^[3]. TUS has a higher spatial resolution than transcranial magnetic stimulation (TMS) ^[1] or direct electric current stimulation ^[2], which are currently used in practice.

Almost a hundred years ago, it was first recognized that ultrasound modulates the electrical activity of cells. Since then, ultrasound neuromodulation in the brain, in the peripheral nervous system of humans and model organisms has been widely covered in the literature ^[3-10]. Despite the long period of research. The fundamental cellular, molecular, and mechanical foundations of ultrasonic neuromodulation still remain largely unknown ^[3-11]. It is noted that ultrasound creates excitatory ^[12,13] and/or inhibitory effects ^[14.15] depending on the system under study and the parameters of the stimulus ^{[3, 13, 14]}. Thus, stimulation of the motor cortex of the brain in animals can induce electromyographic (EMG) signals in the corresponding muscles of the forelimb, hindlimb or tail, which indicates the possibility of TUS to induce action potentials ^[15.16]. The inhibitory effects of TUS are manifested in a long-term weakening of synaptic transmission ^[17]. It is believed that these opposite effects on the activity of neurons are due to differences in the place of stimulation, intensity, frequency of ultrasound and other parameters ^{[18, 28]}.

Ultrasound can have combined thermal and mechanical effects on neurons ^{[19, 20]}. Thermal and cavitation effects of ultrasound, although effectively used to destroy tissue or temporarily open the blood-brain barrier ^[21], require the use of greater power, frequency and/or duration of exposure than those commonly used for neuromodulation ^[3]. Due to compression and expansion in the ultrasonic wave, non-selective currents can occur in cell membranes that change the electrical activity of cells ^[3.11]. Due to the creation of shear stresses in the cell membrane, which increase the tension of the membrane and the appearance of geometric deformation of the lipid bilayer, ultrasound can activate mechanosensitive ion channels ^{[3, 13, 20-26]}.

Ultrasound can have both thermal and mechanical effects on the field of research in brain tissues. However, the temperature increase will be insignificant at low ultrasound intensities ^[27], and the forces of acoustic radiation have a mechanical effect without causing cavitation ^[13]. From the point of view of biophysics, mechanosensitive channels react to the mechanical action of ultrasound ^[28]. For example, changes in the conductivity of channels may be associated with changes in the shape of channels. Ultrasound can do this due to the appearance of shear stresses in the membranes, or by changing the membrane tension when the pressure in the ultrasonic wave changes. These changes widen the channel and make it more cylindrical in the plane of the membrane when opened ^[28]. They are energetically favorable in the presence of membrane tension, and this leads to a tension-dependent energy difference between the states, which contributes to the opening of the channel ^[29,30].

In laboratory conditions, the use of modulated ultrasonic waves can make it possible to identify neural structures that give a frequency-dependent response to external periodic exposure, to identify frequencies at which changes in functional activity will be maximum.

Also, in neurobiological research, the choice of an object is important. In this regard, the Mautner neurons (MN) of bony fish and amphibians serve as a convenient object. They have a significant number of afferent inputs and control the swimming activity of fish by controlling the movement of the caudal fin, initiating a unilateral stroke of the caudal fin, the main mover of fish. This makes it possible to judge functional changes in neurons by the behavior of an animal and to influence it in a targeted manner by various experimental influences. MN are two giant cells in the medulla oblongata of most bony fish and amphibian larvae. They are innervated from the vestibular apparatus through the VIII nerve (Fig.1). In turn, MN contralaterally innervates spinal motor neurons, which control the muscles of the trunk. This point of view has been confirmed by numerous experimental data ^[31-36].

Fig. 1. The layout of Mautner neurons in fish.

One of the aspects of ultrasound application is the induction of rapid functional changes with minimal manifestation of nonspecific reactions of the nervous tissue. The presence of characteristic frequencies in the electrical activity of neurons suggests that exposure to pulse-modulated ultrasound with appropriate pulse repetition frequencies can cause frequency-dependent changes in the functional state of this tissue.

When using ultrasound, as a rule, a significant number of different nerve endings are located in the area of exposure. In the case of changes in their functioning under exposure, it can be assumed that various biochemical processes in organs and tissues innervated by these endings and, consequently, their functional state may also change to varying degrees. This can be most clearly manifested when exposed to identified, specialized neurons responsible for certain functional states of an organ, tissue, and the body as a whole.

The aim of the work is to study the effect of continuous and pulse modulated ultrasound on the morphofunctional state of brain neurons and identified central neurons of vertebrates – Mautner neurons of goldfish. The priority goal of the work is to obtain action spectra for neurons. But in order to obtain them, it is necessary to know how continuous ultrasound affects neurons, since it is used as a factor with a carrier frequency, which is superimposed with modulation frequencies. Therefore, the final effect is the total effect of continuous ultrasound and modulation frequencies.

Materials and methods.

The activity of brain neurons and functional activity of MN was assessed by the behavior of fish in an annular chamber ^[31], counting the number of sectors and turns initiated by activation of MN for 10 minutes. The annular chamber consisted of a circular channel with a width of 20 mm and a water level height of 30 mm. The bottom of the chamber was divided into 8 sectors. In this camera, the fish moves in a circle, periodically changing the direction of movement, making turns. Thus, the speed of its movement and the frequency of turns were quantified. The re-movement of the fish around the ring and the turns of the fish are the standard motor reaction of the fish. The test of motor activity consisted in placing the fish in a camera, and measuring the number of sectors traversed and the frequency of turns or changes of direction of movement. Testing in all experiments was carried out for 10 minutes at the same time, since the motor activity of fish depends on the time of day at which the test is performed.

Irradiation of MN goldfish was carried out on a separate installation, the block diagram of which is shown in Fig. 3, using a focusing ultrasonic emitter connected to a therapeutic generator UZT-1.01F with a carrier frequency of 0.88 MHz with an average spatial and temporal intensity in the range 0.1 - 1 W/^cm2. The irradiation zone of the fish's head was located in the central part of the focal area, with a length of 1 cm and a focal spot radius of 2.5 mm. The ultrasound intensity was evaluated using a differential thermocouple calibrated by ultrasound intensity. The spatial distribution of intensities in the focal area was determined by the paint/paper method, represented as a 2- and 3-dimensional image (Fig.2). The total irradiation time was varied in the range from 15 seconds to 20 minutes. The G6-28 generator was used as a modulator. The fish was fixed in a special thermostatically controlled chamber at a distance of 7 cm from the surface of the radiator. (Fig. 3)

A B

Fig.2. Visualization of ultrasound intensity distributions in the focal area of the emitter. A is a two-dimensional image, B is a three–dimensional image, where Z is the ultrasound intensity scale, W/^cm2.

Fig. 3. A block diagram of an installation for ultrasound exposure to fish neurons.

1. Ultrasound generator, 2 – oscilloscope, 3 - time relay, 4 – emitter, 5 - external camera, 6 - internal camera, 7 - object (fish), 8 – thermostat, 9 - focus indicator, 10 - irradiation area.

The dubbing was performed at a temperature of 18 ° C. The temperature in the chamber was measured before and immediately after irradiation. The efficiency of the thermostating was no worse than 18+ 0.3 ° C.

Morphological changes in MN were studied using a JEM-100B electron microscope after a standard procedure for fixing ultrathin sections of nervous tissue ^[32].

Research results and discussion.

Figure 4 shows changes in the motor activity of fish under the influence of continuous ultrasound with an intensity of 0.1 W/^cm2. The figure shows the stimulating effect of low-intensity ultrasound.

Figure 5 shows the inhibition of the motor activity of fish under the influence of ultrasound with an intensity of 1.0 W/^cm2. This oppression is reversible and disappears: 10 minutes after voicing for 30 seconds, and 30-40 minutes after voicing for 5 minutes. The suppression of activity becomes irreversible after 20 minutes of voicing, with a fatal outcome.

Fig.4. Stimulation of the fish's rotary reaction (the reaction of the Mautner neuron) depending on the time of exposure to low-intensity ultrasound: I _SATA = 0.1 W/^cm2, f = 0.88 MHz, t = 18 ^o C, 100% control level.

After suppression by ultrasound with an intensity of 1 W /^cm2 and an exposure time of 5 minutes, a rapid restoration of the functional activity of fish is observed, followed by ultrasound exposure with an intensity of 0.1 W /^cm2, carried out for 5 minutes.

Figure 6 shows the change in the motor activity of fish relative to the magnitude of the effect of continuous ultrasound, under the influence of amplitude-modulated ultrasound in the modulation frequency range 2-14 Hz, with a depth of 2, a modulation depth of 100%, and an irradiation time of 5 minutes.

but

b

Fig. 5. The dependence of fish swimming activity on the time of exposure to continuous ultrasound: a) suppression of swimming activity, b) suppression of the rotary reaction, A) an area of high mortality of fish (more than 90%). I _SATA = 1 W/^cm2, f = 0.88 MHz, t = 18 ^oh S

It can be seen (see Fig. 6a, b) that the maximum inhibition of motor activity is observed at modulation frequencies of 6 and 10 Hz, and the maximum activation is at a modulation frequency of 8 Hz. Moreover, the rotational reaction of fish changes to a greater extent, while the overall motor reaction changes in the same way, but with with a smaller amplitude. It follows that the identified MN is more susceptible to the action of amplitude-modulated ultrasound than the totality of other neural structures. Functional responses to ultrasound exposure with these modulation frequencies are characteristic both for individual specific neurons and for the neuron system as a whole. The action spectra are given taking into account the effects of the carrier frequency (these effects are subtracted from the spectrum). Moreover, the activation effects caused by a modulation frequency of 8 Hz were canceled by a frequency of 10 Hz, with equal-energy exposure. In this example, one can see the possibility of stabilizing the functional state of the Mautner neuron of fish by using only modulation modes of ultrasonic exposure.

Ultrastructural analysis of electron microscopic images showed that, compared with the norm (Fig.7), voicing MN with ultrasound with an intensity of 1 W/^cm2 for a time from 10 to 20 minutes leads to destructive changes in the ultrastructure of MN, expressed in the stratification of the myelin sheath and ruptures in the axolemma. This is also accompanied by complete desolation of axosomatic synapses and a general decrease in synaptic vesicles. These morphological changes correlate with the behavior of goldfish in the annular chamber, associated with a decrease in the functional activity of MN after exposure to ultrasound, with an intensity of 1 W/^cm2. Ultrasound does not cause visible changes in the myelin sheath or axolemmas of presynaptic fibers when exposed for 5 minutes (ultrasound intensity is average in space and time – ISATA), however, it causes a change in the cytoplasm of MN. These changes consist in the formation of crystal-like structures inside the nucleus (Fig. 8). Similar changes are observed when exposed to pulse-modulated ultrasound with modulation frequencies of 6 and 10 Hz. Crystal-like structures are formed due to the rearrangement of neurofilaments present in the cytoplasm.

But

B

Fig. 6. Dependence of changes in the motor activity of goldfish under the influence of modulated ultrasound on the frequency of modulation: A) rotary reaction, B) general motor activity. I _SATA = 0.35 W/^cm2 (1/2 intensity of continuous ultrasound = 0.7 W/^cm2 – borehole = 2) , f = 0.88 MHz , t = 5 min.

Fig. 7. The structure of goldfish brain tissues before ultrasound exposure. a) the myelin sheath.

Fig. 8. Formation of neurofilament bundles in the brain tissues of goldfish after ultrasound exposure: a) neurofilament bundles. I _SATA =1 W/^cm2, f = 0.88 MHz, t = 5 min.

An essential fact for therapy is that the sounding of fish with ultrasound of low therapeutic intensity 0.1 W/^cm2 leads to the restoration of the structural organization of neurons and the resorption of neurofilament bundles.

The effects of continuous ultrasound depend mainly on the mechanisms of biological action (mechanical, thermal, cavitation). With an increase in the intensity of exposure, one of these mechanisms mainly works. As a rule, with increasing intensity, suppression of the functional state of neurons is observed, correlating with morphological changes in the structures of neurons.

Mechanosensitive ion channels of various types can be one of the targets of modulated ultrasonic exposure. Thus, their activation can be due to an increase in the tension of the cell membrane or geometric deformation of the lipid bilayer. Such channels may include mechanosensitive ion channels (K2Ps), Piezo1, MEC-4, TRPA1, Msc, and voltage-controlled Na⁺ and Ca ²⁺ channels. To date, it is not known exactly how ultrasound affects the activity of these channels.

The effects of modulated ultrasound on neurons may differ significantly from the effects of continuous ultrasound. The presence of active frequencies leads to the transformation of biological effects. At frequencies of activation or suppression of functional activity, effects exceeding the amplitude of the effects of continuous ultrasound can be obtained, with the possibility of changing the sign of the effect with a change in intensity. From the data presented in the article, it can be seen that when exposed to modulated ultrasound with equal energy parameters, frequency-dependent effects of stimulation and suppression of neuronal activity can be obtained at significantly lower energies in the ultrasonic beam than when exposed to continuous ultrasound. Thus, it is possible to significantly reduce the intensity of ultrasound exposure in physiotherapy while maintaining the magnitude of biological effects when using modulated ultrasonic fields. The possibility of controlling the functional state of neurons by modulated ultrasound is shown, using various parts of the action spectra obtained for both individual neurons and complex structures of fish neurons for correction.

Conclusions.

1. For fish neurons, the time ranges of the formation of stimulation effects at an ultrasound intensity of less than 0.1 W/^cm2 and the effects of inhibition of motor activity of fish at an intensity of 1 W/^cm2 are shown.

2. It is shown that it is possible to restore the functional activity of fish using ultrasound stimulation with an intensity of 0.1 W/^cm2 immediately after the depressing effect of ultrasound with an intensity of 0.7 - 1.0 W/^cm2.

3. For the first time, acoustic action spectra in the modulation frequency range of 2-14 Hz for fish brain neurons, including identified Mautner neurons, are shown.

4. It is shown that it is possible to reduce the risk of ultrasonic negative effects while maintaining the magnitude of the biological effect when using modulated ultrasound.

5. For the first time, the possibility of controlling the sign of a biological effect on fish neurons using equal-energy exposure with modulated ultrasound has been shown.