Quantum energy levels of glutamate modulate neural biophotonic signals

Glutamate is the most abundant excitatory neurotransmitter in the brain, and it plays an essential and important role in neural functions. Hypofunction of the glutamatergic pathway and the changes in the glutamate-glutamine cycle function are important neuropathological mechanisms of severe mental disorders including schizophrenia and depression. Current studies have shown that glutamate can induce neural biophotonic activity and transmission, which may involve the mechanism of photon quantum brain; however, it is unclear whether such a mechanism follows the principle of quantum mechanics. Here we show that the action of glutamate on its receptors leads to a decrease in its quantum energy levels, and glutamate then partially or completely loses its function to further induce the biophotonic activity in mouse brain slices. The reduced quantum energy levels of glutamate can be restored by direct-current electrical discharges and the use of energy transfer of chloroplast photosynthesis; hence, the quantum energy recovered glutamate can again induce significant biophotonic activity. Furthermore, the changes in quantum energy levels of glutamate are related to the exchange and transfer of electron energy on its active hydrogen atom. These findings suggest that the glutamate-induced neural biophotonic signals may be involved in the transfer of the quantum energy levels of glutamate, which implies a quantum mechanism of neurotransmitter action. The process of glutamate recycling that is related to the synergism of neurons and glial cells and certain key enzymes may be necessary for the recovery of quantum energy levels of glutamate after completion of the neural signal transmission. These findings may also provide a new idea to develop “quantum drugs”.


Introduction
Analysis of the binding characteristics of biological signaling molecules to their targets or receptors provides an important means to understand their physiological roles and to develop target drugs. However, a large number of studies have found that the effects of signaling molecules on their targets are often unpredictable, for example, the signal molecules or drugs with identical or similar structures such as chiral drugs may produce different effects, while the molecules with completely different structures may induce identical or similar effects (1). For olfactory molecules, human beings can directly experience their differences, and quite different molecules can have similar odors, whereas similar molecules can have dissimilar odors (2).
The theoretical studies speculate that the reasons for these phenomena may be related to the mechanism of quantum biological action (3)(4)(5), however, the theoretical framework based on traditional quantum mechanics, such as quantum coherence, entanglement and superposition, could not explain the existence of phenomena very well (6,7). Recent studies have shown that biophoton activity may play an important role in quantum biological mechanisms, and it was reported that glutamate, the most abundant excitatory neurotransmitter in the brain, could induce biophotonic activities and transmission in neural circuits, which may involve the mechanism of photon quantum brain (8)(9)(10)(11)(12). The characteristic differences such as spectral redshift in glutamate-induced biophotonic activity in different animals may give some evolutionary advantages such as human intelligence (13), but may also allow human beings to be more sensitive to the development of neurological and psychiatric diseases due to the changes in neurotransmitter functions. For example, hypofunction of the glutamatergic pathway and the changes in the glutamate-glutamine cycle function are important neuropathological mechanisms of severe mental disorders including schizophrenia and depression (14)(15)(16)(17)(18)(19)(20)(21). Therefore, we hypothesize that the enhancement or decrease of glutamate function may be related to the changes in quantum energy levels of glutamate, which were experimentally investigated in this study. 4

NMDA receptor mediates glutamate-induced biophotonic activity
Our previous study demonstrated that 50 mM purchased glutamate (p-Glu) can lead to biophotonic activity and transmission (neural biophotonic signals) in neural circuits, presenting four characteristic stages (initiation, maintenance, washing and reapplication) (8). In this study, we further found that N-Methyl-D-aspartate (NMDA), a specific agonist at one of the ionotropic glutamate receptors, could also induce similar patterns of biophotonic activity in mouse brain slices (Fig. 1A), suggesting that the NMDA receptor mediates glutamate-induced biophotonic activity. In addition, the washing effect, which provides a further increase in biophotonic activity, was significantly inhibited by the application of a presynaptic vesicle release inhibitor (H89) at the beginning of washing ( Fig. 1B and Table S1), suggesting that the washing effect is due to the enhanced presynaptic glutamate release. A possible mechanism is that the extracellular application of glutamate can reduce the release of presynaptic vesicles through an inhibitory effect on the presynaptic membrane (22), which results in increased glutamate storage in the presynaptic vesicles and, therefore, a rapid and massive glutamate release from the presynaptic membrane occurs during the early stage of washing.

Decrease in the quantum energy levels of glutamate after its action
Then, we observed long-term (24 h) effects of 50 mM glutamate-induced biophotonic activity. We designed a consumption experiment to evaluate the changing patterns of glutamate-induced biophotonic activity. Mouse brain coronal slices (450 µm in thickness) were cut from the anterior part of the olfactory cortex to the caudal part of cerebellar cortex and all slices were collected. We found that the intensities of glutamate-induced biophotonic activities from a slice for imaging, which was perfused with 100 ml ACSF containing 50 mM glutamate together with four whole mouse brain slices in a bottle, dramatically decreased after perfusion and imaging for approximately 12 h (12.02±0.65 h, Fig. S1). This result allowed us to speculate that the cause of the obvious weakening of glutamate action may be related to the decrease 5 in the quantum energy levels of glutamate after its action on the postsynaptic receptors ( Fig. 2A) because a cyclic perfusion with a fixed volume of ACSF containing 50 mM glutamate was able to partially and almost completely exhaust the action of glutamate by providing appropriate brain tissues and enough perfusion time.
To confirm our results, we designed a cyclic dual perfusion system to conduct further experiments. Two hemisphere slices (left and right, 450 µm in thickness) prepared from a coronal whole brain slice at the anterior level of the hippocampus were placed separately into two perfusion subchambers that were imaged simultaneously (Fig. 2B). We found that the patterns and intensities of 50 mM glutamate-induced biophotonic activity were exactly the same for both slices (Fig. S2), which was consistent with data obtained from a whole brain coronal slice containing left and right hemispheres (8). Then, we tested the effects by only using ACSF containing 50 mM p-Glu after incubation with different quantities of brain slices. All slices prepared from two or four mouse brains were incubated in 100 ml ACSF containing 50 mM p-Glu for 24 h at ~ 28℃. Then, 50 ml incubated solution was collected to perfuse a new prepared brain slice for biophotonic imaging. We found that the intensity of biophotonic activity during the four stages induced by the solution after incubation with two whole brain slices was significantly weaker than that in control solution (Fig. 2C and Table S1), while the effects were almost eliminated after incubation with four whole brain slices, both of which resulted in hypofunctional (partial incompetent, p-Ic) and nonfunctional glutamate or incompetent glutamate (Ic-Glu) ( Fig. 2D and Table S1). The observed results were not due to the change in the molecular structure of glutamate after the action of its receptors or the effects of other active molecules released from the brain slices during incubation. Even if the low concentration of classical neurotransmitters such as acetylcholine, dopamine, 5-hydroxytryptamine (5-HT) and norepinephrine were present in the ACSF after incubation for 24 h, their presence cannot explain the weakening and disappearance of glutamate action because our previous study demonstrated that even high concentrations of 5-HT (> 20 µM) only produced partial inhibitory effects on 50 mM glutamate-induced biophotonic activity, while acetylcholine, dopamine and 6 norepinephrine played reinforcing rather than inhibitory roles (12). Therefore, in essence, these results proved our speculation mentioned above.
Electrical discharge restores the quantum energy levels and the effects of incompetent glutamate.
To test whether there are ways to restore the decreased quantum energy levels of glutamate and reverse its action and function to induce biophotonic activity, we developed two techniques (Fig. 3A). One is called the electrical stimulation assisted quantum energy enhancement technique (ESA-QEET, Fig. 3B，see also SI for details).
One hundred millilitres ACSF containing 50 mM p-Glu was first incubated with four whole brain slices for 24 h at ~ 28℃ and then divided into two parts (50 ml each).
One portion of the solution was treated for 3 h with 12 V direct-current electrical discharges (DC-ED) at a high frequency (~10 k Hz), and another was the control. We found that such treatments significantly restored the biophotonic activity of Ic-Glu compared to the controls without DC-ED treatment and reached to 59.00-118.25% of the effects of ACSF containing 50 mM p-Glu at the different imaging periods ( Fig.   3C and Table S1). The recovery of the quantum energy level of glutamate was not due to the influence of DC-ED on the other components of the ACSF because the ACSF without p-Glu incubated with four whole brain slices for 24 h did not have any effects on the biophotonic activity after treatment with 12 V DC-ED ( Fig. 3D and Table S1).
These findings also imply that the increase in quantum energy levels of glutamate may be involved in the transfer of electron energy on its molecule, and a possible explanation is that the active hydrogen (proton) of glutamate is responsible for the exchange and transfer of quantum energy, which was supported by our findings that, similar to 50 mM p-Glu, 50 mM acetic acid (0.3%) could also induce similar patterns of biophotonic activity in mouse brain slices ( Fig. S3 and Table S1), whereas 50 mM sodium glutamate (gourmet powder), on which the sodium atom is substituted for the active hydrogen atom, could not have any effect on the induction of biophotonic activity in mouse brain slices ( Fig. S4 and Table S1). 7

Chloroplast photosynthesis restores the quantum energy levels and the effects of incompetent glutamate.
Another technique is called the chloroplast-assisted quantum energy enhancement technique (CA-QEET, Fig. 3A, see also SI for details). Chloroplasts can achieve efficient light energy transfer through photosynthesis under light illumination and, therefore, the electron and proton transport between molecules can be realized, in which quantum mechanics is involved (23-26). We explored whether chloroplasts were able to allow the hypofunctional or nonfunctional glutamate to restore its quantum energy levels. Chloroplasts were prepared from a vegetable (spinach) and a comparative analysis showed that the isolated chloroplasts could maintain a certain degree of light transfer activity in the ACSF and the ACSF containing 50 mM p-Glu compared to those under an appropriate condition reported previously (27) (Fig. S5).
We added ~3 g freshly prepared chloroplast precipitate into 50 ml ACSF containing Ic-Glu in two steps (~1.5 g each) and incubated the mixed preparations for 48 h under a combination of day and night illumination (see SI for details). We found that such treatments allowed Ic-Glu to reach 160.25-370.70% of the effects of the 50 mM p-Glu at the different imaging periods ( Fig. 4A and Table S2), showing the super recompetent effects (Fig. 3A). The over-recovery of biophotonic activity was partially because of the acidification effect of light energy transfer of the chloroplasts on ACSF (28-30), which resulted in low pH (from ~7.4 to ~4.0) and induced some degree of biophotonic activity both from the ACSF treated with and without four whole brain slices ( Fig. 4B and Table S2); however, adjusting the acidified ACSF to the original level of pH 7.4 significantly reduced the effects and only induced slight biophotonic activity ( Fig. 4C and Table S2), reinforcing the role of the active hydrogen for the glutamate-induced biophotonic activity mentioned above.

Discussion
This study demonstrated that, after induction of neural biophotonic signals, glutamate lost its action and presented a decline in the molecular quantum energy levels, but it did not involve the changes in its molecular structure. Although the exact 8 quantum biological mechanism, including the atoms involved, is unclear, our findings suggest that this phenomenon may be highly associated with the active hydrogen atom on the glutamate molecule. The change in the quantum states (quantum levels) in a liquid molecule involves the translational level of the molecule, the rotation, vibration and motion of the electrons, and the change in the energy level of the nuclear motion in the molecule (31-33). The mechanism of the decrease in the quantum energy levels after the action of glutamate on its receptors may be mainly related to the change in the electron quantum energy levels in the active hydrogen atom. Therefore, our findings may provide a new idea for further clarifying the importance of the quantum biological mechanism of the functional molecules in the brain. For example, the glutamate released into the synaptic cleft is reused mostly by uptake into surrounding glial cells via the glutamate-glutamine (Glu-Gln) cycle, which is related to the synergism of neurons and glial cells, glutamate transporter and certain key enzymes (34, 35), and this widely-known process of recycling of glutamate seems to violate the energy conservation principle of the organism. Our findings suggest that such a process of recycling is indeed a necessary step for recovering the quantum energy levels of glutamate after its action, possibly as an important mechanism of quantum biology. Second, our findings could lead to a new perspective for elucidating the pathophysiological mechanisms of neuropsychiatric disorders. The changes in the glutamate recirculation, including the changes in transporters and key enzymes, are related to the pathological mechanisms of these diseases (14-21), while the action of an enzyme is considered to be a quantum mechanism, which is involved in the transfer of quantum energy (3). Therefore, the disruption of the Glu-Gln cycle may play an important role in the hypofunction of the glutamatergic pathway and contribute to the pathological mechanisms of neuropsychiatric disorders. Finally, our findings could provide new explanations for some clinical therapies such as local brain electromagnetic stimulations, electroshock, traditional natural medicine and acupuncture treatments (36, 37). One of the possible mechanisms for these treatments is through the restoration of the imbalance of the quantum energy levels of neurotransmitters and other functional molecules. In 9 addition, the differences in the quantum energy levels of odorants may provide a novel way of explaining the diversity of the olfactory sense (2).

Methods
The detailed information for materials and methods is described in Supporting Information (SI).  The intensities of biophotonic activity tended to decrease significantly after the application of H89 compared to those in controls (n=6 for 50 mM p-Glu and 50 mM p-Glu+200 μM H89).
Data are shown as the mean±s. e. m. n=the number of slices from the same number of mice. Significant differences (** p<0.01) are noted from 271-285 min (see Table S1).  also Table S1).  also Table S1).

Experimental mice
Adult male Kunming mice (6-8 weeks), which were originally generated from a Swiss white mouse and has been widely used in biomedical studies in China, were purchased from Hubei Provincial Laboratory Animal Public Service Center (Wuhan, China) and housed in a room with a 12 hrs light/dark cycle (lights on at 7:00AM) with access to food and water adlibitum. The study protocol was approved by the

Committee on the Ethics of Experimental Animals and Biomedicine of South-Central
University for Nationalities.

Preparation of mouse brain slices
Mice were decapitated and the brain was quickly removed and placed in ice-cold The coronal brain slices were cut to 450 µm thickness with a Vibratome (Leica VT1000S, Germany) beginning at the rostral part of the olfactory cortex and ending at the caudal part of the cerebellum. A standardized coronal brain slice or its left and right hemisphere slices at the level of the anterior part of the hippocampus was used for biophoton imaging. The prepared brain slices were kept in ACSF at 0-4℃ for further experiments.

Cyclic dual perfusion system for brain slices
Schematic drawing of the cyclic dual perfusion system is described in details in Fig. 2b in the main text. The perfusion for two parts of the chamber (subchamber) was 21 maintained independently through an input micropump (~4 ml/min) and an output micropump (6-12 ml/min). A mixture of 95% O 2 +5% CO 2 was constantly supplied with a membrane oxygenator placed in the ACSF of glass bottle during the perfusion period. The temperature (~35℃) of the medium in the perfusion chamber was maintained with an electrical heater.

In vitro biophoton imaging system
The in vitro ultra-weak biophoton imaging system (UBIS) was described in our previous studies (1,2). Biophotonic activity (emissions) was detected and imaged with the UBIS using an EMCCD camera (C9100-13 or C9100-23B, Hamamatsu Photonics K. K., Hamamatsu, Japan) in water-cool mode (in this situation, the working temperature at the EMCCD camera can be maintained as low as -95℃) controlled by an image analysis software program (HCImage, Hamamatsu Photonics K. K., Hamamatsu, Japan). The specific steps for biophoton detection and imaging were as follows: (1) the brain slices were transferred to the two subchambers and perfused with ACSF in complete darkness by closing the dark box of the UBIS for approximately 30 min before imaging to exclude the effects of ambient light; (2) real-time imaging was performed by automatically taking an image every 30 s for detection of typical biophotonic activity; (3) the imaging duration was 240-300 min (480-600 series of images) depending on the experimental protocols including an additional 30 min imaging time when the brain slices were only perfused with ACSF before any treatment was carried out; and (4) a regular photograph of brain slices was taken under the normal CCD model before and after the imaging processes were completed to locate its position under the imaging field of view.

Image processing, data extraction and analysis
The specific processes for image processing and data analysis were as follows: (1) all original grey images in TIF format were processed with a program running on the MATLAB platform to eliminate the effect of cosmic rays (white spots) as described before (1)

Acquisition of incompetent glutamate
All slices cut from two or four mouse brains were collected and transferred to 100 ml ACSF containing 50 mM glutamate in a glass bottle and incubated for 24 h at ~28 °C. The incubation temperature was maintained with a thermostat water bath. A mixture of 95% O 2 +5% CO 2 was constantly supplied with a membrane oxygenator placed in the ACSF during the incubation period. The slices were removed from the glass bottle after 24 h incubation and the remaining ACSF was then filtered with a filter paper.

Recovery of the quantum energy level of incompetent glutamate by electrical discharges
A direct-current electrical discharge (DC-ED) device with adjustable voltages (Taiyuan Weiyide Automation Technology Co., Ltd., WD-16A) was used to execute electrical discharges (EDs) using the two same carbon rods as the cathode and anode.
One hundred millilitres ACSF containing 50 mM glutamate after treatment with four whole brain slices for 24 h was equally divided into two parts (50 ml each). One part 23 was then treated for 3 h with 12 V DC-ED at a high frequency (~10 k Hz) and the other was a control without DC-ED treatment. Same treatments were carried out for one hundred millilitres ACSF without 50 mM glutamate.

Isolation of chloroplasts and determination of photochemical activity in the ACSF
Chloroplasts were isolated from spinach according to the methods described approximately 28 h. We found that 1.5 g of freshly prepared chloroplast precipitate in 24 50 ml of ACSF containing 50 mM p-Glu had better efficiency, which remained after incubation for 28 h.

Recovery of the quantum energy level of incompetent glutamate and its activation by chloroplast photosynthesis
One hundred millilitres of ACSF containing 50 mM glutamate was incubated with four mouse brain slices for 24 h and then equally divided into two glass bottles (50 ml each). Freshly prepared chloroplast precipitate (1.5 g) was added to one of two glass bottles and incubated for 24 h under a combination of day and night illumination as described above. Then, the chloroplasts were removed from the solution by centrifugation for 5 min at 3000 rpm, and an additional 1.5 g freshly prepared chloroplast precipitate was added and incubated for another 24 h. After centrifuging the chloroplasts, the remaining ACSF supernatant containing recompetent glutamate was tested for biophotonic activity in mouse brain slices as described above.

Drug preparation
Glutamate (