Consciousness is an unsolved scientific enigma. Diverse quantum models attempt to explain consciousness mechanisms (Fisher, 2015; Hameroff and Penrose, 2014; Jibu and Yasue, 1995; Kumar et al., 2016; Poznanski et al., 2019).

Fisher (2015) first proposed the phosphorus-31 nuclear-spin entanglement model. According to Fisher’s proposal, in bulk water, the survival time of nuclear-spin entanglements for phosphorus-31 is too short (within one second) in free phosphate ions due to proton attack. However, if the phosphorus-31 are the constituents of Ca${}_9$(PO${}_4$)${}_6$, Posner molecules, its unique structure protects phosphorus-31 nuclear-spin entanglements from proton attack. As a result, the decoherent time lasts as long as 21 days or longer for the entanglements (Swift et al., 2018). Long-lived quantum entanglement is central in quantum consciousness processing (Swift et al., 2018). Thus, Posner molecules play a crucial role in processing consciousness (Fisher, 2015; Swift et al., 2018).

However, though attractive, Fisher’s proposal is speculative (Fisher, 2015; Swift et al., 2018) and lacks experimental evidence to support or disprove it. Fisher’s entire proposal cannot be tested experimentally, but a partial test is possible because some verifiable predictions can be derived from the proposal (Fisher, 2015). The testable predictions include varying calcium concentrations and the existence of calcium isotope dependence on consciousness (Fisher, 2015). Since Posner molecules are important in enabling phosphorus-31 nuclear-spin quantum entanglement to mediate consciousness in the brain, it is expected that varying calcium ion concentrations in the extracellular fluid would affect Posner molecules formation and accordingly it will influence the levels of consciousness (Fisher, 2015). So, an increase of ${}^{40}$Ca${}^{2+}$ concentration in the cerebrospinal fluid is expected to increase the number of Posner molecules and increase the level of consciousness. Conversely, a decreased ${}^{40}$Ca${}^{2+}$ concentration is expected to lower the level of consciousness.

Calcium isotope dependence is also expected in Fisher’s proposal (Fisher, 2015). Specifically, ${}^{43}$Ca${}^{2+}$ would disturb and decohere the phosphorus-31 nuclear-spin entanglements if ${}^{43}$Ca${}^{2+}$ replaces the ${}^{40}$Ca${}^{2+}$ within the Posner molecules (Fisher, 2015). Therefore, an increase of ${}^{43}$Ca${}^{2+}$ concentration in the cerebrospinal fluid would lead to a decrease in the animal’s consciousness level, opposite to the effect of ${}^{40}$Ca${}^{2+}$.

We use the loss of righting reflex (LORR) ED${}_{50}$ for mice to sevoflurane to measure consciousness level (Franks and Lieb, 1994; Franks, 2008; Li et al., 2018) to test the predictions mentioned above. LORR ED${}_{50}$ closely correlates to the consciousness level; as described in our previous study, an increased LORR ED${}_{50}$ indicates increased consciousness level and vice versa (Li et al., 2018). By quantifying consciousness levels, we disprove Fisher’s proposal because our findings oppose his predictions.

To investigate the effects of calcium concentrations and calcium isotopes on sevoflurane-induced LORR ED${}_{50}$, sixty C57BL/6 male mice were included. No mice were excluded due to weight loss. Three mice in the saline group and two in each of the other three groups were excluded due to unsuccessful ICV injection. Finally, data of the LORR ED${}_{50}$ of 51 mice, 12 in the saline group and 13 in each of the other three groups were taken into statistical analysis. Two-way repeated-measures ANOVA was used to analyze the data, with one main factor for time and the other for the group. Analyses revealed that regardless of the treatment condition, LORR ED${}_{50}$ values between the first and the second measurements had critical differences (F${}_{1,47}$ = 35.47, P 0.0001). Not surprisingly, the effects of group type on LORR ED${}_{50}$ values were significant (F${}_{3,47}$ = 7.77, P = 0.0003) regardless of the measurement condition. The Bonferroni post hoc multiple-comparison test analyzed the effect of a group within each level of time and the effect of time within each group level. A strong time $\times$ group interaction was found (F${}_{3,47}$ = 34.32, P 0.0001).

3.1 Effects of saline and EGTA on LORR ED${}_{50}$

The basal value of LORR ED${}_{50}$ was determined for the 12 mice to sevoflurane in the saline group, before the ventricular cannulation, was at 1.01 $\pm$ 0.09% (95% CI, 0.95% to 1.07%). The value of LORR ED${}_{50}$ for the same mice to sevoflurane, determined after ICV injection of saline was at 2 $\mu$L, was 0.98 $\pm$ 0.12% (95% CI, 0.91% to 1.06%). The two values were highly comparable (P $>$ 0.9999, Fig. 1). Since the mice were determined twice for their LORR ED${}_{50}$ to sevoflurane, there existed a possibility that the residual sevoflurane of the first determination might influence the second one. However, one day was allowed for the animals to recover between the two determinations. This interval is regarded as adequate for the animals to exhale out the sevoflurane completely. The lateral ventricular catheterization itself might also influence the second LORR ED${}_{50}$. The highly comparable LORR ED${}_{50}$ values before and after saline injection of the mice in the control group ruled out these possibilities, indicating that the two determinations were independent. Therefore, LORR ED${}_{50}$ values of the mice after ICV injections of EGTA, ${}^{40}$CaCl${}_2$ and ${}^{43}$CaCl${}_2$ in the other three groups were respectively independent on their first determinations.

To examine the effect of decreased calcium ion concentration in the cerebrospinal fluid on a consciousness level, EGTA, a calcium chelator, was directly injected into the mice’s lateral ventricle. The basal LORR ED${}_{50}$ for the 13 mice to sevoflurane in the EGTA group was 1.00 $\pm$ 0.15% (95% CI, 0.91% to 1.09%). The LORR ED${}_{50}$ for the mice to sevoflurane after EGTA ICV injection was 1.10 $\pm$ 0.14% (95% CI, 1.02% to 1.19%), significantly higher than the basal value (P 0.001, Fig. 1).

Fig. 2.

Effects of ${}^{\mathrm{40}}$CaCl${}_{\mathrm{2}}$ and ${}^{\mathrm{43}}$CaCl${}_{\mathrm{2}}$ on righting reflex ED${}_{\mathrm{50}}$ for mice to sevoflurane. Pre-ED${}_{50}$ denotes the basal LORR ED${}_{50}$. Post-ED${}_{50}$ presents LORR ED${}_{50}$ after lateral ventricle administration of ${}^{40}$CaCl${}_2$ or ${}^{43}$CaCl${}_2$. The difference in the values of the pre-ED${}_{50}$ between the two groups is not significant. The post-ED${}_{50}$ value is significantly lower than the pre-ED${}_{50}$ in the ${}^{40}$CaCl${}_2$ group or in the ${}^{43}$CaCl${}_2$ group. The post-ED${}_{50}$ values of the two groups are not significantly different. Data are shown as mean (SD), n = 13 mice per group. ****P 0.0001; N.S. = not significantly different (two-way repeated-measures ANOVA followed by the Bonferroni correction as a post hoc test).

Several quantum models based on quantum dynamics have been proposed with attempts to demystify the mechanisms of consciousness. For example, the electric spin entangled neural microtubules (the “Orch OR” theory) (Hameroff and Penrose, 2014), phosphorus-31 nuclear spin entanglements (Fisher, 2015), photon-spin interface (Kumar et al., 2016), and unpaired electron-spin activating nuclear spin-based mind-pixels (Hu and Wu, 2004), all set upon theoretical speculations or simulations. Hence, debates on consciousness models persist because there is no direct experimental evidence to confirm or reject any theory of consciousness (Hameroff and Penrose, 2014), primarily due to a lack of measures to probe consciousness. Anesthesia is a reversible unconscious state induced by general anesthetics. General anesthetics are relatively selective for consciousness while they spare many non-conscious brain activities (Craddock et al., 2015; Hameroff, 2006), anesthesia and consciousness may share the same mechanism, thus making general anesthetics natural probes for consciousness (Craddock et al., 2015; Hameroff, 2006).

In this study, LORR ED${}_{50}$ for mice to sevoflurane was used as a measure to quantify consciousness levels in mice. Thereby, we tested the predictions derived from the proposal that phosphorus-31 nuclear-spin entanglements protected by Posner molecules may mediate consciousness, first proposed by Fisher in 2015 (Fisher, 2015). It is necessary to give a brief introduction to the proposal first. Nine Ca${}^{2+}$ and six PO${}_4^{3-}$ ions form a Posner molecule, Ca${}_9$(PO${}_4$)${}_6$. Two calcium-phosphate “structural clusters” with atomic constituents Ca${}_9$(PO${}_4$)${}_6$, subsequently named “Posner clusters”, were first found by Posner and Betts (Posner and Betts, 1975) while they were examining the X-ray crystal structure of bone mineral-hydroxyapatite. The term “Posner cluster” is expanded to “Posner molecule,” which has been assumed to be central in consciousness due to its unique chemical structure (Fisher, 2015). Posner molecules are assumed to protect the phosphorus-31 nuclear-spin entanglements located within those molecules (Fisher, 2015). The phosphorus-31 nuclear-spin entanglements within the Posner molecules, for encoding information, remain coherent for times of a day, 21 days, or possibly much longer (Swift et al., 2018; Weingarten et al., 2016). This would allow Posner molecules to function as “neural qubits” to transmit quantum information to the spatially separated neurons in the brain (Fisher, 2017; Weingarten et al., 2016). In addition to the phosphorus-31 nuclear-spin entanglements within the molecules, their nuclear spins are further entangled among Posner molecules once two bound Posner molecules start rotating about one another (Weingarten et al., 2016). Once at rest, Posner molecule pairs are susceptible to proton attack and therefore are melted and release their Ca${}^{2+}$ ions into the neuronal cytoplasm (Weingarten et al., 2016). Because the Posner molecules in the spatially separated neurons are entangled, when disentangled, all the molecules are melted simultaneously and subsequently release a swarm of Ca${}^{2+}$ ions (considering nine Ca${}^{2+}$ ions in each Posner molecule) in the neurons. This might modulate intraneuronal Ca${}^{2+}$ levels and thus make multiple neurons spike simultaneously. The simultaneous spiking of spatially separated neurons is considered a mechanism of consciousness (Weingarten et al., 2016).

If the Ca${}^{2+}$ ion concentration in the cerebrospinal fluid is reduced, in line with Fisher’s proposal, a decrease in the number of Posner molecules in the cerebrospinal fluid may occur, and the consciousness level may be lowered (Fisher, 2015). For the same reason, an elevation in the Ca${}^{2+}$ concentration in the cerebrospinal fluid may increase consciousness level. However, our findings in mice contradict these predictions. EGTA reduces Ca${}^{2+}$ concentration in the cerebrospinal fluid after ICV injection (Liang et al., 2004). Contrary to the prediction that the consciousness level of the mice would be reduced after ICV injection of EGTA, instead, an increased consciousness level of the mice, as shown by an increased LORR ED${}_{50}$ value for the mice to sevoflurane in comparison to the basal value, was found after the animals received ICV injection of EGTA in this study. A significantly decreased consciousness level of the mice was found after ICV injection of ${}^{40}$CaCl${}_2$ in this study, again in contradiction to Fisher’s prediction. Unlike calcium-40 with zero nuclear spin, calcium-43 nuclei have 7/2 nuclear spins. In Posner molecules, if calcium-43 ions replace the calcium-40 ions, especially the central ones, the decoherent time for the phosphorus-31 nuclear-spin entanglements is expected to be shortened and thus, the consciousness level of the animals is expected to reduce - i.e., calcium isotope dependence on consciousness is expected (Fisher, 2015). Indeed, we found the consciousness level of the mice was reduced after ICV injections of ${}^{43}$CaCl${}_2$. However, further analysis showed no difference in LORR ED${}_{50}$ between ICV injections of ${}^{40}$CaCl${}_2$ and ${}^{43}$CaCl${}_2$ (P $>$ 0.9999). The identical effects of ${}^{40}$CaCl${}_2$ and ${}^{43}$CaCl${}_2$ on the LORR ED${}_{50}$ indicate no calcium isotope dependence on consciousness.

Our findings are consistent with previous studies (Erickson et al., 1978; Harris, 1979). ICV injection of CaCl${}_2$ increases ethanol-induced sleeping time, whereas EGTA decreases sleeping time in mice, as reported previously (Erickson et al., 1978; Harris, 1979). The inhibitory effect of calcium was also found in in vitro studies, as an increase in extracellular Ca${}^{2+}$ ion concentration suppressed neuronal excitability, while a decrease in extracellular Ca${}^{2+}$ concentration excited neurons (Lu et al., 2010; Ma et al., 2012; Wang et al., 2004). Although exploring the mechanisms of varying calcium concentrations on neural excitability is beyond the scope of this study, it is necessary to point out that varying calcium ion concentrations affect Posner molecules the most and, therefore, affect consciousness more than other possible protein- or ion-involved mechanisms, as Posner molecules are crucial for consciousness, superseding any other potential mechanisms according to the proposal (Fisher, 2015). Therefore, our study is vital to test the predictions derived from Fisher’s proposal (Fisher, 2015) if it overshadows other possible mechanisms.

Consciousness is a challenging problem (Miller, 2005), both in philosophy and in science. Philosophically, consciousness belongs only to humans - consciousness is a feeling of oneness. Scientifically, however, it seems unjustified to claim that consciousness is uniquely specific to humans (Trewavas and Baluška, 2011). For living organisms, even a little bit of consciousness confers sentience and awareness of environment. Since consciousness is a vital function of living organisms, it merits scientific study. However, scientific study on consciousness has methodological problems - consciousness is difficult to define and therefore measure. Fortunately, we have inhaled anesthetics at hand. The potency of inhaled anesthetics can be quantified (Koblin et al., 1998). Therefore, consciousness can be quantified by using inhaled anesthetics in an alternative way.

Our previous study found that the nuclear spin property of ${}^{129}$xenon partly antagonizing its anesthetic property is why the less anesthetic potency of ${}^{129}$xenon than xenon isotopes with zero nuclear spin (Li et al., 2018). The existence of isotopic dependence of xenon anesthesia suggests a possible nuclear-spin mechanism of consciousness in the brain. Therefore, inhaled anesthetics can be applied to investigate nuclear-spin involving the mechanism of consciousness. In Fisher’s theory (Fisher, 2015), Posner molecules can be considered the nuclear-spin property of ${}^{129}$xenon. In our work, sevoflurane was the anesthetic property of ${}^{129}$xenon. The use of sevoflurane to detect Posner molecules’ effect on consciousness is reasonable since Posner molecules partially antagonizable effect is predicted to reduce the anesthetic potency of sevoflurane. Although we disprove Posner molecules in consciousness, it does not mean that nuclear spin is not crucial for consciousness. Nuclear spin may also be implied in sleep. The similarities between sleep and general anesthesia exist (Kelz et al., 2008), as sleep is a natural while general anesthesia is a drug-induced unconscious state.

In conclusion, our findings refute the predictions that phosphorus-31 nuclear-spin entanglements within Posner molecules play a central role in consciousness.

R. C, N. L., H. Q., and R. Z. performed the experiments. N. L. and R. C. analyzed the data and wrote the paper, which all authors read. S. Z. conceived the study, designed the experiments, supervised the overall project. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

All animal operations and experimental protocols conformed to the US National Institutes of Health guide for laboratory animals’ care and use (NIH Publications No. 8023, revised 1978). They were approved by the Institutional Animal Care and Use Committee (approval No: S164) at Tongji Medical College, Huazhong University of Science and Technology.

We thank Prof. Ping Yin, Department of Statistics, Tongji Medical College, at Huazhong University of Science and Technology for statistical assistance. This work was supported by a grant from the National Natural Science Foundation of China (grant 81670068) to S. Z.

The authors declare no conflict of interest.