CO2 instead of O2: how naked mole rats prevent convulsive attacks

Planet Earth is full of amazing creatures, each of which has its own unique feature. Most of these distinguishing features have been known to us for a long time, but there are those that become real discoveries that surprise even the most experienced zoologists. Today we look at a study in which scientists found that naked mole rats not only tolerate high levels of carbon dioxide in the air, but literally need it. What is the reason for such a non-standard need, what is the benefit for diggers in it, and how can this study help modern medicine? We will find answers to these questions in the report of scientists. Go.

Study basis

Despite its appearance far from the ideal of beauty, naked mole rats ( Heterocephalus glaber ) possess a number of amazing talents that many other mammals would envy.

Naked mole rats inhabit the territory of East Africa, more specifically in the dry savannahs and semi-deserts of Kenya, Ethiopia and Somalia. As the name implies, they live underground, eating underground parts of plants, i.e. roots, bulbs, tubers, etc. Curiously, excavators do not drink at all, getting the necessary fluid from food.

Superfluous, at first glance, the skin on the body of the mole rat provides him with more comfortable movement in the tight tunnels of the colony.

Underground tunnels of excavators form real labyrinths, the total length of which can reach 5 km. During construction, excavators form teams of 5-6 individuals: the first individual breaks the tunnel using its long incisors and rakes the ground behind it; the land is transferred from person to person to clear the tunnel from it. Periodically, the role of the excavator passes to another individual in the brigade. During a year, excavators can throw up to 4 tons of land to the surface. This is really a lot, given that the diameter of the tunnel usually does not exceed 5 cm.

This social features do not end there. Excavators, like bees, have their own queen - a female producing offspring, as well as 2-3 males who are engaged in its fertilization. The remaining individuals do not produce offspring, although physiologically capable of this. Their main task is to expand the colonies and protect their relatives. The division into builders and soldiers is based on the dimensions of the individual (larger ones become soldiers).

The female queen surrounded by offspring.

The appearance of naked mole rats is quite unusual. Let's just say that losing it in the crowd will be problematic. However, this is due to lifestyle and habitat. The dimensions of the digger are quite modest: body length up to 10 cm, tail up to 4 cm, and weight up to 35 grams. Females weigh more - up to 80 grams. Excavators have no wool, in addition to thick hairs between the paws, which helps them dig. Given that digging tunnels is a vital activity, the muscle structure has also adapted - more than 25% of the muscle mass of the digger’s body falls on the jaw, which they use during digging.

Again, given the habitat, it is not surprising that diggers are extremely poor at seeing, but they hear very well that they are actively using in communications between individuals, emitting more than 18 different sound signals.

For scientists, naked mole rats have become a real mystery of nature. Firstly, they do not suffer from cancer (only 2 cases were recorded). Secondly, they live for about 30 years, which is incredibly long for rodents (ordinary mice live up to 1.5 years).

Spending their whole lives underground, excavators adapted to the high content of CO ₂ in the air. Previously it was believed that this is where their gas exchange features end, but this is not so. In the work we are considering today, scientists have found that diggers literally need increased CO ₂ in the air.

The basis of all the above features of this species can be called eusociality. Excavators live in colonies, they have a queen, they jointly look after offspring, etc. Add the habitat here and a number of problems arise that needed to be solved by adaptation, in particular the distribution of food and the high content of CO ₂ in the air.

A video that tells about the features of the social life of diggers.

Excavators are cold-blooded, and therefore the ambient temperature is an extremely important indicator for them. In their underground colonies, the temperature is lower (19–28 ° ) than their thermoneutrality (31–34 ° ). When individuals accumulate in the same colony area at thermoneutral temperature, O ₂ consumption and metabolic demand decrease with an increase in the number of individuals. This is probably due to the lack of increased ventilation (caused by hypercapnia, i.e., CO ₂ intoxication ) in this species. Consequently, the accumulation of a large number of individuals in small spaces of the colony can be extremely useful for thermoregulation and for energy conservation.

Adaptation of excavators to increased CO ₂in air is manifested by prolonged survival under extreme hypercapnia, the absence of behavioral and respiratory reactions typical in such conditions, a decrease in the sensitivity of neural gap junctions (intercellular contacts) to CO ₂ , and also insensitivity to certain acids.

The totality of the above adaptations and the fact that adults have a lower than expected brain volume and number of neurons indicates the ability of naked mole rats to limit brain energy consumption. The habitat of this species only contributes to energy savings due to increased hypercapnia, which reduces the activity of neurons. An additional confirmation that hypercapnia suppresses nervous excitability is the powerful anticonvulsant effect of CO ₂on rodents and people.

Based on the above data, scientists have suggested that maintaining the brain in a chronic hypercapnic state promotes the selection of genes that save energy, eliminating other mechanisms to maintain inhibitory tone.

The mammalian brain works close to the theoretical limits of energy consumption, with most of it used to maintain the ionic motive forces needed to transmit electrical signals. Thus, changes in the functions and patterns of expression of ion channels and carriers could occur in excavators in order to protect neurons during the energy crisis.

An important energy-consuming process in the mammalian brain is the extrusion of intraneuronal Cl by the cationic chloride co-transporter KCC2 (protein). As a secondary active transporter, KCC2 indirectly uses ATP * to extrude Cl– through the energy stored in the * K ⁺ gradient generated by Na ⁺ / K ⁺ -ATPase * .

ATP * (adenosine triphosphate) is a universal source of energy for all biochemical processes.

Electrochemical gradient * - a combination of a concentration gradient and membrane potential, which determines the direction of movement of ions through the membrane.

Na⁺/K⁺--* (- ) — , + , Na+ .

KCC2, as a rule, is suppressed during energy crises [36], and inhibition of co-transport of cation-chloride enhances ATP recovery after oxygen-glucose deprivation.

In typical mammals, the ability of GABA to hyperpolarize neurons and inhibit their activity depends on the positive regulation of the development of KCC2, which leads to a decrease in the concentration of neuron Cl.

The abnormal function of KCC2 can interfere with GABA signaling and may be associated with seizures and impaired development of the nervous system, such as epilepsy, autism and schizophrenia.

During this study, scientists observed a colony of naked mole rats under controlled laboratory conditions. During the observations, it was noticed that each of the members of the colony spent a lot of time in the nest (the central part of the colony), where there was an elevated level of CO ₂ .

It is also noteworthy that excavators have demonstrated vulnerability to hypocapnic alkalosis * , which manifests itself as convulsive seizures in environments that mimic above-ground conditions.

Hypocapnic (respiratory / respiratory) alkalosis * - an increase in blood pH that occurs due to hyperventilation of the lungs, leading to excessive removal of CO ₂ .

Analysis of the genome of naked mole rats revealed a species-specific mutation of the histidine point in exon 22, the highly conservative regulatory region of KCC2.

Interestingly, similar mutations were found in people with febrile seizures, idiopathic generalized epilepsy, autism spectrum disorder and schizophrenia.

In accordance with the reduced function of KCC2 in naked mole rats, a decreased efficiency of GABA inhibition is observed, which manifests itself as the launch of seizures in adults at room temperature with diazepam. These seizures are blocked or reversed if the level of CO ₂ rises to what is observed in the colony nest.

Research results

Next, we consider the data obtained in the course of practical experiments, observations and calculations.

An artificial colony was created for experimental diggers equipped with sensors, in particular RFID (radio frequency identification) transponders. The sensors showed that most of the activity occurred within the same camera (socket), the location of which periodically changed. In other words, there were several chambers (nests) in the colony, most of the activity took place in one chamber, therefore individuals moved to another ( 1A ).

Image No. 1

Sensors also showed that all individuals were in the nest, clinging to each other and only occasionally leaving the nest to visit the “toilet” chamber.

Regardless of the caste (the queen, male impregnators, builders, soldiers) or the time of day, all individuals spent most of the time in the nests - 70% of the time for 26 days of observation. At the same time, as expected, the queen and her males spent more time in the nest compared to other castes ( 1B ).

Activity of experimental subjects within an artificial colony.

Measurements of the CO ₂ content in 96 chambers in 5 different colonies showed sharp changes in CO ₂ depending on the purpose of the chamber. So, the toilet chamber showed a minimum value of CO ₂ increase of 0.05% (by volume), and the nesting chamber showed 2.33%.

The nesting chamber of each colony constantly showed the highest levels of CO ₂ (1.15% ± 0.41%, n = 8), followed by the chamber in which food was placed every day (0.49 ± 0.13%, n = 5). Image 1C shows the colony conditions of TT-2 with the average CO ₂ values recorded for each chamber.

From these observations, it follows that excavators spend most of their time in the richest CO ₂ chambers, despite identical temperatures and other factors.

Next, it was necessary to determine whether such behavior is a manifestation of tolerance to CO ₂ or whether excavators consciously give preference to rooms where its content is higher. For this purpose, researchers have conducted a test in which a camera, which is not a socket tickler CO ₂ . After 24 hours, CO ₂ , compressed room air (containing 0.04% CO ₂ ), or pure nitrogen gas (N ₂ ) was introduced into the test chamber . In addition, two more colonies were tested in two other colonies, but with a higher CO content.₂ (2.5% or 11%).

Checking the activity sensor at the entrance of the test chamber has shown increasing visiting this chamber when fed to additional CO ₂ . When pumping regular air or N ₂ , there was no increase in camera attendance ( 1D ).

A similar test was conducted with cameras acting as potential nests. The results showed that most of the time the vast majority of individuals are found in the chamber where the fed CO ₂ . When the supply of carbon dioxide was stopped, the diggers amicably moved their nest to the second chamber by the percentage of CO ₂ .

The combination of these observations clearly indicates that a high content of CO ₂ (but not other gases, for example N ₂ ) increases the attendance of the corresponding premises. It got to the point that the diggers created a nest where, logically, they should not do this, only for the reason that there was more CO ₂ in this section . They made nests near the chambers where gas was supplied, and after the cessation of supply, they immediately moved to the supply chamber itself.

Image No. 2

As you know, excavators do not like to walk in the open air, preferring to spend their whole lives underground. During the study, it was decided to experimentally test the hypothesis that excavators do not tolerate warm with a low content of CO ₂surface-like environment.

Adults were placed in chambers with the following conditions: nest - 2.5% CO ₂ /21% O ₂ / 76.5% N ₂ at 32 ° C; room environment: 0.04% CO ₂ /21% O ₂ / 78.96% N ₂ at 20 ° C and a simulated environment with an air temperature of 42 ° C.

The respiration rate measured in this test showed a direct effect of environmental conditions on the condition of excavators. The respiratory rate doubled in individuals residing in a simulated medium ( 2A ).

The pH and partial pressure of CO ₂ in the blood of the diggers varied from respiratory acidosis *in conditions of imitation of a nest to relative hypocapnic alkalosis in conditions of imitation of conditions on the surface ( 2B ).

Respiratory acidosis * - a shift in the acid-base balance of the body towards an increase in acidity (decrease in pH) caused by hypoventilation or inhalation of air with a high content of carbon dioxide.

Hyperventilation was often accompanied by a period of increased motor activity in the simulated surface environment. This activity usually subsided after about 10 minutes. In 9 out of 10 individuals exposed to surface conditions, signs of convulsive activity appeared after cessation of hyperactivity, on average 14.4 ± 1.2 minutes after the temperature in the chamber reached 42 ° C. The body temperature of the experimental subjects averaged 41.7 ± 0.6 ° C.

Figure 2C shows a representative record of the cortical electroencephalogram (EEG) of seizure activity during exposure to surface conditions (0.04% CO ₂ /21% O ₂ / 78.96% N ₂ at 42 ° C).

Convulsive activity ceased ( 2D ) when air from the “nest” chamber (2.5% ₂ /21% 2 / 76.5% N ₂ ) began to be pumped into the chamber with environmental conditions .

The increased respiratory rate and blood pH measured in naked mole rats under surface conditions ( 2A and 2B ) indicate that respiratory alkalosis is the main cause of the attack, similar to the seizures observed in children and in rats / mice under similar conditions .

In rats and mice, such susceptibility to seizures caused by brain alkalosis decreases by the third week of life. This is due to increased regulation of KCC2 and subsequent maturation of GABA signaling.

In humans, KCC2 is encoded by the SLC12A5 gene. Scientists recently found that the mutation of arginine * and histidine * in region 952 of human KCC2b, located in the highly conservative regulatory region encoded by exon 22, is associated with febrile seizures.

Histidine * is a heterocyclic alpha amino acid, which is one of two conditionally essential amino acids.

Arginine * is an aliphatic basic alpha amino acid, which is one of two conditionally essential amino acids.

A study of the genome of the naked mole rat revealed histidine as a species-specific mutation in the orthologous gene * encoding KCC2.

Orthologous genes * - genes that in various species have evolved from a common predecessor.

Image 3: The

species-specific point mutation KCC2, identified in naked mole rats, can have a strong effect on the physiology of neurons. Studies of overexpression of human KCC2-H952 in muscle hippocampal and neocortical neurons have shown that histidine in this position reduces the expression of KCC2 in the neuronal plasma membrane. This leads to a decreased ability of neurons to extrude Cl ^- .

To investigate the ability of cortical neurons of diggers and mice to extrude Cl ^-, analysis of the GABAA receptor was performed using the local potential fixation method. This method allows you to study the properties of ion channels by isolating a fragment of the cell membrane using a micropipette. This allows you to control the potential difference between the sides of the membrane. The data obtained by this method allow us to estimate the level of intracellular concentration of Cl ^- .

Image No. 4

Analysis confirmed that in naked mole rats, the ability of neurons to extrude Cl- is significantly lower than in mice ( 4A ).

Next, the scientists decided to check whether the standard GABA-potentiating anticonvulsant drug (diazepam) can block the seizures caused by hyperthermia in adult diggers.

Surprisingly, diazepam not only did not prevent convulsions, but actually exacerbated them. Injection of 1 or 5 mg / kg of diazepam (intraperitoneally) to adult males and female diggers (weighing 25 to 57 g, ambient temperature 20 or 32 ° C) caused motor cramps in 10 out of 10 animals. The time to seizure usually was less than 5 minutes after injection of 5 mg / kg of diazepam (n = 5) and more than 20 minutes after 1 mg / kg of diazepam (n = 5).

EEG of two test subjects during injection showed that cramps are indeed accompanied by cortical seizures ( 4B ). With the introduction of 0.5 mg / kg of diazepam, convulsions were not observed, instead there was an increased activity.

For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists andadditional materials to it.

Epilogue

In this study, scientists found that naked mole rats, living almost their entire lives in conditions with a high content of CO ₂ , are not only immune to this, but actually need it.

The secret lies in KCC2, which is a chloride transporter, i.e. it controls the amount of chloride inside the neurons. As a rule, in mammals, chloride in central neurons is kept low. When GABA comes into contact with neurons, the latter takes chloride, which blocks the activity of the neuron.

A decrease in the activity of neurons is essential for the organization of their work. However, in excavators and people with a KCC2 mutation, the efficiency of chloride removal from neurons is reduced. Therefore, inhibition of activity does not occur as expected.

Excavators deal with this problem very unusual, using carbon dioxide. If the CO ₂ content in the air is too low, their brain simply will not be able to function in a normal rhythm, which will manifest itself in the form of seizures.

This work not only allows one to better understand the evolution of such a unique creature as a naked mole rat, but can also help in working with KCC2 mutations in people that cause seizures and the development of idiopathic generalized epilepsy, schizophrenia and autism.

Friday off-top:

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Thank you for your attention, stay curious and have a great weekend everyone, guys! :)

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