"There are about 86 billion neurons in the human brain, a number close to the number of stars in the Milky Way. In a way, everyone's brain is like a universe of depth. ”
For Professor Zhou Ning of ShanghaiTech University, who studies neuroimaging, our brains are both mysterious and romantic.
Figure | Zhou Ning (Source: Zhou Ning)
Recently, she and her team have unearthed a new treasure in this "brain universe": the discovery of a new class of hippocampal neurons.
"These cells seem to be able to connect the external objective information of the organism with the internal subjective intentions. They not only map spatial information, but also synchronously characterize the animal's exploratory intentions. Zhou Ning said.
At the same time, the coding mechanism of these neurons relies on information input from the lateral entorhinal cortex, which provides a new insight into how the brain integrates external environmental information with internal subjective intentions, and also provides a new perspective for understanding the function of located cells in the hippocampus.
It is understood that some scholars have found that hippocampal lesions and hippocampal function are important links with many brain diseases, including Alzheimer's disease, epilepsy and schizophrenia.
Understanding the coding and memory mechanisms of the hippocampus will help develop new diagnostic markers for brain diseases, conduct brain-computer interface research, and develop new drug targets.
The "Navigation Mystery" of the Brain
In the field of neuroscience, people have been plagued by the intriguing question: How does the brain determine the position of an organism in space and use this information to navigate?
In 2014, British scientist Professor John O'Keefe and Norwegian scientists Edvard Moser and May-Britt Moser (now divorced) were jointly awarded the Nobel Prize in Physiology or Medicine for their notable achievements in this field.
As early as 1971, Professor John O'Keeffe discovered a special neuron in the hippocampus while studying rats, and named it "positional cells".
When recording electrodes were implanted into the hippocampus of rats to track the activity of neurons, it was observed that cells at each location only activated when the rat passed through a specific area.
In other words, each location cell corresponds to a specific region in space, and they form an indexing mechanism for the animal brain to map external spatial information.
Interestingly, there are similar localization cells in the human brain, and together these cells form the "cognitive map" of the human brain to the external world.
Over the years, scientists have never stopped exploring the mechanism and function of location-based cell formation.
For example, are hippocampal neurons limited to representational spatial location? Do they also indicate time, or even more abstract concepts?
Do these neurons exist merely as a reflection of the external world in the brain? Or are they also influenced and regulated by the mental state of the organism?
Further, do hippocampal neurons have the ability to encode external objective information and brain subjective intentions at the same time?
It is these questions that continue to inspire people to continue to explore. The answers to these questions may help us to further unravel the deeper mysteries of brain cognition.
How does the brain encode information?
For Ning Zhou, she has been studying life sciences since she was an undergraduate. Previously, after completing his undergraduate studies at the School of Life Sciences at Peking University, Zhou received his Ph.D. in neuroscience from the University of British Columbia (UBC).
In 2011, Ning Zhou established an independent group at China Medical University in Taiwan, China. In 2019, she joined the iHuman Institute of ShanghaiTech University as an independent research group leader.
She has long been committed to conducting basic research in neurophysiology and pathology through fluorescence imaging and electrophysiology.
For example, during his Ph.D., Zhou Ning often used two-photon fluorescence microscopy to image and record brain slices labeled with fluorescent indicators.
She often observed that the fluorescence intensity of calcium ions in the cells of the living brain cells flickered and dimmed with the activity of the brain cells, one after another, like the twinkling of stars in a distant starry sky, which fascinated Zhou Ning very much.
As mentioned earlier, the human brain has about 86 billion neurons. In living animals, the hidden signals behind these intricate networks of neurons are key to understanding how the brain encodes information.
Therefore, Zhou Ning's research direction has gradually focused on the in-depth study of how the brain encodes these complex information through neuroimaging technology in living animals.
"Every time we analyze the activity of calcium ions in neurons, it is like cracking the code of the brain, which is both full of unknowns and extremely exciting." She said.
Previous studies on the hippocampus have revealed that location cells may adjust in response to the animal's foraging motivation or attention state.
For example, when an animal searches for food, the hippocampus may activate more location cells to mark the specific location of the food.
Similarly, when the external environment changes and attracts the animal's attention to the changing signal, the number and activity of the cells in the location may also be adjusted accordingly.
These studies have led Zhou Ning to question whether the subjective will of organisms can be encoded by hippocampal neurons when they are neither driven by food nor attracted by external stimuli?
Imagine this: when we go to work or school every day along a familiar road, we pass by a familiar statue on the corner of the street, and one day we suddenly decide to stop and take a closer look at it, will the neural coding of the hippocampus be different than usual?
Is there a group of neurons that can encode both the location of this statue and our willingness to explore? If the answer is yes, then these neurons have the potential to direct us where and what to do.
This question made Zhou Ning's team full of passion, especially the doctoral student Zeng Yifan showed strong interest in it. After some deliberation, they devised an ingenious experiment to explore the above questions.
Specifically, two behavior boxes with circular tracks were constructed, mice were trained to run in the same direction, and a milk powder ball was given in a fixed position as a reward for each lap.
At the same time, objects of different shapes and colors were placed in the other three positions, allowing the mice to stop and explore when they were close to these objects, or to choose to ignore the objects and continue to run. In both cases, the paths traversed by the mice were highly consistent.
（来源：Nature Communications）
A few weeks before the start of the experiment, they implanted a gradient variable refractive index lens (Grin Lens) in the head of the mice and tagged hippocampal neurons with the calcium ion fluorescence indicator GCaMP6f.
In this way, as mice perform various behaviors, they can record the activity of neurons in the hippocampus in real time through a microscope on the head.
Micromicroscopy is a breakthrough experimental technology that has emerged in the field of neurobiology in recent years. Despite weighing less than 3 grams, it integrates the key functional components of a conventional microscope.
This enables high-speed imaging of brain regions as small as approximately 0.4 square millimeters at the subcellular level, with the ability to simultaneously capture data from more than 200 neurons.
Thanks to its lightweight design, mice can carry the microscope freely in their almost unrestricted natural state, ensuring the naturalness of mouse movements and behavior.
In the behavior box designed by the research group, mice can choose whether to explore approaching objects or not.
While using a camera to film the behavior of the mice, the team used a microscope to record the calcium signaling activity of neurons in the hippocampus.
An in-depth analysis of the collected data was able to identify very typical location cells with characteristics that closely matched those previously reported in traditional locations.
（来源：Nature Communications）
And to the team's excitement, they found a unique group of hippocampal neurons outside of those typical location cells that only activated when mice explored specific locations.
However, these cells are almost silent when mice pass by the same site without any exploratory behavior.
这种特殊的细胞功能此前并未被提及过，因此他们将其命名为探索依赖性位置细胞（oePC，object exploration-dependent place cells）。
（来源：Nature Communications）
Subsequently, they built a series of experiments to study how oePCs jointly encode exploratory intent and spatial information, and to try to explain the mechanisms behind them.
By analyzing the activation time points of oePC and the object-exploring behavior with mice, they observed that the active phase of these cells usually occurs before the animal's exploratory behavior (median around 0.8 seconds) and at about 3 cm away from the object, which means that the activation of oePC actually precedes the actual exploratory behavior.
Upon further study of the positional field properties of oePC, the team found that when an object moved 0.5 cm, 1 cm, 2 cm, or 4 cm from its original position, the activity intensity of the oePC gradually diminished or even disappeared with the distance traveled, indicating that the oePC has the ability to encode at a specific location.
When replacing an old object with a new one, the activity pattern of the oePC does not change significantly.
Curiously, earlier studies have shown that changing objects in the environment will significantly change the activity of cells in traditional locations, so why doesn't oePC change?
At this point, the first thing they have to consider is: Are the old and new objects too similar to the mice for them to distinguish between them?
In order to rule out this possibility, the research team carefully selected a series of complex objects with completely different shapes, colors, etc., to test mice. However, even so, the coding of the oePC still does not produce significant changes.
When further analyzed the traditional location cells with the same position field, they found that these cells did produce significant coding differences in the replacement of objects.
This comparison clearly shows that, unlike traditional positional cells, oePC does not appear to encode the features of the object itself.
In another experimental design, the team cleverly concealed objects behind a partition so that mice could only see them when they showed the initiative to explore and went through a small door to search.
Interestingly, they observed that the oePC was pre-active even when the mice were approaching but not yet seeing objects directly.
（来源：Nature Communications）
When familiar objects are unexpectedly removed, or when objects are suddenly replaced with food in the same position, the activity of the oePC begins to decrease significantly.
This suggests that these cells are not encoding changes in environmental signals or expressing expectations of potential rewards.
In order to further investigate the characteristics of oePC, the research group designed a series of experiments, including imaging observation for several consecutive days, and a behavior box environment transformation test.
At this time, oePC shows a similar pattern to the classical position cells, which means that the neuronal activity of oePC will show some stability in a familiar environment. In the new environment, the potential for reprogramming is revealed.
最后，他们着手探讨了外侧内嗅皮层（LEC，lateral entorhinal cortex）至海马体的输入回路对 oePC 编码能力的影响。
通过在 LEC 表达抑制性的化学遗传学蛋白 hM4Di，并给小鼠注射其特异性配体——叠氮平-N-氧化物（CNO，clozapine-N-oxide），以便来抑制 LEC 神经元的活动。
Experiments have found that when the function of LEC is inhibited, the activity mode of the oePC is significantly disturbed. In contrast, among the mice that expressed the control protein, oePC was not affected.
This finding strongly suggests that signals of intent to explore are transmitted to neurons in the hippocampal region via LEC.
Do-it-yourself "being a welder"
It is also reported that the independent construction of the microscope is one of the key events for the successful completion of this project.
"This microscope is based on the open-source project of the University of California, Los Angeles MiniScope, and its architecture and principles are not new to me. ”
However, in the process of building a miniature microscope, Zhou Ning encountered an unexpected challenge.
Due to the microscope's small size and precise construction, she needed to electronically weld an interface less than a millimeter wide.
At this time, the problem arises: the tip diameter of the soldering iron in the research group is more than a few millimeters, which means that the operating space is extremely small, and the slightest carelessness may lead to a short circuit, a weak solder joint or even a burning chip.
For a researcher with a background in biology, such technical requirements are undoubtedly a great challenge.
To this end, she kept trying various methods, and even considered whether to go to the assembly line of an electronics factory to learn from it. After many trials and failures, Zhou Ning finally mastered the key points of welding.
"Now I am still one of the best in the team, and I am also a skilled worker, so I have to continue to undertake the daily maintenance of the microscope." She said.
In short, she and her team finally revealed the existence of a new group of hippocampal neurons (oePC).
日前，相关论文以《海马体探索意图和空间信息的联合编码》（Conjunctive encoding of exploratory intentions and spatial information in the hippocampus）为题发在 Nature Communications[1]上。
Zeng Yifan is the first author, and Zhou Ning is the corresponding author.
图 | 相关论文（来源：Nature Communications）
Overall, with a series of recent breakthroughs in the field of hippocampal function research, the understanding of this mysterious brain region has gradually deepened.
For example, it has been discovered that hippocampal neurons in mice are able to encode abstract cognitive variables, and experiments in which mice can autonomously control virtual objects to a specified location through hippocampal brain-computer interface technology.
Still, what we know about the hippocampus is the tip of the iceberg.
How the hippocampus maps the external world and translates this information into the subjective consciousness and actions of individuals is a key question to reveal the core mechanisms of cognition and behavior in animals and even humans.
Therefore, Zhou Ning hopes to continue to unravel the mystery of hippocampal function, which will not only help humans understand the working mechanism of the brain, but also may provide new ideas and strategies for the treatment of related neurological diseases.
Specifically, she plans to delve deeper into the role of these neurons in brain disorders, specifically to see if less environmental exploration behavior in individuals with autism is associated with dysfunction of oePC neurons.
In addition, he expects to work with other teams in the field of computational neurobiology to manipulate oePCs in real time using advanced algorithms such as closed-loop control to precisely study their impact on animal behavior.
and hope to work together to refine the neural network model to simulate the complex functions of the hippocampus and further unlock the secrets of brain functioning.
Resources:
1.Zeng, YF., Yang, KX., Cui, Y. et al. Conjunctive encoding of exploratory intentions and spatial information in the hippocampus. Nat Commun 15, 3221 (2024). https://doi.org/10.1038/s41467-024-47570-4
Operation/Typesetting: He Chenlong