Microglial Physiology and Behavior

Since microglia do not have as clear-cut of a relationship with behavior as neurons do, understanding their role is a challenge as many factors need to be considered and measured.

Assessing the function of microglia and behavior requires researchers to take elements into account on the cellular level, such as the proteins and receptors involved during signaling and communication.

In this article, we will take a look at how a few studies have managed to untangle the complex task of assessing the link between microglial physiology and behavior.

To strengthen your background understanding of microglia, check out this introductory article The Behavioral Researcher’s Guide to Microglia  where we discuss what microglia are, including their function and various phenotypes.

Ever since microglial cells have been implicated in influencing behavior, they have gotten special attention from behavioral researchers.

In order to determine exactly how microglia are entwined with behavior, it is crucial to understand their physiology. To do this, regular microglial activity is manipulated through research techniques.

Manipulating microglial activity can be accomplished by manipulating microglial receptors, blocking chemokines which are specific to microglia, or completely eliminating microglia from rodents via chemical induction for a limited number of days.

Thus, the subsequent outcomes on behavior shed light on the relationship between microglial physiology and behavior. Establishing how microglial physiology affects behavior and cognition have many research implications, especially for research that focuses on disease. For a related article by us, check out Microglia, Disease, and Behavior.  

Now, we will take a look at what a few studies have shown us about microglial physiology and behavior.

A study by Kopec et al. quantified microglial expression in adolescent rats and observed behavior. They used adolescent female and male rats to observe how behavior varies along with dopamine D1 receptor (D1r) activity in the nucleus accumbens.

D1r in the nucleus accumbens is implicated in the brain’s “reward” mesolimbic dopaminergic circuitry, a part of the brain which undergoes significant plastic changes throughout adolescence.

Although it is known that D1rs are crucial for social behavior, it still remains a mystery as to how these receptors are regulated in adolescence.

In the study by Kopec et al., microglia were implicated in attenuating the D1rs found in the nucleus accumbens.

Following immunochemistry and brain tissue sampling techniques, the researchers found that:

• Microglia are involved in adolescent brain development.
• Only in male rats do microglia eliminate D1rs via phagocytic activity, ultimately shaping how the nucleus accumbens develops.^[1]

In terms of behavior, the researchers established that:

• Microglia are causally related to changes in adolescent behavior which occur due to development
• Males demonstrate a declining frequency of social play as they go throughout adolescence as measured by the Sociability Chamber
• Only males have an increase in social play when given neutrophil inhibitor factor (NIF), a peptide that binds to microglial receptors and inhibits the receptor from binding with its natural ligands.
• The relationship between microglial activity and social play throughout development depends on gender.

Thus, this experiment demonstrated how manipulation of a specific receptor, important to microglial functioning and physiology, led to tremendous changes in social behavior.

On another note, social behavior is important for development and any manipulation to social behavior itself can also lead to microglial changes. Check out this related article Environmental Effects on Microglia and Behavior  to learn how environmental factors, including early life isolation, can significantly alter microglial activity and behavior.

Healthy neurons influence microglia which in turn can influence behavior. Neurons can release cytokines which can affect microglial efficiency, ultimately leading to significant behavioral changes.

CX3CL1 (also referred to as Fractalkine) is an example of a chemokine expressed by normal, healthy neurons. CX3CL1 specifically affects microglia since the only location it can bind to is  the CX3CL1 receptor which is found exclusively in microglia.

A study by Rogers et al. showed that mice that were deficient in CX3CL1 receptor  (CX3CR1^-/-) had significantly impaired cognitive abilities. The CX3CR1^-/- mice had compromised synaptic plasticity, leading to poor learning and memory.^[2]

When tested for motor learning using the accelerating Rotarod, the CX3CR1^-/- mice displayed:

• Normal motor coordination: On the first day of testing, CX3CR1^-/- mice were able to coordinate and acquire motor skills at the level of wild-type controls, indicating they were physically normal and capable of performing motor tasks.
• Poor motor learning: On the second day of testing, the wild-type mice improved their Rotarod performance, completing the task significantly faster than CX3CR1^-/- Thus, the CX3CR1^-/- mice displayed poor motor learning abilities since they were unable to improve their performance by the second day of testing.

When tested for contextual memory using the Fear Conditioning Chamber, the CX3CR1^-/- mice displayed:

• Normal training/baseline performance: When trained to associate shocking with a stimulus, the CX3CR1^-/- mice performed similar patterns of freezing behavior with controls. This indicates that at baseline the CX3CR1^-/- mice were comparable to controls and were capable of demonstrating similar bouts of freezing in the newly presented task.
• Decreased freezing in learning phase: When tested again 24-h later under the same conditions, the CX3CR1^-/- mice showed impaired learning abilities. The CX3CR1^-/- mice did not freeze as much as the wild-type mice. Thus, the CX3CR1^-/- mice were unable to retain the memory from the previous training and froze less than the controls.
• Normal freezing in a novel environment: However, even though the CX3CR1^-/- mice did not show improved fear conditioning performance when placed in the same setting, they demonstrated normal freezing behavior when placed in a novel environment. The CX3CR1^-/- mice remembered the association between the stimulus and the footshock when tested in a novel environment and performed at the level of controls.
• Possible deficits limited to hippocampal-dependent behaviors: The initial fear conditioning task is a measure of hippocampal-dependent associative learning. The second task (wherein mice are placed in a novel context) is a measure of both hippocampal-dependent and amygdala-dependent associative learning. Since the CX3CR1^-/- mice performed poorly on the first task, this suggests their cognition problem may be limited to tasks that specifically involve the hippocampus.

When tested in the Morris Water Maze, the CX3CR1^-/- mice had:

• Again, normal training/baseline performance: When tested on the hidden-platform water maze task, the CX3CR1^-/- mice performed at the level of controls. Baseline similarity in performance means that the two groups were comparable.
• Significant cognitive impairment in the probe trial: When tested again during the probe trial, the CX3CR1^-/- mice showed significant difficulties in finding the hidden platform. The CX3CR1^-/- mice had a lower amount of crossings into the target platform, suggesting that they could not remember the spatial location of the platform.

Ultimately, this study demonstrated how a receptor that is exclusively on microglia can influence learning and memory in mice when it is knocked out. These findings demonstrate how studying microglial physiology through genetic manipulation techniques can lead to insights on how healthy cognition works.^[3]

Another way to study the relationship between microglia and behavior is to deplete microglia via chemical administration. Then, it is possible to compare behavioral outcomes between rodents with healthy microglia and rodents that lack microglia. This can be accomplished chemically through injections of drugs like liposomal clodronate.

Clodronate is a toxic drug that can induce cell death. Liposomal clodronate is specifically cytotoxic to microglia while sparing other central nervous system cells like neurons and astrocytes.

A study by Nelson and Lenz administered liposomal clodronate to newborn male and female rats on postnatal days 1 and 4, in order to reduce the microglial count.^[4]

Liposomal clodronate treatment led to a large depletion of microglia in the forebrain by postnatal day 6, a deficit that lasted until postnatal day 10. Thus, experimental rats were induced with early life microglial depletion.

Behavior was automatically quantified through instruments like Noldus tracking software.

As a result of the chemical infusion, the researchers observed the following behavioral changes:

• Decreased chase behavior: When examining social play, it was found that experimental juvenile rats with microglial depletion had significantly less chase behavior when compared with vehicle-treated controls. This decrease in chase behaviors persisted in both paired play and group play conditions.
• Increased social avoidance: Adult clodronate-treated rats performed significantly more social avoidance behaviors than controls. When the stimulus rat tries to interact with the experimental rat but the experimental rat moves away, it is known as social avoidance.
• Decreased passive interaction time: Adult experimental rats spent less time interacting passively with other rats. Passive interactions refer to any instance where the experimental rat is within 5 cm of the stimulus rat, but not actively investigating or interacting with it.
• Reduced despair: Although clodronate-treated rats seemed to be less social, they demonstrated less despair than controls in the Forced Swim Test. The experimental rats spent significantly less time being immobile and more time swimming than their vehicle-treated counterparts.
• Reduced anxiety: There was a reduction of anxiety-like behaviors in juvenile and adult rats. In the Elevated Plus Maze, juvenile and adult experimental rats had significantly more open arm entries and spent more time in the open arms than the controls did. In the Open Field Test, juvenile and adult clodronate-treated rats had a significantly greater amount of center entries than controls did.
• Increased locomotor activity: There was also an increase in locomotor activity behaviors. Adult and juvenile clodronate rats crossed significantly more gridlines in the Open Field Test than their respective controls.

These experimental findings show that microglia are not limited to serving only as the brain’s immune cells, but also serve a crucial function for early life programming and development. Microglia can affect juveniles, as well as adults, behavior and motivation.

In this article, we discussed why it’s important to study microglial physiology and covered a few research examples of how scientists have accomplished this.

Since, traditionally, microglia have been viewed as the central nervous system’s leading  immune cells, not a lot of research has focused on assessing their effects on behavior.

In order to understand how microglia are implicated in behavior, it is crucial for researchers to manipulate certain microglial physiological characteristics and study the subsequent effects on behavior.

1. Kopec, Ashley M., et al. “Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats.” Nature communications 9.1 (2018): 3769.
2. Rogers, Justin T., et al. “CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity.” Journal of Neuroscience 31.45 (2011): 16241-16250.
3. Blank, Thomas, and Marco Prinz. “Microglia as modulators of cognition and neuropsychiatric disorders.” Glia 61.1 (2013): 62-70.
4. Nelson, Lars H., and Kathryn M. Lenz. “Microglia depletion in early life programs persistent changes in social, mood-related, and locomotor behavior in male and female rats.” Behavioural brain research 316 (2017): 279-293.