Microglia, Inflammation, and Behavior

Since microglia are essentially in charge of CNS immunity, they are very likely to be involved in mechanisms where inflammation (an immune response) affects behavior.

Thus, researchers that are studying inflammation and behavior should also carefully consider the role that microglia have to play.

In this section, we will take a look at the different behavioral findings that have been established in the context of inflammation and microglia.

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.

Since microglia have been implicated in behavior, all facets that affect microglia and behavior are of interest to behavioral researchers. Since microglia are the central nervous system’s immune cells, they are involved in inflammatory responses which ultimately affect behavior. Thus, inflammation is another piece of the puzzle that explains how behavior works.

To make things more complicated, inflammatory responses, tied to microglial activity, can also be observed as a result of environmental factors. Thus, inflammation and subsequent behavioral alterations can be significantly altered by the environment. For more information, check out our article Microglia, Disease, and Behavior.

Inflammation can modulate the extent of depressive-like and anxiety-like behaviors. When studying anxiety or depression in rodents, it is important to take account of inflammation as it is a factor that can influence the observed behaviors.

A study by Wang et al. showed that when microglia are activated, a state of inflammation is subsequently observed that can attenuate depressive- and anxiety-like behaviors.^[1]

To study the relationship between neuroinflammation and stress-related disorders like depression, the researchers used the chronic mild stress (CMS) induction method. CMS is one of the   models of depression wherein rodents are exposed to a random array of mild stressors (food deprivation, water deprivation, hot environment, radio noise in the room, cage shake, etc.) and subsequently develop depression.

After 12 weeks of CMS, the experimental rats had:

• Hippocampal microglial activation: Proinflammatory mediators like interleukin-1β (IL-1β), IL-6, and IL-18 were upregulated in the hippocampus, indicating that the mice were in a neuroinflammed state.
• Activation of NLRP3 inflammasome: NOD-like receptor protein 3 (NLRP3) inflammasome was found to be activated. The activation of NLRP3 inflammasome is necessary for synthesizing chemotactic and other pro-inflammatory factors related to Thus, traces of this protein are associated with an inflammatory state.
• Significant depressive-like behaviors: In parallel with these microglial observations, significant depressive-like behaviors were established. To do this, the Forced Swim Test was used to measure despair and depressive-like behaviors. This test is also considered to have good predictive value for gaging antidepressants’ effectiveness. CMS rats, during the Forced Swim Test, had decreased struggling and increased immobility time.
• Reduced exploration: Furthermore, CMS rats had a reduction in the performed number of rears, a stance where the mouse is trying to climb the sides of an apparatus. During the Open Field Test, CMS rats did not rear as much as non-stressed controls did, indicating that they were significantly less explorative.
• Reduced locomotor activity: The CMS rats also had reduced locomotor activity in the Open Field Test. They had a significant decrease in the total distance traveled over the apparatus, indicating that they were not as active as the controls were.
• Significant anxiety-like behaviors: When tested in the Elevated-Plus Maze, the maze that is the most commonly used for measuring anxiety-like behaviors, the researchers found that CMS rats performed significantly more anxiety-related behaviors than stress-free controls. The CMS rats avoided open spaces and spent a significantly lower percentage of time in the maze’s open arms. These findings show that CMS rats were more anxious than controls.

The above observations show that CMS rats displayed significant differences in both microglial activity and behavior when compared to stress-free controls. To further explore the relationship between inflammation, microglia, and behavior, researchers administered CMS rats with minocycline, an antibiotic that specifically targets microglial activation.^[2]

Chronic treatment with minocycline reversed the above effects associated with CMS:

• Inhibited hippocampal microglial activation: Minocycline treatment inhibited hippocampal microglial activation, demonstrating that minocycline was effective in significantly lowering the inflammation associated with CMS.
• No activation of NLRP3 inflammasome: Additionally, minocycline inhibited NLRP3 activation, blocking the synthesis of inflammatory factors associated with microglial activity.
• Alleviated depressive-like behaviors: In addition to the inhibited NLRP3 inflammasome and the microglial inactivity, minocycline significantly alleviated the depressive-like behaviors which were initially observed in the CMS condition.
• Decreased immobility: In the Forced Swim Test, CMS rats that were treated with minocycline had decreased levels of immobility (averaging about 150 seconds of immobility) when compared with saline-treated CMS rats (averaging about 240 seconds of immobility time). The reduction of immobility is indicative of alleviated depressive-like behaviors associated with microglial inactivation.
• Increased struggling time: ‘Time spent struggling’ in the Forced Swim Test is the inverse measurement of ‘immobility time.’ The CMS minocycline-treated rats had significantly increased struggling time, averaging about 140 seconds of struggling time compared to the saline-treated CMS controls’ 50 seconds of struggling.
• Increased rearing: Furthermore, in the Open Field Test, minocycline-treated CMS rats reared significantly more (about 10 times in total) than saline-treated CMS rats did (about 2 times in total). However, the minocycline group still did not rear at the rate of the normal group, which reared about 20 times during the observational period. However, the minocycline-treated CMS rats still outperformed the saline-treated CMS rats, suggesting that the drug was able to ameliorate at least some of the locomotive deficits associated with CMS.

In summary, this experiment demonstrated how depressive-like behavior can be observed hand in hand with microglial activity. Furthermore, by studying the relationship between behavior and adiponectin, the researchers also ventured into the relationship between Microglial Physiology and Behavior.

Also, it showed how stress via environmental factors (i.e. the CMS used to induce depression) ultimately led to inflammation and microglial activity that significantly altered behavior. To explore this theme, refer to our article Environmental Effects on Microglia and Behavior.

Alzheimer’s disease is the most common type of neurodegenerative disease, affecting about 6 million Americans in 2019.^[3]

Alzheimer’s disease is characterized by:

• β-amyloid aggregation
• Tau protein hyperphosphorylation

However, neuroinflammation is also an important part of the disease’s pathophysiology, since Alzheimer’s disease is a multifactorial disorder. Traditionally, researchers have focused on targeting tau proteins and β-amyloid. But, neuroinflammation should also be considered since it is a part of the disease profile.

Thus, for researchers interested in developing a cure for Alzheimer’s, or some form of treatment, they must consider as many factors of the disease as possible, including neuroinflammation.

Recent efforts have focused on histone deacetylase inhibitors, a drug that can inhibit histone deacetylase (an enzyme that plays an important role in regulating gene expression). Histone deacetylase inhibitors have been found to have neuroprotective and anti-neuroinflammatory properties, as shown in several behavioral research studies making use of animal models of neurodegenerative diseases.

A study by Zhang et al. showed how MS-275, a benzamide histone deacetylase inhibitor, can significantly reduce neuroinflammation and microglial activity while improving behavioral symptoms in an animal model of Alzheimer’s disease.^[4]

The researchers used APP/PS1 mice, the animal model of cerebral amyloidosis for Alzheimer’s disease. The experimental mice were treated for 10 days with MS-275 while control APP/PS1 mice were given a vehicle solution.

The researchers found the following effects of MS-275 on microglia, neuroinflammation, and behavior:

• Attenuated microglial activation: The mice that were given MS-275 were found to have altered microglial activity when compared with APP-PS1 vehicle-treated controls. The experimental mice had significantly decreased activation in the hippocampus, and a slight reduction in the cortex.
• Reduced Aβ deposition: In parallel with altered microglial activation, there was a reduction in the number of Aβ deposits in the hippocampus and cortex of APP/PS1 mice that were treated with MS-275. This suggests a connection between reduced neuroinflammation (due to altered microglial activity) and subsequent Aβ deposition.
• Improved nesting performance: Behavior was also altered as a result of MS-275 administration. In particular, treated APP/PS1 mice showed improved nesting performance. Nesting is a measure of social behavior and is crucial for survival. While mice with Alzheimer’s disease showed impairments when it comes to building a nest out of provided materials, MS-275 Alzheimer’s showed gradual improvement over the treatment course. In fact, they eventually outperformed and built better (more complex) nests than normal, healthy vehicle-treated controls.

This study demonstrated how neuroinflammation plays an important role in neurodegenerative disease pathophysiology. By altering neuroinflammation, changes were observed in other aspects of the disease (such as Aβ deposition) and behavior.

In the previous example, we saw how neuroinflammation is related to the overall pathophysiology of disease and how targeting microglia with drugs can ameliorate other aspects (such as Aβ deposition) of the disease.

Now, we will take a look at how inflammation can affect the progress of neurodegeneration, how microglia behave when the brain is in a neurodegenerative state and the subsequent effects on behavior.

In a study by Cunningham et al., Me7 prion-diseased mice were used to study the relationship between microglia, inflammation, and behavior. Prion disease is a neurodegenerative disease where proteins accumulate in the brain and have a toxic effect, leading to cognitive and behavioral dysfunction.^[5]

In the experiment, the researchers induced inflammation in the Me7 prion-diseased mice via peripheral injections of bacterial endotoxin (lipopolysaccharide [LPS]), ultimately causing systemic infection.

The Me7 prion-diseased mice which had heightened inflammation due to the bacterial endotoxin (when compared with normal mice and saline-treated prion mice) were found to have:

• Exaggerated inflammatory responses: The prion-diseased LPS-injected mice had heightened levels of IL-1β and TNF-α and microglial IL-1β translation, indicating that microglia were in a proinflammatory state.
• Impaired behavioral responses: The altered microglial activity and inflammatory responses were paired with significant changes in behavior. In the Water Y-Maze, the prion-diseased LPS-challenged mice had more incorrect trials and a significantly higher number of arms entered. This means that the experimental mice were struggling to learn the task at hand and had poorer reference memory than the vehicle-treated Me7 mice.

Vehicle-treated prion-diseased mice did not show differences in behavior and microglial activation when compared with normal saline-treated mice. However, since the LPS-challenged prion-diseased mice did show significant cognitive deficits and increased proinflammatory microglial activity, this suggests that inflammation can modulate the severity of an illness.

Furthermore, even a single injection of LPS was found to have significant effects on prion-diseased mice’s:

• Motor coordination: In an acute challenge, the LPS prion-diseased mice had significant deficits when it came to motor coordination tasks, indicating that acute systematic infection can have significant behavioral effects on behavior after just a single challenge. These effects were established using the Balance Beam where a mouse is elevated on a metal beam and must stay on it as long as possible. The LPS prion-diseased mice spent significantly less time on the beam when compared with non-challenged prion-diseased mice, meaning that they had worse motor coordination and were unable to complete the task correctly.
• Muscle strength: Furthermore, impairments in muscle strength were also observed. When tested in the Inverted Screen Test, also known as the Horizontal Grid Test, the LPS prion-diseased mice were not able to spend as much time on the screen as non-challenged prion-diseased mice. When placed on the mesh screen, and then inverted, the mice were not able to hold on for as long as control Me7 mice did because their muscle strength was compromised as a result of the acute systematic challenge.

Thus, even a single injection of LPS can have significant effects on microglial activity, subsequent inflammatory effects, and, ultimately, behavior.

All in all, this experiment demonstrated the negative behavioral effects that inflammation can have on neurodegenerative disease.

Changes in microglial activity, neuroinflammation, and behavior can be triggered by psychosocial stress. To study these relationships, Sawicki et al., used a repeated social defeat (RSD) mouse model. RSD is induced by exposing aggressive male intruder CD-1 mouse with male C57BL/6 mice.^[6]

The researchers found that RSD-exposed mice had:

• Higher mRNA expression of several proinflammatory genes: The following inflammatory genes had higher mRNA expression in the RSD mice: IL-1β, TNF-α, TLR4, and CCL2. This indicates that RSD mice were in a proinflammatory state and that their microglia were active.
• Microglial activation in nociceptive neurocircuitry: The researchers were able to identify higher microglia activation specifically in the dorsal horn of the lumbar spinal cord. This finding suggests that microglial activation in the spinal cord, combined with the proinflammatory cytokine release, is related to behavioral changes which were simultaneously observed.
• Developed mechanical allodynia: , also known as pain sensitivity, can be quantified through various Pain & Heat Assays. In this experiment, the Electric Von Frey Test was used to establish that the more stress the mice were exposed to, the lower their withdrawal threshold would be. Mechanical allodynia presented itself in the absence of injury and continued for one week after RSD was ceased, so mice continued to have a sensitized response to pain even after the exposure to stress had ceased.

This experiment by Sawicki et al. also had a second phase where the mice were given a drug that can eliminate microglia. To inhibit microglia, researchers administered PLX5622 which is a colony stimulating factor 1 receptor (CSF1R) antagonist.

The RSD-exposed PLX5622-treated mice showed:

• Microglial elimination: After two weeks of treatment with PLX5622, the CSF1R antagonist was able to eliminate microglia from the spinal cord as established by Iba-1 labeling, one of the biomarkers used to measure microglia.
• Attenuated proinflammatory responses: As mentioned previously, the cytokine proteins IL-1β, TLR4, and CCL2 were all increased as a result of RSD. However, treatment with CSF1R led to significant attenuation of their mRNA levels, indicating that these cytokines were reduced.
• Prevented mechanical allodynia development: Furthermore, the pharmacological depletion of microglia ultimately stopped the development of mechanical allodynia. RSD-induced mechanical allodynia was no longer observed in PLX5622-treated mice, as indicated by their withdrawal threshold which was at the level of vehicle-treated non-stressed controls.

This study demonstrated how microglia in the spinal cord are also important for pain-related behavior and that stress can be a factor that increases inflammation and significantly attenuates behavior.

Since microglia are immune cells, they are closely related to the inflammatory response. As a result, research is increasingly focusing on the overlap between microglial activity, inflammation, and subsequent behavioral outcomes.

The inflammatory response is also implicated in pathophysiology. Thus, the relationship between microglia, inflammation, and disease is also carefully studied by many behavioral researchers. For more information on this area of research, check out our related article Microglia, Diseases, and Behavior.

1. Wang, Ya-Lin, et al. “Microglial activation mediates chronic mild stress-induced depressive-and anxiety-like behavior in adult rats.” Journal of neuroinflammation 15.1 (2018): 21.
2. Kobayashi, K., et al. “Minocycline selectively inhibits M1 polarization of microglia.” Cell death & disease 4.3 (2013): e525.
3. Gaugler, Joseph, et al. “2019 Alzheimer’s disease facts and figures.” Alzheimers & Dementia 15.3 (2019): 321-387.
4. Journal of Neuropathology & Experimental Neurology 72.3 (2013): 178-185.
5. Cunningham, Colm, et al. “Systemic inflammation induces acute behavioral and cognitive changes and accelerates neurodegenerative disease.” Biological psychiatry 65.4 (2009): 304-312.
6. Sawicki, Caroline M., et al. “Microglia promote increased pain behavior through enhanced inflammation in the spinal cord during repeated social defeat stress.” Journal of Neuroscience39.7 (2019): 1139-1149.