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Benusa et al. Neuroimmunol Neuroinflammation 2020;7:248-63 I http://dx.doi.org/10.20517/2347-8659.2020.03 Page 253

subclasses. For example, we reported that under certain pathologic conditions, microglia exhibit both an
[79]
increased association with, and disruption of the AIS . It is possible that these microglia are no longer
[78]
providing trophic support for the AIS, a suggested function of AXIS microglia by Baalman et al. during
[81]
homeostatic conditions, but are actively attacking the AIS, perhaps through the release of reactive ions . If
so, then could these microglia, which we characterized using immunocytochemical approaches, in fact, be
“dark microglia”, which are identified by electron microscopy? Studies to address this question are currently
[78]
underway. In addition, are the AXIS microglia, as described by Baalman et al. , the same subclass as the
microglia we have described making AIS contact? Based on work from Baalman et al. , it is likely that
[78]
the AXIS microglia are supporting the neuron and the AIS; but based on our observations, the microglia
may be mediating AIS disruption instead. Answering these questions is essential for accurately classifying
microglia but more importantly, it would help to fully understand the role that these mercurial cells play
under different conditions.

Heterogenic microglial transcriptomes
Surveying vs. reactive (“M1/M2”) microglia
In an effort to more conclusively characterize microglia and to elucidate their functions, morphologic
characterizations have been complemented by molecular classification studies. Initial attempts were based
on presumed states of activity based on limited expression profiles. Simply, microglia were classified as
either “activated” or “resting” but both terms are misleading. Microglia are never “resting” as we now
recognize that they are constantly extending and retracting their processes to survey their surroundings [82,83] .
As a result, the term “surveying”, which more accurately represents the state of activity of microglia, even
under homeostatic conditions, is now used in place of “resting”. Similarly, a more appropriate term for
“activated” is “reactive”. “Activated” implies a lack of activity until microglia are stimulated. Microglia are
constantly active however, and upon detection of changes in the environment, become “reactive”.

Reactive microglia have been further divided into “M1” and “M2” states, referring to the classical (pro-
inflammatory) and alternative (resolving/anti-inflammatory) phenotypes based on expression profiles.
The “M1” and “M2” nomenclature is a naming scheme originally derived from the T cell literature and
applied to macrophages based on their state of activation in vitro following exposure to either the T
helper type 1 (Th1) cytokine interferon gamma (IFN-γ) for the “M1” phenotype, or the T helper type 2
[84]
(Th2) cytokine interleukin 4 (IL-4) for the “M2” phenotype . Based on speculation of similar functions
between macrophages and microglia, the “M1” and “M2” classification was then applied to microglia.
The advantage of the “M1/M2” classification is that it provides a simplified nomenclature to distinguish
between microglia in functionally distinct states. However, these distinct states are frequently identified by
a small subset of surface markers, which limits resolution required for appreciating heterogeneity that is
defined by the entire transcriptome. Moreover, this naming scheme is based on assumptions that cannot be
confirmed under close scrutiny. At best, the “M1/M2” classification is inadequate for accurate description
of the complex functions of these cells (reviewed by [85,86] ). With the recognized inadequacies of the “M1/
M2” nomenclature, it has been postulated that a continuum of activity states exists between the polarized
extremes, resulting in studies presenting “M1” subtypes to better represent the heterogenic nature of these
reactive cells [87,88] . Recent studies however, have shown that factors assigned to either the “M1” or “M2”
phenotype are promiscuous yielding low fidelity to their assigned reactive state [88-90] . Thus, the complexity
of microglia function is undermined by the overly simplistic and polarized naming scheme of “M1/M2”.

Disease-associated microglia
Another subclass of reactive microglia that is specific to non-homeostatic conditions is known as
Disease-Associated Microglia (DAM). First identified in Alzheimer’s disease and amyotrophic lateral
sclerosis models , DAM or microglia with DAM-like phenotypes have now been described in tauopathy
[91]
[94]
models [92,93] multiple sclerosis and aging [91,95] . DAM express typical microglia markers including Iba-1,