Archive for March 2012
The Increasingly Multifaceted Resume Of Microglia, Speculations On What It Might Mean For An Autism Paradox and The Swan Song Of Another Autism Canard
Posted March 26, 2012on:
Hello friends –
I’ve had a couple of interesting papers land in my pubmed feed the past few weeks that seem to be tangentially touching on something that has been at the back of my mind for a long time; namely, the repeated findings of a state of an ongoing immune response in the CNS of the autism population, coupled with a behavioral state that is either static, or in many cases, showing gradual improvement over time. [Discussions of ongoing immune response in the brain in autism, here, here, or here]. This is exactly the opposite of what I expected. Most of the conditions I had generally associated with a state of neuroinflammation, i.e., Alzheimer’s or Parkinson’s show a behavioral profile opposite to autism over time, i.e., a deterioration of skills and cognitive abilities. The diagnosis for these conditions is never a straight line or a gradual curve upwards, but a dispassionately reliable trajectory of a downward spiral.
This is something that has been really bugging me a lot as a riddle, I’ve mentioned it here in comments, and other places on the Internet. While outright signs of neuroinflammation are clearly associated with conditions you would rather not have, as opposed to have, we must admit that the available evidence tells us that we cannot just wave our hands, say ‘neuroinflammation!’, and know much more than the broad strokes. [Note: In my early days of my AutismNet life, my view was somewhat less nuanced.] I think that part of what was bothering me is the result of an oversimplified model in my mind’s eye, but I’d formed that model on top of a set of measurements that had empirical precision but underpowered understandings, alongside a more fundamental lack of knowledge.
We know a little more now.
The first paper that really got me thinking along these lines was Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important step in brain development thought to streamline neural communication by optimizing neuron structure. This was the first paper I’d read that hinted at microglia participation in ‘normal’ brain function; it was only very recently that microglia were considered to have any role in non pathological states. Another paper, Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, also implicated microglia in synaptic pruning.
Then I got myself a copy of The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. It is a review of several recent studies on the non-excited life of microglia.
In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.
This review by Tremblay was published in 2012, evidence of the nascent nature of our available data on microglial involvement in the normal brain environment; Tremblay states that part of the reason this type of finding is so recent is the relative difficulty of measuring microglia in non excited states. They were the electrons of brain measurements; our previous attempts to measure them were capable of causing them to change morphology.
The roles of ‘resting’ or immunologically quiescent microglia have remained relatively unknown (also see Tremblay et al., 2011). This is largely due to the difficulties of studying microglia in their non-activated state. Microglia respond promptly to any changes occurring in their environment, and therefore experimental ex vivo and in vitro preparations inevitably result in transformation of their normally prevailing behavior.
Anyway, some new whizbang technologies (i.e., in vivo two-photon laser scanning microscopy)[?] are allowing researchers to peer into the ho-hum everyday activities of ‘non activated’ microglia, and what they are finding is that the term ‘activated microglia’ might be a bit of a misnomer, microglia have been participating in brain function all along, it is just that our filters were insignificantly powered to detect some of their actions until very recently. Several studies have shown that so called ‘resting’ microglia are constantly evaluating their environment with protusions that seemed to operate rather quickly in relationship to other types of neurons.
This unexpected behavior suggested that resting or surveillant microglia may continuously survey the brain parenchyma as part of their immune function, which would justify the substantial expenditure of energy required to continuously maintain microglial dynamics in the normal brain, without excluding the possibility of an additional, distinct contribution to normal brain physiology
Several papers are reviewed that utilized a couple of highly technical methods, including double roll your own transgenic mouse models to visualize the interactions of microglia in a non excited state and synapses. Specific areas of the brain were measured in different studies, microglia were observed transiently engaging with neurons and seemed to target some dendrites for removal. The authors speculate that this could be a mechanism by which neuronal network maintenance, plasticity, could be affected.
In the mature healthy CNS, neuronal networks are continuously remodeled through the formation, modification and elimination of synaptic structures (see Fortin et al. (2011) for molecular mechanisms of structural plasticity) in relation with behavioral and sensory experience.
To determine a possible role of surveillant microglia in the structural remodeling of synaptic structures under normal physiological conditions, Tremblay et al. (2010b) also examined the size changes of spines and terminals before, during and after microglial contacts. Spines contacted by microglial processes during imaging (30–120 min sessions) were found to be smaller initially than those which remained non-contacted. Spines, but not terminals, also underwent transient increases in size during microglial contact, with smaller spines showing the most pronounced changes. Surprisingly, chronic imaging over 2 days further revealed a statistically significant difference in the elimination rate of microglia-contacted spines: spines contacted by microglia were more frequently eliminated than non-contacted spines (24 versus 7%; P 0.05), and in all cases, only the small spines were seen to disappear. These observations suggest that despite an apparently random sampling of the parenchyma, microglial processes specifically target a subset of small, structurally dynamic and transient dendritic spines.
There is also some description of studies that seemed to indicate that the microglial/synapse interactions could be modified through environmental stimulus, two experiments were described involving sensory deprivation and consequent changes in microglia activity. Other experiments described changes in microglial surveillance as a result of induced changes in neuronal excitability by chemical agonists or antagonists of glutamate receptors. [Perhaps this is the basis of the curious findings in Neuroprotective function for ramified microglia in hippocampal excitotoxicity?]
In their concluding statements, Tremblay provides a good description of just how little we know, and in a style that I love, poses open questions for the newer rounds of literature to address.
Since the recent studies have barely scratched the surface (of the brain in this case), the modalities of microglial interactions with excitatory and inhibitory synapses throughout the CNS, much as their functional significance and particular cellular and molecular mechanisms still remain undetermined. For example, in which contexts do quiescent microglia directly phagocytose axon terminals and dendritic spines, use other mechanisms such as proteolytic remodeling of the extracellular space, or refrain from intervening? How do surveillant microglia recognize and respond to the various molecular signals in their environment, including dynamic changes in neurotransmission and neuronal activity at individual synapses? How do these immune cells cooperate with other glial cells, as well as peripheral myeloid cells, in maintaining or shaping neuronal architecture and activity? And, as in the case of microglial memory of past immune challenges (see Bilbo et al., 2012), do surveillant microglia somehow remember their previous behavioral states, the flux of information processing in the brain, or the structural changes of synaptic elements in recent and not so recent windows of intervention?
The last sentence there, I think, is especially salient considered within a context of developmental programming.
So what we’ve learned is that decades after the discovery of microglia cells as the immune regulators in the CNS, they appear to also be participating in more fundamental maintenance of the neural structure of our brains; there is increasing evidence of direct relationships in synaptic and axonal removal as well as roles in neurotransmission and the regulation of excitability. Is more on the horizon?
But what about autism and our apparent autism paradox of a static or improving behavioral state alongside conditions of immune activation within the CNS?
Well, I have also been thinking about two brain scanning studies that have come out not too long ago, Neuron Number in Children With Autism (Courchesne et all) , which found increased numbers of neurons in the autism cohort, and Differences in White Matter Fiber Tract Development Present From 6 to 24 Months in Infants With Autism (Wolff et all) which found that the autism group showed denser bundled of white matter, so called wiring, between different parts of the brain. In both of these studies mention is made of the fact that it was possible that their findings, increased cell numbers could be the result of inappropriate removal of excess neurons during development.
Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons.
For example, differences in structural organization prior to a period of experience-dependent development related to social cognition (52–54) may decrease neural plasticity through limitations on environmental input, preventing typical neural specialization (52). These alterations could have a ripple effect through decreasing environmental responsiveness and escalating invariance*, thus canalizing a specific neural trajectory that results in the behavioral phenotype that defines ASDs. In typical development, the selective refinement of neural connections through axonal pruning (55) along with constructive processes such as myelination (56) combine to yield efficient signal transmission among brain regions. One or both of these mechanisms may underlie the widespread differences in white matter fiber pathways observed in the current study.
So, we have growing evidence of microglial participation of neural maintenance alongside growing evidence of impaired maintenance in the autism cohort.
Can our autism paradox be explained by microglia converging in the center of these related lines of thought? Is the answer to our riddle that the ongoing immune response in the brain is not sufficiently powered, or targeted, to cause increasing loss of abilities, but instead, was enough to keep critical, once in a lifetime chances for brain organization from occurring? Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain? Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?
That is a pretty neat idea to consider.
Even without the Courchesne and Wolff, the findings that specifically mention impaired network maintenance as possible culprits, the findings of active participation of ‘non-active’ microglia in brain optimization and normal processes is a very problematic finding for another autism canard, the idea that findings of neuroinflammation may not be pathological. The intellectually honest observer will admit that the crux of this defense lay in vaccine count trial testimony presented by John Hopkin’s researchers after their seminal neuroinflammation paper was published. Unfortunately, the vigor with which this testimony is trotted out online does not match the frequency with which such ideas actually percolate into the literature.
But with the data from Tremblay, Paolicelli, and others, such an idea becomes even more difficult to defend, we must now speculate on a mechanism by which either microglia could be in an excited state and continue to perform streamlining of the neural structure, or insist that it is possible that microglia were not excited during development, and something else happened to interfere with neuron numbers, and then, subsequently the microglia became chronically activated.
This is unlikely, and unlikelier still when we consider that anyone proposing such a model must do so with enough robustness to overcome a biologically plausible pathway supported by a variety of studies. And that is only if there was anything underneath the vapor! Make no mistake, if you ever press someone to actually defend, with literature, the mechanisms by which a state of chronic neuroinflammation might be beneficial in autism, or even the result of something else that also causes autism, no further elucidation of that mechanism is ever forthcoming. There isn’t anything there.
At some point, it becomes incumbent of people wishing to make an argument that they propose a biologically plausible mechanism if they wish to continue to be taken seriously. If they cannot, if the literature cannot be probed to make such a case with more empirical support than it might be, the notion so add odds with available evidence should be summarily discarded, unless and until a transcendent set of findings is presented. There should always be room for more findings in our worldview, but precious limited space for faith in the face of contradictory findings.
Considering the Scaffolds of Interconnectedness, “Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus” and How Seeing The Obvious Can Still Be A Pleasant Surprise
Posted March 9, 2012on:
I’ve been thinking a lot lately about the beauty and trials of the tightly coupled systems, the interconnected pathways that keep popping up when pubmed tells me something that might be of interest on journey autism. One theme bubbling to the top of my thoughts is that there is a large set of inputs capable of tweaking the areas we see altered in autism; broken isn’t necessarily appropriate, but the research increasingly tells us that a delicately balanced set of connected processes is readily changed, and the way that the physics work out, there is no way to change just one thing when you have a polygamous marriage of chemical systems.
Imagine a orchestra where all of the musicians were physically bound to one or more of their counterparts, a system of wires, pulleys, springs and levers such that the musicians are actually participating in the playing of each other, not soccer players doing synchronized flips so much as a set of violin-em-cello-em robots, connected to play their instruments in unison, wind them up and create a symphony. Different orchestras might have a tighter wire from one member to another, or an older spring, but when they worked together, you could tell what composition they were playing. In this analogy, you cannot have the drummers start beating harder and faster without also changing how hard the French horn players blow. The situation only gets more complicated if some of our musicians were connected to several other musicians simultaneously. There would still be music if the cellist couldn’t keep a steady rhythm, but it would be different music, not just a different cello.
The communication between a lot of our “systems”, immune, endocrine, stress response and central nervous systems are a lot like musicians in the orchestra, interdependent and intimately connected.
The funny thing is, this same message is being blared to me, and to you, all the time, damn near every time you turn on the TV, but it is hidden in plain sight by legislatively mandated doublespeak. Consider how many advertisements each of us have seen for pharmaceutical drugs where the number of complications and contra-indicated conditions far, far exceed the number of desired effects?
Here is a list of common side effects of Viagra:
Diarrhea, dizziness, flushing, headache, heartburn, stuffy nose, upset stomach
So right off the bat, besides what we are looking for, we can see it is common to expect Viagra to also affect your GI system, immune system, and/ or brain function. These are the types of things that are “common”. (One wonders how Viagra would sell if it always caused headaches and diarrhea, and sometimes transiently ameliorated erectile dysfunction? ) A list of ‘severe’ side effects includes memory loss and a sudden decrease in hearing or vision. Even after decades of work by a lot of exceptionally smart people and hundreds of billions of dollars, the interlocked complexity of our bodies are continuing to prove very difficult to adjust in only the way we’d like, and seemingly minor perturbations in one area can pop up in very unpredictable fashion in other functions.
Trying to put my mind around the implications of this in regards to autism often leaves me with a sense of being profoundly humbled and woefully underprepared, not unlike a lot of my experiences with autism in the real world. Secondarily, again with great similarity to personal experience, I (eventually) come to the (re-)realization that we should rejoice in opportunities to be challenged and learning more about something makes us richer in ways more important than dollars.
A superb example of all of this and more landed in my inbox the other day, Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus (Williamson et all). [Also on this paper, blog favorite, Staci Bilbo]
Williamson reported that animals given a so called ‘enriched environment’ exhibited significantly decreased immune responses in certain portions of the brain following immune challenge, with reduced levels of several chemokines and cytokines in the hippocampus in the treatment group. (A previous discussion about environmental enrichment on this blog can be found here) In this instance, the treatment group got to spend twelve hours a day in a different area, a housing unit with “a running wheel, a PVC tube and various small objects and toys”, while the control group of animals stayed in their drab, Soviet era proletariat cages all day and all night long. Here is the abstract:
Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Environmental enrichment (EE) in rodents increases neurogenesis, enhances cognition, and promotes recovery from injury. However, little is known about the effects of EE on glia (astrocytes and microglia). Given their importance in neural repair, we predicted that EE would modulate glial phenotype and/or function within the hippocampus. Adult male rats were housed either 12h/day in an enriched environment or in a standard home cage. Rats were injected with BrdU at 1week, and after 7weeks, half of the rats from each housing group were injected with lipopolysaccharide (LPS), and cytokine and chemokine expression was assessed within the periphery, hippocampus and cortex. Enriched rats had a markedly blunted pro-inflammatory response to LPS within the hippocampus. Specifically, expression of the chemokines Ccl2, Ccl3 and Cxcl2, several members of the tumor necrosis factor (TNF) family, and the pro-inflammatory cytokine IL-1ß were all significantly decreased following LPS administration in EE rats compared to controls. EE did not impact the inflammatory response to LPS in the cortex. Moreover, EE significantly increased both astrocyte (GFAP+) and microglia (Iba1+) antigen expression within the DG, but not in the CA1, CA3, or cortex. Measures of neurogenesis were not impacted by EE (BrdU and DCX staining), although hippocampal BDNF mRNA was significantly increased by EE. This study demonstrates the importance of environmental factors on the function of the immune system specifically within the brain, which can have profound effects on neural function.
Total interconnectedness kick ass!
Considering the wide ranging and predominantly ‘rather-not-have-than-have’ properties of ‘extra’ TNF-alpha and IL-1beta in the CNS, this is a pretty interesting finding. Not only that, animals ‘protected’ through environmental enrichment also showed increased levels of growth factors known to be altered in autism, again in the hippocampus. In a very real and measurable sense, it was possible to shuffle the neuroimmune cocktail of the brain by changing things like the availability of quality leisure time. As we have seen in other areas, altering the chemical milieu of immunomodulatory factors in the brain isn’t trivial, and is increasingly associated with a variety of conditions classically diagnosed through the study of behaviors.
It should be noted that there were unexpected, and generally negative findings from this study, namely, a relative lack of biomarkers indicative of increased neurogenesis in the environmental enrichment group; something that I think took the authors by a bit of surprise.
There is a short discussion on the possibilities on why the findings of differential neuroimmune responses were found only in the hippocampus, with reference being made to previous studies indicating that this area of the brain has been found to be more susceptible to a variety of insults.
There were some other findings that struck me as particularly intriguing; something that has been hinted at previously in other studies (or transcripts), but not yet well described, likely due to the fact that the area is still largely unknown to us. Specifically, the authors reported a state of glial activation, somewhat the opposite of what they expected.
The data instead suggest that EE changes the phenotype of glia, altering their activation and attenuating their pro-inflammatory response to peripheral LPS, although this remains to be directly tested. Interestingly, the blunted neuroinflammatory response within the DG of EE rats occurring concomitant with the increase in classical glial ‘‘activation’’ markers runs counter to our initial prediction. However, we believe these data simply highlight the fact that little is known about the function of these markers. Moreover, there is a growing literature that distinguishes classical versus alternative activation states in microglia, the latter of which is associated more strongly with repair (Colton, 2009; Colton and Wilcock, 2010).
Thus, it is possible that EE shifts microglia into an alternatively activated phenotype, an intriguing possibility that we are currently exploring.
The authors discuss the fact that their findings were highly spatially specific within the brain, involved a subset of cytokines and chemokines, and environmental enrichment did not seem to affect immune response in the periphery.
The immune response within the hippocampi of EE rats was markedly attenuated for a subset of cytokines and chemokines measured in our study. Importantly, not all measured immune molecules were blunted in the hippocampi of EE rats. Furthermore, the immune response was similar for each housing group in the parietal cortex as well as in the periphery. Within the hippocampus, however, EE rats had an attenuated response of interleukin-1b (IL-1b), the TNF family of genes, and several chemokines involved in the recruitment of leukocytes and monocytes. These families of genes indicate an altered hippocampal milieu in EE rats that may be less pro-inflammatory, more neuroprotective and less permeable to peripheral infiltrating immune cells.
There is a short discussion on the existing knowledge concerning IL-B and TNF-alpha in normal and pathological conditions, and how these findings are consistent with other findings involving environmental enrichment and cognition.
Tumor necrosis factor alpha (TNFa) is well characterized for its roles in inflammation and host defense, sepsis and, most intriguing for this study, apoptosis cascades (for review, see Hehlgans and Pfeffer, 2005). The observed attenuation after an immune challenge of TNFa and several associated genes in EE rats compared to HC controls indicates a potential enduring change in the hippocampal microenvironment of enriched rats, such that one mechanism by which EE may increase neuroprotection following insults to the CNS (Briones et al., 2011; Goldberg et al., 2011; Young et al., 1999) is via altered TNF tone and function, increasing the likelihood of cell survival by reducing apoptotic signaling. In addition to attenuated IL-1b and TNF responses, EE rats showed blunted responses for several chemokines known to influence the recruitment of circulating monocytes and leukocytes to the CNS.
Finally, the authors conclude how their findings add to the literature on environmental enrichment and brain function.
In summary, environmental enrichment is a relatively simple manipulation that results in robust beneficial outcomes for the brain. While previous studies have shown a role in post-insult rehabilitation for EE, our study provides evidence that enrichment need not follow the insult in order to be beneficial. Indeed, neuroinflammatory disease states might be attenuated or delayed in their onset in the face of ongoing EE. The translational reach of this manipulation remains to be explored, but in animal models of neuroinflammation, EE may provide a simple preventative measure for negative outcomes.
The bottom line is that a fuller rat life experience resulted in different neuroimmune profiles, findings with some consistency with previous observations that an enriched rat house resulted in improved behavioral manifestations of cognitive performance. The qualities of these different neuroimmune profiles are also consistent with chemical profiles associated with positive outcomes in several conditions.
There is a deceivingly startling realization hidden in these finding, startling because it reveals the malleable nature of the seemingly different, but basic systems interacting and deceptive because it is so obvious. How many of us have known someone who deteriorated upon entering a nursing home, or even retiring from working? How many of us have kept their children inside for a week due to weather and watched their children go crazy after the already inferior indoor entertainment options are long exhausted? Those changes in emotion, in behaviors and function, just like the findings from this study, are founded by chemistry.
But seeing evidence that relatively simple environmental modifications can rejigger the molecular atmosphere of the brain is still more than a little awe inspiring. Knowing there is machinery underneath the hood is a little different than observing the cogs of cognition swell , shrink, or slow down; nothing less than a deeper understanding of the chemical basis of thought. And that is pretty cool.