Exam 3
Maternal Separation and Cognitive Task Studies: Results
Carried out a cognitive task called the wind shift maze (task of working memory). Looked at levels of IL-10 at postnatal day 35 vs how many errors they made during the wind shift maze task (more errors = poor cognitive performance). When looking at the males, only the males that were maternally separated showed a correlation between lower levels of IL-10 and worst cognitive performance (this was not true of control males). Shows how experience with early life stress might specifically program the immune system such that now it has less anti-inflammatory (and thus more pro-inflammatory) activity that is also affecting the brain. Females showed no correlations; seems as though early life stress is impacting the male immune system more. This makes sense because in early postnatal life male microglia are very primed; the immune system is also differently developed in males vs females; the male immune system is more active during early postnatal life.
Production of Prostaglandins
Cells have membranes comprised of phospholipids. Sometimes, arachadonic acid is released from these membrane-bound phospholipids. COX2 (cyclooxygenase 2) catalyzes the reaction whereby arachadonic acid is converted into prostaglandin. Prostaglandin can become different things based on a second enzyme. This process occurs in microglia and endothelial cells.
COX2 and Early Life Stress Study: Anti-Inflammatory Cytokines
Central treatment with anti-inflammatory cytokines (IL-10) during pre-adolescence also prevents parvalbumin loss and neuronal damage. Parvalbumin is a marker of important cell type in the prefrontal cortex.
Evidence for immune effects of psychological interventions: Compassion Meditation
Compassion meditation is where you not only focus on your own present state, but also concentrating on your relationship with another human being or part of yourself; offering part of yourself or someone else compassion. Looked at effects of compassion meditation on perceived stress after an acute stressor and levels of IL-6. Used an acute stressor called a Trier social stress test; a person is brought into a room in front of a panel of people with a video camera pointed at them and they are told with very little preparation to give a speech on something they don't know much about; panel is trained to have a completely flat affect; after this task they are asked to do mental arithmetic in front of the panel. Sympathetic activation during this task generally causes increases in IL-6 levels in blood. Looked at people and how many meditation sessions they did per week and correlated that with their perceived stress of that particular situation and their levels of IL-6. Both their perceived stress and levels of IL-6 were decreased the more times per week they spent meditating.
Drug Addiction and the Brain: Plasticity
Drug addiction occurs when the circuits controlling impulsivity and compulsivity become rewired to respond preferentially to contexts and cues associated with drugs of abuse. In the brain we have three different circuits at work: -PFC to OFC (involved in repeated thoughts) to hippocampus/insula; this circuit is highlighted in green and is involved in impulsivity (something feels good and I'm going to pay more attention to it and try to obtain it) -Basal ganglia: thalamus, dorsal striatum, globus pallidus, and part of the nucleus accumbens; highlighted in purple; basal banglia regions are recruited after impulsivity and anticipation of the reward already occurred and the salience has already been assigned to the reward; these areas are thus recruited as binging/intoxication occurs in order to cause compulsive behavior; -As you begin doing things compulsively, your reward system (mesolimbic circuit) starts to break so that your set point is lower; this lowered set point starts to cause withdrawal from abstinence from the drug; when you're not able to take the drug the regions in red are recruited; amygdala signals anhedonia (that something is wrong) and negative affect; because of your negative affect you want to get more of the drug and start binging, which starts the whole cycle again. PFC and OFC together regulate what we're thinking about and assigning importance to. Stress very highly contributes to the withdrawal and negative affect. The response to the drug contributes to binging and intoxication. In order to get these systems together, we have to get plasticity.
Negative Reinforcement in Drug Addiction
"Broken" reward circuitry; basal ganglia is beginning to regulate behavioral patterns "Broken" behavior pattern regulators; similar to what is seen in OCD. Looks similar to depression (MDD is often comorbid with addiction). Uncontrolled drug use.
Mechanisms of Glucocorticoid Resistance
-Familial glucocorticoid resistance: some people are just born with more glucocorticoid resistance due to epigenetic factors (can be inherited). Pro-inflammation (i.e. lots of inflammatory cytokines) can lead to more GR resistance in the following ways: -Some inflammatory cytokines (e.g. IL-1B) can lead to lead to the modification of the glucocorticoid receptor; modifications include phosphorylation, nitrosylation, ubiquitination; prevents GR from translocating into the nucleus and causing transcription of anti-inflammatory cytokines or other things that suppress activation of immune cells. -IL-1B can also lead to the expression of an alternate glucocorticoid receptor (GRbeta), which prevents the binding of the GR to DNA Inflammatory cytokines can also activate the enzyme nitric oxide synthase (iNOS), which leads to the production of nitric oxide and binds to GRs. Inflammatory cytokines can activate MAPK, which causes the phosphorylation of GRs. Take home message is that inflammatory cytokines can lead to GR resistance, and the way this happens is through receptor modification or production of alternate GRs. On top of chronic stress and epigenetic/familial inheritance, you can also have immune inflammatory signaling itself causing glucocorticoid resistance (shows downward spiral mentioned previously).
How we study the circuitry of reinforced behavior and reward
-Imaging: take a slice of the brain and look at how the brain changes in response to repeated drug exposure -Lesion different parts of the brain and inactivate them to determine whether certain areas of the brain or certain cell types are necessary for expression of addiction -Central administration of drugs or stimulation; inject drugs into regions of the brain to see what regions are more or less responsive to the drugs -Largely through animal studies and models In animals it's a bit easier to model addiction because we can pretty easily hijack the same circuits in the animals the way we hijack the circuits in our brains with drugs of abuse; it's more complicated in humans because the way stress impacts the circuits is more complicated in humans. In terms of behavior to see whether the animals were actually addicted to the drugs, things like operant conditioning, classical conditioning, and place conditioning were used. Operant conditioning: have an animal press a bar to get an infusion of the drug; how hard will the animal work to get the drug; progressive ratio, second-order schedule. Classical conditioning: animal will form an association between the experience of the drug and the context in which they receive the drug.
Addiction Journal Club
1. Hypothesis: if IKKB activates NFKB and an increase in inflammatory signaling promotes consumption of ethanol, then inhibiting IKKB will decrease ethanol consumption by preventing the activation of NFKB. 2. Drinking in the dark paradigm: allowed mice to drink 3 hours after the dark cycle started for 3 hours; used to measure binge drinking behavior vs general consumption (mice tend to binge drink in the dark); used two bottle preference with ethanol and water; also used a paradigm where they monitored ethanol drinking over a 24 hour period; last paradigm consisted of progressively increasing consumption; basically tried to analyze more than one type of drinking behavior (support the hypothesis that any kind of ethanol preference consumption is affected by this manipulation and is not specific to one type of binge drinking). 3. Methodology and purpose of Figure 1: tested for the effect of IKKB inhibitors TPCA-1 and sulfasalazine on alcohol intake and preference; used 6 hours of 24 hour two bottle choice test; these pharmacological inhibitors work mainly in the periphery (i.e. blocking IKKB in the periphery). 4. Figure 1 results: TPCA-1 treatment was effective, but there was no significant difference in fluid intake or preference; sulfasalazine was effective in reducing addiction-like behavior and and more effective than TPCA-1 - also no significant difference in fluid intake. 5. Differences between Figures 1 and 2 and results of Figure 2: difference between figures 1 and 2 is the bottle conditions and timing; figure 1 has the continuous 24 hour 2 bottle choice whereas figure 2 has the 2 bottle choice test but with limited 3 hour access to ethanol during the dark phase; the results of figure 2 show that TPCA-1 reduced ethanol consumption and preference without affecting total fluid intake; sulfasalazine did not significantly alter ethanol intake but did impact ethanol preference; results of figure 2 are essentially the same as in figure 1. 6. Figure 3: shows IKKB knockdown level of expression in the nucleus accumbens; checking whether IKKB was actually knocked down by Cre; stained brain sections for IKKB in red and GFP; Cre was attached to a gene for GFP, which means that the virus was tagged with GFP. 7. Figure 4: shows the effects of the IKKB knockdown in the nucleus accumbens on ethanol intake and preference during the 24 hour period 2 bottle choice test; mice were either injected or not injected with the virus; there was a significant difference between ethanol consumption and preference, but no difference in total fluid intake between the two groups; IKKB knockdown in the NA reduced ethanol preference. 8. Figure 5: results of IKKB knockdown in central amygdala; preference for ethanol in the 24 hour 2 bottle choice period; knockdown mice have reduced ethanol intake and preference than controls; no change in total fluid intake; higher ethanol percentages also reduced ethanol consumption. 9. Figure 6: results of preference for saccharin at different concentrations and total fluid intake; looked at knockdown in both nucleus accumbens and central amygdala; knockdown mice showed no significant difference in preference for saccharin or any changes in fluid intake; looked at saccharin because they needed to know that any differences in ethanol consumption were not due to change in taste. 10. Figure 7: target verification for the virus they injected that expressed Cre recombinase in both the nucleus accumbens and central amygdala; made sure that the virus was injected in the right area of the brain and that knockdown was complete; found that 100% of the samples had accurate needle placement and viral expression. 11. Figure 8: shows levels of IKKB expression as well as cytokine expression of TNF-a and IL-6 in mice that received Cre injections vs controls; mice that received Cre injections expressed less IKKB, TNF-a, and IL-6; knocking down IKKB blocked cytokine production. 12. Figure 9: stained three different cell types within the central amygdala and nucleus accumbens; stained for neurons, astrocytes, microglia, and IKKB; essentially showed how expressed IKKB is within these different cell types; IKKB is expressed in everything, but is the most expressed in neurons. 13. Figure 10: stained for astrocytes, microglia, and neurons to look at the movement of the viral vector delivery system; found that Cre was expressed primarily in neurons, which means that we were knocking down IKKB mainly in neurons. 14. Table 1: listed target genes that had ethanol-induced changes; shows vast number of genes that could be affected. 15. Hypothesis supported?: the hypothesis was supported by the fact that the two pharmacological inhibitors of IKKB as well as knockdown of IKKB reduced ethanol consumption in mice.
Intervention Journal Club
1. Hypothesis: if regular yoga practice affects systemic biomarkers at the level of the psycho-neuro-immune axis, then a therapeutic effect may be produced at a cellular level by the lowering of the stress response and elicitation of the relaxation response. 2. What is a prospective, single blinded, randomized, trial?: randomized meant the assignment to yoga vs non-yoga was random; the researchers were the ones who were blinded so they didn't know who was receiving which treatment when analyzing data; prospective refers to the fact that they watched a cohort over time and observed what happened. 3. Participants: 66 people from New Delhi; more women, which is characteristic of the disease (more women affected by the disease); disease activity score had to be >2.6 and receiving medical treatment for at least 6 months. 4. What is Rheumatoid Arthritis?: a progressive autoimmune inflammatory disease of the joints; triggers autoantibodies, which activate TLRs and can lead to increased activity of NFKB and production of TNF-a and IL-6 (this is what causes the inflammation of the joints). 5. Experimental design/interventions: participants were split into two groups (yoga vs non-yoga); people in yoga group went through 8 weeks of yoga based lifestyle intervention; yoga was done in sessions that were 120 minutes long 5X a week; participants continued with prescribed medications and medical care; looked at disease activity based on DAS28-ESR; analyzed blood for inflammatory markers and mind-body communicative markers; measured serum TGF-beta levels; RNA extracted from blood samples and PCR was run to quantify expression; looked at quality of life. 6. Table 3 findings: shows before and after data for the 8 weeks for the QOL assessment and levels of biomarkers from blood sample; for the yoga group QOL scores increased and pro-inflammatory biomarkers decreased (TGF-beta is an anti-inflammatory biomarker and it increased); biomarkers for mind-body communication also increased; for the non-yoga group results were mixed, but QOL scores increased. 7. Figure 2: showed post-intervention difference in QOL of the patients; scores for physical, psychosocial, and social domains were higher in the yoga group vs controls. 8. Tables 4 and 5 and Figure 3: showed data about participants' perception about QOL and quality of health pre- and post-intervention; found a significant change in the yoga groups' perception of QOL (increased) after intervention; also a significant increase in the yoga groups' perception of quality of health after intervention. 9. Figure 4: shows differences in gene expression in yoga vs non-yoga groups; found a down-regulation of mRNA specific to pro-inflammatory cytokines (IL-6, CTLA-4, and TNF-a); up-regulation of TGF-B (anti-inflammatory); down-regulation of NFKB was not statistically significant. 10. Figure 5: shows disease assessment between males and females; found a statistically significant difference in the female group between the yoga and non-yoga. 11. Table 6: disease severity scores were negatively correlated with QOL; disease severity scores were positively correlated with pro-inflammatory cytokine levels.
Study on Bacteria and BBB
4 day old rat pups were injected peripherally with E. coli. Researchers looked at serum levels (fraction of blood that's not red blood cells - measure of immune cells and cytokines) to observe their immune response to peripheral injection of E. coli. PBS is the vehicle (no bacteria). Rats had high levels of IL-1B and IL-6 24 hours after being injected with E. coli. Corticosterone was also increased, which is not surprising because there are several ways by which peripheral inflammatory cytokines can induce the HPA axis. An expected immune response was produced in these neonates.
Anti-Inflammatory Treatments for Schizophrenia
A role for inflammatory processes has also been postulated for schizophrenia. Th-2 cytokines (anti-inflammatory) are increased in blood and CSF of schizophrenia patients. Remember that Th-2 cytokines are also increased during pregnancy; evidence has shown that there is increased psychosis during pregnancy in women who are at higher risk for schizophrenia. Monocyte function has been shown to be altered in schizophrenia patients; this dysfunction is different in that there is not a lot of microglial activation in the brain but rather more astrocyte activation. Kynurenic acid is upregulated in schizophrenia patients; quinolinic acid is reduced. Astrocyte activation usually leads to more kynurenic acid production from IDO. Not only does kynurenic acid block NMDA receptors, it also blocks nicotinic acetylcholine receptors (causes cognitive dysfunction). Overall leads to decreased glutamate and dopamine release. If we can rebalance kynurenic and quinolinic acid, we can treat the positive symptoms as well as negative symptoms of schizophrenia. Anti-psychotics only treat positive symptoms of schizophrenia. If you give a healthy person ketamine, some of the symptoms are both the positive and negative symptoms seen in schizophrenia because it works the same way kynurenic acid works. Blocking COX2 in individuals with schizophrenia prevents the increased production of kynurenic acid and aid in some of the symptoms of schizophrenia.
Evidence for immune effects of psychological interventions: Response to Flu Vaccine
All-out decreases in immunity are not what we want, nor what is observed after mindfulness therapies. Training in mindfulness meditation enhanced antibody production following influenza vaccination. Participants mounted a stronger immune response to the flu vaccine after going through mindfulness meditation.
Inflammatory Mediation of Hypertrophy
At the BBB, we have endothelial cells that release prostaglandins and nitric oxide when activated by cytokines. Nitric oxide can induce oxidative distress. Nitric oxide can also act as a signal transduction molecule. In areas (often in GABA neurons) where you have the release of endothelial nitric oxide as a result of inflammatory signaling (i.e. cytokines), the nitric oxide can promote dendritic spine growth, increase glutamate receptor insertion, or promote BDNF activity; these changes lead to increased activity. Therefore, endothelial nitric oxide can promote hypertrophy in places such as the amygdala and certain areas of the nucleus accumbens.
COX2 and Early Life Stress Study: Question
Attempted to answer the question: is there increased COX2 in males that exhibited pro-inflammation (lower levels of IL-10)? In adolescence, there is in fact higher levels of COX2 being produced in the prefrontal cortex.
How microglia and astrocytes are affected by drugs of abuse (study)
Both microglia and astrocytes in reward centers are affected by drugs of abuse. We see increased microglial and astrocyte activation in the nucleus accumbens as a result of exposure to a drug of abuse. This study looked particularly at morphine. Up to 15 minutes after a morphine injection, there were increased levels of CD11B (marker of microglial activation) as well as GFAP (marker of astrocyte activation); over time, these levels decreased. Normally when we see increased astrocyte and microglial activation we would assume that they balance each other out (astrocytes produce more anti-inflammatory cytokines and microglia produce more pro-inflammatory cytokines). However, in drug addiction (and in schizophrenia) even though there is astrocyte activation because of the plasticity that goes on (i.e. the intracellular machinery that gets turned on by drugs of abuse) these astrocytes that are getting activated begin pumping out more pro-inflammatory cytokines than anti-inflammatory cytokines; both microglia and astrocytes are secreting more pro-inflammatory cytokines after chronic drug of abuse administration. We also see that even though there is more astrocyte activity, there is still dysfunctional glutamate transport; even though astrocytes are activated, they are less able to transport and recycle glutamate so there is more extracellular glutamate at the synapses as well as more pro-inflammatory cytokines. Overall, the results show that after chronic exposure to drugs of abuse astrocytes are less able to transport/recycle glutamate and microglia become activated.
Role of Prostaglandins in Sickness
Bottom of image is the periphery and top is in the brain. Prostaglandins are secreted by endothelial cells of the BBB. When prostaglandin is released into the brain we get activation of microglial cells and neurons (both of these have receptors for prostaglandins).
Stress During Adolescence
Brain and microglia are still developing during adolescence. During adolescence, the stressor that is most robustly impactful is social interactions (i.e. disruption of social interactions). Adolescents rely less on caretakers and more on peers; rely on peers for protection and coping. At this stage of development, isolation from peers is the most stressful thing. Can model this in animals through social isolation. If you isolate an animal in a cage during adolescence that is considered a stressor. This can impact the development of microglia. Females have more activated microglia during adolescence, which means they are more vulnerable to challenges; females are more impacted by social stressors during adolescence than males.
Stress and Drug Seeking
CRF is the initiator of the HPA axis. When the stress system is first activated, the hypothalamus releases CRF. There are CRF receptors all over the brain in addition to the pituitary to tell the rest of the brain that there is stress. Withdrawal to drugs of abuse increase the number of these receptors; therefore, withdrawal from drugs of abuse is itself a stressor. Specifically, there is a very large increase in CRF receptors in the amygdala after withdrawal. If you block the CRF receptors with a CRF antagonist, we get decreased withdrawal-like behaviors (i.e. decreased anxiety-like behaviors after withdrawal). This shows how we know that stress is involved in this depression/negative affect that we get from withdrawal; if we know that stress is contributing to drug addiction, then if we can interrupt the stress response that might help.
Mechanisms in Mindfulness Meditation
Breast cancer patients were treated with yoga intervention. Found that patients had increased expression/transcription of glucocorticoid receptors; this means we have enhanced feedback of the system so that cortisol can be better at regulating itself and immune function. There was also reduced NFKB transcription (reduced inflammation). Summary: increased HPA feedback and reduced inflammatory processes. Other evidence shows that mindfulness-based training practices can reduce HPA over-activity. Evidence that mindfulness may subdue HPA responses to stress begin include the fact that training in an integrative meditation was associated with reduced cortisol responses to a 3 minute mental arithmetic stressor (HPA activation was reduced - better stress management). We know that too much cortisol can lead to pro-inflammatory effects and glucocorticoid resistance resulting from constant cortisol can lead to pro-inflammation in the periphery as well. Oxytocin is a pro-social bonding hormone and is good at reducing HPA activity. Mindfulness practices have been shown to increase levels of oxytocin in both men and women (specifically compassion meditation).
Aspirin
COX1 is constitutively expressed in almost all of our cells and is known as a housekeeping enzyme; necessary to promote regular cell health. We don't typically always want to block COX1 because it contributes to cell health. COX1 is also heavily expressed in the gut, which is why when we take too much ibuprofen chronically we can have gastrointestinal dysfunction because we are blocking the COX1 that is expressed in the gut and is necessary for the health of the epithelial cells. COX1 is active in blood clotting; very active in thrombocytes; this is one of the reasons why celecoxib is not very popular right now - it started giving people heart attacks (did not block COX1 at all but blocked COX2, which increased risk for blood clots). However, because COX1 promotes blood clotting if we block COX1 just enough we may be able to prevent blood clotting (and if we can do it in a way that doesn't kill our gut); this is where aspirin comes in. Aspirin blocks both COX1 and COX2, but it is better at blocking COX1 than it is at blocking COX2. This is why people of a certain age who are more vulnerable to blood clots and heart attacks are sometimes prescribed baby aspirin, which is just enough that it won't kill their gut by blocking COX1 but it will block COX1 just enough that it can prevent blood clotting. Therefore, aspirin has been used as an adjuvant treatment for psychiatric disorders because it can block COX2, but because it also blocks COX1 it can also be prescribed to prevent blood clotting. Ibuprofen inactivates COX2 reversibly; does its job and then COX2 is back in action a few hours later. On the other hand, aspirin acetylates (adds an acetyl group) COX1 and COX2 to in activate them; aspirin is irreversible because it adds an acetyl group and hangs on for a day or more (only have to take one aspirin a day). Cannot take ibuprofen at the same time as aspirin because it binds to COX1, and if you take aspirin it won't be able to acetylate COX1; after a couple of hours ibuprofen will let go of COX1 and is now unblocked (aspirin essentially won't work).
Anti-Inflammatory Treatments for Schizophrenia: Research
COX2 inhibitors have been helpful as adjuvant therapy, especially in early stages of the disease; if we can interrupt these pro-inflammatory processes early enough we can prevent the downward spiral into these disorders after which it's harder to treat. One 12 month study by Amminger and colleagues in a group of people at high risk for schizophrenia and already showing prodromal symptoms found a significantly lower transition rate to psychosis in people who received omega-3 fatty acid capsules than in placebo-treated controls. Another recent study reported that omega 3 treatment early in schizophrenia prevented loss of grey matter in cortex (prevented atrophy of PFC). Omega-3 fatty acids prevent oxidative distress and inhibit pro-inflammatory cytokine production.
COX2 Inhibitors
COX2 inhibitors have been used as anti-inflammatory treatment for depression (at least as an adjuvant treatment).
Sickness Behavior
Cytokines administered exogenously induce a large set of symptoms, referred to as sickness behavior. Includes malaise, weakness, fatigue, sleepiness, and disinterest for usual activities such as feeding, drinking, and social interactions. Both in humans and in laboratory animals, activation of the immune system is associated with reduced food intake and altered sleep patterns. In addition, fatigue is frequent in patients suffering from immune-based illnesses as well as in patients treated with cytokines. For instance, 80% of patients undergoing IFNa therapy report fatigue from moderate to severe intensity. 45% of them report depression severe enough to discontinue therapy. Doctors were seeing a general, very similar presentation of behavioral symptoms that didn't seem to line up with any specific illness; realized this was an immune response.
Immune Molecules and Drug Addiction Summary
Cytokines can impact the brain via humoral pathways (directly acting on the BBB endothelial cells or being transported through the BBB) or indirectly through neural communication, such as stimulation of vagal afferents. Pro- and anti-inflammatory cytokines have been shown to differentially affect drug seeking and drug taking. Indomethacin, a COX2 inhibitor, reduces chronic ethanol induction of brain innate immune genes as well as neuronal markers of cell death and addiction-like behavioral dysfunction. We already have seen how blocking microglial activation can prevent reinstatement of CPP for morphine.
Neurobiology of Depressed Mood
Depressed mood is tied to a couple circuits in the brain: prefrontal cortex, nucleus accumbens, hippocampus, VTA, and amygdala. Mesolimbic circuit: dopaminergic projection from VTA to nucleus accumbens; helps regulate whether something that just happened was expected and something we want to do again. Subdivisions of the hippocampus: lower ventral area regulates emotion and mood while dorsal hippocampus (AKA dentate gyrus) is important for cognitive functions and spatial memory. Hippocampus works with other regions in the circuit to communicate whether all of the stimuli that is coming in combined with our experiences and what we know manifest in this perception of some mood (e.g. depression). All of these regions communicate with the prefrontal cortex. The prefrontal cortex helps consolidate everything in a way that you can make a decision based on that. The hypothalamus also plays an important role (e.g. sickness behavior). Amygdala is important in fear regulation and threat detection. The balance and activity of neuromodulators such as serotonin and dopamine is important in helping the communication between these brain areas occur appropriately. These regions have receptors for cytokines, glucocorticoids, and prostaglandins, which allow for pathogens and other dangers in our environment to regulate our perceived mood and associated behaviors. Therefore, regions whose activity is involved in regulation of depressed mood are themselves tightly regulated by glucocorticoids and inflammatory signals.
Effects on Programming: Bad Environments
Early life stress is associated with: -Depression -Drug addiction -Borderline personality disorder -Schizophrenia -Anxiety We know these effects because the CDC did a decades long study called the ACE study that observed children in unstable environments and tracked their mental and physical health throughout their life. Also found that these individuals had many immune-related inflammatory disorders (cardiovascular disease, diabetes, etc.). Most of these dysfunctions manifest later in life, not in children.
TLRs and Drug Addiction
Ethanol largely binds to GABA receptors, but also binds to two different TLRs (TLR2 and TLR4). When ethanol binds to TLR4 it can contribute to the synergistic effect of ethanol and opiates (ethanol and opiates are often abused together); when you block TLR4 you block the synergistic effect. When ethanol binds to both TLR2 and TLR4, it can lead to neuronal apoptosis, release of DAMPs, and activation of microglia; this leads to the induction of the neuroplastic changes that are necessary for these different processes to progress. If you block TLR2 or TLR4, you can block the neuroplastic changes.
NLRP3
Example of how stress can produce an immune response in the brain. DAMPS can either activates TLRs or translocate through the membrane and activate inflammasomes. Inflammasomes are complexes of molecules that can get activated by DAMPs (in this case the inflammasome is NLRP3). ATP is one example of a DAMP. When NLRP3 is activated by a DAMP, it can activate an enzyme called caspase-1. Caspase-1 is necessary for the production of pro-inflammatory cytokines like IL-1B. Through transcription, the inflammasome is expressed and the IL-1B gene is expressed. However, caspase-1 is necessary for the IL-1B protein that was the product of the IL-1B gene expression (pro-IL-1B) to turn into the kind of IL-1B that can be released. Essentially, DAMPs can activate the inflammasome NLRP3 to allow the secretion of IL-1B; direct way of how stress can cause pro-inflammation through microglia. HMGB1 is another important DAMP; has been noted as an important DAMP involved in microglial priming (microglia can be activated by stress through glucocorticoids and if they're activated from stress when they have an immune challenge later they have an increased response to it); HMGB1 is a DAMP produced by microglia when they're activated by glucocorticoids; HMGB1 then comes back to the microglia and activates the inflammasome NLRP3; maybe early life stress can activate microglia through HMGB1 so that they carry that forward into later in life and be more pro-inflammatory (theory).
Early Life Programming
Field of research based on the idea that experiences during the pre- or postnatal period may modulate or "program" the trajectory of a particular developmental process. We have evolved in a way such that developmental processes are there in order to predict what our lifespan is going to be like; tweak our development to prepare for the life that we seem to be living. The idea of programming is that if a process is going in one direction an experience can change the trajectory and allow it to go in a different direction; we see this in our peripheral immune cells and microglia and how that impacts our developing nervous system. Heavily researched areas of fetal programming: -Prenatal stress (i.e. stress to mom that is reaching the fetus) -Nutrition -Maternal infection/early life immune challenge
Control Interventions
Have participants do things like: -Sit quietly and listen to music -Discussion about general good health habits -Sometimes, mind-body movement-based therapies include controls of general aerobic exercise Cannot have participants do nothing because there are a lot of things you're affecting when you do things like yoga, meditation, and CBT.
3 Common Models of Neonatal Immune Challenge
How do we know that viral infections do one thing and bacterial infections do another? There are animal models for each of these things: 1. LPS: endotoxin on bacterial cell walls; bacterial antigen; PAMP that when our immune cells interact with it via their TLR it signifies the presence of bacteria; model of bacterial infection; LPS is expressed on E. coli. 2. Expose animals to E. coli; model of bacterial infection; even though E. coli is also a bacteria, we would use this model instead of just LPS because there could be unknown mechanisms we don't know about that arise from bacterial infections (LPS is also not the only PAMP that exists on bacteria). 3. Poly IC: viral mimetic (double-stranded DNA sometimes expressed on the surface of viruses); viruses sometimes excrete poly IC, which activates TLRs; viral infection model. Different TLRs do differen things in the cell and can induce different processes. Both TLR4 and TLR3 mechanisms converge on NFKB (class of molecules), which means differential transcription of different cytokines.
Anti-Inflammatory Treatments: Background
If we can interrupt pro-inflammatory processes, we can interrupt particular cycles involved in depression, schizophrenia, and drug addiction. A dysregulated immune system can lead to a dysregulated HPA axis (and vice versa); a dysregulated HPA axis can lead to various psychiatric disorders. Pro-inflammatory activity can lead to microglial activation, which can lead to quinolinic acid; can also lead to increased astrocyte activation that can cause increased kynurenic acid; an imbalance in either of these can lead to disorders like depression, schizophrenia, or drug addiction. Peripheral pro-inflammatory activity leads to sickness behavior, which can be maladaptive if we have aberrant pro-inflammatory activity.
Effects of Mindfulness Training on Stress
Managing and coping with our stress response can help us manage and mitigate our baseline immunity. It's not just working through our stress responsivity and things like mindfulness are actually helping our immune function because we know there are things like DAMPs that are expressed when neurons are damaged and HPA turns on immune activation. Just changing the way our brain is perceiving our situation (thinking about the different subregions that are activated) and how there are receptors for neurotransmitters on things like microglia; just expression of different kinds of synaptic activity can induce or not induce certain receptors for DAMPs. Mindfulness meditation is not just reducing our stress.
Treatment for Depression
Image shows a neuron projecting out of the prefrontal cortex. Within the prefrontal cortex there are two main types of neurons: -Glutamatergic -GABAergic GABA neurons act as breaks on the excitatory system. Another neuron (top left) secretes glutamate onto the prefrontal projection neuron. GABA neuron is projecting onto the top left neuron to inhibit it. The GABA neuron has NMDA receptors; excitation of these NMDA receptors via glutamate/quinolinic acid will excite the GABA neuron and allow it to inhibit the top left neuron. If the GABA neuron inhibits the top left neuron, the top left neuron will not be able to excite the prefrontal projection neuron. If we have increased pro-inflammatory signaling in depression, there will be too much activation at the GABA neuron leading to too much inhibition of the top left neuron; wind up having a hypofunctioning prefrontal cortex. If we can somehow block the NMDA receptor on the GABA neuron we can resume the balance, which is what one of the new fast-acting antidepressants does (ketamine). Ketamine is an NMDA antagonist that blocks NMDA receptors. Ketamine acts preferentially on NMDA receptors of GABA neurons. Typical antidepressants take weeks to work because we have to wait for this one kind of receptor (5HT autoreceptor) to desensitize; start by flooding the system with 5HT and wait for the presynaptic autoreceptor to turn itself off. Ketamine works instantaneously and its effect lasts for a long time. What normally happens when there's an appropriate amount of glutamate in the synapse (i.e. not too much)?: -Activation of other glutamate receptors such as AMPA -Activation of AMPA receptors will activate signal transduction molecules (mTOR) -mTOR is also activated by rapamycin (exogenous drug) -When mTOR is activated, it can cause BDNF release -BDNF release can act on receptors of the same neuron and promote nerve health, growth, etc. In the nucleus, mTOR activation can also lead to the transcription and production of new synaptic proteins (new receptors) and thus more ability for excitation. All of these things are necessary, but in a balanced way. When you have too much GABA release and too little astrocyte activation, you can get a disruption of this process in the prefrontal cortex; this is what we see in MDD.
Prostaglandins and HPA Axis
Immune signals from the periphery can induce the HPA axis to turn on. If glucocorticoid receptors are acting properly, they can dampen down the immune response as well as their own activity/production. Prostaglandins can turn on the HPA axis. Prostaglandins are secreted from the BBB, microglia, and vagus nerve and can turn on immune signaling.
Drug Addiction Cycle
Impulsivity: a predisposition toward (i.e. inability to inhibit) rapid, unplanned reactions to internal and external stimuli without regard for the negative consequences; taking drug without thinking about the future consequences; positive reward (going for drug) hijacks the impulsivity circuits in the brain that are in place to obtain rewards; have a preoccupation/craving for the drug and will take it even in the face of consequences; positive reinforcement. Compulsivity: perseveration (trying something over and over again even though you're not getting what you want) in responding in the face of adverse consequences; characterized by a sense of anxiety or stress before committing an act; relief from stress at the time of committing the act; negative reinforcement; repeated binging and intoxication starts to recruit other circuits in the brain more involved in negative affect (i.e. anxiety); when you don't take the drug you have a preoccupation and anticipation/anxiety that causes you to binge, but when you binge you don't get a reward just relief from the anxiety. Impulsivity circuits eventually transition to compulsivity circuits.
Activation of NFKB Pathway by TLRs
In order for NFKB to translocate into the nucleus and start transcription of genes, it must release the IKB it is attached to. In order for the IKB to be released, it must be phosphorylated (add a phosphate group to it) by kinases (IKKB and IKKa). IKKB and IKKa also have to be phosphorylated in order to phosphorylate IKB.TLR activation leads to IKKB and IKKa activation.
Role of Glutamate Receptors in Depression
In the periphery there is inflammation, activation of microglia, COX2 and prostaglandin production, and lack of 5HT being produced by neurons. Get production of either quinolinic or kynurenic acid. Quinolinic acid is an NMDA agonist and can overstimulate NMDA receptors. We could have an increased production of quinolinic acid if we have more microglial activation and less astrocytic activation. We could get more kynurenic acid in the case of too little microglial activation and too much astrocytic activation. In depression we see more quinolinic acid production. In schizophrenia we see more kynurenic acid production.
Evidence for Pro-Inflammatory Profile in MDD
It has been shown in patients with MDD (just a subset - not all of them) appear to have MDD due to an immune pathogenesis (have increased serum levels of pro-inflammatory cytokines). The subset of patients that have MDD and show an increased inflammatory profile are those that have been exposed to early life stressors. It's possible that some kind of early life adversity and/or some kind of familial inheritance leads to one type of depression that is caused by a dysregulated immune response. A lot of MDD patients show heightened Th-1 dominant activity (microglial cytokines in the brain) as well as decreased anti-inflammatory cytokines (IL-10). Many patients that are on antidepressants have a reduction of pro-inflammatory cytokines which makes sense because the reduction of depressive symptoms might decrease the impact of stress on your system which could decrease the amount of pro-inflammatory cytokines; cytokines have been shown to be reduced to normal in a subset of patients after 12 weeks of taking antidepressants. Patients with MDD show decreased kynurenic acid and increased quinolinic acid in the blood as well as decreased astrocytes in the brain (look at this post-mortem). We need to remember when looking at post-mortem samples from patients with MDD that many are from successful suicide attempts; suicide adds another layer of complexity this illness so we should take these results with a grain of salt.
Early Life Bacterial Challenges
Lots of evidence that early life bacterial infection is associated with later brain and behavioral function. The biggest field of research for this kind of exposure is with autism. There is a fairly significant increase in the association of autism when there is maternal exposure to bacterial infection (there is no specific gestational time point - could be first, second, or third trimester). Largely due to prenatal exposure and not early childhood exposure because the mechanisms that underlie autism occur prenatally. When a baby is born, there are no outward signs of autism (or first few years of life either). Bacterial infections lead to cytokine synthesis and exposure that affect at the level of the placenta and increase the risk for autism.
Major Depressive Disorder: Brain
Major depressive disorder is understood to be due to: -Reduced neurogenesis in the hippocampus -Imbalance of excitatory/inhibitory function in the PFC -Hypertrophy of the amygdala; bigger amygdala -Dysregulated neuromodulation by dopamine and serotonin between several of these regions The reason we always thought about depression as being a disorder of dopamine and serotonin is because the first drugs that were tried for depression were those that acted on dopamine and serotonin - and they worked. Because these drugs seemed to help people who were classified as having depression, research all got funneled into the dopamine and serotonin mechanisms of depression; tunnel vision path that only focused on these neurotransmitters. Learning now that while these two neurotransmitters are certainly involved, they are not the end all be all of depression; not everyone is treated successfully with dopamine and serotonin drugs.
Meta-Analysis on Mindfulness Meditation
Mindfulness meditation resulted in: -Decreased NFKB transcription activity -Decreased CRP levels -Increased CD4 cell count and activity -Increased telomerase activity We can't say that mindfulness meditation is directly affecting these mechanisms, but this is fairly good evidence that there could be a causal relationship.
Role of Microglia and Monocytes in Drug Addiction
Microglia and monocytes sense drug abuse and stress. Drugs of abuse (specifically ethanol and opiates) have been shown to bind directly to TLRs on microglia. When drugs of abuse bind to TLRs it induces the transcription of pro-inflammatory cytokines and COX2. Stress leads to taking drugs of abuse; when we're undergoing withdrawal and we have a stressor CRF and glucocorticoid receptors can induce drug seeking. If we're already at a negative affective state, we crave drugs (e.g. I had a long day at work and want a drink); stress can also lead to the initiation of drug taking. Microglia are sensing the exposure to drugs of abuse as well as stress. Pro-inflammatory cytokines can induce the HPA axis and promote glucocorticoid release (which then promotes more drug seeking). This is all part of the negative affect associated with drug taking and drug addiction.
Microglia, Monocytes, and Drug Abuse
Microglia and monocytes sense drug abuse and stress. Drugs of abuse turn on pro-inflammatory activity via TLRs, microglial activation, and NFKB. Pro-inflammatory activity can directly turn on the HPA axis and create a perception of stress. This perception of stress can cause glucocorticoids to further turn on pro-inflammatory activity. Glucocorticoids can also cause drug seeking in response to stress. These pro-inflammatory mechanisms that drive the HPA axis can also drive depression (dampening of reward system and we need drugs to bring us back up to normal). Withdrawal of drugs of abuse is when we feel this dampened reward system because now we need the drug to get back to normal and there's stress involved in this, which drives the circuit (i.e. when we don't have the drug you have a lack of reward and you start to crave which is stressful; this stress turns on this pro-inflammatory system). This circuit essentially restarts over and over again through stress, depression, and withdrawal. This circuit is recruiting the basal ganglia and compulsive behavior mediating parts of the brain.
Role of Microglia During Pre- and Postnatal Development
Microglia are the glue between peripheral inflammation and brain development. Not only is the prenatal and postnatal peripheral immune response changing during typical development, microglia are also developing which means that they are differentially vulnerable to these challenges and can themselves be programmed differentially over development. One primary reason the prenatal and early postnatal periods in rodents are a particularly sensitive period for the later-life vulnerabilities to immune stimuli is because the microglia themselves are functionally different during early brain development. Prenatally, microglia are reaching the brain; go from yolk sac to the brain; look like macrophages. Over the course of prenatal and postnatal life, microglia start to form processes but still look quite amoeboid; around this time males have a perinatal testosterone surge and due to this males have more activated microglia during this time than females. Microglia that have more processes are more immunogenically activated. Microglia begin to look more ramified (like traditional microglia) throughout adolescence. Around puberty, cycling females tend to have more activated microglia than males. In adulthood and aging microglia look even more activated. Inflammaging in older age. Acute stress during any time in development causes a surge in corticosterone. Depending on where on this timeline corticosterone is actually expressed will impact development differently (i.e. will program the system differently); this applies to both immune and stress challenges.
The Subjective Experience of "Self"
Mindfulness involves an acute state of awakening that creates deep transformations of consciousness and behavior. Mindfulness can be thought of in two different ways: top-down or bottom-up.Top-down model of attentive processing: egocentric (everything understood in relation to the self - seeing how emotions and thoughts are affecting you); stimulated by focused attention, intention, and cognitive practices; how the PFC makes sense of different things and making decisions surrounding these things; used a lot in CBT.Bottom-up model of attentive processing: allocentric (frame of reference that is entirely unattached from the self); driven by practices such as meditation and yoga, which are designed to gain awareness without attachment to self; first look at emotions (see them as visceral sensations - stomach fluttering, flushed face, etc.) and then our brain helps us decide what that emotion is based on our experiences in the past; a lot of the times we see how these things have nothing to do with our actual self (can we actually separate these things from who we actually are and look at them as just sensations).
Psychological Interventions
Mindfulness training: continuous focused attention on the breath, bodily sensations, and mental content while in seated postures, yoga, and walking; mindfulness = awareness of the present moment without judgement; we are generally not too bad at being present (we can train ourselves pretty easily to be in the present and not focus too much on the future or past), but as soon as we become present that is flooded with a lot of judgement (do I like it, does this feel good, should I be doing this, etc.); suppressing this judgement is important in mindfulness and is especially helpful in emotion regulation. Music therapy: creating, singing, moving to, and/or listening to music in a therapeutic context to accomplish goals such as stress or pain management. Cognitive behavioral therapy: focuses on exploring relationships among a person's thoughts, feelings, and behaviors. During CBT a therapist will actively work with a person to uncover unhealthy patterns of thought and how they may be causing self-destructive behaviors and beliefs. All of these methods have been shown to be effective in stress management, but have had mixed results in what they do to our immune function; mindfulness training has been shown to be the most effective treatment.
TLRs in Morphine and Ethanol Dependence
Morphine is an exogenous opioid. Morphine binds to and activates TLR2 and TLR4; actions in the nucleus accumbens contributes to increased drug seeking. Actions on TLRs occur through glial activation (NFKB pathways) and results in pro-inflammatory cytokine release. These pro-inflammatory cytokines amplify opioid-induced neuronal activity in drug reward circuitry (affects neuroplasticity). TLR4 activation by itself does not produce behavioral reward (i.e. TLR activation is not involved in how much the animals like the drug); taking the drug still feels just as good, but the plasticity that's necessary for drug seeking and taking in addiction is blocked. This means that the mesolimbic dopamine system in which a drug of abuse is taken and it activates dopamine expression and the nucleus accumbens does not involve a lot of microglial activation or TLR4 activation. Instead, it's when you keep taking drugs or keep activating TLRs and microglia and start getting a pro-inflammatory profile that you start getting neuroplasticity changes; as this is happening you start going into downward spiral and pro-inflammatory cytokines are provoking neuroplasticity changes --> this is what's involved in depression, anti-reward, and recruitment of basal ganglia.
Evidence for immune effects of psychological interventions: CRP and Antibodies
Most human studies rely on salivary or blood markers. A very common marker for immune activity is C Reactive Protein (CRP). CRP is secreted from the liver into the bloodstream in response to pro-inflammatory cytokines. CRP recognizes PAMP and binds to Fc receptors (receptors for antibodies on innate immune cells); in this way, it plays a part in both innate and adaptive immune functions. CRP is elevated rapidly in response to trauma, inflammation, or infection and returns to baseline rapidly upon resolution. The antibodies that are produced by our body in response to the flu vaccine can carry out virus neutralization. Antibodies that are produced are specific for the spike protein sticking out from the virus; by sticking to the spike protein the antibody neutralizes the virus and makes it unable to infect other cells. Another thing antibodies can do is tag the pathogen for other immune cells to come and attack it; examples include opsonization and phagocytosis; CRP can bring the antibody and immune cell together via Fc receptors. Antibodies can also tag the virus for neutrophils to release lysosomes onto the cell and kill it from the outside.
Evidence for immune effects of psychological interventions: Narrative Cognitive Therapy
Narrative cognitive therapy is where we reappraise our narrative. Compared with traditional CBT, which is more of a top-down process. CBT was shown to reduce levels of IL-6 and TNF-a. People who went through narrative cognitive therapy did not show reduced levels of IL-6 and TNF-a, but did show an improvement in depressive symptoms. Shows that top-down approaches can affect immune responses.
Neuroplasticity
Neuroplasticity can hijack the positive and negative reinforcement circuits. Image A: image of a GABA neuron in the nucleus accumbens; rat received saline when it pushed the lever. Image B: image of a GABA neuron in the nucleus accumbens in a rat that received cocaine every time it pushed the lever; the rat was then taught that pressing the lever would no longer give it cocaine; rat had already progressed through addiction process before extinction of cocaine lever extinction; GABA neurons are hugely branched and express many spines; a lot more ready for receptivity than GABA neurons in the brains of normal rats (image A). At the same time, receptors for glutamate are increased.
Anti-Inflammatory Treatments for Depression
Nonsteroidal anti-inflammatory drugs (NSAIDs) - particularly the selective cyclooxygenase 2 (COX2) inhibitor celecoxib and cytokine inhibitors - have shown promising results in clinical trials. NSAIDs are not glucocorticoids because they are nonsteroidal; glucocorticoids are immunosuppressive and can be given to people to suppress their immune system. NSAIDs (e.g. ibuprofen, aspirin, and acetaminophen) inhibit COX2 and produce anti-inflammatory effects. COX2 is the inducible form of COX; only activated in response to pro-inflammatory signals; necessary to produce prostaglandins that activate pro-inflammatory mechanisms.Aspirin (also a COX inhibitor) and statins have also shown effects as adjuvant treatment for depression.These drugs are currently being investigated as adjuvant therapy (supplemental therapy) with SSRIs, but they have been shown to be at least partially effective on their own with a lot of heterogeneity. Shows that neuroinflammatory activity is not the end all be all of mechanisms; if we can interrupt some of the immune mechanisms it may help with the symptoms but it might not be all we need. Not everybody is suffering from the same disorder; not everyone with depression has depression because of pro-inflammatory dysfunction. Overall, these drugs can be helpful for some people, but maybe not everybody and we may need something in addition to it.
Evidence for immune effects of psychological interventions: Tai Chi
One study showed that the most robust decreases in CRP are found after approximately 12 weeks of yoga or tai chi. This effect was more apparent when treatment was done for patients with elevated baseline levels of CRP (e.g. cancer patients, MDD patients). Another study showed mindfulness-based CBT used to treat insomnia (teaching control over arousal mechanisms) has been shown to reduce baseline CRP levels. Tai chi administration over 12 or 16 weeks reversed insomnia-related increase in the percentage of monocytes expressing IL-6 alone, expressing TNF alone, and co-expressing IL-6 and TNF (isolated monocytes challenged with LPS). Keep in mind that there are not that many studies out there yet and it is very hard to have good controls for these studies. Therefore, a lot of this evidence can only be taken with a grain of salt. Can't use animal models (can't get a rat to meditate).
Drug Addiction and the Brain
Our brains are wired for pleasure. We approach and are reinforced by behaviors that lead to rewarding stimuli because are brains are wired such that things that we need for survival are pleasurable. Natural rewards: food, water, sex, and nurturing. These natural rewards have endogenous signals of reward in our brain so that we approach them. We have endogenous pleasure receptors in the brain that are waiting to be activated: -Dopamine (psychostimulants like cocaine) -Enkephalin (opiates like morphine/heroin) -Cannabinoids (marijuana) -Nicotinic acetylcholine (nicotine) These receptors help the circuits become activated when we are engaging in a rewarding/pleasurable act. We happen to have drugs of abuse that are way too good at acting at these receptor (i.e. activate the receptors more than the natural rewards).
Role of Prostaglandins in Sickness: Excitation/Inhibition
Prostaglandins are also secreted by microglia and neurons. The prostaglandin receptors on neurons are called EP. When prostaglandin binds to receptors on GABA neurons, they activate them. When GABA vs glutamate cells are inhibited/activated, we start seeing an influence on the excitation/inhibition balance within that brain region (especially important in the prefrontal cortex). This excitation/inhibition balance is important for appropriate functioning to regulate emotion. There is also activity on CRF neurons, which activates the HPA axis.
Schizophrenia and the Brain
People with schizophrenia often have trouble determining what is safe and what is not (part of paranoia, psychosis, and cognitive dysfunction). Learning about safety signals is one of the core symptoms of schizophrenia. This learning of safety signals is partially regulated by nicotinic acetylcholine receptors that are on GABA neurons and pyramidal neurons in the hippocampus. It has been found that in schizophrenia there is a reduction of acetylcholine receptors on neurons that receive projections from the amygdala (the amygdala regulates what is fearful). People with schizophrenia are often chain smokers; nicotine in cigarettes help them.
Microglia in MDD
Post-mortem anterior cingulate (important in communication between limbic regions and prefrontal cortex) of post-mortem MDD suicides show increased microglial activation (more amoeboid and less ramified). Microglia in animal models of MDD have a more immunogenic profile.
How are there sensitive periods for long-term consequences of immune activation?
Pregnancy is associated with many changes in the maternal immune response. Shift from Th1 to Th2 and Treg - dominant responses; hypothesized to prevent fetal rejection. Maternal immune activation also increases cytokines in the bloodstream that can influence the fetus via the placenta, but they do not typically cross on their own. Maternal antibodies and immune cells contact lining of the placenta which in turn increases cytokine production. The placenta also has its own macrophages that can produce cytokines and chemokines.
Early Life Immune Challenge Effects on Adult Behavior
Put adult rats that were exposed to bacteria when they were neonates in a Morris water maze. Morris water maze is a good test of hippocampal function; spatial memory task; rats have to swim in a tub of water and must find the platform that they can use to get out of the water. Postnatal E. coli exposure didn't affect the amount of time it took to learn where the platform was in Morris water maze. After the rats learned how to reach the platform, researchers waited 2 hours and tested them again to see if they remembered. Postnatal E. coli exposure also did not impact whether rats remembered how to get to the platform after 2 hours as well as after 7 days. The last task was training them to find the platform, and then retraining them by moving the platform. Rats with postnatal E. coli injection learned where the new platform was just as quickly as the vehicle rats, but 2 days after relearning they could not remember where the new platform was whereas vehicle rats could; giving these rats more of a cognitive challenge is where you see the difference. Showed that neonatal bacterial infection can impact cognitive function in adults.
Operant Conditioning: Drugs
Put an animal in a box with a lever they can press to self-administer a drug. There is usually an active lever and inactive lever to make sure the animal isn't just pressing the lever randomly. Over time, animal learns that when they press the lever they get an infusion of the drug. This process measures drug seeking (i.e. what an animal will do to get a drug). Progressive ratio: first ask the animal to press the lever once for a drug, then five times, then 10 times, etc.; over time increase the amount of times it presses the lever to receive drug until you reach the break point; the break point is when the animal doesn't want to work that hard to receive the drug; the more rewarding the drug is and/or the more vulnerable to addiction the animal is will determine how hard the animal works to get the drug and reach the break point. Can use this model to measure the progression through addiction, susceptibility, and abuse liability to drugs. You can also take a certain part of addiction, which is just the rewarding part of the drug (whether a drug feels good; a drug must feel good in order to have any abuse liability); in order for an animal to become addicted to a drug they need to like it first; we can also measure how much the animal likes the drug and can tell whether certain neural/immune mechanisms can affect just that. Drugs may affect progression through addiction or plasticity, but not necessarily reward; these are distinct processes; you can like something a lot and not get addicted to it; if you're more vulnerable to addiction you might get addicted to something that somebody else might not get addicted to because it's not that inherently rewarding to them. Another way to measure progression through addiction (usually for ethanol) is through choice paradigms where you ask an animal how much are you going to drink of ethanol if given the choice of water and ethanol. Drinking in the dark paradigm: rodents drink more in the dark cycle than in the light cycle; started introducing ethanol 3 hours into the dark cycle and get them to binge more on ethanol. Rats and mice (like humans) typically do not like ethanol at first; have to train them to drink it by sweetening it and then progressively decreasing the sweetness.
Maternal Immune Activation Study: Results
Put animals through a social interaction task and found that expected social behavior dysfunction was observed in maternal immune activation animals. To test social behavior they used a three chamber apparatus where one chamber is empty and one has another animal. A typical animal will spend more time interacting with the other animal. Animals that received no poly-IC but had microglial depletion displayed normal social behavior. Animals that received poly-IC and had no microglial activation displayed abnormal social behavior. If you wipe out microglia and then let them repopulate you get a return to normal social behavior. This study shows how if you get rid of some of these microglia that have been programmed by early immune challenges you can prevent or help the aberrant behavior that results from microglial programming.
Microglia Programming
Remember that microglia usually hang around from birth until we die. This means that if you hit a microglia before adolescence that microglia will be affected by that forever (can be programmed to act differently forever). If there's a sensitive period during early development for microglia and you challenge neonates with bacteria during early development (and hence cytokine exposure), microglia can be primed or sensitized to challenges later in life. When you take adult rats that were exposed to E. coli postnatally and challenge them with LPS, the microglia are more activated by LPS. Not only did microglia affect brain development, but they are also still in the brain and ready to respond to a challenge.
Role of Astrocytes in Depression
Remember that one of the main functions of astrocytes is to recycle glutamate so that there is not too much extracellular glutamate floating around and activating various receptors and potentially killing other cells. In depression, we see a reduction of astrocyte activation. Reduction of astrocyte activation can alter the balance between quinolinic and kynurenic acid production. If there's a lack of astrocyte activity, we have a lack of glutamate recycling and reuptake which means there's rogue glutamate floating around. AB in Alzheimer's also reduces astrocyte functioning; there is depression in Alzheimer's. Inflammatory cytokines can act on astrocytes and induce a reduction in glutamate reuptake. When excessive amounts of glutamate bind to extrasynaptic NMDARs (not located on spines), there is a reduction in the synthesis of brain-derived neurotrophic factor (BDNF) with effects on neurogenesis. When eNMDARs are activated by an overflow of glutamate Ca rushes into the cell and induces a calpain molecule, which induces the cleaving of the receptor for BDNF (reduces BDNF signaling). BDNF normally promotes neurogenesis, growth of new spines/synapses, and neuronal health. Summary: lack of astrocytes causes glutamate activation of extrasynaptic NMDARs leading to reduced BDNF functioning leading to reduced neuronal health and atrophy.
Depression and Schizophrenia Study
Remember: in human studies it is difficult to draw any causational conclusions. We know that not everybody who goes through childhood adversity winds up with mental or physical health issues. There are also individuals who have depression and schizophrenia that have had nurturing childhoods. However, we know that early childhood adversities do tend to increase the risk of developing issues later on in life; try to figure out whether it is immune-related mechanisms that link early childhood adversities to later mental health disorders. There is a subset of individuals that are diagnosed with mental health disorders who do have an underlying inflammatory immune-related mechanism that is driving these disorders. This subset of people might have been the ones that underwent early life adversity. Perhaps early life adversity programs the immune system such that it becomes dysregulated and that can lead to mental health disorders. Clinical study illustrated by the graph in the left of the image shows individuals who had a history of familial depression (they themselves were not diagnosed with depression but they had at least one family member who was) - this means they are at a higher genetic risk for depression. Took blood from these individuals and looked at IL-6 levels; brought them back six months later and looked at their depressive symptomology. Only the individuals who had two or more childhood stressors had a positive correlation between IL-6 levels and level of depression. Study on the right of the image shows a subpopulation of people that have schizophrenia that's regulated by immune dysfunction and thus regulated by childhood adversity. Looked at pro-inflammatory cytokines IL-6 and TNFa in people who had been diagnosed with schizophrenia vs those who hadn't. Stratified those who had schizophrenia into those who had experienced childhood adversity and those who had not. The only people who had schizophrenia AND increased pro-inflammatory cytokines were those who experienced childhood adversity.
Role of Cytokines in Sickness
Sickness behavior. If we are sick, there is activation of cytokine receptors and vagal stimulation (activation of vagus nerve). This can affect the hippocampus and hypothalamus.
Effects of Psychological Stress
Sometimes sickness behavior manifests just from being stressed (i.e. not from any immune signaling or infection); this may be a vulnerability to developing MDD or trigger an underlying disorder that already exists. Chronic stress can cause immune suppression. Pro-inflammation can provoke depressed mood. How then can chronic stress precipitate depression via pro-inflammatory signaling (i.e. other than glucocorticoid resistance)? Chronic stress in general is bad for neurons; can induce neural damage or cause them to die/atrophy. These dying/damaged neurons send out DAMPs, which induce pro-inflammatory processes; this can precipitate depression. This can also precipitate any immune mechanisms underlying MDD.
Positive Reinforcement in Drug Addiction
Starting to learn associations between reinforcing stimulus and cues/context/actions that we do to get it. Start to have increased drug use (increased reward). Initial plasticity of reward circuitry.
Stress During Postnatal Development
Stress during postnatal development in any mammal mostly relates to caretaker relationships. A mammal's wellbeing in life is completely reliant on their caretaker's presence, attention, and care. Stress during early development usually means a stress to this relationship. An appropriate level of care and nurturing from our caretakers has a significant ability to buffer almost any trauma in terms of long-term effects on our brain development. If you disrupt the caretaking environment that is when you see a tremendous association with later life emotional and stress regulation disturbances. This means that the way we model early life adversity in animals always involves caretaker disruption; for most rodents this involves maternal care. Negative models of care means that pups are taken away from their mothers and deprived of care; can also make the environment disruptive so the maternal care is more disorganized. Positive models of care involve enhancing maternal care via a paradigm known as neonatal handling; take pups away from mother for a very short period of time and return them back to mother (mild stressor - good for neural development); when the pups are returned the mother is very happy to see them and maternal care is enhanced (NOT helicopter parenting - this is disruptive); this enhances brain/cognitive development. Nuances of stress modeling: we know that it's not corticosterone exposure; it's also that pups need somatosensory stimuli and predictability from their caretaker; the brain is developing in response to these somatosensory stimuli; if somatosensory stimuli are negatively impacted by these disruptive relationships then that can also affect brain development; it's not always stress that's affecting these developmental processes
Early Life Immune Challenges
Strong evidence for neuropsychiatric effects of early life immune challenge: -High prevalence of schizophrenia in babies that were born during the Spanish flu pandemic -Researchers wondered if this surge in schizophrenia had to do with the fact that more moms had the flu while pregnant -There are also more schizophrenia cases in people who were gestated and born in winter months (second or third trimester in spring) Animal research showed that influenza infection in late gestation and/or early childhood led to increased risk for psychotic disorders. It is not the flu infection itself that causes this increased risk - it is the mom's immune response to the infection. Cytokines are produced in the mother OR child when their immune system is activated by the flu. Prenatally, there are also all of the immune cells in the decidua of the placenta that can produce cytokines in response to the flu. This cytokine synthesis during gestation or in early childhood is what causes an increased risk for schizophrenia and bipolar disorders. Even though schizophrenia and bipolar disorder present quite different clinically, what's been found is that the brain circuits controlling the behaviors associated with those disorders are very overlapping.
Maternal Immune Activation Study
Study looked at a maternal immune activation model. Early life immune challenge. Four groups: -Group 1 received no poly-IC -Group 2 received maternal immune activation (i.e. poly-IC prenatally) -Group 3 did not receive poly-IC but their microglia were depleted -Group 4 received poly-IC during gestation and then microglial depletion (wiped out microglial population in the brain) when they were juveniles After microglial depletion, microglia cannot repopulate until about 20 days later. Observed microglia at postnatal day 42 after the completion of microglia depletion (blue is nuclei of other cells, green is microglia). At postnatal day 60, we would expect microglia to be back since repopulation would be complete.
Maternal Separation and Cognitive Task Studies
Study looked at rats that had either undergone maternal separation (separated mother and pups for four hours everyday). Did a developmental profile to see how cytokine levels changed over the course of adolescence. No difference between groups when looking at levels of IL-1B; IL-1B levels decreased throughout adolescence. Also saw no difference between groups in levels of IL-4. Did see a difference between groups when observing levels of IL-10. At postnatal day 35 (right around puberty) they saw that only males showed a robust decrease in IL-10 levels (less anti-inflammatory cytokines means more pro-inflammatory profile).
Effects on Programming: Positive Environments
Study looked at the effect of a stable upbringing. One way to look at good caretaking in rats is to get a very large sample size of mothers and stratify based on caretaking; mark a threshold/cut off for mothers that are considered excellent caretakers and observe them. Can also do a specific manipulation called neonatal handling; every day starting right after birth until about 15 days after birth take the pups away from the mother and return them unharmed after 10 minutes; this is not a severe stressor; when the pups are brought back the mother will engage in a lot more caretaking (NOT hypervigilant); in this way you can mimic a very nurturing caretaking environment. Study used neonatal handling and then looked at microglia at postnatal day 10 to see the effect. First looked at Iba-1 to see if there were more or less microglia, which there was not. Interested in seeing whether microglial activation states and their activity was different; looked at inflammatory cytokine IL-10. Found robust increase in IL-10 gene expression. These results demonstrate how microglia are acting more as brain balancers; increase anti-inflammatory activity and keep themselves quiescent in terms of immunogenic activity with IL-10. Let pups grow up to adulthood (postnatal day 60) and found that even in adulthood the microglia were expressing high levels of IL-10. Then wanted to look at what was causing this increased expression of IL-10; epigenetics. Looked at methylation of IL-10 gene; found decreased methylation in adults that had good maternal care when they were pups. Good maternal care caused less methylation of the IL-10 gene, which means that it was easier for the IL-10 gene to be expressed (and thus more expression of IL-10). The fact that this epigenetic mechanism exists means that it's quite likely the offspring (i.e. offspring of pups who had good maternal care) will also have less methylation of the IL-10 gene and more anti-inflammatory activity of microglia.
Major Depressive Disorder: Diagnosis
Symptoms: -Depressed or sad mood most of the day -Diminished interest or pleasure (anhedonia) -Overeating or undereating -Insomnia or hypersomnia -Psychomotor symptoms (restlessness or slowed down motions) -Fatigue or loss of energy -Feelings of worthlessness or guilt nearly every day -Diminished ability to think or concentrate; indecisiveness -Recurrent thoughts of death and/or suicidal ideation These are taken from the DSM V. In diagnosis, criteria of 5 or more of these symptoms must be seen for at least 2 weeks (NOT due to bereavement or a drug). The good thing about the DSM V is that it allows clinicians to attend to a patient in a specific way and offer available treatments. The bad thing about the DSM V is that is has huge umbrellas over a heterogeneous array of symptoms and puts them all under the same disorder; this means that one person who has depression vs another person who has depression may have very different symptoms and thus likely different mechanisms underlying their pathology, but are being treated the exact same way. Researchers have been using these categories to drive their research for a very long time; trying to develop a mechanism that is explaining all of the criteria, which is impossible.
Drug Withdrawal and Sickness Behavior
The depressive mechanisms involving microglia are the same ones that are brought into the withdrawal phase of addiction. Have increased pro-inflammatory cytokines, microglial activation, sickness behavior, and depressed state. Continued drug use will activate this reward circuitry to temporarily relieve anhedonia whenever the drug is taken. Sickness behavior: anhedonia, loss of energy, and cognitive dampening. Withdrawal from chronic drug use: anhedonia, loss of energy, and cognitive dampening.
Drug Addiction: Downward Spiral
The emergence of drug addiction usually starts when the drug is taken in larger amounts than intended. This feels good and eventually leads to a persistent desire to keep binging on the drug. Repeated binging and intoxication, because of plasticity in reward circuits, leads to a negative affect (the withdrawal of that drug; feel worse than you normally do and now need the drug to bring you back to just baseline/normal not even a high). At this point, the circuits become more tolerable to the drug so you need more drug to get back up to baseline. The brain circuits involved in compulsive behavior start to get recruited and then it becomes a compulsive behavior (looks very similar to OCD - obsessed with a drug and compulsively taking it; anxiety associated with not taking the drug).
Early Life Immune Challenges: Effects of/on the BBB
The fetus has a growing brain, which means it has a developing BBB. BBB begins to develop in gestation and continues to develop throughout early life; this is another way immune challenges/stress can impact development. The BBB is leakier early on in development; tight junctions are present but they're not as prevalent (still being produced), which means that the substances that will be blocked from entering the brain later in development can get in more easily early in development; paracellular transport is easier during development. This means that not only are cytokines/immune molecules getting into the brain more easily, but also bacteria; bacteria do not normally get in the brain but in early development they can.
Role of Astrocytes in Compulsive Side of Addiction Circuit
The same circuit in the preoccupation phase that involve the VTA sending dopamine out to the PFC and NA becomes dysfunctional to the point where the mesolimbic dopamine system that produces rewarding effects is broken and we no longer can feel as much of a high. We then have to keep taking more drug to feel normal, and this winds up recruiting the circuit that involves the basal ganglia where you're getting compulsive behaviors, repeated behaviors, and anxiety. This plasticity involves microglia and all of the neuroplasticity and depressive-like mechanisms discussed previously. Microglia are rearranging the circuit such that the compulsive/anxiety circuit is being more activated and is necessary to bring us back to a normal state; there is no longer a really strong high.
COX2 and Early Life Stress Study: Blocking COX2
Then the question became whether or not COX2 could be blocked. COX2 is easy to block - we can block COX2 when we take ibuprofen. COX2 inhibitors are neuroprotective; they protect from excitotoxicity. COX2 inhibitors are mildly effective as adjuvant therapy for schizophrenia. Put animals through maternal separation and injected a COX2 inhibitor. Looked at their cognitive function during adolescence and found that animals that were given COX2 had less errors during the cognitive task. Blocking COX2 prevented pro-inflammatory activity from damaging cells and disrupting cognitive function.
Study on Bacteria and BBB: CD11b
Took brain samples of the pups and found increased activation of microglia (CD11b). GFAP was not found to be different from vehicle. By 2 hours post E. coli injection bacteria was found in the brain, which is remarkable because if you conducted this experiment on older rats no E. coli would be found in the brain.
Anti-Inflammatory Treatments for Schizophrenia: Progress
Treatments are typically only effective when given in addition to traditional treatments. Not always effective. Some individuals respond well while others do not.
Place Conditioning
Type of classical conditioning used to study associations between contexts and drugs of abuse. Usually use a two chamber apparatus (sometimes 3 chambers). The two chambers have very different contexts (e.g. different patterns on the walls, different floors, different lighting, etc.) Make sure that the animal doesn't prefer one side over the other. Start training the animal to associate one of the contexts/chambers with the drug of abuse and the other context/chamber with saline (control). Do this for multiple days in a row, and on the last test day drop the animal in the center and observe which chamber they go to. How well an animal associates a context with a drug speaks to one of the domains of addiction (i.e. associating your context with drug); this does not measure drug seeking (i.e. what an animal will do in order to obtain the drug).
Statins
Typically used as anti-cholesterol drugs and inhibit the enzyme HMG-CoA reductase, which plays a central role in the production of cholesterol. Statins were never really expected to be effective as a treatment for psychiatric dysfunction. Statins have a lot of anti-inflammatory effects. The way statins were found to be helpful in psychiatric disorders was when a group of men who were at risk for coronary heart disease due to high cholesterol were given statins; the researchers had also measured their depressive symptoms because coronary heart disease is highly comorbid with depression because it's a pro-inflammatory disorder; found that the men who received statins had a significant reduction in depressive symptoms.
Drug Addiction
Uncontrollable desire to take a drug accompanied by diminished control in limiting intake and emergence of an unpleasant emotional state. Drug addiction is not just taking too much of a drug (drug abuse), it is uncontrollable desire and the emergence of an unpleasant emotional state; all of these things are necessary to classify drug addiction.The circuits we look at for drug addiction can also be applied to other behavioral addictions.
What is Depression?
We can talk about the two types of depression: feeling depressed or down vs clinical presentation of major depressive disorder. First: feeling depressed; we feel "down" when we're sick or under stress; this has an immune component.
MDD: A Downward Spiral from Maladaption
We can think of the relationship between stress, immune regulation, and MDD as a vicious cycle. Dysregulated immune responses to stress can lead to increased cytokines in the periphery and circulation. These signals access the CNS via active or passive (if the BBB is leaky) transport, vagal stimulation, or a humoral response (i.e. endothelial cells) and then amplify the initial inflammatory signal (i.e. activate microglia) that can act directly or indirectly on plasticity mechanisms known to contribute to stress susceptibility and depression.Whether or not somebody has a pro-inflammatory profile, they could also have an increased amount of lifetime stress or early life stress that could lead to things later on; if somebody has a history of a lot of lifetime stress or vulnerability that stress can precipitate, we know that stress itself can turn on inflammatory signaling which could lead to this depression spiral. We also know that chronic stress can lead to GR resistance, which can increase the inflammatory profile. Remember: inflammatory cytokines can lead to glucocorticoid receptor resistance. IL-1 has been found to impair glucocorticoid receptor translocation and function, suggesting that cytokines might mediate GR resistance in relevant cell types by direct effects on the GR. You can have a dysregulated immune system causing increased cytokine production and increased microglial activation which itself can lead to a dysregulated HPA axis because (a) immune activation can cause GR resistance and (b) pro-inflammatory signaling can turn on the HPA axis. A dysregulated HPA axis can itself lead to immune signaling because GR resistance means that you get too much inflammation (cortisol leads to immune suppression). Chronic stress is sometimes signaled to the brain as a DAMP, which activates immune signaling in the brain. Shows how it keeps signaling in a circle unless the cycle is interrupted.
Role of Astrocytes in Preoccupation/Anticipation Phase
We know that astrocytes recycle glutamate from the extracellular space. Chronic exposure to drugs of abuse causes dysfunction in astrocytes' ability to recycle glutamate; this happens early on in the addiction cycle during the anticipation phase. The prefrontal cortex tries to make sense of stimuli that are coming in in terms of whether expectations are being met, if things are going to be important to attend to or not, and whether things are going to be important to motivate towards or not. The PFC receives dopamine from the VTA and sends projections down to the nucleus accumbens (also receives information from the NA). The dopamine tone recevied from VTA (how much dopamine and when) allows the PFC to drive information towards the NA to tell the rest of our brain what we're going to be attending to and what we're going to be motivating towards. Within the PFC there are two main cell types: GABAergic interneurons and glutamatergic pyramidal cells (excitatory projection neurons). Both interneurons and pyramidal cells have dopamine receptors on them. GABAergic interneurons inhibit pyramidal glutamatergic projections to the NA. There are different types of dopamine receptors and thus ways to differentially affect the circuit based on which kind of dopamine receptors you hit. The interneurons act as a filter; should we send projections to the NA or should we not; respond in the appropriate way to appropriate stimuli. Normally, information enters the PFC and GABA neurons (because of the dopamine that is coming in) tell glutamatergic projection neurons when they should fire and drive the NA to signal motivation and attentiveness. On the other hand, drugs of abuse activate activate different types of dopamine receptors on both cell types in a perfect way such that it tells the PFC this is exactly what we need to be motivating towards and exactly what we need to be attending over anything else (other information such as friends and family does not get attended to because of the fact that these drugs of abuse hijack the dopamine receptors). Because the GABAergic neurons in the presence of drug use are not turning off the excitatory projection neurons (and only turning off circuit in response to any information besides the drug), a lot of glutamate excitation of the NA is driven by drug use; this drives further drug seeking and the preoccupation/anticipation phase of addiction; we only want drugs and ignore everything else. This is also happening during adolescence when an adolescent will choose an important cue (e.g. friends) and completely block out everything else; this is part of the reason adolescents are so vulnerable to drug addiction.
Mechanisms underlying early life viral infections leading to psychosis
We know that there is no placental transfer of the flu; baby does not get the flu in utero, it only receives the immune signals produced by the mother. Transfer of maternal cytokines across the placenta and/or increased production of immune molecules by the placenta. Evidence: increased maternal IL-6 levels (pro-inflammatory cytokines) during pregnancy predicted greater neonatal amygdala volumes and connectivity, which in turn predicted poorer impulse control at two years of age. This is thought to cause atypical microglial activation, leading to changes in brain development; microglia pruning during development can change the size of the amygdala. The placenta is quite similar to the BBB in that it doesn't let everything in, it's selective, and there's activity of the placental cells (just like the endothelial cells lining the BBB) that receive signals from cytokines and produce their own immune signals. Placental abnormalities that result in hypoxia (not allowing enough oxygen to the fetus) and/or nutritional deficiency or fetal brain growth restriction can also be caused by the virus as well as immune signals; this ia another possible mechanism that contributes to this association. Hypoxia is also linked to schizophrenia. There is no good evidence that there is placental transfer of the virus itself into the fetus. However, maternal autoantibodies to the flu are transported via the placenta and interact with fetal brain antigens to disrupt brain development by attacking different parts of the brain (the teratogenic antibody hypothesis of schizophrenia).
How does early life stress impact microglia?
We're not sure how early life stress directly impacts microglia. There is a little bit of evidence showing that after maternal separation (around postnatal day 15) there is over-activation of microglia and decreased activation of astrocytes.
Immune Pathogenesis of MDD
When you have increased immune signaling, you get production of quinolinic acid and dysregulation of inhibitory/excitatory balance. If you have astrocyte activation, you can balance that out with kynurenic acid activation. Kynurenic acid blocks NMDA; in the case of MDD we don't have this.
Neurobiological Changes in MDD
While there is a reduction of dendrites and neurons in the prefrontal cortex and hippocampus, there is also a decrease of these things in parts of the nucleus accumbens (area involved in reward, pleasure, and motivation). However, in other areas of the nucleus accumbens as well as in the amygdala there is increased dendrites and neurons. Shows how in some areas of the brain we have hypertrophy instead of atrophy (atrophy was seen with excess glutamate and GABA).
