Introduction: The aim of the project was to investigate the effects of antidepressants on brain networks whilst at rest. My hypothesis was that antidepressants work by reversing persistent activity in the brain’s default mode network (DMN). The DMN is implicated in self-reflection and rumination in MDD. The methodologies and results of studies of resting state networks in MDD and the effects of antidepressants are reviewed in the thesis. Increasing evidence implicates glutamate in the action of antidepressant drugs. Whether there are illness related changes in glutamate function is unresolved, largely because of the lack of techniques for assessing it. Ketamine and other NMDA antagonists have improved MDD symptoms within 24 hours though the effects are short lasting. The molecular neural networks involved in ketamine’s putative antidepressant effects are unclear. The thesis reviews the evidence. Much evidence implicates ACC as a site of action of antidepressant effects but whether this is through its regulation of the DMN or other networks is not known. This thesis compares the effect of ketamine and citalopram on ACC-related systems. Method: The thesis combines two systematic reviews of the effects of MDD and antidepressant drugs on i) resting state networks (53 studies) and ii) glutamate neurotransmission (45 studies of clinical efficacy of ketamine). There are two experimental chapters. The first describes investigation into two rapid acting antidepressant drugs acting via glutamate mechanisms. 54 unmedicated cMDD were scanned across two centres on 3T MRI scanners while being infused with placebo (0.5% saline), 0.5mg/kg ketamine or 100mg AZD6765 over 1 hour. fMRI resting state data between drug treatments was compared for the final 25 minutes of the drug infusion and for a 25 minute resting state scan a day later. The second experimental chapter examines whether these effects were shared by citalopram, a standard antidepressant. 67 unmedicated cMDD, rMDD and HC were administered citalopram 7.5mg i.v. and scanned on a 1.5T MRI scanner. In a second study 63 cMDD and HC were administered i.v. citalopram 7.5mg or placebo (0.5% saline). fMRI resting state data for the final 12 ½ minutes following drug infusion was compared. Independent Component Analysis was performed using the Group ICA for fMRI toolbox. The resting component with the highest spatial correlation to the ACC was used. Brain maps of the intensity of the selected component were constructed for each individual. Group averages were calculated and compared using SPM. Regional analysis was performed using Marseille Boite a Regions d'interet. Results: On day 1 AZD6765 significantly increased mean intensity of ACC resting component in the right insula, right IPL and left cingulate gyrus greater than ketamine or placebo. Ketamine increased mean intensity of ACC resting component greater than placebo in the right lentiform nucleus and left mFG. Significantly decreased mean intensity of ACC resting component in the left insula in the AZD6765 group compared to placebo was noted. On day 2 AZD6765 increased mean intensity of ACC resting component greater than ketamine and placebo in the left and right lentiform nuclei. AZD6765 reduced mean intensity of the ACC resting component in the left and right MFG. The first citalopram study revealed reduced mean intensity of ACC resting component in cMDD compared to rMDD and HC in PCC. rMDD had reduced mean intensity of ACC resting component in the precuneus compared to HC. In the second study, citalopram had no effect in HC but normalised precuneus activity in cMDD producing a significant drug x group interaction. Conclusions: The acute antidepressant effects of citalopram are modulated by changes in the bilateral precuneus. The precuneus is central to connectivity with other regions in MDD. It has a prominent role in the DMN and is linked to rumination. The mechanism of the antidepressant effects of AZD6765 is different from those of ketamine and citalopram. The insula, IPL, MFG, cingulate gyrus and lentiform nuclei are all regions implicated in MDD suggesting antidepressant effects. The rapid antidepressant effects of AZD6765 are possibly due to a resetting of the interface between DMN and salience networks.

Autism spectrum disorder (ASD) is associated with functional abnormalities of the anterior cingulate cortex (ACC), a brain area that mediates social behavior. Given evidence of a role of inflammation in ASD, markers of pro-inflammatory and anti-inflammatory microglia were studied using postmortem ACC tissues from ASD and age-matched typically developed control donors. Gene expression levels of pro-inflammatory (CD68, HLA-DRA, IL1B, NOS2, PTGS2) and anti-inflammatory (ARG1, IGF1, MRC1, PPARG) microglial genes were measured using quantitative real-time PCR. Additionally, brain sections were immunohistochemically stained for a microglial marker. Expression levels of IGF1 were modestly higher, while the expression of MRC1 was modestly lower in ASD donors when compared to control donors. No other differences in gene expression levels between the two groups of donors were observed. Statistical significance for changes in expression levels IGF1 and MRC1 did not survive correction for multiple comparisons. Further research on anti-inflammatory microglial involvement in ASD is warranted.

Abstract In the 1990s researchers discovered task-deactivated regions in the human brain. Together, these areas make up the default mode network (DMN). It was originally proposed to act as a balancing mechanism between different brain systems, explaining the deactivations, but is now mostly studied with regards to internal awareness, such as daydreaming and mental imagery. The purpose of this thesis is to present a summary of DMN research, focusing on the network’s suggested role in internal awareness. This will be done by reviewing a wide variety of research that either explicitly or indirectly correlate default mode network features with aspects of consciousness and internal awareness. The subjective experience of being conscious have been a source of argument primarily among philosophers, but the qualities we feel are intimately linked to cognitive functions that are supported by the regions found in the DMN. Cognitive neuroscience may therefore be able to contribute to the concept of internal awareness and consciousness.

Generalized anxiety disorder (GAD) is a prevalent anxiety disorder that is characterized by persistent excessive worrying that is often difficult to control. The pathology of GAD has been associated with abnormal neural activity and functional connectivity. This systematic review has examined the central neural correlates of GAD which are the prefrontal cortex (PFC), the anterior cingulate cortex (ACC) and the amygdala and how activation and functional connectivity in these brain areas differ between patients with GAD and healthy controls. This review also investigated how abnormal functional connectivity and activation in these brain regions relates to worry which is the most prominent psychological symptom in patients with GAD. A systematic review was conducted and seven original functional magnetic resonance imaging (fMRI) studies were included after a literature search on PubMed, Scopus and, Web of Science. The main findings from this review revealed decreased activation in the PFC and ACC and enhanced activation in the amygdala during the viewing of negative stimuli in patients with GAD. Identifying the neural correlates of GAD and how it relates to worry may provide improved treatment in the future such as developing more effective psychotropic drugs or improved psychotherapy. GAD has been associated with lower well-being and life satisfaction and may even be a risk factor for suicidal thoughts. One of the limitations from this review is that several of the included studies recruited patients with comorbidities and for that reason results from these studies cannot be generalized and applied to individuals with GAD.

This study investigated the effects of repeated administrations of nicotine (0.7 mg/kg) on dendritic morphology in the nucleus accumbens (NAcc), prefrontal cortex (Cg 3), and parietal cortex (Par 1). Animals were habituated for 3 days to a locomotor box, and after habituation, every second day for 5 weeks rats were placed into the locomotor chamber immediately after a subcutaneous injection of nicotine or saline. Rats demonstrated tolerance to an initial hypoactive response after each nicotine injection, and this was followed by an increase in activity after each injection (behavioral sensitization). This increase in activity was still present on a nicotine challenge after a 2-week abstinence period. One week after the nicotine challenge day, all rats were perfused and brains were removed. These brains we stained using Golgi-Cox procedures, and dendrites from the nucleus accumbens (N Acc), medial frontal cortex (Cg 3) and parietal cortex (Par 1) were analyzed using the camera lucida procedure. Results showed that rats receiving nicotine demonstrated an increase in dendritic length and spine density relative to controls in the NAcc and Cg3 brain areas, but not Par 1. The increase observed in the NAcc was significantly greater than what has been found with amphetamine or cocaine, and possible underlying mechanisms were discussed.

It is well documented that adolescent substance use is associated with deficits in brain function and behavior. However, possible deficits that predate substance use initiation remain poorly characterized in preadolescents at-risk for developing substance use disorder (SUD). To characterize potential brain and behavioral differences that predate substance use, substance naïve preadolescents, ages 11–12, were recruited into three groups to complete functional magnetic resonance imaging delay discounting: (1) High-risk youth (n=35) with a family history of SUD and externalizing psychiatric disorders, (2) psychiatric controls (n=35) with no family history of SUD, but equivalent externalizing psychiatric disorders as high-risk youth, and (3) healthy controls (n=29) with no family history of SUD and minimal psychopathology. While no behavioral differences between groups were identified, there were group differences in posterior cingulate cortex (PCC) function during decision making. Specifically, the high-risk group showed stronger deactivation of the PCC than healthy controls. These results suggest that high-risk preadolescents may need to suppress activity of key nodes of the default mode network (a task negative network) to a greater extent to properly allocate attention to the task.

Indiana University-Purdue University Indianapolis (IUPUI) / It is well documented that adolescent substance use is associated with deficits in brain function and behavior. However, possible deficits that predate substance use initiation remain poorly characterized in preadolescents at-risk for developing substance use disorder (SUD). To characterize potential brain and behavioral differences that predate substance use, substance naïve preadolescents, ages 11–12, were recruited into three groups to complete functional magnetic resonance imaging delay discounting: (1) High-risk youth (n=35) with a family history of SUD and externalizing psychiatric disorders, (2) psychiatric controls (n=35) with no family history of SUD, but equivalent externalizing psychiatric disorders as high-risk youth, and (3) healthy controls (n=29) with no family history of SUD and minimal psychopathology. While no behavioral differences between groups were identified, there were group differences in posterior cingulate cortex (PCC) function during decision making. Specifically, the high-risk group showed stronger deactivation of the PCC than healthy controls. These results suggest that high-risk preadolescents may need to suppress activity of key nodes of the default mode network (a task negative network) to a greater extent to properly allocate attention to the task.

Att utvärdera samband mellan subjektivt skattad smärta och transmittorsubstanser med magnetresonansspektroskopi Bakgrund: Smärta är en komplex upplevelse, som involverar olika delar av hjärnan. Regionen anterior cingulate cortex (ACC) är kopplad till upplevelsen av smärta och delas in i ett flertal mindre regioner, till exempel den pregenuala regionen (pgACC) och dorsala regionen (dACC). För att studera olika metaboliter och transmittorsubstanser kan magnetresonansspektroskopi (MRS) användas. MRS och sekvensen MEGA-PRESS kan mäta specifika transmittorsubstanser såsom Gamma-AminoButyric Acid (GABA) och glutamin-glutamat (Glx).  Motiv: Det finns kunskapsluckor kring hur individens subjektiva smärtupplevelse i relation till transmittorsubstanser objektivt kan mätas och utvärderas.  Syfte: Att med MRS och MEGA-PRESS undersöka GABA+ och Glx-nivåer i hjärnområdena pgACC och dACC samt undersöka samband mellan smärtkänslighet och GABA+ och Glx i pgACC och dACC.  Metod: En kvantitativ, experimentell pilotstudie genomfördes med tio friska deltagare. Initialt skannades deltagarna i MRT och smärtstimulerades, sedan skattade de den upplevda smärtan med hjälp av Numeric Rating Scale. MRS och tekniken MEGA-PRESS användes för att mäta transmittorsubstansnivåerna.   Resultat: Studien visade att det fanns en statistiskt signifikant negativ korrelation mellan skattad smärtintensitet och uppmätta nivåer av GABA+ i pgACC (Spearman´s rho = -0,67; p = 0,04). Det fanns även ett statistiskt signifikant positivt samband mellan skattad smärtintensitet och uppmätta nivåer av Glx i dACC (Spearman´s rho =0,73; p=0,02). Vidare fanns signifikant skillnad i Glx mellan pgACC och dACC och en icke signifikant skillnad i GABA+.  Konklusion: Sammanfattningsvis visar resultatet att MRS och MEGA-PRESS kan kvantifiera transmittorsubstanser vid utvärdering av smärtkänslighet och att det finns en positiv korrelation mellan Glx och skattad smärtintensitet, samt en negativ korrelation mellan GABA+ och skattad smärtintensitet. Detta kan ge fördjupad insikt i individens smärtupplevelse och kan främja den individuella behandlingen. Genom att ta hänsyn till sambandet mellan smärta och transmittorsubstanser kan det bidra till ökad förståelse kring individens smärtupplevelse. / To evaluate the relation between subjectively estimated pain and neurotransmitters using magnetic resonance spectroscopy  Background: Pain is a complex experience that involves different parts of the brain. The region anterior cingulate cortex (ACC) is connected to the experience of pain and can be divided into several smaller areas, such as the pregenual region (pgACC) and the dorsal region (dACC). To study different metabolites and neurotransmitters, magnetic resonance spectroscopy (MRS) can be used. MRS and the sequence (MEGA-PRESS) can measure specific neurotransmitters such as Gamma-AminoButyric Acid (GABA) and glutamin-glutamate (Glx).  Motive: There are knowledge gaps about how the individual's subjective pain experience in relation to neurotransmitters can be objectively measured and evaluated.  Aim: Using MRS and MEGA-PRESS to examine levels of GABA+ and Glx in the brain regions pgACC and dACC and to examine the relationship between pain sensitivity and GABA+ and Glx in pgACC and dACC.  Methods: A quantitative, experimental pilot study was conducted which included ten healthy participants. The participants were initially scanned in the MRI and subjected to pain-stimulation, thereafter the participants rated the perceived pain using Numeric Rating Scale. MRS and the sequence MEGA-PRESS were used to quantify the neurotransmitters of interest.  Result: There was a significant, negative correlation between rated pain intensity and measured GABA+ levels in pgACC (Spearman´s rho = -0,67; p = 0,04). There was also a significant, positive correlation between rated pain intensity and measured levels of Glx in dACC (Spearman´s rho =0,73; p=0,02). Furthermore, there was a significant difference in Glx between pgACC and dACC as well as a non-significant difference in GABA+ between regions.  Conclusion: In summary, the result shows that MRS and MEGA-PRESS can quantify neurotransmitters when evaluating pain sensitivity and that there is a positive correlation between Glx and estimated pain intensity, and also a negative correlation between GABA+ and estimated pain intensity. This can provide a deeper insight into the individual’s pain experience and promote individual treatment. Further research regarding the meaning of the different brain regions when measuring neurotransmitters is recommended.

The lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC) are two key regions of the frontal executive control network. Ascending cholinergic pathways differentially innervate these two functionally distinct cortices to modulate arousal and motivational signaling for higher-order functions. The action of acetylcholine (ACh) in sensory cortices is constrained by layer, anatomical cell type, and subcellular localization of distinct receptors, but little is known about the nature and organization of frontal-cholinergic circuitry in primates. In this dissertation, we characterized the anatomical localization of muscarinic acetylcholine receptors (mAChRs), m1 and m2–the predominant subtypes in the cortex–and their expression profiles on distinct cell types and pathways in ACC and LPFC of the rhesus monkey, using immunohistochemistry, anatomical tract-tracing, whole cell patch-clamp recordings, and single nucleus RNA sequencing. In the first series of studies (Chapter 2), we used immunohistochemistry and high-resolution confocal microscopy to reveal regional differences in m1 and m2 receptor localization on excitatory pyramidal and inhibitory neuron subpopulations and subcellular compartments in ACC (A24) versus LPFC (A46) of adult rhesus monkeys (Macaca mulatta; aged 7-11 yrs; 4 males and 2 females). The ACC exhibited a greater proportion of m2+ inhibitory neurons and a greater density of presynaptic m2+ receptors localized on inhibitory (VGAT+) terminations on pyramidal neurons compared to the LPFC. This result suggests a greater cholinergic suppression of GABAergic neurotransmission in ACC. In a second set of experiments (Chapter 3), we examined the heterogeneity of m1 and m2 laminar expression in functionally distinct ACC areas A24, A25, and A32. These differ in their connections with higher order cortical areas and limbic structures, such as the amygdala (AMY). The density of m1+ and/or m2 expressing (m1+/m2+) pyramidal neurons was significantly greater in A24 compared to A25 and to A32, while A25 exhibited a significantly greater density of m2+VGAT+ terminals. In addition, we examined the substrates for cholinergic modulation of long-range cortico-limbic processing using bidirectional neural tracers to label one specific subtype, the AMY-targeting projection neurons in these ACC areas. Compared to A24 and A32, the limbic ventral A25 had a greater density of m1+/m2+ AMY-targeting pyramidal neurons across upper layers 2-3 and deep layers 5-6, suggesting stronger cholinergic modulation of amygdalar outputs. Lastly (Chapter 4), we assessed the functional effects of cholinergic modulation on excitatory and inhibitory synaptic activity as well as the molecular signatures related to m1 and m2 receptor expression. In experiments using in vitro whole-cell patch-clamp recordings of layer 3 pyramidal neurons in ACC and LPFC, we found that application of the cholinergic agonist carbachol (CCh) significantly decreased the frequency of excitatory postsynaptic currents (EPSCs) to a greater extent in ACC A24 than in LPFC A46. Using single nucleus RNA sequencing, we found that enriched m1 and m2 transcriptional profiles in distinct cell-types and frontal areas (ACC A24 and LPFC A46) had differentially expressed genes associated with down-stream signaling cascades related to synaptic signaling and plasticity. Together, these data reveal the anatomical, functional, and transcriptomic neural substrates of diverse cholinergic modulation of local excitatory and inhibitory circuits and long-range cortico-limbic pathways in functionally-distinct ACC and LPFC frontal areas that are important for cognitive-emotional integration.

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