Cell Type-Specific Molecular Changes in Autism Spectrum Disorder

Post by Stephanie Williams

What's the science?

Identifying molecular differences that distinguish the brains of individuals diagnosed with autism spectrum disorders (ASD) from non-autistic brains is important for understanding how the brain develops and functions differently in autism. This week in Science, Velmeshev, Kriegstein and colleagues analyzed the transcriptomes of single cells to identify cell-type-specific molecular changes in ASD.

How did they do it?                                            

The authors analyzed the transcriptomes of neural and glial cells from post mortem brain tissue of children and young adults (aged 4 to 22) diagnosed with autism (N=15) and healthy controls (N=16). The authors used single nucleus RNA sequencing (snRNA-seq) to analyze the transcriptomes of single cells in tissue samples from prefrontal cortex and anterior cingulate cortex, two areas known to be affected by ASD. The snRNA-seq technique allowed the authors to analyze the molecular profile of individual cells. Some patients in the ASD cohort had comorbid sporadic epilepsy, which allowed the authors to create an additional age matched group of controls to compare with this group for further analysis. They performed this analysis to tease apart the differences between epilepsy-related molecular changes and ASD-specific molecular changes. The authors then used data from structured interviews to test whether their cell-type specific findings were related to symptom severity.

What did they find?

The authors identified changes in 17 cell types, and found dysregulated development and signaling of upper layer cortical neurons along with activated astrocytes in the ASD group. The genes that the authors found to be most differentially expressed were in layer 2/3 excitatory neurons and vasoactive intestinal polypeptide-expressing interneurons - specifically, genes responsible for synaptic and neurodevelopment. In non-neuronal cells, the top genes differentially expressed were up-regulated in protoplasmic astrocytes and microglia. The authors found that ASD samples contained more protoplasmic astrocytes. Changes in layer 2/3 neurons and microglia were correlated with symptom severity. This correlation suggests that the molecular changes the authors find in the upper layer cortical neurons are responsible for the behavioral symptoms observed in ASD. Analysis of differences between patients who had comorbid ASD and epilepsy with healthy controls revealed changes in L5/6 corticofugal projection neurons and parvalbumin neurons, confirming that the molecular changes observed in the ASD sample were related to ASD pathogenesis and not seizure activity.

What's the impact?

The authors provide a detailed account of specific cell types that contribute to neural pathways affected in the brains of individuals with ASD. Broadly, the authors replicate and extend previous observations about circuit level dysfunction in ASD. Previous work had shown that there was convergence of ASD on specific cell types during development, and the authors extended this finding by showing that there are also convergent transcriptional changes in adult ASD patients. The convergence of the observed molecular changes in the ASD group onto specific cell types in adults has far-reaching implications as it confirms that there may be a common set of targets for therapeutic treatments.

Velmeshev, et al. Single-Cell genomics identifies cell type-specific molecular changes in autism. Science (2019). Access the original scientific publication here.

The authors’ data can be viewed interactively here

Hypothalamic Oxytocin Neurons Represent Fear Engrams in Rats

Post by Lincoln Tracy

What's the science?

Emotional memory representations or engrams (i.e. memory traces, stored in the brain) such as fear, are critical for survival. These engrams allow both animals and humans to sense, evaluate, and respond to dangerous situations in an appropriate manner. Two brain regions involved in the development of fear-related memories—the hypothalamus and the central nucleus of the amygdala (CeA)—are connected by oxytocin neurons. The endogenous hormone oxytocin may play an important role in modulating fear, due to its ability to modulate the salience of social cues and events. However, the exact role of hypothalamic oxytocin neurons in fear conditioning or learning is unknown. This week in Neuron, Hasan and colleagues developed a novel genetic tagging method—virus-delivered genetic activity-induced tagging of cell ensembles, or vGATE—to tag fear-activated oxytocin neurons in rat brains during fear conditioning.

How did they do it?

First, the authors developed the novel genetic method vGATE in a small subset of hypothalamic oxytocin neurons. This method uses a c-fos promoter and three different viruses to identify and permanently tag a small subset of neurons with fluorescent proteins. After confirming that their model worked, they investigated what proportion of the hypothalamic oxytocin neurons contributed to the anxiolytic effect and how these neurons were recruited during fear using a fear conditioning paradigm. They then analyzed brain slices to determine whether the hypothalamic oxytocin neurons projected to the CeA. The authors also used optogenetics—a technique in which neural activity can be controlled by shining light on the vGATE neurons—to investigate whether fear-related behaviors could be controlled. They used histology and electrophysiology to investigate potential anatomical and molecular changes in the brain following fear experience. Finally, they introduced a novel context to the fear conditioning paradigm to investigate the role of hypothalamic oxytocin neurons in fear extinction.

What did they find?

First, the authors found that only a small proportion of the hypothalamic oxytocin neurons—approximately 13 percent—were active during the expression of fear. Second, they found that the majority of vGATE hypothalamic oxytocin neurons projected to the lateral part of the CeA. Third, when the vGATE oxytocin neurons were optogenetically simulated with a blue light there was a substantial reduction in the amount of time the rats were frozen with fear. Fourth, they found that the vGATE oxytocin neurons showed increased glutamatergic—but not oxytocinergic—transmission within the medial CeA during fear exposure. Finally, they found that inhibiting the vGATE oxytocin neurons exclusively impaired fear extinction, suggesting that fear extinction involves blocking oxytocin and glutamate mediated neural modulation in the CeA. These findings suggest that the vGATE oxytocin neurons represent a neuromodulatory memory trace that is a vital contributor to controlling fear-related memories and behaviors.    

What's the impact?

This study is the first to demonstrate that vGATE-assisted hypothalamic oxytocin neurons are adequate to drive fear-related behaviors and are required for extinction of these behaviors. Importantly, experiencing fear leads to large amounts of neural plasticity, bringing about a shift in the lateral CeA from oxytocin signaling to glutamate signaling. These findings have important implications for future investigations of the pathophysiological mechanisms that underlie emotion-based mental disorders (such as PTSD) and their potential treatments, including exogenously administered oxytocin and virus-delivered genetically based therapies.

Hasan et al. A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System. Neuron (2019). Access the original scientific publication here.

Glutamate Receptor Dysregulation and Suicidal Thoughts in PTSD

Post by Deborah Joye

What's the science?

Post-traumatic stress disorder is a condition in which individuals experience persistent anxiety, flashbacks, and intense fear after a traumatic event. People who experience PTSD are at higher risk for developing suicidal thoughts, but the reasons why are not understood. One potential target implicated in mood and stress disorders is metabotropic glutamate receptor type 5 (mGluR5), which moderates activity of other receptors critical for synaptic plasticity and emotional learning. Previous work has identified higher mGluR5 availability and an association between increased mGluR5 gene expression and suicide in individuals with PTSD. This week in PNAS, Davis, Esterlis and colleagues use positron emission tomography (PET) to demonstrate that suicidal PTSD individuals have more mGluR5 availability in frontolimbic regions than individuals with PTSD with no suicidal thoughts, those with major depression and healthy controls, suggesting that mGluR5 dysregulation may serve as a biomarker of suicidality in PTSD specifically.

How did they do it?

The authors recruited 29 individuals with PTSD, 29 individuals with major depression, and 29 healthy controls. Participants completed physical, psychiatric, and neurological examinations during their initial visit to establish their diagnosis and rule out any other major illnesses. Participants also filled out a report on the day of their scan in order to assess suicidal thoughts. Participants were injected with [18F]FPEB, a radioligand with high selectivity and specificity for mGluR5 (e.g., a radioactive substance that selectively binds to mGluR5). Individuals participated in a PET scan and data were analyzed in five key frontolimbic brain regions – the dorsolateral and ventromedial prefrontal cortices, the orbitofrontal cortex, the amygdala, and the hippocampus. The authors analyzed associations between mGluR5 availability and PTSD, depression, suicidal thoughts, and scores from mood and anxiety tests.

What did they find?

Overall, the authors found that mGluR5 availability was higher in individuals with PTSD with suicidal ideation relative to those without suicidal thoughts, those with major depression, and healthy controls. Availability of mGluR5 was not different between those with major depression and healthy controls. Higher mGluR5 availability was associated with suicidal ideation among individuals with PTSD, but not those with major depression. Scores on mood tests were positively correlated with mGluR5 availability in the PTSD group (higher scores mean more mood disturbance). Interestingly, mood test scores were inversely correlated with mGluR5 availability in the major depression group (more mood disturbance was associated with lower mGluR5). Specifically, more mood disturbance was associated with more mGluR5 availability in individuals with PTSD that also reported suicidal thoughts.

What's the impact?

These findings confirm that mGluR5 is upregulated in the frontolimbic regions of individuals with PTSD relative to healthy controls. Notably, this study is the first to demonstrate that higher mGluR5 availability is associated with suicidal ideation specifically in individuals with PTSD but not depression. This study identifies mGluR5 as a possible biomarker and treatment target for intervention and management of suicide risk in individuals with PTSD.

Davis et al., In vivo evidence for dysregulation of mGluR5 as a biomarker of suicidal ideation, PNAS (2019). Access the original scientific publication here.