A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1–2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin

The Acute Stress Response in the Brain: From Neuromodulation to Molecules

All organisms have evolved elaborate systems to mount a stress response, which increases the chances of survival in dangerous situations. The response to acute stress encompasses a myriad of neurochemicals, the stress-mediators, that act in concert throughout the brain and body. One of the most crucial aspects of the stress response is the mobilization and reallocation of energy resources. The brain is the organ that first perceives potential threats and coordinates the stress response, yet the stress response also impacts the brain itself, changing its function and metabolic activity. One brain area with a key role in the stress response is the locus coeruleus (LC), the largest noradrenergic cell population in the central nervous system, located in the pons of the brainstem. The LC sends diffuse projections to the entire brain, thus providing the main supply of norepinephrine (NE) in the brain, and controlling basic functions such as arousal, cognition and vigilance. Theories have proposed that LC activation mediates a rapid shift in the functional connectivity of the brain, to strengthen connections that promote a higher state of alertness, and enhance threat detection. However, causal evidence that the LC is able to directly exert this effect on large-scale brain function was missing. Here, we chemogenetically activated the mouse LC while performing resting-state functional magnetic resonance imaging (fMRI), an approach we termed chemo-connectomics. Our results showed an increase in brain-wide connectivity, and a rapid reconfiguration of the functional connectome that enhanced brain networks tasked with salience processing and threat detection. Even though this experiment was performed in anesthetized mice, these findings mirror the effects observed in fMRI scans of awake human subjects presented with emotionally aversive stimuli. Moreover, we provided clues that alpha1 and beta1 adrenergic receptors might be involved in these effects, by correlating the changes in functional connectivity to the distribution of adrenergic receptors at the mRNA level. These results highlight the role of LC in the stress response, as well as the potential of neuromodulators in regulating whole-brain states. The release of stress-mediators in response to acute stress also affects the brain on a molecular level, in part through the recruitment of intracellular signaling cascades and the regulation of gene expression. Although a large body of literature has focused on such molecular changes in the context of stress-triggered neuropsychiatric disorders, the molecular events that affect cellular function to mediate the healthy, default response to acute stress have not been characterized in detail. Here, we used a multi-omic approach to investigate the molecular response to acute stress in the mouse brain. We focused on the hippocampus (HC), since this area – owing to its connectivity and molecular composition – is at the heart of the stress response. First, we showed that the dorsal (dHC) and ventral HC (vHC), are inherently different at the transcriptome and proteome level. Hence, we performed our analysis in those two regions separately, after an acute swim stress paradigm. We first used phosphoproteomics to identify the signaling events that initiate the cellular response to stress. In addition, we characterized the transcriptional changes that take place over time, as well as the changes on the translatome of excitatory and inhibitory neurons. We found that stress-induced changes in protein phosphorylation and in gene expression are widespread and rapid, but tightly regulated, as they terminate within a few hours after the initiation of stress. Altogether, these results provide the most detailed characterization of the molecular stress response to date, and will hopefully provide a starting point for understanding where the healthy stress-response might be derailed in cases of stress-induced psychopathology

Distinct Proteomic, Transcriptomic, and Epigenetic Stress Responses in Dorsal and Ventral Hippocampus

Background Acutely stressful experiences can trigger neuropsychiatric disorders and impair cognitive processes by altering hippocampal function. Although the intrinsic organization of the hippocampus is highly conserved throughout its long dorsal-ventral axis, the dorsal (anterior) hippocampus mediates spatial navigation and memory formation, whereas the ventral (posterior) hippocampus is involved in emotion regulation. To understand the molecular consequences of stress, detailed genome-wide screens are necessary and need to distinguish between dorsal and ventral hippocampal regions. While transcriptomic screens have become a mainstay in basic and clinical research, proteomic methods are rapidly evolving and hold even greater promise to reveal biologically and clinically relevant biomarkers. Methods Here, we provide the first combined transcriptomic (RNA sequencing) and proteomic (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) profiling of dorsal and ventral hippocampus in mice. We used three different acute stressors (novelty, swim, and restraint) to assess the impact of stress on both regions. Results We demonstrated that both hippocampal regions display radically distinct molecular responses and that the ventral hippocampus is particularly sensitive to the effects of stress. Separately analyzing these structures greatly increased the sensitivity to detect stress-induced changes. For example, protein interaction cluster analyses revealed a stress-responsive epigenetic network around histone demethylase Kdm6b restricted to the ventral hippocampus, and acute stress reduced methylation of its enzymatic target H3K27me3. Selective Kdm6b knockdown in the ventral hippocampus led to behavioral hyperactivity/hyperresponsiveness. Conclusions These findings underscore the importance of considering dorsal and ventral hippocampus separately when conducting high-throughput molecular analyses, which has important implications for fundamental research as well as clinical studies.ISSN:0006-3223ISSN:1873-240

RhoGEF9 splice isoforms influence neuronal maturation and synapse formation downstream of α2 GABAA receptors

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism

Distinct Proteomic, Transcriptomic, and Epigenetic Stress Responses in Dorsal and Ventral Hippocampus

BACKGROUND Acutely stressful experiences can trigger neuropsychiatric disorders and impair cognitive processes by altering hippocampal function. Although the intrinsic organization of the hippocampus is highly conserved throughout its long dorsal-ventral axis, the dorsal (anterior) hippocampus mediates spatial navigation and memory formation, whereas the ventral (posterior) hippocampus is involved in emotion regulation. To understand the molecular consequences of stress, detailed genome-wide screens are necessary and need to distinguish between dorsal and ventral hippocampal regions. While transcriptomic screens have become a mainstay in basic and clinical research, proteomic methods are rapidly evolving and hold even greater promise to reveal biologically and clinically relevant biomarkers. METHODS Here, we provide the first combined transcriptomic (RNA sequencing) and proteomic (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) profiling of dorsal and ventral hippocampus in mice. We used three different acute stressors (novelty, swim, and restraint) to assess the impact of stress on both regions. RESULTS We demonstrated that both hippocampal regions display radically distinct molecular responses and that the ventral hippocampus is particularly sensitive to the effects of stress. Separately analyzing these structures greatly increased the sensitivity to detect stress-induced changes. For example, protein interaction cluster analyses revealed a stress-responsive epigenetic network around histone demethylase Kdm6b restricted to the ventral hippocampus, and acute stress reduced methylation of its enzymatic target H3K27me3. Selective Kdm6b knockdown in the ventral hippocampus led to behavioral hyperactivity/hyperresponsiveness. CONCLUSIONS These findings underscore the importance of considering dorsal and ventral hippocampus separately when conducting high-throughput molecular analyses, which has important implications for fundamental research as well as clinical studies

RhoGEF9 splice isoforms influence neuronal maturation and synapse formation downstream of alpha 2 GABA(A) receptors

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin ( CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABA(A)Rs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of alpha 2-containing GABA(A)Rs during neuron maturation in a Cdc42 dependent mechanism.Peer reviewe

A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1–2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin

Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain and regulates many fundamental brain functions. Studies in humans have suggested that strong LC activation might shift network connectivity to favor salience processing. To causally test this hypothesis, we use a mouse model to study the effect of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term "chemo-connectomics.'' We show that LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. Functional connectivity changes strongly correlate with transcript levels of alpha-1 and beta-1 adrenergic receptors across the brain, and functional network connectivity correlates with NE turnover within select brain regions. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection

Rapid reconfiguration of the functional connectome after chemogenetic locus coeruleus activation

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain and regulates many fundamental brain functions. Studies in humans have suggested that strong LC activation might shift network connectivity to favor salience processing. To causally test this hypothesis, we use a mouse model to study the effect of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term "chemo-connectomics.'' We show that LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. Functional connectivity changes strongly correlate with transcript levels of alpha-1 and beta-1 adrenergic receptors across the brain, and functional network connectivity correlates with NE turnover within select brain regions. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection