Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation

Abstract

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain, regulates many fundamental brain functions, and is implicated in several neuropsychiatric diseases. Although selective manipulation of the LC is not possible in humans, studies have suggested that strong LC activation might shift network connectivity to favor salience processing. To test this hypothesis, we use a mouse model to study the impact 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". LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. We reveal a direct correlation between functional connectivity changes and transcript levels of alpha-1, alpha-2, and beta-1 adrenoceptors across the brain, and a positive correlation between NE turnover and functional connectivity within select brain regions. These results represent the first brain-wide functional connectivity mapping in response to LC activation, and demonstrate a causal link between receptor expression, brain states and functionally connected large-scale networks at rest. 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. RESULTSTo selectively target the LC, we used transgenic mice that express codon-improved Crerecombinase (iCre) under the dopamine-beta-hydroxylase (DBH) promoter (DBH-iCre mice, Figure 1A) 33 . We stereotactically delivered floxed excitatory DREADDs 34 (AAV5-hSyn-DIO-hM3Dq-mCherry; hM3Dq-mCh) or a control virus (AAV5-hSyn-DIO-mCherry; mCh) to the LC, thus restricting virus expression to DBH-positive noradrenergic neurons of the LC (Figure 1B). We assessed successful LC activation using pupillometry, a highly sensitive and clinically relevant readout of LC activation [35][36][37] . After two minutes of baseline recording under light isoflurane anesthesia, we activated LC neurons by administering the potent DREADD activator clozapine at an ultra-low dose (0.03 mg/kg, i.p., Figure 1C) 38 . Within a minute of clozapine injection, we observed a strong increase in pupil diameter in the hM3Dq-mCh group, while pupil diameter of mCh mice did not change in response to clozapine injection and remained stable throughout the 10-minute recording session (Figure 1D-F). To show that our LC activation protocol is behaviorally relevant, we subjected mice to an open field test (OFT) immediately after clozapine injection and recorded their behavior for 30 minutes. In comparison to mCh mice, clozapine injection had profound effects on the behavior of hM3Dq-mCh mice. Several minutes after clozapine administration, hM3Dq-mCh mice showed strongly suppressed locomotor activity ( Figure 1G, H), spent less time in the (more aversive) center of the open field (Figure 1I, J), performed less activity-related supported rears (Figure 1...

What’s That (Blue) Spot on my MRI? Multimodal Neuroimaging of the Locus Coeruleus in Neurodegenerative Disease

“…Better temporal and patterned control is possible with optogenetics, and can be used to elucidate the relative contribution of tonic versus phasic firing patterns to functional connectivity. Moreover, Zerbi et al (2019) provide evidence that the source of LC fMRI signal may be due to NE actions on adrenergic receptors through volume transmission/non-synaptic release from varicosities ( Atzori et al, 2016 ; Mather et al, 2016 ), rather than via LC modulation of vasculature which is primarily vasoconstriction ( Goadsby et al, 1985 ; Bekar et al, 2012 ). Due to emerging evidence of the LC’s modularity ( Chandler et al, 2014 ; Uematsu et al, 2017 ), we also suggest the use of methods that are specific to particular LC projections.…”

“…Notably, PD is diagnosed by motor dysfunction that has largely been localized to the basal ganglia, with LC involvement mostly disregarded due to the sparse noradrenergic input to this region ( Berridge and Waterhouse, 2003 ). However, resting-state fMRI studies have provided evidence of noradrenergic contributions to the striatum ( Zhang et al, 2016 ; Lee et al, 2020 ; Liebe et al, 2020 ), and it was recently reported that the LC directly innervates the caudate and putamen in mice ( Zerbi et al, 2019 ). Stimulation of LC-striatal fibers in mice elicited NE release and increased overall functional connectivity.…”

“…Historically, electrical stimulation and pharmacology have been the manipulations of choice, but these interventions are not cell type-specific and cannot precisely control the rate and timing of stimulation. By combining various circuit-based techniques with MRI, such as optogenetics and chemogenetics, it is now possible to perturb specific brain regions, such as the LC, in a cell type-specific manner to interrogate their effects on brain-wide network activity and connectivity ( Lee et al, 2010 ; Zerbi et al, 2019 ). Methods have also been developed to simultaneously perform MRI and electrophysiology ( Shmuel and Leopold, 2008 ; Pan et al, 2010 , 2013 ), which would be useful for investigating how baseline LC activity correlates with functional connectivity measures.…”

“…A recent, landmark study in rodent LC neuroimaging utilized chemogenetics to stimulate the LC during fMRI in wild-type mice ( Zerbi et al, 2019 ). Notable increases in brain-wide functional connectivity, including the salience, amygdalar, association, hippocampal, striato-motor, and default mode-like networks, suggest that LC activity can rapidly reconfigure brain state.…”

Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications

“…We observed that inhibition of the anterior cingulate area, which has been identified as a hub region (i.e., a highly interconnected brain region), resulted in large‐scale neuronal activity modulations as well as functional connectivity alterations (Peeters et al, ). Furthermore, other recent studies combined chemogenetics with resting‐state functional MRI in order to evaluate functional connectivity alterations after modulation of the excitation–inhibition balance in the somatosensory cortex (Markicevic et al, ) or to assess the effects of selective stimulation of the locus coeruleus with hM3Dq on a large‐scale functional connectivity (Zerbi et al, ).…”