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A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

ABSTRACT: Aim:Selective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine. Methods:The LC of male Sprague-Dawley rats was injected with 10 nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n = 19, DREADD group) or AAV2/7-PRSx8-eGFP (n = 13, Controls). Three weeks later, LC unit recordings were performed in anesthetized rats. We investigated whether clozapine, a drug known to bind to modified neurons expressing hM3Dq receptors, was able to increase the LC firing rate. Baseline unit activity was recorded followed by subsequent administration of 0.01 and 0.1 mg/kg of clozapine in all rats. hM3Dq-mcherry expression levels were investigated using immunofluorescence staining of brainstem slices at the end of the experiment. Results:Unit recordings could be performed in 12 rats and in a total of 12 neurons (DREADDs: n = 7, controls: n = 5). Clozapine 0.01 mg/kg did not affect the mean firing rate of recorded LC-neurons; 0.1 mg/kg induced an increased firing rate, irrespective whether neurons were recorded from DREADD or control rats (p = 0.006). Co-labeling of LC neurons and mCherry-tag showed that 20.6 ± 2.3% LC neurons expressed the hM3Dq receptor. Aspecific expression of hM3Dq-mCherry was also observed in non-LC neurons (26.0 ± 4.1%). Conclusion:LC unit recording is feasible in an experimental set-up following manipulations for DREADD induction. A relatively low transduction efficiency of the used AAV was found. In view of this finding, the effect of injected clozapine on LC-NA could not be investigated as a reliable outcome parameter for activation of chemogenetically modified LC neurons. The use of AAV2/7, a vector previously applied successfully to target dopaminergic neurons in the substantia nigra, leads to insufficient chemogenetic modification of the LC compared to transduction with AAV2/9.

SUBMITTER: Stevens L

PROVIDER: S-EPMC7067893 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

Stevens Latoya L Vonck Kristl K Larsen Lars Emil LE Van Lysebettens Wouter W Germonpré Charlotte C Baekelandt Veerle V Van den Haute Chris C Carrette Evelien E Wadman Wytse Jan WJ Boon Paul P Raedt Robrecht R

Frontiers in neuroscience 20200306

<h4>Aim</h4>Selective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine.<h4>Methods</h4>The LC of male Sprague-Dawley rats was injected with 10 nl of ...[more]

PMID: 32210746

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Project description:BackgroundMost current disease-modifying therapies approved for multiple sclerosis (MS) are immunomodulatory drugs that counteract the aberrant activity of the immune system. Hence, new pharmacological interventions that drive anti-inflammatory activity and neuroprotection would represent interesting alternative therapeutic approaches or complementary strategies to treat progressive forms of MS. There is evidence of reduced noradrenaline levels and alterations to locus coeruleus (LC) noradrenergic neurons in MS patients, as well as in animal models of this disease, potentially factors contributing to the pathophysiology. Drugs that enhance noradrenaline appear to have some beneficial effects in MS, suggesting their potential to dampen the underlying pathology and disease progression.MethodsTherefore, we explored the consequences of chronic LC noradrenergic neurons activation by chemogenetics in experimental autoimmune encephalomyelitis (EAE) mice, the most widely used experimental model of MS. LC activation from the onset or the peak of motor symptoms was explored as two different therapeutic approaches, assessing the motor and non-motor behavioral changes as EAE progresses, and studying demyelination, inflammation and glial activation in the spinal cord and cerebral cortex during the chronic phase of EAE.ResultsLC activation from the onset of motor symptoms markedly alleviated the motor deficits in EAE mice, as well as their anxiety-like behavior and sickness, in conjunction with reduced demyelination and perivascular infiltration in the spinal cord and glial activation in the spinal cord and prefrontal cortex (PFC). When animals exhibited severe paralysis, LC activation produced a modest alleviation of EAE motor symptoms and it enhanced animal well-being, in association with an improvement of the EAE pathology at the spinal cord and PFC level. Interestingly, the reduced dopamine beta-hydroxylase expression associated with EAE in the spinal cord and PFC was reversed through chemogenetic LC activation.ConclusionTherefore, clear anti-inflammatory and neuroprotective effects were produced by the selective activation of LC noradrenergic neurons in EAE mice, having greater benefits when LC activation commenced earlier. Overall, these data suggest noradrenergic LC neurons may be targets to potentially alleviate some of the motor and non-motor symptoms in MS.

Project description:IntroductionThe locus coeruleus noradrenergic (LC-NA) system is studied for its role in various neurological and psychiatric disorders such as epilepsy and Major Depression Dissorder. Chemogenetics is a powerful technique for specific manipulation of the LC to investigate its functioning. Local injection of AAV2/7 viral vectors has limitations with regards to efficiency and specificity of the transduction, potentially due to low tropism of AAV2/7 for LC neurons. In this study we used a canine adenovirus type 2 (CAV2) vector with different volumes and viral particle numbers to achieve high and selective expression of hM3Dq, an excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD), for chemogenetic modulation of LC neurons.MethodsAdult male Sprague-Dawley rats were injected in the LC with different absolute numbers of CAV2-PRSx8-hM3Dq-mCherry physical particles (0.1E9, 1E9, 5E9,10E9, or 20E9 pp) using different volumes (LowV = 3 nl × 300 nl, MediumV = 3 × 600 nl, HighV = 3 × 1200 nl). Two weeks post-injection, double-labeling immunohistochemistry for dopamine β hydroxylase (DBH) and mCherry was performed to determine hM3Dq expression and its specificity for LC neurons. The size of the transduced LC was compared to the contralateral LC to identify signs of toxicity.ResultsAdministration of Medium volume (3 × 600 nl) and 1E9 particles resulted in high expression levels with 87.3 ± 9.8% of LC neurons expressing hM3Dq, but low specificity with 36.2 ± 17.3% of hM3Dq expression in non-LC neurons. The most diluted conditions (Low volume_0.1E pp and Medium Volume_0.1E pp) presented similar high transduction of LC neurons (70.9 ± 12.7 and 77.2 ± 9.8%) with lower aspecificity (5.5 ± 3.5 and 4.0 ± 1.9%, respectively). Signs of toxicity were observed in all undiluted conditions as evidenced by a decreased size of the transduced LC.ConclusionThis study identified optimal conditions (Low and Medium Volume with 0.1E9 particles of CAV2-PRSx8-hM3Dq-mCherry) for safe and specific transduction of LC neurons with excitatory DREADDs to study the role of the LC-NA system in health and disease.

Project description:Excitatory inputs drive burst firing of locus coeruleus (LC) noradrenaline (NA) neurons in response to a variety of stimuli. Though a small number of glutamatergic LC afferents have been investigated, the overall landscape of these excitatory inputs is largely unknown. The current study used an optogenetic approach to isolate three glutamatergic afferents: the prefrontal cortex (PFC), lateral hypothalamus (LH) and periaqueductal grey (PAG). AAV5-DIO-ChR2 was injected into each region in male and female CaMKII-Cre mice and the properties of excitatory inputs on LC-NA cells were measured. Notably we found differences among these inputs. First, the pattern of axonal innervation differed between inputs such that LH afferents were concentrated in the posterior portion of the LC-NA somatic region while PFC afferents were denser in the medial dendritic region. Second, basal intrinsic properties varied for afferents, with LH inputs having the highest connectivity and the largest amplitude excitatory postsynaptic currents while PAG inputs had the lowest initial release probability. Third, while orexin and oxytocin had minimal effects on any input, dynorphin strongly inhibited excitatory inputs originating from the LH and PAG, and corticotrophin releasing factor (CRF) selectively inhibited inputs from the PAG. Overall, these results demonstrate that individual afferents to the LC have differing properties, which may contribute to the modularity of the LC and its ability to mediate various behavioural outcomes. KEY POINTS: Excitatory inputs to the locus coeruleus (LC) are important for driving noradrenaline neuron activity and downstream behaviours in response to salient stimuli, but little is known about the functional properties of different glutamate inputs that innervate these neurons We used a virus-mediated optogenetic approach to compare glutamate afferents from the prefrontal cortex (PFC), the lateral hypothalamus (LH) and the periaqueductal grey (PAG). While PFC was predicted to make synaptic inputs, we found that the LH and PAG also drove robust excitatory events in LC noradrenaline neurons. The strength, kinetics, and short-term plasticity of each input differed as did the extent of neuromodulation by both dynorphin and corticotrophin releasing factor. Thus each input displayed a unique set of basal properties and modulation by peptides. This characterization is an important step in deciphering the heterogeneity of the LC.

Project description:The locus coeruleus (LC) is a brainstem nucleus distinguished by its supply of noradrenaline throughout the central nervous system. Apart from modulating a range of brain functions, such as arousal, cognition and the stress response, LC neuronal excitability also corresponds to the activity of various peripheral systems, such as pelvic viscera and the cardiovascular system. Neurochemically diverse inputs set the tone for LC neuronal activity, which in turn modulates these adaptive physiological and behavioral responses essential for survival. One such LC afferent system which is poorly understood contains the neurohormone arginine-vasopressin (AVP). Here we provide the first demonstration of the molecular and functional characteristics of the LC-AVP system, by characterizing its receptor-specific modulation of identified LC neurons and plasticity in response to stress. High resolution confocal microscopy revealed that immunoreactivity for the AVP receptor 1b (V1b) was located on plasma membranes of noradrenergic and non-noradrenergic LC neurons. In contrast, immunoreactivity for the V1a receptor was exclusively located on LC noradrenergic neurons. No specific signal, either at the mRNA or protein level, was detected for the V2 receptor in the LC. Clusters immunoreactive for V1a-b were located in proximity to profiles immunoreactive for GABAergic and glutamatergic synaptic marker proteins. AVP immunopositive varicosities were also located adjacent to labeling for such synaptic markers. Whole-cell patch clamp electrophysiology revealed that the pharmacological activation of V1b receptors significantly increased the spontaneous activity of 45% (9/20) of recorded noradrenergic neurons, with the remaining 55% (11/20) of cells exhibiting a significant decrease in their basal firing patterns. Blockade of V1a and V1b receptors on their own significantly altered LC neuronal excitability in a similar heterogeneous manner, demonstrating that endogenous AVP sets the basal LC neuronal firing rates. Finally, exposing animals to acute stress increased V1b, but not V1a receptor expression, whilst decreasing AVP immunoreactivity. This study reveals the AVP-V1a-b system as a considerable component of the LC molecular architecture and regulator of LC activity. Since AVP primarily functions as a regulator of homeostasis, the data suggest a novel pathway by modulating the functioning of a brain region that is integral to mediating adaptive responses.

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A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

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A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

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