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The Unlikely Increase of Dopaminergic Signaling in Depression Associated With Chronic Pain

Recent work suggests that the Sp5C-LPBN-VTA pathway is activated during chronic neuropathic pain and results in the increased firing of VTA dopaminergic neurons, leading to depression-like behavior.

By Courtney Bannerman


4 May 2022


PRF News

Depression-Pain-Featured

Recent work suggests that the Sp5C-LPBN-VTA pathway is activated during chronic neuropathic pain and results in the increased firing of VTA dopaminergic neurons, leading to depression-like behavior.

Clinicians have observed the coexistence of chronic pain and depression for many years, and the two can often respond similarly to drug treatments. As in pain research, many papers have reported a decrease in dopaminergic firing in depression. However, Ludi Zhang, working in the lab of Hailin Zhang at Hebei Medical University, China, has shown that depression associated with chronic pain is related to the increased activity of dopaminergic neurons located in the midbrain ventral tegmental area (VTA) and glutamatergic projections that link the spinal trigeminal nucleus caudalis (Sp5C), lateral parabrachial nucleus (LPBN), and VTA. Inhibiting any part of this pathway resulted in a decrease of depression-like behavior.

 

This study was published in Cell Reports on 2 November 2021.

 

Reduced VTA dopaminergic signaling reduces depression-like behavior

Hongbin Yang, a primary investigator at the Ministry of Education Frontiers Science Center for Brain Research and Brain-Machine Integration, Zhejiang University in Hangzhou, China, noted that, “… if their conclusions are right, they’ve added a pretty novel view to the area of chronic pain and depression.”

 

To study this connection between chronic pain and depression, the group used the partial infraorbital nerve transection (pIONT) mouse model, in which neuropathic pain resulting from this nerve transection is quantified by the frequency of face grooming. This grooming started the first day after surgery and continued throughout the duration of testing. Sham surgery was used as a control. In addition, the tail suspension test (TST) and forced swim test (FST) were used to quantify depression-like behavior, the onset of which was observed in the pIONT mice starting at day 28 post-surgery compared to sham mice.

 

Dopaminergic neurons in the VTA have previously been implicated in the development of depression (Chaudhury et al., 2013in vitro cell-attached patch-clamp recordings.

 

Yang noted that, “… the brain region of the VTA is very heterogeneous … and contains dopaminergic, glutaminergic, and GABAergic cells. Just dopaminergic neurons are an anatomically and functionally heterogeneous group of cells as well.” He would have liked to see increased evidence that the VTA dopaminergic neurons recorded were dopaminergic neurons and that the location of recorded cells was in the VTA.

 

Next, Zhang et al. tested whether reducing the firing of dopaminergic neurons could reduce depression-like behavior in these mice. For this experiment, retigabine (a potassium channel activator) was used to inhibit VTA dopaminergic neurons. First, direct administration of retigabine was achieved by implanting into the VTA a multichannel pipette, which allowed for recording dopaminergic neuron firing and delivering retigabine. Retigabine administration successfully reduced the firing activity of VTA dopaminergic neurons in both sham and pIONT mice; however, the decrease in dopaminergic neuron firing in pIONT mice was more significant. Next, a cannula was implanted in the VTA to evaluate depression-like behavior following the administration of retigabine. A reversal of depression-like behavior, measured using the TST, was observed following the administration of retigabine in pIONT mice, but not sham mice. Importantly, retigabine did not affect the display of neuropathic pain behaviors.

 

The group wanted to verify that retigabine was not affecting depression-like behavior indirectly through off-target effects. To demonstrate this, AAV-DIO-hM4D-mCherry was injected into the VTA of a dopamine transporter-Cre mouse line (DAT-Cre) 21 days after pIONT surgery. AAV-DIO-hM4D-mCherry is an adeno-associated virus that specifically targets the hM4D receptor, which is expressed on dopaminergic neurons, and administration of the artificial ligand clozapine-N-oxide (CNO) allows for their selective inhibition. mCherry also allows these neurons to be observed using immunofluorescence. AAV-DIO-mCherry was used as a control. When DAT-Cre + AAV-DIO-hM4D-mCherry mice were treated with CNO (selectively inhibiting dopaminergic neurons), they showed reduced depression-like behavior following the TST and FST.

 

Activation of VTA dopaminergic neurons induces depression-like behavior

Next, the group tested whether the activation of VTA dopaminergic neurons could induce depression-like behavior. In naïve DAT-Cre mice, viral particles carrying the gene for the excitatory DREADD receptor hM3D (AAV-DIO-hM3D-mCherry) were delivered to the VTA. In this model, CNO can excite dopaminergic neurons. Following CNO administration, there was an increase in immobility time after the TST and FST, thus suggesting an increase in depression-like behaviors. In both the hM4D and hM3D models, pain behavior was not affected.

 

An optogenetic strategy was also used to activate the VTA dopaminergic neurons. For this, AAV-DIO-ChR2-mCherry viral particles were delivered to the VTA of DAT-Cre mice. Activation of ChR2 in the VTA dopaminergic neurons with 475-nm laser illumination also increased depression-like behaviors following the TST. However, the group does note that stimulation in naïve mice may be acutely rewarding and may have complicated the behavioral outcomes.

 

Due to neuropathic pain often being observed with depression, the authors hypothesized that a connection between neuropathic pain pathways and the VTA dopaminergic neurons might exist. Therefore, c-Fos was used to determine which neurons could be activated in the brain on day 28 after pIONT surgery. Increased c-Fos immunoreactivity was seen in the medial habenula, the laterodorsal tegmental nucleus, the ventromedial hypothalamic nucleus, the dorsomedial hypothalamic nucleus, the spinal trigeminal tract, the median raphe nucleus, the locus coeruleus, and the VTA. In addition, the spinal trigeminal nucleus caudalis (Sp5C) and the lateral parabrachial nucleus (LPBN), which has been shown to be involved in the chronic pain conduction pathway, were also labeled.

 

Connecting Sp5C, LPBN, and VTA

Neurons projecting to the VTA were traced using retrobeads and Cre-dependent viral labeling (AAV-DIO-GFP). The group identified that the LPBN was one of the regions sending projections to the VTA, that it was activated following pIONT surgery, and that most of the projections were glutamatergic neurons.

 

To determine if the connection between the LPBN-VTA was involved with depression-like behavior, the glutamatergic antagonist AP-5 was applied to the VTA to block glutamatergic input 28 days post-surgery. As a result, AP-5 reversibly reduced the firing of dopaminergic neurons and depression-like behavior. However, like retigabine, AP-5 did not affect pain behavior.

 

Next, the glutamatergic LPBN-VTA pathway was inhibited using the inhibitory opsin NpHR. For these experiments, Vglut2-Cre mice were used, in which the glutamatergic neuron marker Vglut2 is Cre-expressed. pIONT surgery was carried out on Vglut2-Cre mice, and the viral particle AAV-DIO-NpHR-eYFP was injected into the LPBN. LPBN-VTA glutamatergic projections were inhibited through 590-nm laser illumination. A significant reduction in depression-like behaviors was observed in these mice using the TST. When the excitatory ChR2 was used (AAV-DIO-ChR2-mCherry) in naïve mice, and the LPBN-VTA pathway was activated, an increase in depression-like behaviors was observed.

 

Since c-Fos increased in the Sp5C, the researchers wondered if the Sp5C could be involved in the LPBN-VTA pathway, as a link between the Sp5C and the LPBN had previously been reported (Edvinsson et al., 2020). First, retrobeads and AAV-DIO-GFP were injected into the LPBN of Vglut2-Cre mice to verify the Sp5C-LPBN connection. Then,trans-monosynaptic retrograde labeling was used to confirm if there were direct monosynaptic projections from the Sp5C to the LPBN and from the LPBN to the VTA. For this technique, two viral constructs carrying AAV-DIO-TVA-tdTomato and AAV-DIO-RVG were injected into the LPBN of Vglut2-Cre mice. These viral constructs allow for the expression of rabies virus components in glutamatergic cells from the LPBN to the VTA and with future rabies virus infection, tracing to the Sp5C. Four weeks later, the replication-deficient rabies virus (RV-ENVA-DG-eGFP) was injected into the VTA. This allows for tracing direct projections from the LPBN to the VTA and monosynaptic projections from the Sp5C to the LPBN. After another week, GFP was identified at the Sp5C, and tdTomato was detected in VTA. These results confirmed the existence of an Sp5C-LPBN-VTA circuit.

 

To verify that this circuit was involved in the depression-like behavior of pIONT mice, the surgery was performed on Vglut2-Cre mice, and AAV-DIO-NpHR-eYFP was injected into the Sp5C. After a week, NpHR-eYFP was detected in the LPBN. These connections between the Sp5C and LPBN were subsequently inhibited using laser light delivered through the optical fiber implanted in the LPBN. This inhibition resulted in a reduction of depression-like behavior.

 

Next steps and limitations

Yang feels that, although the paper was interesting, more supporting evidence is needed to verify the authors' claims. Yang commented that, “[Zhang et al.] showed that the activation of VTA dopaminergic neurons results in animals' increasing immobility; the mice show freezing-like behavior…. However, dopamine is related to motivated behaviors … motivation can be approaching a goal or avoiding damage, so the mice should move more, not freeze.”

 

This work ultimately suggested that the Sp5C-LPBN-VTA circuit is activated during chronic pain and results in the increased firing of the VTA dopaminergic neurons, leading to depression-like behavior. Hailin Zhang notes, “It is not completely clear from this current study whether this pathway pre-exists before the neuropathic pain, or is newly established and stimulated by the pain signals. This is an interesting question [that] need[s] to be answered further.” The persistent activation of this pathway may exert a plastic effect on the VTA dopaminergic neurons and the downstream targets of these neuron projections, resulting in the observed depression-like behavior. This could explain why there is a disconnect between the onset of chronic pain and depression, a phenomenon also observed in the spared nerve injury model.

 

The authors also note the limitations in using only male mice in the study, as sex hormones can significantly affect pain responses, depression-like behaviors, and underlying mechanisms. However, Hailin Zhang notes that they are using both male and female mice in current ongoing studies.

 

Courtney Bannerman is a PhD Candidate at Queen’s University in Kingston, Ontario, Canada.

 

Graphical abstract: Zhang et al., Cell Reports. 2021 Nov 2;37(5):109936.

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