Abstract / Summary
Adaptive responses to challenging environments depend on optimal function of the locus coeruleus (LC), the brain’s main source of noradrenaline and primary mediator of the initial stress response. Built-in systems that exert regulatory control over the LC are largely unidentified. A good candidate system is neuropeptide Y (NPY), which is traditionally linked to anxiety-relief. Currently, the endogenous source of NPY to the LC, and how NPY-expressing neurons modulate the noradrenergic system to regulate anxiety remain unclear. We here identify, in mice, a novel NPY-expressing neuronal population (peri-LCNPY) neighboring LC noradrenergic neurons that locally innervates the pericoerulean space. Moreover, we demonstrate that stress engages peri-LCNPY neurons, increasing their excitability. Mimicking peri-LCNPY neuronal activation using ex vivo chemogenetics suppresses LC noradrenergic neuron activity, via an NPY Y1 receptor-mediated mechanism. Furthermore, in vivo chemogenetic stimulation of peri-LCNPY neurons results in Y1R-dependent anxiety-relief. Conversely, inhibiting peri-LCNPY neurons increases anxiety-like behaviors. Together, we establish a causal role for peri-LCNPY-mediated neuromodulation of the LC in the regulation of anxiety, providing novel insights in the endogenous mechanisms underlying adaptive responses to adversity.
Introduction
Adaptive responses to stressful experiences are paramount to our survival and well-being. Opposing systems that mediate initiation and termination of the stress response work in tandem to ensure optimal adaptation to challenging environments1,2. Currently, knowledge of the factors that dictate adaptive modulation of the stress response is incomplete, limiting available therapeutics against stress-related afflictions, such as anxiety disorders3,4.
The locus coeruleus (LC) is the brain’s primary source of noradrenaline/norepinephrine (NE), and key regulator of the initial stress response5–7. The LCNE system mediates arousal and allocation of attention, preparing the organism for task-relevant and salience-specific responses, required in novel environments commonly associated with high cognitive and emotional load8–10. To accomplish this, owing to its vast projection network and efferent collaterals11, the LC coordinates myriad functions, from sympathetic responses, such as heart rate and pupil dilation, to complex, high-order cognitive processes, including goal orientation and decision-making9. LC hyperactivity, characterized by high tonic firing, can lead to maladaptive responses to perceived threats, priming the development of pathological anxiety7. In support, optogenetic stimulation of LCNE neurons results in anxiety in mice, whereas chemogenetic inhibition of LCNE cells strongly suppresses stress-driven anxiety-like behaviors12. Given the importance of LCNE activity in shaping anxiety responses, it is important to understand the mechanisms that appropriately regulate its function.
A strong candidate regulator of LCNE activity is the neuropeptide Y (NPY) system, composed of groups of neurons that communicate via NPY release, and others that interpret these signals. NPY, one of the most widely distributed neuropeptides in the central nervous system, is traditionally associated with stress-coping and anxiety relief13–15 and it is dubbed the “stress resilience” molecule3 after early preclinical studies, which employed NPY or NPY receptor agonists, highlighted its anxiolytic effects16–19. In support, NPY gene expression as well as NPY plasma levels have been linked to trait anxiety and stress-related neuropsychological conditions in clinical settings3,20.
NPY immunoreactivity and NPY receptor presence have been observed in the LC21,22. However, few studies have examined the functional relationship between (endogenous) NPY-mediated neuromodulation and the LCNE system. Ex vivo electrophysiological evidence shows that exogenously applied NPY reduces LCNE spontaneous discharge23 and facilitates hyperpolarization of LCNE neurons24. These studies highlight an inhibiting effect of NPY on tonic LC firing, which could be crucial in regulating LC activity during arousal, and under stress. In agreement, exogenous NPY application aiming at the peri-coerulean (peri-LC) space induces anxiolysis in the elevated plus maze (EPM)25, an innately anxiogenic behavioral task that is known to engage the LC26.
Despite these insights, the endogenous source of NPY to the LC remains unidentified. In the rat brain, NPY-like immunoreactivity has been observed in LCNE cell bodies as well as projection fibers21,27–30, suggesting that NPY (co-)released by LCNE neurons constitutes the main endogenous source of NPY to the LC. However, recent RNA sequencing data challenge this, demonstrating NPY presence in the peri-LC space but no co-expression in noradrenergic neurons of the mouse LC31. These contradictory reports have cast doubt on the origins of NPY input to the region. Furthermore, independently of its origins, no studies have addressed how endogenously released NPY modulates LCNE activity to regulate anxiety levels. Understanding the effects of endogenous NPY signaling is critical, as pharmacologically applied NPY engages distinct NPY receptors, resulting in dose-dependent, opposing regulation of anxiety-like behaviors32.
Aiming to address this, we examined NPY organization and function in the mouse LC. We characterize a previously unidentified NPY-expressing, pericoerulean (peri-LCNPY) neuronal population at the anatomical, electrophysiological, and behavioral level. Our data indicate that peri-LCNPY neurons constitute a distinct, non-noradrenergic population that is engaged by exposure to stress. We show that local peri-LCNPY cells suppress LCNE neuronal activity in an NPY-mediated, Y1 receptor-dependent manner. Finally, we demonstrate that activation of peri-LCNPY cells reduces anxiety-like behaviors, via LC Y1 receptors, and conversely, peri-LCNPY inhibition promotes anxiogenesis. Together, we here describe a previously unknown population of NPY-expressing cells that regulates the LC noradrenergic system, thereby promoting adaptive behavioral responses in arousing environments.
Results
Identification of NPY-expressing neurons in the pericoerulean space
To examine the presence of NPY neurons in the peri-LC region, NPY-cre mice33 were crossed with the Ai14 reporter line34, enabling tdTomato (tdT) fluorescence selectively in NPY-expressing cells (Fig. S1A). We observed a clear presence of tdT+ cells neighboring the LC proper, the region occupied by noradrenergic cell bodies (Fig. 1A; Fig. S1B). We performed a systematic mapping of peri-LCNPY neuron location with respect to noradrenergic neurons of the LC (LCNE), identified by tyrosine hydroxylase (TH) expression. In coronal slices immunolabelled for TH, we extracted the coordinates of tdT+ cells at the entire rostrocaudal axis (Anterior-Posterior: -5.80 to -5.25 mm from bregma) containing the LC (Fig. 1A). We then used the distance from LC center to plot peri-LCNPY cell distribution at the mediolateral (Fig. 1B, Fig. S1C) and dorsoventral axes (Fig. 1C, Fig. S1C). Within the pericoerulean region, defined by the extend of reach of LC dendritic processes (Fig. S1B)35, peri-LCNPY neurons congregated largely medially (∼60%) to the LC proper, with ∼20% found dorsal and ∼40% ventral to TH+ cell bodies.
A) Top: Representative examples of tdTomato expression (tdT, red) and noradrenergic immunolabeling (TH, cyan) in coronal slices from NPY-cre:Ai14 mice, depicting the LC at the entire rostrocaudal axis. 4th ventricle is indicated. Bottom: FIJI-processed images containing masks for the two channels (tdT, red; TH, black), on which detection and coordinate extraction per tdT+ cell was based. Scale bar, 100µm; D, dorsal; V, ventral; M, medial; L, lateral. B) Frequency of distribution (%) of peri-LCNPY neurons location in respect to their distance (µm) from LC center at the mediolateral axis. Grey shading depicts the width of LC proper in corresponding coordinates. Across the AP axis, the majority of peri-LCNPY cells, are located medially to LC proper. C) Frequency of distribution (%) of peri-LCNPY neurons location in respect to their distance (µm) from LC center at the dorsoventral axis. Across the AP axis, the majority of peri-LCNPY cells are located ventral to LC proper. D) Left: Representative examples of tdTomato expression (tdT, red) and noradrenergic immunolabeling (TH, cyan) in coronal slices from NPY-cre:Ai14 mice. Right: Quantification of double-immunoreactive NPY+/TH+ cells showed that peri-LCNPY neurons are predominantly TH-lacking. Red arrows, NPY+/TH- cells (98.2%); white arrow, NPY+/TH+ cells (1.8%); Scale bar, 50µm. E) Left: Representative examples of NPY expression (red), detected by in situ hybridization, and noradrenergic immunolabeling (TH, cyan) in coronal slices from wild-type C57BL/6 mice, containing the LC. Right: Quantification of NPY puncta and TH expression showed that the majority of peri-LCNPY neurons are non-noradrenergic (NPY+/TH-, 96.7%, red arrow; NPY+/TH+, 3.3%, white arrow), and vice-versa, only 4% of LCNE cells co-express NPY (TH+/NPY- 96%, red arrows; TH+/NPY+, 4%, white arrow). Scale bar, 50µm. B-C: AP, -5.80mm to -5.60mm, N=2 mice, n=239 cells; AP, -5.60mm to -5.40mm, N=7 mice, n=1004 cells; AP, -5.40mm to -5.25mm, N=6 mice, n=558 cells. D) N=5 mice, n=612 cells. E) NPY: N=2 mice, n=30 cells; TH: N=3 mice, n=125 cells.
Our mapping data indicated that, based on location alone, the peri-LCNPY population is, to a large degree, topographically distinct from LCNE cells. To further validate this, in coronal slices from NPY-cre:Ai14 mice immunolabelled for TH, we quantified the percentage of TH-expressing peri-LCNPY cells in the total peri-LCNPY population (Fig. 1D). In contrast to earlier studies, which showed large overlap between noradrenergic and NPY-expressing neurons21,27, the vast majority of tdT+ cells were TH-devoid, while only 1.8% co-expressed TH, indicating that the absolute number of NPY neurons occupying the pericoerulean space has been largely underestimated31. To control for incomplete Cre-recombinase expression in NPY-cre:Ai14 mice that could potentially confound these results, we used multiplex fluorescent RNAscope in situ hybridization against endogenous NPY, in combination with TH immunolabeling, in brain slices from wild-type C57BL/6 mice (Fig. 1E). We quantified TH expression in NPY+ cells and vice-versa, NPY puncta in LCNE neurons. In agreement with our earlier results, only 3.3% of peri-LCNPY neurons co-expressed TH, while 4% of LCNE cells were NPY+.
Peri-LCNPY neurons respond to acute stress
The LC is an important regulator of the initial stress response, with implications for the development of stress-induced pathology7. To further understand potential contributions of peri-LCNPY neurons in neuromodulation of the LCNE system, we examined their physiological properties under naïve conditions and after stress. For this, we performed whole-cell patch clamp recordings in LC-containing brain slices of control or stressed NPY-cre:Ai14 mice (Fig. 2D, Fig. S2A). First, we established a stress protocol that induces acute and long-lasting anxiety-like phenotypes, following exposure to electrical foot-shocks (Fig. 2A-C). Acutely (30 min) after stress, foot-shocked exposed mice (FS) showed decreased time spent in the open arm of an elevated-plus maze (EPM) compared to non-stressed (NS) controls (NS, 13.2% vs. FS, 6.9% of total exploration time, Fig. 2B). This was accompanied by an increase in anxiety index (NS, 0.76 vs. FS, 0.84), a compound parameter that integrates avoidance and exploratory behaviors36, further supporting direct stress effects on anxiety-like behaviors6. Stress-induced anxiety was long-lasting, as reflected in reduced exploration of an open field one week following exposure to foot-shocks (Fig. 2C). In particular, FS mice spent less time at the center of the open field arena (NS, 4.0% vs. FS, 2.4% of total exploration time), which they visited less frequently compared to controls (NS, 24.5 vs. FS, 17.5 visits).
A) Schematic representation of experimental design. Acute (30 min, elevated plus maze -EPM) and long-lasting (1-week, open field test -OFT) effects of stress (electrical foot-shock: 20 shocks x 2s x 0.3mA) were assessed in wild-type C57BL/6 mice. B) Stress exposure acutely increased anxiety-like phenotypes in the EPM. Stressed mice spent less time in the open arms of the maze when compared to controls (NS, N=9, 13.2% vs. FS, N=9, 6.9% of total EPM time; unpaired t-test, t(16)=2.694, P=0.016). Although decreased, no statistically significant group effect on the frequency of visits to the open arms was seen (NS, 22.7 vs. FS, 14.9; Mann-Whitney U=22, P=0.108). The stress group displayed increased anxiety index, which integrates explorative behavior in addition to avoidance in the assessment of anxiety-like behaviors36 (NS, 0.76 vs. FS, 0.84; unpaired t-test, t(16)=2.306, P=0.035). C) Stress exposure led to long-lasting anxiety-like phenotypes in the OFT. The FS group spent less time in the center of the open field arena (NS, N=15, 4.0% vs. FS, N=15, 2.4% of total OFT time; unpaired t-test, t(28)=2.727, P=0.011) and displayed reduced frequency of visits to the center (NS, 24.5 vs. FS, 17.5; unpaired t-test, t(28)=2.286, P=0.030). Stress had a lingering effect on explorative behavior in the OFT arena (Distance moved: NS, 43m vs. FS, 35.7m; unpaired t-test, t(28)=2.486, P=0.019). B-C: Representative spatial location heatmaps show time spent exploring the arenas. D) Schematic representation of experimental design. Control (NS) or foot-shock-subjected (FS) NPY-cre:Ai14 mice were sacrificed 30 min following stress exposure for electrophysiological recordings. In a different cohort, NS and FS mice were sacrificed 90 min post-stress to examine cFos expression via immunolabeling. E) Whole-cell patch clamp recordings from LCNE neurons. Stress exposure increased LCNE firing frequency, as seen in the number of action potentials fired in response to increasing current injections (2-way RM ANOVA, main stress effect: F(1,26)=9.27, P=0.005). Representative example traces of action potentials fired in response to a 200pA current injection in NS (top) and FS (bottom) mice are depicted. F) Whole-cell patch clamp recordings from tTd+ peri-LCNPY neurons were conducted in current-clamp configuration, to examine their intrinsic electrical properties, and possible effects of acute stress. Stress significantly increased peri-LCNPY neuron firing frequency (Hz), observed in response to increasing current injections (2-way RM ANOVA main group effect, F(1,922)=5.597, P=0.018. Representative example traces of action potentials fired in response to a 175pA current injection in NS (top) and FS (bottom) mice are depicted. G) Quantification of cFos-expressing peri-LCNPY cells showed increased percentage of double-immunoreactive neurons after exposure to stress (NS 7.5%, FS 13.3% of total peri-LCNPY population; unpaired t-test, t(6)=3.169, P=0.019). Representative examples of tdTomato expression (tdT, red) and cFos immunolabeling (yellow) in coronal slices are shown. Scale bar, 50µm. B) NS, N=9; FS, N=9. C) NS, N=15; FS, N=15. E) NS, N=5 mice, n=12 cells, FS, N=6 mice, n=16 cells. F) NS, N=10 mice, n=40 cells; FS, N=13 mice, n=46 cells. G) NS, N=4 mice, n=1572 cells, FS, N=4 mice, n=867 cells. E, F) Scale bar: 20 mV, 100 ms. Data depicted as mean ± SEM. * P <0.05.
We verified that this stress protocol engages LCNE cells. We performed patch-clamp recordings from LCNE cells in brain slices from mice previously subjected to the foot shock paradigm or control conditions. LCNE cells were identified based on their location and morphological characteristics39.
To validate that this approach yielded recordings from putative LCNE neurons, we first confirmed the identity of a subset of biocytin-filled recorded cells using post-hoc TH immunolabeling (Fig. S2A). Our electrophysiological recordings demonstrated that foot shock experience engaged LCNE cells, increasing their excitability. In particular, in slices prepared from stressed mice, we observed a higher number of action potentials in response to increasing current injections, as compared to controls (Fig. 2E). This was accompanied by a reduction in the current necessary for LCNE cells to exceed their action potential threshold and fire (Rheobase: NS, 25pA, FS, 0pA, Fig. S2B), further corroborating increased neuronal excitability after stress.
We next recorded peri-LCNPY intrinsic properties in slices prepared from the same cohort of control and stress-exposed mice (Fig. 2D, S2C). Stress did not affect peri-LCNPY passive properties, such as cell capacitance and membrane resistance (Fig. S2D). Likewise, action potential threshold and resting membrane potential remained unaltered after exposure to stress (Fig. S2D). Control peri-LCNPY neurons showed sustained capacity for high-frequency firing rates (Fig. 2F), corresponding well to the physiological requirements for neuropeptidergic release from dense core vesicles37,38. Notably, in brain slices prepared from stressed mice we detected an even greater number of action potentials in response to increasing current injections, as compared to controls (Fig. 2F), indicating peri-LCNPY neuron engagement in stressful environments. To further validate this, we quantified cFos expression, as proxy for neuronal activation, in peri-LCNPY cells from control and stress-exposed mice (Fig. 2G, Fig. S2E). We found an increased number of cFos+ peri-LCNPY cells in mice subjected to foot-shocks (NS, 7.5%; FS 13.3% of total peri-LCNPY population), corroborating the involvement of peri-LCNPY neurons in the initial stress response.
Peri-LCNPY neurons innervate the pericoerulean space but do not form GABAergic or glutamatergic synaptic connections with LCNE neurons
We demonstrated that exposure to an acute stressor simultaneously engages LCNE and peri-LCNPY neurons, indicating an involvement of the two populations in the stress response. However, the effects of peri-LCNPY activation on the LCNE system remained unknown. To examine whether peri-LCNPY neurons could provide (NPY) input to the LC proper, we employed Cre-dependent, virus-mediated antero-and retrograde labeling of peri-LCNPY cells and mapped their neuroanatomical circuitry40. The vast majority of NE dendritic processes are located outside the nuclear core, where they receive extensive non-noradrenergic synaptic contacts35. Thus, we first investigated whether peri-LCNPY neurons project within the dendritic zone of the LC, to permit signaling from NPY and its co-transmitters in the region.
For this, NPY-cre mice were bilaterally injected with a Cre-dependent AVV (AAV-Syn-FLEX-CoChR-GFP) in the LC and allowed a period of ≥5 weeks of virus incubation. Next, brain slices were collected and a qualitative analysis of labeled NPY+ cell bodies and fibers locally, within the LC, and in selected projection fields, was performed (Fig. 3A, Fig. S3A). While we observed clear innervation of the pericoerulean space, we detected no peri-LCNPY efferents in other evaluated brain regions with known LC outputs (Fig. 3A, Fig. S3A). This suggests that local peri-LCNPY neurons do not possess long-range projection neuron characteristics, as seen for instance in cortical areas41. To further corroborate local connectivity of peri-LCNPY neurons within the region, we next injected a Cre-dependent retrograde HSV virus (HSV-hEF1a-LS1L-mCherry) in the LC. This resulted in retrograde tracing of NPY+ cell bodies throughout the pericoerulean area (Fig. 3B, Fig. S3B), supporting the notion that peri-LCNPY neurons terminate within the region. Moreover, we detected several brain areas containing LC-projecting NPY+ cells, expanding previously reported data on NPY-containing afferents42 (Fig. S3B, Table S1). Both viral tracing strategies indicated that peri-LCNPY projections assemble in the pericoerulean region. To assess whether these fibers are terminating or fibers of passage, we bilaterally injected an AAV construct (AAV-hSyn1-mCBP-EGFP-2A-mSyp1-mRuby) in the LC of NPY-cre mice for Cre-dependent expression of membrane-bound GFP and mRuby-fused synaptophysin, that enables axonal and presynaptic terminal labeling, respectively43 (Fig. 3C). Within the LC dendritic zone, we identified mRuby+ puncta along peri-LCNPY axons, suggesting LCNE synaptic innervation by local peri-LCNPY cells.
A) Left: Schematic of sagittal mouse brain, depicting the location of bilateral virus injections for anterograde labeling in NPY-cre mice. Right: Representative images of GFP expression (yellow) and noradrenergic (TH, cyan) immunolabeling in coronal slices containing the LC, illustrating dense NPY innervation in the peri-LC space. B) Left: Schematic of sagittal mouse brain, depicting the location of bilateral virus injections for retrograde labeling in NPY-cre mice. Right: Representative images of mCherry expression (yellow) and noradrenergic (TH, cyan) immunolabeling in coronal slices containing the LC, illustrating retrogradely-traced NPY+ cell bodies and fibers in the peri-LC space. C) Left: Schematic of sagittal mouse brain, depicting the location of bilateral virus injections for axonal and presynaptic labeling in NPY-cre mice (N=5). Right: Representative images of GFP expression (yellow) and noradrenergic (TH, cyan) immunolabeling in coronal slices containing the LC, illustrating NPY+ cell bodies and axons in the pericoerulean space. Inset: mRuby+ puncta are traced along NPY+ axons (white arrow heads), indicating that peri-LCNPY neurons synapse within the LC dendritic zone. D) Left: Representative examples of Y1R (yellow), Y2R (magenta), and TH (cyan) mRNA detected by in situ hybridization, in coronal slices from wild-type C57BL/6 mice containing the LC. Right: Quantification of Y1R and Y2R puncta showed that the majority of LCNE neurons expressed both NPY receptors (Y1+/Y2+, 74.5% of detected cells, orange arrow and expanded panel). 8% of LCNE neurons contained Y1Rs, 7.4% Y2Rs and 10.1% were YR-lacking. E) Schematic of sagittal mouse brain, depicting the location of bilateral virus injections used to drive hM3Dq expression in NPY-cre mice. After a period of virus incubation (≥ 5 weeks), brain slices were prepared for electrophysiological recordings. Whole-cell patch clamp recordings from LCNE neurons were conducted in current-clamp configuration, in presence of the DREADD actuator C21 (2µM) or vehicle. F) In presence of C21 we detected fewer action potentials in response to increasing current injections, as compared to vehicle (2-way RM ANOVA, main effect of treatment, F(1,30)=10.36, P=0.003). Chemogenetic activation of peri-LCNPY neurons induced membrane hyperpolarization (resting membrane potential: veh, -52.7mV; C21, -63.5mV; unpaired t-test, t(23)=3.30, P=0.003). G) Similar effects of C21 bath application were observed in presence of synaptic blockers against AMPAR (10µM CNQX), NMDAR (50µM D-AP5), GABAAR (100µM picrotoxin) and GABABR (10µM CGP-54626) -mediated currents (2-way RM ANOVA, main effect of treatment, F(1,25)=6.18, P=0.020). H) Pretreatment with the selective Y1R antagonist BIBO-3304 (1µM) blocked the effect of peri-LCNPY chemogenetic activation on LCNE firing frequency (2-way RM ANOVA, main effect of treatment, F(1,24)=0.06, P=0.815) and membrane hyperpolarization (resting membrane potential: veh, -55.0mV; C21, -60.6mV; unpaired t-test, t(16)=1.74, P=0.100). I) Pretreatment with the selective Y2R antagonist BIIE-0246 (1µM) did not preclude the effects of C21 on LCNE excitability (2- way RM ANOVA, main effect of treatment, F(1,21)=7.41, P=0.013). A-C: Scale bar, 100µm and 50µm (inset). D) Scale bar, 50µm and 10µm (expanded panel). F-I) Representative traces of LCNE firing in response to 200pA current injection (scale bar 20mV, 200 ms). A) N=5. B) N=5. C) N=3. D) N=4 mice, n=149 cells. F) Veh, N=4 mice, n=15 cells; C21, N=4 mice, n=17 cells. G) Veh & block, N=2 mice, n=14 cells; C21 & block, N=3 mice, n=13 cells. H) Veh & BIBO-3304, N=3 mice, n=13 cells; C21 & BIBO- 3304, N=4 mice, n=13 cells. I) Veh & BIIE-0246, N=2 mice, n=11 cells; C21 & BIIE-0246, N=3 mice, n=12 cells. F-I). Data depicted as mean ± SEM. * P <0.05; ** P <0.01.
Based on these data, we hypothesized that peri-LCNPY neurons form functional synapses with LCNE cells, necessary for LCNE neuromodulatory control. To test this, we bilaterally injected NPY-cre mice with a Cre-dependent AAV (AAV-Syn-FLEX-CoChR-GFP) in the LC, that enables selective expression of the highly-conducting channelrhodopsin variant CoChR44 in peri-LCNPY neurons (Fig. S4A, B). After a period allowing for virus incubation and CoChR expression in terminals (≥5 weeks), we performed whole-cell patch clamp recordings from putative LCNE neurons (cf., Fig. S2A) To investigate the presence of direct peri-LCNPY◊LCNE synaptic connectivity we opto-stimulated peri-LCNPY neurons and recorded from LCNE cells in brain slices with confirmed CoChR innervation (Fig. S4C, D).
First, to detect AMPAR-mediated or GABAAR-mediated ionotropic currents, we optogenetically stimulated peri-LCNPY neurons with single pulses, while recording from LCNE cells in voltage clamp configuration at -50mV. The fraction of LCNE neurons that displayed perceptible postsynaptic responses to peri-LCNPY optostimulation was negligible (AMPA-mediated, 0/39 cells; GABAAR-mediated, 1/39 cells). Rather, the majority of LCNE neurons remained unresponsive (38/39 cells, Fig. S4C). In these experiments we also applied trains of 20 pulses of 5, 20 or 50 Hz photostimulation, to allow for the possibility of high frequency stimulation requirements in detecting forms of (GABABR or NPY receptor-mediated) metabotropic signaling45,46. As above, no such direct fast onset responsivity was observed between peri-LCNPY and LCNE neurons (Fig. S4C).
Because we performed the experiments above with a potassium gluconate-based intracellular solution, we may have underestimated connectivity at more distal inputs47 to LCNE neurons. To address this, we next recorded a subset of LCNE cells using a cesium chloride (CsCl)-based internal in the absence of synaptic blockers, at -60mV, to allow for the detection of GABAAR or eventual AMPAR-mediated currents47. In agreement with our previous data, no responses to single optical pulse were observed (20/20 cells, non-responsive, Fig. S4D). Finally, to account for the possibility of silent synapses, we performed recordings at +40mV using the CsCl-based internal solution to detect NMDAR-mediated currents. As before, no synaptic responses to peri-LCNPY stimulation were seen (12/12 cells, non-responsive, Fig. S4D). Taken together, these experiments indicate that there is close to null glutamatergic or GABAergic synaptic connectivity (either ionotropic nor fast-onset metabotropic) between peri-LCNPY and LCNE cells.
Pharmacologically applied NPY bidirectionally alters LCNE neuronal excitability, via distinct NPY receptors
Our connectivity data suggest that any potential communication between peri-LCNPY and LCNE neurons is likely mediated by NPY itself. Although we cannot exclude that NPY signaling could manifest in our functional connectivity studies, to our knowledge fast-onset synaptically-driven, direct postsynaptic NPY receptor currents have not been observed before47. Thus, we reasoned that peri-LCNPY-LCNE direct NPY signaling occurs in an alternative manner, the detection of which cannot be achieved by optogenetic circuit mapping approaches. To further investigate this possibility, we first examined NPY receptor presence in LCNE cells, which would be required to mediate direct NPY signaling. Using multiplex fluorescent RNAscope in situ hybridization we identified LCNE neurons that expressed Y1R and/or Y2R puncta in wild-type C57BL/6 mice (Fig. 3D). In accordance with previous reports30, the majority of LCNE neurons expressed both receptors (Y1+/Y2+, 74.5%). Y1R (Y1+/Y2-, 8%) or Y2Rs (Y1-/Y2+, 7.4%) -expressing LCNE subgroups were observed at a lesser extent, while the remaining fraction was YRs-lacking (Y1-/Y2-, 10.1%). Together, our data verified that LCNE neurons express the necessary molecular machinery for NPY-mediated neuromodulation.
Next, we investigated whether YR-mediated NPY signaling alters LCNE intrinsic excitability. For this, we performed whole-cell patch clamp recordings in the presence of different doses of NPY. In brain slices prepared from wild-type C57BL/6 mice, we bath-applied NPY or vehicle, and recorded LCNE firing frequency. In the presence of 30nM NPY we detected fewer action potentials in response to increasing current injections, as compared to vehicle. This effect was mediated by Y1Rs, as NPY-induced decrease in LCNE excitability was blocked in slices pretreated with the selective Y1R antagonist BIBO-330448 (Fig. S5A). Surprisingly, a 10-fold increase in NPY concentration yielded the opposite result, as we observed LCNE hyper-excitability in NPY-treated slices. Notably, LCNE increased firing was abolished in the presence of the selective Y2R antagonist BIIE-024649 (Fig. S5B). Furthermore, 300 nM NPY-induced LCNE hyper-excitability was prevented in slices pre-treated with synaptic blockers (for AMPA/kainate, NMDA, GABAA and GABAB receptors), suggesting that changes in LCNE excitability may occur via an indirect network effect (Fig S5C). Overall, these observations highlight the capacity of LCNE neurons to respond directly and indirectly, via distinct receptors, to pharmacologically applied NPY, with opposing effects on their excitability. This emphasizes the need to understand the mechanisms underlying endogenous NPY-mediated neuromodulation of the LCNE system.
Peri-LCNPY neurons suppress LCNE excitability via postsynaptic Y1Rs
Our pharmacological data demonstrated that NPY has the capacity to bidirectionally alter LCNE excitability states. However, whether endogenous, peri-LCNPY-mediated signaling could affect LCNE firing properties remained unknown. To address this, we combined chemogenetic activation of peri-LCNPY neurons, which allows for protracted peri-LCNPY stimulation, with ex-vivo slice electrophysiology and assessed its effects on LCNE firing patterns. To this end, NPY-cre mice were bilaterally injected with a Cre-dependent AAV (AAV-hSyn-DIO-hM3D(Gq)-mCherry) to drive the expression of a Gq-coupled (excitatory) designer receptor exclusively activated by designer drugs (DREADD) in peri-LCNPY cells (Fig. 3E). This viral construct enabled targeted chemogenetic activation of peri-LCNPY neurons in presence of the DREADD agonist compound 21 (C21)50. First, we validated that C21 activates peri-LCNPY neurons ex vivo (Fig. S6A). Then, we proceeded with whole-cell patch clamp recordings of LCNE neurons in brain slices prepared from NPY-cre mice bilaterally expressing the hM3Dq DREADD (Fig. 3E).
Passive electrophysiological properties and intrinsic excitability of LCNE cells were assessed in current-clamp configuration, after bath application (≥10 min) of C21 (2µM) or vehicle. In the presence of C21 we detected fewer action potentials in response to increasing current injections as compared to vehicle (Fig. 3F), mimicking the effect of low NPY dose application. This effect was accompanied by LCNE membrane hyperpolarization (Resting membrane potential: vehicle, - 52.7mV; C21, -63.5mV) and an increased rheobase (vehicle, 15.0pA; C21, 44.1pA; Fig. 3F, Fig. S6B), indicating that activation of local peri-LCNPY neurons results in reduced LCNE neuronal excitability. Peri-LCNPY chemogenetic stimulation did not alter other electrophysiological parameters of LCNE cells (Fig. S6B). Importantly, in brain slices that lacked hM3Dq expression in the region, we confirmed that C21 did not result in off-target, non-specific effects on LCNE neuronal firing (Fig. S6C).
We next addressed the signaling mechanisms through which peri-LCNPY neuron activity suppresses the excitability of LCNE cells. Particularly, we sought to address (i) the likelihood of the peri-LCNPY stimulation effect being direct or indirect via intermediate (e.g., GABAergic51) neurons; and (ii) whether it could be mediated by peri-LCNPY neurons co-releasing signaling molecules other than NPY, such as GABA or glutamate, despite the latter seeming unlikely (cf., Fig. S4C, D). To that end, we repeated the experiment of chemogenetic peri-LCNPY neuron stimulation, while assessing LCNE neuron excitability, but this time we pharmacologically blocked synaptic network activity (i.e., blocking AMPAR/ kainateR, NMDAR, GABAAR and GABABRs; cf., Fig. S5C). In these conditions, we still observed a clear decrease in LCNE firing in presence of C21 (Fig. 3G), pointing towards direct post-synaptic effects of peri-LCNPY neuron stimulation on LCNE cell excitability.
Finally, to examine whether C21 effects were specifically mediated by NPY signaling, we assessed LCNE excitability in presence of Y1R or Y2R blockers. In slices pretreated with the Y1R antagonist BIBO-3304 (1µM), the effects of C21 on LCNE excitability and membrane hyperpolarization were fully occluded (Resting membrane potential: vehicle, -55.0mV; C21, -60.6mV; Fig. 3H). On the contrary, blocking Y2Rs with BIIE-0246 (1µM) did not prevent the ability of peri-LCNPY stimulation to reduce LCNE firing frequency (Fig. 3I). Synaptic blockers, BIBO-3304 or BIIE-0246 alone did not alter LCNE firing properties in vehicle-treated slices, further highlighting the specificity of the effects of peri-LCNPY chemogenetic activation (Fig. S6D).
Together, our data suggest that sustained stimulation of local peri-LCNPY neurons hyperpolarizes LCNE cells and dampens their excitability. Our observations point to postsynaptic Y1Rs, situated onto LCNE neurons, being the prime mediators of local NPY signaling on the noradrenergic system. Notably, C21-driven activation of peri-LCNPY neurons and exogenous application of 30 nM NPY (but not of 300 nM NPY) were analogous in regard to their effects on LCNE intrinsic excitability, offering insights regarding the potential concentrations of chemogenetically-evoked NPY release from peri-LCNPY cells.
Peri-LCNPY neurons bidirectionally modulate anxiety-like behavior via local Y1Rs
Optogenetic LCNE neuron stimulation is anxiogenic at baseline conditions, and conversely chemogenetic LCNE neuron silencing prevents stress-induced anxiety12,52,53. Based on the observed effects of peri-LCNPY activation on LCNE neuronal excitability, we next hypothesized that local NPY signaling could represent an endogenous mechanism responsible for the regulation of anxiety in novel and/or anxiogenic environments. To test this hypothesis, we assessed the behavioral implications of peri-LCNPY stimulation in vivo. NPY-cre mice were bilaterally injected with a Cre-dependent excitatory DREADD (AAV-hSyn-DIO-hM3D(Gq)-mCherry), or a vector expressing a control fluorescent protein (AAV-hSyn-DIO-mCherry) in the LC. After a period allowing for virus expression (5 weeks), mice were administered C21 (2mg/kg, i.p.) and were subjected to the EPM task (Fig. 4A). In hM3Dq mice, C21 increased the time spent in the open arm of the maze (mCherry, 8.1%, hM3Dq, 11.9% of total exploration time) and the frequency of open arm visits (mCherry, 10.0, hM3Dq, 14.4), indicating a reduction in baseline anxiety levels. In agreement, hM3Dq mice displayed decreased anxiety index (mCherry, 0.80, hM3Dq, 0.74; Fig. 4B). This anxiolytic effect occurred in the absence of C21 effects on general locomotion (Distance moved: mCherry 9.6m, hM3Dq, 10.3m; Fig. S7A). Likewise, controlling for possible off-target effects of C21, in a separate cohort of wild-type C57BL/6 mice, we confirmed that there were no gross behavioral changes (EPM performance, locomotion) after systemic C21 administration (Fig. S7C).
Experimental design for in vivo chemogenetic manipulations: schematic of sagittal mouse brain, depicting the location of bilateral virus injections used to drive hM3D(Gq) (A, C, E), hM4D(Gi) (G) or control vector expression in NPY-cre mice. Representative examples of virus spread, and cannula placement are included. B) After 5 weeks allowing for virus expression, mice were systemically administered the DREADD agonist compound 21 (C21, 2mg/kg, i.p.). Two hours after C21 injection, mice were subjected to the elevated plus maze (EPM) task. The hM3Dq group showed increased time spent in the open arms of the EPM (mCherry, 8.1%, hM3Dq, 11.9% of total exploration time; unpaired t-test, t(40)=2.42, P=0.020) and increased open arm entries (mCherry, 10.0, hM3Dq, 14.4; unpaired t-test, t(40)=2.71, P=0.010) vs. mCherry controls. Furthermore, in hM3Dq mice, C21 decreased anxiety index compared to mCherry controls (mCherry, 0.80, hM3Dq, 0.74; unpaired t-test, t(40)=2.31, P=0.026. D) After 5 weeks allowing for virus expression, cannula-assisted, bilateral, intra-LC micro-infusions of BIBO-3304 (200nM / 0.2µl / side) took place, 20min before systemic C21 administration (2mg/kg, i.p.). Thirty minutes after C21 injection, mice were subjected to the EPM task. A trend for main group effects on time spent in the open arms of the EPM was seen (mCherry & veh, 3.2%; hM3Dq & veh, 18.0%; hM3Dq & BIBO, 5.9% of total exploration time; 1-way ANOVA, F(2,25)=3.16, P=0.060). We detected main group effects for the number of visits to the open arms (mCherry & veh, 3.1; hM3Dq & veh, 12.5; hM3Dq & BIBO, 4.7; 1-way ANOVA, F(2,25)=6.16, P=0.007). This was due to hM3Dq & veh mice displaying significantly more entries vs. the other two groups (P=0.008 vs. mCherry & veh; P=0.042 vs. hM3Dq & BIBO). Likewise, we observed main group effects for anxiety index (mCherry & veh, 0.89; hM3Dq & veh, 0.72; hM3Dq & BIBO, 0.87; 1-way ANOVA, F(2,25)=3.80, P=0.036), driven by decreased anxiety index in hM3Dq & veh group vs. mCherry & veh controls (P=0.042). F, H) After 5 weeks allowing for virus expression, food deprived (24-h) mice were administered C21 (2mg/kg, i.p.). One hour after C21 injection, mice were subjected to the novelty suppressed feeding (NSF) task. F) hM3Dq mice showed increased food intake vs. mCherry controls (mCherry, 0.80 g/kg; hM3Dq, 1.84 g/kg; unpaired t-test, t(40)=3.03, P=0.004. Furthermore, C21 decreased feeding latency in the hM3Dq group (mCherry, 548s; hM3Dq, 343s; Mantel-Cox, χ2(1)=5.52, P=0.019). H) hM4Di mice consumed significantly less food vs. mCherry controls (mCherry, 3.33 g/kg; hM4Di, 1.32 g/kg; unpaired t-test, t(16)=2.66, P=0.017). No effects of C21 on feeding latency were seen (mCherry, 324s; hM4Di, 431s; Mantel-Cox, χ2(1)=1.24, P=0.265). B, D, F, H: Representative spatial location heatmaps show time spent exploring the arenas. B, F) Veh, N=21; hM3Dq N=21. D) mCherry & veh, N=10; hM3Dq & veh, N=10; hM3Dq & BIBO-3304, N=8. H) mCherry, N=10; hM4Di, N=8. Data depicted as mean ± SEM. * P<0.05. ** P <0.01.
Our findings suggest that NPY release by peri-LCNPY neurons induces anxiolysis. We next assessed whether, in accordance with our electrophysiological data (cf., Fig. 3H), this effect was mediated by local LC Y1R activation. To address this, we next performed an EPM experiment combining peri-LCNPY chemogenetic activation with local, intra-LC Y1R antagonism in vivo. For this, NPY-cre mice expressing the excitatory DREADD (hM3D(Gq)) or mCherry control virus in peri-LCNPY cells were bilaterally equipped with intra-cranial cannulas for LC-targeted administration of the selective Y1R antagonist BIBO-3304. After a period allowing for virus expression (≥5 weeks), we micro-infused mice with BIBO (200pmol/ 0.2µl/ side) or vehicle in the LC, before systemically administering C21 (2mg/kg, i.p) and then placed mice in the EPM (Fig 4C, D). As before, vehicle-infused hM3Dq mice (N=10), exhibited reduced anxiety-like behaviors vs. vehicle-infused mCherry controls. Notably, the anxiolytic effect of peri-LCNPY chemogenetic stimulation was abolished in hM3Dq-expressing mice pre-treated with the Y1R antagonist. Particularly, hM3Dq+veh mice spent more time exploring the open arms of the EPM in comparison to the other two groups (mCherry+veh, 3.2%; hM3Dq+veh, 18.0%; hM3Dq+BIBO, 5.9% of total exploration time), although this fell short of statistical significance. Furthermore, a main group effect was observed for open arm entries (mCherry+veh, 3.1; hM3Dq+veh, 12.5; hM3Dq+BIBO, 4.7), driven by increased number of visits as seen in the hM3Dq+veh group compared to other groups (Fig 4D). Lastly, we detected a main group effect for anxiety index (mCherry+veh, 0.89; hM3Dq+veh, 0.72; hM3Dq+BIBO, 0.87), driven by decreased anxiety index in hM3Dq+veh group vs. mCherry+veh controls (Fig 4D). Unlike in the prior, non-cannulated experiment (cf., Fig 4A, B), we here observed group effects on general locomotor activity (Distance moved: mCherry+veh, 5.3m; hM3Dq+veh, 10.4m; hM3Dq+BIBO, 6.8m; Fig. S7B). Nonetheless, our findings confirmed a causal link between peri-LCNPY activity and the regulation of anxiety-like behaviors, where local Y1R-mediated NPY signal tempers LCNE firing and promotes anxiolysis.
Our data suggest that peri-LCNPY-mediated input to the LC sufficiently drives anxiolysis in the EPM. To further corroborate this we evaluated the role of peri-LCNPY neurons in another behavioral assay, namely the novelty-suppressed feeding (NSF) test, which assesses hyponeophagia, the suppression of food intake due to exposure to a novel environment, as a proxy for anxiety54. For this, we used the aforementioned cohort of NPY-cre mice with Cre-dependent excitatory DREADD (hM3Dq), or control virus (mCherry) in peri-LCNPY neurons. Mice were food deprived for 24-h, followed by systemic C21 administration (2mg/kg, i.p.) and assessed in the NSF task (Fig. 4E). In hM3Dq mice, C21 decreased the latency to initiate consumption of a familiar food source placed at the center of an open field arena (mCherry, 548s; hM3Dq, 343s), indicating reduction in anxiety levels. In support, hM3Dq mice spent more time at the center of the arena (Duration Center: mCherry, 7.7%; hM3Dq, 13.4% of total exploration time), where they consumed significantly more food vs. mCherry controls (NSF intake: mCherry, 0.80 g/kg; hM3Dq, 1.84 g/kg; Fig. 4F, Fig. S7D). Controlling for effects of peri-LCNPY chemogenetic stimulation on general consummatory behavior, we observed no group differences on food consumption in a familiar environment (Home-cage intake: mCherry, 6.76 g/kg; hM3Dq, 7.35 g/kg; Fig. S7D). No effects of C21 on general locomotor activity at the NSF were seen (Distance moved: mCherry 41.5m, hM3Dq, 42.7m; Fig. S7D), validating the specificity of the anxiolytic effects of peri-LCNPY activation.
Overall, our data support that peri-LCNPY activation sufficiently drives anxiety relief, however, whether it is also required for anxiolysis remains unresolved. To address this, we examined the effects of chemogenetic peri-LCNPY inactivation on EPM and NSF tasks. For this, we made use of a Cre-dependent AAV (AAV-hSyn-DIO-hM4D(Gi)-mCherry) that drives the expression of an inhibitory DREADD. First, using electrophysiological recordings, we confirmed that hM4Di activation leads to peri-LCNPY cells silencing (Fig. S8A, B). In particular, bath application of C21 (2µM), resulted in reduced peri-LCNPY spontaneous firing, as recorded at current clamp mode. Then, in an independent cohort of NPY-cre mice, we bilaterally injected the inhibitory DREADD (AAV-hSyn-DIO-hM4D(Gi)-mCherry), or a control virus (AAV-hSyn-DIO-mCherry) in the LC. After a period allowing for virus expression (5 weeks), mice were food deprived (24-h), were administered C21 (2mg/kg, i.p.) and were then subjected to the NSF task, as described above (Fig. 4G). In hM4Di mice, C21 administration reduced food intake at the NSF arena, as compared to mCherry controls (NSF intake: mCherry, 3.33 g/kg; hM4Di, 1.32 g/kg), indicative of peri-LCNPY inhibition-triggered anxiogenesis (Fig. 4H). This effect on food intake was specific for anxiogenic contexts, as it did not occur when food was available at the home-cage (Home-cage intake: mCherry, 8.62 g/kg; hM4Di, 8.97 g/kg, Fig. S8C). Peri-LCNPY inhibition by C21 did not affect the latency to initiate food consumption in the NSF arena (mCherry, 324s; hM4Di, 431s), nor the time spent at its center (Duration Center: mCherry, 13.1 %; hM4Di, 13.0% of total exploration time, Fig. 4H, Fig. S8C). As we observed above for NSF, C21 did not alter general locomotion in hM4Di mice (mCherry, 10.7m; hM4Di, 10.8m) (Fig. S8C). Notably, peri-LCNPY inhibition did not affect performance on the EPM (Fig. S8D), indicating differential involvement of peri-LCNPY inhibition in these two distinct anxiety tests. Together, our data support bidirectional control of (certain) anxiety-like behaviors by peri-LCNPY neurons, highlighting a novel endogenous mechanism for the promotion of adaptive responses to challenging environments.
Discussion
Despite decades of research on the basic mechanisms underlying adaptive stress reactivity, our knowledge of the relevant neural circuitries, and molecular substrates remains incomplete. The LCNE system is the brain’s “first responder” to stress and primary coordinator of the global neural processes underlying flight-or-fight7,9,55. Therefore, we here reasoned that elucidating the endogenous systems that curb LC function, could provide us with a mechanistic understanding on how to promote adaptive responses to challenging environments. For this, we examined the endogenous mechanisms underlying NPY-mediated LC neuromodulation. Collectively, we here demonstrate that a previously uncharacterized population of NPY-expressing cells, situated in the pericoerulean region, provides direct, Y1R-mediated NPY tone onto LC noradrenergic neurons, tempering their activity. Under approach-avoidance conflicts, peri-LCNPY-mediated neuromodulation of the LC efficiently lowers anxiety levels, via Y1R-dependent mechanisms. Conversely, suppressing local peri-LCNPY neuronal activity results in anxiogenesis. Together, our findings highlight a role for peri-LCNPY neurons as key mediators of LC function and delineate a novel endogenous circuit for the regulation of (trait) anxiety.
We report that the large majority of peri-LCNPY neurons are situated medially to the LC nuclear core, within a region that is rich both in noradrenergic dendritic fibers and diverse axon terminals targeting them. Convergent signals within this area, mediated by fast neurotransmission or slow peptidergic input12,51,56–58, are proposed to coordinate LC engagement in arousal and under stress. Thus, peri-LCNPY neurons are ideally situated to fine-tune LCNE activity levels when required. Our neuroanatomical tracing data support this notion, as we provide evidence that peri-LCNPY neurons project to the LC dendritic zone, and form functional contacts within the region, and with LCNE neurons themselves. Notably, using viral anterograde tracing we did not detect peri-LCNPY neuronal efferents in other primary LC terminal fields. However, given that peri-LCNPY cells comprise a relatively small population, we cannot exclude that unbundled projection fibers remained undetected in our preparations. Furthermore, our retrograde tracing data revealed several novel brain regions that project to the LC, indicating that peri-LCNPY cells are not the sole source of NPY in the region. Future studies are required to unravel non-LC NPY contributions to the neuromodulation of the LC noradrenergic system.
In contrast to earlier studies in rats21,28,59, we here report that peri-LCNPY neurons constitute a distinct, predominantly non-noradrenergic neuronal population. Although species differences might contribute to the observed discrepancy, it is also plausible that methodological limitations previously overestimated NPY-NE colocalization in the LC proper. Using more advanced techniques, recent single-cell and single-nucleus RNA sequencing data corroborated lack of NPY-expressing noradrenergic neurons, both in the mouse31,60 and in the human61 LC. Notably, similar to our observations, NPY transcripts were detected outside the LC nuclear core31, further validating the existence of a previously undetected discrete pool of NPY-expressing neurons populating the mouse pericoerulean space.
We here show that NPY Y1R activation directly affects LCNE activity. Y1Rs are preferentially localized at post-synaptic sites30,62, ideally placing them to regulate LCNE intrinsic excitability. Notably, the effects we detect here resemble that of exogenous NPY application in the lateral amygdala46. In particular, in amygdalar projection neurons, pharmacologically applied NPY had a hyperpolarizing effect via Y1 (and not Y2) receptors, by activation of inward rectifying potassium channels46. Following peri-LCNPY chemogenetic activation, we did not detect changes in LCNE membrane resistance, calculated near resting membrane potential. However, we cannot exclude differences in potassium channel-regulated membrane resistance at more positive potentials, which we did not assess here, as in our experiment those coincided with action potential-driven active conductances. Likewise, we did not specifically address the contribution of GIRK-mediated currents on C21-induced hyperpolarization of LCNE cells, thus we cannot exclude their involvement in the observed effects.
We here demonstrate that the anxiety-relieving effects of local NPY release in the LC are mediated via Y1Rs, linking our electrophysiological observations with the behavioral outcomes of peri-LCNPY chemogenetic activation. Interestingly, our data are in conflict with an earlier study in rats25, where pharmacologically applied NPY within the LC vicinity resulted in reduced anxiety via Y2Rs. In particular, Kask and colleagues (1998) showed that infusion of NPY or an agonist with affinity for Y2/Y5 receptors in the pericoerulean space had an anxiolytic effect in rats performing the EPM task. On the contrary, a singular low dose of a Y1R/Y5R agonist did not produce this effect. These findings seem at odds with our observations that implicate Y1Rs in the regulation of anxiety-like behaviors following chemogenetically-evoked release by local NPY sources. There are obvious key differences between the two studies. First, exogenously applied NPY can induce widespread recruitment of Y2R-containing efferents outside the LC dendritic zone, with extra-LC contributions accounting for the observed behavioral effects. Instead, we here assess recruitment of local endogenous NPY sources, possibly relying on proximity between pre-and post-synaptic targets, as seen for NE63, resulting in targeted effects on LCNE intrinsic excitability and the ensuing Y1R-dependent anxiolysis.
Moreover, pharmacological dose chosen could contribute to the apparent disparity between the two studies. Indeed, we here report that the pharmacological effects of exogenously applied NPY on LCNE activity are dose-dependent. In fact, bath-applied NPY modulates LCNE neurons in opposite directions (hypo-vs. hyper-excitability), via distinct NPY receptors. Particularly, low NPY dose mimicked the effects of DREADD-induced activation of peri-LCNPY neurons, with both depending on postsynaptic Y1Rs. High NPY dose led to opposite effects, via a network mechanism that relied on Y2Rs and synaptic input onto LCNE cells. While we did not specifically test this, it is possible that presynaptic Y2Rs, expressed on peri-LCNPY cells, play an auto-regulatory role64, in which they moderate peri-LCNPY signaling, leading to disinhibition of LCNE neurons and increased LCNE firing frequency. Exposure to severe stress might require such a mechanism when rapid stress response is the preferable and adaptive choice, and Y2R-mediated signaling is shown to fulfil this role65–68. On the other hand, in mildly anxiogenic environments, prioritizing the regulation of the initial stress response by Y1Rs might preclude the development of maladaptive behavioral patterns. Indeed, in several brain regions other than the LC, Y1R activation results in anxiolysis17,19,69–71. Collectively, these results highlight the intricate outcomes of NPY-mediated neuromodulation of the LCNE system, where NPY tone can fine-tune LCNE activity towards both hypo-and hyper-excitation. Which physiological conditions prompt each requirement in vivo remains to be determined.
In the present study, we addressed the role of peri-LCNPY neurons in the regulation of basal anxiety levels. However, their involvement in modulating behavior after exposure to stress remains unclear. Stress elevates NPY mRNA expression in the LC72, which parallels our findings on stress-induced engagement of peri-LCNPY neurons, as seen in increased firing frequency and cFos-indicated neuronal activation. Moreover, stress enhances LC Y1R and Y2R mRNA levels73, further indicating recruitment of the local NPY system during adversity. In rats, signal intranasal administration of NPY limits the development of hyperarousal and anxiety-like behaviors seen after exposure to stress72. Notably, this is accompanied by attenuated stress-induced increases in LC TH expression, which could reflect curbing of LC function. It is possible that, in a similar manner, peri-LCNPY recruitment and local NPY release contribute to LC silencing and the promotion of adaptive responses under stress.
Combining in vivo chemogenetic manipulations with behavioral assessment, we here showed that modulation of peri-LCNPY neuronal activity exerts bidirectional control over anxiety-like manifestations. Particularly, peri-LCNPY stimulation increased exploration/approach in the EPM and NSF tasks, in agreement with peri-LCNPY-mediated silencing of LCNE cells, and presumably, reduced NE release in terminal fields, promoting anxiolysis. Likewise, we demonstrate that peri-LCNPY inhibition, which is expected to relieve an NPY brake on LCNE neurons, led to anxiogenesis at the NSF. Notably, we report the lack of effect of in vivo peri-LCNPY inhibition in the EPM, implying divergent requirements for peri-LCNPY function in each task conditions. Of note, in the NSF, but not the EPM, mice were subjected to prior food deprivation, which is shown to increase LC neuronal activity during food approach53. Increased LC engagement could set the stage for enhanced peri-LCNPY recruitment upon fasting. Thus, our NSF data might reflect an adaptive mechanism aimed to counteract increased LCNE tonic activity-induced avoidance/ aversion12,53, in favor of exploration of / foraging in new -and potentially unsafe environments-under conditions of limited food availability. In accordance with this notion, whereas peri-LCNPY activity levels influenced food-drive and consumption in the anxiogenic NSF task, it left consummatory behavior in the familiar (safe) context of the home-cage unaltered.
In the current study, we performed cell type-specific dissection of a novel circuitry mediating anxiety relief. In particular, we provide mechanistic data for the anxiolytic effects of a previously unidentified NPY-expressing neuronal population. Using combinatory approaches, we showcase anatomical, electrophysiological and behavioral evidence that peri-LCNPY neurons exert neuromodulatory control over the LCNE system, promoting its adaptive engagement in arousing environments, and the maintenance of low baseline anxiety levels. Collectively, these data expand our understanding of how endogenous peptidergic influences regulate the brain’s stress systems and highlight peri-LCNPY neurons as a possible new target for the modulation of anxiety-like behaviors.
Methods
Animals
In all experiments naïve adult male or female mice were used (20–35 g, ≥4 weeks). C57BL/6J (Charles River, France), NPY-Cre (Jax #027851) and Ai14 (Jax #007914) mice were bred in house after purchasing founders from the original breeding colonies. NPY-cre:Ai14 were bred in house. All mice were group-housed (2–5 per cage) unless otherwise specified. Mice were housed in a 12:12 light/dark cycle (lights on at 07:00 a.m.) at 22 ± 2 °C (60–65% humidity). Unless otherwise specified, animals had access to ad libitum water and lab chow. Experiments were approved by the Animal Ethics Committee of Utrecht University and the Dutch Central Authority for Scientific Procedures on Animals (CCD) and were conducted in agreement with the Dutch law (Wet op de Dierproeven, 2014) and European regulations (Guideline 86/609/EEC).
Stereotactic surgeries
Mice (≥ 5 weeks at the time of surgery) were anesthetized with ketamine (75 mg/kg i.p.; Narketan, Vetoquinol) and dexmedetomidine (1 mg/kg i.p.; dexdomitor, Vetoquinol). Lidocaine (0.1 ml; 10% in saline; B. Braun) was injected under the skull skin and eye ointment cream (CAF, Ceva Sante Animale B.V.) was applied. Animals were then fixed on a stereotactic frame (UNO B.V. model 68U801 or 68U025), where they kept on a heat pad (33 °C) during surgery. For cannula implantations, the skull surface was scratched with a scalpel and phosphoric acid (167-CE, ultra-Etch, ultradent, USA) was applied for 5 min to roughen the surface at the start of surgery. Viral infusions were done using a 31 G metal needle (Coopers Needleworks) attached to a 10 µl Hamilton syringe (model 801RN) via flexible tubing (PE10, 0.28 mm ID, 0.61 mm OD, Portex). The Hamilton syringe was controlled by an automated pump (UNO B.V., model 220). Injections were done bilaterally at 250 nl per side, at an injection rate of 100 nl/min. The injector was gradually retracted during the last minute of a 10 min long diffusion period. Next, the skin was sutured (V926H, 6/0, VICRYL, Ethicon) and animals were administered the dexmedetomidine antagonist atipamezole (50 mg/kg; Atipam, Dechra, s.c.), carprofen (5 mg/kg, Carporal, s.c.) and 1 ml of saline (s.c.). Mice were left to recover on a heat plate (36 °C) while being monitored and moved to the housing stables when fully awake. Carprofen (0.025 mg/L) was provided in the drinking water during the first post-operative week. Post-surgery, animals were single-housed before rejoining their cage-mates at three days post-op. Animals with cannulas were single-housed for the remainder of the experiment.
For ex vivo optogenetic or chemogenetic-assisted electrophysiology experiments and ELISA, NPY-cre mice were bilaterally injected with rAAV5-Syn-FLEX-CoChR-GFP (4.4*10^12 gc/ml; UNC Vector Core) or rAAV5-hSyn-DIO-hM3D(Gq)-mCherry (4.2*10^12 gc/ml; Addgene) in the Locus Coeruleus (LC, AP: -5.45 mm; ML: ± 1.59 mm; and DV: -3.96 mm from bregma) under a 10 degree angle. All animals were allowed to recover for a minimum of four weeks before brain collection for electrophysiological recordings and other biochemical assays.
For in vivo chemogenetic experiments, NPY-cre mice were bilaterally injected with rAAV5-hSyn-DIO-hM3D(Gq)-mCherry, AAV5-hSyn-DIO-hM4D(Gi)-mCherry or AAV5-hSyn-DIO-mCherry (all viruses: 4.2*10^12 gc/ml; Addgene) in the LC (AP: -5.45 mm; ML: ± 1.59 mm; DV: -3.96 mm or AP: -5.3 mm; ML: ± 1.95 mm; DV: 3.5 mm from bregma) under a 10 degree angle. All animals were allowed to recover for five weeks before participating in behavioral assays.
For cannula implantations, NPY-cre mice were bilaterally implanted with a guide cannula (4 mm; C315GMN/Spc; Bilaney) above the LC (AP: -5.35 mm; ML: ± 1.63 mm; and DV: -3.76 mm or AP: - 5.35 mm; ML: ± 1.77 mm; and DV: -3.86 mm from bregma) under a 10 degree angle. The cannulas were secured by adding a layer of adhesive luting cement (C&B Metabond; Parkell, Edgewood, NY, USA) around them. Viral infusions were performed immediately after cannula placement, by inserting the injector through the guide, and lowering it to DV -3.96 mm. After infusions, dummy internal injectors (4 mm; C315FD/Spc) were locked onto the guides to avoid blockage, and remained there until experiment completion. All animals were allowed to recover for five weeks before participating in behavioral assays.
Behavioral assays
All behavioral manipulations and tests were performed during the light phase, between 12-6pm. Only male mice were included in the behavioral assays described below. Animals were transported to the behavioral room at least 2 weeks before the start of the experiment, for acclimatization. Real-time data collection and offline analysis for EPM and NSF tasks were done with Ethovision video tracking (version XT 9; Noldus).
Foot-shock stress
Exposure to foot-shock stress was conducted in operant chambers (29.53 × 24.84 × 18.67 cm, Med Associates) modified for foot shock delivery, via the grid floor. The chambers were contained in a soundproofing box. Mice were placed in the chamber and given 5 min of habituation to the novel environment. A total of 20 foot-shocks (1 s duration, 0.3 mA intensity) were delivered with an interval of 60s for the next 20 min of the session. The last 5 min of the session (30min of total duration) were shock-free. Control (no-stress) mice were exposed to the operant chamber (novel environment) in absence of electrical foot-shocks. After the session, all mice were transferred back to their home-cage.
Open Field
The open field (OF) task was performed using a standard arena (round, diameter 80 cm). Light intensity in the center of the arena was 25 Lux. At the start of the session, mice were placed next to the walls of the arena and allowed to freely explore for 10 min. Behavior was scored for the following variables: time spent in the center or wall zone (% of total exploration time); frequency of visits to the center or wall zone; and total distance moved. The OF arena was cleaned with 70% ethanol solution in-between animals.
Elevated Plus Maze
The elevated plus maze (EPM) task was performed using a standard apparatus (arm length: 65 cm × 65 cm) elevated at 65 cm above ground, equipped with 15 cm high walls delimiting the enclosed arms. Light intensity in the center of the maze was 40 Lux. At the start of the session, mice were placed at one of the closed arms, facing the EPM center, and allowed to freely explore the maze for 10 min. Behavior was scored for the following variables in the first 5 minutes of the EPM: time spent in the open or closed arms (% of total exploration time); frequency of visits to the open or closed arms; total distance moved; and anxiety index36, calculated as: 1 - [(time in OA / (time in OA + time in CA)) + (entries to OA / (entries to OA + entries to CA)) / 2], where values closer to 1 delineate increased anxiety. EPM was cleaned with 70% ethanol solution in-between animals.
Novelty Suppressed Feeding
The novelty suppressed feeding (NSF) task was performed using the open field arena as described above. A metal disk containing standard lab chow (1 pellet) was firmly affixed at the center of the arena. The pellet itself was attached to the disk, so that the animals could not move it during the test. At the start of the session, mice were placed next to the walls of the arena and allowed to freely explore for 10 min. Behavior was scored for the following variables: time spent in the center or wall zone (% of total exploration time); frequency of visits to the center or wall zone; and total distance moved. In addition, latency to initiate feeding and food consumption in the arena, calculated as: food pellet weight at start - end of session / mouse weight (g/kg), were recorded. A different disk, and chow pellet were used per mouse, and the NSF arena was cleaned with 70% ethanol solution in-between animals.
In vivo chemogenetic experiments
All animals were habituated to restrain for intraperitoneal (i.p.) injections with at least 3x handling sessions that included “sham” injections before the start of the experiments. For intracranial infusions, mice were extensively handled for at least a week (1x day) for familiarization with infusion procedures (dummy removal, injector/ tubing plug-in), before the start of the experiments. For all experiments, only mice with confirmed viral expression within the LC and, when applicable, correct cannula placement after histological control were included in data analysis.
For peri-LCNPY stimulation experiments, two separate batches of NPY-cre mice were used, with 2 months interval between experiments. Both cohorts (Batch 1: mCherry, N=11; hM3D(Gq), N=11; Batch 2: mCherry, N=10; hM3D(Gq), N=10) displayed similar performance, thus data were pooled.
For intra-LC Y1 antagonism, two separate batches of NPY-cre mice were used, with 2.5 months interval between experiments. Both cohorts (Batch 1: mCherry + veh, N=4; hM3D(Gq) + veh, N=5; hM3D(Gq) + BIBO, N=5; Batch 2: mCherry + veh, N=6; hM3D(Gq) + veh, N=5; hM3D(Gq) + BIBO, N=3) displayed similar performance, thus data were pooled.
Elevated Plus Maze
For peri-LCNPY stimulation or inhibition experiments, all mice were administered C21 (2mg/kg, i.p.). One hour after injection, animals were exposed to a novel environment (MED apparatus, as described above) for 30min, before being transferred to a clean cage, where they remain single-housed for the rest of the experimental procedures. Thirty minutes later, mice were subjected to the EPM test, before returning to their home-cage.
For intra-LC Y1 antagonism experiments, internal guides (1 mm projection; C315IMN/Spc; Bilaney) attached to tubing (PE10, 0.28 mm ID, 0.61 mm OD, Portex) were bilaterally inserted in the cannulas. The tubing was attached to 10 µl Hamilton syringe (model 801RN), controlled by an automated pump (UNO B.V., model 220). While the animals were in their home cage, 200pmol BIBO-3304 or vehicle was infused in a volume of 200 nl (1% DMSO, 99% PBS) in each hemisphere, at a rate of 100 nl/min. The injector was left in place for an additional minute. Twenty minutes after intra-cannula infusions, animals were administered C21 (2mg/kg, i.p.). Thirty-five minutes after C21 injections, mice were subjected to the EPM task, as described above.
Novelty suppressed feeding
For peri-LCNPY stimulation or inhibition experiments, mice were subjected to the NSF task, 48 hours after participating in the EPM and following 24-h of food deprivation. All mice were administered C21 (2mg/kg, i.p.) and NSF took place one hour after injection. Immediately after the end of the session, mice were returned to their home-cage where food (chow pellet) intake was recorded for 5-min.
Patch-clamp electrophysiology
Animals were anesthetized with pentobarbital (Euthasol 20%, 0.1ml, i.p.) between 8.30 a.m. and 10 a.m. and transcardially perfused with ice-cold carbogenated (95% O2, 5% CO2) slicing solution containing (in mM): choline chloride 92; ascorbic acid 10; CaCl2 0.5; glucose 25; HEPES 20; KCl 2.5; N-Acetyl L Cysteine 3.1; NaHCO3 25; NaH2PO4 1.2; NMDG 29; MgCl2 7; sodium pyruvate 3; Thiourea 2. Brains were quickly extracted, placed on a vibratome (1200 VTs, Leica) and sliced in the coronal plane at 250 μm thickness, in ice-cold slicing solution. Slices recovery was performed for 30 min at 36 °C in carbogenated solution of identical composition. Thereafter, slices were maintained at room temperature in carbogenated incubation solution containing (in mM): ascorbic acid 3; CaCl2 2; glucose 25; HEPES 20; KCl 2.5; NaCl 92; NaHCO3 20; NaH2PO4 1.2; NMDG 29; MgCl2 2; sodium pyruvate 3 and Thiourea 2. During recordings, slices were immersed in artificial cerebrospinal fluid (ACSF) containing (in mM): CaCl2 2.5; glucose 11; HEPES 5; KCl 2.5; NaCl 124; NaHCO3 26; NaH2PO4 1; MgCl2 1.3 and were continuously superfused at a flow rate of 2 ml min-1 at 28-30 °C.
Peri-LCNPY or LCNE neurons were patch-clamped using borosilicate glass pipettes (2.7–4.5 MΩ; glass capillaries, GC150-10, Harvard apparatus, UK), under a TH4-200 Olympus microscope (Olympus, France). For voltage or current clamp recordings, signals were amplified and digitized using a HEKA EPC-10 patch-clamp amplifier (HEKA Elektronik GmbH). Data were acquired using PatchMaster v2×90.2 software. Access resistance was continuously monitored with a − 4 mV step delivered at 0.1 Hz. Experiments were discarded if the access resistance increased by more than 20% during the recording. All electrophysiological measures are recorded with a 10 s inter sweep interval (0.1 Hz).
Intrinsic excitability
Recordings were made in a potassium gluconate-based internal containing (in mM): Kglu 139; HEPES 10; EGTA 0.2; creatine phosphate 10; KCl 5; Na2ATP 4; Na3GTP 0.3; MgCl2 2. Upon break-in, cells were kept at -50mV for 10 min prior to the onset of current-clamp recordings. To assess passive membrane properties and cell firing patterns neurons were subjected to 17 consecutive current steps of 800 ms length, starting from −150 to +250 pA, with a 25 pA inter-step increment.
For stress effects on peri-LCNPY or LCNE excitability, LC-containing slices from NPY-cre:Ai14 mice were obtained 30 min after foot-shock stress or exposure to the novel environment (no-stress controls). LCNPY cells were identified by tdTomato expression under the microscope. LCNE neurons were identified based on location and morphological characteristics.
For ex vivo chemogenetic experiments, effects of bath-applied C21 were examined in LC-containing slices from NPY-cre mice at ≥5 weeks after DIO-hM3D(Gq) injection. Recordings were made in continuous perfusion of C21 (2 µM) or vehicle (0.1% DMSO). When applicable, slices were pretreated with synaptic blockers (in µM: CNQX 10, D-AP5 50, picrotoxin 100, CGP-54626 10), Y1R (BIBO-3304, 1 µM) or Y2R (BIIE-0246, 1 µM) antagonists for 10 min before being transferred in ACSF containing C21 or vehicle and the correspondent antagonist mix. All slices for chemogenetic experiments were controlled for virus expression at the end of recordings. Only data from slices with strong hM3D(Gq) expression within the peri-LC were taken along for analysis.
For NPY effects on LCNE excitability, LC-containing slices were obtained from wild-type C57BL/6 mice. Recordings were made in continuous perfusion of NPY (30 or 300 nM) or vehicle (0.1% DMSO). When applicable, slices were pretreated (10 min) with synaptic blockers as described above, before being transferred in ACSF containing NPY or vehicle.
For validation of DREADD constructs, effects of bath-applied C21 were examined in LC-containing slices from NPY-cre mice at ≥5 weeks after DIO-hM3D(Gq) or DIO-hM4D(Gi) injection. Peri-LCNPY cells were identified by mCherry expression under the microscope. Peri-LCNPY spontaneous activity was recorded before and after C21 (2 µM) bath application.
Peri-LCNPY to LCNE connectivity experiments
At ≥5 weeks after virus injection of FLEX-CoChR in the LC of NPY-cre mice, recordings were made in voltage clamp in a potassium gluconate-based internal solution containing (in mM): Kglu 139; HEPES 10; EGTA 0.2; creatine phosphate 10; KCl 5; Na2ATP 4; Na3GTP 0.3; MgCl2 2, at -50 mV for the detection of inward glutamatergic and outward GABAergic currents. Alternatively, recordings were performed with a cesium chloride-based internal solution containing (in mM): CsCl 139; HEPES 10; EGTA 0.2; creatine phosphate 10; NaCl 5; Na2ATP 4; Na3GTP 0.3; MgCl2 2; spermine 0.1, at -60 mV or +40 mV for detecting inward GABA-or outward NMDA-mediated currents, in correspondence. Responses to single optical pulse (470 nm, 1 ms, 1–2 mW) or trains of 5, 20 and 50 Hz delivered through the light path of the microscope powered by a light-emitting diode driver (LEDD1B; Thorlabs, Newton, NJ) were recorded. Connectivity was determined based on whether a neuron showed an opto-evoked synaptic response ≥ 5 pA, over an average of 10-20 sweeps. In a portion of recordings, internal solutions contained 3% biocytin (B4261, Sigma-Aldrich), for cell-filling and post-hoc identification. All slices for optogenetic experiments were controlled for virus expression at the end of recordings. Only data from slices with strong CoChR expression within the peri-LC were included in the connectivity analysis.
Histology & Immunolabelling
All mice were anesthetized with pentobarbital (Euthasol, 0.1 ml, i.p.) and transcardially perfused with PBS followed by freshly-made ice-cold 4%PFA. Brains were extracted and post-fixated in 4% PFA overnight, before being transferred to an anti-freeze solution (30% sucrose) until they sank. Thereafter sections measuring 35 µm (colocalization studies) or 50 µm (histological control of virus targeting) were collected using a cryostat (Leica CM 1950) at -12°C and stored in PBS & 0.01% NaN3 until immunolabelling. Brains from animals participating in cannula experiments were post-fixated for 48-h and transferred to 10% sucrose before being embedded to a 10% sucrose/ 10% gelatin solution. Next, embedded brains were re-fixated in 10% sucrose/ 4% PFA solution overnight and stored in 30% sucrose until slicing. Sections (50 µm) were collected with a vibrotome (Leica VT 1000S) at room temperature (RT). In most cases, every 5th consecutive section was collected for further processing, to ensure LC representation in the entire rostrocaudal axis. For immunohistochemical stainings, sections were washed in 1× PBS, and blocked (1-h, RT) in a solution containing 5% normal goat or donkey serum, 2.5% bovine serum albumin and 0.2% Triton X-100. Primary antibodies against NPY (Novus Biologicals, NBP1-46535), TH (LNC1, Millipore, MAB318), GABA (Sigma-Aldrich, A2052), and cFos (9F6, Cell Signaling Technology, 2250S) were incubated overnight at 4°C. Secondary antibodies (Alexa Fluor 488, 568 or 647, Invitrogen) were incubated for 2-h (RT). Slices were mounted in 0.2% gelatin and cover-slipped with DABCO antifading medium (Merck, 10981).
For NPY mapping studies, sections were imaged at 10x magnification using a confocal microscope (LSM 880, Zeiss). Tiling was performed to include the mediolateral space to LC, up to 1 mm from the LC proper. Images were processed with FIJI74 and an in-house macro was used for detection of tdT+ and TH-expressing cells. Location of NPY-expressing neurons was determined based on X (mediolateral) and Y (dorsoventral) coordinates as calculated against an ROI representing LC center of mass per image. Frequency distributions for NPY location in ML and DV axes were calculated for three different rostrocaudal ranges, namely-5.80mm to -5.60mm, -5.60mm to -5.40mm and -5.40mm to -5.25mm, and averaged over the corresponding images.
For co-localization studies, sections were imaged by confocal microscope at 20x or 40x magnification and z-stacks (≥ 8 images) were processed with FIJI. An in-house macro was used for detection of tdT+, TH+, and cFos+ cells, which overlaid detected ROIs in one channel over the second. For quantification of tdT+/cFos+ double-expressing cells the following inclusion criteria were used: 1) cFos channel intensity ≥50% of a positive, cFos-expressing “example” cell and 2) cFos channel intensity ≥ 150% of background.
RNA-scope in situ hybridization assay and analysis
In situ hybridization (ISH) was performed as per manufacturer’s instructions (ACDBio, RNAscope Multiplex Fluorescent Reagent kit v2, 317621). Briefly, animals were transcardially perfused with sterile 4% PFA and post-fixated ovenight at 4 °C. Brains were transferred to 10% sucrose until they sank. This step was repeated with 20 and 30% sucrose. Brains were then embedded in optimal cutting temperature media (Tissue-Tek, VWR, The Netherlands) and placed in the cryostat at −20 °C for 1 h to equilibrate. Sections measuring 10 μm containing the LC were then mounted on SuperFrost Plus slides (VWR, The Netherlands) and allowed to dry at −20 °C for 2 h. For ISH, sections were pre-treated with hydrogen peroxide for 10 min before target retrieval at 99 °C for 5 min and treatment with Protease III for 30 min at 40 °C. Hybridization to probes against NPY (313321), Y1R (427021), Y2R (315951) or TH (317621) was carried out at 40 °C for 2 h. HRP signals against each channel (C1–C3) were then sequentially amplified and developed using TSA Vivid fluorophores (520, 570 and 650) at a dilution of 1:1500 or 1:3000. Positive-(3-plex PN 320881), and negative-control probes (3-plex PN 320871) were included in each experiment to assess sample RNA quality and optimal permeabilization conditions. In some cases, immunolabeling was combined with ISH. For this, sections were blocked with 0.1% BSA in TBS (30min, RT) before incubation of primary antibody against TH (1.5-h, RT). Sequential secondary HRP (30 min, RT) and TSA (10 min, RT) incubations were performed next. Sections were counterstained with DAPI for 30 s, coverslipped with ProLong Gold Antifade Mountant and allowed to dry overnight at room temperature. Confocal mages (40x magnification) were processed with FIJI and an in-house macro for detection of TH-or NPY-expressing cells.
Data analysis, statistics and reproducibility
Data were analyzed with Noldus (Ethovision V9.0), GraphPad Prism (9.5.1), Igor Pro-8 (Wavemetrics, USA) and FIJI. Sample size was predetermined on the basis of published studies, experimental pilots and in-house expertise. Animals were randomly assigned to experimental groups. Compiled data are always reported and represented as mean +SEM, with single data points plotted (single cell for electrophysiology and single animal for behavioral experiments). Animals or data points were not excluded from analyses unless noted. When applicable, statistical comparisons were conducted using unpaired t-tests, and 1-way or 2-way repeated measure ANOVAs. When applicable post hoc comparisons were performed in case of significant main (interaction) effects. Normality distribution was confirmed with the Kolmogorov-Smirnov test and in case of violation non-parametric Mann-Whitney test was performed. Two-tailed testing was performed with significance level α set at 0.05.
Authors Contributions
D.R., F.J.M., study conceptualization, project administration, supervision, manuscript writing, review and editing, funding acquisition. D.R., data acquisition, analysis and visualization. K.R data acquisition and analysis. I.G.W-D. technical assistance.
Conflicts of Interest
The authors declare no competing interests.
Acknowledgements
We thank the Meye and Adan labs for their support during this project. We thank Barbara Sakic and Nicky van Kronenburg for technical support. We thank Manuel Mameli, Salvatore Lecca and Stephane Ciocchi for critical review of the manuscript. This work was supported by the Dutch Research Council with NWO Veni (016.Veni.192.188) and XS (OCENW.XS21.3.076) grants to D.R.; by the European Union’s Horizon 2020 research and innovation program ERC Starting grant (804089; ReCoDE) to F.J.M.; and by the Dutch Research council (NWO) via an ENW-VIDI (VI.Vidi.203.102) grant to F.J.M.