Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo

Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo View ORCID Profile Ahmed A.A. Aly , View ORCID Profile Hongbo Jia , View ORCID Profile Michael R. Kreutz , View ORCID Profile Anna Karpova doi: https://doi.org/10.1101/2025.09.23.677999 Ahmed A.A. Aly 1 RG Neuroplasticity, Leibniz Institute for Neurobiology , Magdeburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ahmed A.A. Aly Hongbo Jia 2 Combinatorial NeuroImaging Core Facility, Leibniz Institute for Neurobiology , Magdeburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hongbo Jia Michael R. Kreutz 1 RG Neuroplasticity, Leibniz Institute for Neurobiology , Magdeburg, Germany 3 Leibniz Group ‘Dendritic Organelles and Synaptic Function’, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf , Hamburg, Germany 4 German Center for Neurodegenerative Diseases (DZNE) , Magdeburg, Germany 5 Center for Behavioral Brain Sciences, Otto von Guericke University , Magdeburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael R. Kreutz Anna Karpova 1 RG Neuroplasticity, Leibniz Institute for Neurobiology , Magdeburg, Germany 5 Center for Behavioral Brain Sciences, Otto von Guericke University , Magdeburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna Karpova For correspondence: anna.karpova{at}lin-magdeburg.de Abstract Full Text Info/History Metrics Preview PDF SUMMARY The postmitotic nature, exceptional longevity, and elaborate cytoarchitecture of neurons exert extraordinary demands on proteostasis and autophagy regulation. Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. Previous work suggests that neuronal amphisomes also serve as signalling platforms, though their physiological relevance in vivo remains largely unexplored. Here, we demonstrate dynamic trafficking of amphisomes within the long-range, highly branched axons of locus coeruleus norepinephrine ( LC-NE ) neurons. Using in vivo photoconversion, we show that amphisomes originating in distal axons can traverse the entire axonal length to reach the soma. Two-photon imaging of LC-NE projections to the prefrontal cortex revealed that velocity and directionality of trafficking are tightly regulated by LC activity states, behavioural context, and autocrine norepinephrine signalling. Activation of Gi-coupled receptor signalling unifies directionality of transport, enhances somatic cargo delivery, whereas prolonged distal immobilization correlates with increased norepinephrine release, consistent with a signalling function. Together, these findings establish LC amphisomes as dual-function organelles that integrate degradative transport with activity-dependent signalling in vivo . Highlights - Amphisomes are present within the distal axons of locus coeruleus norepinephrine neurons - Distally generated amphisomes traverse the entire axonal length to reach the soma in vivo - Velocity and directionality of trafficking are regulated by LC activity, behavioural context, and autocrine norepinephrine signalling - Prolonged distal amphisome pausing correlates with increased norepinephrine release INTRODUCTION In cultured neurons, autophagic vesicles form at distal axons and engage diverse mechanisms to reach somatic lysosomes ( Lee et al., 2011 ; Maday et al., 2012 ; Maday & Holzbaur, 2014 , 2016 ; Cheng et al., 2015 ; Jordens et al., 2001 ; Wijdeven et al., 2016 ; Fu et al., 2014 ; Wong & Holzbaur, 2014b; Andres-Alonso et al., 2019 ; Cai et al., 2010 ; Cason et al., 2021 ; Cason & Holzbaur, 2023; Kulkarni & Maday, 2018). Neuronal amphisomes are hybrid organelles formed by the fusion of autophagosomes with late endosomes ( Cheng et al., 2015 ) and are endowed with specialized signalling properties (Kononenko et al., 2017; Andres-Alonso et al., 2019 ). In previous work, we found that trafficking and signalling of TrkB-containing amphisomes in pyramidal neurons is tightly controlled by the RapGAP SIPA1L2 ( Andres-Alonso et al., 2019 ). Our data show that dynein-snapin complexes mediate amphisome retrograde trafficking and that LC3 binding to SIPA1L2 enhances its RapGAP activity, thereby attenuating TrkB-induced Rap signalling upstream of ERK activation, ultimately leading to a slowdown of organelle transport ( Figure 1A ) . At highly active presynaptic sites, PKA phosphorylation of snapin and SIPA1L2 immobilizes amphisomes at synaptic boutons, terminates RapGAP activity, and thereby allows TrkB/Rap1 signalling that will facilitate glutamate release ( Figure 1A ) . While this study provided evidence for signalling amphisomes in hippocampal pyramidal neurons, key questions remained whether such mechanisms operate in vivo in a physiologically relevant context. Download figure Open in new tab Figure 1. SIPA1L2/LC3B-positive amphisomes localize to LC axons projecting to the PFC and exhibit three distinct motility patterns (A) . Neuronal amphisomes are hybrid organelles on the autophagy pathway that combine degradative and signalling functions as they travel along long-distance projecting axons. The Rap GTPase–activating protein SIPA1L2 binds LC3/ATG8 and Snapin, thereby linking amphisomes to the dynein motor. Through this interaction, SIPA1L2 governs the speed of retrograde trafficking and modulates local signalling at synaptic boutons ( Andres-Alonso et al., 2019 , Karpova et al., 2025 ). (B) Confocal image showing a virally expressed GFP-labelled LC axon with endogenous SIPA1L2 and LC3B immunoreactivity within an axonal varicosity resembling a synaptic bouton, and (C) the corresponding intensity profile. (D) . Schematic of in vivo two-photon imaging of mNeonGreen-LC3B-labeled amphisomes in LC axons projecting to the PFC-M1 . (E) . Representative in vivo time-lapse frames acquired through a transcranial window over the PFC-M1 . Dashed circles highlight amphisomes moving along LC axons, whereas asterisks indicate mNeonGreen-LC3B puncta that remained immobile during the image acquisition. (F) . Linear correlation between the average and maximum velocities of LC3B-positive amphisomes, reflecting the distance each vesicle travels over time. (G) . Correlation between the total track duration and the displacement ratio of LC3B-positive amphisomes. (H) . Correlation between displacement ratio and average velocity, illustrating the heterogeneity of amphisome behaviour during spontaneous LC activity. (I) . PCA of three key features identifies three principal clusters of LC3B-positive amphisomes, each exhibiting distinct trafficking patterns: slow unidirectional, slow bidirectional, and fast bidirectional. Despite their low number - less than ∼3,000 neurons in rodents ( Sara, 2009 ) and fewer than 50,000 in humans ( Mouton et al., 1994 ) - locus coeruleus norepinephrine neurons exert widespread modulatory influence on arousal, attention, affect, nociception, stress reactivity, and circadian cycles, thereby shaping behaviour and multiple forms of learning and memory (Samuels et al., 2008; Sara, 2009 ; Wagatsuma et al., 2018 ; Suárez-Pereira et al., 2022; reviewed in Berridge & Waterhouse, 2003 ; Sara & Bouret, 2012 ; Chandler et al., 2019 ; Ross & Van Bockstaele, 2021 ). Their activity is tightly coupled to sleep-wake dynamics, with oscillatory fluctuations in norepinephrine release and switches between tonic and burst firing modulating sleep microarchitecture and memory consolidation ( Kjaerby et al., 2022 ). Morphologically, LC-NE neurons exhibit relatively small somata and exceptionally branched axons that modulate neuronal activity in several brain regions via “local volume transmission” ( McKinney et al., 2023 ; Chandler et al., 2019 ; Fuxe et al., 2015 ; Toyoda et al., 2022 ). The extreme length and fine calibre ( Descarries et al., 1977 ) of these distal processes pose a significant challenge for autophagy-mediated proteostasis. These challenging conditions and the accessibility of LC-NE fibres for in vivo imaging led us to choose norepinephrine axons projecting to the prefrontal cortex ( PFC) as a model system to study the regulation of amphisome trafficking and their signalling capacity in the intact mouse brain. RESULTS SIPA1L2/LC3-positive amphisomes are present in LC-NE axons projecting to the PFC We have previously shown in hippocampal neurons that amphisomes have dual degradative and signalling functions and that the RapGAP SIPA1L2 regulates both trafficking and synaptic positioning to facilitate synaptic vesicle release ( Andres-Alonso et al., 2019 ; Karpova et al, 2025 / Figure 1A ) . To investigate whether amphisomes exist in LC-NE axons, we unilaterally injected a Cre-inducible adeno-associated virus (AAV) expressing GFP as a cytoplasmic fill into the LC of dopamine beta hydroxylase Cre (Dbh-Cre: B6.Cg-Dbh^tm3.2(cre)Pjen/J; Tillage et al., 2020 ) mice, expressing Cre recombinase from the endogenous Dbh locus, thereby specifically labelling LC - NE neurons and enabling visualization of their distal projections ( Figure S1A-B ) . Consistent with previous work, subsequent immunochistochemical analysis revealed that GFP-labelled LC-NE neurons were indeed positive for LC3B and SIPA1L2 ( Figure S1C ) , with both proteins co-localized in GFP-labelled LC-NE axonal varicosities resembling synaptic boutons ( Figure 1B, C ) . Furthermore, LC3B puncta in LC-NE neurons co-localized with snapin present at synaptic boutons ( Figure S1D, E ) . Finally, the hybrid identity of these vesicles as amphisomes was corroborated by immunodetection of the late endosome marker Rab7 in close proximity to LC3/SIPA1L2 clusters ( Figure S1F, G ) . To assess mobility of LC3B-positive amphisomes in distal LC-NE axons in vivo , we established a Cre-dependent, LC -specific labelling strategy ( Figure S1H, I ) via viral expression of mNeonGreen-LC3 (AAV-EF1α-DIO-mNeonGreen-LC3B) and performed two-photon imaging through a transcranial window over the PFC-M1 ( Figure 1D ) , a canonical LC-NE target ( Sara, 2009 ; Chandler et al., 2014 ; Totah et al., 2018 ). Time-lapse imaging revealed that, during spontaneous (basal) LC activity, LC3B-positive amphisomes exhibited clear motility along axons ( Figure 1D, E ) , likely mediated by SIPA1L2 and its interaction with the dynein adaptor protein snapin ( Andres-Alonso et al., 2019 ). Subsequent quantitative analysis of LC3B-positive amphisome trajectories in vivo , based on track velocity and mean displacement ratio during basal LC-NE neuronal activity, revealed pronounced heterogeneity in both speed and motility patterns ( Figure 1F-H ) . Some amphisomes moved unidirectionally at ∼0.1 µm/s, whereas others displayed bidirectional trajectories with fluctuating velocities ( Figure 1F-H , S1J ) . To determine whether these differences define distinct vesicle subpopulations, we performed principal component analysis (PCA) using average velocity, maximum velocity, and displacement ratio. PCA identified three primary clusters of LC3B-positive amphisomes: slow unidirectional, slow bidirectional, and fast bidirectional, each with distinct trafficking properties ( Figure 1I , S1J ) . These results indicate that distal LC axons projecting to the PFC-M1 harbour mobile LC3B-positive amphisomes with very different motility patterns and trafficking velocities during basal LC-NE neuronal activity. Amphisomes originating in distal axons traverse the entire distance to reach the LC soma in vivo Distal LC-NE axons might lack the capacity for local cargo degradation, thereby potentially requiring retrograde transport of autophagic cargo to the soma for disposal. To test this hypothesis in vivo , we first examined the abundance of lysosomal machinery within distal LC-NE axons projecting to the PFC . LC-NE axons were first labelled with GFP ( Figure S1A ) , followed by immunohistochemical detection of the lysosomal marker LAMP2a in the PFC and subsequent three-dimensional GFP isosurface masking ( Figure 2A, B ; Figure S2A ) . Quantitative image analysis revealed that LAMP2a puncta were only sporadically detected within distal LC axons, with their frequency being negligible compared to the surrounding cellular environment ( Figure 2A-C ; Figure S2A ) . These findings suggest that distal LC-NE axons are largely devoid of lysosomal machinery and that under such conditions, amphisomes might be rather stable organelles during retrograde transport. We, therefore, next determined whether amphisomes originating in distal axons can traverse the full distance from the PFC to LC-NE somata in vivo and employed a photoconversion-based amphisome labelling strategy. Specifically, LC-NE amphisomes in Dbh-CRE mice were labelled with the photoconvertible fluorophore mEOS4 fused to the N-terminus of LC3B (AAV-EF1α-DIO-mEOS4-LC3B, Figure 2D ), and an optical fiber was implanted in the mPFC to enable local photoconversion ( Figure 2D ; Figure S2B, C ) , as previously described (Yizhar et al., 2011). This approach allowed selective photoconversion of mEOS4-LC3B-labeled amphisomes in distal LC axons, followed by subsequent detection of photoconverted puncta in the LC-NE neurons after brain sectioning. Both qualitative and quantitative analyses revealed significantly elevated levels of photoconverted “red” mEOS4 fluorescence in LC-NE neurons of animals subjected to UV light stimulation in the PFC , whereas non-stimulated controls displayed negligible red fluorescence ( Figure 2E ; Figure S2B-D ) . mEOS4-LC3B-puncta were indeed present at axonal varicosities ( Figure S2E ) and, finally, multiple photoconverted puncta were detected in TH-positive neurites adjacent to LC-NE somata after photoconversion, providing compelling evidence that distal amphisomes can indeed reach neuronal somata within a 4-6-hour timeframe in vivo ( Figure 2F ). To examine whether LC-NE neuronal activity influences this process, we exposed animals to a novel environment during photoconversion, a behavioural paradigm known to reliably enhance LC activity and norepinephrine release (Yizhar et al., 2011; Llorca-Torralba et al., 2019 ). Notably, exposure to the open arena significantly reduced the number of mEOS4-LC3B-positive puncta detected in LC-NE somata, suggesting that enhanced neuronal activity might modulate amphisome dynamics by reducing their somatic delivery ( Figure 2G-I ) . Finally, the colocalization of somatic mEOS4-LC3B puncta with SIPA1L2 confirmed the identity of mEOS4-LC3B-expressing particles as amphisomes ( Figure 2J, K ) . Download figure Open in new tab Figure 2. LAMP2a-positive lysosomes are sparsely distributed in LC axons projecting to the PFC , and amphisomes originating in distal axons traverse long distances to the LC soma (A) . Representative confocal maximum-intensity projection of GFP-labelled LC axons in the PFC co-immunolabeled with anti-TH and anti-LAMP2a antibodies. (B) . Three-dimensional reconstructions generated with Imaris ( Bitplane ) software. Left: 3D isosurface rendering of GFP-labelled LC axonal projections, illustrating the dense LC network in the PFC . Middle: Colocalization of GFP-labelled LC axons (cyan) with LAMP2a immunoreactivity (yellow). Right: LAMP2a immunoreactivity masked by GFP-defined ROIs (cyan). Box size: 5 µm. (C) . Quantification of LAMP2a-positive puncta within GFP-labelled LC axons compared with the surrounding PFC neuropil. Data are presented as mean ± SEM; ***p < 0.001, Nested two-tailed t-test. (D) . Schematic of the experimental strategy for in vivo photoconversion. (E) . Confocal tile-scan depicting photoconverted mEOS4-LC3B fluorescence in LC neurons following distal photoconversion in the PFC . Anti-TH immunostaining labels LC-NE neurons. (F). Representative confocal image showing photoconverted mEOS4-LC3B puncta in the vicinity of LC somata. (G) . Video snapshots acquired during photoconversion under different behavioural conditions: home cage (familiar environment) or novel open-field arena. (H) . Representative confocal images showing photoconverted mEOS4-LC3B (excitation 568 nm) puncta and non-photoconverted soluble mEOS4-LC3B (excitation 488) within LC somata. (I) . Quantification of somatic photoconverted mEOS4-LC3B puncta across behavioural conditions. N represents the average number of somatic “red” puncta obtained from 6 animals: home cage – 69 neurons from 6 animals; novel arena – 97 neurons from 6 animals. *p < 0.05, Unpaired t-test. (J) . Confocal images of LC neurons expressing mEOS4-LC3B co-labelled with antibodies against SIPA1L2. (K) . Line-intensity profile demonstrating colocalization of somatic mEOS4-LC3B and SIPA1L2. Chemogenetic inhibition via Gi signalling in LC-NE neurons increases amphisome speed and unifies their directionality α2-adrenergic receptors (α2-ARs), which couple to Gi and inhibit cAMP-PKA signalling ( Jewell-Motz et al., 1998 ; Brown et al., 2022), mediate the auto-suppression of NE release and contribute to the functional fatigue of LC-NE neurons ( Silverman et al., 2025 ). Our previous work and work from others indicate that immobilization of amphisomes at synaptic boutons critically depends on cAMP-PKA signalling ( Chheda et al., 2001 ; Di Giovanni & Sheng, 2015 ; Andres-Alonso et al., 2019 ). We therefore hypothesized that activation of Gi signalling in LC-NE neurons may regulate amphisome trafficking in distal LC-NE axons by reducing their immobilization and accelerating retrograde transport ( Figure 3A ) . To test this hypothesis, we combined in vivo two-photon imaging of Cre-inducible LC3B-labeled amphisomes through a cranial window ( Figure 1D, E ) with chemogenetic modulation of LC-NE activity using the inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD), an engineered variant of the muscarinic acetylcholine receptor hM4D (AAV-hSyn-DIO-hM4D(Gi)-mCherry) that signals via Gi ( McCall et al., 2015 ; Wagatsuma et al., 2018 ; Figure 3A , S3A ). Intriguingly, systemic administration of the selective DREADD agonist JHU37160 (J60; Bonaventura et al., 2019 ) significantly increased the average speed of amphisomes ( Figure 3B-D ) . We next examined whether Gi signalling activation via DREADDs alters amphisome directionality by preventing PKA-dependent immobilization ( Andres-Alonso et al., 2019 ). Analysis of mobile amphisomes in LC-NE axons projecting to the PFC prior to JHU37160 administration revealed that spontaneously active LC-NE axons contained comparable proportions of unidirectional and bidirectional vesicles ( Figure 3E, G ) . Strikingly, neuronal Gi activation reduced the fraction of bidirectional amphisomes to ∼34%, resulting in a pronounced shift toward unidirectional trafficking ( Figure 3F, G ) . Consistent with prior evidence that LC3B-labeled amphisomes segregate into distinct trafficking subpopulations during basal LC-NE activity ( Figure 1I ) , subsequent PCA revealed major shifts in clustering patterns after JHU37160 treatment ( Figure 3H, I ) . The three principal clusters observed under baseline conditions, slow bidirectional, fast bidirectional, and slow unidirectional, were replaced by new populations characterized as slow unidirectional, fast unidirectional, and extra-fast unidirectional vesicles ( Figure 3I ) . Collectively, these findings demonstrate that Gi signalling, through inhibition of the cAMP/PKA pathway, diminishes amphisome immobilization and bidirectional switching, thereby enforcing unidirectional trafficking and accelerating transport along distal LC-NE axons ( Figure 3J ) . Download figure Open in new tab Figure 3. Chemogenetic activation of Gi signalling enhances amphisome speed and biases trafficking direction in LC-NE neurons (A) . Schematic of the experimental timeline for in vivo assessment of amphisome trafficking speed and motility patterns during chemogenetic LC-NE silencing. (B) . Representative in vivo two-photon time-lapse frames acquired before and after ip administration of JHU37160. Dashed circles highlight automatically detected and tracked mNeonGreen-LC3B-positive amphisomes using the TrackMate plugin with manual annotation in ImageJ. The drawing illustrates the distance travelled by a representative amphisome before and after JHU37160 administration. (C) . Averaged amphisome speed across multiple LC-NE axons before and after Gi activation. Data represent mean ± SEM; ***p < 0.001, unpaired t-test. (D). Quantification of average amphisome speed within LC-NE axons before and after JHU37160 administration. *p < 0.05, paired t-test. (E, F). Cluster analysis of individual amphisome trajectories before (E) and after (F) JHU37160 treatment. Each point represents a single trajectory; the grey dashed line indicates mean trafficking velocity, and the yellow-shaded area denotes the standard deviation. The distribution of mean trafficking speed was broadened following Gi activation (F) . Magenta circles represent unidirectional amphisomes (displacement ratio ≥ 0.8); grey circles represent bidirectional vesicles. (G). Pie chart quantifying the proportion of amphisomes exhibiting unidirectional vs. bidirectional motility before and after JHU37160 treatment. Gi activation decreased the proportion of bidirectional amphisomes by ∼34% and increased unidirectional trafficking (n = number of amphisomes analysed before and after treatment, from 5 animals per group). (H, I) PCA plot visualization of trafficking velocities and motility patterns before and after J60 administration. (H) Three distinct clusters were observed: slow unidirectional (yellow), slow bidirectional (blue), and fast bidirectional (cyan). The vesicle pool acquired prior to J60 administration was merged with the vesicle pool recorded during spontaneous LC activity for a more comprehensive representation of clusters and is included in Fig. 1I . (I) Following chemogenetic activation of Gi signalling in LC-NE neurons with J60 administration, the three primary clusters were replaced by newly emerged clusters characterized as slow unidirectional, fast unidirectional, and extra-fast unidirectional. Ellipses represent the spatial distribution of data points for each cluster, summarizing within-cluster variation. Dashed lines indicate PCA axes representing feature variance. In (I) , newly emerged patterns are overlaid with pre-silencing cluster areas for comparison. (J) Schematic representation illustrating how Gi signalling activation, via inhibition of the cAMP/PKA pathway, reduces amphisome immobilization and bidirectional switching, thereby enforcing unidirectional trafficking and accelerating transport along distal LC-NE axons. Stop-over of amphisomes in LC-NE axons correlates with enhanced NE release in vivo To assess the correlation between amphisome motility patterns and spontaneous NE release, we utilized the recently developed genetically encoded fluorescent NE sensor, GRABNE2h, for rapid, specific in vivo detection of norepinephrine ( Feng et al., 2019 ; Feng et al., 2024). GRABNE2h, which emits within the EGFP-comparable spectral range, enabled simultaneous monitoring of autocrine NE dynamics in LC-NE axons and amphisome trafficking labelled with mRuby3.LC3B, using dual-colour, two-photon time-lapse imaging through a transcranial window ( Figure 4A, B ; Figure S4A-D ) . Spatiotemporal analysis showed that unidirectional amphisome mobilization, following the activation of Gi signalling ( Figure 3 ) , in LC-NE axons projecting to the M1-PFC coincided with decreased GRABNE2h intensity ∼3 s prior to the event (valley values, Figure S4E-G ) . Peak values before and after mobilization were unchanged, indicating no NE increase upon amphisome mobilization ( Figure S4H ) . Some amphisomes switched directionality during spontaneous LC-NE activity ( Figure 1I , Figure S1J ) , raising the question of whether changes in NE release might influence the switch in amphisome trafficking. While valley values of GRABNE2h intensity remained unchanged, peak values of GRABNE2h intensity increased significantly following the “trigger”, suggesting an elevation in NE release associated with directional switching ( Figure S4L ) . We next asked whether prolonged amphisome stationary pauses, following active motility and disrupting fast trafficking, followed by Gi-activated signalling, might enhance neurotransmitter release comparable to the previously reported effect on glutamate release in hippocampal primary neurons ( Andres-Alonso et al., 2019 ). Peak values of GRABNE2h fluorescence increased significantly within a two-second window around the “trigger” event (–1 to +1 s) relative to the preceding two seconds, indicating that NE release was elevated before and during the amphisome stalling ( Figure 4F, G ) . Thus, our results indicate that amphisome stalling in vivo correlates with a prolonged increase in spontaneous GRABNE2h intensity, as indicated by the peak values of ΔF/F₀%, likely reflecting prolonged NE release from LC boutons. Download figure Open in new tab Figure 4. Enhanced NE release correlates with amphisome stop-overs within distal LC-NE axons (A). AAV-hSyn-GRABNE2h and AAV-EF1α-DIO-mRuby3-LC3B were injected into the LC, followed by transcranial window implantation over the PFC. (B-C). Representative two-photon images showing GRABNE2h expression and signal fluctuations in distal LC-NE axons within the PFC . (D). Schematic representation of the trigger selection for identifying stationary pauses of amphisomes. (E) . Averaged GRABNE2h signal (ΔF/F₀%) aligned to the trigger, showing elevated NE release within a 2-second window around the stationary pauses. (F, G) . Statistical analysis of GRABNE2h peak and valley values of ΔF/F₀%. n.s – non-significant, *p < 0.05, paired t-test. DISCUSSION LC-NE neurons are among the most metabolically burdened cells of the brain and are highly vulnerable to degeneration in Alzheimer’s and Parkinson’s disease. Single-nucleus RNA sequencing of the human LC shows strong enrichment of the autophagy marker MAP1LC3B, highlighting a pronounced reliance on autophagic processes ( Weber et al., 2023 ). Multiple studies highlight the autophagy pathway as a key mechanism for clearing polymerized catecholamine derivatives such as neuromelanin, an early biomarker of Parkinson’s disease (Ferrucci et al., 2013; Zucca et al., 2018 ; Yamaguchi et al., 2018 ; reviewed in Lu et al., 2020 ; Rubinsztein & Nixon, 2024). Beyond degradation, emerging evidence shows that neuronal autophagy also serves as a signalling hub, exemplified by the existence of amphisomes that can retain signalling function during retrograde transport in the absence of fusion with lysosomes ( Cheng et al., 2015 ; Kononenko et al., 2017; Andres-Alonso et al., 2019 ). In this study, we identify determinants of autophagic vesicle trafficking in vivo and demonstrate that SIPA1L2/LC3B/Rab7/Snapin-positive amphisomes are present in LC-NE axons, where their motility and velocity are tightly regulated by animal behaviour, neuronal activity, and autocrine norepinephrine signalling. Selective activation of Gi-coupled high-affinity α2-adrenergic receptors signalling in LC-NE neurons is associated with distinct physiological states of the LC , including specific phases of sleep ( Hansen & Manahan-Vaughan, 2015 ; Nguyen & Connor, 2019; Lim et al., 2010 ; Lemon et al., 2009 ; Maity et al., 2015 ). Intriguingly, chemogenetic activation of Gi-coupled signalling via hM4D-DREADD expression and JHU37160 administration shifted amphisome trafficking clusters from slow bidirectional, fast bidirectional, and slow unidirectional to slow unidirectional, fast unidirectional, and extra-fast unidirectional. This chemogenetic manipulation also increased the proportion of unidirectional amphisomes from 52.5% to 86.7% and elevated the overall average velocity by over 50%. Fast-moving and retrogradely transported amphisomes are likely to reach neuronal somata efficiently, promoting the delivery of cargo for degradation. These findings are consistent with previous observations in acute optic nerve preparations, where two-photon imaging under deep anaesthesia revealed that the majority (∼85%) of LC3-positive vesicles were transported retrogradely ( Luo et al., 2024 ). In contrast, vesicles exhibiting slower dynamics and low displacement ratios may serve in local cargo collection or signalling, consistent with previously described roles of amphisomes ( Andres-Alonso et al., 2019 , 2021; Karpova et al., 2025 ). The locus coeruleus is the principal source of norepinephrine in the brain (Swanson & Hartman, 1975; Loughlin et al., 1986; Berridge & Waterhouse, 2003 ; Szabadi, 2013 ). To monitor autocrine NE release from LC-NE axons in vivo , we employed the next-generation genetically encoded fluorescent sensor GRABNE2h, which enables rapid and specific detection of NE (Feng et al., 2024). Intriguingly, reducing NE levels in distal LC axons projecting to the PFC facilitated unidirectional amphisome mobilization, consistent with chemogenetic inhibition of LC-NE neuronal activity, suggesting a shared regulatory mechanism controlling unidirectional trafficking. Additionally, frequent directional switches of LC3B-positive amphisomes during spontaneous neuronal activity were associated with transient increases in NE release, which were significantly elevated prior to the switch. These switches likely reflect differential activity-dependent association of amphisome with a dynein or kinesin motor ( Cai et al., 2010 ; Cheng et al., 2015 ; Andres-Alonso et al., 2019 ; Maday et al., 2012 ; Fu et al., 2014 ; Fu & Holzbaur, 2014). In previous work we found that stationary pauses of SIPA1L2-positive amphisomes are regulated by two molecular mechanisms downstream of cAMP and PKA activation ( Andres-Alonso et al., 2019 ): (a) modulation of amphisome interactions with the dynein motor complex, facilitated by the motor adaptor protein snapin ( Cai et al., 2010 ; Xie et al., 2015; Cheng et al., 2015 ; Andres-Alonso et al., 2019 ); and (b) regulation of motor processivity through the GTPase-activating protein (GAP) activity of RapGAP SIPA1L family members, which associate with the outer membrane of autophagosomes ( Andres-Alonso et al., 2019 ; Goldsmith et al., 2022 ). Elevated NE levels appear to induce amphisome immobilization. In addition, the subsequent prolonged stationary pauses of amphisomes seem to further promote high NE release, possibly via a positive feedback loop associated with the signalling function of amphisomes. Collectively, these findings align with observations that higher LC-NE activity during anxiety-like behaviour reduces the number of LC3B-positive amphisomes photoconverted in PFC axons reaching the soma, and further support the idea that stationary pauses of LC3B-positive vesicles contribute to local activation of synaptic vesicle release machinery ( Andres-Alonso et al., 2019 ). An open question is the nature of the signalling machinery of LC-NE amphisomes and the mechanism of docking in proximity of NE release sites. In previous work, we identified BDNF/TrkB signalling amphisomes ( Andres-Alonso et al., 2019 ). BDNF/TrkB signalling has been shown in multiple neuronal types, including LC-NE neurons ( Matsunaga et al., 2004 ; Traver et al., 2006 ; Liu et al., 2015 ; Suto et al., 2019 ), but alternative signalling amphisomes, distinct from TrkB-containing ones, may also exist in LC-NE neurons via association with the multidomain protein SIPA1L2. Finally, what might be the advantage of combining a degradative with a signalling function in one organelle? We speculate that in the absence of mature lysosomes in axons, this enables a tight control of trafficking and potentially cargo uptake at those release sites that have been particularly active. In volume transmission, neurotransmitters are released from multiple axonal varicosities rather than single synapses, allowing NE to diffuse broadly and reach numerous target cells. Our data suggest that a dedicated feedback mechanism at these varicosities regulates amphisome trafficking, signalling, and potentially formation. Thus, it is tempting to speculate that due to enhanced release that comes along with high metabolic activity and increased protein damage, efficient protein removal by amphisome formation takes place. We further speculate that physiological states, such as the sleep-wake cycle, might be critically involved in amphisome biogenesis and trafficking. During sleep, amphisomes may be transported rapidly to the soma without pausing at NE release sites due to decreased cAMP/PKA signalling ( Figure 3J ) , efficiently removing damaged protein cargos. During wakefulness, amphisomes may pause at varicosities to support NE release and, presumably, cargo loading from late endosomes (Andres-Alonso et al., 2021), a mechanism tightly linked to neurotransmitter release. Overall, our findings indicate that LC-NE neurons employ activity- and autocrine-regulated mechanisms to coordinate autophagic vesicle trafficking for somatic delivery depending upon varying functional states. Limitations of the study Studying axonal amphisome trafficking in vivo is technically challenging due to the difficulty of axon-specific labelling and limited access to deep axonal segments. Transcranial two-photon imaging permits visualization of distal axons but does not capture trafficking directionality, and dual fluorescent probes limit simultaneous tracking of axonal orientation with microtubule plus-end markers such as EB3. Although the GRABNE2h sensor reliably reports electrically evoked norepinephrine release, spontaneous release produces only modest ΔF/F₀ signals, complicating correlative analyses. RESOURCE AVAILABILITY Lead contact Further details and requests for reagents and resources should be directed to the lead contact ( anna.karpova{at}lin-magdeburg.de ) and will be fulfilled upon request. Materials availability Materials can be made available upon specific request. Data and code availability This study does not report original code. Additional information to reanalyse the data is available from the lead contact upon request. AUTHOR CONTRIBUTIONS A.K. conceptualized and supervised the project; A.A.A.A. and A.K. performed the experiments and analysed the data; H.J. and M.R.K. provided equipment and materials; A.K. wrote the manuscript and prepared all figures; and all authors contributed to commenting on and revising the final version. DECLARATION OF INTERESTS Authors declare no conflict of interest DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGY IN THE WRITING PROCESS The authors used Grammarly and Microsoft Copilot to assist with grammar refinement and take full responsibility for the final content. STAR*Methods Detailed methods are provided online and include: KEY RESOURCES TABLE View this table: View inline View popup EXPERIMENTAL MODEL Animals All animal experimental procedures were approved by the responsible authorities of Saxony-Anhalt (SA, Landesverwaltungsamt für Verbraucherschutz und Veterinärangelegenheiten, Referat 203; License number 42502-2-1638 LIN). B6.Cg-Dbh^tm3.2(cre)Pjen/J (Strain #:033951 RRID: IMSR_JAX:033951) mouse line was obtained from the Jackson Laboratory and maintained at the animal facility at the LIN. The average age of the animals for surgical procedures was between 10-12 weeks old, and all animals were treated according to the animal welfare guidelines of the Leibniz Institute for Neurobiology. METHOD DETAILS Cloning of viral constructs and viral partial production Open reading frames (ORFs) for mNeonGreen-LC3B, mRuby3-LC3B, and mEOS4-LC3B were synthesized as gBlocks (Integrated DNA Technologies) and subcloned into the AAV-EF1α-DIO backbone (Addgene #50462) using SgsI/AscI-NheI restriction sites. AAV particles expressing fluorescently tagged LC3B were produced as DJ and AAV2/9 serotypes. Additional AAV particles were obtained as ready-to-use preparations from Addgene or the Zurich Viral Vector Facility (see key resource table for details). For AAV production, the standard iodixanol gradient ultracentrifugation protocol provided by Addgene was used with minor modifications. Briefly, HEK293T cells were plated in three 150-mm petri dishes and co-transfected the following day with the expression plasmid, the packaging plasmid (pAAV-DJ or pAAV2/9), and pHelper at a 1:1:1 molar ratio using polyethylenimine (PEI). Cells were harvested three days post-transfection, washed with 10 mL prewarmed PBS, resuspended in 10 mL PBS, pooled from the three dishes, and centrifuged at 1,000 × g for 30 min at 4 °C. The cell pellet was stored at −70°C overnight, while the supernatant was transferred into fresh tubes. A PEG precipitation was performed by adding 4 g of polyethylene glycol and 2,32 g of NaCl per 40 ml of supernatant, followed by shaking for 3 h in an overhead shaker at 4 °C and then resting overnight at 4 °C. The solution was subsequently centrifuged at 3,000 × g for 30 min at 4 °C. The resulting pellet was resuspended in 2 ml of Tris-HCl buffer (50 mM TRIS base, 150 mM NaCl, pH 8.5). The initial cell pellet was resuspended in 3 mL of the same TRIS-HCl buffer and subjected to three freeze-thaw cycles. The lysate was combined with the PEG-precipitated solution and treated with Benzonase (50 U/mL) for 1 h at 37 °C to degrade residual nucleic acids. The mixture was clarified by centrifugation at 8,000 × g for 30 min at 4 °C, and the supernatant was filtered through a 0.2 μm membrane before AAV purification. AAV particles were purified by ultracentrifugation in an iodixanol step gradient consisting of 8 ml 15% (prepared in 1 M NaCl/PBS-MK buffer: 5.84 g NaCl, 26.3 mg MgCl₂, and 14.91 mg KCl in 100 ml PBS), 6 ml 25%, 5 mL 40%, and 5 mL 60% (both prepared in PBS-MK buffer: 26.3 mg MgCl₂ and 14.91 mg KCl in 100 ml PBS). The 25% and 60% fractions were supplemented with phenol red for visualization. Gradients were centrifuged in 38.6 mL Ultra-Clear tubes (SW32 Ti rotor, Beckman) at 170,000 × g for 16 h at 4 °C. For washing and concentration of AAV particles, the 40% fraction was applied to centrifugal filter units preconditioned with 0.1% Pluronic F-68 in PBS for 10 min at room temperature. Filters were subsequently rinsed with 0.01% and 0.001% Pluronic F-68 in PBS containing 200 mM NaCl, followed by iterative centrifugation (3,000–3,500 rpm, 4 °C) until the viral preparation was concentrated to 200-250 μL. Viral concentrates were recovered by rinsing the filter walls and stored at 4 °C for short-term use (≤2 weeks) or aliquoted and stored at –80 °C for long-term use. Viral titres were determined by quantitative PCR using the following primers: forward ITR primer, 5′-GGAACCCCTAGTGATGGAGTT-3′ and reverse ITR primer, 5′-CGGCCTCAGTGAGCGA-3′ ( Aurnhammer et al., 2012 ). AAV injection into the Locus coeruleus For stereotactic injections, animals were anesthetized with ketamine/xylazine (100/5 mg/kg, intraperitoneally) and placed in a stereotactic frame. The skull was exposed and cleaned, and the bregma and lambda landmarks were identified. For targeting the locus coeruleus, stereotaxic coordinates relative to bregma were anterior-posterior (AP) −5.4 mm, medial–lateral (ML) ±0.93 mm, and dorsal-ventral (DV) -3.6 mm (Allen Mouse Brain Atlas; available at http://mouse.brain-map.org ). A small craniotomy (0.1– 0.2 mm) was drilled, and a Hamilton syringe mounted on a nanolitre injector was lowered slowly (≤0.2 mm/s). After a 5-minute pause, 500 nL of virus was injected at 75 nL/min ( Cearley and Wolfe, 2007 ). The needle remained in place for 10 min post-injection before being withdrawn slowly (0.1-0.2 mm/s) to minimize tissue damage. The incision was closed with silk sutures and reinforced with dental cement. Animals were monitored during recovery and received postoperative analgesia (carprofen, 5 mg/kg, s.c.). Surgical sites were inspected daily, with wound healing confirmed 3 days post-surgery, and animals were monitored until tissue collection. Optical fiber implantation and optical fiber-mediated photoconversion in the home cage versus the open arena Following viral expression, an optical fiber was implanted above the medial PFC to enable in vivo photoconversion. Anaesthesia and preparatory procedures were identical to AAV injections. A 300-µm craniotomy was drilled above the right mPFC (AP +1.8 mm, ML +0.3 mm relative to bregma), and a 300-µm fiber cannula mounted in a ceramic ferrule was lowered to DV −2.2 mm at 1 μm/min and secured with dental cement. Wounds were sutured, and animals received identical postoperative care and analgesia. Four to six weeks post-surgery, the implanted fiber was coupled to a 405 nm laser (5 mW output) through a flexible fiber cord (300 μm core, ∼85% transmission), allowing unrestricted movement of the animal. Photoconversion of mEOS-LC3B in the PFC was performed in awake mice under two conditions: a familiar environment (home cage) and a novel environment (40 × 40 × 40 cm white-walled arena; McCall et al., 2017). Identical stimulation parameters were applied across contexts (405 nm, 5 mW, 600 s total, 1 s pulses with 10 s intervals). Mice were video recorded throughout the procedure to monitor locomotion and exploration, enabling correlation of behavioural state with photoconversion outcomes. Six hours after photoconversion, mice were anesthetized and perfused with 4% PFA. Brains were dissected, postfixed, and sectioned (40-45 μm) under low-light conditions to minimize stochastic photoconversion. Coronal LC-NE sections were collected in PBS and processed for immunohistochemistry. Confocal microscopy was used to quantify photoconverted LC3B puncta within LC-NE somata. Transcranial window implantation For cranial window implantation, animals were prepared as described above. A larger portion of the skull was exposed, and connective tissue was gently removed to prepare the bone surface. A 5 mm circular craniotomy was drilled over the prefrontal-motor cortex region with continuous saline irrigation to prevent heating (Zuluaga-Ramirez et al., 2015; Goldey et al., 2014 ). The skull flap was removed carefully without disrupting the dura, and a 5 mm glass coverslip was placed over the exposed cortex. The coverslip was sealed with cyanoacrylate adhesive and stabilized with dental cement ( Xu et al., 2007 ; Mostany & Portera-Cailliau, 2008 ; Goldey et al., 2014 ; Holtmaat et al., 2009 ; Yang et al., 2010). A custom-made metal headplate was affixed over the cranial window using SuperBond and dental cement to enable stable head fixation during two-photon imaging. Animals recovered under standard postoperative care. Two-photon imaging in vivo of LC axons projecting to PFC Two to four weeks after AAV transduction and window implantation, in vivo two-photon imaging was performed using a Bergamo microscope (Thorlabs, Newton, NJ, USA) equipped with a tunable Ti: sapphire laser (Chameleon Vision S, Coherent Inc.) and a fixed-wavelength laser (HighQ-2, Spectra-Physics Inc.). Both lasers were aligned to the same foci and operated simultaneously to enable dual-colour imaging in 4D (XYZT), whereas the wavelength of the Vision S laser was used in the range between 920 nm and 960 nm for green-emitting fluorophores (mNeonGreen-LC3B, GRABNEh), and the HighQ-2 laser (wavelength 1040 nm) was used for red fluorophores (mRuby3-LC3B). Actual power for in-vivo imaging with each laser was set to be less than 30 mW to minimize photobleaching or damage in long imaging sessions. Prior to imaging, animals were handled and habituated to the setup for five days to reduce stress and ensure their comfort during the procedure. On the first day of imaging, animals were briefly anesthetized with isoflurane (30 seconds) and head-fixed under the objective. Imaging areas were first localized with a 4x objective under one-photon excitation (488 nm LED, X-CITE 200) and detected with a camera (1500M-GE, a 1.4 MP) to capture an overview image and adjust the focal plane for the region of interest. High-resolution data were acquired with a 40x water-immersion objective (NA 0.8) in two-photon mode. Distal axons in the M1-mPFC region were imaged with a digital zoom of 10-13x. Optical sectioning was performed in 1 µm Z-steps, with 30 Hz frame rate, collecting 100-150 repeated stacks per axon. Fifteen frames were averaged per plane, resulting in acquisition times of 20-30 min with ∼2-3 µm resolution for axon thickness. Data and metadata were saved automatically for subsequent analysis. Combined two-photon imaging and chemogenetic silencing For combined two-photon imaging and chemogenetic activation of Gi-signalling in LC-NE neurons, B6.Cg-Dbh^tm3.2(cre)Pjen/J mice received stereotactic co-injections of AAV-EF1a-DIO-mNeonGreen-LC3B and AAV-EF1a-DIO-hM4D(Gi)-mCherry ( Armbruster et al., 2007 ) into the LC-NE , followed by implantation of a transcranial window and headplate, as described above. Axonal stretches were imaged sequentially under basal conditions and following chemogenetic activation of Gi signalling. The DREADD agonist JHU37160 ( Bonaventura et al., 2019 ) was administered subcutaneously at 0.5 mg/kg immediately after the initial imaging session. After a 20–30 min post-injection interval to allow sufficient blood-brain barrier penetration (Lawson et al., 2024), the same axonal stretch was re-imaged. This design enabled direct comparison of LC3B-positive vesicle trafficking during spontaneous activity versus Gi - signalling activation. Two-photon imaging was conducted using the parameters described above. Z-stacks were acquired with a 1 μm step size, 10-13x digital zoom, and 30 Hz acquisition rate, averaging 15 frames per Z-plane to reduce motion artifacts. For each axon, 100-150 repeated stacks were collected. Immunohistochemistry Animals were perfused transcardially with PBS followed by 4% PFA. Extracted brains were post-fixed in 4% PFA for at least 8 hours and cryoprotected in 30% sucrose for 48–72 hours. Coronal brain sections (40 μm) were prepared using a freezing microtome (HM440E, Microm). Heat-based antigen retrieval was performed by incubating sections in 10 μM sodium citrate (pH 9, adjusted with 5M NaOH) for 30 min at 80 °C. Sections were then permeabilized in 0.2% Triton X-100 in PBS for 1 hour and blocked in blocking buffer (2% glycine, 0.2% gelatine, 2% BSA, and 50 mM NH 3 Cl (pH 7.4)). Primary antibodies were incubated for 24-48h at 4°C in blocking buffer, followed by three 10-minute PBS washes and incubation with fluorophore-conjugated secondary antibodies for 1 hour at room temperature. After a final series of PBS washes and a brief rinse in distilled water, sections were mounted with Mowiol 4-88 (Merck Chemicals GmbH) and imaged using confocal microscopy. Confocal laser scanning microscopy and image analysis Confocal images of fixed tissue samples were acquired using a Leica TCS SP8 STED 3X confocal laser scanning microscope equipped with a 405 nm diode laser and a white-light laser (WLL) (Leica Microsystems, Mannheim, Germany). For imaging LC-NE neurons and their terminals in the PFC, 63×/1.4 NA (HCX PL APO, Leica) or 100×/1.4 NA (HC PL APO, Leica) oil-immersion objectives were used. Images were acquired at 400-600 Hz with a lateral resolution of 1024 × 1024 pixels and a Z-stack step size of ∼160-180 nm employing Hybrid (HyD) detectors. Image analysis Amphisome detection and characterization analysis in Z-stack acquired in vivo Image processing and analysis were performed using ImageJ/FIJI, following a multi-step workflow optimized for amphisome quantification. Pre-processing included sequential digital filters: rolling ball background subtraction (radius optimized to feature size), median filtering (2-3-pixel radius), and Gaussian smoothing (σ = 1-2 pixels) to reduce noise while preserving biological structures and improving signal-to-noise ratio ( Schindelin et al., 2012 ; Berg et al., 2019 ). Contrast enhancement employed histogram-based methods, with parameters documented to maintain data integrity (Aaron & Chew, 2021). Regions of Interest (ROIs) were selected based on standardized anatomical criteria, incorporating morphological features and fluorescence intensity profiles. Automated ROI detection algorithms, supplemented by manual verification, ensured consistent selection across experimental conditions. Multi-channel analysis isolated specific vesicle populations via channel splitting and colocalization, with Max-Entropy thresholding applied using machine learning-optimized parameters for accurate segmentation across the full Z-stack (Arganda-Carreras et al., 2017; Wiesmann et al., 2020). Amphisome quantification utilized the StarDist deep learning-based object detection algorithm, trained on manually annotated vesicle datasets. StarDist employs a U-Net architecture with star-convex polygon representation, enabling accurate separation of closely packed vesicles while preserving morphological integrity ( Schmidt et al., 2018 ; Weigert et al., 2020 ; Völker et al., 2020; Fazeli et al., 2021). The pipeline generates vesicle counts per ROI, size distributions (μm²), and shape parameters, with automated circularity measurements (4π × area/perimeter²) and variance statistics. Quality control included merging thresholded images with pre-processed data for visual verification. This standardized workflow ensures reproducible vesicle detection and characterization with high sensitivity and specificity. Image processing and amphisome tracking analysis Time-lapse in vivo imaging of amphisome dynamics in axons requires precise processing to preserve spatial and temporal accuracy. Motion correction was performed in ImageJ/FIJI using the FAST4DREG plugin, which incorporates the NanoJ-Core algorithm for sub-pixel registration across all dimensions (x, y, z, t) ( Laine et al., 2019 ; Culley et al., 2018 ). Motion correction parameters were optimized iteratively for each dataset to maximize registration accuracy while minimizing artifacts, enabling reliable tracking of small, rapidly moving vesicles in living tissue. Following motion correction, a multi-step noise reduction protocol was applied to optimize signal-to-noise ratio while preserving biological dynamics. Rolling ball background subtraction (50-pixel diameter) removed large-scale intensity variations, followed by Gaussian filtering (σ = 2 pixels) to reduce high-frequency noise (Schindelin et al., 2019; Berg et al., 2020). Parameters were empirically optimized to maintain vesicle edge definition while minimizing background fluctuations. Maximum intensity projections of z-planes were then generated to produce high-contrast 2D representations, facilitating robust tracking of vesicular movement over time. Vesicle tracking was performed using the TrackMate algorithm, enhanced with machine learning-based detection and Linear Assignment Problem (LAP) trackers ( Tinevez et al., 2017 ; Simon Youssef et al., 2011). Spot detection employed Laplacian of Gaussian filtering, followed by feature extraction of intensity, size, and contrast, with track linking incorporating gap closing and split/merge handling. Automated tracking results were validated against manual analysis, demonstrating high correlation (r > 0.90) for key movement parameters (Nicolas Chenouard et al., 2014 ; Yichen Wu et al., 2019). The pipeline extracted multiple movement parameters, including instantaneous and mean velocities (μm/s), maximum speed and acceleration, total displacement, path length, directional persistence, and pause frequency and duration. For chemogenetic experiments involving hM4D(Gi) receptor activation, trafficking parameters were compared within the same axons pre- and post-J60 administration, enabling paired analysis using paired t-tests in GraphPad Prism. Quality control was implemented throughout the pipeline, including semi-automated removal of out-of-focus frames, validation of drift correction via stationary landmarks, monitoring of signal-to-noise ratio across time series, and manual spot checking for tracking accuracy. Image processing, GRABNE2h signal analysis, combined with amphisome tracking analysis Initial image preprocessing was performed in ImageJ-FIJI and Jupyter Lab, incorporating the NoRMCorre algorithm for motion correction with sub-pixel accuracy (Zhou et al., 2020), an essential step for maintaining spatial precision, particularly for tracking small, rapidly moving vesicles. Noise reduction combined wavelet-based denoising with adaptive thresholding, optimized for neural imaging to preserve biological signal while minimizing background fluctuations ( Giovannucci et al., 2019 ). Vesicle tracking was performed using the TrackMate algorithm, enhanced with machine learning-based detection and Linear Assignment Problem (LAP) trackers ( Tinevez et al., 2017 ). This semi-automated approach enabled precise quantification of multiple movement parameters, including instantaneous and mean velocities, displacement vectors, directional persistence, and pause characteristics. Tracking accuracy was validated against manual analysis in subset datasets, confirming the reliability of automated measurements ( Chenouard et al., 2014 ). This approach allows detailed comparison of vesicular behaviour across experimental conditions while maintaining consistent measurement criteria. Norepinephrine dynamics were analysed via GRABNE2h fluorescence using specialized ROI selection algorithms ( Feng et al., 2019 ; Feng et al., 2025). Signal processing included adaptive baseline correction (8th percentile F0), photobleaching compensation via exponential fitting, and movement artifact correction through cross-correlation analysis, ensuring signal fidelity for temporal analysis of neurotransmitter dynamics (Patriarchi et al., 2020). Feature extraction and event detection were implemented using custom Python scripts, including hierarchical clustering for stop-point identification and wavelet transform analysis for directional changes. Movement patterns were classified via machine learning, with statistical validation through bootstrapping (Pachitariu et al., 2018). Data visualization employed Z-score normalization, hierarchical clustering of temporal patterns, and event-triggered averages, while statistical inference used mixed-effects models to account for both fixed and random effects ( Saxena et al., 2020 ). This integrated analytical pipeline enables robust detection of both broad trends and subtle variations in amphisome trafficking and norepinephrine release. Plotting Scatter plots represent mean ± standard error of the mean (SEM). Plots were generated in FiJi, Python using SciPy, matplotlib, and NumPy packages or GraphPad Prism 9. Figures were assembled and annotated in Adobe Illustrator 2025. QUANTIFICATION AND STATISTICAL ANALYSIS All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Sample sizes (n) are reported either in the figure panels or in the corresponding legends. Depending on the study design and data distribution, appropriate parametric tests were applied. Data are presented as mean ± SEM, and statistical significance was defined as * p < 0.05, *** p < 0.001. SUPPLEMENTAL FIGURES AND LEGENDS Download figure Open in new tab Figure S1. In vivo , cell-type-specific labelling of LC neurons and axons combined with LC3B-mediated detection of amphisomes (A). Experimental timeline and AAV-DIO-GFP injections into the LC of Dbh-Cre mice, expressing Cre recombinase from the endogenous Dbh locus. (B) . Representative confocal tile-scan image showing specific GFP expression in LC-NE neurons. D, dorsal; V, ventral. (C) . Representative confocal images of LC neurons illustrating LC3B and SIPA1L2 expression. (D) . Confocal image of a GFP-labelled LC axon with endogenous LC3B and Snapin immunoreactivity, and (E) the corresponding line intensity profile. (F, G) . Confocal images showing endogenous SIPA1L2, LC3B, and Rab7 co-localized in LC neurons. (H) . Confocal tile-scan image showing LC-NE neurons expressing mNeonGreen-LC3B. (I) . Tile scan confocal image of fluorescently labelled LC axonal terminals in the mPFC , showing dense labelling in layers I and II. Inset highlight LC3B-positive puncta in distal LC axons (arrows). (J). Semi-automated image analysis of distal LC axons showing three distinct tracks over time (1, 2, and 3), representing different trafficking behaviours of LC3B-positive amphisomes in vivo . Download figure Open in new tab Figure S2. Optical fiber-mediated photoconversion of mEOS4-LC3B in LC axons projecting to the PFC . (A). GFP-positive axons in the PFC used for LAMP2a detection are TH-positive. Colocalization of GFP-labelled LC axons (cyan) with TH immunoreactivity (magenta) confirms LC -specific labelling. (B) . Experimental timeline: animals were perfused four to six hours after photostimulation, and LC sections were immunostained for tyrosine hydroxylase. The photoconverted group underwent a UV-light stimulation protocol consisting of 1-second pulses delivered at 10-second intervals for 10 minutes (C) . Representative optical fiber trajectory targeting the mPFC . (D) . Representative confocal tile-scan image of the LC without photostimulation in the PFC . (E) . Representative confocal image showing mEOS4-LC3B-positive puncta localized within LC axonal varicosities. Download figure Open in new tab Figure S3. Co-expression of mNeuonGreen-LC3B and hM4D(Gi)-mCherry in LC neurons (A). Confocal tail-scan microscopy revealed that both mNeonGreen-LC3B and hM4D(Gi)-mCherry were co-expressed in TH-positive LC-NE neurons. Expression of hM4D(Gi) was not LC -, but neuron-specific (AAV-hSyn-hM4D(Gi)-mCherry. Download figure Open in new tab Figure S4. Amphisome mobilization occurs with a decrease in NE release, while a switch in amphisome directionality correlates with a rapid increase in NE within LC axons (A). Schematic of the experimental approach using AAV-EF1a-DIO-mRuby3-LC3B expression in LC-NE neurons of DbH-Cre expressing animal to track amphisomes in combination with GRABNEh. (B). Confocal tile-scan image of LC-NE neurons expressing mRuby3-LC3B. (C) . mRuby3-LC3B expression in LC-NE axons (arrowheads). (D) . Magnified confocal image of LC-NE neurons labelled with anti-TH antibodies and expressing mRuby3-LC3B. (E). Schematic representation of the trigger selection of amphisome trafficking. (F). Averaged GRABNE2h signal (ΔF/F₀%) aligned to the “trigger” time point (amphisome mobilization), showing a reduction in NE release approximately 3 seconds before mobilization . (G). Quantification reveals no significant change in peak values, while valley values were significantly lower before the “trigger” compared to the values after the “trigger”. (H) . Analysis of GRABNE2h peak values of ΔF/F₀% before and after the “trigger” event. n.s. – non-significant. *p < 0.05, paired t-test. (I) . Schematic representation of the extracted “trigger” point for identifying the switch in amphisome directionality. (J, K). Averaged GRABNE2h intensity (ΔF/F₀%) aligned to the “trigger”, showing no significant fluctuations (n.s.) beyond normal spontaneous activity. Statistical analysis of valley (K) and peak values. *p < 0.05, paired t-test. (L). Lookup table bars indicate the average GRABNE2h intensities aligned to the “trigger.” ACKNOWLEDGMENTS The authors acknowledge the professional technical assistance of I. Herbert for AAV production and M. Marunde for help with brain sectioning. We also thank O. Kobler for assistance with image processing, J. Pakan for sharing the cranial window implantation technique, and M. Prigge for providing the Dbh-Cre mouse line and for helping with animal procedures. We further acknowledge A. M. Oelschlege and J. Kaufhold for assistance with animal licensing procedures, and U. Thomas, M. Andres-Alonso, and E. D. Gundelfinger for helpful discussions. Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) FOR5228 RP06, Project-ID 447288260 (to AK and MRK), LIN special project, and Schram Stiftung to AK. Funder Information Declared Deutsche Forschungsgemeinschaft (DFG) FOR5228 RP06 , Project-ID 447288260 LIN special project Schram Stiftung Footnotes ↵ # Lead contact References 1. ↵ Andres-Alonso , M. , Ammar , M.R. , Butnaru , I. , Gomes , G.M. , Acuña Sanhueza , G. , Raman , R. , Yuanxiang , P. , Borgmeyer , M. , Lopez-Rojas , J. , Raza , S.A. , et al. ( 2019 ). SIPA1L2 controls trafficking and local signaling of TrkB-containing amphisomes at presynaptic terminals . Nat Commun 10 , 5448 . doi: 10.1038/s41467-019-13224-z OpenUrl CrossRef PubMed 2. ↵ Armbruster , B.N. , Li , X. , Pausch , M.H. , Herlitze , S. , and Roth , B.L . ( 2007 ). Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand . 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