Intact-brain analyses reveal distinct information carried by SNc dopamine subcircuits

“Seminal early studies into the information encoded by midbrain DA neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al.”

“We expressed GCaMP6f (Chen et al., 2013) in DMS- or DLS-projecting SNc DA neurons by injecting CAV-cre into the DMS or the DLS, followed by injection of a cre-dependent GCaMP6f construct into the SNc.”

“…neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al., 1996, 2004; Steinberg et al., 2013; Sutton, 1988).”

“…that the DA neurons, which increase their firing in response to these stimuli, may signal salience rather than value (Brischoux et al., 2009; Bromberg-Martin et al., 2010; Matsumoto and Hikosaka, 2009), though controversy on this point remains (Cohen et al., 2012; Fiorillo et al., 2013,…”

“…neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al., 1996, 2004; Steinberg et al., 2013; Sutton, 1988).”

“We began by demonstrating that novel circuit-tracing and -recording methodologies can be effectively combined with whole-brain CLARITY/COLM imaging (Chung et al., 2013; Tomer et al., 2014) to provide a uniquely informative overview of richly defined cell types and their global connectivity motifs…”

“This information notably enriches the ascending spiral model proposed by Haber et al. (2000), which could not make definitive statements about striatal inputs onto DAergic versus non-DAergic midbrain cells.”

“…monitored activity from projection-defined SNc DA neurons during behavior using fiber photometry, a method for collecting population intracellular [Ca2+] fluorescent signals from a genetically encoded Ca2+ indicator such as GCaMP through a single chronic fiber optic implant (Gunaydin et al., 2014).”

“Another study used recordings in awake monkeys to demonstrate a correlation between the depth at which a DA neuron was recorded along the electrode (indicating a more ventromedial location in the midbrain) and the likelihood that it would decrease rather than increase its firing in response to an aversive cue (Matsumo…”

“…target-defined subpopulations are located in different subregions of the VTA, their excitatory synapses are differentially modulated by rewarding and aversive stimuli, and they receive distinct inputs, which can elicit opposite behaviors when selectively recruited (Lammel et al., 2008, 2011, 2012).”

“…functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate in separable circuits.”

“For example, a recent viral circuit tracing study showed that VTA and SNc DA neurons receive different proportions of inputs from key brain regions (Watabe-Uchida et al., 2012).”

“…utilizing a GFP-expressing rabies virus (RVdGGFP) that both lacks the glycoprotein needed to spread transsynaptically and is pseudotyped with EnvA (so that it can infect only neurons expressing TVA, an avian receptor protein normally absent in mammalian cells [Wickersham et al., 2007]).”

“…functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate in separable circuits.”

“Afferents to projection-defined SNc DA neurons also differ; in particular, we here identify new principles of afferent-projection complexity in SNc subcircuits (see Beier et al., 2015 for additional illuminating information regarding VTA subcircuits).”

“…potassium channel antagonist 4-AP (100 mM) to isolate monosynaptic inputs by preventing disynaptic disinhibitory responses through the GABAergic cells of the SNr (with TTX) while enabling ChR2 to drive neurotransmitter release in the absence of action potentials (with 4-AP) (Petreanu et al., 2009).”

“Seminal early studies into the information encoded by midbrain DA neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al.”

“…target-defined subpopulations are located in different subregions of the VTA, their excitatory synapses are differentially modulated by rewarding and aversive stimuli, and they receive distinct inputs, which can elicit opposite behaviors when selectively recruited (Lammel et al., 2008, 2011, 2012).”

“…neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al., 1996, 2004; Steinberg et al., 2013; Sutton, 1988).”

“…neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al., 1996, 2004; Steinberg et al., 2013; Sutton, 1988).”

“…CLARITYmonomer solution containing 1% acrylamide, 0.0125% bis-acrylamide, and 4% PFA and then polymerized at 37 C for 6–7 hr. Brains were passively cleared in SDS Borate Buffer (pH 8.5) at 37 C for 4–5 weeks, equilibrated in Focusclear for imaging and imaged using COLM methods (Tomer et al., 2014).”

“Seminal early studies into the information encoded by midbrain DA neurons suggested that a key function of DA is to transmit reward prediction error signals (Mirenowicz and Schultz, 1996; Schultz et al., 1997; Waelti et al., 2001), a hypothesis concordant with temporal difference learning models (Montague et al.”

“…functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate in separable circuits.”

“However, not all midbrain DA neurons appear to encode similar information in their activity patterns (Bromberg-Martin et al., 2010; Lammel et al., 2014; Roeper, 2013).”

“…leading to the hypothesis that the DA neurons, which increase their firing in response to these stimuli, may signal salience rather than value (Brischoux et al., 2009; Bromberg-Martin et al., 2010; Matsumoto and Hikosaka, 2009), though controversy on this point remains (Cohen et al., 2012;…”

“…which increase their firing in response to these stimuli, may signal salience rather than value (Brischoux et al., 2009; Bromberg-Martin et al., 2010; Matsumoto and Hikosaka, 2009), though controversy on this point remains (Cohen et al., 2012; Fiorillo et al., 2013, 2013; Ungless et al., 2004).”

“…target-defined subpopulations are located in different subregions of the VTA, their excitatory synapses are differentially modulated by rewarding and aversive stimuli, and they receive distinct inputs, which can elicit opposite behaviors when selectively recruited (Lammel et al., 2008, 2011, 2012).”

“Further development of high-throughput processing of these wholebrain datasets will be needed (Chung and Deisseroth, 2013; Kim et al., 2013, 2015) to fully capitalize on this opportunity for the rapid advancement of understanding structure-function relationships in the brain.”

“…CLARITY-optimized light-sheet microscopy (COLM) (Chung et al., 2013; Tomer et al., 2014), along with a variation of rabies-based circuit mapping (Schwarz et al., 2015); the lattermethod (TRIO) operates similarly to previously published rabies-based circuit mapping technologies, utilizing a…”

“d Intact, inclusive approaches to classifying neuronal cell types d Differential brain-wide circuit incorporation of SNc dopamine neuron subpopulations d Opposite valence encoding of shock by projection target- defined SNc neurons d Independently controlled information streams from the SNc to the DMS and DLS Lerner et…”

“…functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate in separable circuits.”

“These regions of the dorsal striatum have been functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate i…”

“Differences in Ih currents are strongly correlated with action potential rebound delays and can influence pacemaking activity (Neuhoff et al., 2002), particularly in the case of calbindin-negative SNc DA neurons (which constitute the majority of the SNc DA neuron population).”

“However, not all midbrain DA neurons appear to encode similar information in their activity patterns (Bromberg-Martin et al., 2010; Lammel et al., 2014; Roeper, 2013).”

“Although the specificity of the TH-GFP line has been questioned (Lammel et al., 2015), we found that GFP expression is specific for TH+ neurons in the SNc (Figure S3).”

“We injected CAV-cre into either the DMS or DLS and then injected AAVs expressing cre-dependent rabies glycoprotein (G) and TC, a high efficiency version of the TVA receptor fused to mCherry (Miyamichi et al., 2013), into the SNc.”

“(legend continued on next page) 636 Cell 162, 635–647, July 30, 2015 ª2015 Elsevier Inc. to identify such connections (Chuhma et al., 2011; Xia et al., 2011) or found them to be very weak (Bocklisch et al., 2013).”

“Further development of high-throughput processing of these wholebrain datasets will be needed (Chung and Deisseroth, 2013; Kim et al., 2013, 2015) to fully capitalize on this opportunity for the rapid advancement of understanding structure-function relationships in the brain.”

“(legend continued on next page) 636 Cell 162, 635–647, July 30, 2015 ª2015 Elsevier Inc. to identify such connections (Chuhma et al., 2011; Xia et al., 2011) or found them to be very weak (Bocklisch et al., 2013).”

“In fact, SNc DA neurons could in principle even be similar to VTA DA neurons in terms of the valence of information represented because mice will self-stimulate for SNc DA neuron activity just as for VTA DA neuron activity (Ilango et al., 2014; Rossi et al., 2013).”

“In TRIO, cre is delivered from the retrograde CAV-cre virus (Hnasko et al., 2006; Soudais et al., 2001), which transduces axon terminals and thereby defines ‘‘starter cells’’ (fromwhich rabies tracingwill occur) by virtue of their projection target.”

“These regions of the dorsal striatum have been functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas might also participate i…”

“Indeed, combined with other recent lines of evidence (Lammel et al., 2008, 2011; Margolis et al., 2008), we have found that projection-defined subpopulations of DA neurons display different intrinsic properties.”

“Similarly, for mice in which DA neuron TH expression was restricted to neurons projecting to either the DLS or the ventromedial striatum (VMS), DLS-projecting neurons were located more laterally than VMS-projecting neurons (Darvas and Palmiter, 2009; 2010; see also Schiemann et al., 2012).”

“In TRIO, cre is delivered from the retrograde CAV-cre virus (Hnasko et al., 2006; Soudais et al., 2001), which transduces axon terminals and thereby defines ‘‘starter cells’’ (fromwhich rabies tracingwill occur) by virtue of their projection target.”

“In fact, SNc DA neurons could in principle even be similar to VTA DA neurons in terms of the valence of information represented because mice will self-stimulate for SNc DA neuron activity just as for VTA DA neuron activity (Ilango et al., 2014; Rossi et al., 2013).”

“These regions of the dorsal striatum have been functionally distinguished by previous studies (Castañé et al., 2010; Faure et al., 2005; Featherstone and McDonald, 2004; Yin and Knowlton, 2004; Yin et al., 2004, 2005a, 2009, 2005b), leading us to hypothesize that SNc projections to these areas…”

“…which increase their firing in response to these stimuli, may signal salience rather than value (Brischoux et al., 2009; Bromberg-Martin et al., 2010; Matsumoto and Hikosaka, 2009), though controversy on this point remains (Cohen et al., 2012; Fiorillo et al., 2013, 2013; Ungless et al., 2004).”

“Similarly, for mice in which DA neuron TH expression was restricted to neurons projecting to either the DLS or the ventromedial striatum (VMS), DLS-projecting neurons were located more laterally than VMS-projecting neurons (Darvas and Palmiter, 2009; 2010; see also Schiemann et al., 2012).”

“Similarly, for mice in which DA neuron TH expression was restricted to neurons projecting to either the DLS or the ventromedial striatum (VMS), DLS-projecting neurons were located more laterally than VMS-projecting neurons (Darvas and Palmiter, 2009; 2010; see also Schiemann et al., 2012).”

“Further development of high-throughput processing of these wholebrain datasets will be needed (Chung and Deisseroth, 2013; Kim et al., 2013, 2015) to fully capitalize on this opportunity for the rapid advancement of understanding structure-function relationships in the brain.”

“A similar input-output architecture also applies to SNc-DA circuitry (Lerner et al., 2015 [in this issue of Cell]).”

“In vivo imaging with fiber photometry Fiber photometry was performed as in (28, 63, 64).”

“Those techniques are now starting to enable the streamlined analysis of axonal projections or cell positions in intact mouse brains in a manual or semi-automated way (Lerner et al., 2015; Menegas et al., 2015; Schwarz et al., 2015).”

“Regions of the caudate receive inputs from nearly the entire cortex [11,17]; and, additionally, via various connections with the midbrain [13], the associative striatum acts as a moderator of communication between limbic and motor regions.”

“, 2015) and resembles more closely those of the midbrain dopamine systems (Beier et al., 2015; Lerner et al., 2015), with biased input and segregated output.”

“Instead, they increase their firing to both rewarding and aversive events (Fiorillo et al. 2013, Horvitz 2000, Joshua et al. 2008, Lerner et al. 2015, Matsumoto & Hikosaka 2009b).”

“…their anatomic and functional heterogeneity and the intricate wiring of their target neuropils (Beier et al., 2015; Fiorillo et al., 2013; Lammel et al., 2014; Lerner et al., 2015) have made it difficult to resolve how they can selectively alter synaptic signaling between different neural pathways.”

“Fiber photometry Fiber photometry was performed using modulated 405 nm and 490 nm LEDs (54).”

“Such parallel channels for information flow are reminiscent of the parallel nigrostriatal DA subcircuits recently described for different subregions of the substantia nigra (Lerner et al., 2015).”

“We used fiber photometry (20) to examine whether ARN neurons exhibited intermittent synchronous activity in awake freely behaving mice.”

“Previous work has shown that when excited with 410 nm light as opposed to 488 nm light, the fluorescence emission of GCaMP is essentially calcium-independent (Lerner et al., 2015; Zalocusky et al., 2016).”

“We monitored astrocyte Ca2+ levels in the NAc using a fiber-photometry system in freely behaving mice (Lerner et al., 2015; Saunders et al., 2018) and specifically stimulated dopaminergic afferents to the NAc using optogenetics (Figures 1A and 1B).”

“Light collection, filtering, and demodulation were as previously described (45, 46).”

“The COLM system (described earlier in Tomer et al., 2014; Lerner et al., 2015) is optimized for high-resolution imaging and is most useful for studies requiring sub-cellular information, while the approach outlined here is optimized for speed and practicality in cellular resolution work, most…”

“However, there is some disagreement about the extent of collateralization of dopamine axons (Yetnikoff et al., 2014); some dopamine neurons may project to multiple brain areas (Fallon and Loughlin, 1982; Loughlin and Fallon, 1984; Takada and Hattori, 1986).”

“Our photometry setup utilized a 490 nm beam to activate Ca-dependent GCaMP6 fluorescence, as well as a 405 nm beam to image an isosbestic reference signal, which compensates for photo-bleaching and movement-related artifacts (Lerner et al., 2015).”

“These striatal circuits further serve to regulate the output of the midbrain dopamine system, which extends a widespread neuromodulatory influence throughout the brain (Kupchik et al., 2015; Lerner et al., 2015).”

“Fiber photometry data processing and analysis (mouse): Pre-processing of fiber photometry data was performed as previously described (Cho et al., 2017; Lerner et al., 2015).”

“Genetically guided tracing experiments have facilitated the identification of input and output wiring of DA neurons in the VTA and SNc (Beier et al., 2015; Lerner et al., 2015; Watabe-Uchida et al., 2012).”

“Prominent pathways that are implicated but unverified in reward and aversion are also delineated ( gray) (Beier et al. 2015; Britt et al. 2012; Humphries & Prescott 2010; Kirouac et al. 2004; Lammel et al. 2012; Lerner et al. 2015; Liu et al. 2014; Luo et al. 2015; McDevitt et al. 2014; Namburi et al. 2015a,b; Nieh et…”

“signals were subtracted with and then divided by the average signal in a 1 s period before CS in each trial) in this study, additional methods using activityindependent wavelength of excitation (Kudo et al., 1992; Lerner et al., 2015) or examination of emission spectrum (Cui et al.”

“A–B) Comparison of pS129 aSyn expression in the ipsilateral midbrain after two site striatal injections using (A) original, traditionally-used coordinates [34] or (B) optimized coordinates guided by recent findings revealing distinct SNpc-striatal innervation patterns [54].”

“…tracing of neuromodulatory systems has been used in the noradrenergic, dopaminergic and serotonergic systems (Beier et al., 2015; Faget et al., 2016; Lerner et al., 2015; Ogawa et al., 2014; Pollak Dorocic et al., 2014; Schwarz et al., 2015; Watabe-Uchida et al., 2012; Weissbourd et al., 2014).”

“…our fibers to areas where this specificity problem is reduced (Lammel et al., 2015) and that are known to contain the classical DA neurons that show RPE activity and are involved in reward processing (Lammel et al., 2011, 2012; Matsumoto and Hikosaka, 2009; Lerner et al., 2015; Kim et al., 2016).”

“…Keller et al., 2015), developmental biology (Huisken et al., 2004; Keller et al., 2008; Preibisch et al., 2010; Reynaud et al., 2015; Wu et al., 2013), cell biology (Gao et al., 2012; Planchon et al., 2011) and high-resolution whole brain neuroanatomy (Lerner et al., 2015; Tomer et al., 2014).”

“Projection specific approaches typically involve injection of viruses that show retrograde capabilities, into a target region of choice, allowing the efficient segregation of DA neuron subtypes based on their projections; indeed, these approaches have been used to segregate functionally distinct subpopulations within…”

“Neural circuits involved in associative learning integrate sensory signals with rewarding or punishing reinforcement cues, frequently encoded by neuromodulatory dopaminergic neurons (DANs; Aso et al., 2014a; Bromberg-Martin et al., 2010; Lerner et al., 2015; Schultz et al., 1997; Waddell, 2016).”

“, 2009), and can thus be used to estimate Ca2+-independent changes in measured fluorescence (Lerner et al., 2015), which presumably largely come from hemodynamic contamination (Ma et al.”

“One hypothesis motivated by the excitationrelease coupling principle in neurobiology would suggest that DA waves may be inherited from the sequential firing of topographically projectingmidbrain DA cells (Lerner et al., 2015).”

“This is not likely to be due to the potential diversity of dopamine neurons because subpopulations of dopamine neurons projecting to specific targets nonetheless receive inputs from many brain areas (Beier et al., 2015; Lerner et al., 2015; Menegas et al., 2015).”

“Changes in calcium-dependent GcaMP6s fluorescence (490 nm) signal were compared with calcium-independent GCaMP6s fluorescence (405 nm), providing internal control for movement and bleaching artifacts (Lerner et al., 2015; Su et al., 2017).”

“We measured calcium-dependent fluorescence (excited at 490 nm) as a measure of AgRP neuron activity (Cui et al., 2013; Gunaydin et al., 2014) and calcium-independent fluorescence (excited at 405 nm) as a control for movement and bleaching artifacts (Figure 1B) (Lerner et al., 2015).”

“Furthermore, like the fly DANs, mammalian DANs can be anatomically divided into those that generate aversion and different types of reward (Lammel et al., 2011, 2012; Cohen et al., 2012; Lerner et al., 2015; Beier et al., 2015; Tellez et al., 2016).”

“…roles of SNc and VTA neurons in exploratory activity, habituation, reinforcement, aversion (Bromberg-Martin et al., 2010; Friedman et al., 2014; Lerner et al., 2015; Roeper, 2013; Walsh et al., 2014), and the involvement of multiple nuclei in locomotion and reward (Benarroch, 2013;…”

“Around 50% of synapses made with SNc neurons are GABAergic (41), and these originate from numerous sources including the substantia nigra pars reticulata (SNr), the globus pallidus, the superior colliculus, the rostromedial tegmental nucleus, and SPNs located in striosomes (33, 42).”

“One way to acquire 3D fluorescence information is by mechanical scanning through the sample volume to obtain images at multiple focal planes, such as confocal [124], two-photon [125], light-sheet [126,127] and other various super-resolution microscopy techniques [103, 128 – 132].”

“Dual-wavelength Fiber Photometry (FP): Dual-wavelength FP was performed as we and others have previously described (Lerner et al., 2015; Su et al., 2017; Zalocusky et al., 2016).”

“Previous work has used Rb-based approaches to assess celltype-specific afferent inputs to midbrain dopamine neurons (Beier et al., 2015; Lerner et al., 2015; Menegas et al., 2015; Watabe-Uchida et al., 2012).”

“It is only when dopamine neurons are viewed from this structural-output-defined perspective that observations of opposite-valence responses to stimuli by different subsets of dopamine neurons become interpretable (Kim et al., 2014a; Lerner et al., 2015; Matsumoto and Hikosaka, 2009).”

“Consistent with this heterogeneity, dorsal raphe 5-HT neuron manipulations and recordings suggest complex roles in a range of behaviors related to cognition and motivation (Liu et al. 2014; Cohen et al. 2015; Brodsky et al. 2016; Li et al. 2016; You et al. 2016; Ren et al. 2018).”

“Recent studies suggest that DA neurons are heterogeneous in terms of their functions (Brischoux et al., 2009; Lerner et al., 2015; Matsumoto and Hikosaka, 2009).”

“To control for motion artifacts, we excited GCaMP6s with its calcium-dependent excitation wavelength of 470 nm and at its calcium-independent isosbestic excitation wavelength of 405 nm (Lerner et al., 2015).”

“GuPPy is programmed to use a control isosbestic wavelength recorded by users to remove smaller movement artifacts as well as bleaching artifacts when calculating the ΔF/F (as described in Lerner et al, 2015; Fig.”

“Critically, a calcium-independent fluorescence in the UV spectra was used in all recordings to control for movement and bleaching artifacts (Figures S5B–S5D) (Lerner et al., 2015).”

“We engineered mice to express the calcium indicator GCaMP6s in AgRP or POMCneurons andmeasured calcium-dependent fluorescence as a proxy for neural activity and calcium-independent fluorescence as a control for bleaching and movement artifacts (Figures 1A and 1B) (Lerner et al., 2015; Su et al., 2017).”