Anatomically defined and functionally distinct dorsal raphe serotonin sub-systems

May combine two claims.

The DRTph2→SC and DRTph2→AC clusters preferentially occupied dorsal and ventral DR, respectively, with partial overlaps accounting for 17.4% of the SC volume and 39.6% of the AC volume.

“DRTph2→SC and DRTph2→AC clusters preferentially occupied dorsal and ventral DR, respectively, albeit with partial overlaps that accounted for 17.4% of the DRTph2→SC volume and 39.6% of the DRTph2→AC volume”

• AssumptionRests on an assumption: Tph2 staining reliably identifies serotonin-producing neurons.

May combine two claims.

DRSert→OFC and DRSert→CeA axons exhibit complementary whole-brain projection patterns, preferentially innervating superficial cortical and deep subcortical regions.

“3D rendering suggested that DRSert→OFC and DRSert→CeA axons exhibited complementary whole-brain projection patterns, preferentially innervating superficial (cortical) and deep (subcortical) regions”

May combine two claims.

The OFC and OB were intensively innervated by DRSert→OFC axons but lacked DRSert→CeA axons.

“the OFC and OB were intensively innervated by DRSert→OFC axons but lacked DRSert→CeA axons”

May combine two claims.

The DRVglut3&Tph2 cluster overlaps 66%±0.6% with the DRTph2→AC cluster and 19%±1.1% with the DRTph2→SC cluster.

“we found that it had 66%±0.6% (mean±SEM) overlap with the DRTph2→AC but only 19%±1.1% overlap with the DRTph2→SC cluster”

May combine two claims.

Activation of DRSert→OFC neurons did not affect center entry or center time in the open field, or open-arm entry or time in the EPM.

“activation of DRSert→OFC neurons did not affect center entry or center time in the open field, or open-arm entry or time in open arms of the EPM”

• AssumptionRests on an assumption: Avoidance of the center in the open field and of the open arms in the elevated plus maze indicates anxiety-like behavior.

May combine two claims.

Neither activation nor Tph2 depletion of DRSert→OFC neurons affected fear learning and memory recall.

“Neither activation nor Tph2 depletion of DRSert➔OFC neurons affected fear learning and memory recall”

May combine two claims.

All recordings from the DRSert group showed activation after onset of licking, and evoked activity persisted during reward consumption.

“all recordings from the DRSert group showed an activation after the onset of licking, and the evoked activity persisted during the reward consumption period”

• cites Li et al., 2016Not checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: Sucrose water is a rewarding stimulus.

May combine two claims.

DRSert→OFC neurons, but not DRSert→CeA neurons, mediate the effect of DR serotonin neuron manipulations on forced-swim immobility.

“Both our gain- and loss-of-function manipulations in the forced-swim test suggest that DRSert→OFC neurons, but not DRSert→CeA neurons, mediate this effect (Figure 7).”

May combine two claims.

The seven mice from the DRSert group showed more varied responses to foot-shock, including one biphasic response.

“The seven mice from the DRSert group showed more varied responses, including one that exhibited a biphasic response composed of a transient elevation followed by long-lasting depression”

• AssumptionRests on an assumption: Mild electrical foot-shock is an aversive or punishing stimulus.

The quote only partly states this claim.

The DRSert→OFC and DRSert→CeA groups showed similar activation pattern to the DRSert group after reward.

“The DRSert→OFC and DRSert→CeA groups showed similar activation pattern”

• AssumptionRests on an assumption: Sucrose water is a rewarding stimulus.

May combine two claims.

Presynaptic inputs to DRSert→OFC and DRSert→CeA neurons originated from similar brain regions but had striking quantitative differences.

“While presynaptic inputs to these two DR serotonin subpopulations originated from similar brain regions, there were striking quantitative differences.”

May combine two claims.

Chemogenetic activation of both DRSert→OFC and DRSert→CeA neurons significantly decreased locomotion compared with controls.

“chemogenetic activation of both DRSert→OFC and DRSert→CeA neurons significantly decreased locomotion compared with controls”

May combine two claims.

In all eight mice from the DRSert→CeA group, foot-shock induced a transient elevation of Ca2+ signals, followed by a small depression in a subset.

“in all eight mice from the DRSert→CeA group, foot-shock induced a transient elevation of Ca2+ signals, followed by a small depression in a subset”

• AssumptionRests on an assumption: Mild electrical foot-shock is an aversive or punishing stimulus.

May combine two claims.

Tph2 depletion from DRSert→OFC neurons caused a significant decrease in center time and center entry compared with controls, and a significant decrease in open-arm time in the EPM.

“Tph2 depletion from DRSert→OFC neurons caused a significant decrease in center time and center entry compared with controls (Figure 6B), as well as a significant decrease in open-arm time in the EPM”

• AssumptionRests on an assumption: Avoidance of the center in the open field and of the open arms in the elevated plus maze indicates anxiety-like behavior.

May combine two claims.

Activation of DRSert→CeA neurons promoted anxiety-like behavior, causing significantly decreased center entry and center time in the open field and decreased open arm entry and open arm time in the EPM compared to all controls except one case.

“activation of DRSert→CeA neurons promoted anxiety-like behavior, causing significantly decreased center entry and center time in the open field (Figure 6C) and activation significantly decreased open arm entry and open arm time (Figure 6G) compared to all controls except one case.”

• AssumptionRests on an assumption: Avoidance of the center in the open field and of the open arms in the elevated plus maze indicates anxiety-like behavior.

May combine two claims.

Tph2 depletion from DRSert→CeA neurons appeared anxiolytic, causing a significant increase in center entry in the open field and open arm time in the EPM.

“Tph2 depletion from DRSert→CeA neurons appeared anxiolytic, causing a significant increase in center entry in the open field (Figure 6D) and open arm time in EPM”

• AssumptionRests on an assumption: Avoidance of the center in the open field and of the open arms in the elevated plus maze indicates anxiety-like behavior.

May combine two claims.

For nine of ten datasets, one cluster had the same expressive power as multiple clusters, and only DRSert→OB might be better described as two clusters.

“We found that, for nine out of ten datasets, one cluster had the same expressive power as multiple clusters and the only sub-system that might be better described as two clusters is DRSert→OB.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Previous studies showed that DR serotonin neurons, when treated as a whole, were activated during reward consumption in freely moving mice.

“Previous studies showed that DR serotonin neurons, when treated as a whole, were activated during reward consumption in freely moving mice (Li et al., 2016).”

• cites Li et al., 2016Not checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: Sucrose water is a rewarding stimulus.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The DR contains about 35% of the approximately 26,000 serotonin-producing neurons in the mouse brain and is the predominant source of serotonergic innervation of the forebrain.

“The dorsal raphe nucleus (DR) contains ~35% of ~26,000 serotonin-producing neurons in the mouse brain and is the predominant source of serotonergic innervation of the forebrain (Ishimura et al., 1988).”

• cites Ishimura et al., 1988 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

DR serotonin neurons as a whole receive monosynaptic inputs from diverse brain regions.

“DR serotonin neurons as a whole receive monosynaptic inputs from diverse brain regions (Ogawa et al., 2014; Pollak Dorocic et al., 2014; Weissbourd et al., 2014).”

• cites Ogawa et al., 2014 sourceNot checked yet

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• cites Pollak Dorocic et al., 2014 sourceNot checked yet

  Not checked in the free run.

• cites Weissbourd et al., 2014 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Recent studies on acute activation of DR serotonin neurons have reported divergent findings, including reinforcement, promotion of waiting for delayed reward, promotion of anxiety-like behaviors and suppression of locomotion, and suppression of locomotion without effects on reinforcement or anxiety-like behaviors.

“recent studies on the effects of acute activation of DR serotonin neurons have reported divergent findings, including reinforcement (Liu et al., 2014), promotion of waiting for delayed reward rather than reinforcement (Fonseca et al., 2015; Miyazaki et al., 2012), promotion of anxiety-like behaviors and suppression of locomotion (Teissier et al., 2015; Urban et al., 2016), and suppression of locom”

• cites Liu et al., 2014 sourceNot checked yet

  Not checked in the free run.

• cites Teissier et al., 2015 sourceNot checked yet

  Not checked in the free run.

• cites Correia et al., 2017 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The serotonin system is the most widely used pharmacological target for treating depression and anxiety.

“It is the most widely used pharmacological target for treating depression and anxiety (Belmaker and Agam, 2008; Ravindran and Stein, 2010)”

• cites Belmaker and Agam, 2008 sourceNot checked yet

  Not checked in the free run.

• cites Ravindran and Stein, 2010 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

“These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.”

• Also stated in: § Discussion · plain

  “Here we provide evidence for the existence of parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.”

Classified as probably not a substantial claim.

A physiological and circuitry-based theory of how the serotonin system is organized to carry out its diverse functions remains elusive.

“However, a physiological and circuitry-based theory of how the serotonin system is organized to carry out its diverse functions remains elusive (Dayan and Huys, 2015; Muller and Jacobs, 2010).”

• cites Dayan and Huys, 2015Not checked yet

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• cites Muller and Jacobs, 2010Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Whole-brain input mapping suggested heterogeneity of DR serotonin neurons with respect to presynaptic inputs.

“Whole-brain input mapping suggested heterogeneity of DR serotonin neurons has also been implicated with respect to presynaptic inputs (Weissbourd et al., 2014).”

• cites Weissbourd et al., 2014 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Based on retrograde tracing, DR subregions may preferentially project to different targets.

“Based on retrograde tracing DR subregions may preferentially project to different targets (reviewed in Waselus et al., 2011).”

• cites Waselus et al., 2011 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

About 60% of DR serotonin neurons co-express the vesicular glutamate transporter Vglut3 in the rat and co-release glutamate in the mouse.

“~60% of DR serotonin neurons co-express the vesicular glutamate transporter Vglut3 in the rat (Gras et al., 2002) and co-release glutamate in the mouse (Liu et al., 2014; Sengupta et al., 2017).”

• cites Gras et al., 2002 sourceNot checked yet

  Not checked in the free run.

• cites Liu et al., 2014 sourceNot checked yet

  Not checked in the free run.

• cites Sengupta et al., 2017 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The DR serotonin system has a coordinated spatial and neurochemical organization with respect to its projection targets, although these subdivisions are not absolute.

“Thus, the DR serotonin system has a coordinated spatial and neurochemical organization with respect to its projection targets, although these subdivisions are not absolute.”

May combine two claims.

DRSert→OFC neurons and frontal cortex→DRSert neurons may form a reciprocal loop that promotes active coping under challenge.

“Thus, DRSert→OFC neurons and frontal cortex→DRSert neurons may constitute a reciprocal loop to promote active coping in the face of challenge.”

• cites Warden et al., 2012 sourceNot checked yet

  Not checked in the free run.

• cites Weissbourd et al., 2014 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

Immobility in the forced-swim test is a passive coping strategy and is often used as an indicator of a depression-like state in rodents.

“Immobility in the forced-swim test represents a passive coping strategy in the face of challenge and is often used to indicate a depression-like state in rodent models (Petit-Demouliere et al., 2005).”

• cites Petit-Demouliere et al., 2005 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

DRSert→OFC and DRSert→CeA neurons respond similarly to reward, differently while anticipating an action that leads to reward, and oppositely to punishment.

“These data indicate that DRSert→OFC and DRSert→CeA neurons respond similarly to reward, differently while anticipating an action that leads to reward, and oppositely to punishment.”

• AssumptionRests on an assumption: Mild electrical foot-shock is an aversive or punishing stimulus.
• AssumptionRests on an assumption: Sucrose water is a rewarding stimulus.

Probably not a substantial claim: may not pass S3 (load-bearing).

Selective serotonin reuptake inhibitors are effective in treating depression.

“A recent large-scale meta-analysis provided strong evidence for the efficacy of selective serotonin reuptake inhibitors in treating depression (Cipriani et al., 2018).”

• cites Cipriani et al., 2018 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Our retrograde tracing combined with image registration provides a more comprehensive view of the spatial organization of DR serotonergic projections in the mouse.

“Our retrograde tracing combined with image registration provides a more comprehensive view of the spatial organization of DR serotonergic projections in the mouse (Figure 1).”

May combine two claims.

Collateralization of individual DR serotonin neurons can be extremely broad yet highly specific.

“These analyses further indicate that the collateralization of individual DR serotonin neurons can be extremely broad (e.g., DRSert→OFC neurons also innervate OB anteriorally and ENT posteriorally) yet highly specific (e.g., DRSert→OFC neurons innervate cortical amygdala but avoid the nearby basolateral amygdala).”

May combine two claims.

Activation of DRSert→OFC (but not DRSert→CeA) neurons promotes active coping in the face of challenge.

“Taken together, these data suggest that activation of DRSert→OFC (but not DRSert→CeA) neurons promotes active coping in the face of challenge”

• AssumptionRests on an assumption: Immobility and struggle in the forced-swim test indicate passive and active coping, respectively.

May combine two claims.

The input-output architecture of the DR serotonin system differs from the locus coeruleus norepinephrine system with more homogeneous input and output, and resembles midbrain dopamine systems with biased input and segregated output.

“Thus, the input-output architecture of the DR serotonin system differs from that of the locus coeruleus norepinephrine system with more homogeneous input and output (Schwarz et al., 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.”

• cites Schwarz et al., 2015 sourceNot checked yet

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• cites Beier et al., 2015 sourceNot checked yet

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• cites Lerner et al., 2015 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Each sub-system characterized may still be heterogeneous in composition.

“We want to emphasize that each sub-system we characterized may still be heterogeneous in their composition.”

May combine two claims.

The DR serotonin system contains at least two, and likely more, parallel sub-systems with distinct innervation patterns.

“Thus, the DR serotonin system contains at least two, and likely more, parallel sub-systems with distinct innervation patterns.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Glutamate released by DRSert→OFC neurons might also add to the suppression of movement by DR serotonin neurons.

“Glutamate released from DRSert→OFC neurons may also contribute to this effect.”

May combine two claims.

DRSert→CeA neurons inhibit escape behavior, although a secondary effect on movement cannot be ruled out.

“this suggests that DRSert→CeA neurons inhibit escape behavior, although we cannot rule out this being a secondary effect on movement”

• AssumptionRests on an assumption: Immobility and struggle in the forced-swim test indicate passive and active coping, respectively.

Probably not a substantial claim: may not pass S1 (asserted). May combine two claims.

Activation of DRSert→OFC neurons releases both glutamate and serotonin, and these transmitters have opposing effects on anxiety-like behavior.

“one interpretation is that activation of DRSert→OFC neurons results in release of both glutamate and serotonin that have opposing effects on anxiety-like behavior.”

May combine two claims.

Anatomically segregated DR subsystems have distinct and sometimes opposing functions.

“In conclusion, our behavioral analyses demonstrate that anatomically-segregated DR subsystems have distinct, and sometimes even opposing, functions (Figure 7E).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Other amygdala nuclei, BNST, and PVH have been linked to anxiety.

“DRSert→CeA neurons collateralize to other amygdala nuclei, BNST, and PVH (Figure 2), all of which have been implicated as anxiety-related regions (Calhoon and Tye, 2015).”

• cites Calhoon and Tye, 2015Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

A Bartlett's test indicated that group variances did not differ significantly.

“Bartlett’s test shows no significant difference among variance of groups.”

Classified as probably not a substantial claim.

Axonal labeling density values for individual brain regions were normalized to the region volume and total sample labeling density to remove injection-volume variability.

“Reported values of axonal labeling density for individual brain regions were normalized both to the volume of the region itself and the total labeling density for that sample to eliminate variability due to injection volume.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At the start of each recording session, fluorescence values in the 490-nm and 405-nm channels were approximately matched to ensure accurate fitting and subtraction during analysis.

“At the start of each recording session, fluorescence values in the 490-nm and 405-nm channels were approximately matched to ensure accurate fitting and subtraction during analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The Holm-Sidak procedure was used to adjust p-values for multiple comparisons where needed.

“Multiple comparisons were corrected when appropriate by adjusting pvalues using the Holm-Sidak method.”

May combine two claims.

At a CNO dose of 1 mg/kg, there are no side effects on locomotion/anxiety-related behavior tests, but at 10 mg/kg there are significant side effects.

“According to the most recent report regarding the CNO and clozapine’s influence on locomotion/anxiety related behavior test, there is no side effect when the dose of CNO is 1 mg/kg, but significant side-effect when the dose of CNO is 10 mg/kg.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Histograms of the dependent variables indicated that the data met the normality assumption.

“According to histogram of dependent variable, the distributions of data meet normality assumption.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Animals were randomly assigned to experimental groups.

“Animals were randomly assigned to experimental groups.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CSD analysis uses the second spatial derivative of the LFP to estimate where synaptic current is, its direction, and its size.

“A current source density (CSD) analysis was performed by calculating the second spatial derivative of the LFP (Nicholson and Freeman, 1975). This was used to estimate the magnitude, direction, and location of synaptic current recorded in the LFPs.”

• cites Nicholson and Freeman, 1975Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

One mouse died before completing all the assays.

“One mouse died before all the assays were completed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All male mice used in gain- and loss-of-function behavioral experiments were test naïve for individual behavioral paradigms.

“All male mice used in gain- and loss-of-function behavioral experiments were test naïve for individual behavioral paradigms, and were individually housed with one female partner.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Every mouse that survived the surgical procedure contributed to the analyzed data.

“All the mice that survived the surgery were included in the data analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Decisions about collapsing brain areas were made before analysis and agreed upon by four separate anatomical experts.

“These decisions were made prior to analysis and were agreed upon by four separate anatomical experts.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The analysis did not adjust for possible double counting of cells from consecutive sections, which could overestimate counts depending on cell size.

“We did not adjust for the possibility of double counting cells from consecutive sections, which would result in overestimates/tion, the extent of which would depend on the size of the cells in the regions quantified.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All procedures followed animal care and biosafety guidelines approved by Stanford University's Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety in accordance with NIH guidelines.

“All procedures followed animal care and biosafety guidelines approved by Stanford University’s Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety in accordance with NIH guidelines.”

Classified as probably not a substantial claim.

Optogenetic activation of prefrontal cortical neuron terminals in the DR promotes active coping.

“Optogenetic activation of prefrontal cortical neuron terminals in the DR has previously been shown to promote active coping (Warden et al., 2012).”

• cites Warden et al., 2012 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

In the forced-swim test, sessions were recorded on video and scored independently by two researchers who were blind to genotype.

“The sessions were videotaped and independently analyzed by two researchers blind to genotype.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sample sizes were selected on the basis of those reported in earlier papers.

“Sample sizes were chosen based on those used in previous papers.”

Classified as probably not a substantial claim.

Chemogenetic activation of DR serotonin neurons reduces immobility in the forced-swim test.

“Chemogenetic activation of DR serotonin neurons has recently been reported to reduce immobility in the forced-swim test (Teissier et al., 2015).”

• cites Teissier et al., 2015 sourceNot checked yet

  Not checked in the free run.