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-Cori

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Peer review for Alex P.

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By Alex Tomceac

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https://drive.google.com/file/d/1MkqtvzfJ9wpLo0sogvdNzKJR0Ol2fqbk/view?usp=sharing

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Ashley Vega Rough Draft

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Please fill in the table.

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Cells within this brain area release endogenous opioids to inhibit pain. Deep brain stimulation of this area is sometimes used as a way to treat chronic pain.

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https://www.neuroscientificallychallenged.com/blog/know-your-brain-reticular-formation

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Graphical Abstract

INTRODUCTION

Dopamine (DA) neuron dysfunction has been implicated in numerous brain disorders, including addiction, depression, schizophrenia, and Parkinson's disease. Our incomplete understanding of the complex brain circuits in which dopamine neurons participate represents a major obstacle to developing more sophisticated hypotheses and improved treatments for these disorders. In the mammalian brain, the majority of DA neurons are clustered in two adjacent midbrain regions, the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Initial in vivo electrophysiological studies suggested that midbrain DA neurons were a homogenous population, uniformly excited by rewards or reward-predictive cues, and unaffected or transiently inhibited by aversive events ( Mirenowicz and Schultz 1996; Ungless et al., 2004). These findings were consistent with behavioral evidence demonstrating an unequivocal relationship between DA transmission and the reinforcing effects of natural and drug rewards ( Wise and Rompré, 1989). However, the simplifying assumption that DA neurons functioned as a homogenous unit was called into question as data emerged that were inconsistent with this account. Although most DA neurons were activated by rewards and reward-predictive cues, some were activated by noxious or salient stimuli ( Brischoux et al., 2009; Matsumoto and Hikosaka, 2009; Zweifel et al., 2011). Furthermore, important roles for DA neurons were uncovered in numerous behavioral or psychological processes other than reward, including salience, aversion, fear, working memory, and movement coordination ( Bromberg-Martin et al., 2010; Jin and Costa, 2010; Zweifel et al., 2011; Lammel et al., 2012; Matsumoto and Takada, 2013). Heterogeneity was also detected at the cellular level, as subpopulations of DA neurons were found to have distinct intrinsic molecular and electrophysiological properties ( Margolis et al., 2006a, 2008; Lammel et al., 2008, 2011).

While a consensus is emerging that DA neurons are best conceptualized as functionally heterogeneous subpopulations capable of influencing diverse behavioral states ( Bromberg-Martin et al., 2010; Roeper, 2013; Marinelli and McCutcheon, 2014), the underlying organizational principles that account for this heterogeneity remain unclear. Such principles would be especially useful in understanding the function of the VTA, which is cytochemically more diverse than the SNc ( Margolis et al., 2006b). In addition to DA neurons, the VTA contains neurons that release GABA, glutamate, and their various combinations, all of which form local and long-range connections ( Swanson, 1982; Fields et al., 2007; Morales and Root, 2014; Root et al., 2014). The anatomical location of a DA neuron's synaptic inputs and/or outputs may be a key determinant of its intrinsic properties and behavioral roles ( Lammel et al., 2008; Margolis et al., 2008; Lammel et al., 2011, 2012). Thus, a comprehensive map detailing both the input and output connections of VTA-DA neurons would be of great value in deducing principles of midbrain circuit function.

Previous studies have sought to separately identify either the sources of synaptic inputs to VTA neurons ( Phillipson, 1979; Carr and Sesack, 2000; Zahm et al., 2011; Watabe-Uchida et al., 2012), or the projection targets of these cells ( Beckstead et al., 1979; Swanson, 1982). However, these experiments have two major limitations. First, synaptic inputs to the two main classes of VTA neurons (DA and GABA neurons) have not been comparatively evaluated at the whole-brain level. VTA-DA and VTA-GABA neurons have distinct firing patterns in vivo ( Cohen et al., 2012), and their optogenetic activation produces opposing behavioral effects ( Tsai et al., 2009; Tan et al., 2012). Thus, a systematic, comparative analysis of synaptic input onto VTA-DA and VTA-GABA neurons could be informative in elucidating general organizational principles that permit interdigitated neurons to make distinct contributions to brain function.

A second limitation has been an inability to systematically link information about a VTA-DA neuron's inputs with its projection targets. While such input-output relationships can be established in ultrastructural studies (e.g., Carr and Sesack, 2000), the methods involved are labor-intensive and thus cannot practically be scaled up to elucidate whole-brain input-output maps. Accordingly, only a handful of three-node circuits involving VTA neurons have been identified, undoubtedly representing a small fraction of such connections.

To address these limitations, we utilized rabies virus-based and cell type-specific monosynaptic tracing techniques ( Wickersham et al., 2007; Callaway and Luo, 2015), as well as a viral-genetic strategy that permits whole-brain mapping of input-output relationships of genetically defined neuronal populations ( Schwarz et al., 2015). These methods allowed for the generation of a comprehensive, high-resolution map of the input and output connectivity of VTA-DA neurons, thereby revealing previously unappreciated connections, including an anterior cortex→VTA-DA→nucleus accumbens (NAc) circuit. Using optogenetics, electrophysiology, and in vivo pharmacology, we verify the functional relevance of this circuit by demonstrating its role in positive reinforcement. Collectively, our data demonstrate that the input-output relationships of VTA-DA neuron subpopulations are complex yet decipherable. They provide a critical roadmap for the development of more sophisticated and testable hypotheses about the neural circuit substrates that mediate the diverse behavioral functions of the VTA, in particular its DA neurons, in adaptive and pathological behaviors.

RESULTS

VTA-DA and VTA-GABA Neurons Receive Direct Input from Similar Brain Regions

Rabies-mediated transsynaptic tracing has previously been used to map direct inputs to DA neurons in two major midbrain nuclei, the SNc and VTA ( Watabe-Uchida et al., 2012). To comprehensively map input and output through the VTA, we first extended these studies by comparing direct inputs to both VTA-DA and VTA-GABA neurons, and then examined the cell types in the input regions that synapse onto these two VTA populations.

To restrict our analysis specifically to VTA-DA and VTA-GABA neurons we used DAT-Cre mice, in which Cre mimics the expression pattern of the plasma membrane dopamine transporter ( Bäckman et al., 2006; Lammel et al., 2015), and GAD2-Cre mice, in which Cre mimics the expression of glutamic acid decarboxylase-2 ( Taniguchi et al., 2011), an enzyme that coverts glutamate to GABA ( Figure S1). Two adeno-associated viruses (AAVs), expressing respectively a Cre-dependent TVA (the receptor for viruses containing the avian EnvA envelope glycoprotein) fused with mCherry (TC) and a Cre-dependent rabies glycoprotein (G), were injected into the VTA of these mice () ( Miyamichi et al., 2013). Two weeks later, we injected EnvA-pseudotyped, G deleted, and GFP-expressing rabies virus (RV dG). RV dG could only infect TC-expressing mammalian cells due to the EnvA pseudotype. Co-expression of G enabled TC-expressing neurons to complement RV dG and produce infectious rabies viruses that spread to their presynaptic partners. Almost all starter cells (defined by co-expression of TC and GFP) in the DAT-Cre mice expressed tyrosine hydroxylase (TH, a marker for DA neurons) (; quantified in inset) whereas few starter cells in the VTA of GAD2-Cre mice co-expressed TH (; quantified in inset). Control experiments demonstrated that the rabies-mediated transsynaptic tracing depended on Cre recombinase, TC, and G expression ( Figure S2). Together, these results validated the cell-type specificity for input tracing.

We comparatively evaluated the sources of synaptic inputs to VTA-DA and VTA-GABA neurons by examining the distribution of GFP-positive neurons across different brain regions. Consistent with previous results ( Watabe-Uchida et al., 2012), VTA-DA ( Figure 1D-O ) and VTA-GABA (data not shown) neurons received direct synaptic inputs from diverse neuronal types in a wide range of brain regions. We quantified input neurons in 22 brain regions (see Supplemental Experimental Procedures for details), and calculated the proportion of inputs from each region for both DAT-Cre- and GAD2-Cre-based tracing (; Table S1). VTA-GABA neurons received proportionally more inputs from the anterior cortex and central amygdala (CeA), while VTA-DA neurons received more inputs from the paraventricular hypothalamus (PVH) and lateral hypothalamus (LH), although none of these differences were significant when corrected for multiple comparisons. These results suggest that VTA-DA and VTA-GABA neurons receive largely similar input from different brain regions.

VTA-DA and VTA-GABA Neurons Receive Input from Diverse Cell Types

To explore whether differences may exist at the cellular level within each input region, we combined in-situ hybridization (ISH) with rabies tracing ( Takatoh et al., 2013; Weissbourd et al., 2014) on three selected input regions: the PVH, LH, and dorsal raphe (DR), each of which contains heterogeneous neuronal populations that are implicated in reward-related behaviors.

In the PVH (), we used probes for genes encoding the precursors for the neuropeptides oxytocin and arginine vasopressin (AVP), which have been implicated in several behavioral processes including reward, fear, and anxiety ( Stoop, 2012). However, it is unknown whether neurons producing these peptides directly synapse onto VTA neurons. Co-labeling of RV dG-GFP with probes corresponding to oxytocin or AVP was observed when tracing inputs to both VTA-DA and VTA-GABA neurons (). For those GFP⁺ PVH neurons that synapsed onto VTA-DA neurons, ~6% each were oxytocin⁺ and AVP⁺ (). These fractions increased to ~13% each for oxytocin⁺ and AVP⁺ neurons that synapse onto VTA-GABA neurons ().

In the LH (), we used probes corresponding to the neuropeptides neurotensin (NT) and hypocretin (orexin), which also have been linked to reward-related behaviors ( Korotkova et al., 2003; Kempadoo et al., 2013). NT⁺ and orexin⁺ neurons were labeled by RV dG-GFP in both DAT-Cre and GAD2-Cre mice, indicating that they synapse directly onto VTA-DA and VTA-GABA neurons (). Interestingly, both NT⁺ and orexin⁺ neurons exhibited a two-fold preference for DA neurons ().

The DR contains heterogeneous neuronal populations that utilize different neurotransmitters including serotonin, GABA, and glutamate ( Weissbourd et al., 2014) and has recently been shown to send a reward signal to the VTA, largely through neurons that co-express both vGluT3 (a marker for a subset of glutamatergic neurons) and Tph2 (a serotonin neuron marker) ( Liu et al., 2014; McDevitt et al., 2014). To determine the neurochemical identity of DR cells that synapse onto VTA-DA and VTA-GABA neurons, we used ISH probes designed to detect either (1) all 3 vesicular glutamate transporters (vGluT1/2/3), markers of excitatory glutamatergic neurons, (2) both glutamic acid decarboxylases (GAD1/2), markers of inhibitory GABAergic neurons, or (3) Tph2. All three populations contributed a large fraction of DR input to both VTA-DA and VTA-GABA neurons, with similar relative preferences ( Figure 2G-I ).

Taken together, these results indicate that VTA neurons receive complex synaptic inputs from excitatory, inhibitory, and modulatory (serotonergic and peptidergic) neurons. These inputs synapse onto both VTA-DA and VTA-GABA neurons with some quantitative differences in their preferences.

TRIO and cTRIO Analyses Reveal Distinct Inputs onto VTA Neurons Based on Their Output

Previous studies have demonstrated considerable heterogeneity in the properties of VTA-DA neurons that correlates with the location to which VTA-DA neurons project ( Lammel et al., 2008; Margolis et al., 2008). To investigate whether DA neurons projecting to different targets receive inputs from different brain areas, we utilized recently developed TRIO (for Tracing Relationships between Input and Output) and cTRIO (for cell-type-specific TRIO) techniques ( Schwarz et al., 2015). TRIO relies on the axonal uptake of CAV-Cre, a canine adenovirus vector expressing Cre recombinase that efficiently transduces axon terminals ( Soudais et al., 2001). RV dG-mediated input tracing based on CAV-Cre injected at a projection site can reveal inputs to projection-defined VTA subpopulations ( Figure S3A). cTRIO further refines input tracing to VTA-DA neurons that project to a specific output site by utilizing CAV-FLExloxP-Flp that expressed a Cre-dependent Flp recombinase in conjunction with AAVs expressing Flp-dependent TC and G in DAT-Cre mice (). Four major targets of DA neurons were selected for CAV injections: two subdivisions of the ventral striatum-lateral or medial nucleus accumbens (NAcLat, NAcMed), medial prefrontal cortex (mPFC), and amygdala (Amy) (; Figure S3B-E).

The starter cell locations in TRIO and cTRIO experiments were consistent with a topographic organization of VTA-DA neurons based on their projection targets ( Lammel et al., 2008). Along the medial-lateral axis, starter cells for NAcLat-projecting neurons were more laterally distributed within the VTA compared to starter cells for NAcMed-projecting neurons, for example (, S3F, S4). Quantification of VTA starter cells based on TH staining revealed that these four groups contained different fractions of DA neurons in the TRIO experiments, ranging from ~90% for NAcLat-projecting to <50% for mPFC- and Amy-projecting VTA neurons ( Figure S3C), consistent with previous studies using conventional tracers ( Swanson, 1982). By contrast, >90% of the starter cells for all four groups were TH+ in cTRIO experiment (), validating the neurochemical specificity of the cTRIO strategy.

Both TRIO and cTRIO analyses revealed quantitative differences in the inputs onto VTA neurons according to their outputs ( Figure S3H, ); the variation in cTRIO experiments was smaller than for TRIO ( Table S2) likely because starter cells were defined not only by projection but also cell type. We focus our discussion on cTRIO experiments because of the clear DA neuron identity. Notably, NAcLat-projecting VTA-DA neurons received proportionally more input from the anterior cortex, dorsal striatum, and nucleus accumbens core (NAcCore) and lateral shell (NAcLatS), but less input from the DR, than the three other VTA-DA populations. In contrast, NAcMed-projecting DA neurons received preferential input from the NAc medial shell (NAcMedS) at the expense of dorsal striatum (). These experiments demonstrate that VTA-DA neurons that project to different output sites receive biased input from selective regions, notably dorsal raphe and different striatal regions.

NAcMed- and NAcLat-projecting DA Neurons Have Distinct Arborization Patterns

A striking finding from the TRIO and cTRIO analyses is that VTA-DA neurons that project to subdivisions of the ventral striatum (NAcMed vs. NAcLat) receive highly biased inputs from different brain regions. This suggests that these projections originate from largely different populations of DA neurons. Indeed, individual VTA-DA neurons have been suggested to innervate one selective projection target based on lack of co-labeling of retrograde tracers injected into different VTA projection sites ( Fallon, 1981; Swanson, 1982; Margolis et al., 2006a; Lammel et al., 2008). However, this methodology may not detect all axonal collateralizations because potential target regions can only be sampled 1-3 at a time. Additionally, incomplete coverage of the retrograde tracer within each sampled region may reduce the probability of detecting axon collateralization. To overcome these limitations, we employed an intersectional strategy to label VTA-DA neurons that project to a specific output target () that allowed us to visualize directly axonal arbors of anatomically-defined DA neurons, permitting a more comprehensive analysis of the axonal collateralization (or lack thereof) of specific DA neuron subpopulations.

We injected CAV-FLExloxP-Flp into the NAcLat or NAcMed of DAT-Cre mice and an AAV expressing Flp-dependent membrane-tethered GFP (mGFP) into the VTA. This allowed examination of the axonal arborization pattern of NAcLat- or NAcMed-projecting VTA-DA neurons. Quantification of these axonal projections in 10 brain regions that contained projections from at least one of the four DA neuron subtypes examined revealed that the axon arbors of the NAcLat-projecting DA neurons largely avoided the NAc medial shell, while projecting more laterally and broadly in the striatum including the dorsal striatum ( Figure 4B-C ; quantified in ). By contrast, axonal arbors of NAcMed-projecting VTA-DA neurons were highly enriched in the NAc medial shell (, quantified in ) but largely avoided the NAc lateral shell. In total, NAcLat-projecting DA neurons had three-fold broader arborizations in the striatum than NAcMed-projecting DA neurons (). Very few axonal arbors were observed in the mPFC or central amygdala from either condition. Interestingly, while the axonal arbors of NAcLat-projecting DA neurons were largely confined to the striatum, NAcMed-projecting DA neurons sent significant collaterals outside the striatum, including the septum and ventral pallidum, indicating that this DA subpopulation is capable of simultaneously influencing neural activity in multiple brain regions.

Electrophysiological Examination of Synaptic Connections Predicted by cTRIO

cTRIO analysis of the input-output relationships of VTA-DA neurons revealed several previously unrecognized connectivity patterns. To examine whether they represent functional connections, we performed ex vivo electrophysiological assays, in which we optically activated inputs expressing channelrhodopsin-2 (ChR2) while recording from identified DA neurons projecting to specific targets labeled with retrobeads ( Lammel et al., 2012).

Previous work suggested that lateral habenula (LHb) neurons do not make synaptic connections onto VTA-DA neurons projecting to the NAcLat ( Lammel et al., 2012) whereas cTRIO analysis suggests that LHb inputs comprised ~3% of all inputs to this population. To investigate this discrepancy, we injected an AAV expressing ChR2 into the LHb and red retrobeads into the NAcLat of transgenic mice in which DA neurons were GFP⁺ (). Whole-cell recordings were made from acute slices containing VTA neurons that were both green (DA⁺) and red (NAcLat-projecting). Photostimulation-evoked synaptic responses were observed in 2 of 26 recorded neurons ( Figure 5B-D ), which were blocked by the AMPA receptor antagonist CNQX. These observations are consistent both with previous observations that the LHb input to NAcLat-projecting VTA-DA neurons is sparse (0 of 4 cells; Lammel et al., 2012) and with our cTRIO results showing that such a connection exists. The location of responding neurons may explain why previous electrophysiology studies may have missed this connection, as the responding cells were located more ventrally () than expected for NAcLat-projecting DA neurons ( Lammel et al., 2008). This example also illustrates that rabies-based transsynaptic techniques have the sensitivity to uncover relatively rare connections that could be missed using other techniques.

Another surprising cTRIO result is that the anterior cortex, including the mPFC, makes monosynaptic connections onto NAc-projecting DA neurons (). Previous electron microscopy studies using classical tracers suggested that mPFC inputs to the VTA synapse onto mPFC-projecting but not onto NAc-projecting DA neurons ( Carr and Sesack, 2000). To investigate this discrepancy, we performed similar electrophysiological recordings to those just described but injected the AAV expressing ChR2 into the mPFC. 5 of 37 NAc-projecting VTA-DA neurons showed a time-locked response to photostimulation ( Figure 5B-D ), functionally validating a pathway identified by cTRIO. As our input tracing data demonstrated that anterior cortical input to VTA-DA neurons also arose from other frontal cortical regions besides the mPFC ( Figure S5), in separate experiments we injected the AAV expressing ChR2 into broader anterior cortical regions, including the insular and orbitofrontal cortices in addition to the mPFC. In these mice, 19 of 36 NAc-projecting VTA-DA neurons showed a time-locked response to photostimulation that was blocked by CNQX. These connections also displayed a higher mean EPSC amplitude (28.4±7.7 pA for anterior cortex versus 14.9±3.0 pA for mPFC, ), likely due to more anterior cortical cells expressing ChR2. Thus, signals arising from the anterior cortex are capable of influencing neural activity in a large proportion of NAcLat-projecting VTA-DA neurons.

The Anterior Cortex→VTA-DA→NAcLat Circuit Is Reinforcing

We next asked what role the anterior cortex→VTA-DA→NAcLat circuit could play in behavior. As anterior cortex provided excitatory input onto NAcLat-projecting VTA-DA neurons in our slice recordings, we hypothesized that activation of anterior cortical inputs to the VTA in vivo would lead to DA release in the NAcLat, which would be reinforcing. To test this hypothesis, we injected mice with an AAV expressing either ChR2-eYFP (n=7) or eYFP alone (n=8) into the anterior cortex (including the mPFC, insular, and orbitofrontal cortices), and implanted optical fibers targeted just dorsal to the VTA ( Figure 6A-B ; Figure S6). After waiting 2-3 months, mice were subjected to an intracranial self-stimulation (ICSS) procedure during which they were allowed to respond freely at 5 adjacent nosepoke ports for photostimulation. Responses at 4 ports resulted in photostimulation of anterior cortex→VTA projections at varying stimulation frequencies (1, 5, 10, or 20 Hz) for 2s; responses at a 5 th control port did not result in any stimulation (). ChR2-eYFP-expressing mice developed a strong preference for the 20-Hz port over successive training days, making significantly more responses at this port than at the control port that did not produce stimulation (). In contrast, eYFP-expressing control mice did not show this preference ( Figure 6E-F ).

To determine whether the reinforcing effects of the anterior cortex→VTA projection stimulation required DA release in the NAcLat, we combined site-specific pharmacology with optogenetic manipulation. In a subset of high-responding ChR2-eYFP-expressing mice (n=5), we infused varying doses of flupenthixol, a non-selective DA receptor antagonist, or saline vehicle, into the NAcLat via previously implanted cannulas targeting this area (see Supplemental Experimental Procedures; ; Figure S6). Flupenthixol infusion into the NAcLat prior to ICSS behavioral sessions resulted in a dose-dependent decrease in responding for 20-Hz stimulation of anterior cortex→VTA projections (), consistent with our hypothesis that optically-evoked DA release in NAcLat was a critical mediator of the ICSS behavior we observed. Flupenthixol infusion also reduced locomotion during ICSS sessions (). However, the latency to initiate response at the 20-Hz nosepoke port was unaffected (), indicating that mice were physically capable of making nosepoke responses under the influence of flupenthixol. Importantly, because behavioral sessions did not begin until >10 min after drug infusions were complete, DA receptors in the NAcLat were likely to be fully blocked at the beginning of the session when latency measurements were recorded.

Taken together, these data suggest that the anterior cortex makes direct, excitatory synaptic connections onto VTA-DA neurons projecting to the NAcLat, and that activation of this circuit is reinforcing.

DISCUSSION

We have utilized rabies-mediated transsynaptic tracing and TRIO/cTRIO to dissect the complex circuits in which midbrain VTA-DA neurons participate. Our unbiased anatomical mapping revealed the global input-output architecture of the majority of VTA-DA neurons. We validated two previously unknown circuits with electrophysiology and identified a behavioral function of one of the new circuits. Below we discuss the technical and conceptual implications of these findings.

Technical Considerations

Recent transsynaptic tracing techniques have permitted systematic, high-resolution analyses of inputs based on the location, genetic identity (cell type), or axonal projection of starter cells ( Callaway and Luo, 2015). cTRIO simultaneously leverages all of these targeting strategies, thereby allowing access to more specific starter cell populations for dissecting complex circuits, such as those involving VTA-DA neurons. A previous method of labeling inputs based on outputs of VTA-DA neurons relied on RV dG injection directly into an output site, and trans-complementing the virus in VTA-DA neurons by expressing G under the control of TH-Cre ( Lammel et al., 2012). A caveat of this method is that if RV dG spread among DA neurons, input-output specificity would be compromised, as all TH-Cre-expressing neurons would be competent to initiate transsynaptic tracing. As TRIO and cTRIO limit TC and G expression only to neurons projecting directly to the target site, the input-output relationship is preserved even if VTA-DA neurons are locally connected. In addition, TRIO and cTRIO produced about ten-fold more efficient labeling than the previous method ( Lammel et al., 2012), thus identifying more comprehensive monosynaptic inputs to projection-based VTA-DA subpopulations.

The electrophysiological validations of new connections identified in our cTRIO experiments highlight the sensitivity of rabies-mediated transsynaptic tracing techniques. While previous electrophysiological ( Lammel et al., 2012) and EM studies ( Carr and Sesack, 2000) did not identify the LHb→VTA-DA→NAcLat or the mPFC→VTA-DA→NAcLat circuit, their presence in our cTRIO experiments motivated a further examination. Our observations support the hypothesis that the LHb sends a weak input to the NAcLat-projecting VTA-DA neurons. We also identified and validated direct synaptic connections from the mPFC onto VTA-DA neurons projecting to the NAc.

While EM and slice electrophysiology are powerful methods for studying circuit architecture and function, they are less suited for large-scale connectivity analyses. TRIO/cTRIO provide a global "scaffold" to identify input-output architecture of a region and/or cell types of interest. The properties and function of the circuits can be further investigated using electrophysiology and optogenetics, as exemplified in our study. These combined approaches can be applied to many other cell types and circuits in the mammalian brain.

Architecture of VTA-DA Circuitry

Our whole-brain transsynaptic tracing from VTA-DA and VTA-GABA neurons have extended previous findings ( Watabe-Uchida et al., 2012). We observed that both types of neurons receive inputs from a wide variety of brain regions, with overall similar distributions (). This is reminiscent of our recent input-tracing study in the dorsal raphe (DR), where serotonin and GABA neurons receive qualitatively similar input from diverse brain regions ( Weissbourd et al., 2014). This common feature could be used for input to VTA or DR GABA neurons to serve as feed-forward inhibition to regulate the activity of DA or serotonin neurons. As is the case for DR-serotonin and -GABA neurons, VTA-DA and -GABA neurons receive diverse direct input from excitatory, inhibitory, and modulatory neurons, including prominent direct input from DR serotonin and GABA neurons (), highlighting the complex regulation of these neuromodulatory systems and their rich interactions.

In addition to the complex input patterns, VTA-DA neurons project to diverse output sites that appear to correlate with their intrinsic properties, cell body location, and, in one specific example, input preference ( Lammel et al., 2008, 2011, 2012). Our TRIO/cTRIO analyses have provided a comprehensive overview of the input-output relationships of multiple subtypes of VTA-DA neurons projecting to four principal output sites: the nucleus accumbens (NAcMed, NAcLat), medial prefrontal cortex, and amygdala. Our data confirmed a correlation between VTA output sites and the location of the cell soma within the VTA ( Figure S4). At the same time, these data have produced a more nuanced picture of the input-output relationships of this complex structure than the discrete pathways suggested by a previous study ( Lammel et al., 2012), likely reflecting the sensitivity of the TRIO techniques (see above). Qualitatively, VTA-DA neurons projecting to all output sites receive input from all 22 brain regions examined in this study. Quantitatively, however, input patterns can be highly biased depending on the output sites ( & S3). NAcLat-projecting VTA-DA neurons have the most distinct input patterns compared with all three other groups of VTA-DA neurons. Furthermore, our axonal arborization analysis () demonstrates that NAcLat-projecting VTA-DA neurons have little overlap with NAcMed-projecting VTA-DA neurons in their striatal target regions, and that NAcMed-projecting VTA-DA neurons have previously unknown extra-striatal collateralization. Thus, our data support a biased-input/discrete output architecture for VTA-DA circuitry. A similar input-output architecture also applies to SNc-DA circuitry ( Lerner et al., 2015; companion paper).

As the major output site of VTA-DA neurons and a crucial structure implicated in reward processing and drug addiction, the nucleus accumbens has been intensively studied. However, NAc-projecting DA neurons have been considered a homogeneous population. Our data demonstrate that NAcMed- and NAcLat-projecting VTA-DA neurons represent two discrete populations with distinct targets and receive differential inputs. NAcLat-projecting VTA-DA neurons preferentially receive input from the anterior cortex and striatal regions, whereas NAcMed-projecting VTA-DA neurons preferentially receive input from the dorsal raphe (). Together with the differential gene expression and firing patterns, these observations strongly suggest that these DA neuron subpopulations are integrated into different circuits and serve distinct biological functions. These findings highlight the importance of differentiating DA neuron subpopulations and NAc sub-regions where possible in future studies.

The observations of discrete outputs and specific input-output relationships for VTA-DA neurons contrast with the architecture of locus coeruleus norepinephrine (LC-NE) circuits. LC-NE neurons received similar inputs regardless of where they project, and projection-defined subpopulations of LC-NE neurons also project to all brain regions examined ( Schwarz et al., 2015). While these catecholamine circuits have sometimes been treated as complementary systems, with the DA neurons projecting to the striatum and NE neurons elsewhere, their input-output structures are markedly different. The NE circuit architecture is consistent with a role to "broadcast" a general message throughout the brain, which may be optimal for coordinating brain states such as sleep and arousal. The DA system is better suited for sending discrete signals. For example, subpopulations of VTA-DA neurons have previously been found to differentially encode reward and aversion in mice ( Lammel et al., 2012) and non-human primates ( Matsumoto and Hikosaka, 2009). Indeed, the circuit architecture of using parallel channels to encode discrete information may be evolutionarily conserved; recent studies have identified unique populations of Drosophila DA neurons carrying appetitive or aversive signals that project to discrete zones of the mushroom body, an insect center for learning and memory ( Aso et al., 2014).

A Top-Down Reinforcement Signal

In this study, we identified a behavioral function of a previously unappreciated connection from the anterior cortex to NAcLat-projecting VTA-DA neurons. While prior work demonstrated that altering activity in the PFC changes DA levels in the striatum (e.g., Karreman and Moghaddam, 1996), other data suggested that a direct mPFC→VTA-DA→NAcLat circuit did not exist ( Carr and Sesack, 2000). Our cTRIO studies revealed a monosynaptic connection from the anterior cortex to NAcLat-projecting DA neurons, a finding that we confirmed with ex vivo electrophysiological recordings from identified NAcLat-projecting VTA-DA neurons. Using optogenetics and behavioral pharmacology, we identified a functional role for this circuit in mediating positive reinforcement. Thus, our data suggest that frontal cortical regions are capable of exerting top-down control of midbrain neurons that contribute to motivated behaviors via DA release in the NAc, bypassing other intermediary regions such as the LDT and striatum to directly engage DA neurons and exert executive control over a powerful neuromodulatory system.

In summary, our data add to a growing body of evidence in favor of a model wherein functionally distinct midbrain DA neuron subtypes encode different signals and participate in largely separate yet interconnected circuits through their biased input and discrete output pathways. However, much remains to be understood. For example, investigating the circuit properties of NAc inputs onto both DA and GABA neurons (feedback and feed-forward inhibition), and defining the message encoded in the firing patterns of each of these DA neuron subpopulations in response to various natural stimuli, will be necessary to understand the normal and pathophysiological functions of these important circuits.

EXPERIMENTAL PROCEDURES

Mice and Viral Procedures

DAT-Cre ( Bäckman et al., 2006), tdTomato Cre reporter Ai14 ( Madisen et al., 2010), and C57Bl/6 mice were obtained from the Jackson Laboratories, and GAD2-Cre mice ( Taniguchi et al., 2011) were from Josh Huang. Mice were housed on a 12-hour light/dark cycle with food and water ad libitum. Viral vectors were prepared as previously described ( Schwarz et al., 2015). Detailed procedures for rabies tracing, TRIO, cTRIO and axonal arborization are described in Supplemental Experimental Procedures. All procedures followed animal care and biosafety guidelines approved by Stanford University's Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety.

Electrophysiology

Six-week old DAT-Cre mice were injected with AAV₁-CMV-FLEx-GFP into the VTA, rhodamine-conjugated retrobeads into NAcLat, and AAVDJ-hsyn1-ChR2-H134R-eYFP into the LHb, mPFC, or anterior cortex. 8 weeks later, recordings were done as previously described ( Lammel et al., 2011). Excitatory postsynaptic currents (EPSCs) were recorded in whole-cell voltage clamp; series resistance and input resistance were monitored on-line with a 4-mV hyperpolarizing step (50 ms). Neurons were voltage-clamped at -60 mV to record AMPAR EPSCs. For drug application, CNQX (10 μM) was added to the ACSF. Connections were defined as neurons that exhibited an average current (over 5 sweeps) of ≥10 pA recorded either at −60 mV or +40 mV.

Behavior

AAV₅-hsyn1-eYFP or AAV₅-hsyn1-ChR2-eYFP (0.5 μL) was infused bilaterally into the anterior cortex of C57Bl/6 mice. Optical fibers were implanted dorsal to the VTA, and cannulas were implanted targeting the NAcLat. >8 weeks following virus infusions, ChR2-eYFP mice and eYFP controls were given 5 daily ICSS training sessions during which they could respond at 5 nosepoke ports. A response at each port produced 2s of 473nm light, pulsed at a particular frequency (20, 10, 5, 1, or 0 Hz). Following ICSS training, a subset of high-responding ChR2-eYFP mice was used for drug infusion studies. These mice received bilateral 0.25 μL infusions of either flupenthixol (F114, Sigma, dissolved in saline) or saline vehicle through the implanted guide cannula 10 minutes prior to ICSS sessions.

Statistical Analysis

Results of all statistical analyses for tracing experiments are provided in Table S2. For each input brain region, paired t-tests were used for input tracing and ISH experiments, and one-way ANOVAs were used for TRIO and cTRIO data, with correction for multiple comparisons. (We did not use 2-way ANOVA because the data from different brain regions did not meet equal variance assumption.) For starter cell distributions, t-tests were used with corrections for multiple comparisons. Behavioral data were analyzed with parametric or non-parametric one-way repeated-measures ANOVAs followed by Holm-Sidak or Tukey post-hoc tests.

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Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping

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Dopamine (DA) neuron dysfunction has been implicated in numerous brain disorders, including addiction, depression, schizophrenia, and Parkinson's disease. Our incomplete understanding of the complex brain circuits in which dopamine neurons participate represents a major obstacle to developing more sophisticated hypotheses and improved treatments for these disorders.

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Function: part of the basal ganglia. Works with the striatum to inhibit movement.

Projections:
- Input goes into pars reticula. Received from striatum, globus pallidus (indirect), subthalamic nucleus.
- Output originates from pars compacta. Goes to thalamus (GABA) & striatum (dopamine).

Cell types:
- Initial brief trough cells: rapid firing
- Initial prolonged trough cells: longer inactivity troughs, dopaminergic
- Bursty cells: fire burts of variable length, probably interneurons.

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Function: Descending motor tracts (corticospinal ,cotricobulbar) for motor movement
Special Cell Types: Tracts are myelinated (oligodendrocytes) which come from pyramidal cells
Cortical Projections: Corticospinal: Primary motor cortex to spinal cord
Corticobulbar: Pre-central gyrus to cranial nerve nuclei

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Stellate cells provide excitatory input to other superior colliculus (SC) cell types, including wide-field (WF) cells, and Rorb-Cre horizontal cells make local inhibitory connections

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Stellate cells provide excitatory input to other superior colliculus (SC) cell types, including wide-field (WF) cells, and Rorb-Cre horizontal cells make local inhibitory connections

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visual, auditory and somatosensory information are integrated to initiate motor commands
receives projections from 85% to 90% of the retinal ganglion cells

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The PAG is the gray matter located around the cerebral aqueduct within the tegmentum of the midbrain. Projects to the nucleus raphe magnus and contains descending autonomic tracts. Receives ascending pain and temperature information by the spinomesencephalic tract from the spinothalamic tract

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-Function: able to receive information from the environment and then use that information to initiate a behavioral response appropriate to the current environmental context

1. A structure on the tectum that receives neural projections directly from the retina.

2. The primary area of the brain thought to be involved in saccadic eye movements.

3. The area of the brain thought to be involved in hand-eye coordination

Multi-sensory orientation

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Autonomic function, motivated behavior, and behavioral responses to threatening stimuli.

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Cell types: multipolar, fusiform, stellate, and pyramidal neurons
2 words: Pain Management

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- Function:
motor control: control and coordinate gait, subcortical centers of the extrapyramidal motor system. Contains iron, hemoglobin, and ferritin which gives it its red color. Integrates signals from motor cortex and cerebellum and projects to spinal cord interneurons and motor neurons through the rubrospinal tract.
- Cell Types:
stellate or fusiform cells (small, medium, and large spiny neurons)
-Subcortical and Cortical Projections:

Ventrolateral RN: Neurons projecting to the sacral cord

Dorsomedial RN: Neurons projecting to the upper part of the spinal cord

Dorsal RN: Neurons projecting to the medullary lateral tegmentum

Caudal & Rostral RN: Contralaterally projecting rubro-bulbospinal neurons to the medullary lateral tegmental field

Recieves projections from the trigeminal nucleus and the interpositus nucleus of the cerebellum

-2 words: Gait Coordination

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(highlighted in red)

Function:
Primarily serves for alertness (attention) and sleep; promotes arousal and consciousness.

Special Cell Types:
Noradrenergic neurons (locus ceruleus), cholinergic neurons (pedunculopontine nucleus, laterodorsal tegmental nucleus), Raphe nuclei (Raphe cells)

Projections:

Afferent: Spinothalamic tract, spinoreticular tract, medial lemniscus

Efferent: Reticulospinal tract, thalamus

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Functions:
Reward system
Behavior Reinforcement
Motivation
Addiction

Cell types: Dopaminergic neurons

Subcortical and Cortical Projections:  nucleus accumbens (NAc) and medial prefrontal cortices (mPFC)

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Great job putting together the study guide!!

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Great job putting together the study guide!!

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Here is a link to a really good video library from Dr. Susan Stensaas.  Check out the one on the unfixed brain.  It'll surprise you how soft the brain actually is.

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More About the Neuroanatomy Video Lab: Brain Dissections
The series originated when teaching with a neurologist in Ghana and Kenya where it was clear when anatomy was taught and separated from the clinic by many years, much was not retained. Here Dr. Stensaas presents just enough anatomy to localize the patient's problem. These videos can be used before or after a clinical case presentation. Apologies are offered for a few minor errors that could not be corrected due to the one time recording opportunity as well as an unscripted personal style. The audience for these videos can be nurses, physician assistants, medical students, residents, registrars, house officers, or anyone interested in the brain. All of the videos have English captions to assist those for whom English is not their native language or to visualize the many new terms. Videos can be downloaded and repurposed in a variety of formats for those lacking bandwidth or Internet connectivity or who wish to embed the videos in other presentation software. They may be shared, copied, distributed and put on local servers, but not republished to commercial video distribution systems without permission. Dr. Stensaas is indebted to the Eccles Health Sciences Library of the School of Medicine for the videography and server support, to Pat Brennan for additional videography, and to Paul Burrows from Teaching and Learning Technologies at the University of Utah for creating and maintaining this NeuroLogic Exam website. Finally I acknowledge neurologists Dr. David Renner and Dr. Leonard Jarcho who showed which anatomy was most important in their practice of medicine. Suzanne S. Stensaas, Ph.D.
Professor Emeritus
Department of Neurobiology and Anatomy
University of Utah School of Medicine

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correlate human brain structure and function

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Brain Dissections: Neuroanatomy Video Lab

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Neuroanatomy Video Lab: Brain Dissections -- This series of Neuroanatomy video lessons with brain dissections has two principal objectives. The first is to provide viewers access to human brain specimens, something lacking in many places. The second is to simplify the anatomy, omitting some details, and making numerous generalizations.

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Here is a nice mini review on pericytes in response to Forrest's question in class today.

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Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, Bethesda, MD, United States

Robert W. Franz Cancer Center, Providence Portland Medical Center, Earle A. Chiles Research Institute, Portland, OR, United States

Pericytes are mural cells surrounding blood vessels, adjacent to endothelial cells. Pericytes play critical roles in maturation and maintenance of vascular branching morphogenesis. In the central nervous system (CNS), pericytes are necessary for the formation and regulation of the blood-brain barrier (BBB) and pericyte deficiency accompanies CNS diseases including multiple sclerosis, diabetic retinopathy, neonatal intraventricular hemorrhage, and neurodegenerative disorders. Despite the importance of pericytes, their developmental origins and phenotypic diversity remain incompletely understood. Pericytes express multiple markers and the origin of pericytes differs by tissue, which may cause difficulty for the identification and understanding of the ontogeny of pericytes. Also, pericytes have the potential to give rise to different tissues in vitro but this is not clear in vivo. These studies indicate that pericytes are heterogeneous in a tissue- and context- dependent manner. This short review focuses on recent studies about identification of pericytes, heterogeneous origin of pericytes during development and in adults, and the differentiation capacity of pericytes, and pericytes in pathological settings.

Introduction

Pericytes are mural cells surrounding blood vessels and embedded within the basement membrane of the vasculature and adjacent to endothelial cells ( 1). Pericytes cover microvessels such as arterioles, venules and capillaries, while large-diameter vessels like arteries and veins are covered by vascular smooth muscle cells (VSMCs), the other type of mural cells. Mural cells are known to play fundamental roles in vascular network formation: pericyte coverage is critical for vascular stability and structure. In the central nervous system (CNS), pericytes play an essential role in the maturation and maintenance of the blood brain barrier (BBB) ( 2- 4), which is established by the interaction between the microvessels (endothelial cells and pericytes) and surrounding astrocytes within the neurovascular unit. The BBB is a diffusion barrier which blocks the inflow of various molecules and toxins from blood to brain, but not all CNS vessels equally contribute to the BBB: several areas of the brain including the pituitary gland, pineal gland, subventricular zones and choroid plexus are not protected by the BBB. The diversity of the BBB architecture and function remains largely unexplored. Given that pericyte density, morphology, and function vary in different vascular beds ( 5), these observations may reflect their heterogeneous characteristics. In this review, we give an overview of pericytes with a focus on their heterogeneity. A more objective definition of different subsets of pericytes may help to shed light on novel therapeutic approaches for neuro-vascular diseases associated with pericyte loss such as stroke and Alzheimer's disease.

Identification of Pericytes

Morphological characteristics of pericytes were defined by electron microscopy: pericytes possess a cell body with a prominent nucleus and contain a small amount of cytoplasm with several long processes covering the endothelial wall. Pericytes are embedded in the basement membrane where they interact with endothelial cells: pericyte-endothelial cell interaction enhances basement membrane assembly (Figure 1).

Figure 1. Morphology of pericytes. (A) Schematic image of pericyte-endothelial cell interaction. (B) Whole-mount immunostaining of mouse embryonic skin with antibodies to pericytes (NG2, red) and endothelial cells (PECAM-1, blue) at embryonic day (E) 15.5. Pericytes are semicircular shape and cover blood vessels. (C) Triple immunostaining of E15.5 mouse embryonic brain with antibodies to pericytes (NG2, red; PDGFRβ, green) and endothelial cells (PECAM-1, blue). Scale bars, 50 μm.

Although the ultrastructural characteristics of pericytes has been well studied using electron microscopy, pericytes remain a relatively poorly defined cell type, without highly specific markers available for their identification ( 6). The following markers have been used as pericyte markers, but no general pan-pericyte molecular marker has been discovered because of their heterogeneous distributions and functions in various tissues ( 2, 5, 7). We should note that these markers are also expressed by other cell types including perivascular fibroblasts, VSMCs and macrophages. Chondroitin sulfate proteoglycan4/neural glial antigen 2 (NG2) is expressed on the surface of pericytes during angiogenesis ( 8, 9), but is also expressed on glial precursor O2A cells in the CNS, which generate either oligodendrocytes or astrocytes in vitro. Platelet-derived growth factor receptor beta (PDGFRβ) ( 10, 11) is one of the most widely studied molecular marker expressed in pericytes. PDGF-B/PDGFRβ signaling is essential for pericyte proliferation and recruitment to blood vessels. Alpha smooth muscle actin (αSMA) ( 12), desmin ( 13), and vimentin ( 14) are contractile filaments. Regulator of G protein signaling 5 (RGS5) ( 15, 16) is a GTPase activating protein for Giα and Gqα, and the expression pattern of RGS5 overlaps with expression pattern of NG2 and PDGFRβ. CD146, also known as melanoma cell adhesion molecule (MCAM) has been used as a marker for pericytes and VSMCs as well as endothelial cells ( 17). Recent studies suggest that CD146 regulates PDGFRβ activation and involves in BBB integrity ( 18) and is essential for pericyte recruitment ( 19). CD13/aminopeptidase N (APN) ( 20) is a membrane-bound metalloprotease which was originally identified as a myeloid cell marker ( 21). CD13 has been used as a surface marker for brain pericytes ( 22, 23). Overall, current immunohistochemical approaches to identify pericytes use antibodies against these proteins, depending on tissue and microvessel types. The expression of these markers varies depending on developmental stages, organs, pathological situations, and in vitro or in vivo conditions. Therefore, anatomical characteristics combined with at least two molecular markers are important to define pericytes. Indeed, recent studies have revealed that double PDGFRβ- EGFP and NG2-DsRed fluorescence reporter expression patterns are valuable for pericyte/mural cell identification of the CNS tissues ( 24, 25). Although the expression of αSMA is known as a marker for both pericytes and VSMCs, capillary pericytes do not express αSMA ( 12): capillary pericytes in embryonic skin express only NG2 and PDGFRβ but not αSMA ( 26). In contrast, capillary pericytes in tumors do express αSMA ( 27).

Genetic mouse models including transgenic markers, fluorescent reporters and lineage tracing lines are valuable tools to trace the pericyte lineage during development and in pathological conditions: nuclear β-galactosidase reporter ( 28) as well as emerging fluorescent reporter and lineage tracing lines using the promoter of PDGFRβ ( 24, 29), NG2 ( 24, 30, 31), or Tbx18 ( 32) are available. Alternatively, a fluorescent Nissl dye specifically labels brain pericytes and enables the imaging in the live mouse ( 33).

Developmental Origin of Pericytes

The developmental origin of pericytes is heterogeneous, and much remains to be deciphered. Most commonly described, and best understood is their origin from mesenchymal stem cells ( 34). Chick-quail chimera analysis ( 35, 36) and genetic lineage tracing experiments using neural crest-specific Cre recombinase lines such as Wnt-1-Cre and Sox10-Cre mice in combination with a Cre-mediated reporter line demonstrate that neural crest contributes to pericytes in the face, brain, and thymus ( 36- 40). Using similar genetic lineage tracing experiments, the origin of pericytes in the gut ( 41), lung ( 42), and liver ( 43) in mice has been traced to the mesothelium, a single layer of squamous epithelium. In the heart, the epicardial mesothelium gives rise to coronary pericytes and VSMCs ( 44- 46). Recent studies have demonstrated that some endocardium also contributes to coronary pericytes ( 47). These studies clearly indicate that the origin of pericytes is heterogeneous in a tissue- and context- dependent manner.

We have recently revealed that myeloid progenitor cells differentiate into a subset of pericytes in the ectoderm-derived skin and brain during development (Figure 2) ( 26). Using high-resolution whole-mount imaging and a series of genetic lineage tracing experiments with hematopoietic cell-specific Vav-Cre and myeloid cell-specific CD11b-Cre lines in combination with a Cre-mediated fluorescent reporter line, we found that the developmental sources of pericytes are heterogeneous and some pericytes are derived from myeloid progenitors in the developing skin and brain. Mutant mice lacking myeloid lineage exhibit defective pericyte development. Moreover, TGF-β promotes the differentiation of myeloid progenitors into pericytes in vitro and in vivo. In a similar line of research, some CD31⁺ F4/80⁺ macrophages contribute to cerebrovascular pericytes during embryogenesis ( 48). Insight into the heterogeneous origins of pericytes will have important implications for understanding the establishment of the organ-specific vascular networks during embryonic angiogenesis. Moreover, whether pericytes of different origins have different functions in these tissues remains to be elucidated.

Figure 2. Heterogeneity in the origin of pericytes. Pericytes in both endoderm and mesoderm-derived organs, such as lung, liver, gut, and heart have mesoderm origin (orange). Pericytes in the aorta region have several origins: secondary heart field, neural crest, and somite (asterisk). Previous studies have demonstrated that ectoderm-derived neural crest give rise to pericytes in brain and thymus (green). Our group has recently shown that mesoderm-derived myeloid progenitor cells differentiate into pericytes in ectoderm-derived organs such as skin and brain through TGF-β signaling during development.

It is an intriguing question of whether pericytes of heterogeneous origins at embryonic stages remain in adult: one possible scenario is that a subtype of pericytes may become dominant in adult. Moreover, adult tissue-resident progenitors contribute to pericytes in pathological situations. Indeed, mesenchymal stem cells generate pericytes after radiation therapy ( 49), while mesenchymal tumors such as bone and soft tissue sarcomas can be derived from pericytes ( 50). Glioblastoma stem cells generate pericytes to support tumor growth ( 51). Defining the origin of adult tissue pericytes needs the development of a cell-type specific inducible Cre recombinase line such as CreER. Transient Cre activity produces fluorescent reporter positive cells at a defined developmental time point and then allows for the tracking of their progeny in adults. The inducible lineage tracing experiment also allows us to examine whether pericytes of different origins could differentially contribute to the neovascularization processes in pathological conditions such as tumor angiogenesis and wound healing.

Pericytes in Adult and Diseases

Pericytes have been reported as a component of stem cell niches and mesenchymal stem cells. In the bone marrow, NG2⁺ pericytes in arterioles promote hematopoietic stem cell (HSC) quiescence and are important for HSC maintenance ( 52). Likewise, NG2⁺/Nestin⁺ pericytes associate with portal blood vessels in fetal liver and are required for the HSC niche ( 53). In the adult brain, neural stem cells (NSCs) located in the largest germinal region of the forebrain, the ventricular-subventricular zone (V-SVZ). Endothelial cells and pericytes in the V-SVZ form the NSC niche, and V-SVZ pericytes secrete diffusible factors that increase the proliferation and enhance neuronal differentiation ( 22). In vitro differentiation capacity of pericytes into mesenchymal cell types (e.g., adipocytes, chondrocytes, osteoblasts, fibroblasts, VSMCs) has been proven in a multitude of studies ( 54- 58). Pericytes facilitate repair process after myocardial infarction in the heart ( 59, 60), while pericytes regenerate injured and dystrophic skeletal muscles ( 61). In vivo lineage tracing experiments have reported pericytes as progenitors of white adipocytes ( 62), follicular dendritic cells ( 63), odontoblasts (mesenchyme-derived dentine producing cells) ( 64), and skeletal muscle ( 61). Differentiation capacities of pericytes into neurons, astrocytes, and oligodendrocytes have been also reported ( 56). In addition, pericytes have been reported to play a major role as fibroblast progenitors in fibrotic responses ( 65).

Cardiac pericytes have been studied as a therapeutic target after injury. Cardiac pericytes account for up to 5% of the total non-cardiomyocyte cell population ( 66). Recent studies identified microvascular pericytes in the human ventricular myocardium and demonstrated that human cardiac pericytes express mesenchymal stem/stromal cell markers including CD44, CD73, CD90, and CD105 ( 58). Indeed, cardiac pericytes have the capacity to differentiate into mesodermal lineage: osteo-, chondro-, and adipogenesis, but no potential for skeletal myogenesis in vitro ( 58). Many cardiac diseases are associated with fibrosis, an accumulation of fibroblasts and an excess of extracellular matrix proteins which may affect the architecture and function of the organ or the tissue. Pericytes have been shown to contribute to fibrosis organ-dependently ( 67): NG2⁺/Nestin − type 1 pericytes are recruited and accumulated in the ischemic interstitial space around fibrotic tissue but do not contribute to fibrosis ( 67).

However, a series of lineage tracing experiments with Tbx18CreERT2 line, which is selectively expressed by pericytes and VSMCs in multiple adult organs, has revealed that adult pericytes of heart, brain, skeletal muscle and fat depots do not behave as multipotent progenitors in aging and different pathological situations such as a high-fat diet and injury. In addition, pericytes do not contribute to fibroblasts in fibrotic responses ( 32). These results are in contrast to the previous studies that demonstrated the multipotent potential of pericytes. The discrepancies might be explained by the differing methods used in the studies to identify pericytes as well as the different behavior of in vitro and in vivo pericytes. The population of pericytes which can differentiate into mesenchymal cell types may vary by organ and developmental stage in vivo. It is also possible that pericyte differentiation requires stem cell-like cells of non-pericyte origin ( 64).

Conclusion

The absence of any single marker to identify pericytes complicates the study of the ontogeny and the differentiation capacity of pericytes. Much remains to be elucidated regarding how and when the origin of pericytes in an organ is determined and whether pericytes have potential for differentiation in vivo. There are questions about how pericytes derived from different origins behave when they co-exist in a tissue: do they show identical gene expression patterning and behavior in adult tissues under normal and pathological conditions? Future studies using pericyte-specific lineage tracing mice and precise gene-expression profiling will pave the way for the understanding of the spatial-temporal pericyte behavior in vivo and may contribute to new therapeutic strategies for diseases associated with pericyte loss and/or dysfunction.

Author Contributions

Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Funding

YM is supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute (HL005702-11). TY was supported by the Japan Society for the Promotion of Science (JSPS) NIH-KAITOKU.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank K. Gill for editorial help and laboratory management, and R. Reed for administrative assistance.

References

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29. Cuervo H, Pereira B, Nadeem T, Lin M, Lee F, Kitajewski J, et al. PDGFRbeta-P2A-CreER T2 mice: a genetic tool to target pericytes in angiogenesis. Angiogenesis (2017) 20:655-62. doi: 10.1007/s10456-017-9570-9

31. Hill RA, Patel KD, Goncalves CM. Grutzendler J, Nishiyama A, Modulation of oligodendrocyte generation during a critical temporal window after NG2 cell division. Nat Neurosci. (2014) 17:1518-27. doi: 10.1038/nn.3815

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35. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development (2001) 128:1059-68.

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57. Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res. (1998) 13:828-38. doi: 10.1359/jbmr.1998.13.5.828

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Tissue Specific Origin, Development, and Pathological Perspectives of Pericytes

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Pericytes are mural cells surrounding blood vessels, adjacent to endothelial cells. Pericytes play critical roles in maturation and maintenance of vascular branching morphogenesis. In the central nervous system (CNS), pericytes are necessary for the formation and regulation of the blood-brain barrier (BBB) and pericyte deficiency accompanies CNS diseases including multiple sclerosis, diabetic retinopathy, neonatal intraventricular hemorrhage, and neurodegenerative disorders.

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The corpus callosum gets its blood supply from a branch of the anterior communicating artery (median callosal or subcallosal artery), from the pericallosal artery (distal part of the anterior cerebral artery) and from the posterior pericallosal artery (usually a branch of the posterior cerebral artery)

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Study Guide is finished for all lectures we did so far. 

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Study Guide is finished for all lectures we did so far. 

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04 - The Meninges (15 minutes): The epidural, subdural and subarachnoid spaces are demonstrated and discussed with respect to trauma and disease. The relationship of the brainstem and cerebellum to the tentorium demonstrates the vulnerability of the brain stem to increased supratentorial pressure and herniation. Arachnoid granulations and the sagittal sinus are shown. A subdural hematoma specimen as well as sections from a ruptured aneurysm complete the demonstration. (Suzanne S. Stensaas, Ph.D., University of Utah)

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04 - The Meninges (15 minutes): The epidural, subdural and subarachnoid spaces are demonstrated and discussed with respect to trauma and disease. The relationship of the brainstem and cerebellum to the tentorium demonstrates the vulnerability of the brain stem to increased supratentorial pressure and herniation. Arachnoid granulations and the sagittal sinus are shown.

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Coronal view

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Coronal and sagittal view of corpus callosum

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Executive functions:
-impulse control
-adjust to complex behaviors
-focus
-planning

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Memory and Learning

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Feeling guilt or remorse, and to interpret reality, are dependent on a well-functioning prefrontal cortex

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Integrates motor, sensory, and cognitive performance between the two hemispheres of the brain

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Allows sounds to be distinguished and the sources of sounds to be identified.

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Involved in:
- Arousal
- Autonomic responses associated with fear
- Emotional responses
- Hormonal secretions
- Memory

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Responsible for voluntary motor control