The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.

The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.

Embracing diversity in the 5-HT neuronal system.

Neurons that synthesize and release 5-hydroxytryptamine (5-HT; serotonin) express a core set of genes that establish and maintain this neurotransmitter phenotype and distinguish these neurons from other brain cells. Beyond a shared 5-HTergic phenotype, these neurons display divergent cellular properties in relation to anatomy, morphology, hodology, electrophysiology and gene expression, including differential expression of molecules supporting co-transmission of additional neurotransmitters. This diversity suggests that functionally heterogeneous subtypes of 5-HT neurons exist, but linking subsets of these neurons to particular functions has been technically challenging. We discuss recent data from molecular genetic, genomic and functional methods that, when coupled with classical findings, yield a reframing of the 5-HT neuronal system as a conglomeration of diverse subsystems with potential to inspire novel, more targeted therapies for clinically distinct 5-HT-related disorders.

Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2-high versus r2-Pet1Vglut3-high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1Tph2-high and r2-Pet1Vglut3-high neurons likely regulate distinct brain regions and behaviors. Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+/5-HT+ identity; this suggests that some r2-Pet1Vglut3-high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.SIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1+ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1 boutons-those that are VGLUT3+ or VGLUT3+/5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.

Reciprocal Lateral Hypothalamic and Raphe GABAergic Projections Promote Wakefulness.

The lateral hypothalamus (LH), together with multiple neuromodulatory systems of the brain, such as the dorsal raphe nucleus (DR), is implicated in arousal, yet interactions between these systems are just beginning to be explored. Using a combination of viral tracing, circuit mapping, electrophysiological recordings from identified neurons, and combinatorial optogenetics in mice, we show that GABAergic neurons in the LH selectively inhibit GABAergic neurons in the DR, resulting in increased firing of a substantial fraction of its neurons that ultimately promotes arousal. These DRGABA neurons are wake active and project to multiple brain areas involved in the control of arousal, including the LH, where their specific activation potently influences local network activity leading to arousal from sleep. Our results show how mutual inhibitory projections between the LH and the DR promote wakefulness and suggest a complex arousal control by intimate interactions between long-range connections and local circuit dynamics.SIGNIFICANCE STATEMENT: Multiple brain systems including the lateral hypothalamus and raphe serotonergic system are involved in the regulation of the sleep/wake cycle, yet the interaction between these systems have remained elusive. Here we show that mutual disinhibition mediated by long range inhibitory projections between these brain areas can promote wakefulness. The main importance of this work relies in revealing the interaction between a brain area involved in autonomic regulation and another in controlling higher brain functions including reward, patience, mood and sensory coding.

Altered oligodendroglia and astroglia in chronic traumatic encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive tauopathy found in contact sport athletes, military veterans, and others exposed to repetitive head impacts. White matter rarefaction and axonal loss have been reported in CTE but have not been characterized on a molecular or cellular level. Here, we present RNA sequencing profiles of cell nuclei from postmortem dorsolateral frontal white matter from eight individuals with neuropathologically confirmed CTE and eight age- and sex-matched controls. Analyzing these profiles using unbiased clustering approaches, we identified eighteen transcriptomically distinct cell groups (clusters), reflecting cell types and/or cell states, of which a subset showed differences between CTE and control tissue. Independent in situ methods applied on tissue sections adjacent to that used in the single-nucleus RNA-seq work yielded similar findings. Oligodendrocytes were found to be most severely affected in the CTE white matter samples; they were diminished in number and altered in relative proportions across subtype clusters. Further, the CTE-enriched oligodendrocyte population showed greater abundance of transcripts relevant to iron metabolism and cellular stress response. CTE tissue also demonstrated excessive iron accumulation histologically. In astrocytes, total cell numbers were indistinguishable between CTE and control samples, but transcripts associated with neuroinflammation were elevated in the CTE astrocyte groups compared to controls. These results demonstrate specific molecular and cellular differences in CTE oligodendrocytes and astrocytes and suggest that white matter alterations are a critical aspect of CTE neurodegeneration.

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex.

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei.SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO2Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.

cKit+ cardiac progenitors of neural crest origin.

The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKit(CreERT2/+) and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNC(kit)). CNC(kit) possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKit(CreERT2)-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNC(kit) is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit(+) cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNC(kit) contribution to myocardium.

Partial Raphe Dysfunction in Neurotransmission Is Sufficient to Increase Mortality after Anoxic Exposures in Mice at a Critical Period in Postnatal Development.

Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hßactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk. SIGNIFICANCE STATEMENT: Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.

THSD1 (Thrombospondin Type 1 Domain Containing Protein 1) Mutation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: A ruptured intracranial aneurysm (IA) is the leading cause of a subarachnoid hemorrhage. This study seeks to define a specific gene whose mutation leads to disease. METHODS: More than 500 IA probands and 100 affected families were enrolled and clinically characterized. Whole exome sequencing was performed on a large family, revealing a segregating THSD1 (thrombospondin type 1 domain containing protein 1) mutation. THSD1 was sequenced in other probands and controls. Thsd1 loss-of-function studies in zebrafish and mice were used for in vivo analyses and functional studies performed using an in vitro endothelial cell model. RESULTS: A nonsense mutation in THSD1 was identified that segregated with the 9 affected (3 suffered subarachnoid hemorrhage and 6 had unruptured IA) and was absent in 13 unaffected family members (LOD score 4.69). Targeted THSD1 sequencing identified mutations in 8 of 507 unrelated IA probands, including 3 who had suffered subarachnoid hemorrhage (1.6% [95% confidence interval, 0.8%-3.1%]). These THSD1 mutations/rare variants were highly enriched in our IA patient cohort relative to 89 040 chromosomes in Exome Aggregation Consortium (ExAC) database (P<0.0001). In zebrafish and mice, Thsd1 loss-of-function caused cerebral bleeding (which localized to the subarachnoid space in mice) and increased mortality. Mechanistically, THSD1 loss impaired endothelial cell focal adhesion to the basement membrane. These adhesion defects could be rescued by expression of wild-type THSD1 but not THSD1 mutants identified in IA patients. CONCLUSIONS: This report identifies THSD1 mutations in familial and sporadic IA patients and shows that THSD1 loss results in cerebral bleeding in 2 animal models. This finding provides new insight into IA and subarachnoid hemorrhage pathogenesis and provides new understanding of THSD1 function, which includes endothelial cell to extracellular matrix adhesion.

The choroid plexus and cerebrospinal fluid: emerging roles in development, disease, and therapy.

Although universally recognized as the source of cerebrospinal fluid (CSF), the choroid plexus (ChP) has been one of the most understudied tissues in neuroscience. The reasons for this are multiple and varied, including historical perceptions about passive and permissive roles for the ChP, experimental issues, and lack of clinical salience. However, recent work on the ChP and instructive signals in the CSF have sparked new hypotheses about how the ChP and CSF provide unexpected means for regulating nervous system structure and function in health and disease, as well as new ChP-based therapeutic approaches using pluripotent stem cell technology. This minisymposium combines new and established investigators to capture some of the newfound excitement surrounding the ChP-CSF system.

Islet1 derivatives in the heart are of both neural crest and second heart field origin.

RATIONALE: Islet1 (Isl1) has been proposed as a marker of cardiac progenitor cells derived from the second heart field and is utilized to identify and purify cardiac progenitors from murine and human specimens for ex vivo expansion. The use of Isl1 as a specific second heart field marker is dependent on its exclusion from other cardiac lineages such as neural crest. OBJECTIVE: Determine whether Isl1 is expressed by cardiac neural crest. METHODS AND RESULTS: We used an intersectional fate-mapping system using the RC::FrePe allele, which reports dual Flpe and Cre recombination. Combining Isl1(Cre/+), a SHF driver, and Wnt1::Flpe, a neural crest driver, with Rc::FrePe reveals that some Isl1 derivatives in the cardiac outflow tract derive from Wnt1-expressing neural crest progenitors. In contrast, no overlap was observed between Wnt1-derived neural crest and an alternative second heart field driver, Mef2c-AHF-Cre. CONCLUSIONS: Isl1 is not restricted to second heart field progenitors in the developing heart but also labels cardiac neural crest. The intersection of Isl1 and Wnt1 lineages within the heart provides a caveat to using Isl1 as an exclusive second heart field cardiac progenitor marker and suggests that some Isl1-expressing progenitor cells derived from embryos, embryonic stem cultures, or induced pluripotent stem cultures may be of neural crest lineage.

Potentiation of active locomotor state by spinal-projecting serotonergic neurons.

Animals produce diverse motor actions that enable expression of context-appropriate behaviors. Neuromodulators facilitate behavioral flexibility by altering the temporal dynamics and output of neural circuits. Discrete populations of serotonergic (5-HT) neurons target circuits in the brainstem and spinal cord, but their role in the control of motor behavior is unclear. Here we define the pre- and post-synaptic organization of the spinal-projecting serotonergic system and define a role in locomotor control. We show that while forebrain-targeting 5-HT neurons decrease their activity during locomotion, subpopulations of spinal projecting neurons increase their activity in a context-dependent manner. Optogenetic activation of ventrally projecting 5-HT neurons does not trigger initiation of movement, but rather enhances the speed and duration of ongoing locomotion over extended time scales. These findings indicate that the descending serotonergic system potentiates locomotor output and demonstrate a role for serotonergic neurons in modulating the temporal dynamics of motor circuits.

Altered 5-HT2A/C receptor binding in the medulla oblongata in the sudden infant death syndrome (SIDS): Part II. Age-associated alterations in serotonin receptor binding profiles within medullary nuclei supporting cardiorespiratory homeostasis.

The failure of chemoreflexes, arousal, and/or autoresuscitation to asphyxia may underlie some sudden infant death syndrome (SIDS) cases. In Part I, we showed that some SIDS infants had altered 5-hydroxytryptamine (5-HT)2A/C receptor binding in medullary nuclei supporting chemoreflexes, arousal, and autoresuscitation. Here, using the same dataset, we tested the hypotheses that the prevalence of low 5-HT1A and/or 5-HT2A/C receptor binding (defined as levels below the 95% confidence interval of controls-a new approach), and the percentages of nuclei affected are greater in SIDS versus controls, and that the distribution of low binding varied with age of death. The prevalence and percentage of nuclei with low 5-HT1A and 5-HT2A/C binding in SIDS were twice that of controls. The percentage of nuclei with low 5-HT2A/C binding was greater in older SIDS infants. In >80% of older SIDS infants, low 5-HT2A/C binding characterized the hypoglossal nucleus, vagal dorsal nucleus, nucleus of solitary tract, and nuclei of the olivocerebellar subnetwork (important for blood pressure regulation). Together, our findings from SIDS infants and from animal models of serotonergic dysfunction suggest that some SIDS cases represent a serotonopathy. We present new hypotheses, yet to be tested, about how defects within serotonergic subnetworks may lead to SIDS.

A Phox2b::FLPo transgenic mouse line suitable for intersectional genetics.

Phox2b is a transcription factor expressed in the central and peripheral neurons that control cardiovascular, respiratory, and digestive functions and essential for their development. Several populations known or suspected to regulate visceral functions express Phox2b in the developing hindbrain. Extensive cell migration and lack of suitable markers have greatly hampered studying their development. Reasoning that intersectional fate mapping may help to overcome these impediments, we have generated a BAC transgenic mouse line, P2b::FLPo, which expresses codon-optimized FLP recombinase in Phox2b expressing cells. By partnering the P2b::FLPo with the FLP-responsive RC::Fela allele, we show that FLP recombination switches on lineage tracers in the cells that express or have expressed Phox2b, permanently marking them for study across development. Taking advantage of the dual-recombinase feature of RC::Fela, we further show that the P2b::FLPo transgene can be partnered with Lbx1(Cre) as Cre driver to generate triple transgenics in which neurons having a history of both Phox2b and Lbx1 expression are specifically labeled. Hence, the P2b::FLPo line when partnered with a suitable Cre driver provides a tool for tracking and accessing genetically subsets of Phox2b-expressing neuronal populations, which has not been possible by Cre-mediated recombination alone.

Development of brainstem 5-HT1A receptor-binding sites in serotonin-deficient mice.

The sudden infant death syndrome is associated with a reduction in brainstem serotonin 5-hydroxytryptamine (5-HT) and 5-HT(1A) receptor binding, yet it is unknown if and how these findings are linked. In this study, we used quantitative tissue autoradiography to determine if post-natal development of brainstem 5-HT(1A) receptors is altered in two mouse models where the development of 5-HT neurons is defective, the Lmx1b(f/f/p) , and the Pet-1⁻/⁻ mouse. 5-HT(1A) receptor agonist-binding sites were examined in both 5-HT-source nuclei (autoreceptors) and in sites that receive 5-HT innervation (heteroreceptors). In control mice between post-natal day (P) 3 and 10, 5-HT(1A) receptor binding increased in several brainstem sites; by P25, there were region-specific increases and decreases, refining the overall binding pattern. In the Lmx1b(f/f/p) and Pet-1⁻/⁻ mice, 5-HT(1A)-autoreceptor binding was significantly lower than in control mice at P3, and remained low at P10 and P25. In contrast, 5-HT(1A) heteroreceptor levels were comparable between control and 5-HT-deficient mice. These data define the post-natal development of 5-HT(1A)-receptor binding in the mouse brainstem. Furthermore, the data suggest that 5-HT(1A)-heteroreceptor deficits detected in sudden infant death syndrome are not a direct consequence of a 5-HT neuron dysfunction nor reduced brain 5-HT levels. To elucidate the developmental relationship between serotonin (5-HT) levels and 5-HT(1A) receptors in the brainstem, we examined 5-HT(1A) binding in two 5-HT-deficient mouse models. In nuclei containing 5-HT neurons, 5-HT(1A) binding was decreased (autoreceptors), while binding was maintained in projection sites (heteroreceptors). Thus, brainstem 5-HT(1A)-heteroreceptor-binding sites do not appear developmentally sensitive to reduced brain 5-HT levels.

Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation.

The hindbrain roof plate and choroid plexus are essential organizing centers for inducing dorsal neuron fates and sustaining neuron function. To map the formation of these structures, we developed a broadly applicable, high resolution, recombinase-based method for mapping the fate of cells originating from coordinates defined by intersecting combinations of expressed genes. Using this method, we show that distinct regions of hindbrain roof plate originate from discrete subdomains of rhombencephalic neuroectoderm expressing Wnt1; that choroid plexus, a secretory epithelium important for patterning later-formed hindbrain structures and maintaining neuron function, derives from the same embryonic primordium as the hindbrain roof plate; and that, unlike the floor plate, these dorsal organizing centers develop in a patterned, segmental manner, built from lineage-restricted compartments. Our data suggest that the roof plate and choroid plexus may be formed of functional units that are capable of differentially organizing the generation of distinct neuronal cell types at different axial levels.

The knockout mouse project.

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Identification of an essential spinoparabrachial pathway for mechanical itch.

The sensation of itch is a protective response that is elicited by either mechanical or chemical stimuli. The neural pathways for itch transmission in the skin and spinal cord have been characterized previously, but the ascending pathways that transmit sensory information to the brain to evoke itch perception have not been identified. Here, we show that spinoparabrachial neurons co-expressing Calcrl and Lbx1 are essential for generating scratching responses to mechanical itch stimuli. Moreover, we find that mechanical and chemical itch are transmitted by separate ascending pathways to the parabrachial nucleus, where they engage separate populations of FoxP2PBN neurons to drive scratching behavior. In addition to revealing the architecture of the itch transmission circuitry required for protective scratching in healthy animals, we identify the cellular mechanisms underlying pathological itch by showing the ascending pathways for mechanical and chemical itch function cooperatively with the FoxP2PBN neurons to drive chronic itch and hyperknesis/alloknesis.

Projections and interconnections of genetically defined serotonin neurons in mice.

Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets.

Redefining the serotonergic system by genetic lineage.

Central serotonin-producing neurons are heterogeneous-differing in location, morphology, neurotoxin sensitivity and associated clinical disorders-but the underpinnings of this heterogeneity are largely unknown, as are the markers that distinguish physiological subtypes of serotonergic neurons. Here we redefined serotonergic subtypes on the basis of genetic programs that are differentially enacted in progenitor cells. We uncovered a molecular framework for the serotonergic system that, having genetic lineages as its basis, is likely to have physiological relevance and will permit access to genetically defined subtypes for manipulation.