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Serotonergic neurons modulate a wide range of behaviors and functions, from mood and aggression to vital autonomic processes like heart rate, respiratory dynamics, and body temperature. We hypothesize that this broad scope reflects the collective actions of many functionally and molecularly distinct subtypes of serotonergic neurons, each with specialized roles in different neural processes. Supporting this idea are examples of heterogeneity among serotonergic neurons with respect to developmental origin, biophysical properties, and molecular expression; yet deciphering the functional and behavioral relevance of these differences has been challenging. In order to better understand serotonergic system organization, we have developed and applied a set of mouse genetic tools to subdivide serotonergic neurons into groups based on molecular criteria, and then to query these subtypes for differences with respect to biophysical properties, hodology, gene expression, and whole animal function. We applied these tools in a stage-wise fashion, from neural system en masse, as reference, and then to specific serotonergic neuron subtypes. From this, we have established that serotonergic neurons play key roles in at least two life-sustaining reflexes - the respiratory chemoreflex (breathing modulation to keep tissue PCO2/pH within physiological limits) and body temperature regulation. We found that chemoreflex modulation, but not body temperature regulation, maps to a specific serotonergic neuron subtype - that subtype with a developmental history of Egr2 gene expression. Further, in brain slice preparations, we found that this subtype is chemosensitive, increasing firing rate in response to conditions of hypercapnic acidosis. Thus, in vivo, Egr2-serotonergic neurons likely transduce chemosensory information into action potential firing to increase respiratory drive and ultimately breathing. Further, we found that Egr2-serotonergic neurons project selectively to respiratory nuclei involved in PCO2/pH sensory signal transduction, but not primary respiratory motor nuclei. This indicates that the serotonergic system has distinct sensory and motor divisions - another unexpected finding. In summary, these results establish a previously unappreciated functional modularity and organization to the serotonergic system, and open up potential for tailored function-specific therapeutic strategies, for example here as relates to disorders of respiratory homeostasis or thermoregulation.

The mammalian neocortex is an exquisite, highly organized brain structure composed of hundreds of subpopulations of neurons and glia, precisely connected to enable motor control, sensory perception, information integration, and planning. Unique molecular, structural, and anatomical neuronal properties underlie diverse functionality, endowing much of the neocortex’s complex processing power. Neocortical size correlates with information processing capacity, suggesting that increased neuronal number and diversity begets increased sophistication. One excitatory projection neuron type, callosal projection neurons (CPN), has disproportionately expanded with cortical size increase. CPN directly connect homotypic regions of the two neocortical hemispheres by sending axons via the largest white matter fiber tract in the brain, the corpus callosum (CC), allowing quick relay, integration, and comparison of information. In humans, the CC contains over 300,000 axons, CPN have been centrally implicated in autism spectrum disorders, and absence or surgical disruption of CPN connectivity in humans is associated with defects in abstract reasoning, problem solving, and generalization. Therefore, CPN are critical to complex brain functions, and their diversity likely contributes to these roles. Work presented in this dissertation addresses molecular controls over CPN development, specifically genes that are expressed by, and function in, particular subpopulations of CPN. While much progress has been made in identifying molecular controls over neocortical arealization, lamination, and broad subtype specification, CPN diversity has remained largely unaddressed. Therefore, this work begins by identifying genes more highly expressed in CPN than other closely related projection neuron populations, and uncovers molecular diversity within CPN. From this molecular diversity, functional analysis of three candidate molecular controls over CPN subtype diversity follows. Cited2 acts broadly in neocortical progenitor development and postnatally in refining somatosensory CPN identity. Caveolin1 identifies a population of CPN with dual axonal projections. Tmtc4 is mutated in human CC disease and can function in CPN axonal development. These analyses of CPN molecular diversity in mouse then expand to an investigation of which molecular subpopulations are conserved, expanded, or uncommon between rodent and primate, allowing both for comparative evolutionary theories of CPN function, and indicating which CPN populations critical for human brain function can be best studied in rodent models.

The Met receptor tyrosine kinase (RTK) is expressed in the mammary gland under both normal and neoplastic conditions. Overexpression of the Met receptor is found in 15--20% of human breast cancers and is correlated with shortened disease-free interval and overall survival. In order to explore the role of dysregulated Met receptor signaling on the development of mammary tumors I have characterized a transgenic mouse model that expresses either wild type or a dysregulated Met receptor in the mammary epithelium under the control of the mouse mammary tumor virus promoter/enhancer (MMTV-Met). The Met receptor variants contained a mutation that results in decreased receptor ubiquitination and prolonged receptor signaling (Y1003F) or an activating mutation that was originally observed in patients with papillary renal carcinoma (M1250T) or both mutations (YF/MT). In vitro and in vivo transformation assays demonstrated that each mutation singly is weakly transforming, however, there was an additive effect on transformation when both mutations were present. This additive effect was observed in the transgenic mice where multiparous MMTV-Met-YF/MT mice developed tumors earlier and with much greater penetrance than did mice expressing either of the single mutants. This provides the first in vivo model that demonstrates a role for ubiquitination in suppression of transforming activity of an RTK. MMTV-Met-YF/MT tumors displayed a range of histological phenotypes but were mainly comprised of luminal lineage cells. Notably, MMTV-Met-M1250T tumors contained cells from both the basal and luminal populations, suggesting transformation of a progenitor cell. Progenitor cell transformation in RTK transgenic mouse models is uncommon and highlights distinct signaling differences and potentially lineage specificity of the two Met mutants. / Through assays of overexpression in vivo and inhibition in vitro, Met receptor signaling has been correlated with the development of the mammary gland. To examine the effects of loss of Met receptor signaling on mammary gland development I have utilized the Cre/LoxP1 recombination system to knock-out the Met receptor from the mammary epithelium. Mammary-specific Cre recombinase efficiently excised floxed DNA as visualized by activation of a beta-galactosidase reporter In Met+/+ glands, however, few beta-galactosidase positive cells are retained In the Mefl/fl glands and an intermediate number are retained in the Met fl/+ glands. This indicates that Met-null cells are selected against and supports a role for Met in the development of the mammary gland.

Glutamate is the essential neurotransmitters in pain pathways. The discovery of the vesicular glutamate transporters (VGLUT1-3) has been a fundamental step on the way to describe glutamate-dependent pain pathways. We used the Cre-lox system to construct conditional knockouts with deficient Vglut2 transmission in specific neuronal populations. We generated a Vglut2f/f;Ht-Pa-Cre line to selectively delete Vglut2 from the peripheral nervous system. These Vglut2 deficient mice showed decreased acute nociceptive responses and were less prone to develop an inflammatory state. They did not develop cold allodynia, or heat hyperalgesia and were less hypersensitive to mechanical stimuli in the PSNL chronic pain model. Further analyses of genes with altered expression after nerve injury, revealed candidates for future studies of chronic pain biomarkers. Interestingly, the Vglut2f/f;Ht-Pa-Cre mice developed an elevated itch behavior. To investigate more specific neuronal populations, we analyzed mice lacking Vglut2 in the Nav1.8 population, as inflammatory hyperalgesia, cold pain, and noxious mechanosensation have been shown to depend upon Nav1.8Cre positive sensory neurons. We showed that deleting Vglut2 in Nav1.8Cre positive neurons abolished thermal hyperalgesia in persistent inflammatory models and responses to noxious mechanical stimuli. We also demonstrated that substance P and VGLUT2-dependent glutamatergic transmission are co-required for the development of formalin-induced inflammatory pain and heat hyperalgesia in persistent inflammatory states. Deletion of Vglut2 in a subpopulation of neurons overlapping with the vanilloid receptor (TRPV1) primary afferents in the dorsal root ganglia resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. Substance P signaling and VGLUT2-mediated glutamatergic transmission in TRPV1 neurons was co-required for the development of inflammatory pain states. Analyses of an itch phenotype uncovered the pathway within TRPV1 neurons, with VGLUT2 playing a regulatory role and GRPR neurons, which are to plausible converge the itch signal in the spinal cord. These studies confirmed the essential role of VGLUT2-dependent glutamatergic transmission in acute and persistent pain states and identified the roles of specific subpopulations of primary afferent neurons. Additionally, a novel pain and itch transmission pathway in TRPV1/VGLUT2 positive neurons was identified, which could be part of the gate control of pain.

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No description available.

Neuronal networks are the central functional units of the nervous system. Knowledge about the identity of participating neurons and the assembly of these during development is crucial for the understanding of CNS function. A promising system to dissect the development and functionalities of a neuronal network is the central pattern generator (CPG) for locomotion. We used screening approaches to identify spinal neuronal subpopulations by their specific gene expression, potentially involved in CPG function. Amongst others we found paired-like homeodomain transcription factor 2 (Pitx2) as a cholinergic interneuron marker for partition cells, with a possible role in the spinal network for locomotion. In addition, we present two genes, Chondrolectin (Chodl) and Estrogen-related receptor beta (ERRβ) as novel markers for fast and slow motor neurons, respectively. The neuronal components of the CPG integrate three key functions; rhythm generation, ipsilateral flexors/extensors coordination and bilateral coordination over the midline. Commissural interneurons (CINs) are considered to participate in the latter. During development axons are guided to their targets by the help of axon guidance molecules. Netrin-1 and its receptor DCC (Deleted in Colorectal Cancer) have been shown to play an important role for spinal cord neurons in axon-pathfinding and migration towards the midline. We show that loss of netrin-1 functionally results in a switch from alternating to synchronous left-right locomotor activity and deletion of DCC surprisingly leads to a different phenotype, best described as uncoordination. Thus, during development, netrin-1 and DCC play a critical role for the establishment of a functional balanced CPG. Further we show a selective loss of CINs, predominantly from dorsally originating subtypes, not affecting the ventral-most V3 subtype in netrin-1 mutant mice, but a loss of CINs from all progenitor domains in Dcc mutant mice. Together, our data suggest a netrin-1-independent mechanism for DCC in axon guidance and a role of the most ventral originating CINs as part of the neuronal network controlling synchronous activities over the midline. Another pair of axon guidance molecules, EphA4 and ephrinB3, has been shown to cooperate in preventing ipsilateral interneurons from crossing the spinal midline and if either molecule is deleted in mice, this will result in a defect in left-right coordination of locomotion. We provide in vivo and in vitro evidence that the GTPase-activating protein α2-chimerin, as a downstream molecule of EphA4 signaling, is essential in axon guidance decisions involved in the correct formation of the spinal circuitry for locomotion.

Vitamin D3 must be metabolically activated by the liver to 25-hydroxyvitamin D3 (25OHD3) and then by the kidney 1alphahydroxylase (1alphaOHase) to become 1,25dihydroxyvitamin D 3 (1,25(OH)2D3). 1,25(OH)2 D3 is a potent inhibitor of tumor growth in vitro and in vivo. Recent studies indicate that metabolic activation of 1,25(OH) 2D3 also occurs in cancer cells such as breast cancer. Consequently, the major objective of this project was to determine if tumoral 25OHD 3-1alphahydroxylase modulates any or all of the stages of breast tumor progression without inducing the hypercalcemic side effects of 1,25(OH) 2D3. For this purpose we used the PyVMT breast cancer mouse model in which the oncoprotein, polyomamiddle T antigen (PyMT) is under the control of mouse mammary tumor virus LTR (MMTV LTR). Mice exhibited tumors restricted to the mammary epithelium progressing to the various stages of breast cancer. Animals were treated with either vehicle, 25OHD3 (2000 pM/24h) or 1,25(OH)2D3 (12pM/24h). Mice treated with the vitamin D precursor, 25OHD3, exhibited a marked reduction in tumor onset and growth comparable to the 1,25(OH)2D3 treated group. Furthermore, biomarkers of tumor progression were markedly reduced in 25OHD3 and 1,25(OH)2D3 animals as compared to vehicle-treated animals. However, mean circulating calcium concentrations remained unchanged in 25OHD3 treated animals but increased significantly in 1,25(OH)2D3 treated animals as compared to controls. Tumoral levels of 1,25(OH)2D3 in mice treated with 25OHD3 were increased 79% in comparison to vehicle control mice. Additionally, 25OHD3 and 1,25(OH)2D 3 treated animals had a significant decrease in the mean number of lung metastases per animal as compared to vehicle treated control animals. This study therefore suggests an important autocrine role of 1alphaOHase expression in breast tumor cells. Furthermore, accumulation of intra-tumoral 1,25(OH) 2D3 in response to 25OHD3 administration strongly suggests that locally produced 1,25(OH)2D3 plays a significant role in restraining tumor growth without inducing the hypercalcemic side effects associated with 1,25(OH)2D3.

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