Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.Cre recombinase ͉ pet-1 ͉ transgenic ͉ yellow fluorescent protein ͉ monoamine S erotonin (5HT) is a transmitter of broad relevance to nervous system development and function (1-4). Serotonergic pathways innervate most cytoarchitectonic structures of the CNS, and accordingly they have been implicated in the modulation of circuitry involved in nearly all behaviors and physiological processes (3, 5-7). Additionally, 5HT neurotransmission is modulated through abundant afferent information arising from, for example, other monoaminergic systems (8, 9) and from orexinergic, glutamatergic, and GABAergic pathways (10-12). The remarkably expansive neuromodulatory influence of 5HT is the product of a complex transcriptional cascade that generates 5HT-synthesizing neurons in the ventral hindbrain (13-18). 5HT also figures prominently in mental health disorders as a number of lines of evidence provide strong support for the hypothesis that altered serotonergic signaling contributes to neurological and psychiatric pathogenesis (19)(20)(21)(22). Despite considerable progress in understanding the importance of 5HT neurotransmission, however, the mechanisms governing 5HT neuron development and the precise physiological roles of 5HT in the modulation of CNS circuitry are not yet clear.Studies of 5HT neurons are hind...

The molecular architecture of developing serotonin (5HT) neurons is poorly understood yet its determination is likely to be essential for elucidating functional heterogeneity of these cells and the contribution of serotonergic dysfunction to disease pathogenesis. Here, we describe the purification of postmitotic embryonic 5HT neurons by flow cytometry for whole genome microarray expression profiling of this unitary monoaminergic neuron type. Our studies identified significantly enriched expression of hundreds of unique genes in 5HT neurons thus providing an abundance of new serotonergic markers. Furthermore, we identified several hundred transcripts encoding homeodomain, axon guidance, cell adhesion, intracellular signaling, ion transport, and imprinted genes associated with various neurodevelopmental disorders that were differentially enriched in developing rostral and caudal 5HT neurons. These findings suggested a homeodomain code that distinguishes rostral and caudal 5HT neurons. Indeed, verification studies demonstrated that Hmx homeodomain and Hox gene expression defined an Hmx+ rostral subtype and Hox+ caudal subtype. Expression of engrailed genes in a subset of 5HT neurons in the rostral domain further distinguished two subtypes defined as Hmx+En+ and Hmx+En-. The differential enrichment of gene sets for different canonical pathways and gene ontology categories provided additional evidence for heterogeneity between rostral and caudal 5HT neurons. These findings demonstrate a deep transcriptome and biological pathway duality for neurons that give rise to the ascending and descending serotonergic subsystems. Our databases provide a rich, clinically relevant, resource for definition of 5HT neuron subtypes and elucidation of the genetic networks required for serotonergic function.

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or -inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer (ePet-EYFP) or cortical neurons were cultured on low amounts of laminin +/− relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of GAP-43 and/or β1 integrin than cortical neurons. Blocking β1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.

Summary:Purpose: This study was designed to quantify the relation between expressions of NMDA receptor (NMDAR) subunits (1 and 2NB) and the epileptogenicity in human focal cortical dysplasia.Methods: Immunoblotting and immunoprecipitation were used to quantify these receptor subunits in tissue resected from EEG-verified epileptic and distal nonepileptic frontal cortical areas in each of three patients as determined by chronic subdural electrode recordings. In each patient, adjacent sections were immunostained to verify that the numbers of dysplastic neurons were greater in epileptic than in nonepileptic cortex.Results: In all patients, NMDAR2A/B expressions and their coassemblies with NMDARl were increased in epileptic dysplastic cortex compared with the relatively normal appearing nonepileptic cortex. For all three patients, there were no significant differences in NMDARl protein expressions between the two EEG groups.Conclusions: These results suggest that increased NMDARl-N M D A R 2 m coassembly contributes to hyperexcitability in dysplastic cortical neurons and focal seizure onsets. Key Words: Cortical dysplasia-NMDAR-Epilepsy-Immuno-Human cortical dysplasia (CD), which is characterized by disorganization of the cortical architecture, abnormalities of neuronal size, shape, and lamination (1,2), is frequently associated with partial epilepsy (3,4). Recently, glutamate receptors such as AMPA-GluR2/3 and NMDAR immunoreactivity have been reported to increase in human dysplastic neurons (2,5). NMDA receptors modulate glutamate postsynaptic neurotransmission by generating long-lasting channel openings for longer depolarizations. These data are consistent with the hypothesis that excitatory postsynaptic NMDA receptors are essential in epileptogenicity associated with CD. NMDA receptors are composed of two classes of subunits: one subunit of NMDAR1 (NRI), which has at least eight splice variants, and four subunits of NMDAR2 (NR2A-NR2D). It was shown that in frog oocytes, the NRl gene could express a functional receptor with only a weak response activated by glutamate (6), whereas none of the NR2 subunits alone was shown to be functional. Recent studies have shown that the NMDAAccepted June I , 1999. Address correspondence and reprint requests to Dr. T. L. Babb at Department of Neurosciences NC-30, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A. E-mail: babbt@ccf.org receptor complex is a membrane-spanning polypeptide whose physiologic response to glutamate is potentiated by the coagonist glycine in cultured mouse brain cells (7); and glycine binding is essential for activation of NMDA receptors in frog oocytes (8). The kinetics of NMDA cultured hippocampal receptors, using wholecell voltage clamp (9) and outside-out patches (lo), identified the active receptor as consisting of a tetramer of two glycine and two glutamate binding sites. Brose et al.(1 1) demonstrated that in rat brain, the NMDA subunit NR1 messenger RNA (mRNA) is broadly distributed, must be coexpressed for functional...

Embryonic CNS neurons can migrate from the ventricular zone to their final destination by radial glial-guided locomotion. Another less appreciated mechanism is somal translocation, where the young neuron maintains its primitive ventricular and pial processes, through which the cell body moves. A major problem in studying translocation has been the identification of neuronal-specific markers that appear in primitive, radially shaped cells. We used enhanced yellow fluorescent protein under control of the Pet-1 enhancer/promoter region (ePet-EYFP), a specific marker of early differentiated serotonergic neurons, to study their migration via immunohistology and time-lapse imaging of living slice cultures. As early as E10.0, ePet-EYFP-expressing neurons were axonless, radially oriented, and spanned the entire neuroepithelium. The soma translocated within the pial process toward the pial surface and could also translocate through its neurites, which sprouted from the pial process. The dynamin inhibitor dynasore significantly reduced translocation velocity, while the nonmuscle myosin II inhibitor blebbistatin and the kinesin inhibitor AMP-PNP had no significant effect. Here we show for the first time that serotonergic neurons migrate by somal translocation mediated, in part, by dynamin.

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Summary:Purpose: The cellular mechanisms that may contribute to epilepsy in resected human cortical dysplasia (CD) were compared with the in utero radiated rat CD model. In human and rat focal hippocampal epilepsy, postsynaptic Nmethyl-D-aspartate receptors are up-regulated and presynaptic axon collaterals hyperinnervate them. We hypothesized that in both human and rat CD: (a) the N-methyl-D-aspartate receptor subunits NR1 and NR2A/B would be increased and coassembled, and (b) aberrant axons would be in regions of CD.Methods: Tests for presynaptic and postsynaptic changes in human and rat CD included the following: (a) cytology, (b) immunocytochemistry, (c) coimmunoprecipitation, (d) doublelabeled immunofluorescence, and (e) Timm histochemistry of hippocampal mossy fibers. Within-patient comparisons were made between epileptic tissue, identified by subdural electroencephalographic seizure onsets, and nonepileptic tissue remote from the focus but within the therapeutic resection. Rats were radiated at embryonic day 17, and offspring were studied postnatally. Statistical comparisons were made against normal rats matched for age and tissue processing.Results: In focal CD patients, NR2A/B subunits and their coassemblies with NR1 were increased significantly more than for the remote nonepileptic cortex. Confocal microscopy showed that NRI -NR2A/B colabeled single dysplastic neurons in both human and rat. In CD rats, mossy fibers innervated the anomalously oriented hippocampal neurons.Conclusions: Human epileptic CD exhibits a spectrum of abnormal cell orientations and laminations that must require plastic axodendritic changes during development. These altered circuits and receptors could account for the seizures and cognitive deficits found in patients with CD. The radiated rat CD model with cortical dyslaminations and NR2A/B subunit increases would allow the development and testing of drugs targeted at only the NR2A/B subunit or at decoupling the NRI-NR2 coassembly, which could provide a specific antiepileptic drug for dysplastic circuits without inducing general depression of all brain neurons. Key Words: Central dysplasia-NMDA receptor-Gamma radiation-Development.Neuronal migration disorders that occur prenatally are termed cortical dysplasia (CD) to characterize abnormal laminar organizations. Dysplastic regions in humans develop focal neocortical epilepsy early in life, are mostly drug-refractory, but are treated by cortical resections. Human dysplastic neurons have increased N-methyl-Daspartate (NMDA) receptor subunits NR1 and NR2A/B (1). The NR1 coassembles with NR2 in populations of dysplastic neurons (coprecipitation) (2), and NR1-NR2A/B are coexpressed in single neurons only in dysplastic cortex ( 3 ) . These altered stoichiometrics of NMDA receptor subunits represent a hypothetical model for focal seizures arising from networks of single dysplastic neurons hyperexcitable to glutamate. It is known from studies of NR1 and NR2 subunits differentially Address correspondence and reprint requests to Dr. Thomas L...

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