Modeling Activity and Target-Dependent Developmental Cell Death of Mouse Retinal Ganglion Cells Ex Vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death

EphrinA5 Signaling Is Required for the Distinctive Targeting of Raphe Serotonin Neurons in the Forebrain

International audienceSerotonin (5-HT) neurotransmission in the brain relies on a widespread axon terminal network originating from the hindbrain raphe nuclei. These projections are topographically organized such that the dorsal (DR), and median raphe (MnR) nuclei have different brain targets. However, the guidance molecules involved in this selective targeting in development are unknown. Here, we show the implication of ephrinA5 signaling in this process. We find that the EphA5 gene is selectively expressed in a subset of 5-HT neurons during embryonic and postnatal development. Highest coexpression of EphA5 and the 5-HT marker Tph2 is found in the DR, with lower coexpression in the MnR, and hardly any colocalization of the caudal raphe in the medulla. Accordingly, ephrinA induced a dose-dependent collapse response of 5-HT growth cones cultured from rostral but not caudal raphe. Ectopic expression of ephrinA3, after in utero electroporation in the amygdala and piriform cortex, repelled 5-HT raphe fiber ingrowth. Conversely, misplaced DR 5-HT axons were found in ephrin A5 knockout mice in brain regions that are normally only targeted by MnR 5-HT axons. This causes an overall increase in the density of 5-HT innervation in the ventromedial hypothalamus, the suprachiasmatic nucleus, and the olfactory bulb. All these brain areas have high expression of ephrinAs at the time of 5-HT fiber ingrowth. Present results show for the first time the role of a guidance molecule for the region-specific targeting of raphe neurons. This has important implications to understand how functional parsing of central 5-HT neurons is established during development

The number of Brn3a-expressing RGCs undergoes a reduction during the first postnatal week.

<p>(A) P0, P3, and P7 retinal sections were immunostained with a Brn3a antibody. The inner surface of the retina (RGC layer) is down. The density of Brn3a-expressing cells decreases from P0 to P7 and the retinal ganglion cell layer reorganizes from a multiple cell layer at P0 to a single cell layer at P7. (B) The total number of Brn3a-positive RGCs in the retina declines between P0 and P7. n≥3 for each age. Error bar, s.e.m.; scale bar 50 µm; * p<0.05, ** p<0.01, Kruskall-Wallis test.</p

Blockade of ephrin-A signaling does not affect Brn3a-expressing cell number reduction.

<p>(A) Ephrin-A5 treatment prevents the retinotopic organization of axonal arbors <i>ex vivo</i>, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031105#pone.0031105-Nicol2" target="_blank">[22]</a>. Temporal axons arborize in the rostral SC in control conditions. In contrast, overall activation of ephrin-A/EphA signaling with ephrin-A5 abolishes the preference of axons from the temporal retina for the anterior SC. Grey circles symbolize Brn3a-positive RGCs and their density is representative of the number of Brn3a-labeled cells in each condition. (B, C) The density of Brn3a-immunoreactive cells in retinal explants is not affected by application of ephrin-A5 between DIV1 and DIV12 (0.5 µg/µl). Error bar, s.e.m.; Scale bar 200 µm; n≥40 cultures per condition; ns, not significant, ANOVA.</p

Blockade of electrical activity limits the reduction in the number of Brn3a-expressing cells.

<p>(A) TTX treatment prevents the retinotopic organization of axonal arbors <i>ex vivo</i>. Temporal axons arborize in the rostral SC in control conditions. In contrast, electrical activity blockade with TTX abolishes the preference of axons from the temporal retina for the anterior SC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031105#pone.0031105-Nicol1" target="_blank">[19]</a>. Grey circles symbolize Brn3a-positive RGCs and their density is representative of the number of Brn3a-labeled cells in each condition. (B) Incubation of retino-collicular co-cultures in the sodium channel blocker TTX increases the number of Brn3a-immunoreactive cells in retinal explants at DIV12. (C) TTX treatment causes a ∼64% increase in the number of Brn3a-expressing cells at DIV12. Error bar, s.e.m.; Scale bar 200 µm; n≥22 cultures per condition; *** p<0.001, ANOVA.</p

Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain

International audienceSerotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system

Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5–B9) to the forebrain and brainstem

International audienceSerotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1–B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5–B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system

Dorsal raphe serotonin neurotransmission is required for the expression of nursing behavior and for pup survival

International audienceProper maternal care is an essential factor of reproductive success in mammals, involving a repertoire of behaviors oriented toward the feeding and care of the offspring. Among the neurotransmitters involved in the initiation of these behaviors, serotonin (5-HT) seems to play an important role. Here we compared pup-oriented maternal behaviors in mice with constitutive 5-HT depletion, the tryptophan hydroxylase 2-knock-out (Tph2-KO) and the Pet1-KO mice. We report that the only common pup-oriented defect in these 2 hyposerotoninergic models is a defective nursing in parturient mice and altered nursing-like (crouching) behavior in virgin mice, while pup retrieval defects are only present in Tph2-KO. Despite a normal mammary gland development and milk production, the defect in appropriate nursing is responsible for severe growth retardation and early lethality of pups born to hyposerotonergic dams. This nursing defect is due to acute rather constitutive 5-HT depletion, as it is reproduced by adult knockdown of Tph2 in the dorsal raphe nucleus in mothers with a prior normal maternal experience. We conclude that 5-HT innervation from the dorsal raphe is required for both the initiation and maintenance of a normal nursing behavior. Our findings may be related to observations of reduced maternal/infant interactions in human depression