Tania Vitalis

Lack of Barrels in the Somatosensory Cortex of Monoamine Oxidase A–Deficient Mice: Role of a Serotonin Excess during the Critical Period

In a transgenic mouse line (Tg8) deficient for the gene encoding monoamine oxidase A (MAOA), we show that the primary somatosensory cortex (S1) lacks the characteristic barrel-like clustering of layer IV neurons, whereas normal pattern formation exists in the thalamus and the trigeminal nuclei. No barrel-like patterns were visible with tenascin or serotonin immunostaining or with labeling of thalamocortical axons. An excess of brain serotonin during the critical period of barrel formation appears to have a causal role in these cortical abnormalities, since early administration of parachlorophenylalanine, an inhibitor of serotonin synthesis, in Tg8 pups restored the formation of barrels in S1, whereas inhibition of catecholamine synthesis did not. Transient inactivation of MAOA in normal newborns reproduced a barrelless phenotype in parts of S1.

Effects of Monoamine Oxidase A Inhibition on Barrel Formation in the Mouse Somatosensory Cortex: Determination of a Sensitive Developmental Period

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High‐pressure liquid chromatography measures of brain serotonin (5‐HT) showed three‐ to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral‐to‐caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially.

The Role of Serotonin in Early Cortical Development

The cerebral cortex is widely innervated by serotonin (5-HT)-containing axons originating from neurons in the raphe nuclei. The early development of this monoamine system in the cortex prompted speculation long ago that it has important functions in cortical maturation and plasticity. Here we review evidence, derived from a plethora of studies and from our recent unpublished work, that supports an important role for 5-HT in a number of major events in the developing cortex, especially at the early stages. This evidence points to a regulatory role for 5-HT in neuronal proliferation, migration and differentiation, and in preventing apoptotic cell death.

Embryonic depletion of serotonin affects cortical development.

Compelling evidence suggests that serotonin (5‐HT) is necessary for the refined organization of the cerebral cortex. Here we sought to analyse the short‐ and long‐term consequences of embryonic 5‐HT depletion on the development of the cerebral neocortex of the rat. We focused on the migration and differentiation of the pyramidal (projection) and nonpyramidal (interneuron) neuronal populations. Our paradigm used daily injection of DL‐P‐chlorophenylalanine (PCPA), a reversible inhibitor of 5‐HT synthesis, during the E12–17 stage of embryonic development, when major events in corticogenesis take place. We monitored the 5‐HT depletion induced by this treatment and showed that it led to subtle alterations in both the pyramidal and nonpyramidal neuronal populations. We found that E12–17 PCPA treatment altered the maturation of pyramidal neurons of layers III and V of the somatosensory cortex, with these cells displaying reduced dendritic arborization and complexity. These long‐lasting alterations were not associated with modification of cortical BDNF levels at postnatal stages. We also showed that PCPA treatment transiently altered the incorporation in the cortical plate of interneurons derived from the caudal ganglionic eminence, and persistently affected the differentiation of a subpopulation expressing calretinin and/or cholecystokinin.

Serotonin 3A Receptor Subtype as an Early and Protracted Marker of Cortical Interneuron Subpopulations

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT3A promoter (5-HT3A:GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT3A:GFP mice, we identified 2 populations of 5-HT3A-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT3A mRNA detection showed that cortical 5-HT3A interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT3A receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT3A interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT3A subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ.

During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCOs basal processes which normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants suggesting a role for cadherins in SCO development.

The N-terminal region of reelin regulates postnatal dendritic maturation of cortical pyramidal neurons

Cajal-Retzius cells, located in layer I of the cortex, synthesize and secrete the glycoprotein reelin, which plays a pivotal role in neuronal migration during embryonic development. Cajal-Retzius cells persist after birth, but their postnatal role is unknown. Here we show that Cajal-Retzius cells receive a major excitatory synaptic input via serotonin 5-HT3 receptors. Blocking this input using pharmacological tools or neutralization of reelin signaling results in hypercomplexity of apical, but not basal, dendrites of cortical layer II/III pyramidal neurons. A similar hypercomplexity is observed in the cortex of the 5-HT3A receptor knockout mouse. The increased dendritic complexity can be rescued by application of recombinant full-length reelin or its N-terminal fragment, but not by the central fragment of reelin, and involves a signal transduction pathway independent of the activation of the canonical reelin receptors. Taken together, our results reveal a novel role of serotonin, Cajal-Retzius cells, and reelin in the postnatal maturation of the cortex.

Developmental expression of monoamine oxidases A and B in the central and peripheral nervous systems of the mouse

Monoamine oxidases A (MAOA) and B (MAOB) are key players in the inactivation pathway of biogenic amines. Their cellular localization has been well established in the mature brain, but nothing is known concerning the localization of both enzymes during development. We have combined in situ hybridization and histochemistry to localize MAOA and MAOB in the developing nervous system of mice. Our observations can be summarized as five key features. (1) MAOA is tightly linked to catecholaminergic traits. MAOA is expressed in all noradrenergic and adrenergic neurons early on, and in several dopaminergic cell groups such as the substantia nigra. MAOA is also expressed in all the neurons that display a transient tyrosine hydroxylase expression in the brainstem and the amygdala and in neurons with transient dopamine‐β‐hydroxylase expression in the cranial sensory ganglia. (2) MAOA and MAOB are coexpressed in the serotoninergic neurons of the raphe from E12 to P7. During postnatal life, MAOA expression declines, whereas MAOB expression remains stable. (3) MAOA is transiently expressed in the cholinergic motor nuclei of the hindbrain, and MAOB is expressed in the forebrain cholinergic neurons. (4) MAOA‐ and MAOB‐expressing neurons are also detected in structures that do not contain aminergic neurons, such as the thalamus, hippocampus, and claustrum. (5) Starting at birth, MAOB expression is found in a variety of nonneuronal cells, the choroid plexus, the ependyma, and astrocytes. These localizations are of importance for understanding the effects of monoaminergic transmission during development. J. Comp. Neurol. 442:331–347, 2002.

Conserved pattern of tangential neuronal migration during forebrain development.

Origin, timing and direction of neuronal migration during brain development determine the distinct organization of adult structures. Changes in these processes might have driven the evolution of the forebrain in vertebrates. GABAergic neurons originate from the ganglionic eminence in mammals and migrate tangentially to the cortex. We are interested in differences and similarities in tangential migration patterns across corresponding telencephalic territories in mammals and reptiles. Using morphological criteria and expression patterns of Darpp-32, Tbr1, Nkx2.1 and Pax6 genes, we show in slice cultures of turtle embryos that early cohorts of tangentially migrating cells are released from the medial ganglionic eminence between stages 14 and 18. Additional populations migrate tangentially from the dorsal subpallium. Large cohorts of tangentially migrating neurons originate ventral to the dorsal ventricular ridge at stage 14 and from the lateral ganglionic eminence from stage 15. Release of GABAergic cells from these regions was investigated further in explant cultures. Tangential migration in turtle proceeds in a fashion similar to mammals. In chimeric slice culture and in ovo graft experiments, the tangentially migrating cells behaved according to the host environment - turtle cells responded to the available cues in mouse slices and mouse cells assumed characteristic migratory routes in turtle brains, indicating highly conserved embryonic signals between these distant species. Our study contributes to the evaluation of theories on the origin of the dorsal cortex and indicates that tangential migration is universal in mammals and sauropsids.

The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitro

In the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB1Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB1R expression as well as effects of CB1R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB1Rs are strongly expressed in both reelin‐expressing Cajal‐Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB1R protein is localized first to somato‐dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB1Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP‐55940 results in the relocation of receptors from axons to somato‐dendritic endosomes, indicating the functional competence of embryonic CB1Rs. The adult phenotype of CB1R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB1R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB1R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB1R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption.