Developmental genes controlling neural circuit formation are expressed in the early postnatal hypothalamus and cellular lining of the third ventricle.

The arcuate nucleus of the hypothalamus is central in the regulation of body weight homeostasis through its ability to sense peripheral metabolic signals and relay them, through neural circuits, to other brain areas, ultimately affecting physiological and behavioural changes. The early postnatal development of these neural circuits is critical for normal body weight homeostasis, such that perturbations during this critical period can lead to obesity. The role for peripheral regulators of body weight homeostasis, including leptin, insulin and ghrelin, in this postnatal development is well described, yet some of the fundamental processes underpinning axonal and dendritic growth remain unclear. Here, we hypothesised that molecules known to regulate axonal and dendritic growth processes in other areas of the developing brain would be expressed in the postnatal arcuate nucleus and/or target nuclei where they would function to mediate the development of this circuitry. Using state-of-the-art RNAscope® technology, we have revealed the expression patterns of genes encoding Dcc/Netrin-1, Robo1/Slit1 and Fzd5/Wnt5a receptor/ligand pairs in the early postnatal mouse hypothalamus. We found that individual genes had unique expression patterns across developmental time in the arcuate nucleus, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, dorsomedial nucleus of the hypothalamus, median eminence and, somewhat unexpectedly, the third ventricle epithelium. These observations indicate a number of new molecular players in the development of neural circuits regulating body weight homeostasis, as well as novel molecular markers of tanycyte heterogeneity.

The enigmatic fetal subplate compartment forms an early tangential cortical nexus and provides the framework for construction of cortical connectivity.

The most prominent transient compartment of the primate fetal cortex is the deep, cell-sparse, synapse-containing subplate compartment (SPC). The developmental role of the SPC and its extraordinary size in humans remain enigmatic. This paper evaluates evidence on the development and connectivity of the SPC and discusses its role in the pathogenesis of neurodevelopmental disorders. A synthesis of data shows that the subplate becomes a prominent compartment by its expansion from the deep cortical plate (CP), appearing well-delineated on MR scans and forming a tangential nexus across the hemisphere, consisting of an extracellular matrix, randomly distributed postmigratory neurons, multiple branches of thalamic and long corticocortical axons. The SPC generates early spontaneous non-synaptic and synaptic activity and mediates cortical response upon thalamic stimulation. The subplate nexus provides large-scale interareal connectivity possibly underlying fMR resting-state activity, before corticocortical pathways are established. In late fetal phase, when synapses appear within the CP, transient the SPC coexists with permanent circuitry. The histogenetic role of the SPC is to provide interactive milieu and capacity for guidance, sorting, "waiting" and target selection of thalamocortical and corticocortical pathways. The new evolutionary role of the SPC and its remnant white matter neurons is linked to the increasing number of associative pathways in the human neocortex. These roles attributed to the SPC are regulated using a spatiotemporal gene expression during critical periods, when pathogenic factors may disturb vulnerable circuitry of the SPC, causing neurodevelopmental cognitive circuitry disorders.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

Perinatal programming of metabolic diseases: role of insulin in the development of hypothalamic neurocircuits.

It is increasingly accepted that the metabolic future of an individual can be programmed as early as at developmental stages. For instance, offspring of diabetic mothers have a greater risk of becoming obese and diabetic later in life. Animal studies have demonstrated that hyperinsulinemia and/or hyperglycemia during perinatal life permanently impair the organization and long-term function of hypothalamic networks that control appetite and glucose homeostasis. This review summarizes the main findings regarding the key regulatory roles of perinatal insulin and glucose levels on hypothalamic development and on long-term programming of metabolic diseases reported in different rodent models.

Slit2 activity in the migration of guidepost neurons shapes thalamic projections during development and evolution.

How brain connectivity has evolved to integrate the mammalian-specific neocortex remains largely unknown. Here, we address how dorsal thalamic axons, which constitute the main input to the neocortex, are directed internally to their evolutionary novel target in mammals, though they follow an external path to other targets in reptiles and birds. Using comparative studies and functional experiments in chick, we show that local species-specific differences in the migration of previously identified "corridor" guidepost neurons control the opening of a mammalian thalamocortical route. Using in vivo and ex vivo experiments in mice, we further demonstrate that the midline repellent Slit2 orients migration of corridor neurons and thereby switches thalamic axons from an external to a mammalian-specific internal path. Our study reveals that subtle differences in the migration of conserved intermediate target neurons trigger large-scale changes in thalamic connectivity, and opens perspectives on Slit functions and the evolution of brain wiring.

Molecular pathways controlling development of thalamus and hypothalamus: from neural specification to circuit formation.

The embryonic diencephalon gives rise to the vertebrate thalamus and hypothalamus, which play essential roles in sensory information processing and control of physiological homeostasis and behavior, respectively. In this review, we present new steps toward characterizing the molecular pathways that control development of these structures, based on findings in a variety of model organisms. We highlight advances in understanding how early regional patterning is orchestrated through the action of secreted signaling molecules such as Sonic hedgehog and fibroblast growth factors. We address the role of individual transcription factors in control of the regional identity and neural differentiation within the developing diencephalon, emphasizing the contribution of recent large-scale gene expression studies in providing an extensive catalog of candidate regulators of hypothalamic neural cell fate specification. Finally, we evaluate the molecular mechanisms involved in the experience-dependent development of both thalamo-cortical and hypothalamic neural circuitry.

Embryonic and postnatal development of the layer I-directed ("matrix") thalamocortical system in the rat.

Inputs to the layer I apical dendritic tufts of pyramidal cells are crucial in "top-down" interactions in the cerebral cortex. A large population of thalamocortical cells, the "matrix" (M-type) cells, provides a direct robust input to layer I that is anatomically and functionally different from the thalamocortical input to layer VI. The developmental timecourse of M-type axons is examined here in rats aged E (embryonic day) 16 to P (postnatal day) 30. Anterograde techniques were used to label axons arising from 2 thalamic nuclei mainly made up of M-type cells, the Posterior and the Ventromedial. The primary growth cones of M-type axons rapidly reached the subplate of dorsally situated cortical areas. After this, interstitial branches would sprout from these axons under more lateral cortical regions to invade the overlying cortical plate forming secondary arbors. Moreover, retrograde labeling of M-type cell somata in the thalamus after tracer deposits confined to layer I revealed that large numbers of axons from multiple thalamic nuclei had already converged in a given spot of layer I by P3. Because of early ingrowth in such large numbers, interactions of M-type axons may significantly influence the early development of cortical circuits.

Removal of GABA within adult modulatory systems alters electrical coupling and allows expression of an embryonic-like network.

The maturation and operation of neural networks are known to depend on modulatory neurons. However, whether similar mechanisms may control both adult and developmental plasticity remains poorly investigated. To examine this issue, we have used the lobster stomatogastric nervous system (STNS) to investigate the ontogeny and role of GABAergic modulatory neurons projecting to small pattern generating networks. Using immunocytochemistry, we found that modulatory input neurons to the stomatogastric ganglion (STG) express GABA only after metamorphosis, a time that coincides with the developmental switch from a single to multiple pattern generating networks within the STNS. We demonstrate that blocking GABA synthesis with 3-mercapto-propionic acid within the adult modulatory neurons results in the reconfiguration of the distinct STG networks into a single network that generates a unified embryonic-like motor pattern. Using dye-coupling experiments, we also found that gap-junctional coupling is greater in embryos and GABA-deprived adults exhibiting the unified motor pattern compared with control adults. Furthermore, GABA was found to diminish directly the extent and strength of electrical coupling within adult STG networks. Together, these observations suggest the acquisition of a GABAergic phenotype by modulatory neurons after metamorphosis may induce the reconfiguration of the single embryonic network into multiple adult networks by directly decreasing electrical coupling. The findings also suggest that adult neural networks retain the ability to express typical embryonic characteristics, indicating that network ontogeny can be reversed and that changes in electrical coupling during development may allow the segregation of multiple distinct functional networks from a single large embryonic network.

Molecular mechanisms of thalamocortical axon targeting.

The thalamocortical (TC) projection in the mammalian brain is a well characterized system in terms of laminar specificity of neocortical circuits. To understand the mechanisms that underlie lamina-specific TC axon targeting, we studied the role of extracellular and cell surface molecules that are expressed in the upper layers of the developing cortex in in vitro culture techniques. The results demonstrated that multiple upper layer molecules co-operated to produce stop behaviour of TC axons in the target layer. Activity dependency of TC axon branching was also investigated in organotypic co-cultures of the thalamus and cortex. TC axon branches were formed dynamically by addition and elimination during the second week in vitro, when spontaneous firing increased in thalamic and cortical cells. Pharmacological blockade of firing or synaptic activity reduced the remodelling process, in particular branch addition, in the target layer. Together, these findings suggest that TC axon targeting mechanisms involve the regulation with multiple lamina-specific molecules and modification of the molecular mechanisms via neural activity.

The dopamine-synthesizing cells in the swimming larva of the tunicate Ciona intestinalis are located only in the hypothalamus-related domain of the sensory vesicle.

Dopamine is a major neuromodulator synthesized by numerous cell populations in the vertebrate forebrain and midbrain. Owing to the simple organization of its larval nervous system, ascidian tunicates provide a useful model to investigate the anatomy, neurogenesis and differentiation of the dopaminergic neural network underlying the stereotypical swimming behaviour of its chordate-type larva. This study provides a high-resolution cellular analysis of tyrosine hydroxylase (TH)-positive and dopamine-positive cells in Ciona intestinalis embryos and larvae. Dopamine cells are present only in the sensory vesicle of the Ciona larval brain, which may be an ancestral chordate feature. The dopamine-positive cells of the ascidian sensory vesicle are located in the expression domain of homologues of vertebrate hypothalamic markers. We show here that the larval coronet cells also arise from this domain. As a similar association between coronet cells and the hypothalamus was reported in bony and cartilaginous fishes, we propose that part of the ascidian ventral sensory vesicle is the remnant of a proto-hypothalamus that may have been present in the chordate ancestor. As dopaminergic cells are specified in the hypothalamus in all vertebrates, we suggest that the mechanisms of dopamine cell specification are conserved in the hypothalamus of Ciona and vertebrates. To test this hypothesis, we have identified new candidate regulators of dopaminergic specification in Ciona based on their expression patterns, which can now be compared with those in vertebrates.

In utero development of central ANG-stimulated pressor response and hypothalamic fos expression.

Central renin-angiotensin system (RAS) is as important as the peripheral RAS in the control of the cardiovascular homeostasis in the adult. However, previous fetal studies on angiotensin II (ANG II)-induced cardiovascular responses focused exclusively on the peripheral side. Thus, few data exist characterizing the in utero development of central angiotensin-mediated pressor responses. The present study determined cardiovascular responses to central application of ANG II in the chronically prepared near-term ovine fetus, and determined the action sites marked by c-fos expression in the fetal hypothalamus following intracerebroventricular (icv) injection of ANG II in utero. ANG II significantly increased fetal systolic, diastolic, and mean arterial pressure (MAP) within 5 min after injection of this peptide into the brain. Adjusted fetal MAP against amniotic pressure was also increased by icv ANG II, associated with increased c-fos in the central putative cardiovascular area--the paraventricular nuclei (PVN). Application of ANG II also induced intense c-fos expression in the supraoptic nuclei (SON), accompanied by a significant increase of fetal plasma vasopressin (AVP) levels, while maternal blood pressure (BP) and plasma AVP concentration were not changed. These results indicate that the central ANG II-mediated pressor response is functional at the last third of gestation, acting at the sites consistent with the cardiovascular neural network in the hypothalamus.

Maturation of rhythmic neural network: role of central modulatory inputs.

Modulatory systems are well known for their roles in tuning the cellular and synaptic properties in the adult neuronal networks, and play a major role in the control of the flexibility of functional outputs. However far less is known concerning their role in the maturation of neural networks during the development. In this review, using the stomatogastric nervous system of lobster, we will show that the neuromodulatory system exerts a powerful influence on developing neural networks. In the adult the number of both motor target neurons and their modulatory neurons is restricted to tens of identifiable cells. They are therefore well characterized in terms of cellular, synaptic and morphological properties. In the embryo, these target cells and their neuromodulatory population are already present from mid-embryonic life. However, the motor output generated by the system is quite different: while in the embryo all the target neurons are organized into a single network generating unique motor pattern, in the adult this population splits into two distinct networks generating separate patterns. This ontogenetic partitioning does not rely on progressive acquisition of adult properties but rather on a switch between two possible network operations. Indeed, adult networks are present early in the embryonic life but their expression is repressed by central modulatory neurons. Moreover, embryonic networks can be revealed in the adult system again by altering modulatory influences. Therefore, independently of the developmental age, two potential network phenotypes co-exist within the same neuronal architecture: when one is expressed, the other one is hidden and vice versa. These transitions do not necessarily need dramatic changes such as growth/retraction of processes, acquisition of new intra-membrane proteins etc. but rather, as shown by modelling studies, it may simply rely on a subtle tuning of pre-existing intercellular electrical coupling. This in turn suggests that progressive ontogenetic alteration may not take place at the level of the target network but rather at the level of modulatory input neurons.

Electrical coupling can prevent expression of adult-like properties in an embryonic neural circuit.

Electrical coupling is widespread in developing nervous systems and plays a major role in circuit formation and patterning of activity. In most reported cases, such coupling between rhythmogenic neurons tends to synchronize and enhance their oscillatory behavior, thereby producing monophasic rhythmic output. However, in many adult networks, such as those responsible for rhythmic motor behavior, oscillatory neurons are linked by synaptic inhibition to produce rhythmic output with multiple phases. The question then arises whether such networks are still able to generate multiphasic output in the early stage of development when electrical coupling is abundant. A suitable model for addressing this issue is the lobster stomatogastric nervous system (STNS). In the adult animal, the STNS consists of three discrete neural networks that are comprised of oscillatory neurons interconnected by reciprocal inhibition. These networks generate three distinct rhythmic motor patterns with large amplitude neuronal oscillations. By contrast, in the embryo the same neuronal population expresses a single multiphasic rhythm with small-amplitude oscillations. Recent findings have revealed that adult-like network properties are already present early in the embryonic system but are masked by an as yet unknown mechanism. Here we use computer simulation to test whether extensive electrical coupling may be involved in masking adult-like properties in the embryonic STNS. Our basic model consists of three different adult-like STNS networks that are built of relaxation oscillators interconnected by reciprocal synaptic inhibition. Individual model cells generate slow membrane potential oscillations without action potentials. The introduction of widespread electrical coupling between members of these networks dampens oscillation amplitudes and, at moderate coupling strengths, may coordinate neuronal activity into a single rhythm with different phases, which is strongly reminiscent of embryonic STNS output. With a further increase in coupling strength, the system reaches one of two final states depending on the relative contribution of inhibition and inherent oscillatory properties within the networks: either fully synchronized and dampened oscillations, or a complete collapse of activity. Our simulations indicate that, beginning from either of these two states, the emergence of distinct adult networks during maturation may arise from a developmental decrease in electrical coupling that unmasks preexisting adult-like network properties.

Ontogeny of modulatory inputs to motor networks: early established projection and progressive neurotransmitter acquisition.

Modulatory information plays a key role in the expression and the ontogeny of motor networks. Many developmental studies suggest that the acquisition of adult properties by immature networks involves their progressive innervation by modulatory input neurons. Using the stomatogastric nervous system of the European lobster Homarus gammarus, we show that contrary to this assumption, the known population of projection neurons to motor networks, as revealed by retrograde dye migration, is established early in embryonic development. Moreover, these neurons display a large heterogeneity in the chronology of acquisition of their full adult neurotransmitter phenotype. We performed retrograde dye migration to compare the neuronal population projecting to motor networks located in the stomatogastric ganglion in the embryo and adult. We show that this neuronal population is quantitatively established at developmental stage 65%, and each identified projection neuron displays the same axon projection pattern in the adult and the embryo. We then combined retrograde dye migration with FLRFamide-like, histamine, and GABA immunocytochemistry to characterize the chronology of neurotransmitter expression in individual identified projection neurons. We show that this early established population of projection neurons gradually acquires its neurotransmitter phenotype complement. This study indicates that (1) the basic architecture of the known population of projection inputs to a target network is established early in development and (2) ontogenetic plasticity may depend on changes in neurotransmitter phenotype expression within preexisting neurons rather than in the addition of new projection neurons or fibers.

Central inputs mask multiple adult neural networks within a single embryonic network.

It is usually assumed that, after construction of basic network architecture in embryos, immature networks undergo progressive maturation to acquire their adult properties. We examine this assumption in the context of the lobster stomatogastric nervous system. In the lobster, the neuronal population that will form this system is at first orgnanized into a single embryonic network that generates a single rhythmic pattern. The system then splits into different functional adult networks controlled by central descending systems; these adult networks produce multiple motor programmes, distinctively different from the single output of the embryonic network. We show here that the single embryonic network can produce multiple adult-like programmes. This occurs after the embryonic network is silenced by removal of central inputs, then pharmacologically stimulated to restore rhythmicity. Furthermore, restoration of the flow of descending information reversed the adult-like pattern to an embryonic pattern. This indicates that the embryonic network possesses the ability to express adult-like network characteristics, but descending information prevents it from doing so. Functional adult networks may therefore not necessarily be derived from progressive ontogenetic changes in networks themselves, but may result from maturation of descending systems that unmask preexisting adult networks in an embryonic system.

Structural study of the development of ocularity domains using a neural network model.

We present a model for the development of ocularity domains in the visual cortex of mammals during the embryonic stage. We model the thalamo-cortical pathway with a self-organising neural network with two source layers, each of them serving different retinae, and one target layer, where the connections end. The connectivity between the source layers and the target layer is driven by Hebbian learning. In both the source layers and the target layer we assume excitatory lateral signal diffusion between proximal neurons that causes them to be correlated. According to the developmental state being modelled, we do not consider either correlation or anti-correlation between the signals originated in neurons of different retinae. The basic assumptions made are proved to be sufficient to attain a distribution of connections arranged in ocularity domains. The dependence of the geometry of the ocularity domains on the parameters of the model is analysed and a correlation between the width of the signal diffusion and the extent of the domains is found. The generality of the assumptions made allows an easy translation of the model to explain the development of other elements of the sensory nervous system.

Functional differentiation of adult neural circuits from a single embryonic network.

The stomatogastric nervous system (STNS) of adult lobsters and crabs generates a number of different rhythmic motor patterns which control different regional movements of the foregut. Since these output patterns are generated by discrete neural networks that, in the adult, are well characterized in terms of synaptic and cellular properties, this system constitutes an ideal model for exploring the mechanisms underlying the ontogeny of neural network organization. The foregut and its rhythmic motor patterns were studied in in vitro STNS nerve-muscle preparations of the embryo and different larval stages of the lobster Homarus gammarus. The development of Homarus comprises a long embryonic stage in ovo followed by three pelagic larval stages prior to the onset of benthic life. During these stages the foregut itself develops slowly from a simple ectodermal invagination that occurs in the embryo. During successive larval stages it progressively acquires all the specialized structures and shape of the adult foregut. In contrast, the STNS is morphologically recognizable at early embryonic stages. In all recorded stages the STNS spontaneously expresses rhythmic motor activity. During development, this activity is progressively restructured, beginning with a single rhythmic motor pattern in the embryo where all the stomodeal muscles are strongly coordinated. In subsequent stages, however, this single pattern is progressively subdivided to give rise eventually to the three discrete rhythmic motor patterns characteristic of the adult STNS. Our data suggest that rather than a dismantling of redundant embryonic and larval neural networks, the different adult networks emerge as a progressive partitioning of discrete circuits from a single embryonic network.