Ontogenetic rules for the molecular diversification of hypothalamic neurons.

Developmental Switch in Neuropeptide Y and Melanocortin Effects in the Paraventricular Nucleus of the Hypothalamus

Terminal differentiation of neurons is accompanied by irreversible exit from the cell cycle and expression of neuronal phenotypes. The molecular mechanism whereby committed neuronal progenitors lose their ability to reenter the cell cycle is largely unknown. Here, we report that the nuclear transport system is rapidly remodeled in primary cortical progenitor cells (CPCs) at the very beginning of neuronal terminal differentiation. High levels of Ran GTPase-activating protein 1 (RanGAP), a key regulator of the Ran GTP-GDP cycle, in primary CPCs are drastically reduced upon neuronal induction. Small ubiquitin-like modifier (SUMO)-2/3-conjugated RanGAP undergoes desumoylation and degradation in neuronally committed CPCs, where reduced RanGAP levels impede the nuclear import of nucleocytoplasmic shuttling proteins including the DNA replication initiation factor Cdc6. Furthermore, RNAi-mediated down-regulation of RanGAP expression in undifferentiated CPCs induces neuronal phenotypes including cell cycle exit. Our data suggest that remodeling of the RanGAP-mediated nuclear transport system plays a key role in cell cycle exit for terminal differentiation of cortical neurons.

Although numerous data showing severe morphological impairment of magnocellular and parvocellular hypothalamic neurons due to chronic alcoholic consumption have been gathered from animal experiments, only one study (Harding et al., 1996) was performed on POST MORTEM human brain. This study showed a reduction in the number of vasopressin (VP)-immunoreactive neurons in the supraoptic (SON) and paraventricular (PVN) nuclei, but did not provide any data regarding the effect of chronic alcohol intake on human parvocellular neurons. In order to assess whether the changes observed in the animal model also occur in humans and provide a structural basis for the results of clinical tests, we performed immunohistochemical and morphometric analysis of magnocellular (VP and oxytocin, OT) and parvocellular (corticotropin-releasing hormone, CRH) neurons in post-mortem brains of patients afflicted with chronic alcoholic disease. We analyzed 26-male alcoholics and 22 age-matched controls divided into two age groups--"young" (< 40 yr) and "old" (> 40 yr). Hypothalamic sections were stained for OT, VP, and CRH. The analysis revealed: 1) decrease in VP-immunoreactivity in the SON and PVN as well as OT-immunoreactivity in the SON in alcoholic patients; 2) increase in OT-immunoreactivity in the PVN; 3) increase in CRH-immunoreactivity in parvocellular neurons in the PVN. Furthermore, the proportion of cells containing CRH and VP was increased in alcoholics. These findings indicate that chronic alcohol consumption does indeed impair the morphology of magnocellular neurons. The enhancement of CRH-immunoreactivity and increased co-production of CRH and VP in parvocellular neurons may be due to a decline in glucocorticoid production, implied by the hypoplasic impairment of adrenal cortex we observed in alcoholics during the course of this study.

Within the rat paraventricular nucleus of the hypothalamus two types of neurons have been distinguished based on morphological appearance, i.e., parvocellular and magnocellular neurons. The parvocellular neurons play a key role in regulating the activity of the hypothalamo-pituitary-adrenal axis, which is activated, e.g., after stress exposure. These neurons receive humoral negative feedback via the adrenal hormone corticosterone but also neuronal inhibitory input, either directly or transsynaptically relayed via GABAergic interneurons. In the present study we examined to what extent the neuronal GABAergic input is influenced by the humoral signal. To this end, miniature inhibitory postsynaptic currents (mIPSCs) were recorded in parvo- and magnocellular neurons of adrenalectomized rats, which lack corticosterone, and in sham-operated controls. Under visual control neurons in coronal slices containing the paraventricular nucleus were designated as putative parvocellular or magnocellular neurons: the former were located in the medial part of the nucleus and displayed a small fusiform soma; the latter were mostly located in the lateral part and were recognized by their large round soma. Compared with putative magnocellular neurons, parvocellular neurons generally exhibited a lower membrane capacitance, lower mIPSC frequency, and smaller mIPSC amplitude. Following adrenalectomy, the mIPSC frequency was significantly enhanced in parvo- but not magnocellular neurons. Other properties of the cells were not affected. In a second series of experiments we examined whether the increase in mIPSC frequency was due to the absence of corticosterone or caused by other effects related to adrenalectomy. The data support the former explanation since implantation of a corticosterone releasing pellet after adrenalectomy fully prevented the change in mIPSC frequency. We conclude that, in the absence of humoral negative feedback, local GABAergic input of parvocellular neurons in the paraventricular nucleus is enhanced. This may provide a compensatory mechanism necessary for maintaining controllable network activity.

The mature enteric nervous system (ENS) is composed of many different neuron subtypes and enteric glia, which all arise from the neural crest. How this diversity is generated from neural crest-derived cells is a central question in neurogastroenterology, as defects in these processes are likely to underlie some paediatric motility disorders. Here we review the developmental appearance (the earliest age at which expression of specific markers can be localized) and birthdates (the age at which precursors exit the cell cycle) of different enteric neuron subtypes, and their projections to some targets. We then focus on what is known about the mechanisms underlying the generation of enteric neuron diversity and axon pathfinding. Finally, we review the development of the ENS in humans and the etiologies of a number of paediatric motility disorders.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.

Vestibular and spiral ganglion neurons (VGNs and SGNs) developed in the inner ear, where they extend fibers to innervate the vestibular and cochlear hair cells and project centrally to the vestibular and cochlear nuclei. This review focuses on representative molecular factors that regulate key processes in the development of inner ear neurons, including their specification, differentiation, axon targeting, and functional diversification. A temporal regulatory cascade defines the initial precursors through factors such as Smarca4, Six1, Eya1, followed by Sox2. While Sox2 deletion abolishes hair cell formation, a subset of inner ear neurons transiently develops but undergoes apoptosis before birth. In contrast, Neurog1 deletion eliminates all ear-derived neurons but results in differential reductions in cochlear and vestibular hair cells. The development and survival of inner ear neurons depend on TrkB and TrkC signaling. Although deletion of TrkB and TrkC results in a complete loss of neurons, each shows distinct effects on VGN and SGN survival and innervation. Downstream of early transcriptional regulators, Neurod1 and Isl1 promote neuronal differentiation, survival, migration, and the formation of peripheral and central projections. The development of VGNs depends on at least two progenitor populations that give rise to three neuronal subtypes that differ in their innervation of vestibular hair cells but show incomplete segregation in the vestibular nuclei. In contrast, SGNs develop later and exhibit sequential segregation into four neuronal subtypes, corresponding to the two types of cochlear hair cells, with tonotopically organized projections to both the cochlea and cochlear nuclei.

In both invertebrates such as Drosophila and vertebrates such as mouse or human, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila, there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. In contrast to T1 neuroblasts, T2 neuroblasts generate intermediate neural progenitors (INPs) that expand the number and diversity of cell types. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of neurons derived from T2 neuroblasts is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster (presumptive neuron subtype). This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

Transcriptional activation of Histone H1° during neuronal terminal differentiation

The brain RAS plays a central role in cardiovascular homeostasis by modulating sympathetic nerve activity and hormone secretion. Still, the relevance of intrinsic brain RAS function is questioned because renin levels are too low to have an impact on angiotensin II (AngII) formation. The discovery of the prorenin receptor (PRR) brought new support to the brain RAS involvement in neurohumoral regulation. However, the mechanisms underlying PRR-mediated actions remain unknown. We used whole cell patch-clamp electrophysiology in hypothalamic slices to examine the effects of prorenin (PR) on the excitability of magnocellular neurosecretory neurons (MCNs) of the supraoptic nucleus and parvocellular RVLM-projecting presympathetic neurons of the paraventricular nucleus (PVN-RVLM), which play integral roles in cardiovascular control, and have been implicated in the pathophysiology of neurogenic hypertension. PR application (2.5nM) increased the firing activity of both MCNs (basal 1.3±0.4 Hz vs PR 3.3±0.7 Hz; p=0.0086) and parvocellular neurons (basal 0.5±0.1 Hz vs PR 2.1±0.4 Hz; p=0.0006), including a subset of PVN-RVLM neurons (basal 0.5±0.1 Hz vs PR 1.4±0.4 Hz; p=0.048). In MCNs, this effect was blocked by a PRR antagonist ( PRO20 ; 250nM; basal 1.9±0.6 Hz vs PRO20 1.3±0.5 Hz; p=0.017), but persisted in the presence of the AT1 receptor blocker, losartan (50μM; basal 0.8±0.2 Hz vs losartan 2.3±0.5 Hz; p=0.0018). Conversely, PR effects on PVN neurons persisted with PRO20 (basal 0.9±0.2 Hz vs PRO20 1.6±0.3 Hz; p=0.013), but were largely (~75%) blunted with losartan (basal 0.2±0.04 Hz vs losartan 0.6±0.1 Hz; p=0.0193; Δ basal 1.6±0.4 Hz vs Δ losartan 0.4±0.1 Hz; p=0.0175). These results suggest that while PR actions are primarily PRR-mediated in MCNs, they are predominantly AngII-dependent in PVN cells. Lastly, PR effects were abolished in both cell types by dialyzing neurons with the Ca 2+ chelator BAPTA (10mM; MCNs: basal 0.6 ± 0.1 Hz vs BAPTA 0.3 ± 0.1 Hz; p=0.0277; PVN: basal 0.4±0.05 Hz vs BAPTA 0.5±0.2 Hz; p=0.6468). Our results show for the first time that PR stimulates firing activity in both hypothalamic neurosecretory and presympathetic neurons, and support distinct AngII-independent and dependent signaling mechanisms in each neuronal population.

Mammalian somatosensory mechanotransduction

Human midbrain dopamine (DA) neurons are a heterogeneous group of cells that share a common neurotransmitter phenotype and are in close anatomical proximity but display different functions, sensitivity to degeneration, and axonal innervation targets. The A9 DA neuron subtype controls motor function and is primarily degenerated in Parkinson’s disease (PD), whereas A10 neurons are largely unaffected by the condition, and their dysfunction is associated with neuropsychiatric disorders. Currently, DA neurons can only be reliably classified on the basis of topographical features, including anatomical location in the midbrain and projection targets in the forebrain. No systematic molecular classification at the genome-wide level has been proposed to date. Although many years of scientific efforts in embryonic and adult mouse brain have positioned us to better understand the complexity of DA neuron biology, many biological phenomena specific to humans are not amenable to being reproduced in animal models. The establishment of human cell-based systems combined with advanced computational single-cell transcriptomics holds great promise for decoding the mechanisms underlying maturation and diversification of human DA neurons, and linking their molecular heterogeneity to functions in the midbrain. Human pluripotent stem cells have emerged as a useful tool to recapitulate key molecular features of mature DA neuron subtypes. Here, we review some of the most recent advances and discuss the current challenges in using stem cells, to model human DA biology. We also describe how single cell RNA sequencing may provide key insights into the molecular programs driving DA progenitor specification into mature DA neuron subtypes. Exploiting the state-of-the-art approaches will lead to a better understanding of stem cell-derived DA neurons and their use in disease modeling and regenerative medicine.

In both Drosophila and mammals, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila , there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of T2-derived neurons is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster. This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

Acute hypernatremia promotes natriuresis in part by increasing activity of oxytocinergic magnocellular neurons in the hypothalamus that promote synaptic release of oxytocin (OT) from axon terminals in the neurohypophysis. Acute hypernatremia also blunts stress‐induced activation of the hypothalamic‐pituitary‐adrenal axis via a mechanism that we believe is likely to depend on central paracrine (rather than synaptic) release of OT from magnocellular dendrites. Interestingly, prior work from other groups indicates that acute hypernatremia produces an increase in the concentration of circulating OT significantly more rapidly than it creates an increase in the concentration of central OT. The current project was designed to develop a better understanding of the underlying cellular mechanisms through which osmotic stressors may plausibly promote both synaptic and paracrine release of OT from the same population of osmosensitive hypothalamic neurons, but with distinctly different temporal dynamics. Towards that end, we used the Cre‐LoxP system to generate OT‐reporter mice that express tdTomato in neurons that synthesize OT. Initial studies indicated a strong colocalization of tdTomato with OT/neurophysin1 throughout the hypothalamus. Whole‐cell patch clamp recordings from tdTomato positive magnocellular neurons in the paraventricular nucleus of the hypothalamus revealed expected intrinsic properties, including transient outward rectification produced by a prominent A‐type K+ current (IA). Initial imaging studies revealed that the dendrites of OT positive magnocellular neurons are passive conductors, with robust activity dependent dendritic calcium influx that diminishes substantially with increasing distance from the soma. Bath application of an external osmolyte (15 mM mannitol) produced a clear increase in the frequency of spontaneous action potentials observed in OT positive magnocellular neurons, and yet intriguingly, also produced a clear decrease in activity dependent dendritic calcium influx. This effect was also much more prominent in distal dendrites than in proximal dendrites, consistent with a mechanism that depends on decreased dendritic input resistance. Also consistent with that interpretation, we found that acute application of 15 mM mannitol significantly reduces the amplitude of evoked glutamatergic EPSCs recorded at the soma, as produced by a small theta glass stimulator placed near the distal dendrites. Interestingly, preliminary data suggest that parvocellular OT neurons lack similar osmosensitivty. Collectively, these results provide a partial mechanistic explanation for why acute osmotic stress is likely to initially favor synaptic over paracrine release of OT from osmosensitive hypothalamic magnocellular neurons.Support or Funding InformationSupported by NIMH R0104641This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.