Jessica L. Ables

Dynamic Contribution of Nestin-Expressing Stem Cells to Adult Neurogenesis

Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreERT2/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreERT2/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreERT2/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreERT2/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.

Neurod1 is essential for the survival and maturation of adult-born neurons

The transcriptional program that controls adult neurogenesis is unknown. We generated mice with an inducible stem cell–specific deletion of Neurod1, resulting in substantially fewer newborn neurons in the hippocampus and olfactory bulb. Thus, Neurod1 is cell-intrinsically required for the survival and maturation of adult-born neurons.

Not(ch) just development: Notch signalling in the adult brain

The Notch pathway is often regarded as a developmental pathway, but components of Notch signalling are expressed and active in the adult brain. With the advent of more sophisticated genetic manipulations, evidence has emerged that suggests both conserved and novel roles for Notch signalling in the adult brain. Not surprisingly, Notch is a key regulator of adult neural stem cells, but it is increasingly clear that Notch signalling also has roles in the regulation of migration, morphology, synaptic plasticity and survival of immature and mature neurons. Understanding the many functions of Notch signalling in the adult brain, and its dysfunction in neurodegenerative disease and malignancy, is crucial to the development of new therapeutics that are centred around this pathway.

Notch1 Is Required for Maintenance of the Reservoir of Adult Hippocampal Stem Cells

Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreERT2/R26R-YFP/Notch1loxP/loxP [Notch1inducible knock-out (iKO)] mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild-type (WT) mice (nestin-CreERT2/R26R-YFP/Notch1w/w) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared with WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 d post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 d post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 d of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli.

IκB Kinase Regulates Social Defeat Stress-Induced Synaptic and Behavioral Plasticity

The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase–related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat–induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.Depression involves plasticity of brain reward neurons, although the mechanisms and behavioral relevance are unknown. Transcriptional profiling of nucleus accumbens (NAc) for RhoGTPase related genes, known regulators of synaptic structure, following chronic social defeat stress, revealed a long-term reduction in Rac1 transcription. This was marked by a repressive chromatin state surrounding its proximal promoter. Inhibition of class 1 HDACs with MS-275 rescued both decreased Rac1 transcription and social avoidance behavior. A similar repressive chromatin state was found surrounding the Rac1 promoter in human postmortem NAc from depressed subjects, which corresponded with reduced Rac1 transcription. We show Rac1 is necessary and sufficient for social avoidance and anhedonia, and the formation of stubby excitatory spines by redistributing synaptic cofilin, an actin severing protein downstream of Rac1. Our data identifies epigenetic regulation of Rac1 in NAc as a bona fide disease mechanism in depression and reveals a functional role in regulating stress-related behaviors.

AKT Signaling within the Ventral Tegmental Area Regulates Cellular and Behavioral Responses to Stressful Stimuli

BACKGROUND The neurobiological mechanisms by which only a minority of stress-exposed individuals develop psychiatric diseases remain largely unknown. Recent evidence suggests that dopaminergic neurons of the ventral tegmental area (VTA) play a key role in the manifestation of stress vulnerability. METHODS Using a social defeat paradigm, we segregated susceptible mice (socially avoidant) from unsusceptible mice (socially interactive) and examined VTA punches for changes in neurotrophic signaling. Employing a series of viral vectors, we sought to causally implicate these neurotrophic changes in the development of avoidance behavior. RESULTS Susceptibility to social defeat was associated with a significant reduction in levels of active/phosphorylated AKT (thymoma viral proto-oncogene) within the VTA, whereas chronic antidepressant treatment (in mice and humans) increased active AKT levels. This defeat-induced reduction in AKT activation in susceptible mice was both necessary and sufficient to recapitulate depressive behaviors associated with susceptibility. Pharmacologic reductions in AKT activity also significantly raised the firing frequency of VTA dopamine neurons, an important electrophysiologic hallmark of the susceptible phenotype. CONCLUSIONS These studies highlight a crucial role for decreases in VTA AKT signaling as a key mediator of the maladaptive cellular and behavioral response to chronic stress.

Ascl1 (Mash1) Defines Cells with Long-Term Neurogenic Potential in Subgranular and Subventricular Zones in Adult Mouse Brain

Ascl1 (Mash1) is a bHLH transcription factor essential for neural differentiation during embryogenesis but its role in adult neurogenesis is less clear. Here we show that in the adult brain Ascl1 is dynamically expressed during neurogenesis in the dentate gyrus subgranular zone (SGZ) and more rostral subventricular zone (SVZ). Specifically, we find Ascl1 levels low in SGZ Type-1 cells and SVZ B cells but increasing as the cells transition to intermediate progenitor stages. In vivo genetic lineage tracing with a tamoxifen (TAM) inducible Ascl1CreERT2 knock-in mouse strain shows that Ascl1 lineage cells continuously generate new neurons over extended periods of time. There is a regionally-specific difference in neuron generation, with mice given TAM at postnatal day 50 showing new dentate gyrus neurons through 30 days post-TAM, but showing new olfactory bulb neurons even 180 days post-TAM. These results show that Ascl1 is not restricted to transit amplifying populations but is also found in a subset of neural stem cells with long-term neurogenic potential in the adult brain.

In vivo contribution of nestin- and GLAST-lineage cells to adult hippocampal neurogenesis.

Radial glia‐like cells (RGCs) are the hypothesized source of adult hippocampal neurogenesis. However, the current model of hippocampal neurogenesis does not fully incorporate the in vivo heterogeneity of RGCs. In order to better understand the contribution of different RGC subtypes to adult hippocampal neurogenesis, we employed widely used transgenic lines (Nestin‐CreERT2 and GLAST::CreERT2 mice) to explore how RGCs contribute to neurogenesis under basal conditions and after stimulation and depletion of neural progenitor cells. We first used these inducible fate‐tracking transgenic lines to define the similarities and differences in the contribution of nestin‐ and GLAST‐lineage cells to basal long‐term hippocampal neurogenesis. We then explored the ability of nestin‐ and GLAST‐lineage RGCs to contribute to neurogenesis after experimental manipulations that either ablate neurogenesis (i.c.v. application of the anti‐mitotic AraC, cytosine‐β‐D‐arabinofuranoside) or stimulate neurogenesis (wheel running). Interestingly, in both ablation and stimulation experiments, labeled RGCs in GLAST::CreERT2 mice appear to contribute to neurogenesis, whereas RGCs in Nestin‐CreERT2 mice do not. Finally, using NestinGFP reporter mice, we expanded on previous research by showing that not all RGCs in the adult dentate gyrus subgranular zone express nestin, and therefore RGCs are antigenically heterogeneous. These findings are important for the field, as they allow appropriately conservative interpretation of existing and future data that emerge from these inducible transgenic lines. These findings also raise important questions about the differences between transgenic driver lines, the heterogeneity of RGCs, and the potential differences in progenitor cell behavior between transgenic lines. As these findings highlight the possible differences in the contribution of cells to long‐term neurogenesis in vivo, they indicate that the current models of hippocampal neurogenesis should be modified to include RGC lineage heterogeneity.

Reexposure to nicotine during withdrawal increases the pacemaking activity of cholinergic habenular neurons

Significance According to the World Health Organization, tobacco consumption causes the death of close to 6 million people each year, yet successful attempts to quit smoking are very rare. The present study identifies a group of neurons in the brain that respond differently to nicotine after a period of abstinence, suggesting that altered activity of these neurons may contribute to difficulties with smoking cessation. The discovery of genetic variants in the cholinergic receptor nicotinic CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula–interpeduncular axis as a critical relay circuit in the control of nicotine dependence. Although clear roles for α3, β4, and α5 receptors in nicotine aversion and withdrawal have been established, the cellular and molecular mechanisms that participate in signaling nicotine use and contribute to relapse have not been identified. Here, using translating ribosome affinity purification (TRAP) profiling, electrophysiology, and behavior, we demonstrate that cholinergic neurons, but not peptidergic neurons, of the medial habenula (MHb) display spontaneous tonic firing of 2–10 Hz generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels and that infusion of the HCN pacemaker antagonist ZD7288 in the habenula precipitates somatic and affective signs of withdrawal. Further, we show that a strong, α3β4-dependent increase in firing frequency is observed in these pacemaker neurons upon acute exposure to nicotine. No change in the basal or nicotine-induced firing was observed in cholinergic MHb neurons from mice chronically treated with nicotine. We observe, however, that, during withdrawal, reexposure to nicotine doubles the frequency of pacemaking activity in these neurons. These findings demonstrate that the pacemaking mechanism of cholinergic MHb neurons controls withdrawal, suggesting that the heightened nicotine sensitivity of these neurons during withdrawal may contribute to smoking relapse.