Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca2+) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.

Norepinephrine (NE), and specific adrenoceptors, have been reported to influence distinct aspects of adult hippocampal neurogenesis, including latent stem cell activation, progenitor proliferation, and differentiation. These findings are predominantly based on the use of pharmacological approaches in both in vitro and in vivo systems. Here, we sought to assess the consequences of genetic ablation of NE on adult hippocampal neurogenesis, by examining dopamine β hydroxylase knockout (Dbh -/-) mice, which lack NE from birth. We find that Dbh -/- mice exhibit no difference in adult hippocampal progenitor proliferation and survival. Further, the number of immature newborn neurons, labeled using stage-specific developmental markers within the hippocampal neurogenic niche, was also unaltered in Dbh -/- mice. In contrast, the noradrenergic neurotoxin DSP-4, which had previously been shown to reduce adult hippocampal neurogenesis in rats, also resulted in a decline in hippocampal progenitor proliferation in C57/Bl6N mice. These findings indicate that pharmacological lesioning of noradrenergic afferents in adulthood, but not the complete genetic loss of NE from birth, impairs adult hippocampal neurogenesis in mice.

Motor symptoms in Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons of the substantia nigra (SN), while early non-motor symptoms such as anxiety and sleep disturbances are likely mediated by dysfunction of locus coeruleus (LC) norepinephrine (NE) neurons. The LC develops α-synuclein pathology prior to SN DA neurons in PD, and later undergoes degeneration, but the mechanisms responsible for its vulnerability are unknown. The SN and LC are the only structures in the brain that produces appreciable amounts of neuromelanin (NM), a dark cytoplasmic pigment. It has been proposed that NM initially plays a protective role by sequestering toxic catecholamine metabolites and heavy metals, but may become harmful during aging and PD as they overwhelm cellular machinery and are released during neurodegeneration. Rodents do not naturally produce NM, limiting the study of causal relationships between NM and PD-associated LC pathology. Adapting a viral-mediated approach for expression of human tyrosinase, the enzyme responsible for peripheral melanin production, we successfully promoted pigmentation in mouse LC neurons that recapitulates key features of endogenous NM found in primates, including eumelanin and pheomelanin, lipid droplets, and a double-membrane encasement. Pigment expression results in mild neurodegeneration, reduced NE levels, transcriptional changes, and novelty-induced anxiety phenotypes as early as 1-week post-injection. By 6-weeks, NM accumulation is associated with severe LC neurodegeneration and a robust neuroinflammatory response. These phenotypes are reminiscent of LC dysfunction in PD, validating this model for studying the consequences of pigment accumulation in the LC as it relates to neurodegenerative disease.

RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro . As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.

Mineralocorticoid receptors (MRs) in the brain play a role in learning and memory, neuronal differentiation, and regulation of the stress response. Within the hippocampus, the highest expression of MRs is in area CA2. CA2 pyramidal neurons have a distinct molecular makeup resulting in a plasticity-resistant phenotype, distinguishing them from neurons in CA1 and CA3. Thus, we asked whether MRs regulate CA2 neuron properties and CA2-related behaviors. Using three conditional knockout methods at different stages of development, we found a striking decrease in multiple molecular markers for CA2, an effect mimicked by chronic antagonism of MRs. Furthermore, embryonic deletion of MRs disrupted afferent inputs to CA2 and enabled synaptic potentiation of the normally LTP-resistant synaptic currents in CA2. We also found that CA2-targeted MR knockout was sufficient to disrupt social behavior and alter behavioral responses to novelty. Altogether, these results demonstrate an unappreciated role for MRs in controlling CA2 pyramidal cell identity and in facilitating CA2-dependent behaviors.

Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

In rodents, exposure to predator odors such as cat urine acts as a severe stressor that engages innate defensive behaviors critical for survival in the wild. The neurotransmitters norepinephrine (NE) and dopamine (DA) modulate anxiety and predator odor responses, and we have shown previously that dopamine β-hydroxylase knockout (Dbh -/-), which reduces NE and increases DA in mouse noradrenergic neurons, disrupts innate behaviors in response to mild stressors such as novelty. We examined the consequences of Dbh knockout (Dbh -/-) on responses to predator odor (bobcat urine) and compared them to Dbh-competent littermate controls. Over the first 10 min of predator odor exposure, controls exhibited robust defensive burying behavior, whereas Dbh -/- mice showed high levels of grooming. Defensive burying was potently suppressed in controls by drugs that reduce NE transmission, while excessive grooming in Dbh -/- mice was blocked by DA receptor antagonism. In response to a cotton square scented with a novel "neutral" odor (lavender), most control mice shredded the material, built a nest, and fell asleep within 90 min. Dbh -/- mice failed to shred the lavender-scented nestlet, but still fell asleep. In contrast, controls sustained high levels of arousal throughout the predator odor test and did not build nests, while Dbh -/- mice were asleep by the 90-min time point, often in shredded bobcat urine-soaked nesting material. Compared with controls exposed to predator odor, Dbh -/- mice demonstrated decreased c-fos induction in the anterior cingulate cortex, lateral septum, periaqueductal gray, and bed nucleus of the stria terminalis, but increased c-fos in the locus coeruleus and medial amygdala. These data indicate that relative ratios of central NE and DA signaling coordinate the type and valence of responses to predator odor.

The human genome contains vast genetic diversity as naturally occurring coding variants, yet the impact of these variants on protein function and physiology is poorly understood. RGS14 is a multifunctional signaling protein that suppresses synaptic plasticity in dendritic spines of hippocampal neurons. RGS14 also is a nucleocytoplasmic shuttling protein, suggesting that balanced nuclear import/export and dendritic spine localization are essential for RGS14 functions. We identified genetic variants L505R (LR) and R507Q (RQ) located within the nuclear export sequence (NES) of human RGS14. Here we report that RGS14 encoding LR or RQ profoundly impacts protein functions in hippocampal neurons. RGS14 membrane localization is regulated by binding Gαi-GDP, whereas RGS14 nuclear export is regulated by Exportin 1 (XPO1). Remarkably, LR and RQ variants disrupt RGS14 binding to Gαi1-GDP and XPO1, nucleocytoplasmic equilibrium, and capacity to inhibit long-term potentiation (LTP). Variant LR accumulates irreversibly in the nucleus, preventing RGS14 binding to Gαi1, localization to dendritic spines, and inhibitory actions on LTP induction, while variant RQ exhibits a mixed phenotype. When introduced into mice by CRISPR/Cas9, RGS14-LR protein expression was detected predominantly in the nuclei of neurons within hippocampus, central amygdala, piriform cortex, and striatum, brain regions associated with learning and synaptic plasticity. Whereas mice completely lacking RGS14 exhibit enhanced spatial learning, mice carrying variant LR exhibit normal spatial learning, suggesting that RGS14 may have distinct functions in the nucleus independent from those in dendrites and spines. These findings show that naturally occurring genetic variants can profoundly alter normal protein function, impacting physiology in unexpected ways.

Exposure to unfamiliar odorants induces an array of repetitive defensive and non-defensive behaviors in rodents which likely reflect adaptive stress responses to the uncertain valence of novel stimuli. Mice genetically deficient for dopamine β-hydroxylase (Dbh-/-) lack the enzyme required to convert dopamine (DA) into norepinephrine (NE), resulting in globally undetectable NE and supranormal DA levels. Because catecholamines modulate novelty detection and reactivity, we investigated the effects of novel plant-derived odorants on repetitive behaviors in Dbh-/- mice and Dbh+/- littermate controls, which have catecholamine levels comparable to wild-type mice. Unlike Dbh+/- controls, which exhibited vigorous digging in response to novel odorants, Dbh-/- mice displayed excessive grooming. Drugs that block NE synthesis or neurotransmission suppressed odorant-induced digging in Dbh+/- mice, while a DA receptor antagonist attenuated grooming in Dbh-/- mice. The testing paradigm elicited high circulating levels of corticosterone regardless of Dbh genotype, indicating that NE is dispensable for this systemic stress response. Odorant exposure increased NE and DA abundance in the prefrontal cortex (PFC) of Dbh+/- mice, while Dbh-/- animals lacked NE and had elevated PFC DA levels that were unaffected by novel smells. Together, these findings suggest that novel odorant-induced increases in central NE tone contribute to repetitive digging and reflect psychological stress, while central DA signaling contributes to repetitive grooming. Further, we have established a simple method for repeated assessment of stress-induced repetitive behaviors in mice, which may be relevant for modeling neuropsychiatric disorders like Tourette syndrome or obsessive-compulsive disorder that are characterized by stress-induced exacerbation of compulsive symptoms.

The noradrenergic locus coeruleus (LC) is among the earliest sites of tau and α-synuclein pathology in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The onset of these pathologies coincides with loss of noradrenergic fibers in LC target regions and the emergence of prodromal symptoms including sleep disturbances and anxiety. Paradoxically, these prodromal symptoms are indicative of a noradrenergic hyperactivity phenotype, rather than the predicted loss of norepinephrine (NE) transmission following LC damage, suggesting the engagement of complex compensatory mechanisms. Because current therapeutic efforts are targeting early disease, interest in the LC has grown, and it is critical to identify the links between pathology and dysfunction. We employed the LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which preferentially damages LC axons, to model early changes in the LC-NE system pertinent to AD and PD in male and female mice. DSP-4 (two doses of 50 mg/kg, one week apart) induced LC axon degeneration, triggered neuroinflammation and oxidative stress, and reduced tissue NE levels. There was no LC cell death or changes to LC firing, but transcriptomics revealed reduced expression of genes that define noradrenergic identity and other changes relevant to neurodegenerative disease. Despite the dramatic loss of LC fibers, NE turnover and signaling were elevated in terminal regions and were associated with anxiogenic phenotypes in multiple behavioral tests. These results represent a comprehensive analysis of how the LC-NE system responds to axon/terminal damage reminiscent of early AD and PD at the molecular, cellular, systems, and behavioral levels, and provides potential mechanisms underlying prodromal neuropsychiatric symptoms.