Stress coping stimulates hippocampal neurogenesis in adult monkeys

Coping with intermittent social stress is an essential aspect of living in complex social environments. Coping tends to counteract the deleterious effects of stress and is thought to induce neuroadaptations in corticolimbic brain systems. Here we test this hypothesis in adult squirrel monkey males exposed to intermittent social separations and new pair formations. These manipulations simulate conditions that typically occur in male social associations because of competition for limited access to residency in mixed-sex groups. As evidence of coping, we previously confirmed that cortisol levels initially increase and then are restored to prestress levels within several days of each separation and new pair formation. Follow-up studies with exogenous cortisol further established that feedback regulation of the hypothalamic-pituitary-adrenal axis is not impaired. Now we report that exposure to intermittent social separations and new pair formations increased hippocampal neurogenesis in squirrel monkey males. Hippocampal neurogenesis in rodents contributes to spatial learning performance, and in monkeys we found that spatial learning was enhanced in conditions that increased hippocampal neurogenesis. Corresponding changes were discerned in the expression of genes involved in survival and integration of adult-born granule cells into hippocampal neural circuits. These findings support recent indications that stress coping stimulates hippocampal neurogenesis in adult rodents. Psychotherapies designed to promote stress coping potentially have similar effects in humans with major depression.     

Phenotypic deficits in mice expressing a myosin binding protein C lacking the titin and myosin binding domains

The majority of familial hypertrophic cardiomyopathy patients carrying a mutation in the cardiac myosin binding protein C gene show low penetrance, late onset of the disease and a relatively benign phenotype. Sudden death in these patients, if it occurs, usually takes place after the fifth or sixth decade of life and can be precipitated by stress. Previously, we prepared mice carrying a mutated MyBP-C lacking both the titin and myosin binding sites at the carboxyl terminus. This mutation is found in some familial hypertrophic cardiomyopathy patients and the mice develop some symptoms that are consistent with the disease. In the present study, we wished to determine the response of these animals to various forms of cardiovascular stress. Consistent with the human disease presentation, only a mild cardiac hypertrophy was detected in unstressed animals. Although there are no complementary human data with which to compare the mice, molecular signs of stress were apparent in the animals, as increased levels of the intermediate filament protein, desmin and the chaperone protein, alpha-B-crystallin, were present in the hearts. To determine whether the animals were sensitive to stress, they were subjected to sub-maximal treadmill exercise or to chronic isoproterenol infusion. The affected mice were significantly compromised in their exercise capacity and showed an impaired response during isoproterenol infusion. Increased mortality was observed during the exercise regimen, with some animals experiencing sudden death. We conclude that the mouse model recapitulates some of the known aspects of the human disease, particularly its late onset and benign phenotype. However, cardiac stress can lead to severe bradycardia and death.     

Mice lacking the calcineurin inhibitor Rcan2 have an isolated defect of osteoblast function

Calcineurin-nuclear factor of activated T cells signaling controls the differentiation and function of osteoclasts and osteoblasts, and regulator of calcineurin-2 (Rcan2) is a physiological inhibitor of this pathway. Rcan2 expression is regulated by T(3), which also has a central role in skeletal development and bone turnover. To investigate the role of Rcan2 in bone development and maintenance, we characterized Rcan2(-/-) mice and determined its skeletal expression in T(3) receptor (TR) knockout and thyroid-manipulated mice. Rcan2(-/-) mice had normal linear growth but displayed delayed intramembranous ossification, impaired cortical bone formation, and reduced bone mineral accrual during development as well as increased mineralization of adult bone. These abnormalities resulted from an isolated defect in osteoblast function and are similar to skeletal phenotypes of mice lacking the type 2 deiodinase thyroid hormone activating enzyme or with dominant-negative mutations of TRα, the predominant TR isoform in bone. Rcan2 mRNA was expressed in primary osteoclasts and osteoblasts, and its expression in bone was differentially regulated in TRα and TRβ knockout and thyroid-manipulated mice. However, in primary osteoblast cultures, T(3) treatment did not affect Rcan2 mRNA expression or nuclear factor of activated T cells c1 expression and phosphorylation. Overall, these studies establish that Rcan2 regulates osteoblast function and its expression in bone is regulated by thyroid status in vivo.     

Mice lacking Mrp1 have reduced testicular steroid hormone levels and alterations in steroid biosynthetic enzymes

The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ABC active transporter family that can transport several steroid hormone conjugates, including 17β-estradiol glucuronide, dehydroepiandrosterone sulfate (DHEAS), and estrone 3-sulfate. The present study investigated the role that MRP1 plays in maintaining proper hormone levels in the serum and testes. Serum and testicular steroid hormone levels were examined in both wild-type mice and Mrp1 null mice. Serum testosterone levels were reduced 5-fold in mice lacking Mrp1, while testicular androstenedione, testosterone, estradiol, and dehydroepiandrosterone (DHEA) were significantly reduced by 1.7- to 4.5-fold in Mrp1 knockout mice. Investigating the mechanisms responsible for the reduction in steroid hormones in Mrp1−/− mice revealed no differences in the expression or activity of enzymes that inactivate steroids, the sulfotransferases or glucuronosyltransferases. However, steroid biosynthetic enzyme levels in the testes were altered. Cyp17 protein levels were increased by 1.6-fold, while Cyp17 activity using progesterone as a substrate was also increased by 1.4- to 2.0-fold in mice lacking Mrp1. Additionally, the ratio of 17β-hydroxysteroid dehydrogenase to 3β-hydroxysteroid dehydrogenase, and steroidogenic factor 1 to 3β-hydroxysteroid dehydrogenase were significantly increased in the testes of Mrp1−/− mice. These results indicate that Mrp1−/− mice have lowered steroid hormones levels, and suggests that upregulation of steroid biosynthetic enzymes may be an attempt to maintain proper steroid hormone homeostasis.     

Norbin ablation results in defective adult hippocampal neurogenesis and depressive-like behavior in mice

Adult neurogenesis in the hippocampus subgranular zone is associated with the etiology and treatment efficiency of depression. Factors that affect adult hippocampal neurogenesis have been shown to contribute to the neuropathology of depression. Glutamate, the major excitatory neurotransmitter, plays a critical role in different aspects of neurogenesis. Of the eight metabotropic glutamate receptors (mGluRs), mGluR5 is the most highly expressed in neural stem cells. We previously identified Norbin as a positive regulator of mGluR5 and showed that its expression promotes neurite outgrowth. In this study, we investigated the role of Norbin in adult neurogenesis and depressive-like behaviors using Norbin-deficient mice. We found that Norbin deletion significantly reduced hippocampal neurogenesis; specifically, the loss of Norbin impaired the proliferation and maturation of newborn neurons without affecting cell-fate specification of neural stem cells/neural progenitor cells (NSCs/NPCs). Norbin is highly expressed in the granular neurons in the dentate gyrus of the hippocampus, but it is undetectable in NSCs/NPCs or immature neurons, suggesting that the effect of Norbin on neurogenesis is likely caused by a nonautonomous niche effect. In support of this hypothesis, we found that the expression of a cell–cell contact gene, Desmoplakin, is greatly reduced in Norbin-deletion mice. Moreover, Norbin-KO mice show an increased immobility in the forced-swim test and the tail-suspension test and reduced sucrose preference compared with wild-type controls. Taken together, these results show that Norbin is a regulator of adult hippocampal neurogenesis and that its deletion causes depressive-like behaviors.Major depressive disorder (MDD) is one of the most common and debilitating psychiatric illnesses with a lifetime prevalence of ∼17% (1). Clinical symptoms of MDD include anhedonia, depressed mood, helplessness, and cognitive disruption. Because of the clinical and etiological heterogeneity of MDD, the pathophysiology of MDD remains elusive (2), and the biological underpinnings remain unclear. Decades of research have clearly established that various neurotransmitters, especially monoamine neurotransmitters, as well as neurotrophic factors, contribute to MDD (3). More recently glutamatergic signaling also has been linked to MDD. With the confirmation of continuous neurogenesis in adult brains of most mammals, including humans, a neurogenic hypothesis of MDD has been put forward and continues to drive research (4).The neurogenic hypothesis of MDD is based on several correlative studies. Brain imaging and postmortem studies of MDD patients suggest that reduction in hippocampal volume could be reflective of reduced neurogenesis in addition to mature neuronal cell loss (5, 6). Antidepressant treatment in animals increases hippocampal neurogenesis with a lag time that is reminiscent of the delayed onset of antidepressant efficacy typically observed in humans (7). In addition, stress, a common risk factor for depression, inhibits neurogenesis in nonhuman primates, and this phenomenon can be corrected by antidepressant treatment (8). In rodents, ablation of hippocampal neurogenesis causes a depressive phenotype and abolishes the effect of certain antidepressants (9). Therefore, it is hypothesized that reduced adult hippocampal neurogenesis may underlie the pathological mechanism of MDD and that an up-regulation of neurogenesis would oppose the action of stress and/or depression, having a potential therapeutic benefit for MDD.In the hippocampus, adult neurogenesis involves the five following steps: (i) proliferation of neural stem/progenitor cells (NSCs/NPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG); (ii) fate specification (neurons versus astrocytes); (iii) massive loss of progenitor cells; (iv) neuronal differentiation and maturation; and (v) ultimate integration to the existing neuronal circuitry of the DG (10). Each step is modulated by both physiological stimuli and pathophysiological conditions. Two important determinants, cell-autonomous factors and neurogenic niche, play a key role in determining the ultimate outcome of adult neurogenesis. Cell-autonomous factors are intrinsic NSC/NPC genetic traits that dictate their fate, and the neurogenic niche corresponds to their local microenvironment defined by various parameters (e.g., neuronal inputs, vasculature, and glia composition) that interact with NSCs/NPCs and also influence their fate (11).Previous studies indicated that neurotransmitter glutamate regulates DG neurogenesis (12, 13). Of the eight metabotropic glutamate receptors (mGluRs), mGluR5 is most highly expressed in NSCs (14, 15). In vivo activation of mGluR5 receptors benefits the proliferation and/or survival of NSCs in the hippocampus. In vitro, pharmacological blockade of mGluR5 reduced NSC proliferation and survival, whereas activation of mGluR5 receptors substantially enhanced cell proliferation, suggesting that mGluR5 has a cell-autonomous role of in neurogenesis (16, 17).We previously identified Norbin as a positive regulator of mGluR5 (18). Forebrain-specific Norbin-KO mice display phenotypic characteristics that resemble phenotypes observed in cases of reduced mGluR5 activity, including impaired synaptic plasticity and sensitivity to psychostimulants. In the CNS, Norbin is a neuronal gene whose expression is up-regulated concomitantly with long-term potentiation, the electrophysiological mechanism underlying learning and memory. Norbin expression correlates with neurite outgrowth and also was found to have an impact on both the quantity and the length of neurites in cultured neuroblastoma N2a cells. In summary, Norbin (i) regulates mGluR5, whose activity affects DG neurogenesis; (ii) promotes neurite outgrowth; (iii) is highly expressed in the DG of the hippocampus, the niche for adult DG neurogenesis. Norbin-KO mice also display impaired cognitive functions. For all these reasons, we investigated the role of Norbin in adult hippocampal neurogenesis and whether the loss of Norbin could contribute to depression-like behaviors.We found here that neuron-specific Norbin ablation in mice, using two distinct Cre systems, results in a significant reduction in hippocampal neurogenesis. Both the proliferation and survival of newborn neurons are impaired. This impairment is likely the result of a nonautonomous niche effect because Norbin expression is limited to mature granular neurons in the DG. In addition, gene-expression analysis of Norbin-KO mice compared with wild-type controls indicated that Norbin deletion results in a significant reduction in desmoplakin (DSP) expression. In light of the observed reduction in adult hippocampal neurogenesis in Norbin-KO mice, we used three well-established behavioral paradigms to determine if there also was a depressive-like consequence of Norbin KO. Results from the forced-swim test (FST), the tail-suspension test (TST), and the sucrose preference test indicated that Norbin-KO mice display depressive-like characteristics.     

Melatonin's stimulatory effect on adult hippocampal neurogenesis in mice persists after ovariectomy

In this study, we examined whether melatonin treatment would increase new cell formation in the hippocampus in ovariectomized (OVX) mice. Chronic exogenous melatonin administration increased bromodeoxyuridine (BrdU) (OVX-sham 72 ± 3.2 versus OVX-mel 122 ± 12.0; P < 0.05) and doublecortin (DCX) (OVX-sham 88 ± 3.1 versus OVX-mel 176 ± 9.9; P < 0.05) immunoreactive cells in the hippocampus of ovariectomized mice. This neuronal development was correlated with synaptic plasticity, identified using the Golgi impregnation method to quantify dendritic spines in mouse dentate gyrus (DG). Finally, the antidepressant-like state of the animals was evaluated by the tail suspension test. The results indicate that melatonin acts on birth, survival, and differentiation of new neurons in the hippocampus, stimulates maturation of spines, and exerts an antidepressant-like action under estrogen-deprived conditions, in both a strain- and gender-independent manner, suggesting that this indoleamine may be useful in improving brain functions.     

Chronic treatment with melatonin stimulates dendrite maturation and complexity in adult hippocampal neurogenesis of mice

In the course of adult hippocampal neurogenesis, the postmitotic maturation and survival phase is associated with dendrite maturation. Melatonin modulates the survival of new neurons with relative specificity. During this phase, the new neurons express microtubule-associated protein doublecortin (DCX). Here, we show that the entire population of cells expressing DCX is increased after 14 days of treatment with melatonin. As melatonin also affects microtubule polymerization which is important for neuritogenesis and dendritogenesis, we studied the consequences of chronic melatonin administration on dendrite maturation of DCX-positive cells. Treatment with melatonin increased the number of DCX-positive immature neurons with more complex dendrites. Sholl analysis revealed that melatonin treatment lead to greater complexity of the dendritic tree. In addition, melatonin increased the total volume of the granular cell layer. Besides its survival-promoting effect, melatonin thus also increases dendritic maturation in adult neurogenesis. This might open the opportunity of using melatonin as an adjuvant in attempts to extrinsically stimulate adult hippocampal neurogenesis in neuropsychiatric disease, dementia or cognitive ageing.     

Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.     

Persistent impairment of hippocampal neurogenesis in young adult rats following early postnatal alcohol exposure

Background: Prenatal alcohol exposure can cause damage to the developing fetus with outcomes including growth deficiency, facial dysmorphology, brain damage, and cognitive and behavioral deficits. Smaller brains in children with FASD have been linked both with reduced cell proliferation in the developing CNS and with apoptotic cell loss of postmitotic neurons. Prenatal alcohol exposure in rodents during the period of brain development comparable to that of the first and second trimesters of human pregnancy persistently alters adult neurogenesis. Long‐term effects of alcohol exposure during the third trimester equivalent, which occurs postnatally in the rat, on adult neurogenesis have not been previously reported. The goal of this study was to examine the effect of postnatal binge‐like alcohol exposure on cell proliferation and neurogenesis in hippocampal dentate gyrus during adolescence and young adulthood. Methods: Male Long‐Evans rat pups were assigned to 3 groups: alcohol‐exposed (AE), sham‐intubated (SI) or suckle control (SC). AE pups received ethanol in a milk formula in a binge manner (2 feedings, 2 hours apart, total dose 5.25 g/kg/day) on postnatal days (PD) 4–9. BrdU was injected every other day on PD30–50. Animals were perfused either on PD50 to examine cytogenesis and neurogenesis in hippocampal dentate gyrus at the end of BrdU injections or on PD80 to evaluate new cell survival. Dorsal hippocampal sections were immunostained for BrdU, a marker for proliferating cells, Ki67, endogenous marker of proliferation, and NeuN, a marker for mature neurons. Results: Binge‐like alcohol exposure on PD4–9 significantly reduced the number of mature neurons in adult hippocampal dentate gyrus (DG) both on PD50 and PD80, without altering cumulative cytogenesis on PD50. In addition, the number of new neurons, that were generated between PD30 and 50, was further reduced after 30 days of survival in all 3 groups (SC, SI, and AE). Conclusions: These observations suggest that early postnatal binge alcohol exposure results in long‐term deficits of adult hippocampal neurogenesis, providing a potential basis for the deficits of hippocampus‐dependent behaviors reported for this model.     

Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures

Mutations in doublecortin (DCX) are associated with intractable epilepsy in humans, due to a severe disorganization of the neocortex and hippocampus known as classical lissencephaly. However, the basis of the epilepsy in lissencephaly remains unclear. To address potential functional redundancy with murin Dcx, we targeted one of the closest homologues, doublecortin-like kinase 2 (Dclk2). Here, we report that Dcx; Dclk2-null mice display frequent spontaneous seizures that originate in the hippocampus, with most animals dying in the first few months of life. Elevated hippocampal expression of c-fos and loss of somatostatin-positive interneurons were identified, both known to correlate with epilepsy. Dcx and Dclk2 are coexpressed in developing hippocampus, and, in their absence, there is dosage-dependent disrupted hippocampal lamination associated with a cell-autonomous simplification of pyramidal dendritic arborizations leading to reduced inhibitory synaptic tone. These data suggest that hippocampal dysmaturation and insufficient receptive field for inhibitory input may underlie the epilepsy in lissencephaly, and suggest potential therapeutic strategies for controlling epilepsy in these patients.