Reviving through human hippocampal newborn neurons.

Recent contradictory data has renewed discussion regarding the existence of adult hippocampal neurogenesis (AHN) in humans, i.e., the continued production of new neurons in the brain after birth. The present review revisits the debate of AHN in humans from a historical point of view in the face of contradictory evidence, analyzing the methods employed to investigate this phenomenon. Thus, to date, of the 57 studies performed in humans that we reviewed, 84% (48) concluded in favor of the presence of newborn neurons in the human adult hippocampus. Besides quality of the tissue (such as postmortem intervals below 26hours as well as tissue conservation and fixation), considerations for assessing and quantify AHN in the human brain require the use of stereology and toxicological analyses of clinical data of the patient.

Elevated alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors.

Substantial evidence links alcohol drinking and serotonin (5-HT) functioning in animals. Lowered central 5-HT neurotransmission has been found in a subgroup of alcoholics, possibly those with more aggressive, assaultive tendencies. Several rodent studies have also suggested that intact 5-HT systems are important determinants of sensitivity and/or tolerance to ethanol-induced ataxia and hypothermia. Null mutant mice lacking the 5-HT1B receptor gene (5-HT1B-/-) have been developed that display enhanced aggression and altered 5-HT release in slice preparations from some, but not all, brain areas. We characterized these mice for sensitivity to several effects of ethanol. Mutant mice drank twice as much ethanol as wild-type mice, and voluntarily ingested solutions containing up to 20% ethanol in water. Their intake of food and water, and of sucrose, saccharin and quinine solutions, was normal. Mutants were less sensitive than wild-types on a test of ethanol-induced ataxia and, with repeated drug administration, tended to develop tolerance more slowly. In tests of ethanol withdrawal and metabolism, mutants and wild-type mice showed equivalent responses. Our results suggest that the 5-HT1B receptor participates in the regulation of ethanol drinking, and demonstrate that serotonergic manipulations lead to reduced responsiveness to certain ataxic effects of ethanol without affecting dependence.

Adult hippocampal neurogenesis: an actor in the antidepressant-like action.

Depression and anxiety are psychiatric illnesses that are major burdens in society and affect as much as 7% of the world's population. The heterogeneous nature of depression suggests an involvement of multiple distinct brain regions including amygdala, prefrontal cortex and the hippocampus, which may be responsible for the diversity of the symptoms. Besides its critical role in learning and memory, the hippocampus is one of only two areas in mammalian brain where adult neurogenesis occurs. Of the current leading hypotheses of the pathophysiology and treatment of depression, the neurogenesis hypothesis of depression deserves particular attention because changes in neurogenesis are only seen after chronic, but not acute, antidepressant treatment. This review revisits the role of adult hippocampal neurogenesis in the pathophysiology of mood disorders, especially anxiety/depression, and also in the antidepressant-like responses, especially in stressed rodents.

Antidepressants recruit new neurons to improve stress response regulation.

Recent research suggests an involvement of hippocampal neurogenesis in behavioral effects of antidepressants. However, the precise mechanisms through which newborn granule neurons might influence the antidepressant response remain elusive. Here, we demonstrate that unpredictable chronic mild stress in mice not only reduces hippocampal neurogenesis, but also dampens the relationship between hippocampus and the main stress hormone system, the hypothalamo-pituitary-adrenal (HPA) axis. Moreover, this relationship is restored by treatment with the antidepressant fluoxetine, in a neurogenesis-dependent manner. Specifically, chronic stress severely impairs HPA axis activity, the ability of hippocampus to modulate downstream brain areas involved in the stress response, the sensitivity of the hippocampal granule cell network to novelty/glucocorticoid effects and the hippocampus-dependent negative feedback of the HPA axis. Remarkably, we revealed that, although ablation of hippocampal neurogenesis alone does not impair HPA axis activity, the ability of fluoxetine to restore hippocampal regulation of the HPA axis under chronic stress conditions, occurs only in the presence of an intact neurogenic niche. These findings provide a mechanistic framework for understanding how adult-generated new neurons influence the response to antidepressants. We suggest that newly generated neurons may facilitate stress integration and that, during chronic stress or depression, enhancing neurogenesis enables a dysfunctional hippocampus to restore the central control on stress response systems, then allowing recovery.

Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products.

The products of the adenovirus-2 (Ad2) immortalizing oncogene E1A repress the activity of the SV40, polyoma virus and E1A enhancers. Evidence is presented that Ad2 infection of MPC11 plasmocytoma cells results in an inhibition of transcription of both the gamma 2b heavy chain (IgH) and the kappa light chain immunoglobulin genes. This inhibition is caused by the Ad2 E1A products. Furthermore, the Ad2 E1A products repress transcription activated by the immunoglobulin heavy chain enhancer in chimeric recombinants, which are either stably integrated in the genome of lymphoid cells or are present as episomes. The implications of negative regulation of cellular enhancers are discussed.

Enhanced aggressive behavior in mice lacking 5-HT1B receptor.

The neuromodulator serotonin (5-hydroxytryptamine, 5-HT) has been associated with mood disorders such as depression, anxiety, and impulsive violence. To define the contribution of 5-HT receptor subtypes to behavior, mutant mice lacking the 5-HT1B receptor were generated by homologous recombination. These mice did not exhibit any obvious developmental or behavioral defects. However, the hyperlocomotor effect of the 5-HT1A/1B agonist RU24969 was absent in mutant mice, indicating that this effect is mediated by 5-HT1B receptors. Moreover, when confronted with an intruder, mutant mice attacked the intruder faster and more intensely than did wild-type mice, suggesting the participation of 5-HT1B receptors in aggressive behavior.

Increased exploratory activity and altered response to LSD in mice lacking the 5-HT(5A) receptor.

In order to determine the distribution and function of the 5-HT5A serotonin receptor subtype, we generated knockout mice lacking the 5-HT5A gene. Comparative autoradiography studies of brains of wild-type (wt) and 5-HT5A knockout (5A-KO) mice revealed the existence of binding sites with high affinity for [125I]LSD that correspond to 5-HT5A receptors and that are concentrated in the olfactory bulb, neocortex, and medial habenula. When exposed to novel environments, the 5A-KO mice displayed increased exploratory activity but no change in anxiety-related behaviors. In addition, the stimulatory effect of LSD on exploratory activity was attenuated in 5A-KO mice. These results suggest that 5-HT5A receptors modulate the activity of neural circuits involved specifically in exploratory behavior and suggest that some of the psychotropic effects of LSD may be mediated by 5-HT5A receptors.

Behavioral and serotonergic consequences of decreasing or increasing hippocampus brain-derived neurotrophic factor protein levels in mice.

Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRI) act as indirect agonists of serotonin (5-HT) receptors. Although these drugs produce a rapid blockade of serotonin transporters (SERTs) in vitro, several weeks of treatment are necessary to observe clinical benefits. This paradox has not been solved yet. Recent studies have identified modifications of intracellular signaling proteins and target genes that could contribute to antidepressant-like activity of SSRI (e.g., increases in neurogenesis and BDNF protein levels), and may explain, at least in part, their long delay of action. Although these data suggest a positive regulation of 5-HT on the expression of the gene coding for BDNF, the reciprocal effects of BDNF on brain 5-HT neurotransmission remains poorly documented. To study the impact of BDNF on serotonergic activity, a dual experimental strategy was used to analyze neurochemical and behavioral consequences of its decrease (strategy 1) or increase (strategy 2) in the brain of adult male mice. (1) In heterozygous BDNF+/- mice in which brain BDNF protein levels were decreased by half, an enhancement of basal extracellular 5-HT levels (5-HText) that induced a down-regulation of SERT, i.e., a decrease in its capacity to reuptake 5-HT, was found in the hippocampus. In addition, the SSRI, paroxetine, failed to increase hippocampal 5-HText in BDNF+/- mice, while it produces robust effects in wild-type littermates. Thus, BDNF+/- mice can be viewed as an animal model of genetic resistance to serotonergic antidepressant drugs. (2) In wild-type BDNF+/+ mice, the effects of intra-hippocampal (vHi) injection of BDNF (100 ng) in combination with a SSRI was examined by using intracerebral microdialysis and behavioral paradigms that predict an antidepressant- and anxiolytic-like activity of a molecule [the forced swim test (FST) and the open field paradigm (OF) respectively]. BDNF induced a rapid and transient increase in paroxetine response on 5-HText in the adult hippocampus, which was correlated with a potentiation of its antidepressant-like activity in the FST. The effects of BDNF were selectively blocked by K252a, an antagonist of its high-affinity TrkB receptor. Such a correlation between neurochemical and behavioral effects of [BDNF+SSRI] co-administration suggests that its antidepressant-like activity is linked to the activation of 5-HT neurotransmission in the adult hippocampus. BDNF also had a facilitatory effect on anxiety-like behavior in the OF test, and paroxetine prevented this anxiogenesis. What was the mechanism by which BDNF exerted these latter effects? Surprisingly, by using zero net flux method of quantitative microdialysis in vivo, we found that an intra-hippocampal BDNF injection in wild-type mice decreased the functional activity of SERT as observed in BDNF+/- mice. However, the decreased capacity of SERT to reuptake 5-HT was not associated to an increase in basal 5-HText in the hippocampus of WT mice. Interestingly, using in situ hybridization experiments indicated that TrkB receptor mRNA was expressed in the hippocampus and dorsal raphe nucleus in adult mice suggesting that the neurochemical and behavioral effects of intra-hippocampal BDNF injection can mobilize both pre- and post-synaptic elements of the brain 5-HT neurotransmission. Taken together, these set of experiments unveiled a relative opposition of neurochemical and behavioral responses following either a decrease (in BDNF+/- mutant mice) or an increase in brain BDNF levels (bilateral intra-hippocampal injection) in adult mice. In view of developing new antidepressant drug strategy, a poly-therapy combining BDNF with a chronic SSRI treatment could thus improve the efficacy of current medications.

Regionally selective changes in neurotransmitter receptors in the brain of the 5-HT1B knockout mouse.

The serotonin1B receptor knockout (5-HT1B KO) mouse is a valuable animal model of addiction to psychostimulants. We previously found selective increases in dopamine (DA) turnover in the nucleus accumbens of these mice, in addition to several changes in their central serotonin system. Here, we searched for further DA adaptations by measuring D1 and D2 receptor as well DA plasma membrane transporter (DAT) sites by ligand binding autoradiography, and G-protein coupling to D1 and D2 receptors by [35S]GTP gamma S autoradiography. Except for a slight increase in the lateral septum, D1 receptor binding did not differ from wild-type in twenty-one other neocortical, limbic or basal ganglia regions examined in the KO. Nor were there changes in D1 agonist-stimulated G-protein coupling in any of these regions, including the lateral septum. Increases in D2 binding sites, presumably involving GABAergic projection neurons, were measured in the nucleus accumbens, olfactory tubercle and ventral tegmental area of the 5-HT1B KO. However, no activation of the efficacy of D2 receptor coupling to G-protein could be measured in these and other brain regions. Binding to DAT was unchanged throughout brain. Because of their implication in cocaine addiction, the functionality of mu-opioid and GABAB receptors was also assessed by [35S]GTP gamma S autoradiography. 5-HT1B KO showed selective decreases in G-protein coupling to mu-opioid receptors in the paraventricular thalamic nucleus, and to GABAB receptors in the basolateral nucleus of amygdala. It is likely that these latter changes underlie some aspects of the addictive behavior of the 5-HT1B KO mouse.

Serotonin receptors. Genetic insights into serotonin function.

Targeted disruption of the genes for the 5-HT1B and 5-HT2C serotonin receptors and monoamine oxidase A have confirmed pharmacological experiments and revealed unexpected behavioral roles for serotonin.

Risk of facial splashes in four major surgical specialties in a multicentre study.

This study analyses the results of face-shield blood spatter contamination at six medical facilities to determine exposure risk when facial protection is not used. Blood spatter exposure was evaluated on the basis of overall incidence, location of spatter on face shields, surgical specialty, risk for operating room staff, length of surgery and volume of blood loss. Six hundred face shields were evaluated for blood spatter contamination by visual inspection as well as by staining with leucomalachite green. The face shield was divided into three regions: Orbital (O-region), Paraorbital (P-region) and Mask (M-region). Visual examination detected blood spatter contamination in 50.5% (303/600) of the face shields, whereas leucomalachite green staining detected blood contamination in 66.0% (396/600). Blood contamination was 36.6% (220/600) in the O-region, 37.8% (227/600) in the P-region and 57.0% (342/600) in the M-region. Among operating room staff, the incidence of blood spatter was greatest among lead surgeons at 83.5% (167/200), followed by the first assistant at 68.5% (137/200) and the scrub nurse at 46.0% (92/200). By specialty, cardiovascular surgery was at highest risk with an incidence of 75.3% (113/150) followed by neurosurgery at 69.3% (104/150), gastrointestinal at 60.0% (90/150) and orthopaedic surgery at 60.0% (90/150).

A novel conditional knockout strategy applied to serotonin receptors.

Here we demonstrate the feasibility of a doubly regulatable transgenic mouse design that allows for gene manipulation by both Cre-recombinase and the tetracycline inducible system. Using a knock-in strategy to insert both elements of the tetracycline inducible system and a neomycin (neo) cassette flanked by loxP sequences (floxed) into the wild-type locus, we generated mice that express the 5-HT(1B) receptor in a conditional manner. In the presence of a floxed neo-cassette, receptor expression was silenced. Removal of this cassette by Cre-mediated recombination led to 5-HT(1B) receptor expression, which was highly regulatable when doxycycline, a derivative of tetracycline, was administered to the mice. This system allowed for a determination of an in vivo time course of receptor half-life and recovery. Physiological studies also demonstrated that rescued 5-HT(1B) receptors were functional, and that this functionality was reversible upon treatment with doxycycline. Crossing mice where the 5-HT(1B), or the 5-HT(1A), receptors were silenced by the neo-cassette, with mice expressing either Cre-recombinase or the tetracycline transactivator (tTA) under the control of tissue-specific promoters, led to tissue-specific re-expression of these receptors. Our studies thus demonstrate the potential of this strategy for achieving both a classic knockout, as well as subsequent tissue-specific and/or inducible knockouts.

The broken mouse: the role of development, plasticity and environment in the interpretation of phenotypic changes in knockout mice.

With the advent of gene knockout technology has arisen the problem of how to interpret the resulting phenotypic changes in mice lacking specific genes. This problem is especially relevant when applied to behavioral phenotypes of knockout mice, which are difficult to interpret. Of particular interest are the roles of development and compensatory changes, as well as other factors, such as the influence of the gene knockout on nearby genes, the effect of the genetic background strain, maternal behavioral influences, and pleiotrophy.

Molecular heterogeneity along the dorsal-ventral axis of the murine hippocampal CA1 field: a microarray analysis of gene expression.

There has been increasing interest in functional heterogeneity along the septotemporal, dorsal-ventral (D-V) axis of the hippocampus. Although anatomical connectivity and lesion studies point to discrete roles for these sub-regions, the contribution of differential gene expression across this axis has not been systematically studied. Here we present findings from an Affymetrix microarray screen aimed at identifying genes in the CA1 region of the adult murine hippocampus that show significant differential expression along the D-V axis. Our results indicate that the vast majority of monitored genes (>90%) had tissue expression levels that differed by less than 20% between regions, while less than 0.1% of genes had expression levels that varied more than three-fold by sub-region. Only 23 probes showed a CA1 dorsoventral signal intensity ratio greater than three: 18 enriched dorsally and five enriched ventrally. Probes with the greatest difference in expression levels represent a range of genes with known functions in patterning and signaling, as well as genes without known function. Selective screening with digoxigenin-labeled in situ hybridization confirms the existence of CA1 sub-regionalized expression, with some genes exhibiting a graded expression pattern across the D-V axis, and others restricted to a discrete region. Our findings demonstrate that there are gene expression differences across the D-V axis of the adult murine hippocampus within traditionally recognized cytoarchitecturally defined boundaries. Combined with the previously recognized differences in connectivity and results from lesion studies, our data further confirm the existence of functional heterogeneity along the D-V axis.

The functional serotonin 1a receptor promoter polymorphism, rs6295, is associated with psychiatric illness and differences in transcription.

The G/C single-nucleotide polymorphism in the serotonin 1a receptor promoter, rs6295, has previously been linked with depression, suicide and antidepressant responsiveness. In vitro studies suggest that rs6295 may have functional effects on the expression of the serotonin 1a receptor gene (HTR1A) through altered binding of a number of transcription factors. To further explore the relationship between rs6295, mental illness and gene expression, we performed dual epidemiological and biological studies. First, we genotyped a cohort of 1412 individuals, randomly split into discovery and replication cohorts, to examine the relationship between rs6295 and five psychiatric outcomes: history of psychiatric hospitalization, history of suicide attempts, history of substance or alcohol abuse, current posttraumatic stress disorder (PTSD), current depression. We found that the rs6295G allele is associated with increased risk for substance abuse, psychiatric hospitalization and suicide attempts. Overall, exposure to either childhood or non-childhood trauma resulted in increased risk for all psychiatric outcomes, but we did not observe a significant interaction between rs6295 and trauma in modulating psychiatric outcomes. In conjunction, we also investigated the potential impact of rs6295 on HTR1A expression in postmortem human brain tissue using relative allelic expression assays. We found more mRNA produced from the C versus the G-allele of rs6295 in the prefrontal cortex (PFC), but not in the midbrain of nonpsychiatric control subjects. Further, in the fetal cortex, rs6295C allele exhibited increased relative expression as early as gestational week 18 in humans. Finally, we found that the C:G allelic expression ratio was significantly neutralized in the PFC of subjects with major depressive disorder (MDD) who committed suicide as compared with controls, indicating that normal patterns of transcription may be disrupted in MDD/suicide. These data provide a putative biological mechanism underlying the association between rs6295, trauma and mental illness. Moreover, our results suggest that rs6295 may affect transcription during both gestational development and adulthood in a region-specific manner, acting as a risk factor for psychiatric illness. These findings provide a critical framework for conceptualizing the effects of a common functional genetic variant, trauma exposure and their impact on mental health.

5-HT1B receptor knock-out mice exhibit increased exploratory activity and enhanced spatial memory performance in the Morris water maze.

In an attempt to characterize the contribution of the 5-HT1B receptor to behavior, 5-HT1B knock-out (KO) mice were subjected to a battery of behavioral paradigms aimed at differentiating various components of cognitive and emotional behaviors. In an object exploration task, wild-type (WT) and 5-HT1B KO mice did not differ in locomotor activity. 5-HT1B KO mice, however, displayed lower thigmotaxis (an index of anxiety) associated with a higher level of object exploratory activity, but no genotype differences were observed in the elevated plus maze. 5-HT1B KO mice also displayed a lack of exploratory habituation. In the spatial version of the Morris water maze, 5-HT1B KO mice showed higher performances in acquisition and transfer test, which was not observed in the visual version of the task. No genotype differences were found in contextual fear conditioning, because both WT and 5-HT1B KO mice were able to remember the context where they had received the aversive stimulus. The deletion of the 5-HT1B receptor, associated with appropriate behavioral paradigms, thus allowed us to dissociate anxiety from response to novelty, and perseverative behavior (lack of habituation) from adaptive behavioral inhibition underlying cognitive flexibility (transfer stage in the water maze). The deletion of the 5-HT1B receptor did not result in significant developmental plasticities for other major 5-HT receptor types but may have influenced other neurotransmission systems. The 5-HT1B receptor may be a key target for serotonin in the modulation of cognitive behavior, particularly in situations involving a high cognitive demand.

Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease.

Neuronal intranuclear inclusions are a histopathological hallmark of Huntington's disease. Nevertheless, the precise mechanism by which they are formed and their relevance to neuronal cell death and/or dysfunction remains unclear. We recently generated a conditional mouse model of Huntington's disease (HD94) in which silencing expression of mutated huntingtin led to the disappearance of intranuclear aggregates and amelioration of the behavioral phenotype. Here, we analyze primary striatal neuronal cultures from HD94 mice to explore the dynamics of aggregate formation and reversal, the possible mechanisms involved, and the correlation between aggregates and neuronal death. In parallel, we examine symptomatic adult HD94 mice in similar studies and explored the relationship between aggregate clearance and behavioral reversal. We report that, in culture, aggregate formation and reversal were rapid processes, such that 2 d of transgene expression led to aggregate formation, and 5 d of transgene suppression led to aggregate disappearance. In mice, full reversal of aggregates and intranuclear mutant huntingtin was more rapid than reported previously and preceded the motor recovery by several weeks. Furthermore, the proteasome inhibitor lactacystin inhibited the aggregate clearance observed in culture, thus indicating that aggregate formation is a balance between the rate of huntingtin synthesis and its degradation by the proteasome. Finally, neither expression of the mutant huntingtin nor aggregates compromised the viability of HD94 cultures. This correlated with the lack of cell death in symptomatic HD94 mice, thus demonstrating that neuronal dysfunction, and not cell loss, triggered by mutant huntingtin underlies symptomatology.

Excessive activation of serotonin (5-HT) 1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-ht transporter knock-out mice.

Deficiency in the monoamine degradation enzyme monoamine oxidase A (MAOA) or prenatal exposure to the monoamine uptake inhibitor cocaine alters behavior in humans and rodents, but the mechanisms are unclear. In MAOA knock-out mice, inhibiting serotonin synthesis during development can prevent abnormal segregation of axons in the retinogeniculate and somatosensory thalamocortical systems. To investigate this effect, we crossed MAOA knock-outs with mice lacking the serotonin transporter 5-HTT or the 5-HT1B receptor, two molecules present in developing sensory projections. Segregation was abnormal in 5-HTT knock-outs and MAOA/5-HTT double knock-outs but was normalized in MAOA/5-HT1B double knock-outs and MAOA/5-HTT/5-HT1B triple knock-outs. This demonstrates that the 5-HT1B receptor is a key factor in abnormal segregation of sensory projections and suggests that serotonergic drugs represent a risk for the development of these projections. We also found that the 5-HT1B receptor has an adverse developmental impact on beam-walking behavior in MAOA knock-outs. Finally, because the 5-HT1B receptor inhibits glutamate release, our results suggest that visual and somatosensory projections must release glutamate for proper segregation.

Of mice and flies: commonalities among 5-HT receptors.

Pharmacological studies as well as molecular cloning of 5-HT receptors have revealed a multiplicity of receptor subtypes, not only in mammals but, as discussed in this review by René Hen, also in molluscs and arthropods. Most of these receptors belong to the G protein-coupled receptor family, and their mechanism of action involves modulating levels of second messengers such as cAMP, IP3 and Ca2+. Rather than being specialized in a particular physiological function, a given receptor may be expressed in multiple neurons throughout the brain but always in the same compartment in these neurons. The 5-HT1B receptor, for example, is generally localized presynaptically on neuronal terminals, where it inhibits neurotransmitter release. A widespread distribution of 5-HT receptors might explain how 5-HT can modulate the multiple neuronal circuits that underlie complex behaviours.