Several stressors fail to reduce adult hippocampal neurogenesis

Neurogenesis in the dentate gyrus of the hippocampus of adult laboratory animals has been widely reported to be vulnerable to many psychological and physical stressors. However, we have found no effects of acute restraint stress, acute or subchronic tailshock stress, or acute, subchronic, or chronic resident-intruder stress on neural progenitor cell (NPC) proliferation, short or long term survival of newborn cells, or brain-derived neurotrophic factor (BDNF) mRNA expression in adult rats. In addition, we did not observe any effect of chronic resident-intruder stress on NPC proliferation in adolescent rats. A selectively bred stress-sensitive line was also found to exhibit no alterations in NPC proliferation following tailshock stress, although this line did exhibit a lower proliferation rate under baseline (unstressed) conditions when compared with non-selected rats. These results challenge the prevailing hypothesis that any stressor of sufficient intensity and duration has a marked negative impact upon the rate of hippocampal neurogenesis, and suggest that some yet unidentified factors related to stress and experimental conditions are crucial in the regulation of neurogenesis.     

卡马西平对匹罗卡品诱导成年癫癎大鼠海马齿状回神经发生影响的研究

目的研究卡马西平对成年癫癎大鼠海马齿状回神经发生的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫癎模型,于干预后第6d腹腔注射5-溴脱氧尿核苷嘧啶标记海马齿状回内源性神经前体细胞的增殖情况;用免疫组织化学方法及免疫荧光双标方法观察海马齿状回新生细胞的增殖、存活、分化及迁移情况。结果 （1）卡马西平可明显抑制癫癎大鼠海马齿状回新生细胞增殖;（2）卡马西平可明显促进癫癎大鼠海马齿状回新生细胞的存活;（3）卡马西平可增加癫癎大鼠海马齿状回新生神经元的数量,但不增加新生细胞分化为成熟神经元的比例;（4）卡马西平对新生神经细胞的异位迁移无抑制作用。结论卡马西平对癫癎大鼠海马齿状回神经发生的影响是其控制癫癎临床症状的可能机制之一。     

Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats

PURPOSE: Mossy fiber sprouting is a common abnormality found in patients and models of temporal lobe epilepsy. The role of mossy fiber sprouting in epileptogenesis is unclear, and its blockade would be useful experimentally and perhaps therapeutically. Results from previous attempts to block mossy fiber sprouting have been disappointing or controversial. In some brain regions, prolonged application of the sodium channel blocker tetrodotoxin prevents axon sprouting and posttrauma epileptogenesis. The present study tested the hypothesis that prolonged, focal infusion of tetrodotoxin would block mossy fiber sprouting after an epileptogenic treatment. METHODS: Adult rats were treated with pilocarpine to induce status epilepticus. Several hours to 3 days after pilocarpine treatment, a pump with a cannula directed toward the dentate gyrus was implanted to deliver 10 microM tetrodotoxin or vehicle alone at 0.25 microl/h. This method blocks local EEG activity in the hippocampus (Galvan et al. J Neurosci 2000; 20:2904-16). After 28 days of continuous infusion, rats were perfused with fixative, and their hippocampi analyzed anatomically with stereologic techniques. RESULTS: Tetrodotoxin infusion was verified immunocytochemically in tetrodotoxin-treated but not vehicle-treated hippocampi. Tetrodotoxin-infused and vehicle-infused hippocampi displayed similar levels of hilar neuron loss. The Timm stain revealed mossy fiber sprouting regardless of whether hippocampi were treated with tetrodotoxin infusion, vehicle infusion, or neither. CONCLUSIONS: Prolonged infusion of tetrodotoxin did not block mossy fiber sprouting. This finding suggests that sodium channel-mediated neuronal activity is not necessary for mossy fiber sprouting after an epileptogenic treatment.     

Single alcohol exposure in early life damages hippocampal stem/progenitor cells and reduces adult neurogenesis

Alcohol exposure during pregnancy may cause fetal alcohol syndrome (FAS), characterized by impaired cognitive functions. Neurogenesis occurs in the adult hippocampus and is functionally associated with learning, memory, and mood disorders. However, whether early postnatal exposure to alcohol impairs neurogenesis and through which mechanisms it occurs is poorly understood. Here, we report that a single episode of alcohol exposure in postnatal day 7 (P7) decreases neurogenesis in the adult hippocampus. Furthermore, we demonstrate a co-localization of glial fibrillar acidic protein, nestin, and vimentin with activated caspase-3 12 h after ethanol treatment. Finally, we show that the number of primary neurospheres derived from the hippocampi of alcohol-exposed mice is reduced compared to controls. These findings suggest that alcohol exposure in postnatal mice reduces the pool of neural stem/progenitor cells in the DG, and subsequently results in a decrease of adult neurogenesis. This may explain certain aspects of impaired hippocampal functions in FAS.     

Increased granule cell neurogenesis in the adult dentate gyrus following mossy fiber stimulation sufficient to induce long-term potentiation

Neurons are continually added at a low rate to the granule cell layer of the dentate gyrus during adulthood in rats. The functional significance of this unusual feature is not completely understood, although recent studies suggest continued granule cell neurogenesis is essential for normal learning and memory. We report here that, in the adult rat, stimulation of the granule cell mossy fibers sufficient to induce long-term potentiation (LTP) increases the number of newly formed granule cells in the dentate gyrus, indicating that granule cell neurogenesis is regulated by efferent activity and, possibly, the induction of LTP.     

Long lasting changes in the spontaneous activity of hippocampal neurons following stimulation of the entorhinal cortex

The spontaneous activity of hippocampal and denate units was investigated in acute and chronic experiments on adult rats. Spontaneous unitary discharges from both regions were measured before and after brief tetanic stimulation of the entorhinal cortex. An increase in the frequency of spontaneous unit activity was obtained in units recorded from the dentate gyrus and CA3 field of the hippocampus for up to 20 min following stimulation. In chronic recordings of physiologically identified dentate units from freely moving rats similar results were obtained. The findings are discussed with reference to long lasting potentiation of synaptic activity in the perforant path-dentate granule cell connection.     

Absence of hippocampal mossy fiber sprouting in transgenic mice overexpressing brain-derived neurotrophic factor

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2--3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus.     

Prolonged infusion of cycloheximide does not block mossy fiber sprouting in a model of temporal lobe epilepsy

PURPOSE: The role of protein synthesis in mossy fiber sprouting is unclear. Conflicting reports exist on whether a single dose of the protein synthesis-blocker cycloheximide administered around the time of an epileptogenic injury can block the eventual development of mossy fiber sprouting. METHODS: In rats, osmotic minipumps and cannulae were implanted to deliver 8 mg/ml cycloheximide to one dentate gyrus and vehicle to the other. This method has been used to block protein synthesis in the infused region for up to 5 days with minimal neurotoxic effects (Taha and Stryker, Neuron 2002;34:425-36). After 2 days of infusion, rats were treated with pilocarpine to induce status epilepticus. Pumps were removed 3 days later. Thirty days after pilocarpine treatment, rats were perfused, and hippocampal sections were processed for Timm staining. RESULTS: Timm staining revealed aberrant mossy fiber sprouting in the inner molecular layer regardless of whether hippocampi were treated with cycloheximide or vehicle. Cycloheximide-treated hippocampi displayed more aberrant Timm staining and more tissue damage around the infusion site than did vehicle-treated hippocampi. CONCLUSIONS: Prolonged infusion of cycloheximide, spanning the period of pilocarpine treatment, did not block mossy fiber sprouting. This finding suggests that protein-dependent mechanisms around the time of an epileptogenic injury are not necessary for the eventual development of synaptic reorganization.     

Transient elevation of adult hippocampal neurogenesis after dopamine depletion

Degeneration of the midbrain dopaminergic neurons during Parkinson's disease (PD) may affect remote regions of the brain that are innervated by the projections of these neurons. The dentate gyrus (DG), a site of continuous production of new neurons in the adult hippocampus, receives dopaminergic inputs from the neurons of the substantia nigra (SN). Thus, depletion of the SN neurons during disease or in experimental settings may directly affect adult hippocampal neurogenesis. We show that experimental ablation of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydopyridine (MPTP) mouse model of PD results in a transient increase in cell division in the subgranular zone (SGZ) of the DG. This increase is evident for the amplifying neural progenitors and for their postmitotic progeny; our results also indicate that MPTP treatment affects division of the normally quiescent stem cells in the SGZ. We also show that l-DOPA, used in the clinical treatment of PD, while attenuating the MPTP-induced death of dopaminergic neurons, does not alter the effect of MPTP on cell division in the DG. Our results suggest that a decrease in dopaminergic signaling in the hippocampus leads to a transient activation of stem and progenitor cells in the DG.     

Maturation of granule cell dendrites after mossy fiber arrival in hippocampal field CA3

Most granule neurons in the rat dentate gyrus are born over the course of the first 2 postnatal weeks. The resulting heterogeneity has made it difficult to define the relationship between dendritic and axonal maturation and to delineate a time course for the morphological development of the oldest granule neurons. By depositing crystals of the fluorescent label Dil in hippocampal field CA3, we retrogradely labeled granule neurons in fixed tissue slices from rats aged 2-9 days. The results showed that all labeled granule cells, regardless of the age of the animal, exhibited apical dendrites. On day 2, every labeled neuron had rudimentary apical dendrites, and a few dendrites on each cell displayed immature features such as growth cones, varicosities, and filopodia. Some cells displayed basal dendrites. By day 4, the most mature granule neurons had longer and more numerous apical branches, as well as various immature features. Most had basal dendrites. On days 5 and 6, the immature features and the basal dendrites had begun to regress on the oldest cells, and varying numbers of spines were present. On day 7, the first few adult-like neurons were seen: immature features and basal dendrites had disappeared, all dendrites reached the top of the molecular layer, and the entire dendritic tree was covered with spines. These data show that dendritic outgrowth occurs before, or concurrent with, axon arrival in the CA3 target region, and that adult-like granule neurons are present by the end of the first week.     

卡马西平对匹罗卡品诱导成年癫间疒大鼠海马齿状回神经前体细胞增殖的影响

目的研究卡马西平对成年癫大鼠海马齿状回内源性神经前体细胞增殖的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫模型,将癫大鼠随机分为癫对照组和癫卡马西平组,正常大鼠随机分为正常对照组和正常卡马西平组。癫对照组和正常对照组给以蒸馏水灌胃,同时癫卡马西平组和正常卡马西平组给予卡马西平灌胃。于灌胃后第6d腹腔注射5-溴脱氧尿苷嘧啶(BrdU)标记海马齿状回的内源性神经前体细胞的增殖情况;用免疫组化方法观察各组大鼠在注射BrdU后第1d、第7d齿状回BrdU阳性细胞数量的表达。结果①注射BrdU后第1d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组减少(P<0.05);②注射BrdU后第7d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组明显减少(P<0.05)。结论卡马西平抑制癫大鼠海马齿状回内源性神经前体细胞增殖。     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat.The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus.During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

戊四氮诱发癫痫大鼠海马齿状回颗粒细胞树突发芽

目的利用化学点燃大鼠癫痫模型,观察癫痫发作后大鼠海马齿状回颗粒细胞树突的形态学变化,探讨癫痫敏感性形成机制。方法利用戊四氮（PTZ）制作化学点燃大鼠癫痫模型。采用点燃成功后3d、7d两个时间点,应用神经元高尔基染色法,在光镜下观察和定量分析大鼠海马齿状回颗粒细胞的树突改变。结果颗粒细胞的形态学参数（树突总长度、树突分支点数、树突野最大伸展距离和树突棘密度）在PTZ点燃后3d时均显著降低（P〈0.05）,而在PTZ点燃后7d时明显回升,并明显高于生理水平（P〈0.05）。结论在PTZ点燃大鼠癫痫发作后,颗粒细胞树突出现明显的可塑性改变,并在PTZ点燃后7d时出现发芽现象,这可能与癫痫敏感性的形成有关     

Mice with ablated adult brain neurogenesis are not impaired in antidepressant response to chronic fluoxetine

The neurogenesis hypothesis of major depression has two main facets. One states that the illness results from decreased neurogenesis while the other claims that the very functioning of antidepressants depends on increased neurogenesis. In order to verify the latter, we have used cyclin D2 knockout mice (cD2 KO mice), known to have virtually no adult brain neurogenesis, and we demonstrate that these mice successfully respond to chronic fluoxetine. After unpredictable chronic mild stress, mutant mice showed depression-like behavior in forced swim test, which was eliminated with chronic fluoxetine treatment, despite its lack of impact on adult hippocampal neurogenesis in cD2 KO mice. Our results suggest that new neurons are not indispensable for the action of antidepressants such as fluoxetine. Using forced swim test and tail suspension test, we also did not observe depression-like behavior in control cD2 KO mice, which argues against the link between decreased adult brain neurogenesis and major depression.     

Decreased numerical density of CA3 hippocampal mossy fiber synapses in schizophrenia

The CA3 region of the hippocampus is unique in its connectivity, its role in cognitive maintenance, and its great vulnerability in schizophrenia. The down regulation of the expression and binding activity of glutamate receptors was revealed in the CA3 hippocampal region and may be attributed to cognitive disturbances in schizophrenia. Our previous study demonstrated that only schizophrenics with predominantly positive (but not predominantly negative) symptoms had smaller-sized branched spines (thorny excrescences) of CA3 pyramidal neurons and fewer synaptic contacts formed by dentate mossy fiber terminals (MFT-synapses). In the present study, we used an unbiased stereological physical dissector method to verify whether the numerical density of MFT-synapses is altered in schizophrenia. A morphometric study was performed in 10 normal controls and eight age-matched cases with chronic schizophrenia, including five cases with predominantly positive and three with predominantly negative symptoms. Schizophrenic cases had a significantly reduced numerical density of MFT-synapses (-25%, P < 0.01) compared with the control group. The decrease was similar in schizophrenic subgroups with predominantly positive and predominantly negative symptoms. No effects of postmortem delay, age, duration of disease, and neuroleptic exposure were found. Taken together with our previous results, the data suggest that the decrease of numerical density of MFT-synapses may be the result of different mechanisms in schizophrenics with predominantly positive and predominantly negative symptoms.     

Association between mossy fiber sprouting and expression of semaphorin-3f protein in dentate gyrus of hippocampus in lithium-pilocarpine-induced status epilepticus mouse model

Objectives: Mossy fiber sprouting is involved in the pathogenesis of mesial temporal lobe epilepsy. But the exact mechanism of formation of mossy fiber sprouting is still unclear. Semaphorin-3f protein could inhibit the growth of neuron axons. The aim of this research is to evaluate the association between semaphorin-3f expression and mossy fiber sprouting.

Methods: We established pilocarpine-induced status epilepticus (PISE) models firstly. Then, mossy fiber sprouting in the hippocampus of PISE models was examined by Timm staining. Expression of semaphorin-3f was evaluated by western blot analysis and immunohistochemical examination. Expression of semaphorin-3f protein in different subregions of hippocampus and its relationship with mossy fiber sprouting were studied.

Results: We found that in PISE group, mossy fiber sprouting appeared in dentate gyrus (DG) region. It started to develop in the latent phase of PISE group and increased significantly in the chronic phase. Expression of semaphorin-3f protein in DG region started to decrease in the latent phase, and stayed at low level in the chronic phase. No such change was found in the other groups.

Conclusions: These results indicate that the decrease in semaphorin-3f expression in DG region was in parallel to the change of mossy fiber sprouting in PISE models, suggesting that mossy fiber sprouting is closely associated with reduced expression of semaphorin-3f in this model.     

Acute exposure to predator odor elicits a robust increase in corticosterone and a decrease in activity without altering proliferation in the adult rat hippocampus

Stress has long been implicated as a major cause of depression in humans and more recently has been suggested to decrease neurogenesis, which may be a contributing factor to depression development. Animal models of stress may be a relevant tool for investigating links between neurogenesis and depression. This has largely been investigated using chronic stress models in rodents. However, stress may be chronic or experienced in discrete episodes. Acute stress may be particularly relevant to humans experiencing unexpected societal pressures and obligations. Our study examined the effect of acute stress on the proliferative phase of adult hippocampal neurogenesis. Young adult rats were exposed for 20 min to the predator odor TMT, a natural stressor for rodents with significant ethological relevance. BrdU IP injections were concurrent with TMT exposure to assess proliferation effects with animal sacrifice 2 h after BrdU injection. Robust stress responses were evident following TMT exposure as detected by elevated corticosterone (CORT) levels and a significant reduction in exploratory behavior. Exposure to TMT did not alter the number of BrdU-positive cells in the hippocampus despite physiological and behavioral evidence of stress. CORT level elevation has long been accepted as a marker of stress; however, this study indicates that increases in CORT level may not always correlate with diminished neurogenic proliferation. This study further suggests that various stressors may not operate through the same biological substrates resulting in a differential ability to modulate neurogenesis.     

Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABA(A) receptor ligand binding in a model of temporal lobe epilepsy

The disabling seizures associated with mesial temporal lobe epilepsy (TLE) are often resistant to antiepileptic drugs (AEDs). The biological basis of this refractoriness is unknown but may include alterations in AED targets in the epileptogenic brain tissue, reduced AED penetration to the seizure focus, and neuropathological brain alterations such as hippocampal sclerosis typically found in patients with refractory TLE. In the present study, we used a rat model of TLE to examine whether AED responders differ from non-responders in their structural alterations and GABA(A) receptor characteristics in the hippocampal formation. In this model, spontaneous recurrent seizures develop after a status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala. The frequency of these seizures was recorded by continuous video/EEG monitoring before, during, and after daily treatment with phenobarbital, which was given at maximum tolerated doses for 2 weeks. Based on their individual response to phenobarbital, rats were grouped into responders and non-responders. The severity or duration of the initial brain insult (the status epilepticus) did not differ between responders and non-responders, indicating that the difference between the two subgroups is genetically determined. Subsequent histological examination showed a significant loss of neurons in the CA1, CA3c/CA4, and dentate hilus of non-responders, whereas responders did not differ in this respect from non-epileptic controls. The morphological alterations in the non-responders were associated with striking alterations in autoradiographic imaging of diazepam-sensitive and diazepam-insensitive GABA(A) receptor binding in the dentate gyrus with a significant shift to enhanced diazepam-insensitive binding. The present data indicate that neurodegeneration and associated GABA(A) receptor changes in the dentate gyrus are critically involved in the mechanisms underlying refractoriness of seizures in TLE.     

Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats

The mossy fiber pathway in the dentate gyrus undergoes sprouting and synaptic reorganization in response to seizures. The types of new synapses, their location and number, and the identity of their postsynaptic targets determine the functional properties of the reorganized circuitry. The goal of this study was to characterize the types and proportions of sprouted mossy fiber synapses in kindled and kainic acid-treated rats. In normal rats, synapses labeled by Timm histochemistry or dynorphin immunohistochemistry were rarely observed in the supragranular region of the inner molecular layer when examined by electron microscopy. In epileptic rats, sprouted mossy fiber synaptic terminals were frequently observed. The ultrastructural analysis of the types of sprouted synapses revealed that 1) in the supragranular region, labeled synaptic profiles were more frequently axospinous than axodendritic, and many axospinous synapses were perforated; 2) sprouted mossy fiber synaptic terminals formed exclusively asymmetric, putatively excitatory synapses with dendritic spines and shafts in the supragranular region and with the soma of granule cells in the granule cell layer; 3) in contrast to the large sprouted mossy fiber synapses in resected human epileptic hippocampus, the synapses formed by sprouted mossy fibers in rats were smaller; and 4) in several cases, the postsynaptic targets of sprouted synapses were identified as granule cells, but, in one case, a sprouted synaptic terminal formed a synapse with an inhibitory interneuron. The results demonstrate that axospinous asymmetric synapses are the most common type of synapse formed by sprouted mossy fiber terminals, supporting the viewpoint that most sprouted mossy fibers contribute to recurrent excitation in epilepsy.