Neurons of the Dentate Molecular Layer in the Rabbit Hippocampus

The molecular layer of the dentate gyrus appears as the main entrance gate for information into the hippocampus, i.e., where the perforant path axons from the entorhinal cortex synapse onto the spines and dendrites of granule cells. A few dispersed neuronal somata appear intermingled in between and probably control the flow of information in this area. In rabbits, the number of neurons in the molecular layer increases in the first week of postnatal life and then stabilizes to appear permanent and heterogeneous over the individuals’ life span, including old animals. By means of Golgi impregnations, NADPH histochemistry, immunocytochemical stainings and intracellular labelings (lucifer yellow and biocytin injections), eight neuronal morphological types have been detected in the molecular layer of developing adult and old rabbits. Six of them appear as interneurons displaying smooth dendrites and GABA immunoreactivity: those here called as globoid, vertical, small horizontal, large horizontal, inverted pyramidal and polymorphic. Additionally there are two GABA negative types: the sarmentous and ectopic granular neurons. The distribution of the somata and dendritic trees of these neurons shows preferences for a definite sublayer of the molecular layer: small horizontal, sarmentous and inverted pyramidal neurons are preferably found in the outer third of the molecular layer; vertical, globoid and polymorph neurons locate the intermediate third, while large horizontal and ectopic granular neurons occupy the inner third or the juxtagranular molecular layer. Our results reveal substantial differences in the morphology and electrophysiological behaviour between each neuronal archetype in the dentate molecular layer, allowing us to propose a new classification for this neural population.     

人参皂甙对缺氧大鼠海马DG区神经细胞DNA损伤保护作用的研究

目的:用单细胞凝胶电泳技术(SCGE)研究急慢性缺氧大鼠海马DG区神经细胞细胞DNA损伤和人参皂甙对缺氧大鼠海马细胞DNA的保护作用.方法:健康成年SD大鼠随机分为急、慢性缺氧正常对照组、急性缺氧组和慢性缺氧组(分别在模拟海拔5000米高原环境连续缺氧暴露0d、3d和30d)、急性缺氧人参皂甙干预组、慢性缺氧人参皂甙干预组.应用SCGE检测海马DG区神经细胞DNA损伤.结果:随着缺氧时间的增加,海马DG区神经细胞DNA的损伤程度加重,尾长、尾部DNA百分含量和尾距显著增加(P＜0.05).人参皂甙能使缺氧损伤的海马DG区神经细胞的尾长、尾部DNA百分含量和尾距均较缺氧组减少(P＜0.05).结论:人参皂甙能有效地减轻缺氧引起的海马组织细胞DNA的断裂损伤.     

Age-Related Changes of Elements and Relationships Among Elements in Human Hippocampus, Dentate Gyrus, and Fornix

To elucidate compositional changes of the limbic system with aging, the authors investigated age-related changes of elements in the hippocampus, dentate gyrus, and fornix and the relationships among elements by direct chemical analysis. After ordinary dissections at Nara Medical University were finished, the hippocampi, dentate gyri, and fornices were resected from identical cerebra of the subjects which consisted of 23 men and 23 women, ranging in age from 70 to 101 years. After ashing with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. The average contents of P, Zn, and Na were significantly less in both the hippocampi and dentate gyri compared with the fornices. It was found that the Ca and Mg contents increased significantly in the hippocampus with aging; the P content increased significantly in the dentate gyrus with aging, whereas the Na content decreased in the dentate gyrus with aging; and the Mg content increased significantly in the fornix with aging. Regarding the relationships among elements, a significant direct correlation between Ca and Fe contents and an extremely significant inverse correlation between P and Zn contents were found in both the hippocampi and dentate gyri. In addition, a significant direct correlation between P and Mg contents was found in both the hippocampi and fornices. Pearson's correlation was used to examine whether there were elements with significant correlation among the hippocampus, dentate gyrus, fornix, and mammillary body. Significant correlations were found in five elements of Ca, P, Mg, Zn, and Fe except for S and Na among the hippocampus, dentate gyrus, and mammillary body with one exception. Regarding the fornix, significant correlations were found in two elements of P and Fe between the fornix and hippocampus, dentate gyrus, or mammillary body.     

Hypoxic Excitability Changes and Sodium Currents in Hippocampus CA1 Neurons

1. The objective of the present study was to distinguish if inhibition of neuronal activity by hypoxia is related to a block of voltage-gated Na+ channels. 2. The effect of chemical hypoxia induced by cyanide (0.5 mM, 10 min perfusion) was studied with patch-clamp technique in visualized intact CA1 pyramidal neurons in rat brain slices. Action potentials were elicited in whole cell current-clamp recordings and the threshold was estimated by current pulses of 50-ms duration and incremental amplitudes (n = 31). The effect of cyanide on the Na+ current and conductance was studied in voltage clamp recordings from cell-attached patches (n = 13). 3. Cyanide perfusion during 10 min increased the threshold for excitation by 73 +/- 79 pA (p = 0.001), which differed from the effect in control cells (11 +/- 41 pA, ns). The change in current threshold was correlated to a change in membrane potential (r = -0.88, p < 0.0001). Cyanide had no significant effect on the peak amplitude, duration, or rate of rise of the action potential. 4. Cyanide perfusion did not change the Na+ current size, but caused a small decrease in ENa (-17 +/- 22 mV, ns) and a slight increase in Na+ conductance (+14 +/- 26%, ns), which differed (p = 0.045) from controls (-19 +/- 23 %, ns). 5. In conclusion, chemical hypoxia does not cause a decrease in Na+ conductance. The decreased excitability during hypoxia can be explained by an increase in the current threshold, which is correlated with the effect on the membrane potential.     

Structural Remodeling of the Neuronal Network in the Dentate Gyrus of the Epileptically Transformed Murine Hippocampus

Immunohistochemical analysis of the hippocampus of a transgene mutant line of the mice (genetic model of temporal epilepsy) demonstrated a progressive increase in the level of brain-derived neurotrophic factor (BDNF) in granular cells of the dentate gyrus and their axons; this increase correlated with an enhancement of the level of epileptic activity. Epileptogenic reprojection of mossy fibers toward an internal zone of the molecular layer of the hippocampus was also observed. In addition, we observed excessive spreading of the zone of axon branching of GABA-ergic basket cells, as compared with the norm; their maximum collateralization was found within an internal zone of the molecular layer of the dentate gyrus. This allows us to hypothesize that the processes of sprouting of the mossy fibers and recovery of recurrent inhibition of the granular cells are interrelated.     

Sensorimotor Intervention Recovers Noradrenaline Content in the Dentate Gyrus of Cortical Injured Rats

Nowadays, a consensus has been reached that designates the functional and structural reorganization of synapses as the primary mechanisms underlying the process of recovery from brain injury. We have reported that pontine noradrenaline (NA) is increased in animals after cortical ablation (CA). The aim of the present study was to explore the noradrenergic and morphological response after sensorimotor intervention (SMI) in rats injured in the motor cortex. We used male Wistar adult rats allocated in four conditions: sham-operated, injured by cortical ablation, sham-operated with SMI and injured by cortical ablation with SMI. Motor and somatosensory performance was evaluated prior to and 20 days after surgery. During the intervening period, a 15-session, SMI program was implemented. Subsequently, total NA analysis in the pons and dentate gyrus (DG) was performed. All groups underwent histological analysis. Our results showed that NA content in the DG was reduced in the injured group versus control, and this reduction was reverted in the injured group that underwent SMI. Moreover, injured rats showed reduction in the number of granule cells in the DG and decreased dentate granule cell layer thickness. Notably, after SMI, the loss of granule cells was reverted. Locus coeruleus showed turgid cells in the injured rats. These results suggest that SMI elicits biochemical and structural modifications in the hippocampus that could reorganize the system and lead the recovery process, modulating structural and functional plasticity.     

Prenatal Stress Causes Oxidative Damage to Mitochondrial DNA in Hippocampus of Offspring Rats

Mitochondrion, the primary source of reactive oxygen species (ROS), is also the target of ROS. 8-Hydroxy-2′-deoxyguanosine (8-OH-dG) is the major end-product of damaged DNA caused by ROS. In our previous studies, we showed that prenatal stress (PNS) preferentially caused cognitive dysfunction and increased ROS in the hippocampus of female offspring rats. The present study aimed to determine 8-OH-dG level of mitochondria in order to elucidate the mechanism of hippocampal pyramidal neuronal damage and cognitive dysfunction induced by PNS. Pregnant rats were divided into two groups: control group (undisturbed) and PNS group (exposed to a restraint stress for 7 days at the late stage of gestation). Offspring rats were divided into four groups: female-control group, male-control group, female-stress group, male-stress group and used at 30-day-old after their birth. The content of 8-OH-dG was determined by high performance liquid chromatography-electrochemical detection (HPLC-ECD). The results showed that the contents of 8-OH-dG in female and male prenatal stressed offspring were significantly higher than that in their respective controls (P < 0.001). 8-OH-dG level was significantly higher in the female-stress group than in the male-stress group (P < 0.05), whereas there was no any gender-dependent difference in the control groups. These results suggest that accumulation of oxidative mitochondrial DNA damage may play an important role in PNS-induced cognitive dysfunction in female offspring rats. Special issue article in honor of Dr. Akitane Mori.     

Relationships Among the Hippocampus,Dentate Gyrus,Mammillary Body,Fornix, and Anterior Commissure from a Viewpoint of Elements

To elucidate the relationships among the brain regions belonging to the limbic system, the authors investigated the relationships among the hippocampus, dentate gyrus, mammillary body, and fornix, using the anterior commissure as a control, from a viewpoint of elements. After ordinary dissections at Nara Medical University were finished, the hippocampi, dentate gyri, mammillary bodies, fornices, and anterior commissures were resected from identical cerebra of the subjects. The subjects consisted of 23 men and 23 women, ranging in age from 70 to 101 years (average age = 83.5 ± 7.5 years). After ashing with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. With regard to seven elements of Ca, P, S, Mg, Zn, Fe, and Na, it was examined whether there were significant correlations among the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure. It was found that there were extremely or very significant direct correlations among all of the five brain regions of the hippocampus, dentate gyrus, mammillary body, fornix, and anterior commissure in the P content. Likewise, with regard to the Fe content, there were significant direct correlations among the four brain regions belonging to the limbic system, except for the anterior commissure. In both the Ca and Zn contents, there were extremely or very significant direct correlations among the hippocampus, dentate gyrus, and mammillary body of the gray matter.     

Comparison of Ionized Calcium-binding Adapter Molecule 1 Immunoreactivity of the Hippocampal Dentate Gyrus and CA1 Region in Adult and Aged Dogs

Similarities between age-related changes in the canine and human brain have resulted in the general acceptance of the canine brain as a model of human brain aging. The hippocampus is essentially required for intact cognitive ability and appears to be particularly vulnerable to the aging process. We observed changes in ionized calcium-binding adapter molecule 1 (Iba-1, a microglial marker) immunoreactivity and protein levels in the hippocampal dentate gyrus and CA1 region of adult (2-3 years) and aged (10-12 years) dogs. We also observed the interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, protein levels in these groups. In the dentate gyrus and CA1 region of the adult dog, Iba-1 immunoreactive microglia were well distributed and their processes were highly ramified. However, in the aged dog, the processes of Iba-1 immunoreactive microglia were hypertrophied in the dentate gyrus. Moreover, Iba-1 protein level in the dentate gyrus in the aged dog was higher than in the adult dog. IFN-gamma expression was increased in the dentate gyrus homogenates of aged dogs than adult dogs. In addition, we found that some neurons were positive to Fluoro-Jade B (a marker for neuronal degeneration) in the dentate polymorphic layer, but not in the hippocampal CA1 region in the aged dog. These results suggest that Iba-1 immunoreactive microglia are hypertrophied in the dentate gyrus in the aged dog.     

Experimental Post-traumatic Stress Disorder Decreases Astrocyte Density and Changes Astrocytic Polarity in the CA1 Hippocampus of Male Rats

Post-traumatic stress disorder (PTSD) is a psychiatric condition resulting from exposure to a traumatic event. It is characterized by several debilitating symptoms including re-experiencing the past trauma, avoidance behavior, increased fear, and hyperarousal. Key roles in the neuropathology of PTSD and its symptomatology have been attributed to the hippocampus and amygdala. These regions are involved in explicit memory processes and context encoding during fear conditioning. The aim of our study was to investigate whether PTSD is capable of altering the morphology, density and expression of glial fibrillary acidic protein (GFAP) in astrocytes from the CA1 region of the hippocampus and the medial amygdala and correlate the data obtained with the orientation index of the polarity of astrocytes. Thirty male rats were divided in two groups: control (n = 15) and PTSD (n = 15). The inescapable shock protocol, in which the animals are exposed to a single episode of footshock, was used to induce PTSD. Our results show that, in the hippocampus, PTSD is capable of decreasing the density of GFAP+ astrocytes as well as altering astrocytic morphology, as shown by the reductions observed in the total number of primary processes, in the number of primary processes in the lateral quadrants, and the degree of branching in the lateral quadrants. The analysis of the orientation index indicates that PTSD alters the polarity of hippocampal astrocytes. No alterations were observed in the amygdala astrocytes. Therefore, this study demonstrates notable changes in hippocampal astrocytes, supporting the concept that these cells play an important role in PTSD symptomatology.

     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.     

IQGAP1 Expression in Spared CA1 Neurons After an Excitotoxic Lesion in the Mouse Hippocampus

Repeated seizures induce permanent alterations in the hippocampal circuits in experimental models with intractable temporal lobe epilepsy. Sprouting and synaptic reorganization induced by seizures has been well-studied in the mossy fiber pathway. However, studies investigating sprouting and synaptic reorganization beyond the mossy fiber pathway are limited. The present study examined the biochemical changes of CA1 pyramidal neurons undergoing morphological changes after excitotoxicity-induced hippocampal CA3 neuronal death. IQ-domain GTPase-activating proteins (IQGAP1), is an effector of Rac1 and Cdc42 and an actin-binding protein, was upregulated in CA1 pyramidal neurons after kainic acid-induced hippocampal CA3 neuronal degeneration. IQGAP1 + cells were colocalized with Nestin, but not in astrocytes or mature neurons. Furthermore, IQGAP1 did not originate from newly divided local precursors or NG2 + cells. IQGAP1 and adenomatous polyposis coli localized in CA1 pyramidal neurons, and Cdc42 activation was followed by IQGAP1 recruitment. These findings suggest that IQGAP1 is upregulated in pre-existed sparing neurons of the CA1 layer undergoing morphological changes after excitoxicity-induced hippocampal CA3 neuronal death. It demonstrates the utility of IQGAP1 as a possible marker for spared pyramidal neurons, which may contribute to structural and functional alternations responsible for the development of epilepsy.     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

Content and Concentration of Taurine,Hypotaurine, and Zinc in the Retina,the Hippocampus,and the Dentate Gyrus of the Rat at Various Postnatal Days

Taurine and zinc possess neurotrophic and neuroprotective properties, and they have been demonstrated to interact in the central nervous system (CNS). The aim of this work was to determine taurine, hypotaurine, and zinc levels during postnatal development and any possible significant correlation between them in selective areas of the CNS with differential taurine level regulation and intrinsic capacity to proliferate. Taurine and hypotaurine content (nM/region) and concentration (nM/mg protein) and total zinc levels were determined in the retina, hippocampus, and dentate gyrus of the rat at postnatal days 5, 10, 15, 20, 30, and 50. Taurine and hypotaurine increased during development in the retina without significant correlation between them. In the hippocampus there was a progressive decrease, and in the dentate gyrus there was an initial increase and a posterior decrease of taurine and hypotaurine levels. Correlation between the two amino acids was observed at P10, P15, and P50 for the hippocampus and at P15, P30, and P50 for the dentate gyrus. The variations in total zinc levels followed a biphasic behavior, with an early decrease and later increase. Significant and positive correlation of zinc and taurine was only observed in the hippocampus at P30 and P50 and negative in the dentate gyrus at P30. No significant correlation was obtained for the retina. The maintenance of taurine levels in specific CNS areas does not seem to be related to the availability of the precursor, hypotaurine, which might have a role by itself. There are critical postnatal periods during which there is a preservation of taurine, hypotaurine, or zinc levels. It seems that these requirements could be related to zinc-taurine interactions.     

Prenatal Stress Produces Social Behavior Deficits and Alters the Number of Oxytocin and Vasopressin Neurons in Adult Rats

The present study investigated the long-lasting effects of prenatal repeated restraint stress on social behavior and anxiety, as well as its repercussions on oxytocin (OT) and vasopressin (VP)-positive neurons of the paraventricular (PVN) and supraoptic (SON) nuclei from stressed pups in adulthood. Female Wistar rats were exposed to restraint stress in the last 7 days of pregnancy. At birth, pups were cross-fostered and assigned to the following groups: prenatally non-stressed offspring raised by prenatally non-stressed mothers (NS:NS), prenatally non-stressed offspring raised by prenatally stressed mothers (S:NS), prenatally stressed offspring raised by prenatally non-stressed mothers (NS:S), prenatally stressed offspring raised by prenatally stressed mothers (S:S). As adults, male prenatally stressed offspring raised both by stressed mothers (S:S group) and non-stressed ones (NS:S group) showed impaired social memory and interaction. In addition, when both adverse conditions coexisted (S:S group), increased anxiety-like behavior and aggressiveness was observed in association with a decrease in the number of OT-positive magnocellular neurons, VP-positive magnocellular and parvocellular neurons of the PVN. The NS:S group exhibited a reduction in the amount of VP-positive magnocellular neurons compared to the S:NS. Thus, the social behavior deficits observed in the S:S and NS:S groups may be only partially associated with these alterations to the peptidergic systems. No changes were shown in the OT and VP cellular composition of the SON nucleus. Nevertheless, it is clear that a special attention should be given to the gestational period, since stressful events during this time may be related to the emergence of behavioral impairments in adulthood.     

Effects of Treadmill Exercise Combined with MK 801 Treatment on Neuroblast Differentiation in the Dentate Gyrus in Rats

N-methyl-d-aspartate receptor (NR) is involved in activity-dependent synaptic plasticity, such as associative long-term potentiation, and in related central functions, such as learning and memory. In this study, we observed effects of treadmill exercise on NR1 and doublecortin (DCX, a marker for neuroblast differentiation) in the subgranular zone of the dentate gyrus (DG). At 6 weeks of age, rats were put on a treadmill with or without running for 1 h/day for 5 consecutive days at 22 m/min for 5 weeks. Exercise increased NR1 immunoreactivity and protein level in the hippocampus. To identify the correlations between NR and neuroblasts, we intraperitoneally administered a NR antagonist, MK-801, to the exercised rats. MK-801 treatment reduced NR1 protein level in the hippocampus of the exercised rats. In addition, in the MK-801-treated group, the number of DCX cells was significantly decreased in the subgranular zone of the DG. These results suggest that NR may be one of the important factors that modulate neuroblast differentiation during exercise in rats.     

Effects of Sensitive to Apoptosis Gene Protein on Cell Proliferation, Neuroblast Differentiation, and Oxidative Stress in the Mouse Dentate Gyrus

Sensitive to apoptosis gene (SAG) protein is a redox-inducible protein that protects cells against apoptosis induced by redox agents. In this study, we observed effects of SAG on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus (DG) using Ki67 and doublecortin (DCX), respectively. For easy penetration into neurons, Tat-SAG expression vector was constructed by ligation with SAG and expression vector, Tat, in-frame with six histidine open-reading frames to generate the expression vector, and cloned into E. coli DH5α cells. One or 5?mg/kg Tat-SAG fusion protein (Tat-SAG) was intraperitoneally administered to mice once a day for 3?weeks. The administration of Tat-SAG significantly increased the number of 5-bromodeoxyuridine positive cells, Ki67 positive cells and DCX immunoreactive neuroblast in the mouse DG: Especially, in the 5?mg/kg Tat-SAG-treated mice, DCX positive neuroblasts showed a well-developed arborization of tertiary dendrites in the DG. On the other hand, we examined that the administration of Tat-SAG significantly reduced the DNA damage and lipid peroxidation judging from 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal immunohistochemistry: The decrease was much more distinct in the 5?mg/kg Tat-SAG-treated mice than 1?mg/kg Tat-SAG-treated mice. This result suggests that SAG significantly increases cell proliferation, neuroblast differentiation and oxidative stress in normal states.     

Pharmacotherapy with Fluoxetine Restores Functional Connectivity from the Dentate Gyrus to Field CA3 in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a high-incidence genetic pathology characterized by severe impairment of cognitive functions, including declarative memory. Impairment of hippocampus-dependent long-term memory in DS appears to be related to anatomo-functional alterations of the hippocampal trisynaptic circuit formed by the dentate gyrus (DG) granule cells - CA3 pyramidal neurons - CA1 pyramidal neurons. No therapies exist to improve cognitive disability in individuals with DS. In previous studies we demonstrated that pharmacotherapy with fluoxetine restores neurogenesis, granule cell number and dendritic morphology in the DG of the Ts65Dn mouse model of DS. The goal of the current study was to establish whether treatment rescues the impairment of synaptic connectivity between the DG and CA3 that characterizes the trisomic condition. Euploid and Ts65Dn mice were treated with fluoxetine during the first two postnatal weeks and examined 45–60 days after treatment cessation. Untreated Ts65Dn mice had a hypotrophyc mossy fiber bundle, fewer synaptic contacts, fewer glutamatergic contacts, and fewer dendritic spines in the stratum lucidum of CA3, the terminal field of the granule cell projections. Electrophysiological recordings from CA3 pyramidal neurons showed that in Ts65Dn mice the frequency of both mEPSCs and mIPSCs was reduced, indicating an overall impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons. In treated Ts65Dn mice all these aberrant features were fully normalized, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The positive effects of fluoxetine on the DG-CA3 system suggest that early treatment with this drug could be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions.