Forniceal deep brain stimulation rescues hippocampal memory in Rett syndrome mice.

Deep brain stimulation (DBS) has improved the prospects for many individuals with diseases affecting motor control, and recently it has shown promise for improving cognitive function as well. Several studies in individuals with Alzheimer disease and in amnesic rats have demonstrated that DBS targeted to the fimbria-fornix, the region that appears to regulate hippocampal activity, can mitigate defects in hippocampus-dependent memory. Despite these promising results, DBS has not been tested for its ability to improve cognition in any childhood intellectual disability disorder. Such disorders are a pressing concern: they affect as much as 3% of the population and involve hundreds of different genes. We proposed that stimulating the neural circuits that underlie learning and memory might provide a more promising route to treating these otherwise intractable disorders than seeking to adjust levels of one molecule at a time. We therefore studied the effects of forniceal DBS in a well-characterized mouse model of Rett syndrome (RTT), which is a leading cause of intellectual disability in females. Caused by mutations that impair the function of MeCP2 (ref. 6), RTT appears by the second year of life in humans, causing profound impairment in cognitive, motor and social skills, along with an array of neurological features. RTT mice, which reproduce the broad phenotype of this disorder, also show clear deficits in hippocampus-dependent learning and memory and hippocampal synaptic plasticity. Here we show that forniceal DBS in RTT mice rescues contextual fear memory as well as spatial learning and memory. In parallel, forniceal DBS restores in vivo hippocampal long-term potentiation and hippocampal neurogenesis. These results indicate that forniceal DBS might mitigate cognitive dysfunction in RTT.

Robust increase of microglia proliferation in the fornix of hippocampal axonal pathway after a single LPS stimulation.

Microglia are resident immunocompetent cells having important roles in innate immunity in the brains. In the present study, we found that a single lipopolysaccharide (LPS) administration significantly increased microglial proliferation in the fornix and dentate gyrus (DG) but not the cerebral cortex and corpus callosum of adult mice. LPS-induced microglial proliferation was especially robust at the white matter of the fornix. The density of microglia increased in the fornix and DG for roughly one week and returned to basal levels at least 20days after a single LPS administration. Consecutive LPS administration did not induce such dramatic increase of microglial proliferation in the fornix. The inhibition of vascular endothelial growth factor signaling by AZD2171 largely suppressed LPS-induced increase of microglial proliferation in the fornix. In conclusion, the present study indicates that the hippocampal neuronal system has a higher proliferative microglial capability against LPS-induced inflammatory administration compared with other brain regions.

Choice of diffusion tensor estimation approach affects fiber tractography of the fornix in preterm brain.

BACKGROUND AND PURPOSE: Neonatal DTI enables quantitative assessment of microstructural brain properties. Although its use is increasing, it is not widely known that vast differences in tractography results can occur, depending on the diffusion tensor estimation methodology used. Current clinical work appears to be insufficiently focused on data quality and processing of neonatal DTI. To raise awareness about this important processing step, we investigated tractography reconstructions of the fornix with the use of several estimation techniques. We hypothesized that the method of tensor estimation significantly affects DTI tractography results. MATERIALS AND METHODS: Twenty-eight DTI scans of infants born <29 weeks of gestation, acquired at 30-week postmenstrual age and without intracranial injury observed, were prospectively collected. Four diffusion tensor estimation methods were applied: 1) linear least squares; 2) weighted linear least squares; 3) nonlinear least squares, and 4) robust estimation of tensors by outlier rejection. Quality of DTI data and tractography results were evaluated for each method. RESULTS: With nonlinear least squares and robust estimation of tensors by outlier rejection, significantly lower mean fractional anisotropy values were obtained than with linear least squares and weighted linear least squares. Visualized quality of tract reconstruction was significantly higher by use of robust estimation of tensors by outlier rejection and correlated with quality of DTI data. CONCLUSIONS: Quality assessment and choice of processing methodology have considerable impact on neonatal DTI analysis. Dedicated acquisition, quality assessment, and advanced processing of neonatal DTI data must be ensured before performing clinical analyses, such as associating microstructural brain properties with patient outcome.

Fimbria-fornix (FF)-transected hippocampal extracts induce the activation of astrocytes in vitro.

Hippocampus is one of the neurogenesis areas in adult mammals, but the function of astrocytes in this area is still less known. In our previous study, the fimbria-fornix (FF)-transected hippocampal extracts promoted the proliferation and neuronal differentiation of radial glial cells in vitro. To explore the effects of hippocampal extracts on gliogenesis, the hippocampal astrocytes were treated by normal or ff-transected hippocampal extracts in vitro. The cells were immunostained by brain lipid-binding protein (BLBP), nestin, and SOX2 to assess their state of activation. The effects of astrocyte-conditioned medium on the neuronal differentiation of hippocampal neural stem cells (NSCs) were also investigated. After treatment of FF-transected hippocampal extracts, the number of BLBP, nestin, and Sox-positive cells were obviously more than the cells which treated by normal hippocampal extracts, these cells maintained a state of activation and the activated astrocyte-conditioned medium also promoted the differentiation of NSCs into more neurons. These findings suggest that the astrocytes can be activated by FF-transected hippocampal extracts and these activated cells also can promote the neuronal differentiation of hippocampal NSCs in vitro.

Short-term learning induces white matter plasticity in the fornix.

Magnetic resonance imaging (MRI) has greatly extended the exploration of neuroplasticity in behaving animals and humans. Imaging studies recently uncovered structural changes that occur in gray and white matter, mainly after long-term training. A recent diffusion tensor imaging (DTI) study showed that training in a car racing game for 2 h induces changes in the hippocampus and parahippocampal gyri. However, the effect of short-term training on the white matter microstructure is unknown. Here we investigated the influence of short learning tasks on structural plasticity in the white matter, and specifically in the fornix, in humans and rats. Human subjects performed a 2 h spatial learning task, and rats underwent training for 1 d in a Morris water maze. Between tasks, subjects were scanned with DTI, a diffusion MRI framework sensitive to tissue microstructure. Using tract-based spatial statistics, we found changes in diffusivity indices in both humans and rats. In both species, changes in diffusion in the fornix were correlated with diffusion changes in the hippocampus, as well as with behavioral measures of improvement in the learning tasks. These results, which provide the first indication of short-term white matter plasticity in the human brain, suggest that the adult brain white matter preserves dynamic characteristics and can be modified by short-term learning experiences. The extent of change in white matter was correlated with their extent in gray matter, suggesting that all components of the neural network are capable of rapid remodeling in response to cognitive experiences.

Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.

Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.

Organotypic slice co-cultures reveal that early postnatal hippocampal axons lose the ability to grow along the fimbria, while retaining the ability to invade and arborise in septal neuropil.

The failure of cut axons to grow along fibre tracts in the adult CNS contrasts with their ability to do so in development. Organotypic slices culture of a number of areas enables the time of failure to be pinpointed to around the second week of postnatal life in the rat. 'Heterochronic' co-culture of slices above and below this age shows that the failure is due to the inability of the older axons to grow into either the same age or younger targets. Using hippocampo-septal slices the present experiments show that this failure is due to an inability to recognise the glial pathway of the fimbria, even when this is of a younger age. However, the older hippocampal neurons retain the ability to grow axons into septal target tissue when they are placed in direct contact with it. This exactly mirrors the inability of cut central axons to regenerate along their previous fibre pathways while they retain their ability to reinnervate neuropil.

A double dissociation of subcortical hippocampal efferents for encoding and consolidation/retrieval of spatial information.

CA3 lesions impair encoding, whereas CA1 lesions impair retrieval during learning of a Hebb-Williams maze. CA3 efferents in the fimbria were transected, taking care to spare cholinergic and GABAergic afferents. CA1 efferents in the dorsal fornix were similarly transected. Fimbria transections, but not dorsal fornix transections, resulted in deficits for the encoding of spatial information during learning of a Hebb-Williams maze. Dorsal fornix, but not fimbria, transections resulted in deficits for retrieval of spatial memory during learning of a Hebb-Williams maze. These results reveal a double dissociation for the roles of CA3 and CA1 subcortical efferents in encoding and retrieval processes that mirror the double dissociation seen after excitotoxic lesions of CA1 and CA3. These data provide support for the theory that the cholinergic projections from the septal nuclei modulate the dynamics for encoding and consolidation/retrieval in the hippocampus.

Influence of 5,7-dihydroxytryptamine lesions of the rat fornix-fimbria and cingulum bundles on spontaneous activity in an aversive maze.

Exposure to aversive environmental stimuli stimulates the serotonergic neurones that project to the forebrain and inhibit spontaneous activity when studied in a simple maze. This study explored the putative role of the principal 5-hydroxytryptamine (5-HT) neurones that project to the hippocampus from the median raphe nucleus in this response to an aversive environment by lesioning the 5-HT fibres that project through the fornix/fimbria and cingulum bundles. The effects of the lesions were investigated in independent groups of animals tested in an enclosed four-arm maze and a more aversive elevated maze of the same dimensions composed entirely of four open arms. The rats were significantly less active in the open maze, the principal effect of maze design being observed during the first 5 min sub-trial of a 15 min trial. This response to the more aversive environment was totally abolished by the lesion. It is concluded that exposure to an explicitly aversive environment elicits a brief stimulation of the 5-HT neurones that project to the hippocampus from the median raphe nucleus and that this stimulation inhibits the initial burst of exploratory activity that is observed in animals placed in a less aversive novel environment.

Attention and executive systems abnormalities in adults with childhood ADHD: A DT-MRI study of connections.

Attention-deficit/hyperactivity disorder (ADHD) is hypothesized to be due, in part, to structural defects in brain networks influencing cognitive, affective, and motor behaviors. Although the current literature on fiber tracts is limited in ADHD, gray matter abnormalities suggest that white matter (WM) connections may be altered selectively in neural systems. A prior study (Ashtari et al. 2005), using diffusion tensor magnetic resonance imaging (DT-MRI), showed alterations within the frontal and cerebellar WM in children and adolescents with ADHD. In this study of adults with childhood ADHD, we hypothesized that fiber pathways subserving attention and executive functions (EFs) would be altered. To this end, the cingulum bundle (CB) and superior longitudinal fascicle II (SLF II) were investigated in vivo in 12 adults with childhood ADHD and 17 demographically comparable unaffected controls using DT-MRI. Relative to controls, the fractional anisotropy (FA) values were significantly smaller in both regions of interest in the right hemisphere, in contrast to a control region (the fornix), indicating an alteration of anatomical connections within the attention and EF cerebral systems in adults with childhood ADHD. The demonstration of FA abnormalities in the CB and SLF II in adults with childhood ADHD provides further support for persistent structural abnormalities into adulthood.

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex.

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.

Habenula lesions alter synaptic plasticity within the fimbria-accumbens pathway in the rat.

Both the habenula and the nucleus accumbens, and especially the glutamatergic innervation of the latter from the hippocampus, have been hypothesized to be involved, in different ways, in the pathophysiology of cognitive disturbances in schizophrenia. Lesions of the habenula produce disturbances of memory and attention in experimental animals. As the habenular nuclei have been shown to influence the release of many neurotransmitters, both in the hippocampus and the nucleus accumbens, we examined in this study the effects of bilateral habenula lesions on the plasticity of the fimbria-nucleus accumbens pathway, by means of the long-term depression phenomenon in freely moving rats. Long-term depression, induced within the shell region of the nucleus accumbens by low-frequency stimulation of the fimbria, was exaggerated and showed greater persistence in habenula-lesioned rats compared with sham-operated animals. These results indicate that plasticity in the fimbria-nucleus accumbens pathway is altered by habenula lesions in a way similar to previously-reported effects of stress and the psychosis-provoking agent ketamine. Moreover, they strengthen the views that the habenula belongs to systems, mediating higher cognitive functions, which involve the hippocampus and the nucleus accumbens. Finally, this study suggests that dysfunction of the habenula could contribute to cognitive alterations in diseases such as schizophrenia, where the habenula is reported to exhibit exaggerated calcification.

Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated in male and female rodents.

Sexual dimorphism of neurons and astrocytes has been demonstrated in different centers of the brain, but sexual dimorphism of oligodendrocytes and myelin has not been examined. We show, using immunocytochemistry and in situ hybridization, that the density of oligodendrocytes in corpus callosum, fornix, and spinal cord is 20-40% greater in males compared with females. These differences are present in young and aged rodents and are independent of strain and species. Proteolipid protein and carbonic anhydrase-II transcripts, measured by real-time PCR, are approximately two to three times greater in males. Myelin basic protein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase, measured by Western blots, are 20-160% greater in males compared with females. Surprisingly, both generation of new glia and apoptosis of glia, including oligodendrocytes, are approximately two times greater in female corpus callosum. These results indicate that the lifespan of oligodendrocytes is shorter in females than in males. Castration of males produces a female phenotype characterized by fewer oligodendrocytes and increased generation of new glia. These findings indicate that exogenous androgens differentially affect the lifespan of male and female oligodendrocytes, and they can override the endogenous production of neurosteroids. The data imply that turnover of myelin is greater in females than in males. Mu-calpain, a protease upregulated in degeneration of myelin, is dramatically increased at both transcriptional and translational levels in females compared with males. These morphological, molecular, and biochemical data show surprisingly large differences in turnover of oligodendrocytes and myelin between sexes. We discuss the potential significance of these differences to multiple sclerosis, a sexually dimorphic disease, whose progression is altered by exogenous hormones.

Cell proliferation in the adult hippocampal formation of rodents and its modulation by entorhinal and fimbria-fornix afferents.

New granule neurons are produced in the dentate gyrus (DG) of rodents throughout adult life. Recent studies have also reported adult neurogenesis in the cerebral cortex in normal animals or after brain injury. However, few of these studies focused on the hippocampal formation (HF), a cortical area involved in learning and memory in which extensive cell death occurs in neurodegenerative diseases. Thus, we studied cell proliferation in the HF of rodents and the intrinsic putative neurogenic potential of entorhinal cortex (EC) progenitors. We show that only the DG generates new neurons in the HF. In addition, neurospheres from the EC differentiate into neurons and glia in vitro and after transplantation in the adult DG. We also analyzed whether the absence of the synaptic input from the main hippocampal afferents induces neuronal generation in the HF outside the DG and/or regulates the proliferation of DG neuroprogenitors. We show that the denervation of the hippocampus does not induce neurogenesis in HF regions other than the DG. However, neuroprogenitor proliferation in the DG is reduced after fimbria-fornix lesions but not after entorhinal deafferentation, which supports the view that neuroprogenitor proliferation and/or differentiation in the DG are controlled from basal forebrain/septal neurons.

Specific activation of histaminergic neurons during daily feeding anticipatory behavior in rats.

When food is available during a restricted and predictable time of the day, animals show increased locomotor and food searching behaviors before the anticipated daily meal. We had shown that histamine-containing neurons are the only aminergic neurons related to arousal that become active in anticipation of an upcoming meal. To further map, the brain regions involved in the expression of the feeding-anticipatory behavior, we quantified the expression of Fos in hypothalamic areas involved in arousal. We found that nearly 35% of the histamine neurons from the tuberomammillary nucleus were Fos-immunoreactive immediately before mealtime. One hour before this transient increase in Fos-immunoreactivity, we found a similarly brief increase of fos mRNA in the tuberomammillary nucleus. In contrast, the activation of two types of perifornical hypothalamic neurons followed meal onset by 1-2 h. One neuron type was orexin/hypocretin-immunoreactive, while the other type was neither orexin nor melanin concentrating hormone-immunoreactive. The present work indicates that the increased locomotor activity that anticipates mealtime coincides with the activation of the tuberomammillary nucleus, and that the behavioral activation during the consummatory phase of feeding coincides more closely with the delayed activation of the perifornical hypothalamic area.

Distribution and origin of vesicular glutamate transporter 2-immunoreactive fibers in the rat hippocampus.

This study examined the distribution of vesicular glutamate transporter 2 (VGLUT2)-immunoreactive neuronal structures in the ipsilateral and contralateral hippocampi of unilateral fimbria/fornix transected, unilateral entorhinal cortex ablated, and intact female and male rats. In the hippocampi of intact animals, the highest density of VGLUT2-positive boutons was observed in the supragranular layer of the dentate gyrus, followed by the CA2 pyramidal and oriens layers, and the stratum lacunosum-moleculare of the CA1 field. This staining pattern was identical both in males and in females. Electron microscopic examination revealed that the immunolabeling was confined to axon terminals forming exclusively asymmetric synaptic contacts. The quantitative analysis of the synaptic targets of VGLUT2-positive terminals showed that in the dentate gyrus, 59% of the synaptic targets were dendritic spines, followed by dendritic shafts (22%) and granule cell somata (19%). In the pyramidal layer of the CA2 field, VGLUT2-immunoreactive boutons contacted mostly dendritic shafts (85%), only some of which (15%) synapsed with spines. The synaptic targets of VGLUT2-positive varicosities were dendritic spines (71%) and shafts (29%) in the stratum lacunosum-moleculare of the CA1 field. The fimbria/fornix transection caused a significant reduction in the density of VGLUT2-positive boutons only in the CA2 field, while entorhinal cortex ablation elicited no change in fiber density in any of the areas analyzed. Furthermore, our latest experiments on colchicine-treated animals revealed a large population of VGLUT2-positive neurons in the hippocampus that may be a possible intrinsic source of hippocampal VGLUT2 boutons. Our results suggest that the most likely sources of VGLUT2-positive boutons in the dentate supragranular layer, the CA2 area, as well as in the stratum lacunosum-moleculare of the CA1 field, might be the mossy cells, the supramammillary area, and the nucleus reuniens thalami, respectively.

Msx1 expression in the adult mouse brain: characterization of populations of beta-galactosidase-positive cells in the hippocampus and fimbria.

We have analyzed Msx1 expression in the mature mouse brain using in situ hybridization and beta-galactosidase activity in Msx1(nLacZ) mice. The study revealed that Msx1 is strongly expressed in the circumventricular organs, such as the subcommissural organ and choroid plexus, and in some epithelia, such as that of the dorsal, but not the ventral part of the third ventricle. Immunohistochemical analysis revealed that the Msx1-expressing cells of the hippocampus and fimbria are astrocytes, oligodendrocytes or immature oligodendrocytes. In contrast, no co-expression was detected in these structures using several neuronal markers. These results were confirmed, using transmission electron microscopy, by the presence of 5-bromo-3-indolyl-beta-D-galactopyranosideprecipitates in astrocytes and oligodendrocytes in both sites. Moreover, using an anti-glial fibrillary acidic protein antibody (GFAP), our study reveals two populations of astrocytes in the adult hippocampus and other areas, such as the fimbria, namely Msx1+/GFAP+ and Msx1-/GFAP+. Beta-galactosidase activity was also observed in endothelial cells of hippocampal fissure blood vessels. We also observed co-localization of polysialic acid neural cell adhesion molecule, a marker of the polysialylated form of the neural cell adhesion molecule, in Msx1-expressing cells in the fimbria. These cells may be precursors of glial cells and originate from the epithelium of the fimbria. The present study indicates, in the mature mouse brain, that Msx1 may be linked to secretory activity in circumventricular organs, and to glial proliferation and differentiation in the hippocampus and fimbria, and presumably also in other cerebral areas. We suggest that Msx1 could be associated with brain homeostasis and blood-brain barrier function.

A novel mouse brain slice preparation of the hippocampo-accumbens pathway.

The nucleus accumbens (NAc) is an important component of circuitry that underlies reward related behaviors and the rewarding properties of drugs of abuse. Glutamatergic afferents to the nucleus are critical for its normal function and for behaviors related to drug addiction. An angled, sagittal mouse brain slice preparation has been designed to facilitate concurrent stimulation of two major glutamatergic afferent pathways to the nucleus accumbens. Medium spiny neurons at the medial core/shell boundary of the accumbens were depolarized by stimulation of either hippocampal or limbic cortical afferents through activation of AMPA-type glutamate receptors. High frequency but not low frequency stimulation of hippocampal afferents depolarized medium spiny neurons to a membrane potential that resembled the up state observed upon high frequency stimulation in vivo. The magnitude of the membrane depolarization was positively correlated with the amplitude of the stimulus-evoked EPSP. Concurrent stimulation of hippocampal and limbic cortical afferents at theta frequency selectively induced a long-term depression (LTD) in the magnitude of stimulus-evoked EPSPs on the hippocampal afferent only. These data suggest that this brain slice preparation can be used to study mechanisms underlying synaptic plasticity at two of the critical glutamatergic afferent synapses in the nucleus accumbens as well as characterizing potential interactions between afferents. Additionally, LTD at hippocampo-accumbens synapses can be induced at a stimulus frequency known to support reinstatement of drug seeking behavior.

Coordinated functions of Netrin-1 and Class 3 secreted Semaphorins in the guidance of reciprocal septohippocampal connections.

An essential characteristic of the CNS function is the formation of reciprocal connections between brain areas. Although the mechanisms controlling the establishment of neuronal connections are being determined, very little is known about the development of reciprocal connections, which often course along identical pathways. Here, we show that Netrin-1, expressed along the fimbria, chemoattracts both septohippocampal and hippocamposeptal fibers. Moreover, we show that both Semaphorins 3A and 3F expressed in regions nearby the septum prevent the growth of septal axons into these regions. Blocking experiments with recombinant ecto-Neuropilins indicate that both Semaphorins 3A and 3F act cooperatively in the repulsion of septal axons. Furthermore, netrin-1-deficient mice develop a reduced septohippocampal projection. We conclude that the coordinated actions of Netrin-1 and Semaphorins 3A and 3F cooperate in the development of septohippocampal and hippocamposeptal connections, indicating that the same molecular cues serve the construction of reciprocal connections in both directions of growth.