Mouse Hippocampal Formation Research Articles

Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling.

To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of 'starter' AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.

Global Exploration of RNA-Binding Proteins in Exercise-Induced Adult Hippocampal Neurogenesis: A Transcriptome Meta-analysis and Computational Study.

Voluntary physical exercise is a robust enhancer of adult hippocampal neurogenesis (AHN). A complete understanding of the molecular regulation of AHN is important in order to exploit the benefits of the process toward therapeutic approaches. Several factors such as epigenetic modifiers, non-coding RNAs, and transcription factors have been reported to regulate AHN. However, there is a limited understanding of the impact of RNA-binding proteins (RBPs) on exercise-mediated AHN, in spite of their well-documented significance in embryonic neurogenesis. The present study is the first global analysis to catalog the potential RBPs influencing exercise-mediated AHN. Here, a transcriptome meta-analysis was conducted to study exercise-mediated gene expression modulation in hippocampi of adult mice. Next, potential RBPs influencing transcriptome-wide expression changes via untranslated regions (UTRs) were identified. Among other RBPs, MATR3, Musashi, TIA1, and FXR2 (known critical modulators of neurogenesis) were found to potentially regulate gene expression patterns. Subsequently, binding sites of known neurogenesis-regulating RBPs were identified in the UTRs of AHN-associated genes modulated by exercise. Finally, a number of RBPs including RBFOX1, RBFOX3, and QKI (known regulators of neurogenesis) were found to be highly expressed in mouse hippocampal formation and also potentially interact with other RBPs, suggesting their combinatorial functioning in exercise-induced AHN. Thus, the present meta-analysis-based computational study identified several RBPs potentially important in exercise-induced AHN, which could form a foundation for further experiments to unravel RBP-mediated regulation of AHN.

A Taxonomy of Transcriptomic Cell Types Across the Isocortex and Hippocampal Formation

The isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Using single-cell RNA-sequencing approaches, we profiled ~1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation. The cell types are organized hierarchically and exhibit varying degrees of discrete or continuous variations. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, shared across multiple regions, or part of one or more gradients. Glutamatergic neuron types display much greater diversity than GABAergic neuron types, both molecularly and spatially, and define regional identities as well as inter-region relationships. Our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

Similar nicotinic excitability responses across the developing hippocampal formation are regulated by small-conductance calcium-activated potassium channels.

The hippocampal formation forms a cognitive circuit that is critical for learning and memory. Cholinergic input to nicotinic acetylcholine receptors plays an important role in the normal development of principal neurons within the hippocampal formation. However, the ability of nicotinic receptors to stimulate principal neurons across all regions of the developing hippocampal formation has not been determined. We show in this study that heteromeric nicotinic receptors mediate direct inward current and depolarization responses in principal neurons across the hippocampal formation of the young postnatal mouse. These responses were found in principal neurons of the CA1, CA3, dentate gyrus, subiculum, and entorhinal cortex layer VI, and they varied in magnitude across regions with the greatest responses occurring in the subiculum and entorhinal cortex. Despite this regional variation in the magnitude of passive responses, heteromeric nicotinic receptor stimulation increased the excitability of active principal neurons by a similar amount in all regions. Pharmacological experiments found this similar excitability response to be regulated by small-conductance calcium-activated potassium (SK) channels, which exhibited regional differences in their influence on neuron activity that offset the observed regional differences in passive nicotinic responses. These findings demonstrate that SK channels play a role to coordinate the magnitude of heteromeric nicotinic excitability responses across the hippocampal formation at a time when nicotinic signaling drives the development of this cognitive brain region. This coordinated input may contribute to the normal development, synchrony, and maturation of the hippocampal formation learning and memory network. NEW & NOTEWORTHY This study demonstrates that small-conductance calcium-activated potassium channels regulate similar-magnitude excitability responses to heteromeric nicotinic acetylcholine receptor stimulation in active principal neurons across multiple regions of the developing mouse hippocampal formation. Given the importance of nicotinic neurotransmission for the development of principal neurons within the hippocampal formation, this coordinated excitability response is positioned to influence the normal development, synchrony, and maturation of the hippocampal formation learning and memory network.

Reduction of dynamin 1 in the hippocampus of aged mice is associated with the decline in hippocampal‑dependent memory.

Dynamin1 is a known synaptic protein, which has is key in the presynaptic regulation of endocytosis. The present study investigated the association between age and the observed changes in Morris water maze performance, and immunoreactivity and protein levels of dynamin1 in the mouse hippocampal formation. In addition, the effects of dynasore, an inhibitor of dynamin 1, on the hippocampal dependent memory were determined to elucidate the correlation between dynamin 1 and memory. In the training phase of the Morris water maze task, the mean escape latency of the aged group (24months old) was significantly longer, compared with that of the adult group (4months old), although the average swimming speed and the total distance traveled during the probe trial were similar in the two groups. In the aged group, the time spent locating the target platform was significantly longer and the time spent in the correct quadrant was significantly shorter, compared with those in the adult group. In the adult group, a moderate level of dynamin1 was detected in the hippocampal CA1 and CA3 regions, and in the dentate gyrus. In the aged group, the immunoreactivity of dynamin1 was almost eliminated in the CA3 region and the dentate gyrus. In addition, the protein levels of dynamin1 in the brain were significantly lower in the aged group, compared with those in the adult group. The direct infusion of dynasore, significantly reduced the contextual memory, compared with that of animals in the vehicle‑treated group. These results suggested that dynamin1 was susceptible to the aging process, and that a reduction in dynamin1 may result in hippocampal‑dependent memory deficits by disrupting endocytosis and the release of neurotransmitters.

Prenatal Alcohol Exposure Is Associated with Altered Subcellular Distribution of Glucocorticoid and Mineralocorticoid Receptors in the Adolescent Mouse Hippocampal Formation

BackgroundAccumulating evidence indicates that several of the long-term consequences of prenatal alcohol exposure (PAE) are the result of changes in the development and function of cortico-limbic structures, including the hippocampal formation. The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are key regulators of hippocampal formation development, structure, and functioning and, thus, are potential mediators of PAE’s effects on this brain region. In the present studies, we assessed the impact of PAE on components of corticosteroid signaling pathways in the mouse hippocampal formation.MethodsThroughout pregnancy, mouse dams were offered either 10% (w/v) ethanol sweetened with 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-hour) access, drinking-in-the-dark paradigm. The hippocampal formation was isolated from naïve postnatal day 40 to 50 offspring, and subcellular fractions were prepared. Using immunoblotting techniques, we measured the levels of GR, MR, 11-β-hydroxysteroid dehydrogenase 1 (11β-HSD1), and the FK506-binding proteins 51 (FKBP51, FKBP5) and 52 (FKBP52, FKBP4). Finally, we determined the effect of PAE on context discrimination, a hippocampal-dependent learning/memory task.ResultsPAE was associated with reduced MR and elevated GR nuclear localization in the hippocampal formation, whereas cytosolic levels of both receptors were not significantly altered. FKBP51 levels were reduced, while FKBP52 levels were unaltered, and 11β-HSD1 levels were increased in postnuclear fractions isolated from PAE mouse hippocampal formation. These neurochemical alterations were associated with reduced context discrimination.ConclusionsThe data support a model in which PAE leads to increased nuclear localization of GRs secondary to reductions in FKBP51 and increases in 11β-HSD1 levels in the adolescent mouse hippocampal formation. Persistent dysregulation of GR subcellular distribution is predicted to damage the hippocampal formation and may underlie many of the effects of PAE on hippocampal-dependent functioning.

Imaging neurite development of adult-born granule cells

Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in the mammalian hippocampus. Newborn granule cells mature over several weeks to functionally integrate into the pre-existing neural circuitry. Even though an increasing number of genes that regulate neuronal polarization and neurite extension have been identified, the cellular mechanisms underlying the extension of neurites arising from newborn granule cells remain largely unknown. This is mainly because of the current lack of longitudinal observations of neurite growth within the endogenous niche. Here we used a novel slice culture system of the adult mouse hippocampal formation combined with in vivo retroviral labeling of newborn neurons and longitudinal confocal imaging to analyze the mode and velocity of neurite growth extending from immature granule cells. Using this approach we show that dendritic processes show a linear growth pattern with a speed of 2.19±0.2 μm per hour, revealing a much faster growth dynamic than expected by snapshot-based in vivo time series. Thus, we here identified the growth pattern of neurites extending from newborn neurons within their niche and describe a novel technology that will be useful to monitor neuritic growth in physiological and disease states that are associated with altered dendritic morphology, such as rodent models of epilepsy.

Imaging neurite development of adult-born granule cells

Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in the mammalian hippocampus. Newborn granule cells mature over several weeks to functionally integrate into the pre-existing neural circuitry. Even though an increasing number of genes that regulate neuronal polarization and neurite extension have been identified, the cellular mechanisms underlying the extension of neurites arising from newborn granule cells remain largely unknown. This is mainly because of the current lack of longitudinal observations of neurite growth within the endogenous niche. Here we used a novel slice culture system of the adult mouse hippocampal formation combined with in vivo retroviral labeling of newborn neurons and longitudinal confocal imaging to analyze the mode and velocity of neurite growth extending from immature granule cells. Using this approach we show that dendritic processes show a linear growth pattern with a speed of 2.19±0.2 μm per hour, revealing a much faster growth dynamic than expected by snapshot-based in vivo time series. Thus, we here identified the growth pattern of neurites extending from newborn neurons within their niche and describe a novel technology that will be useful to monitor neuritic growth in physiological and disease states that are associated with altered dendritic morphology, such as rodent models of epilepsy.

Neuronal degeneration and gliosis time-course in the mouse hippocampal formation after pilocarpine-induced status epilepticus

Temporal lobe epilepsy (TLE) is the most common type of human epilepsy and has been related with extensive loss of hippocampal pyramidal and dentate hilar neurons and gliosis. Many characteristics of TLE are reproduced in the pilocarpine model of epilepsy in mice. This study analyzed the neuronal damage, assessed with Fluoro-Jade (FJB) and cresyl violet, and gliosis, investigated with glial fibrilary acidic protein (GFAP) immunohistochemistry, occurring in the hippocampal formation of mice at 3, 6, 12 and 24h, 1 and 3 weeks after the pilocarpine-induced status-epilepticus (SE) onset. The maximum neuronal damage score and the FJB-positive neurons peak were found in the hilus of dentate gyrus 3 and 12h after SE onset (P<0.05), respectively. At 1 week after SE onset, the greatest neuronal damage score was detected in the CA1 pyramidal cell layer and the greatest numbers of FJB-positive neurons were found both in the CA1 and CA3 pyramidal cell layers (P<0.05). The molecular, CA3 and CA1 pyramidal cell layers expressed highest presence of GFAP immunoreaction at 1 and 3 weeks after SE onset (P<0.05). Our findings show that, depending on the affected area, neuronal death and gliosis can occur within few hours or weeks after SE onset. Our results corroborate previous studies and characterize short time points of temporal evolution of neuropathological changes after the onset of pilocarpine-induced SE in mice and evidences that additional studies of this temporal evolution may be useful to the comprehension of the cellular mechanisms underlying epileptogenesis.

Estradiol acts via estrogen receptors alpha and beta on pathways important for synaptic plasticity in the mouse hippocampal formation

Estradiol affects hippocampal-dependent spatial memory and underlying structural and electrical synaptic plasticity in female mice and rats. Using estrogen receptor (ER) alpha and beta knockout mice and wild-type littermates, we investigated the role of ERs in estradiol effects on multiple pathways important for hippocampal plasticity and learning. Six hours of estradiol administration increased immunoreactivity for phosphorylated Akt throughout the hippocampal formation, whereas 48 h of estradiol increased immunoreactivity for phosphorylated TrkB receptor. Estradiol effects on phosphorylated Akt and TrkB immunoreactivities were abolished in ER alpha and ER beta knockout mice. Estradiol also had distinct effects on immunoreactivity for post-synaptic density 95 (PSD-95) and brain derived-neurotrophic factor (BDNF) mRNA in ER alpha and beta knockout mice. Thus, estradiol acts through both ERs alpha and beta in several subregions of the hippocampal formation. The different effects of estradiol at 6 and 48 h indicate that several mechanisms of estrogen receptor signaling contribute to this female hormone's influence on hippocampal synaptic plasticity. By further delineating these mechanisms, we will better understand and predict the effects of endogenous and exogenous ovarian steroids on mood, cognition, and other hippocampal-dependent behaviors.

Segmentation of the mouse hippocampal formation in magnetic resonance images

The hippocampal formation plays an important role in cognition, spatial navigation, learning, and memory. High resolution magnetic resonance (MR) imaging makes it possible to study in vivo changes in the hippocampus over time and is useful for comparing hippocampal volume and structure in wild type and mutant mice. Such comparisons demand a reliable way to segment the hippocampal formation. We have developed a method for the systematic segmentation of the hippocampal formation using the perfusion-fixed C57BL/6 mouse brain for application in longitudinal and comparative studies. Our aim was to develop a guide for segmenting over 40 structures in an adult mouse brain using 30 μm isotropic resolution images acquired with a 16.4 T MR imaging system and combined using super-resolution reconstruction.

Distribution of Phosphorylated TrkB Receptor in the Mouse Hippocampal Formation Depends on Sex and Estrous Cycle Stage

Tropomyosin-related kinase B receptor (TrkB) is a neurotrophin receptor important for the synaptic plasticity underlying hippocampal-dependent learning and memory. Because this receptor is widely expressed in hippocampal neurons, the precise location of TrkB activation is likely important for its specific actions. The goal of this study was to identify the precise sites of TrkB activation in the mouse hippocampal formation and to determine any changes in the distribution of activated TrkB under conditions of enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal excitability. Using electron microscopy, we localized TrkB phosphorylated at tyrosine 816 (pTrkB) in the hippocampal formation of male and female mice under conditions of naturally low circulating estradiol and naturally high circulating estradiol, when BDNF expression, TrkB signaling, and synaptic plasticity are enhanced. To compare relative amounts of pTrkB in each group, we counted profiles containing pTrkB-immunoreactivity (pTrkB-ir) in all hippocampal subregions. pTrkB-ir was in axons, axon terminals, dendrites, and dendritic spines of neurons in the hippocampal formation, but the majority of pTrkB-ir localized to presynaptic profiles. pTrkB-ir also was abundant in glial profiles, which were further identified as microglia using immunofluorescence and confocal microscopy. Axonal and glial pTrkB-ir and pTrkB-ir in the CA1 stratum radiatum were more abundant in high-estradiol states (proestrus females) than low-estradiol states (estrus and diestrus females and males). These findings suggest that presynaptic TrkB is positioned to modulate estradiol-mediated and BDNF-dependent synaptic plasticity. Furthermore, they suggest a novel role for TrkB in microglial function in the neuroimmune system.

Cellular and subcellular localization of estrogen and progestin receptor immunoreactivities in the mouse hippocampus

Estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), and progestin receptor (PR) immunoreactivities are localized to extranuclear sites in the rat hippocampal formation. Because rats and mice respond differently to estradiol treatment at a cellular level, the present study examined the distribution of ovarian hormone receptors in the dorsal hippocampal formation of mice. For this, antibodies to ERalpha, ERbeta, and PR were localized by light and electron immunomicroscopy in male and female mice across the estrous cycle. Light microscopic examination of the mouse hippocampal formation showed sparse nuclear ERalpha and PR immunoreactivity (-ir) most prominently in the CA1 region and diffuse ERbeta-ir primarily in the CA1 pyramidal cell layer as well as in a few interneurons. Ultrastructural analysis additionally revealed discrete extranuclear ERalpha-, ERbeta-, and PR-ir in neuronal and glial profiles throughout the hippocampal formation. Although extranuclear profiles were detected in all animal groups examined, the amount and types of profiles varied with sex and estrous cycle phase. ERalpha-ir was highest in diestrus females, particularly in dendritic spines, axons, and glia. Similarly, ERbeta-ir was highest in estrus and diestrus females, mainly in dendritic spines and glia. Conversely, PR-ir was highest during proestrus, mostly in axons. Except for very low levels of extranuclear ERbeta-ir in mossy fiber terminals in mice, the labeling patterns in the mice for all three antibodies were similar to the ultrastructural labeling found previously in rats, suggesting that regulation of these receptors is well conserved across the two species.

Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3

The neural cell recognition molecule NB-3, which is also referred to as contactin-6, is a member of the contactin subgroup molecules that are expressed prominently in the developing nervous system after birth. In mice, an NB-3 deficiency impairs motor coordination and reduces the synaptic density between parallel fibers and Purkinje cells in the cerebellum. Here, we studied the role of NB-3 in the formation of glutamatergic synapses in the hippocampal formation. At postnatal day 5, NB-3 immunoreactivity was detected in the subiculum, the stratum lacunosum–moleculare of the CA1 region and the hilus of the dentate gyrus. NB-3 expression in the strata radiatum and oriens was weak, and it was very weak in the granule cell layer of the dentate gyrus, the pyramidal cell layer of regions CA3 to CA1 and the stratum lucidum. NB-3-positive puncta partially overlapped with vesicular glutamate transporter 1 (VGLUT1) and 2 (VGLUT2), excitatory presynaptic markers, but not with vesicular GABA transporter (VGAT), an inhibitory presynaptic marker. The density of VGLUT1 and VGLUT2 puncta in the regions where NB-3 was strongly expressed in wild-type mice was reduced by ∼20–30% in NB-3 knockout mice relative to wild-type mice, whereas that of VGAT puncta was not affected by NB-3 deficiency. Thus, NB-3 has key roles in the formation of glutamatergic, but not GABAergic, synapses during postnatal development of the hippocampal formation as well as the cerebellum.

Hippocampal N‐Methyl‐d‐Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol Exposure

Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.

P.1.12 Interaction between neuropeptide Y, Y1 and Y2 receptor protein in the mouse hippocampal formation and amygdaloid complex

P.1.12 Interaction between neuropeptide Y, Y1 and Y2 receptor protein in the mouse hippocampal formation and amygdaloid complex

Region and lamina-specific distribution of extracellular matrix proteoglycans, hyaluronan and tenascin-R in the mouse hippocampal formation

The extracellular matrix is known to show region-specific characteristics in the adult brain. Our comparative cytochemical study is focused on the laminar organisation of major extracellular matrix constituents in the murine hippocampal formation, including the regions CA1, CA2 and CA3 of the hippocampus proper, the dentate gyrus, the subiculum and the presubiculum. Components related to chondroitin sulphate proteoglycans were detected by N-acetylgalactosamine-binding Wisteria floribunda agglutinin, colloidal iron staining, and antibodies to different proteoglycan domains, including the Cat-301 and Cat-315 epitopes of aggrecan, as well as neurocan, brevican and phosphacan. The distribution patterns of these components were correlated with the patterns revealed for hyaluronan and the brain-specific extracellular matrix glycoprotein, tenascin-R, known to be ligands of extracellular matrix proteoglycans. Lectin binding clearly labelled perineuronal nets of the extracellular matrix around interneurons, which were preferentially located within or near the principal cell layers in all regions. In the hippocampus proper, the CA2 subfield showed an intense labelling of the neuropil around pyramidal cell bodies and the neuropil zones in the strata oriens and radiatum. These patterns were also seen after immunoreaction for chondroitin proteoglycan domains, brevican and phosphacan, as well as after detection of hyaluronan and tenascin-R. Characteristic laminar and intralaminar patterns were additionally expressed in the neuropil in all regions. In the dentate gyrus, the staining intensity for brevican, phosphacan and tenascin-R was predominant in the middle molecular layer, and for Cat-315 in the inner molecular layer, whereas immunoreactivity for neurocan increased within the outer molecular layer towards the hippocampal fissure. Our findings indicate that proteoglycans, hyaluronan and tenascin-R show differential patterns of co-expression in the individual regions and laminae of the hippocampal formation. The inhomogeneous composition of these major components suggests that the extracellular matrix is specifically adapted to the functional domains of intrahippocampal connections and afferent fibre systems.

Perforant path lesioning induces sprouting of CA3-associated fibre systems in mouse hippocampal formation.

In comparison to the rat, the anatomy of the mouse hippocampus, and in particular the response to entorhinal cortex lesioning, is less well characterised. Here we studied the axonal sprouting response after lesioning of the entorhinodentate perforant path projection in young adult SJL/J and C57BL/6 mice. We found that lesioning led to translaminar sprouting of Timm stained regio inferior hippocampus (CA3)-associated fibre systems into the denervated termination zones of the CA3 and dentate gyrus, from the adjacent non-denervated stratum radiatum of CA3. Differences were seen in the Timm staining pattern of the two strains of mice, while the response to lesioning appeared similar albeit less pronounced than that observed in the rat. We also observed an intensified acetylcholine esterase staining reflective of cholinergic sprouting in the denervated perforant path termination zones, which was particularly prominent in areas with sprouting of Timm stained CA3-associated fibres. Finally, we showed that some of the sprouting fibres within the CA3 were myelinated, due to an increased density of silver impregnated myelinated fibres in this region after lesioning. These results show that the basic characteristics of the response to perforant path lesioning in mice are similar to those in the rat, but suggest that the magnitude of the response in the two species is different.

Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation.

Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental differentiation in vivo. Here we have investigated the expression of HARP mRNA and protein in the perforant path lesioned C57B1/6 mouse hippocampal formation from 1 to 35 days after surgery. This type of lesion induces a dense anterograde and terminal axonal degeneration, activation of glial cells, and reactive axonal sprouting within the perforant path zones of the fascia dentata and hippocampus as well as axotomy-induced retrograde neuronal degeneration in the entorhinal cortex. Analysis of sham- and unoperated control mice showed that HARP mRNA is expressed in neurons and white and gray matter glial cells as well as vascular and pial cells throughout the normal, adult brain. Lesioning induced high levels of HARP mRNA in astroglial-like cells in the denervated zones of fascia dentata and hippocampus as soon as day 2 postlesion. This expression reached maximum at day 4, and declined toward normal at day 7-14. Combined HARP in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemical staining and double immunofluorescent stainings for GFAP and HARP at day 4 postlesion showed colocalization of HARP mRNA and protein to hypertrophic GFAP-immunopositive astrocytes in the denervated areas. Finally, the axotomized entorhinal layer II neurons, which expressed high levels of HARP mRNA in the normal brain, exhibited a marked decline in hybridization signal after axotomy. The induction of high levels of HARP mRNA and protein in astrocytes in the denervated areas of fascia dentata and hippocampus is of particular interest as astrocytes and astrocyte-derived factors are known to be implicated in axonal growth and regeneration and in rescuing injured neurons.

Differential effect of dehydroepiandrosterone and its steroid precursor pregnenolone against the behavioural deficits in CO-exposed mice

The neuroactive steroids pregnenolone (3β-hydroxy-5-pregnen-20-one) and dehydroepiandrosterone (DHEA, 3α-hydroxy-5-androstene-17-one) are negative allosteric modulators of the GABA A receptors and positive modulators of acetylcholine, NMDA and σ 1 receptors. Pregnenolone was recently shown to potentiate the neuronal damage induced by excessive glutamate in cell culture models, whereas dehydroepiandrosterone was reported to present some neuroprotective activity. The in vivo relevance of these effects was investigated in mice submitted to an hypoxic insult, the repeated exposure to carbon monoxide (CO) gas, a model that leads to neurodegeneration in the CA 1 hippocampal area and learning deficits. Recording spontaneous alternation behaviour in the Y-maze assessed short-term memory and long-term memory was examined using a passive avoidance task. After exposure to CO, mice showed a progressive deterioration of their learning ability, reaching significance after 3 days and being maximal after 7 days. Pregnenolone administered before CO significantly facilitated the hypoxia-related deficits, which could be measured 1 day after CO and appeared maximal after 3 days. Dizocilpine blocked the deficits in vehicle- and pregnenolone-treated CO-exposed animals, showing that pregnenolone selectively facilitated the NMDA receptor-dependent excitotoxicity. Dehydroepiandrosterone blocked the appearance of the CO-induced deficits, even after 7 days. Interestingly, the σ 1 receptor antagonist N, N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)ethylamine (NE-100) failed to affect the dehydroepiandrosterone-induced protection, showing the lack of involvement of σ 1 receptors. Cresyl violet-stained sections of the mouse hippocampal formation showed that the neurodegeneration observed in the CA 1 area after exposure to CO was augmented by pregnenolone and blocked by dehydroepiandrosterone. These results show that pregnenolone and dehydroepiandrosterone, although being similarly involved in modulating the excitatory/inhibitory balance in the brain, do not equally affect the extent of excitotoxic insults.