Short- and long-term effects of chronic toluene exposure on spatial memory in adolescent and adult male Wistar rats.

Addiction to toluene-containing volatile inhalants is of significant medical and social concern, particularly among youth. These concerns are underscored by the fact that the majority of adult abusers of toluene started as teenagers. Surprisingly, however, the lasting effects of chronic toluene exposure, especially in various age groups, have not been well investigated. Recently, we reported that adolescent and adult male Wistar rats show differential responses to chronic toluene exposure in recognition memory tasks. Since different cognitive functions may be differentially affected by drugs of abuse, we used the same model to evaluate the short- and long-term effects of chronic toluene on spatial learning and memory using Morris water maze. Daily exposure to toluene (2000 ppm) for 40 days (5 min/day) resulted in age-dependent behavioral changes. For example, only adolescent animals showed a decrease in time and distance travelled to find the hidden platform 24 h after the last toluene exposure. In contrast, only adult rats exhibited a decrease in acquisition time and distance travelled at 90 days' post toluene exposure. Our data provide further support for the contention that age-dependent responses should be taken into consideration in interventional attempts to overcome specific detrimental consequences of chronic toluene exposure.

Myo-Inositol Limits Kainic Acid-Induced Epileptogenesis in Rats.

Epilepsy is a severe neurological disease characterized by spontaneous recurrent seizures (SRS). A complex pathophysiological process referred to as epileptogenesis transforms a normal brain into an epileptic one. Prevention of epileptogenesis is a subject of intensive research. Currently, there are no clinically approved drugs that can act as preventive medication. Our previous studies have revealed highly promising antiepileptogenic properties of a compound-myo-inositol (MI) and the present research broadens previous results and demonstrates the long-term disease-modifying effect of this drug, as well as the amelioration of cognitive comorbidities. For the first time, we show that long-term treatment with MI: (i) decreases the frequency and duration of electrographic SRS in the hippocampus; (ii) has an ameliorating effect on spatial learning and memory deficit associated with epileptogenesis, and (iii) attenuates cell loss in the hippocampus. MI treatment also alters the expression of the glial fibrillary acidic protein, LRRC8A subunit of volume-regulated anion channels, and protein tyrosine phosphatase receptor type R, all expected to counteract the epileptogenesis. All these effects are still present even 4 weeks after MI treatment ceased. This suggests that MI may exert multiple actions on various epileptogenesis-associated changes in the brain and, therefore, could be considered as a candidate target for prevention of epileptogenesis.

Age-related cognitive decline in rats is sex and context dependent.

Previously, we had observed age-related cognitive decline in male rats compared to adolescent and adult rats. This was shown in both a multi-branched maze test (MBM), as well as in the Morris water maze test (MWM). In the present study, we compared the behavior of similar age groups in both male and female rats using the same paradigms. The results confirmed the increase in errors and time spent in MBM in aged male rats compared to other age groups. However, no such differences were observed in female rats. In the acquisition phase of MWM, aged male rats did not differ significantly from the other two groups in terms of time spent in quadrants, whereas aged female rats spent significantly more time in quadrants compared to the other 2 age groups. Aged male rats also travelled significantly more than the other 2 age groups during the acquisition phase, whereas no such differences were observed in female rats. In both short term (30 min post acquisition) and long term (24 h after acquisition) retrieval phases of MWM, significant gender-related differences were also observed in all age groups. These findings suggest gender- and context-dependent alterations in cognitive functions during aging.

Aging affects cognition and hippocampal ultrastructure in male Wistar rats.

It is now well established that aging is associated with emotional and cognitive changes. Although the basis of such changes is not fully understood, ultrastructural alterations in key brain areas are likely contributing factors. Recently, we reported that aging-related anxiety in male Wistar rats is associated with ultrastructural changes in the central nucleus of amygdala, an area that plays important role in emotional regulation. In this study, we evaluated the cognitive performance of adolescent, adult, and aged male Wistar rats in multi-branch maze (MBM) as well as in Morris water maze (MWM). We also performed ultrastructural analysis of the CA1 region of the hippocampus, an area intimately involved in cognitive function. The behavioral data indicate significant impairments in few indices of cognitive functions in both tests in aged rats compared to the other two age groups. Concomitantly, a total number of presynaptic vesicles as well as vesicles in the resting pool were significantly lower, whereas postsynaptic mitochondrial area was significantly higher in aged rats compared to the other age groups. No significant differences in presynaptic terminal area or postsynaptic mitochondrial number were detected between the three age groups. These results indicate that selective ultrastructural changes in specific hippocampal region may accompany cognitive decline in aging rats.

Gut neurotoxin p-cresol induces differential expression of GLUN2B and GLUN2A subunits of the NMDA receptor in the hippocampus and nucleus accumbens in healthy and audiogenic seizure-prone rats.

Mislocalization and abnormal expression of N-methyl-D-aspartate glutamate receptor (NMDAR) subunits is observed in several brain disorders and pathological conditions. Recently, we have shown that intraperitoneal injection of the gut neurotoxin p-cresol induces autism-like behavior and accelerates seizure reactions in healthy and epilepsy-prone rats, respectively. In this study, we evaluated the expression of GLUN2B and GLUN2A NMDAR subunits, and assessed the activity of cAMP-response element binding protein (CREB) and Rac1 in the hippocampi and nucleus accumbens of healthy and epilepsy-prone rats following p-cresol administration. We have found that subchronic intraperitoneal injection of p-cresol induced differential expression of GLUN2B and GLUN2A between the two brain regions, and altered the GLUN2B/GLUN2A ratio, in rats in both groups. Moreover, p-cresol impaired CREB phosphorylation in both brain structures and stimulated Rac activity in the hippocampus. These data indicate that p-cresol differently modulates the expression of NMDAR subunits in the nucleus accumbens and hippocampi of healthy and epilepsy-prone rats. We propose that these differences are due to the specificity of interactions between dopaminergic and glutamatergic pathways in these structures.

Behavioural and brain ultrastructural changes following the systemic administration of propionic acid in adolescent male rats. Further development of a rodent model of autism.

Short chain fatty acids, produced as gut microbiome metabolites but also present in the diet, exert broad effects in host physiology. Propionic acid (PPA), along with butyrate and acetate, plays a growing role in health, but also in neurological conditions. Increased PPA exposure in humans, animal models and cell lines elicit diverse behavioural and biochemical changes consistent with organic acidurias, mitochondrial disorders and autism spectrum disorders (ASD). ASD is considered a disorder of synaptic dysfunction and cell signalling, but also neuroinflammatory and neurometabolic components. We examined behaviour (Morris water and radial arm mazes) and the ultrastructure of the hippocampus and medial prefrontal cortex (electron microscopy) following a single intraperitoneal (i.p.) injection of PPA (175 mg/kg) in male adolescent rats. PPA treatment showed altered social and locomotor behaviour without changes in learning and memory. Both transient and enduring ultrastructural alterations in synapses, astro- and microglia were detected in the CA1 hippocampal area. Electron microscopic analysis showed the PPA treatment significantly decreased the total number of synaptic vesicles, presynaptic mitochondria and synapses with a symmetric active zone. Thus, brief systemic administration of this dietary and enteric short chain fatty acid produced behavioural and dynamic brain ultrastructural changes, providing further validation of the PPA model of ASD.

Concentration- and time-dependent effects of myo-inositol on evoked epileptic afterdischarge in the hippocampus in vivo.

Epilepsy is one of the most widespread neurological diseases characterized by spontaneous recurrent seizures. There is no cure for epilepsy, and available pharmacological treatments with anti-seizure drugs are only symptomatic. Moreover, about third of epilepsy patients are resistant to the anti-seizure drugs. Thus, it is essential to discover new anti-epilepsy drugs. Recently, myo-inositol has been identified as a promising antiepileptic compound. In the present study, using electrophysiological method, we examined for the first time, the effect of myo-inositol on the generation of epileptic afterdischarges in the hippocampus evoked by a local electrical stimulation. This was achieved by implanting two electrodes with a cannula into the same dorsal hippocampus, which allowed for simultaneous local injection of myo-inositol or saline and afterdischarges induction and recording from the same hippocampus. We found that myo-inositol has time- and concentration-dependent effects on the evoked afterdischarges. Specifically, 5 minutes after 1 M myo-inositol infusion, the afterdischarges duration was significantly decreased as compared to preinjection durations in the same animals and also as compared to preinjection level durations in saline injected or contralateral hippocampus myo-inositol infused animals. Further, 0.055 M myo-inositol significantly decreased afterdischarges duration at 5 minutes as compared to 40 minutes post-injection. At both concentrations, the afterdischarges duration recovered to the pre-injection value at 40 minutes after the myo-inositol injection. The present data, taken together with our previous results, strongly suggest that myo-inositol has significant local seizure-suppressant effect.

Effect of propionic acid on the morphology of the amygdala in adolescent male rats and their behavior.

Autism spectrum disorder is a group of life-long developmental syndromes, characterized by stereotypic behavior, restricted, communication deficits, cognitive and social impairments. Autism spectrum disorder is heritable state, provided by the mutations of well-conserved genes; however, it has been increasingly accepted, that most of such states are the result of complex interaction between individual's genetic profile and the environment that he/she is exposed to. Gut microbiota plays one of the central roles in the etiology of autism. Propionic acid is one of the most abundant short-chain fatty acids, made by enteric bacteria. Propionic acid has many positive functions and acts as the main mediator between nutrition, gut microbiota and brain physiology. However, increased level of propionic acid is associated with various neurological pathologies, including autism. It is proposed that some types of autism might be partially related with alterations in propionic acid metabolism. The amygdala, the main component of social brain, via its large interconnections with fronto-limbic neural system, plays one of the key roles in social communications, emotional memory and emotional processing. Social behavior is a hot topic in autism research. As to anxiety, it is not the main characteristics of ASD, but represents one of the most common its co morbidities. Several theoretical reasons compatible with amygdala dysfunction have been suggested to account for socio-emotional disturbances in autism. In the present study, using adolescent male Wistar rats, the effect of acute administration of low dose of propionic acid on social behavior, anxiety-like behavior and the structure/ultrastructure of central nucleus of amygdale was described. In addition to qualitative analysis, on electron microscopic level the quantitative analysis of some parameters of synapses was performed. Behavior was assessed 2, 24 and 48 hours after treatment. The results revealed that even single and relatively low dose of propionic acid is sufficient to produce fast and relatively long lasting (48 h after treatment) decrease of social motivation, whereas asocial motivation and emotional sphere remain unaffected. Morphological analyses of propionic acid-treated brain revealed the reduced neuron number and the increase of the number of glial cells. Electron microscopically, in some neurons the signs of apoptosis and chromatolysis were detected. Glial alterations were more common. Particularly, the activation of astrocytes and microglia were often observed. Pericapillary glia was the most changed. Neuronal, glial and presynaptic mitochondria showed substantial structural diversities, mainly in terms of size and form. Total number of the area of presynaptic profile was significantly decreased. Some axons were moderately demyelinated. In general, the data indicate that even low dose of propionic acid produces in adolescent rodents immediate changes in social behavior, and structural/ultrastructural alterations in amygdala. Ultrastructural alterations may reflect moderate modifications in functional networks of social brain.

In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

BACKGROUND: MicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response. METHODS: Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO. RESULTS: In the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-ß and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls. CONCLUSIONS: Based on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.

Myoinositol Attenuates the Cell Loss and Biochemical Changes Induced by Kainic Acid Status Epilepticus.

Identification of compounds preventing or modifying the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action are of great importance. We have previously shown that myoinositol (MI) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (KA) induced status epilepticus (SE). However in these studies we have not detected any effects of MI on the first day after SE. In the present study we broadened our research and focused on other molecular and morphological changes at the early stages of SE induced by KA and effects of MI treatment on these changes. The increase in the amount of voltage-dependent anionic channel-1 (VDAC-1), cofilin, and caspase-3 activity was observed in the hippocampus of KA treated rats. Administration of MI 4 hours later after KA treatment abolishes these changes, whereas diazepam treatment by the same time schedule has no significant influence. The number of neuronal cells in CA1 and CA3 subfields of hippocampus is decreased after KA induced SE and MI posttreatment significantly attenuates this reduction. No significant changes are observed in the neocortex. Obtained results indicate that MI posttreatment after KA induced SE could successfully target the biochemical processes involved in apoptosis, reduces cell loss, and can be successfully used in the future for translational research.

In Vivo Inhibition of miR-155 Promotes Recovery after Experimental Mouse Stroke.

A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery. Significance statement: In the present study, we investigated an effect of the in vivo inhibition of a microRNA, miR-155, on brain recovery after experimental cerebral ischemia. To our knowledge, this is the first report describing the efficiency of intravenous anti-miRNA injections in a mouse model of ischemic stroke. The role of miRNAs in poststroke revascularization has been unexplored and in vivo regulation of miRNAs during the subacute phase of stroke has not yet been proposed. Our investigation introduces a new and unexplored approach to cerebral regeneration: regulation of poststroke angiogenesis and recovery through direct modulation of specific miRNA activity. We expect that our findings will lead to the development of novel strategies for regulating neurorestorative processes in the postischemic brain.

Electron microscopic morphometry of isolated rat brain porosome complex.

Porosomes are the universal secretory portals at the cell plasma membrane where secretory vesicles dock and transiently fuse via the kiss-and-run mechanism of cellular secretion, to release intravesicular cargo to the outside of the cell. During last two decades discovery of porosome and a great volume of work from different laboratories provide molecular insights on the structure, function, and composition of the porosome complex, especially the neuronal porosome. In rat neurons 12-17 nm cup-shaped lipoprotein porosomes present at presynaptic membrane. They possess a central plug and sometimes are with docked synaptic vesicles. Although earlier studies have greatly progressed our understanding of the morphology and the proteome and limited lipidome of the neuronal porosome complex, the current study was carried out to determine the morphology of the bare protein backbone of the neuronal porosome complex. Results from our study demonstrate that although the eight-fold symmetry of the immunoisolated porosome is maintained, and the central plug is preserved in the isolated structures, there is a loss in the average size of the porosome complex, possibly due to a loss of lipids from the complex.

Ultrastructural changes to rat hippocampus in pentylenetetrazol- and kainic acid-induced status epilepticus: A study using electron microscopy.

A pentylenetetrazol (PTZ)-induced status epilepticus model in rats was used in the study. The brains were studied one month after treatment. Ultrastructural observations using electron microscopy performed on the neurons, glial cells, and synapses, in the hippocampal CA1 region of epileptic brains, demonstrated the following major changes over normal control brain tissue. (i) There is ultrastructural alterations in some neurons, glial cells and synapses in the hippocampal CA1 region. (ii) The destruction of cellular organelles and peripheral, partial or even total chromatolysis in some pyramidal cells and in interneurons are observed. Several astrocytes are proliferated or activated. Presynaptic terminals with granular vesicles and degenerated presynaptic profiles are rarely observed. (iii) The alterations observed are found to be dependent on the frequency of seizure activities following the PTZ treatment. It was observed that if seizure episodes are frequent and severe, the ultrastructure of hippocampal area is significantly changed. Interestingly, the ultrastructure of CA1 area is found to be only moderately altered if seizure episodes following the status epilepticus are rare and more superficial; (iv) alterations in mitochondria and dendrites are among the most common ultrastructural changes seen, suggesting cell stress and changes to cellular metabolism. These morphological changes, observed in brain neurons in status epilepticus, are a reflection of epileptic pathophysiology. Further studies at the chemical and molecular level of neurotransmitter release, such as at the level of porosomes (secretory portals) at the presynaptic membrane, will further reveal molecular details of these changes.

The effect of kainic acid on hippocampal dendritic spine motility at the early and late stages of brain development.

Dendrites and spines undergo dynamic changes in physiological conditions, such as learning and memory, and in pathological conditions, such as epilepsy. Abnormalities in dendritic spines have commonly been observed in brain specimens from epilepsy patients and animal models of epilepsy. However, the functional implications and clinical consequences of this dendritic pathology for epilepsy are uncertain. Motility of dendritic spines and axonal filopodia has been recently discovered by the advanced imaging techniques, and remains to a large degree an exciting phenomenology in search of function. Here we demonstrate the effect of kainic acid (KA), which is a structural analog of glutamate, on dendritic spine motility in hippocampal CA1 area at the different stages of brain development. In order to reveal the changes that take place in spine and filopodial motility in the epileptic model of brain, time-lapse imaging of acute hippocampal slices treated with various concentrations of KA after different incubation time points was performed. The effects of KA exposure were tested on the slices from young (postnatal day (P)7-P10) and adolescent (P28-P30) Thy1-YFPH transgenic mice. Slices were treated with either 50 µM or 100 µM of KA, for either 30 or 100 min. The results obtained in our experiments show diverse effects of KA in 2 different age groups. According to our results, 100 µM/100 min KA treatment increases spine motility at early stage of brain development (P10) by 41.5%, while in P30 mice spine motility is increased only by 3%. Our findings also indicate that effect of KA on hippocampal dendritic spine motility is predominantly time- rather than concentration-dependent.

Hypokinetic stress and neuronal porosome complex in the rat brain: the electron microscopic study.

Porosomes are the universal secretory machinery in cells, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside of the cell during cell secretion. Studies using atomic force microscopy, electron microscopy, electron density and 3D contour mapping, provided rich nanoscale information on the structure and assembly of proteins within the neuronal porosome complex in normal brain. However it remains uncertain whether pathological conditions that alter process of neurotransmission, provoke alterations in the porosome structure also. To determine if porosomes are altered in disease states, the current study was undertaken for first time using high resolution electron microscope. One of pathologies that produce subtle alteration at the presynaptic terminals has been demonstrated to be hypokinetic stress. The central nucleus of amygdale is the brain region, where such alterations are mostly expressed. We have examined the width and depth of the neuronal porosome complex and their alterations provoked by chronic hypokinetic stress in above mentioned limbic region. Specifically, we have demonstrated that despite alterations in the presynaptic terminals and synaptic transmission provoked by this pathological condition in this region, the final step/structure in neurosecretion--the porosome--remains unaffected: the morphometric analysis of the depth and diameter of this cup-shaped structure at the presynaptic membrane point out to the heterogeneity of porosome dimensions, but with unchanged fluctuation in norm and pathology.

Immediate and persisting effect of toluene chronic exposure on hippocampal cell loss in adolescent and adult rats.

Abuse of toluene-containing volatile inhalants has become widespread among adolescents. Besides, because toluene is usually used as an industrial solvent in manufacturing of chemical pharmaceuticals and multiple commonly used household and commercial products, it has high potential for abuse for adults also. Long-term exposure to toluene vapor has a severe impact on the central nervous system, resulting in numerous neurological, neurobiological and behavioral impairments. Recently in the hippocampus some molecular and biochemical changes as a result of toluene chronic exposure were described. Such data point out the involvement of this area in the toluene addiction. However it remains uncertain whether toluene provokes structural alterations in the hippocampus. In this study we exposed male Wistar rats to 2000 ppm inhaled toluene for 40 days in rats at ages P 28-32 (adolescents) and P 70-75 (adults). The immediate and delayed effects of toluene chronic exposure (immediately after the end of toluene chronic inhalation and 90-day after the end of toluene chronic inhalation, correspondingly) on pyramidal cell loss in adolescent and adult rats was investigated. The results reveal that (i) chronic exposure to 2000 ppm of toluene chronic exposure alters the structure of hippocampus in adolescent and adult rats provoking both, immediate and delayed effects; (ii) the character of structural alterations depends upon the postnatal age of testing of the animals.

Bergmann glial ensheathment of dendritic spines regulates synapse number without affecting spine motility.

In the cerebellum, lamellar Bergmann glial (BG) appendages wrap tightly around almost every Purkinje cell dendritic spine. The function of this glial ensheathment of spines is not entirely understood. The development of ensheathment begins near the onset of synaptogenesis, when motility of both BG processes and dendritic spines are high. By the end of the synaptogenic period, ensheathment is complete and motility of the BG processes decreases, correlating with the decreased motility of dendritic spines. We therefore have hypothesized that ensheathment is intimately involved in capping synaptogenesis, possibly by stabilizing synapses. To test this hypothesis, we misexpressed GluR2 in an adenoviral vector in BG towards the end of the synaptogenic period, rendering the BG α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) Ca2+-impermeable and causing glial sheath retraction. We then measured the resulting spine motility, spine density and synapse number. Although we found that decreasing ensheathment at this time does not alter spine motility, we did find a significant increase in both synaptic pucta and dendritic spine density. These results indicate that consistent spine coverage by BG in the cerebellum is not necessary for stabilization of spine dynamics, but is very important in the regulation of synapse number.

Morphogenesis and regulation of Bergmann glial processes during Purkinje cell dendritic spine ensheathment and synaptogenesis.

Astrocytes have an important role in synaptic formation and function but how astrocytic processes become associated with synaptic structures during development is not well understood. Here we analyzed the pattern of growth of the processes extending off the main Bergmann glial (BG) shafts during synaptogenesis in the cerebellum. We found that during this period, BG process outgrowth was correlated with increased ensheathment of dendritic spines. In addition, two-photon time-lapse imaging revealed that BG processes were highly dynamic, and processes became more stable as the period of spine ensheathment progressed. While process motility was dependent on actin polymerization, activity of cytoskeletal regulators Rac1 and RhoG did not play a role in glial process dynamics or density, but was critical for maintaining process length. We extended this finding to probe the relationship between process morphology and ensheathment, finding that shortened processes result in decreased coverage of the spine. Furthermore, we found that areas in which BG expressed dn-Rac1, and therefore had a lower level of synaptic ensheathment, showed an overall increase in synapse number. These analyses reveal how BG processes grow to surround synaptic structures, elucidate the importance of BG process structure for proper development of synaptic ensheathment, and reveal a role for ensheathment in synapse formation.

Amyloid beta peptide adversely affects spine number and motility in hippocampal neurons.

Elevated levels of amyloid-beta peptide (Abeta) are found in Down's syndrome patients and alter synaptic function during the early stages of Alzheimer's disease. Dendritic spines, sites of most excitatory synaptic contacts, are considered to be an important locus for encoding synaptic plasticity. We used time-lapse two-photon imaging of hippocampal pyramidal neurons in organotypic slices to study the effects of Abeta on the development of dendritic spines. We report that exposure of hippocampal neurons to sub-lethal levels of Abeta decreased spine density, increased spine length and subdued spine motility. The effect of Abeta on spine density was reversible. Moreover, Abeta's effect on dendritic spine density was blocked by rolipram, a phosphodiesterase type IV inhibitor, suggesting the involvement of a cAMP dependent pathway. These findings raise the possibility that Abeta-induced spine alterations could underlie the cognitive defects in Alzheimer's disease and Down syndrome.

Purkinje cell dendrites grow in alignment with Bergmann glia.

The pattern of growth of Purkinje cell dendrites has been analyzed and related to their interactions with Bergmann glial radial processes. In cerebellar slice cultures from mice expressing green fluorescent protein (GFP) under the glial fibrillary acidic protein (GFAP) promoter, Purkinje cells were transfected and imaged with two-photon microscopy over 2 days. We report that while the Purkinje cell dendritic tree grows, individual dendrites increase or decrease in length. Importantly, we demonstrate that vertical growth of Purkinje cell dendrites occurs primarily in alignment with radial glial processes. These findings suggest that radial glial processes provide a structural substrate for the directional growth of Purkinje cell dendrites, thus influencing the shape of the dendritic tree.