Heather A. Davies

Stress suppresses and learning induces plasticity in CA3 of rat hippocampus: a three-dimensional ultrastructural study of thorny excrescences and their postsynaptic densities

Chronic stress and spatial training have been proposed to affect hippocampal structure and function in opposite ways. Previous morphological studies that addressed structural changes after chronic restraint stress and spatial training were based on two-dimensional morphometry which does not allow a complete morphometric characterisation of synaptic features. Here, for the first time in such studies, we examined these issues by using three-dimensional (3-D) reconstructions of electron microscope images taken from thorny excrescences of hippocampal CA3 pyramidal cells. Ultrastructural alterations in postsynaptic densities (PSDs) of thorny excrescences receiving input from mossy fibre boutons were also determined, as were changes in numbers of multivesicular bodies (endosome-like structures) within thorny excrescences and dendrites. Quantitative 3-D data demonstrated retraction of thorny excrescences after chronic restraint stress which was reversed after water maze training, whilst water maze training alone increased thorny excrescence volume and number of thorns per thorny excrescence. PSD surface area was unaffected by restraint stress but water maze training increased both number and area of PSDs per thorny excrescence. In restrained rats that were water maze trained PSD volume and surface area increased significantly. The proportion of perforated PSDs almost doubled after water maze training and restraint stress. Numbers of endosome-like structures in thorny excrescences decreased after restraint stress and increased after water maze training. These findings demonstrate that circuits involving contacts between mossy fibre terminals and CA3 pyramidal cells at stratum lucidum level are affected conversely by water maze training and chronic stress, confirming the remarkable plasticity of CA3 dendrites. They provide a clear illustration of the structural modifications that occur after life experiences noted for their different impact on hippocampal function.

Rapid reversal of stress induced loss of synapses in CA3 of rat hippocampus following water maze training.

The impact was examined of exposing rats to two life experiences of a very different nature (stress and learning) on synaptic structures in hippocampal area CA3. Rats were subjected to either (i) chronic restraint stress for 21 days, and/or (ii) spatial training in a Morris water maze. At the behavioural level, restraint stress induced an impairment of acquisition of the spatial response. Moreover, restraint stress and water maze training had contrasting impacts on CA3 synaptic morphometry. Chronic stress induced a loss of simple asymmetric synapses [those with an unperforated postsynaptic density (PSD)], whilst water maze learning reversed this effect, promoting a rapid recovery of stress‐induced synaptic loss within 2–3 days following stress. In addition, in unstressed animals a correlation was found between learning efficiency and the density of synapses with an unperforated PSD: the better the performance in the water maze, the lower the synaptic density. Water maze training increased the number of perforated synapses (those with a segmented PSD) in CA3, both in stressed and, more notably, in unstressed rats. The distinct effects of stress and learning on CA3 synapses reported here provide a neuroanatomical basis for the reported divergent effects of these experiences on hippocampal synaptic activity, i.e. stress as a suppressor and learning as a promoter of synaptic plasticity.

Behavioural, physiological and morphological analysis of a line of apolipoprotein E knockout mouse

Using apolipoprotein E knockout mice derived from the Maeda source [Piedrahita J. A. et al. (1992) Proc. natn. Acad Sci. US.A. 89, 4471 4475], we have studied the influence of apolipoprotein E gene deletion on normal CNS function by neurological tests and water maze learning, hippocampal ultrastructure assessed by quantitative immunocytochemistry and electron microscopy, CNS plasticity, i.e. hippocampal long-term potentiation and amygdaloid kindling, and CNS repair, i.e. synaptic recovery in the hippocampus following deafferentation. In each study there was little difference between the apolipoprotein E knockout mice and wild-type controls of similar age and genetic background. Apolipoprotein E knockout mice aged eight months demonstrated accurate spatial learning and normal neurological function. Synaptophysin and microtubule-associated protein 2 immunohistochemistry and electron microscopic analysis of these animals revealed that the hippocampal synaptic and dendritic densities were similar between genotypes. The induction and maintenance of kindled seizures and hippocampal long-term potentiation were indistinguishable between groups. Finally, unilateral entorhinal cortex lesions produced a marked loss of hippocampal synaptophysin immunoreactivity in both groups and a marked up-regulation of apolipoprotein E in the wild-type group. Both apolipoprotein E knockout and wild-type groups showed immunohistochemical evidence of reactive synaptogenesis, although the apolipoprotein E knockout group may have initially shown greater synaptic loss. It is suggested that either apolipoprotein E is of no importance in the maintenance of synaptic integrity and in processes of CNS plasticity and repair, or more likely, alternative (apolipo)proteins may compensate for the loss of apolipoprotein E in the knockout animals.

Remodelling of synaptic morphology but unchanged synaptic density during late phase long-term potentiation(ltp): A serial section electron micrograph study in the dentate gyrus in the anaesthetised rat

In anaesthetised rats, long-term potentiation (LTP) was induced unilaterally in the dentate gyrus by tetanic stimulation of the perforant path. Animals were killed 6 h after LTP induction and dendritic spines and synapses in tetanised and untetanised (contralateral) hippocampal tissue from the middle molecular layer (MML) were examined in the electron microscope using stereological analysis. Three-dimensional reconstructions were also used for the first time in LTP studies in vivo, with up to 130 ultrathin serial sections analysed per MML dendritic segment. A volume sampling procedure revealed no significant changes in hippocampal volume after LTP and an unbiased counting method demonstrated no significant changes in synapse density in potentiated compared with control tissue. In the potentiated hemisphere, there were changes in the proportion of different spine types and their synaptic contacts. We found an increase in the percentage of synapses on thin dendritic spines, a decrease in synapses on both stubby spines and dendritic shafts, but no change in the proportion of synapses on mushroom spines. Analysis of three-dimensional reconstructions of thin and mushroom spines following LTP induction revealed a significant increase in their volume and area. We also found an increase in volume and area of unperforated (macular) and perforated (segmented) postsynaptic densities. Our data demonstrate that whilst there is no change in synapse density 6 h after the induction of LTP in vivo, there is a considerable restructuring of pre-existing synapses, with shaft and stubby spines transforming to thin dendritic spines, and mushroom spines changing only in shape and volume.

Long-term potentiation in the rat dentate gyrus is associated with enhanced Arc/Arg3.1 protein expression in spines, dendrites and glia.

Electron microscopic immunocytochemical methods were used to determine the localization, subcellular distribution and expression of activity‐regulated cytoskeletal protein (Arc/Arg3.1) in dentate gyrus after unilateral induction of long‐term potentiation (LTP) in the perforant pathway of anaesthetized rats. At 2 h post‐induction, immunoreaction product was visible in the dentate gyrus in both the granule cell and molecular layers. Arc expression was higher in the potentiated than the unstimulated contralateral hemisphere. Single‐section electron microscopy analysis in unstimulated tissue and in tissue prepared 2 and 4 h after LTP induction showed Arc immunoreactivity (Arc‐IR) in dendrites, dendritic spines and glia. Arc‐IR was associated with synaptic and non‐synaptic plasma membrane apposed to axon terminals and with cytoplasmic organelles, including the cytoskeleton. Arc‐IR was also present in neuronal perikarya and there was occasional labelling of nuclei and axons. At 2 h post‐LTP induction, there were significant increases in Arc‐IR within the granule cell and molecular layers of the dentate gyrus and particularly within the middle molecular layer relative to the inner and outer molecular layers. This increase in Arc expression 2 h after LTP induction was blocked by the N‐methyl‐d‐aspartate receptor antagonist (RS)‐3‐2‐carboxypiperazin‐4‐yl‐propyl‐1‐phosphonic acid. In animals killed 4 h after LTP induction, Arc expression had declined and differences between the potentiated and unpotentiated hemispheres were no longer significant. Our data provide ultrastructural evidence for a transient LTP‐associated increase in the expression of Arc protein in the middle molecular layer of the dentate gyrus, with preferential targeting to dendrites, dendritic spines and glia.

Ultrastructural synaptic correlates of spatial learning in rat hippocampus.

Memory formation is believed to alter neural circuitry at the synaptic level. Although the hippocampus is known to play an important role in spatial learning, no experimental data exist on the synaptic correlates of this process at the ultrastructural level. Here, we have employed quantitative electron microscopy in order to compare the density, size and spatial arrangement of synapses in the dentate gyrus, and in area CA1, of spatially trained (water maze, invisible platform) versus control (visible platform) rats. No training-associated changes of hippocampal volume were found using a stereological estimaion (disector) of the volume density of dentate granule, or CA1 pyramidal cells. Nor were changes found in either density, or sizes of synapses (spinous or dendritic), in CA1 or dentate gyrus. However, analysis of synaptic spatial distribution showed a training-associated increase in the frequency of shorter distances (i.e. clustering) between synaptic active zones in CA1, but not dentate, thus indicating alterations in local neural circuitry. This finding indicates subtle changes in synaptic organization in area CA1 of the hippocampus following a learning experience, suggesting that spatial memory formation in mammalian hippocampus may involve topographical changes in local circuitry without synapse formation de novo.

Engineered neural tissue for peripheral nerve repair.

A new combination of tissue engineering techniques provides a simple and effective method for building aligned cellular biomaterials. Self-alignment of Schwann cells within a tethered type-1 collagen matrix, followed by removal of interstitial fluid produces a stable tissue-like biomaterial that recreates the aligned cellular and extracellular matrix architecture associated with nerve grafts. Sheets of this engineered neural tissue supported and directed neuronal growth in a co-culture model, and initial in vivo tests showed that a device containing rods of rolled-up sheets could support neuronal growth during rat sciatic nerve repair (5 mm gap). Further testing of this device for repair of a critical-sized 15 mm gap showed that, at 8 weeks, engineered neural tissue had supported robust neuronal regeneration across the gap. This is, therefore, a useful new approach for generating anisotropic engineered tissues, and it can be used with Schwann cells to fabricate artificial neural tissue for peripheral nerve repair.

Mitochondria form a filamentous reticular network in hippocampal dendrites but are present as discrete bodies in axons: a three-dimensional ultrastructural study

The fine structure of mitochondria and smooth endoplasmic reticulum (SER) was studied via electron microscopy in dendritic and axonal neuronal segments of hippocampal areas CA1, CA3, and dentate gyrus (DG) of both ground squirrels in normothermic and hibernating conditions, and rats. Ultrathin serial sections of ∼60 nm (up to 150 per series) were taken and three‐dimensional (3D) reconstructions made of dendritic segments, up to 36 μm in length. Mitochondria were demonstrated to be present in filamentous form in every dendrite examined, in each of the hippocampal regions studied, whether in rat or ground squirrel. In addition, apparent continuity between the outer mitochondrial membrane and that of SER was observed by 3D reconstructions of very ultrathin (20 nm) serial sections prepared from dendritic segments. It is believed that SER penetrate into the heads of thin and mushroom spines but mitochondria do not enter the heads of these types of spines in dentate gyrus or CA1 of either rat or ground squirrel. However, in CA3 we have shown here that mitochondria penetrate into the base of the large thorny excrescences. Mushroom dendritic spines (but not thin spines) contained puncta adherentia, formed between pre‐ and postsynaptic membranes. In contrast to dendrites, the mitochondrial population of axonal processes in the same hippocampal regions were found only in the form of discrete bodies no more than 3 μm in length. The issue of the likely function of this network in dendrites and its potential role in calcium movement is discussed. J. Comp. Neurol. 492:50–65, 2005.

Role of Caspases in Cytokine-Induced Barrier Breakdown in Human Brain Endothelial Cells

During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood–brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.

Chronic restraint stress down-regulates amygdaloid expression of polysialylated neural cell adhesion molecule.

The amygdala is a brain area which plays a decisive role in fear and anxiety. Since exposure to chronic stress can induce profound effects in emotion and cognition, plasticity in specific amygdaloid nuclei in response to prior stress has been hypothesized to account for stress-induced emotional alterations. In order to identify amygdala nuclei which may be affected under chronic stress conditions we evaluated the effects of 21-days chronic restraint stress on the expression of a molecule implicated crucially in alterations in structural plasticity: the polysialylated neural cell adhesion molecule. We found that polysialylated neural cell adhesion molecule-immunoreactivity within the amygdala, present in somata and neuronal processes, has a regional gradient with the central medial and medial amygdaloid nuclei showing the highest levels. Our results demonstrate that chronic restraint stress induced an overall reduction in polysialylated neural cell adhesion molecule-immunoreactivity in the amygdaloid complex, mainly due to a significant decrease in the central medial amygdaloid and medial amygdaloid nuclei. Our data suggest that polysialylated neural cell adhesion molecule in these nuclei may play a prominent role in functional and structural remodeling induced by stress, being a potential mechanism for cognitive and emotional modulation. Furthermore, these finding provide the first clear evidence that life experiences can regulate the expression of polysialylated neural cell adhesion molecule in the amygdaloid complex.