Mzia G. Zhvania

Structure, isolation, composition and reconstitution of the neuronal fusion pore.

Neuronal communication is dependent on the fusion of 40–50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5–8 Å resolution, the presence of 8–10 nm in diameter cup‐shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.

Membrane-directed molecular assembly of the neuronal SNARE complex

Since the discovery and implication of N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full‐length recombinant t‐SNAREs and v‐SNARE present in opposing liposomes, leading to the assembly of a t‐/v‐SNARE ring complex. Using high‐resolution electron microscopy, the electron density maps and 3D topography of the membrane‐directed SNARE ring complex was determined at nanometre resolution. Similar to the t‐/v‐SNARE ring complex formed when 50 nm v‐SNARE liposomes meet a t‐SNARE‐reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t‐SNARE‐reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane‐directed t‐/v‐SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome‐reconstituted recombinant t‐/v‐SNARE proteins, and native v‐SNARE present in isolated SV membrane, the membrane‐directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.

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.

Effect of myo-inositol on convulsions induced by pentylenetetrazole and kainic acid in rats

We studied the effect of myo-inositol on pentylenetetrazole and kainic acid-induced seizures in rats. Myo-inositol significantly reduced seizure activity.

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.

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.

White noise and neuronal porosome complex: transmission electron microscopic study

In the present electron microscopic study the effect of continuous white noise on the morphology of synapses and neuronal porosome complex (the neurotransmitter-release or secretory machinery) in two subcortical auditory brain regions - colliculus inferior and medial geniculate body in cat, were investigated. Several morphological alterations in some synapses were detected in both subcortical areas. These alterations mainly indicate to the decrease of functional activity of synapses. Rarely important pathological modifications in pre- and post-synaptic regions were detected. In addition to descriptive studies, the morphometric analysis of porosome diameter and depth was performed in colliculus inferior and medial geniculate body. The results revealed that while white noise has no effect on the porosome diameter and depth in colliculus inferior, it provokes significant alterations in the morphology of porosome complex in medial geniculate body. In particular, the significant increase of porosome depth in this nucleus may reflect the alteration in neurotransmission.

Effect of Different Forms of Hypokinesia on the Ultrastructure of Limbic, Extrapyramidal and Neocortical Areas of the Rat Brain: Electron Microscopic Study

The effect of chronic restraint stress and chronic hypokinesia “without stress” on the ultrastructure of central and lateral nuclei of amygdala, CA1 and CA3 area of the hippocampus, cingular cortex, nucleus caudatus and motor cortex of adult male rats were elucidated. In some neurons and synapses of abovementioned regions pathological modifications were revealed. More significant alterations provokes chronic restraint stress. Alterations are mostly concentrated: first—in the nuclei of amygdala, then in the CA1 and CA3 areas. Moderate alterations were observed in cingular cortex and nucleus caudatus. In comparing with it, hypokinesia “without stress” provokes only moderate modifications: predominantly in the nucleus caudatus, in lesser degree—in the hippocampus and amygdalae.

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.

Chronic Toluene Exposure and the Hippocampal Structure in Adolescent and Adult Brains

Abstract Toluene and toluene-containing volatile substances are the most commonly abused solvents with a demonstrative addictive potential for humans. The misuse of these substances is associated with the activation of brain areas involved in both addiction and learning. As potential substrates for both processes, the regions of the mesocorticolimbic system have been identified. A number of clinical and experimental studies indicate that the hippocampus is involved in toluene addiction. In particular, toluene affects learning and memory and provokes various molecular, biochemical, and structural alterations in the hippocampus of adolescents and adults. The character of these alterations depends upon the age the organism tested. In this chapter we review immediate and persistent effects of chronic toluene exposure on the structure of the hippocampus in organisms of different ages.