I. B. Mikheeva

Ultrastructural Changes in the Mixed Synapses of Mauthner Neurons Related to Long-Term Potentiation and Natural Modification of the Motor Function

We examined changes in the ultrastructure of afferent mixed synapses on the membrane of Mauthner neurons (M cells) of the goldfish, which were related to two functional states, long-term potentiation (LTP) of the electrotonic response (a model form of the memory trace) and adaptation (resistivity to fatigue resulting from long-lasting motor training and considered a natural form of the memory trace manifested on the neuronal level). LTP was induced in medullary slices using high-frequency electrical stimulation of the afferent input. Adaptation was produced using natural vestibular stimulation (everyday motor training, which modified motor behavior of the fish and function of the M cell). It was supposed that if the LTP phenomenon is involved in the formation of natural memory, both the adaptation and the LTP states should be accompanied by similar specific structural modifications. Indeed, it was found that in both cases the number of fibrillar bridges in the gaps of desmosome-like contacts (DLC) in the mixed synapses on the M cell surface demonstrated an about twofold increase. These bridges are known to include actin filaments, which function as conductors of cationic signals; thus, the LTP-related increase in the density of bridges corresponds to increased efficacy of electrotonic coupling via mixed synapses. Such a structural correlate of LTP, which probably has the same functional significance in mixed synapses of the “adapted” M cells, allows us to suppose that LTP is a natural property of the nervous system. The LTP-type intensification of the relay function of mixed synapses, which corresponds to adaptation, is probably a compensatory rearrangement allowing M cells to maintain some balance of the synaptic influences and, at the same time, to remain in a stable and plastic state; this is necessary for stable functioning under changing environmental conditions.

Cytoskeletal regulation of the cellular function by dopamine

The interaction of dopamine with model membranes, isolated G-actin, and living cells, such as Mauthner neurons and fibroblast-like BHK-21 cells has been studied. It was found that in vitro dopamine passes through the phospholipid membrane and directly polymerizes G-actin due to incorporation into threads as their integral part. In in vivo conditions, it penetrates inside the cell and induces the appearance of a network of actin filaments in loci rich in globular actin. The data suggest that there exists a mechanism of dopamine interaction with living cells, which is based on direct polymerization of cytosolic G-actin as its cellular target. The reorganization of the actin cytoskeleton leads to changes in the morphofunctional status of cells.

Visual input controls the functional activity of goldfish Mauthner neuron through the reciprocal synaptic mechanism

Goldfish are known to exhibit motor asymmetry due to functional asymmetry of their Mauthner neurons that induce the turns to the right or left during free swimming. It has been previously found that if the less active neuron is subjected to prolonged aimed visual stimulation via its ventral dendrite, the motor asymmetry of goldfish is inverted, testifying that this neuron becomes functionally dominant, while the size of the ventral dendrite under these conditions is reduced 2-3 times compared to its counterpart in mirror neuron. Earlier it has been also revealed that training optokinetic stimulation induces adaptation, a substantial resistance of both fish motor asymmetry and morphofunctional state of Mauthner neurons against prolonged optokinetic stimulation. The aim of this work was to study the cellular mechanisms of the effect of an unusual visual afferent input on goldfish motor asymmetry and Mauthner neuron function in norm and under adaptation. It was shown that serotonin applied onto Mauthner neurons greatly reduces their activity whereas its antagonist ondansetron increases it. Against the background of visual stimulation, serotonin strengthens functional asymmetry between neurons whereas ondansetron smoothes it. Taken together these data suggest the involvement of serotonergic excitatory synaptic transmission in the regulation of Mauthner neurons by vision. Ultrastructural study of the ventral dendrites after prolonged optokinetic stimulation has revealed depletions of numeral axo-axonal synapses with specific morphology, identified by means of immunogold label as serotonergic ones. These latter in turn are situated mainly on shaft boutons, which according to specific ultrastructural features are assigned to axo-dendritic inhibitory synapses. Thus, the excitatory serotonergic synapses seem to affect Mauthner neuron indirectly through inhibitory synapses. Further, it was morphometrically established that adaptation is accompanied by the significant decrease of active zones dimensions in both serotonergic and inhibitory synapses. Finally, it was determined in model experiments that the interaction of globular actin with glycine, a main inhibitory neurotransmitter supposedly directly and chronically affecting the ventral dendrite, results in actin filaments formation. It is assumed that glycine-induced cytosolic actin polymerization is a cause of reduction in the ventral dendrite size under stimulation. Thus, it was established that a rather small group of synapses situated on an individual dendrite of the neuron determines the execution of the important form of animal behavior.

Fatty acid amide hydrolase inhibitor URB597 may protect against kainic acid–induced damage to hippocampal neurons: Dependence on the degree of injury

OBJECTIVE Status epilepticus (SE) provokes changes, which lead to neuronal alterations. Endocannabinoids (eCBs) can affect the neuronal survival during excitotoxicity and brain damage. Using a kainic acid (KA)-induced experimental SE model, we investigated whether cellular changes entail damage to endoplasmic reticulum (ER), mitochondria, and nuclei in hippocampal cells (CA1 field), and whether these alterations can be diminished by treatment with URB597, an inhibitor of eCB enzymatic degradation. MATERIAL AND METHODS SE was induced in Wistar rats by the microinjection of KA into the lateral ventricle. URB597 or a vehicle (10% DMSO) were injected in the same way into the brain of animals 24h after the KA infusion and then daily for the next nine days. The behavior of animals was controlled visually and recorded with a video system. The intensity of SE significantly varied in different animals. Convulsive (stages 3-5 according to the Racine scale) and nonconvulsive seizures (mainly stages 1, 2 and rarely 3, 4) were recognized. RESULTS Two weeks after SE, a significant loss of hippocampal cells occurred in animals with KA injections. In survived cells, ultrastructural alterations in ER, mitochondria, and nuclei of hippocampal neurons were observed. The degree of cell injury depended on the severity of SE. Alterations evoked by moderate seizures were prevented or diminished by URB597, but strong seizures induced mostly irreversible damage. CONCLUSIONS The beneficial impact of the FAAH inhibitor URB597 can give impetus to the development of novel neuroprotective strategies.

On the regulative role of the glutamate receptor in mitochondria.

Abstract The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system – succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia.

Ultrastructure of Mauthner Neurons in Optokinetic Stimulation and Enucleation of the Eye

Previous studies have shown that contralateral (to the preferred turning side) optokinetic stimulation and ipsilateral enucleation of the eye lead to significant (by a factor of 2–4) decreases in the volume of the ventral dendrite (VD) in one of the two Mauthner neurons (MN) of the goldfish, which becomes functionally more active. We report here our studies of MN ultrastructure after these unilateral influences from the visual system. In both cases, the whole length of the shrunken VD showed emptying of synapses and compaction of its cytoskeleton as compared with the cytoskeleton of the VD of the contralateral MN and the cytoskeleton of the lateral dendrites and bodies of both neurons. It is suggested that emptied synapses are part of the excitatory visual input and that the control of the functional activity of MN via VD involves both cytoskeletal and synaptic mechanisms.

The structure of mixed synapses in Mauthner neurons during exposure to substances altering gap junction conductivity.

The aim of the present work was to study the effects of dopamine, ecdysone, and chlorpromazine, substances which alter the conductivity of gap junctions (GJ), on the ultrastructure of mixed synapses in goldfish Mauthner neurons. These studies showed that dopamine, which increased the electrical conductivity of mixed synapses, appeared to target desmosome-like contacts (DLC). Hypertrophy of DLC, along with increases in the numbers of bridges within their clefts, showed that the mechanism by which dopamine increased electrical conductivity involved neuronal actin. This was indicated by the transformation of isolated monomeric muscle actin into polymerized actin in the presence of dopamine. Conversely, GJ were degraded by dopamine. Ecdysone, which also increased GJ conductivity, altered GJ structure, increasing the numbers of GJ at the attachment zone and decreasing the sectional length, but had virtually no effect on DLC structure. Ecdysone also showed no interaction with DLC in in vitro conditions. The mechanism of action of ecdysone is thus associated primarily with GJ function. Chlorpromazine, which decreased GJ conductivity, partially or completely degraded the fibrillar juxtamembrane material of DLC, preventing actin polymerization, with corresponding in vitro effects, but produced no changes in GJ. The mechanism of its action therefore appears to be based on changes in the state of neuronal actin.

Morphofunctional Changes in Incubated Mauthner Neurons in Goldfish Treated with Peptides from Scorpion Venom

Electron microscopy with negative contrast showed that direct interaction of one of the peptide fractions of scorpion venom with monomeric chromatographically pure actin led to polymerization of actin, transforming it from the globular form to the fibrillar form. The effects of prolonged orthodromic stimulation on the evoked electrical activity and ultrastructure of Mauthner neurons (MN) were studied in incubated slices of goldfish medulla oblongata in the presence of this actin-polymerizing venom fraction. Peptides in this fraction were found to stabilize the amplitude of the electrical response of MN to exhaustion and to protect the ultrastructure of afferent chemical synapses and the neurons themselves from damage induced by stimulation. Enhancements in morphofunctional resistance were accompanied by stabilization of actin-containing specialized synaptic structures – desmosome-like contacts. The data obtained here provide evidence that peptides of this fraction of scorpion venom have direct actions on the actin component of the MN cytoskeleton and demonstrate potential for its use as a pharmacological tool able to penetrate living cells with value for studying the role of actin in the mechanisms of adaptation and memory.

Structural differences between desmosome-like contacts in afferent chemical and mixed synapses of Mauthner neurons in the goldfish.

the cellular level on the ability of synapses to undergo stable, long-term changes in their conductivity or efficiency. Signal conduction in synapses occurs mainly in the area at which the presynaptic bouton makes contact with the postsynaptic neuron via specialized contact structures. It is generally recognized that signal transmission in synapses of the chemical type takes place within an active zone (AZ) [17, 21]. In mixed-type synapses, where transmission includes chemical and electrotonic components, gap junctions (GJ) are present along with the AZ [12], to provide for electrotonic communication [13]. It has been suggested that long-term changes in synapse conductivity are associated with longterm structural changes in the corresponding conduction zones [8, 19, 20]. These two types of specialized junctions in the contact zones of both chemical and mixed synapses are supplemented by desmosome-like contacts (DLC), also termed puncta adhaerentia ; the structure of these is based, as demonstrated previously [11, 15], on filamentous actin (F-actin) and they have a characteristic distribution in the contact zone. In chemical synapses, they are located beside or around AZ [14, 23]. In mixed synapses, they surround GJ [20, 24]. In terms of their structural organization, the DLC of chemical and mixed synapses are very similar. The presumptive adhesional, mechanical function which they have in synapses and, it seems, their identical compositions, give no reason to expect any differences to exist between the DLC of different synapses. Therefore, the question of the existence of any kind of difference between them has not even been posed to date. However, new experimental data which we have obtained in studies of mixed synapses suggest that the DLC of these synapses perform not only an adhesional function, but may also have a role in the transmission of the transsynaptic electrotonic signal [7, 19]. This suggestion is also supported by data showing that F-actin can conduct an electric current along its molecules both in water [18] and when it is inserted into a bilayer membrane [1, 16]. The association of two functions in a single type of DLC in mixed synapses should be reflected in some way in their overall structure and should lead to differences from the DLC of chemical synapses, which do not have electrotonic transmission [25] and have only one – the adhesional – function. The aim of the present work was to perform comparative electron-microscopic investigations of the two types of DLC in afferent synapses of the chemical and mixed types in Mauthner neurons (MN) in the goldfish.

Ultrastructural localization of the ROMK potassium channel in rat liver and heart

The intracellular localization and distribution of the ROMK protein in rat liver and heart was studied by the electron microscopy of ultrathin sections using the antibodies against the ROMK channel protein, one of the contenders for the role of mitochondrial ATP-dependent potassium channel. In rat heart and liver tissues, the ROMK protein is localized on the membranes of mitochondrial cristae but differently distributed in hepatocytes and cardiomyocytes. In hepatocytes, colloidal gold particles were rather evenly distributed on the membranes of mitochondrial cristae. In cardiomyocytes, the number of granules was considerably lower than in hepatocytes, and they were also localized on the membranes of mitochondrial cristae and confined only by the center of these organelles.