Propionic acid affects the synaptic architecture of rat hippocampus and prefrontal cortex.

It is well documented that propionic acid (PPA) produces behavioral, morphological, molecular and immune responses in rats that are characteristic of autism spectrum disorder in humans. However, whether PPA affects the ultrastructure and synaptic architecture of regions of autistic brain has not been adequately addressed. Earlier we show that single intraperitoneal (IP) injection of PPA (175 mg/kg) produces superficial changes in the spatial memory and learning of adolescent male Wistar rats. However, in neurons, synapses and glial cells of hippocampal CA1 area and medial prefrontal cortex transient (mainly) or enduring alterations were detected. In this study, we used electron microscopic morphometric analysis to test the effect of PPA on different structural parameters of axodendritic synapses of the hippocampus and prefrontal cortex. The animals were treated with a single IP injection of PPA (175 mg/kg). The length and width of synaptic active zone, the area of presynaptic and postsynaptic mitochondria, the distance between presynaptic mitochondria and the synapse active zone, the distance between postsynaptic mitochondria and postsynaptic density and the depth and opening diameter of neuronal porosome complex were evaluated. Our results show that synaptic mitochondria of the hippocampus and prefrontal cortex are the most vulnerable to PPA treatment: in both regions, the area of postsynaptic mitochondria were increased. In general, our results show that even small dose of PPA, which produces only superficial effects on spatial memory and learning is able to alter the synapse architecture in brain regions involved in cognition and autism pathogenesis. Therefore, the microbiome may be involved in the control of neurotransmission in these regions.

Differential effects of aging on hippocampal ultrastructure in male vs. female rats.

Age-related decline in physical and cognitive functions are facts of life that do not affect everyone to the same extent. We had reported earlier that such cognitive decline is both sex- and context-dependent. Moreover, age-associated ultrastructural changes were observed in the hippocampus of male rats. In this study, we sought to determine potential differences in ultrastructural changes between male and female rats at various stages of life. We performed quantitative electron microscopic evaluation of hippocampal CA1 region, an area intimately involved in cognitive behavior, in both male and female adolescent, adult and old Wistar rats. Specifically, we measured the number of docking synaptic vesicles in axo-dendritic synapses, the length of active zone as well as the total number of synaptic vesicles. Distinct age- and sex-dependent effects were observed in several parameters. Thus, adult female rats had the lowest synaptic active zone compared to both adolescent and old female rats. Moreover, the same parameter was significantly lower in adult and old female rats compared to their male counterparts. On the other hand, old male rats had significantly lower number of total synaptic vesicles compared to both adolescent and adult male rats as well as compared to their female counterparts. Taken together, it may be suggested that age- and sex-dependent ultrastructural changes in the hippocampus may underlie at least some of the differences in cognitive functions among these groups.

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.

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

Abuse of toluene-containing volatile inhalants, particularly among youth, is of significant medical and social concern worldwide. Teenagers constitute the most abundant users of toluene and the majority of adult abusers of toluene started as teenagers. Although the euphoric and neurotoxic effects of acute toluene have been widely studied, lasting effects of chronic toluene exposure, especially in various age groups, have not been well investigated. In this study, we used adolescent and adult male Wistar rats to evaluate the short- and long-term effects of chronic toluene on various behaviors including cognitive function. Daily exposure to toluene (2000 ppm) for 40 days (5 min/day) resulted in age-dependent behavioral impairments. Specifically, adolescent animals showed recognition memory impairment the day after the last exposure, which had normalized by day 90 post- exposure, whereas such impairment in adult animals was still evident at day 90 post-exposure. Our data suggest that age-dependent responses should be taken into consideration in interventional attempts to overcome specific detrimental consequences of chronic toluene exposure.

Anxiety and ultrastructural consequences of chronic mild stress in rats.

Detrimental consequences following exposure to severe stress, either acute or chronic are well recognized. Chronic mild stress (CMS) is also a leading cause of emotional distress and neuropsychiatric conditions such as anxiety disorders. However, the neurobiological substrates of the latter, particularly at the ultrastructural levels have not been adequately investigated. In this study, adult male Wistar rats were subjected to 4 h daily mild restraint for 20 days and their behavior in open field and elevated plus maze (EPM) were evaluated 24 h after the last restraint. Anxiety-like behavior was evident in CMS exposed rats by increases in rearing and grooming in the open field and the avoidance of open arms in the EPM. Concomitant ultrastructural alterations such as chromatolysis, agglutination of synaptic vesicles or mitochondrial damage were also observed in the central nucleus of amygdala (CNA), an area intimately involved in emotional and fear response, in CMS exposed rats. These results while confirming detrimental consequences of CMS, also suggest that ultrastructural alterations in CNA may be a basis for CMS-induced anxiety.

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.

Electron microscopy demonstrating noise exposure alters synaptic vesicle size in the inferior colliculus of cat.

CONTEXT: White noise is known to have detrimental effects on different brain regions, especially auditory regions, including inferior colliculus. Although the basis for such alterations has been hypothesized to result from abnormalities in neurotransmitter release, the mechanism is unclear. The final step in neurotransmission is the docking and transient fusion of synaptic vesicles at the base of cup-shaped lipoprotein structures called porosomes at the presynaptic membrane and the consequent release of neurotransmitters. Earlier studies in cat brain document altered morphology of the secretory portal the porosome at nerve terminals in the inferior colliculus following white noise exposure. The current study was performed to test the hypothesis of possible changes to synaptic vesicle size in the colliculus, following white noise exposure. MATERIAL AND METHODS: Electron microscopic morphometry of synaptic vesicles size in axo-dendritic synapses at the colliculus region of the cat brain was performed. RESULTS: We report, for first time, decreased size of both docked and undocked vesicles in high-intensity white noise-exposed animals. In both control and experimental animals, docked vesicles are demonstrated to be smaller than undocked vesicles, suggesting fractional discharge of vesicular contents via porosome-mediated kiss-and-run mechanism. CONCLUSION: These studies advance our understanding of neurotransmitter release and the impact of white noise on brain function.

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.

Gender differences in anxiety response to high intensity white noise in rats.

Prolong exposure to high intensity white noise (HIWN), defined as a heterogeneous mixture of sound waves extending over a wide frequency range, has detrimental peripheral and central consequences including cardiovascular and emotional effects. Anxiety is a common manifestation of HIWN. Although gender-dependent differences in manifestation of anxiety and/or response to treatment of this condition has been amply documented, potential differences in response to HIWN, a common exposure in combat, construction and rave disco, has not been adequately investigated. In this study, both male and female Wistar rats were subjected to HIWN for 10 consecutive days, 1 h/day. On day 11, a day after the last exposure, the performance of the rats in open field (OF) and elevated plus maze (EPM) was evaluated. Male rats showed a higher anxiety-like response to HIWN as evidenced by: lower number of entries into the open arm of the EPM, lower number of entries into central zone of OF, excess grooming in OF and more boluses in closed arm of EPM. These results indicate that gender-related differences in anxiety in general, and in response to HIWN, in particular, has to be taken into consideration when investigating the neurobiological components and/or treatment modalities.

Age-related behavioral and ultrastructural changes in the rat amygdala.

Although the relationships between brain structure and emotions may alter across the life span, this relationship is of particular importance during aging when significant alterations in emotions may be manifested. Understanding the structural-behavioral relationship could not only provide a neurobiological basis of these changes, but could also suggest potential intervention. Since anxiety is commonly observed in aging population, we undertook this study to determine the extent of this behavioral manifestations as well as the associated ultrastructural changes in the amygdala. Rats of various age groups, adolescent, adult, and aged were tested for anxiety-like behavior and the ultrastructure/presynaptic architecture of the central nucleus of amygdala (CNA) were evaluated using transmission electron microscopy (EM). Aged rats were consistently more anxious than the other groups as evidenced by their scores in the elevated plus maze. Morphometric EM analysis of axodendritic synapses revealed that the aged rats had a lower presynaptic area as well as number of synapses, but unexpectedly a higher number of presynaptic mitochondria in CNA. Since presynaptic mitochondria are known to provide the energy for neurotransmission, it may be concluded that compensatory mechanisms are still operational during aging, and hence, may be a target for therapeutic intervention at this stage of life span.

Behavioral and neuroanatomical effects on exposure to White noise in rats.

Noise pollution is a severe public health problem as continuous exposure to even moderate noise levels between 55-65 dB can lead to various pathologies, including neurological states. In the present study, we assessed the ultrastructural alterations in selective auditory pathways of the rat brain following high intensity white noise exposure. In addition, learning, anxiety-like behavior and locomotor activity were assessed. Adult male rats were exposed to 100 dB noise, one hour daily, for 10 consecutive days. The evaluations were performed on day 11. Exposure to noise did not affect learning or the components of locomotor activity. However, it induced anxiety-like behavior as evidenced by time spent in the closed arm of elevated-plus maze. Concomitantly, ultrastructural changes in medial geniculate body, considered an integral component of classical auditory pathway, as well as in the hippocampus and basolateral amygdala, considered important structures of non-classical auditory pathway were noted. Specifically, noise resulted in neuronal apoptosis, chromatolysis, cytoplasmic organelle destruction, and glial activation in medial geniculate body and hippocampus, as well as mild alterations in amygdala. These results provide further evidence of detrimental consequences following exposure to loud noise.

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.

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.

Neuronal Porosome Complex: Secretory Machinery at the Nerve Terminal.

Neuronal porosomes are 15 nm cup-shaped lipoprotein secretory machines composed of nearly 30 proteins present at the presynaptic membrane, that have been investigated using multiple imaging modalities, such as electron microscopy, atomic force microscopy, and solution X-ray. Synaptic vesicles transiently dock and fuse at the base of the porosome cup facing the cytosol, by establishing a fusion pore for neurotransmitter release. Studies on the morphology, dynamics, isolation, composition, and reconstitution of the neuronal porosome complex provide a molecular understanding of its structure and function. In the past twenty years, a large body of evidence has accumulated on the involvement of the neuronal porosome proteins in neurotransmission and various neurological disorders. In light of these findings, this review briefly summarizes our current understanding of the neuronal porosome complex, the secretory nanomachine at the nerve terminal.

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.

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.

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.

The protective effect of myo-inositol on hippocamal cell loss and structural alterations in neurons and synapses triggered by kainic acid-induced status epilepticus.

It is known that myo-inositol pretreatment attenuates the seizure severity and several biochemical changes provoked by experimentally induced status epilepticus. However, it remains unidentified whether such properties of myo-inositol influence the structure of epileptic brain. In the present light and electron microscopic research we elucidate if pretreatment with myo-inositol has positive effect on hippocampal cell loss, and cell and synapses damage provoked by kainic acid-induced status epilepticus. Adult male Wistar rats were treated with (i) saline, (ii) saline + kainic acid, (iii) myo-inositol + kainic acid. Assessment of cell loss at 2, 14, and 30 days after treatment demonstrate cytoprotective effect of myo-inositol in CA1 and CA3 areas. It was strongly expressed in pyramidal layer of CA1, radial and oriental layers of CA3 and in less degree-in other layers of both fields. Ultrastructural alterations were described in CA1, 14 days after treatment. The structure of neurons, synapses, and porosomes are well preserved in the rats pretreated with myo-inositol in comparing with rats treated with only kainic acid.

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.