Vesicular Amine Transporter VMAT2 in the Rat Ileum: from Principal Mechanism to Clinical Applications

Besides the role of a classical neurotransmitter, histamine plays a key role in the immune/inflammatory processes in the gastrointestinal tract. Specific transport proteins pack the amine neurotransmitters into vesicles so that their release can be regulated by neural activity. Recently, two vesicular amine transporters (VMAT1 and VMAT2), essential components of monoaminergic neurons and endocrine cells were identified. In our study we investigated VMAT2 distribution in the rat distal ileum using an immunocytochemical technique. VMAT2- immunreactivity was found in neurons of the submucosal and myentericplexuses, as well as in a dense network of nerve fibers.VMAT2-containing varicosities were numerous in the circular muscle layer and around the blood vessels. Some fibers and nerve terminals were observed beneath the epithelial cells. This data provide important information about the amine-handling structures in the distal gut. Moreover, it raises the possibility for development of new pharmacotherapeutic approach to inflammatory bowel diseases

Ghrelin accelerates synapse formation and activity development in cultured cortical networks

Background: While ghrelin was initially related to appetite stimulation and growth hormone secretion, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of those processes is related to synaptic efficacy and plasticity. Previous studies have shown that ghrelin not only stimulates synapse formation in cultured cortical neurons and hippocampal slices, but also alters some of the electrophysiological properties of neurons in the hypothalamus, amygdala and other subcortical areas. However, direct evidence for ghrelin's ability to modulate the activity in cortical neurons is not available yet. In this study, we investigated the effect of acylated ghrelin on the development of the activity level and activity patterns in cortical neurons, in relation to its effect on synaptogenesis. Additionally, we quantitatively evaluated the expression of the receptor for acylated ghrelin - growth hormone secretagogue receptor-1a (GHSR-1a) during development. Results: We performed electrophysiology and immunohistochemistry on dissociated cortical cultures from neonates, treated chronically with acylated ghrelin. On average 76 � 4.6% of the cortical neurons expressed GHSR-1a. Synapse density was found to be much higher in ghrelin treated cultures than in controls across all age groups (1, 2 or 3 weeks). In all cultures (control and ghrelin treated), network activity gradually increased until it reached a maximum after approximately 3 weeks, followed by a slight decrease towards a plateau. During early developmental stages (1-2 weeks), the activity was much higher in ghrelin treated cultures and consequently, they reached the plateau value almost a week earlier than controls. Conclusions: Acylated ghrelin leads to earlier network formation and activation in cultured cortical neuronal networks, the latter being a possibly consequence of accelerated synaptogenesis

Acute stimulation of dissociated cortical neurons of newborn rats with orexin A:Effect on the network activity

Orexin A (OXA) and B are hypothalamic neu-ropeptides with recognized importance in the physiological regulation of various brain activities, including sleep/wakefulness, learning and memory, locomotion, auto-nomic control. Orexin activity is mediated by two types of receptors; OR1 binds OXA with higher affinity, while OR2 binds both ligands equally. There is a growing interest in OXA role in neurodegenerative diseases with respect to the non-motor symptoms such as sleep and attention disorders. Recent studies in Parkinson's patients found 40% lower concentration of OXA in the frontal cortex and 25% reduction in the cere-brospinal fluid. Both the number of orexinergic neurons in the hypothalamus and the levels of orexin in the cerebrospinal fluid are reduced by 72% in the end-stage in mouse model of Huntington's disease. Despite the extensive information about OXA expression and function in the nervous system of adults, data about the orexin system and its role in the functioning of developing neuronal networks are still insufficient. Neuronal cell cultures are often used as a model for brain inquiries; therefore, we undertook our study to investigate immunocyto-chemically the expression of OR1 in dissociated cortical neu-rons at various ages, and the acute effect of OXA application on the network activity of neurons, cultured on multi electrode arrays. Initially, control recordings were made after refreshing 50% (300 microliters) of the culture medium. After 2 hours, 300 microliters of medium were replaced again either with medium containing OXA (0.5-2 micromolar, n=8) or plain medium (n=5). Paired t-tests indicated a significant increase in the network activity after acute OXA application (p<0.003), but not after plain medium treatment (p>0.32), supporting the hypothesis that OXA stimulates neuronal activity in the cortex. These results indicate that potential drugs, based on OXA are attractive candidates for prevention and treatment of disor-ders associated with neuronal activity declin

Acyl Ghrelin Improves Synapse Recovery in an In Vitro Model of Postanoxic Encephalopathy

Comatose patients after cardiac arrest have a poor prognosis. Approximately half never awakes as a result of severe diffuse postanoxic encephalopathy. Several neuroprotective agents have been tested, however without significant effect. In the present study, we used cultured neuronal networks as a model system to study the general synaptic damage caused by temporary severe hypoxia and the possibility to restrict it by ghrelin treatment. Briefly, we applied hypoxia (pO2 lowered from 150 to 20 mmHg) during 6 h in 55 cultures. Three hours after restoration of normoxia, half of the cultures were treated with ghrelin for 24 h, while the other, non-supplemented, were used as a control. All cultures were processed immunocytochemically for detection of the synaptic marker synaptophysin. We observed that hypoxia led to drastic decline of the number of synapses, followed by some recovery after return to normoxia, but still below the prehypoxic level. Additionally, synaptic vulnerability was selective: large- and small-sized neurons were more susceptible to synaptic damage than the medium-sized ones. Ghrelin treatment significantly increased the synapse density, as compared with the non-treated controls or with the prehypoxic period. The effect was detected in all neuronal subtypes. In conclusion, exogenous ghrelin has a robust impact on the recovery of cortical synapses after hypoxia. It raises the possibility that ghrelin or its analogs may have a therapeutic potential for treatment of postanoxic encephalopathy

Ghrelin: a link between ageing, metabolism and neurodegenerative disorders

Along with the increase in life expectancy over the last century comes the increased risk for development of age-related disorders, including metabolic and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. These chronic disorders share two main characteristics: 1) neuronal loss in motor, sensory or cognitive systems, leading to cognitive and motor decline; and 2) a strong correlation between metabolic changes and neurodegeneration. In order to treat them, a better understanding of their complexity is required: it is necessary to interpret the neuronal damage in light of the metabolic changes, and to find the disrupted link between the peripheral organs governing energy metabolism and the CNS. This review is an attempt to present ghrelin as part of molecular regulatory interface between energy metabolism, neuroendocrine and neurodegenerative processes. Ghrelin takes part in lipid and glucose metabolism, in higher brain functions such as sleep–wake state, learning and memory consolidation; it influences mitochondrial respiration and shows neuroprotective effect. All these make ghrelin an attractive target for development of biomarkers or therapeutics for prevention or treatment of disorders, in which cell protection and recruitment of new neurons or synapses are needed

The neural code in developing cultured networks: experiments and advanced simulation models

Understanding the neural code of cultured neuronal networks may help to forward our understanding of human brain processes.The most striking property of spontaneously firing cultures is their regular bursting activity, a burst being defined as synchronized firing of groups of neurons spread throughout the entire network. The regularity of bursting may change gradually with time, typically being stable over hours (Stegenga et al. 2008). Cultured cortical networks composed of many thousands of neurons show bursting behavior starting from the end of the first week in vitro. Bursts can be characterized by both intraburst parameters (burst shape, maximum firing rate, leading and trailing edge steepness, etc.) and interburst parameters (statistics, stability of burst rates) (Van Pelt et al. 2004; Wagenaar et al. 2006). Not only the temporal burst characteristics develop with time. Also, spatial burst propagation patterns, socalled ‘’burst waves’’, change with age of the network

Regulatory effects of orexin A on neuronal networks formation and activity in vitro

Orexin A (OXA) is a neuropeptide, isolated from neurons in the hypothalamus, which regulates various brain activities, including wakefulness and higher brain functions like learning and memory. There is a growing interest in OXA’s role in neurodegenerative diseases with respect to non-motor symptoms such as sleep-, attention- and cognitive- disorders. Recent studies in Parkinson’s and Alzheimer’s patients found lower concentrations of OXA in the prefrontal cortex and cerebro-spinal fluid. It is widely assumed that deteriorated cognitive processes are related to impaired network connectivity. However, little is known about the effects of OXA on the network activity and synaptogenesis. Therefore, we investigated the development of activity in dissociated cortical neurons of rat chronically treated with 0.5 µM OXA for three weeks. Network activity was recorded with multi electrode arrays. Additionally, after one-, two- or three weeks cultures were stained immunocytochemically for detection of the presynaptic marker synaptophysin. OXA treated cultures become spontaneously active earlier, and the plateau of their activity was higher than in controls. Immunostaining revealed that the synaptic density was much higher in OXA treated cultures in all age groups. Hence, OXA has a strong stimulating effect on network formation and activity, the latter probably being a consequence of the accelerated synaptogenesis. These results indicate that drugs, based on OXA are potential candidates for prevention and treatment of disorders associated with neuronal connectivity and activity decline

Role of Ectonucleotidases in the Synapse Formation During Brain Development: Physiological and Pathological Implications

Extracellular adenine nucleotides and nucleosides, such as ATP and adenosine, are among the most recently identified and least investigated diffusible signaling factors that contribute to the structural and functional remodeling of the brain, both during embryonic and postnatal development. Their levels in the extracellular milieu are tightly controlled by various ectonucleotidases: ectonucleotide pyrophosphatase/phosphodiesterases (E-NPP), alkaline phosphatases (AP), ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eN). During central nervous system development and in adulthood all ectonucleotidases have diverse expression pattern, cell specific localization and function. Formation, maturation, and refinement of synaptic contacts are influenced by neurotransmitters and neuromodulators, and control of extracellular adenine nucleotide levels by ectonucleotidases are important for understanding the role of purinergic signaling in developing tissues and potential targets in developmental disorders such as autism