Modulation of gene expression in contextual fear conditioning in the rat.

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning.

Acetyl-l-carnitine prevents serotonin-induced behavioural sensitization and dishabituation in Hirudo medicinalis.

Several studies suggest that acetyl-l-carnitine (ALC) might influence learning processes. Along this line of investigation, we have previously shown that ALC impaired sensitization and dishabituation induced by nociceptive stimulation of the dorsal skin of the leech Hirudo medicinalis, in the behavioural paradigm of the swim induction (SI). In previous works we showed that 5HT was involved in both sensitization and dishabituation of SI acting through the second messenger cAMP. In this work, we have reported that for given doses and temporal ranges ALC was able to block sensitization and to impair dishabituation mimicked by the injection of 5-HT or 8Br-cAMP, a membrane permeable analogue of cAMP. Our results show that a single treatment with 2mM ALC was the most effective concentration to block the onset of sensitization induced by 5-HT injection and its major effects occurred 11 days after ALC treatment. 2mM ALC also blocked sensitization induced by 8Br-cAMP injection, whereas, ALC did not completely abolish dishabituation induced by 5-HT or 8Br-cAMP injection at the tested concentrations and at every time point.

Differentially expressed genes in Hirudo medicinalis ganglia after acetyl-L-carnitine treatment.

Acetyl-L-carnitine (ALC) is a naturally occurring substance that, when administered at supra-physiological concentration, is neuroprotective. It is involved in membrane stabilization and in enhancement of mitochondrial functions. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer's disease and painful neuropathies. ALC is known to improve the cognitive capability of aged animals chronically treated with the drug and, recently, it has been reported that it impairs forms of non-associative learning in the leech. In the present study the effects of ALC on gene expression have been analyzed in the leech Hirudo medicinalis. The suppression subtractive hybridisation methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the leech nervous system after ALC treatment. The method detects differentially but also little expressed transcripts of genes whose sequence or identity is still unknown. We report that a single administration of ALC is able to modulate positively the expression of genes coding for functions that reveal a lasting effect of ALC on the invertebrate, and confirm the neuroprotective and neuromodulative role of the substance. In addition an important finding is the modulation of genes of vegetal origin. This might be considered an instance of ectosymbiotic mutualism.

Lipofuscin accumulation and gene expression in different tissues of mnd mice.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage diseases characterized by neurological impairment and blindness. NCLs are almost always due to single mutations in different genes (CLN1-CLN8). Ubiquitous accumulation of undigested material and of a hydrophobic inner mitochondrial membrane protein, the subunit c of mitochondrial ATP synthase, has been described. Although protein mutation(s) in the endoplasmic reticulum-lysosomes axis can modify the trafficking and the recycling of different molecules, one of the upstream targets in these diseases may be represented by the balance of gene expression. To understand if and how neurons modify the levels of important genes during the first phases of the disease, it is important to characterize the mechanisms of neurodegeneration. Due to the impossibility of performing this analysis in humans, alternative models of investigation are required. In this study, a mouse model of human NCL8, the mnd mouse has been employed. The mnd mice recapitulate many clinical and histopathological features described in NCL8 patients. In this study, we found an altered expression of different genes in both central and peripheral organs associated with lipopigment accumulation. This is a preliminary approach, which could also be of interest in providing new diagnostic tools for NCLs.

Molecular mechanisms of short-term habituation in the leech Hirudo medicinalis.

Although habituation is ubiquitous in the animal kingdom, its underlying mechanisms remain poorly understood. In this study, we began to explore the molecular cascades underlying short-term habituation in the leech Hirudo medicinalis. In H. medicinalis, a training paradigm, consisting of low-frequency repetitive electrical stimulation of the skin, produces a gradual increase in the latency to swim that spontaneously recovers within 20-30 min. As first step in determining the molecular pathways in short-term habituation, we examined the role of Ca(2+). Both Ca(2+) influx through voltage-gated channels and Ca(2+) release from intracellular stores were found to contribute to short-term habituation. The analysis of the downstream targets of elevated cytosolic Ca(2+) revealed that the activation of the phosholipase A(2) was required for the induction of short-term habituation. Finally, we reported that the recruitment of arachidonic acid metabolites, generated by the 5-lipoxygenase pathway, was also necessary for the induction of swim induction habituation. These results provide the framework for a comprehensive characterization of the molecular underpinnings of habituation. This outcome will allow us to compare the mechanisms of habituation with those underlying other forms of nonassociative learning in the leech, such as sensitization and dishabituation, and, more in general, with those governing habituation in different vertebrate and invertebrate model systems.

Prolonged hypotensive and bradycardic effects of passive mandibular extension: evidence in normal volunteers.

Various procedures involving stimulations of facial regions are known to induce so-called trigemino-cardiac reflexes that entail a decrease of heart rate and blood pressure. We here report the effects of a specific stimulation that consists in a submaximal passive mandibular extension obtained by means of a dilatator applied for 10 minutes between the upper and lower incisor teeth, associated with partial active masticatory movements. Blood pressure and heart rate were determined in 18 young normal volunteers by Omron M4, before (20 minutes), during (10 minutes) and after mandibular extension (80 minutes) and under control conditions (same overall duration without stimulation). While control values remained stable, mandibular extension was followed by a progressive decline of both blood pressure (up to about 12/11 mmHg) and heart rate (up to about 13 bpm), statistically confirmed by ANOVA both on absolute values and on changes from basal values. The decline of systolic blood pressure and heart rate significantly correlated with basal values. The present findings indicate that submaximal opening of the mouth, associated to partial masticatory movements, induces a prolonged reduction of blood pressure and heart rate in normotensive volunteers.

Modulation of myelin basic protein gene expression by acetyl-L-carnitine.

Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring molecule which plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions, demonstrating efficacy and high tolerability in the treatment of neuropathies of various etiologies. ALC is a molecule of considerable interest for its clinical application in various neural disorders, although little is known regarding its effects on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after chronic ALC treatments. We provided evidence for a downregulation of the expression of all of the isoforms of myelin basic protein gene following prolonged ALC treatment, indicating a possible role in the modulation of myelin basic protein turnover, stabilizing and maintaining myelin integrity.

Cytoprotective effect of acetyl-L-carnitine evidenced by analysis of gene expression in the rat brain.

Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring substance that when administered at supraphysiological concentrations is neuroprotective. ALC plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions. ALC has demonstrated efficacy and high tolerability in the treatment of neuropathies of various etiologies, and it is a molecule of considerable interest for its clinical application in various neural disorders, such as Alzheimer's disease and painful neuropathies, although little is known regarding the effects of ALC on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after a chronic ALC treatment. In the present paper, we provide evidences for the up-regulation of the expression of prostaglandin D(2) synthase, brain-specific Na(+)-dependent inorganic phosphate transporter, and cytochrome b oxidase, bc1 complex induced in the rat brain by ALC. On the contrary, ALC treatment down-regulates the expression of the gene of ferritin-H. Altogether, these results suggest that ALC might play a cytoprotective role against various brain stressors.

Up-regulation of kinesin light-chain 1 gene expression by acetyl-L-carnitine: therapeutic possibility in Alzheimer's disease.

We investigated the effects of acetyl-l-carnitine on gene expression by means of the suppression subtractive hybridization method. The approach gives the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with acetyl-l-carnitine for 21 days. We observed that acetyl-l-carnitine increases the light-chain subunit of kinesin-1 gene expression. Recent evidences reported a link between kinesin-1 light-chain and Alzheimer's disease. Pathological hallmarks of Alzheimer's disease are potentially linked to alterations of the axonal compartments. Amyloid-beta peptide is a principal component of senile plaques and is considered to be central in the pathogenesis of the disease. The fast anterograde axonal transport of amyloid-beta peptide is mediated by direct binding to the light-chain subunit of kinesin-1. In this scenario, our results are of relevant importance for possible therapeutic intervention, suggesting a pathway for the treatment of Alzheimer's disease.

In the rat brain acetyl-L-carnitine treatment modulates the expression of genes involved in neuronal ceroid lipofuscinosis.

Acetyl-L-carnitine (ALC) is a naturally occurring substance that, when administered at supraphysiological concentration, is neuroprotective. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer's disease and painful neuropathies. Suppression subtractive hybridization methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after ALC treatment. The method generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes which are regulated by ALC. We report that ALC treatment: (1) upregulates lysosomal H(+)/ATPase gene expression and (2) downregulates myelin basic protein gene expression. The expression of these genes is altered in some forms of neuronal ceroid lipofuscinosis (NCL) pathologies. In this case, ALC might rebalance the disorders underlying NCL disease represented by a disturbance in pH homeostasis affecting the acidification of vesicles transported to lysosomal compartment for degradation. This study provides evidence that ALC controls genes involved in these serious neurological pathologies and provides insights into the ways in which ALC might exert its therapeutic benefits.

Stochastic models for the in silico simulation of synaptic processes.

BACKGROUND: Research in life sciences is benefiting from a large availability of formal description techniques and analysis methodologies. These allow both the phenomena investigated to be precisely modeled and virtual experiments to be performed in silico. Such experiments may result in easier, faster, and satisfying approximations of their in vitro/vivo counterparts. A promising approach is represented by the study of biological phenomena as a collection of interactive entities through process calculi equipped with stochastic semantics. These exploit formal grounds developed in the theory of concurrency in computer science, account for the not continuous, nor discrete, nature of many phenomena, enjoy nice compositional properties and allow for simulations that have been demonstrated to be coherent with data in literature. RESULTS: Motivated by the need to address some aspects of the functioning of neural synapses, we have developed one such model for synaptic processes in the calyx of Held, which is a glutamatergic synapse in the auditory pathway of the mammalia. We have developed such a stochastic model starting from existing kinetic models based on ODEs of some sub-components of the synapse, integrating other data from literature and making some assumptions about non-fully understood processes. Experiments have confirmed the coherence of our model with known biological data, also validating the assumptions made. Our model overcomes some limitations of the kinetic ones and, to our knowledge, represents the first model of synaptic processes based on process calculi. The compositionality of the approach has permitted us to independently focus on tuning the models of the pre- and post- synaptic traits, and then to naturally connect them, by dealing with "interface" issues. Furthermore, we have improved the expressiveness of the model, e.g. by embedding easy control of element concentration time courses. Sensitivity analysis over several parameters of the model has provided results that may help clarify the dynamics of synaptic transmission, while experiments with the model of the complete synapse seem worth explaining short-term plasticity mechanisms. CONCLUSIONS: Specific presynaptic and postsynaptic mechanisms can be further analysed under various conditions, for instance by studying the presynaptic behaviour under repeated activations. The level of details of the description can be refined, for instance by further specifying the neurotransmitter generation and release steps. Taking advantage of the compositionality of the approach, an enhanced model could then be composed with other neural models, designed within the same framework, in order to obtain a more detailed and comprehensive model. In the long term, we are interested, in particular, in addressing models of synaptic plasticity, i.e. activity dependent mechanisms, which are the bases of memory and learning processes. More on the computer science side, we plan to follow some directions to improve the underlying computational model and the linguistic primitives it provides as suggested by the experiments carried out, e.g. by introducing a suitable notion of (spatial) locality.

Inhibition of Na+/K+ ATPase potentiates synaptic transmission in tactile sensory neurons of the leech.

Increasing evidence indicates that modulation of Na(+)/K(+) ATPase activity is involved in forms of neuronal and synaptic plasticity. In tactile (T) neurons of the leech Hirudo medicinalis, Na(+)/K(+) ATPase is the main determinant of the afterhyperpolarization (AHP), which characterizes the firing of these mechanosensory neurons. Previously, it has been reported that cAMP (3',5'-cyclic adenosine monophosphate), which mediates the effects of serotonin (5HT) in some forms of learning in the leech, negatively modulates Na(+)/K(+) ATPase activity, thereby reducing the AHP amplitude in T neurons. Here, we show that a transient inhibition of Na(+)/K(+) ATPase can affect the synaptic connection between two ipsilateral T neurons. Bath application of 10 nm dihydroouabain (DHO), an ouabain analogue, causes an increase in the amplitude of the synaptic potential (SP) recorded in the postsynaptic element when a test stimulus is applied in the presynaptic neuron. Iontophoretic injection of cAMP into the presynaptic T neuron also produces an increase of SP. Simulations carried out by using a computational model of the T neuron suggest that a reduction of the pump rate and a consequent depression of the AHP might facilitate the conduction of action potentials to the synaptic terminals. Moreover, nearly intact leeches injected with 10 nm DHO respond with a swimming episode more quickly to an electrical stimulation, which selectively activates T neurons exhibiting sensitization of swimming induction. Collectively, our results show that inhibition of Na(+)/K(+) ATPase is critical for short-term plasticity.

Acetyl-L-carnitine up-regulates expression of voltage-dependent anion channel in the rat brain.

Acetyl-L-carnitine (ALC) exerts unique neuroprotective, neuromodulatory, and neurotrophic properties, which play an important role in counteracting various pathological processes, and have antioxidative properties, protecting cells against lipid peroxidation. In this study, suppression subtractive hybridization (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with ALC. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated after ALC treatment. In the present paper, we report the identification of the gene of mitochondrial voltage-dependent anion channel (VDAC) protein which is positively modulated by the ALC treatment. VDAC is a small pore-forming protein of the mitochondrial outer membrane. It represents an interesting tool for Ca(2+) homeostasis, and it plays a central role in apoptosis. In addition, VDAC seems to have a relevant role in the synaptic plasticity.

Role for calcium signaling and arachidonic acid metabolites in the activity-dependent increase of AHP amplitude in leech T sensory neurons.

Previous studies have revealed a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) neurons of the leech Hirudo medicinalis. The firing of T cells is characterized by an AHP, which is mainly due to the activity of the Na+/K+ ATPase. Low-frequency repetitive stimulation of T neurons leads to a robust increment of the AHP amplitude, which is correlated with a synaptic depression between T neuron and follower cells. In the present study, we explored the molecular cascades underlying the AHP increase. We tested the hypothesis that this activity-dependent phenomenon was triggered by calcium influx during neural activity by applying blockers of voltage-dependent Ca2+ channels. We report that AHP increase requires calcium influx that, in turn, induces release of calcium from intracellular stores so sustaining the enhancement of AHP. An elevation of the intracellular calcium can activate the cytosolic isoforms of the phosholipase A2 (PLA2). Therefore we analyzed the role of PLA2 in the increase of the AHP, and we provide evidence that not only PLA2 but also the recruitment of arachidonic acid metabolites generated by the 5-lipoxygenase pathway are necessary for the induction of AHP increase. These data indicate that a sophisticated cascade of intracellular signals links the repetitive discharge of T neurons to the activation of molecular pathways, which finally may alter the activity of critical enzymes such as the Na+/K+ ATPase, that sustains the generation of the AHP and its increase during repetitive stimulation. These results also suggest the potential importance of the poorly studied 5-lipoxygenase pathway in forms of neuronal plasticity.

Computational model of touch sensory cells (T Cells) of the leech: role of the afterhyperpolarization (AHP) in activity-dependent conduction failure.

Bursts of spikes in T cells produce an AHP, which results from activation of a Na+/K+ pump and a Ca2+-dependent K+ current. Activity-dependent increases in the AHP are believed to induce conduction block of spikes in several regions of the neuron, which in turn, may decrease presynaptic invasion of spikes and thereby decrease transmitter release. To explore this possibility, we used the neurosimulator SNNAP to develop a multi-compartmental model of the T cell. The model incorporated empirical data that describe the geometry of the cell and activity-dependent changes of the AHP. Simulations indicated that at some branching points, activity-dependent increases of the AHP reduced the number of spikes transmitted from the minor receptive fields to the soma and beyond. More importantly, simulations also suggest that the AHP could modulate, under some circumstances, transmission from the soma to the synaptic terminals, suggesting that the AHP can regulate spike conduction within the presynaptic arborizations of the cell and could in principle contribute to the synaptic depression that is correlated with increases in the AHP.

Identification of differentially expressed genes induced in the rat brain by acetyl-L-carnitine as evidenced by suppression subtractive hybridisation.

Acetyl-L-carnitine (ALC) is a molecule widely present in the central nervous system (CNS) formed by the reversible acetylation of carnitine. It acts by stimulating energy metabolism. Reported neurobiological effects of this substance include modulation of brain energy and phospholipid metabolism; cellular macromolecules (including neurotrophic factors and neurohormones); synaptic transmission of multiple neurotransmitters. ALC is of considerable interest for its clinical application in Alzheimer's disease and in the treatment of painful neuropathies. There are experimental data that it affects attention and antagonizes deterioration of ability to learn, improving long-term memory. Moreover, ALC influences nonassociative learning of sensitization type in Hirudo medicinalis. These findings are suggesting that ALC might exert its effects by means of new protein synthesis. ALC or saline solution was injected intraperitoneally each day for 21 days in rats. Poly(A)+ RNAs were isolated from control and treated rat brain. Suppression subtractive hybridisation (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatments. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance, and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated or switched off/on after ALC treatment. We identified two modulated genes, the isoform gamma of 14-3-3 protein and a precursor of ATP synthase lipid-binding protein, and one gene switched on by the treatment, the heat shock protein hsp72.

Sensitization and dishabituation of swim induction in the leech Hirudo medicinalis: role of serotonin and cyclic AMP.

In this paper the role of serotonin (5HT) and cyclic AMP (cAMP) in sensitization and dishabituation of swim induction (SI) has been investigated in the leech Hirudo medicinalis. Electrical stimulation of the body wall evokes swimming activity with a constant latency. In animals with a disconnection between head ganglion and segmental ganglia, repetitive stimulation induces habituation of swimming whereas brushing on the dorsal skin provokes sensitization of a naïve response or dishabituation of a previously habituated response. Our findings indicate that 5HT is the neurotransmitter underlying both sensitization and dishabituation of SI. Injection of the 5HT receptor blocking agent methysergide impaires the onset of sensitization and dishabituation induced by brushing. Moreover, injection of 5HT mimics these forms of nonassociative learning, whereas injection of dopamine does not. Finally, the effects of 5HT are mediated by cAMP: (1) after injections of specific adenylate cyclase inhibitors such as MDL 12.330A or SQ22536, brushing becomes ineffective in facilitating the SI in either non-habituated or habituated animals. (2) 8Br-cAMP application mimics both sensitization and dishabituation of SI.

Activity-dependent increase of the AHP amplitude in T sensory neurons of the leech.

We identified a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) sensory neurons of the leech Hirudo medicinalis. Repetitive intracellular stimulation with 30 trains of depolarizing impulses at 15-s inter-stimulus interval (ISI) led to an increase of the AHP amplitude (~60% of the control). The enhancement of AHP lasted for >/=15 min. The AHP increase was also elicited when a T neuron was activated by repetitive stimulation of its receptive field. The ISI was a critical parameter for the induction and maintenance of AHP enhancement. ISI duration had to fit within a time window with the upper limit of 20 s to make the training effective to induce an enhancement of the AHP amplitude. After recovery from potentiation, AHP amplitude could be enhanced once again by delivering another training session. The increase of AHP amplitude persisted in high Mg(2+) saline, suggesting an intrinsic cellular mechanism for its induction. Previous investigations reported that AHP of leech T neurons was mainly due to the activity of the Na(+)/K(+) ATPase and to a Ca(2+)-dependent K(+) current (I(K/Ca)). In addition, it has been demonstrated that serotonin (5HT) reduces AHP amplitude through the inhibition of the Na(+)/K(+) ATPase. By blocking the I(K/Ca) with pharmacological agents, such as cadmium and apamin, we still observed an increase of the AHP amplitude after repetitive stimulation, whereas 5HT application completely inhibited the AHP increment. These data indicate that the Na(+)/K(+) ATPase is involved in the induction and maintenance of the AHP increase after repetitive stimulation. Moreover, the AHP increase was affected by the level of serotonin in the CNS. Finally, the increase of the AHP amplitude produced a lasting depression of the synaptic connection between two T neurons, suggesting that this activity-dependent phenomenon might be involved in short-term plasticity associated with learning processes.

Time-dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat.

The effects of contextual fear-learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one-trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear-learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short-term (STP) and long-term potentiation (LTP) when compared to control "naïve" subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long-term but not short-term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from "shock-only" rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings - no conditioned freezing during retention testing). In "exploration" preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired-pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animal's prior experience.