Assessing autophagy in sciatic nerves of a rat model that develops inflammatory autoimmune peripheral neuropathies

The rat sciatic nerve has attracted widespread attention as an excellent model system for studying autophagy alterations in peripheral neuropathies. In our laboratory, we have developed an original rat model, which we used currently in routine novel drug screening and to evaluate treatment strategies for chronic inflammatory demyelinating polyneuropathy (CIDP) and other closely related diseases. Lewis rats injected with the S-palmitoylated P0(180-199) peptide develop a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology and several typical features of CIDP. We have set up a series of techniques that led us to examine the failures of autophagy pathways in the sciatic nerve of these model rats and to follow the possible improvement of these defects after treatment. Based on these newly introduced methods, a novel area of investigation is now open and will allow us to more thoroughly examine important features of certain autophagy pathways occurring in sciatic nerves

Hippocampal amino acid concentrations after raphe and/or septal cell suspension grafts in rats with fimbria-fornix lesions

Two weeks after infracallosal electrolytic fimbria-fornix lesions, Long-Evans female rats received intrahippocampal suspension grafts of either fetal septal or mesencephalic raphe tissue, or a mixture of both. Ten months after lesion surgery, the concentrations of alanine, aspartate, GABA, glutamate, glutamine, glycine, serine and taurine were determined in a dorsal, a "middle" and a ventral region of the hippocampus. We found neither the lesions nor the grafts to have significantly modified the concentration of these amino acids which, in all groups, presented a regional heterogeneity in their hippocampal distribution. GABA, glutamate and glutamine were highest in the ventral hippocampus, whereas the other amino acids were highest in the dorsal region. Our results (i) show that fimbria-fornix lesions do not result in lasting effects on hippocampal concentrations of the assessed amino acids, (ii) confirm the regional heterogeneity in the distribution of these amino acids in the hippocampus and (iii) demonstrate that cell suspension grafts of fetal septal or mesencephalic raphe tissue, as well as grafts of a mixture of both of these tissues, do not exert a non-specific effect on either of the amino acid concentrations measured. These data complete those of the preceeding paper [Kiss et al. (1990) Neuroscience 36, 61-72] concerning the effects of the same grafts on hippocampal cholinergic, serotonergic and noradrenergic markers, as well as on several behavioural variables

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones

Effects of MDL 73005 on water-maze performances and locomotor activity in scopolamine-treated rats

The stimulation of 5-HT1A receptors in the raphe or their blockade in the hippocampus can reduce cognitive deficits induced by blockade of muscarinic receptors in the hippocampus. We investigated the effects of MDL 73005 (8-[2-(2,3-dihydro-1,4-benzodioxin-2-ylmethylamino) ethyl]-8-azaspiro[4,5] decane-7,9-dione methyl sulphonate), an agonist at 5-HT1A somatodendritic autoreceptors and an antagonist at postsynaptic 5-HT1A receptors in rats treated systemically with scopolamine. Spatial memory was assessed in a water maze using protocols testing reference and working memory. Home cage locomotor activity was also determined. Working memory and locomotor activity were evaluated before and after para-chlorophenylalanine (pCPA) treatment. Scopolamine produced a weak impairment of reference memory at 0.5 mg/kg, and a more pronounced impairment of working memory at 0.25 and 0.5 mg/kg. MDL 73005 alone (2 mg/kg, i.p.) had no effect, but prevented the memory impairments induced by 0.25 mg/kg of scopolamine. Scopolamine induced hyperlocomotion. MDL 73005 alone did not affect locomotor activity, but exacerbated the hyperlocomotion induced by 0.5 mg/kg of scopolamine. pCPA did not abolish the effects of MDL 73005, suggesting that these effects were not due to an action at presynaptic receptors, or even that they involved receptors other than serotonergic ones (e.g., D2). In conclusion, MDL 73005 is able to antagonise moderate spatial memory dysfunctions induced by systemic muscarinic blockade

Combined 192 IgG-saporin and 5,7-dihydroxytryptamine lesions in the male rat brain

In a previous experiment [Eur J Neurosci 12 (2000) 79], combined intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT; 150 microg) and 192 IgG-saporin (2 microg) in female rats produced working memory impairments, which neither single lesion induced. In the present experiment, we report on an identical approach in male rats. Behavioral variables were locomotor activity, T-maze alternation, beam-walking, Morris water-maze (working and reference memory) and radial-maze performances. 192 IgG-saporin reduced cholinergic markers in the frontoparietal cortex and the hippocampus. 5,7-DHT lesions reduced serotonergic markers in the cortex, hippocampus and striatum. Cholinergic lesions induced motor deficits, hyperactivity and reduced T-maze alternation, but had no other effect. Serotonergic lesions only produced hyperactivity and reduced T-maze alternation. Beside the deficits due to cholinergic lesions, rats with combined lesions also showed impaired radial-maze performances. We confirm that 192 IgG-saporin and 5,7-DHT injections can be combined to produce concomitant damage to cholinergic and serotonergic neurons in the brain. In female rats, this technique enabled to show that interactions between serotonergic and basal forebrain cholinergic mechanisms play an important role in cognitive functions. The results of the present experiment in male rats are not as clear-cut, although they are not in obvious contradiction with our previous results in females

Involvement of the basal cholinergic forebrain in the mediation of general (Propofol) anesthesia:

BACKGROUND: Recent studies have pointed out the involvement of the basal forebrain gamma-aminobutyric acid-mediated system in mediating the effects of general anesthesia. In this study, the authors asked whether the basal forebrain cholinergic system is also involved in mediating the effects of general anesthetics such as propofol. METHODS: Cholinergic lesions were produced by administration of the selective immunotoxin 192 immunoglobulin G-saporin into the lateral ventricles, the medial septum, or the nucleus basalis magnocellularis. The anesthetic potency of propofol was determined using an anesthetic score with a crossover counterbalanced design. Animals were given intraperitoneal propofol (25 or 50 mg/kg) repeatedly every 15 min to set up a subanesthetic (low-dose) or anesthetic (high-dose) state. The anesthetic score was assessed for each cumulative dose. Control of the cholinergic depletion was performed using histochemical acetylcholinesterase staining on brain slices. RESULTS: A shift from a subanesthetic state to an anesthetic state was observed mainly in the rats with the immunotoxin injected into the lateral ventricles or the medial septum and vertical diagonal band of Broca, compared with controls. In those rats, the density of acetylcholinesterase reaction products was normal in the striatum and the thalamus, but reduced in the cortex and the hippocampus. CONCLUSION: The anesthetic potency of propofol was increased in all rats with hippocampal lesions, whatever the injection sites, compared with controls. These results demonstrate that a cholinergic dysfunction in the basal forebrain potentiates the anesthetic effects of propofol

The double-H maze test, a novel, simple, water-escape memory task: Acquisition, recall of recent and remote memory, and effects of systemic muscarinic or NMDA receptor blockade during training

To explore spatial cognition in rodents, research uses maze tasks, which differ in complexity, number of goals and pathways, behavioural flexibility, memory duration, but also in the experimenter's control over the strategy developed to reach a goal (e.g., allocentric vs. egocentric). This study aimed at validating a novel spatial memory test: the double-H maze test. The transparent device made of an alley with two opposite arms at each extremity and two in its centre is flooded. An escape platform is submerged in one arm. For experiments 1-3, rats were released in unpredictable sequences from one of both central arms to favour an allocentric approach of the task. Experiment 1 (3 trials/day over 6 days) demonstrated classical learning curves and evidence for recent and nondegraded remote memory performance. Experiment 2 (2 days, 3 trials/day) showed a dose-dependent alteration of task acquisition/consolidation by muscarinic or NMDA receptor blockade; these drug effects vanished with sustained training (experiment 3; 4 days, 3 trials/day). Experiment 4 oriented rats towards a procedural (egocentric) approach of the task. Memory was tested in a misleading probe trial. Most rats immediately switched from response learning-based to place learning-based behaviour, but only when their initial view on environmental cues markedly differed between training and probe trials. Because this simple task enables the formation of a relatively stable memory trace, it could be particularly adapted to study consolidation processes at a system level or/and the interplay between procedural and declarative-like memory systems

The Effects of Intrahippocampal Grafts, Training, and Postoperative Housing on Behavioral Recovery After Septohippocampal Damage in the Rat

This study examined whether the expression of behavioral effects of grafts rich in cholinergic neurons placed into the hippocampus of rats with septohippocampal damage may be modulated by postoperative housing or training conditions. Among 91 Long-Evans female rats, 61 sustained a bilateral aspirative lesion of the fimbria-fornix fibers and all overlying tissue, while 30 were given sham operations. Ten days after surgery, fetal septal suspension grafts were performed in the hippocampus of half the lesioned rats. Two days later, all rats were randomly assigned to one of three housing or training conditions: standard, standard with daily training, and enriched. Two and 5 months later, the rats were tested for learning using a Hebb-Williams maze. At both these delays, performance was clearly impaired in lesioned rats and was found to be ameliorated by grafts only in rats which had received daily training. Cresyl violet staining and acetylcholinesterase histochemistry showed that, irrespective of the housing or training conditions, all grafts had survived and provided the denervated hippocampus with a substantial cholinergic reinnervation. Our results suggest that the beneficial behavioral effects of intrahippocampal suspension grafts of septal cells may depend on the postsurgical training or handling conditions of the graft recipients. This result might be of importance for interpreting some behavioral effects of grafts, since in most studies in which grafts were found to induce beneficial behavioral effects (especially on learning capacity), these effects were generally observed at the end of a rather long testing period. Moreover, the present findings show that this delay, before graft function is expressed, might be linked not only to the time needed by grafts to establish a functional reinnervation in the host brain, but also to the training and/or handling conditions of the graft recipient

5,7-dihydroxytryptamine lesions enhance and serotonergic grafts normalize the evoked overflow of acetylcholine in rat hippocampal slices: Serotonin depletion facilitates acetylcholine release

Adult rats were subjected to intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT; 150 micro g) and, 15 days later, to intrahippocampal grafts of fetal raphe cell suspensions. About 11 months later, we assessed baseline and electrically evoked release of tritium ([3H]) in hippocampal slices, preloaded with tritiated ([3H])choline or [3H]serotonin (5-HT), in the presence or absence of the 5-HT1B receptor agonist CP-93,129 and the 5-HT receptor antagonist methiothepine. HPLC determinations of monoamine concentrations were also performed. The lesions reduced the concentration of 5-HT (-90%) and the accumulation (-80%) as well as the evoked release (-90%) of [3H]5-HT. They also decreased the inhibitory effects of CP-93,129 on the evoked release of [3H]5-HT. Most interestingly, they facilitated the evoked release of [3H]acetylcholine (+20%). In slices from rats subjected to lesions and grafts, the responsiveness of the serotonergic autoreceptors (presumably located on the terminals of the grafted neurons) and the release of acetylcholine were close to normal. These results confirm that grafts rich in serotonergic neurons may partially compensate for the dramatic effects of 5,7-DHT lesions on serotonergic hippocampal functions. The lesion-induced reduction of the 5-HT1B autoreceptor-mediated inhibition of evoked 5-HT release may be an adaptation enhancing serotonergic transmission in the (few) remaining terminals. The facilitated release of acetylcholine is probably caused by a reduced serotonergic tone on the inhibitory 5-HT1B heteroreceptors of the cholinergic terminals. When related to data in the literature, this facilitation may be of particular interest in terms of transmitter-based strategies developed to tackle cognitive symptoms related to neurodegenerative diseases

Autophagy in neuroinflammatory diseases.

Autophagy is a metabolically-central process that is crucial in diverse areas of cell physiology. It ensures a fair balance between life and death molecular and cellular flows, and any disruption in this vital intracellular pathway can have consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases. Recent pharmacological studies have shown evidence that small molecules and peptides able to activate or inhibit autophagy might be valuable therapeutic agents by down- or up-regulating excessive or defective autophagy, or to modulate normal autophagy to allow other drugs to repair some cell alteration or destroy some cell subsets (e.g. in the case of cancer concurrent treatments). Here, we provide an overview of neuronal autophagy and of its potential implication in some inflammatory diseases of central and peripheral nervous systems. Based on our own studies centred on a peptide called P140 that targets autophagy, we highlight the validity of autophagy processes, and in particular of chaperone-mediated autophagy, as a particularly pertinent pathway for developing novel selective therapeutic approaches for treating some neuronal diseases. Our findings with the P140 peptide support a direct cross-talk between autophagy and certain central and peripheral neuronal diseases. They also illustrate the fact that autophagy alterations are not evenly distributed across all organs and tissues of the same individual, and can evolve in different stages along the disease course.journal articlereview2017 Aug2017 05 29importe