Catherine Vidal

Nicotinic and muscarinic modulations of excitatory synaptic transmission in the rat prefrontal cortexin vitro

The importance of the cholinergic innervation of the neocortex in cognitive functions has been shown in a number of clinical and animal studies. Until recently, attempts to study the mode of action of acetylcholine in the neocortex have concentrated on muscarinic effects, whereas cholinergic actions mediated by nicotinic receptors have been difficult to demonstrate. The present work was undertaken to study the mechanism of action of nicotinic agents on cortical neurons and compare it to muscarinic effects by means of intracellular recordings in a slice preparation. The study was performed in the prelimbic area of the rat prefrontal cortex, a cortical region particularly involved in cognitive processes. Recordings were made from pyramidal cells located in layers II/III and synaptic potentials were evoked by stimulation of superficial cortical layers. Iontophoretic applications of nicotinic agonists (nicotine, dimethylphenylpiperazinium, cytisine) increased the amplitude of the monosynaptic excitatory postsynaptic potential mediated by non-N-methyl-D-aspartate glutamate receptors in 14% (22/159) of cells. This effect was abolished by the selective nicotinic blocker, neuronal bungarotoxin (IC50 = 0.6-0.7 microM) and by dihydro-beta-erythroidine (IC50 = 20-30 microM), whereas hexamethonium, mecamylamine, curare and alpha-bungarotoxin were ineffective. The nicotinic agonists did not change resting membrane potential, input resistance or current-voltage relationship. They also did not affect the depolarizations produced by glutamate applied by iontophoresis in the somatic or dendritic area. In contrast, the muscarinic agonists (muscarine, acetyl-beta-methylcholine) decreased the amplitude of the excitatory postsynaptic potential in 100% of the neurons tested. Atropine was more effective (IC50 = 0.08 microM) than pirenzepine (IC50 = 2 microM) to antagonize the muscarinic action. These effects were observed in the absence of any direct postsynaptic change in membrane potential or input resistance, provided that the site of the iontophoretic application was more than 100 microM distant from the soma. The muscarinic agonists did not influence the actions of iontophoretically applied glutamate. These results suggest that nicotinic and muscarinic agonists modulate excitatory synaptic transmission mediated at dendritic sites by non-N-methyl-D-aspartate glutamate receptors, possibly through a presynaptic action. Thus ascending cholinergic systems may take part in information processing in the prefrontal cortex through the control of ongoing excitation to pyramidal cells.

Working memory, response selection, and effortful processing in rats with medial prefrontal lesions.

This study examined the effects of lesions of the prelimbic area of the rat prefrontal cortex on acquisition and retention of nonmatching (NMTS) and matching-to-sample (MTS) tasks. Both tasks involved a reference and a working memory component, but only working memory was impaired by the lesions. A comparison of the 2 tasks revealed quantitatively similar deficits in postoperatively trained rats. In preoperatively trained rats, however, the deficits were more important in the MTS task than in the NMTS task. In addition, an effect of interference between successive trials was observed in the NMTS task but not in the MTS task. Perseverative tendencies were observed in the MTS task only. These results suggest that prefrontal lesions induce working memory deficits as a result of poor temporal encoding and increased susceptibility to interference and impair effortful processing, such as that engaged in response selection mechanisms.

In Vitro and In Vivo Neurotoxicity of Prion Protein Oligomers

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.

Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system

Neuronal nicotinic ACh receptors (nAChRs) are present in the rat medial habenula (MHB) and interpeduncular nucleus (IPN), two brain regions connected through the fasciculus retroflexus (FR). The goal of the present study was to compare the electrophysiological and pharmacological characteristics of nAChRs located at pre- and postsynaptic sites within the MHB-IPN system. nAChRs located on the soma of IPN neurons were studied using patch-clamp techniques and a preparation of acutely isolated neurons. Whole-cell currents evoked by Ach and nicotine showed an intense rectification at positive membrane potentials. nAChR channels were relatively nonselective for cations, had a unitary conductance of 35 pS, and were activated by several nicotinic agonists with the following rank order: cytisine greater than ACh greater than nicotine greater than dimethylphenylpiperazinium (DMPP). They were blocked by mecamylamine, hexamethonium, curare, and dihydro-beta-erythroidine (DHBE), but were insensitive to alpha- bungarotoxin and neuronal bungarotoxin. In contrast, nAChRs recorded on the soma of MHB neurons under equivalent experimental conditions exhibited different characteristics for single-channel conductance and agonist and antagonist sensitivity. The pharmacological properties of presynaptic nAChRs in the IPN were analyzed in a rat brain slice preparation. Stimulation of the FR produced a presynaptic afferent volley recorded in the rostral subnucleus of the IPN. Nicotinic agonists decreased the amplitude of the afferent volley with different efficacies: nicotine greater than cytisine greater than ACh greater than DMPP. The action of nicotine was insensitive to alpha-bungarotoxin and to neuronal bungarotoxin, but was blocked by mecamylamine, hexamethonium, curare, and DHBE, with IC50 values different from those reported for IPN postsynaptic nAChRs. This study thus demonstrates the functional diversity of nAChRs in the rat CNS.

Nicotinic and muscarinic receptors in the rat prefrontal cortex: Differential roles in working memory, response selection and effortful processing

The aim of the present study was to evaluate the effects of cholinergic receptor blockade in the rat prefrontal cortex on cognitive processes. The nicotinic antagonists neuronal bungarotoxin and dihydro-β-erythroidine and the muscarinic antagonist scopolamine were injected into the prelimbic area of the prefrontal cortex. Their behavioural effects were assessed in a T-maze to test reference memory (visual discrimination task) and working memory in delayed matching (MTS) and non-matching to sample (NMTS) tasks. Neuronal bungarotoxin produced a significant decrease in working memory performance in the MTS task but not in the NMTS task. In contrast, scopolamine impaired working memory in both MTS and NMTS tasks. Reference memory was not altered by any of the cholinergic antagonists. These results demonstrate a differential role of nicotinic and muscarinic receptors in the rat prefrontal cortex. Nicotinic transmission appears to be important in delayed response tasks requiring effortful processing for response selection, while the muscarinic system is involved in general working memory processes.

Nicotinic receptors in the brain. Molecular biology, function, and therapeutics.

Although the psychological and physiological effects of nicotine have long suggested that nicotine exerts specific actions in the brain, the identification of neuronal nicotinic receptors (nAChRs) only began in the past few years with the development of molecular genetics. It is now clear that neuronal nAChRs form a family of highly heterogenous receptor subtypes, as evidenced by the number of genes encoding nAChR subunits, the diversity of immunopurified receptor proteins, and the multiple functional types of ligand-gated ion channels. Neuronal nAChRs have discrete localizations within the brain, and are involved in modulating neuronal firing and transmitter release. Cumulative evidence from animal and human studies indicates that nicotinic systems play a major role in higher cognitive functions and dysfunctions. In particular, the loss of cortical nAChRs is a neurochemical hallmark of Alzheimer (AD) and Parkinson (PD) diseases. In addition, nicotine improves memory and attention in AD and PD. Our recent studies using electrophysiological biochemical and behavioral approaches suggest that the prefrontal cortex is a major target site for the cognitive actions of nicotine.

Pharmacological profile of nicotinic acetylcholine receptors in the rat prefrontal cortex: an electrophysiological study in a slice preparation.

The specificity of nicotinic receptors in the neocortex has been questioned previously because: (i) electrophysiological responses to nicotine could not be blocked by nicotinic antagonists, and (ii) the effect of nicotine was not mimicked by acetylcholine. In the present study, the presence of functional nicotinic receptors in rat neocortex has been assessed in a slice preparation of prefrontal cortex, using evoked field potential and unit recordings. Nicotine and the nicotinic agonists, dimethylphenylpiperazinium, cytisine, acetylthiocholine, applied by iontophoresis, produced an increase in the negative wave of field potentials, reflecting an increased excitability of cortical neurons. This effect was blocked by the selective probe for neuronal nicotinic receptors Toxin F (1.4 microM in the perfusion medium) and by dihydro-beta-erythroidine (100 microM). Alpha-bungarotoxin, the blocker of skeletal muscle acetylcholine receptor had no effect. Iontophoretically applied acetylcholine, muscarine and pilocarpine, on the other hand, produced a decrease in the field potential amplitude, which was blocked by atropine and scopolamine (1-10 microM). In the presence of eserine (10 microM), the muscarinic effect of acetylcholine was dramatically altered, leading to the development of a nicotinic response sensitive to Toxin F. Thus, the physiological activation of nicotinic receptors in rat prefrontal cortex appears to require higher concentrations of acetylcholine than do muscarinic receptors. Our results show that: (i) the rat prefrontal cortex possesses functional nicotinic receptors with a pharmacological profile clearly distinct from muscle receptors, and (ii) a nicotinic effect of acetylcholine can be revealed when its degradation by acetylcholinesterase is inhibited.

Evidence for distinct spinal locomotion generators supplying respectively fore- and hindlimbs in the rabbit

Evidence for a central rhythm generator of locomotion at the spinal level has been provided in the acute spinal cat2, 6. Such a generator also occurs in therabbi t preparation 16,1s. These results are mainly related to the properties of the lumbosacral cord (low spinal preparations) since, in decapitate animals, forelimb stepping activities are either absent, very weak or irregular 1°,~,~4. Nonetheless, Miller and van der Meche 12 described the coordinated stepping of all 4 limbs in the high spinal cat after administration of nialamide and DOPA. EMGs were recorded in the cat moving on a treadmill and, in these conditions, the only proximal muscles of the forelimb displayed locomotor bursts, the more distal being silent. More recently, foreand hindlimb movements have been obtained in high spinal cats deprived of phasic afferent inputs 3. In these previous studies, the purpose was not to evaluate whether or not the cervical spinal cord, on its own, had the capability of generating locomotor bursts. It is not known from these experiments to what extent ascending propriospinal inputs from the lumbosacral levels could operate in forelimb locomotor activation. In the curarized rabbit, evidence has been given of independent generation of locomotor activity at fore and hind levels ~7,2°. In the present study we compare the locomotor capabilities of the isolated cervicothoracic cord with those of the entire spinal cord in curarized rabbit preparations injected with nialamide and DOPA, and bring data about intergirdle coordination in fictive locomotion. Experiments were carried out in decorticate, unanaesthetized and curarized rabbits. Nerves to forelimb flexor muscles (left and right deep radial nerves, DR, supplying wrist flexor muscles) and to hindlimb flexor muscles (left and right anterior tibial muscle nerves, TA, supplying ankle flexor muscles) were prepared. Central locomotor activities were so recorded in every limb from nerves to flexor muscles working at corresponding joints (wrist or ankle). Left and right superficial radial nerves (i.e. forelimb cutaneous nerves) were also prepared for stimulating their central cut ends. Stimulus intensity and duration could be adjusted so as to excite either all A fibre group (short shocks: 0.1 msec) or both A and C fibre groups (longer shocks: 1 msec), the afferent

The effect of medial hypothalamus lesions on pain control

In the rat, discrete electrolytic lesions located in 6 different parts of the medial hypothalamus (MH) are shown to induce clearcut hyperalgesia. During a time limit of 14 days following the lesions, no other obvious deficits were noticed (in sensory and/or motor functions, affectivity towards conspecifics, food and water intake). Three nociceptive reactions (tail withdrawal, vocalization, vocalization after-discharge) were tested and their thresholds measured following electrical stimulation of the tail. The lesions aimed at the rostral part of the arcuate nucleus, as well as at ventromedial and dorsomedial nuclei, produced the most profound hyperalgesia. The possible involvement of the endorphinergic and enkephalinergic systems known to be located in the MH is discussed. The relation between the hyperalgesic effects of MH lesions and various structures (limbic areas, descending pain control system, pituitary) is also considered.

Histopathological and cognitive defects induced by Nef in the brain

Complex mechanisms of human immunodeficiency virus type‐1 (HIV‐1) brain pathogenesis suggest the contribution of individual HIV‐1 gene products. Among them, the Nef protein has been reported to harbor a major determinant of pathogenicity in AIDS‐like disease. The goal of the present study was to determine whether Nef protein expressed in vivo by primary macrophages could induce a brain toxicity also affecting the behavior of the rat. To achieve this goal we grafted Nef‐transduced macrophages into the rat hippocampus. Two months post‐transplantation, we observed that Nef induces monocyte/macrophage recruitment, expression of TNF‐α, and astrogliosis. No apoptotic event was detected. We further demonstrated that Nef neurotoxicity is associated with cognitive deficits.—Mordelet, E., Kissa, K., Cressant, A., Gray, F., Ozden, S., Vidal, C., Charneau, P., Granon, S. Histopathological and cognitive defects induced by Nef in the brain. FASEB J. 18, 1851‐1861 (2004)